issn 131fl-d5d7 Pages 1-266 ■ Year 2017, Vol. 64, No. 1 SkpYrnnkii hrvri*flJu> druitro StavmiMn Chrmk*J Strcirtp ActaChimicaSlo Acta Chimica Slo Slovenica ActaC t. ESpM- V * ■ k * r* ■ < , " 20 um- W A * tC v 20 um V y Q " ^^ . iVi 64/2017 Review: Theoretical Purge Factor Determination as a Control Strategy for Potential Mutagenic Impurities in the Synthesis of Drug Substances ■ The Lock is the Key: Development of Novel Drugs through Receptor Based Combinatorial Chemistry Scientific papers: Forward Osmosis in Wastewater Treatment Processes ■ Influence of Thermal and Bacterial Pretreatment of Microalgae on Biogas Production in Mesophilic and Thermophilic Conditions ■ Monodispersed Gold Nanoparticles as a Probe for the Detection of Hg2+ Ions in Water http://acta.chem-soc.si 9771318020004 EDITOR-IN-CHIEF ALEKSANDER PAVKO Slovenian Chemical Society, Hajdrihova 19, SI-1000 Ljubljana, Slovenija, E-mail: ACSi@fkktMni-lj.si, Telephone: (+386)-1-476-0252; Fax: (+386)-1-1-476-0300 ASSOCIATE EDITORS Marija Bešter-Rogač, University of Ljubljana, Slovenia Janez Cerkovnik, University of Ljubljana, Slovenia Krištof Kranjc, University of Ljubljana, Slovenia Franc Perdih, University of Ljubljana, Slovenia Helena Prosen, University of Ljubljana, Slovenia Damjana Rozman, University of Ljubljana, Slovenia Melita Tramšek, Jožef Stefan Institute, Slovenia Irena Vovk, National Institute of Chemistry, Slovenia ADMINISTRATIVE ASSISTANT Marjana Gantar National Institute of Chemistry, Slovenia EDITORIAL BOARD Wolfgang Buchberger, Johannes Kepler University, Austria Alojz Demšar, University of Ljubljana, Slovenia Stanislav Gobec, University of Ljubljana, Slovenia Marko Goličnik, University of Ljubljana, Slovenia Günter Grampp, Graz University of Technology, Austria Wojciech Grochala, University of Warsaw, Poland Danijel Kikelj, Faculty of Pharmacy, Slovenia Ksenija Kogej, University of Ljubljana, Slovenia Janez Košmrlj, University of Ljubljana, Slovenia Blaž Likozar, National Institute of Chemistry, Slovenia Mahesh K. Lakshman, The City College and The City University of New York, USA Janez Mavri, National Institute of Chemistry, Slovenia Friedrich Srienc, University of Minnesota, USA Walter Steiner, Graz University of Technology, Austria Jurij Svete, University of Ljubljana, Slovenia Ivan Švancara, University of Pardubice, Czech Republic Jiri Pinkas, Masaryk University Brno, Czech Republic Gašper Tavčar, Jožef Stefan Institute, Slovenia Christine Wandrey, EPFL Lausanne, Switzerland Ennio Zangrando, University of Trieste, Italy, Chairman Branko Stanovnik, Slovenia Members Josef Barthel, Germany Udo A. Th. 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Tisk - Printed by: Tiskarna Skušek, Ljubljana © Copyright by Slovenian Chemical Society Graphical Contents ActaChimicaSlc ActaChimicaSlc Slovenica ActaC Year 2017, Vcl. 64, Nc. 1 REVIEW 1—14 Organic chemistry Theoretical Purge Factor Determination as a Control Strategy for Potential Mutagenic Impurities in the Synthesis of Drug Substances Nevenka Kragelj Lapanja, Renata Toplak Casar, Sabina Jurca and Bojan Doljak 15—39 Organic chemistry The Lock is the Key: Development of Novel Drugs through Receptor Based Combinatorial Chemistry Nikola Marakovic and Goran Sinko SCIENTIFIC PAPER 40-44 Physical chemistry Computational Investigation of the Dissociative Adsorption of Dichloroacetylene (C2Cl2) on N Functionalized Carbon and Carbon Germanium (CGe) Nanocone Sheets in the Gas Phase and Dimethyl Sulfoxide Meysam Najafi Graphical Contents 45—54 Physical chemistry Infrared Spectroscopy for Analysis of Co-processed Ibuprofen and Magnesium Trisilicate at Milling and Freeze Drying Manoj Acharya, Satyaki Mishra, Rudra N. Sahoo and Subrata Mallick 55-62 Materials science MnO2 Submicropartides from Chinese Brush and Their Application in Treatment of Methylene Blue Contaminated Wastewater Qi Wang, Chunlei Ma, Wanjun Li, Meng Fan, Songdong Li and Lihua Ma 63—72 Chemical education Development of Chemistry Pre-Service Teachers During Practical Pedagogical Training: Self-Evaluation vs. Evaluation by School Mentors Vesna Ferk Savec and Katarina S. Wissiak Grm 73-82 Materials science Preparation and Catalytic Study on a Novel Amino-functionalized Silica-coated Cobalt Oxide Nanocomposite for the Synthesis of Some Indazoles Mohammad Ali Ghasemzadeh, Bahar Molaei, Mohammad Hossein Abdollahi-Basir and Farzad Zamani 83—94 Chemical, biochemical and environmental engineering Forward Osmosis in Wastewater Treatment Processes Jasmina Korenak, Subhankar Basu, Malini Balakrishnan, Claus Helix-Nielsen and Irena Petrinic Graphical Contents 95-101 Physical chemistry A Novel High-performance Electrospun Thermoplastic Polyurethane/Poly(vinylidene fluoride)/Polystyrene Gel Polymer Electrolyte for Lithium Batteries Yuanyuan Deng, Zeyue He, Qi Cao, Bo Jing, Xianyou Wang and Xiuxiang Peng Lithium-ion rechargeable battery Discharge mechanism .....— ........- <■' ■ _ tlRCbolyta 102—116 Organic chemistry Synthesis, Characterization and Cytotoxicity of Substituted [1]Benzothieno[3,2-e][1,2,4]triazolo [4,3-a]pyrimidines Samir Botros, Omneya M. Khalil, Mona M. Kamel and Yara S. El-Dash 117—128 Physical chemistry Synthesis, Cytotoxic and Anti-proliferative Activity of Novel Thiophene, Thieno[2,3-ö]pyridine and Pyran Derivatives Derived from 4,5,6,7-tetrahydrobenzo[ö] thiophene Derivative Rafat Milad Mohareb, Nadia Youssef Megally Abdo and Fatma Omar Al-farouk 129-143 Materials science Green Biosynthesis of Spherical Silver Nanoparticles by Using Date Palm (Phoenix Dactylifera) Fruit Extract and Study of Their Antibacterial and Catalytic Activities Saeed Farhadi, Bahram Ajerloo and Abdelnassar Mohammadi 144—158 Chemical, biochemical and environmental engineering Utilization of Corn Cob and TiO2 Photocatalyst Thin Films for Dyes Removal Hui-Yee Gan, Li-Eau Leow and Siew-Teng Ong Graphical Contents 159—169 Organic chemistry Synthesis of Some Unique Carbamate Derivatives bearing 2-Furoyl-1-piperazine as a Valuable ... Muhammad Athar Abbasi, Ghulam Hussain, Aziz-ur-Rehman, Sabahat Zahra Siddiqui, Syed Adnan Ali Shah, Muhammad Arif Lodhi, Farman Ali Khan, Muhammad Ashraf, Qurat-ul-Ain, Irshad Ahmad, Rabia Malik, Muhammad Shahid and Zahid Mushtaq 170—178 Materials science Nitrogen Doped Graphene Nickel Ferrite Magnetic Photocatalyst for the Visible Light Degradation of Methylene Blue Rajinder Singh, Jigmet Ladol, Heena Khajuria and Haq Nawaz Sheikh 179-185 Inorganic chemistry Synthesis, Structures, and Antimicrobial Activities of Two Cobalt(II) Complexes [Co(L1)2(OH2)2] and [Co(L\] Yong-Jun Han, Li Wang, Qing-Bin Li and Ling-Wei Xu 186—192 Chemical, biochemical and environmental engineering Monodispersed Gold Nanoparticles as a Probe for the Detection of Hg2+ Ions in Water Bindhu Muthunadar Rajam, Parimaladevi Ramasamy and Umadevi Mahalingam 193-201 Analytical chemistry Poly-Dianix Blue/Multi-Walled Carbon Nanotube Modified Electrode for Detection of Levodopa in the Presence of High Concentrations of Ascorbic and Uric Acids Abdolhamid Hatefi-Mehrjardi, Mohammad Ali Karimi, Azam Barani and Mahdiyeh Soleymanzadeh Graphical Contents 202—207 Organic chemistry Mn(II), Zn(II) and Cd(II) Complexes Based on Oxa-diazole Backbone Containing Carboxyl Ligand: Synthesis, Crystal Structure, and Photoluminescent Study Li-Na Wang, Lin Fu, Jia-Wei Zhu, Yu Xu, Meng Zhang, Qi You, Peng Wang and Jie Qin 208—214 Inorganic chemistry Synthesis and Structure of [Cu(Hapn)]NO3]NO3, [Cu(Hapn)(H2O)2]SiF6, [Cu(Hapn)(H2O)BF4]BF4 • H2O and [Cu(Hapn)(NH2SO3)2] n-complexes (apn = 3-(prop-2-en-1-ylamino)propanenitrile) Mykhailo Luk'yanov, Evgeny Goreshnik, Vasyl Kinzhybalo, and Marian Mys'kiv 215—220 Inorganic chemistry Three 1D cyanide-bridged M(Ni, Pd, Pt)-Mn(II) Coordination Polymer: Synthesis, Crystal Structure and Magnetic Properties Jingwen Shi, Chongchong Xue, Lingqian Kong and Daopeng Zhang 221—226 Inorganic chemistry Phase Equilibria and some Properties of Solid Solutions in The Tl5Te3-Tl9SbTe6-Tl9GdTe6 System Samira Zakir Imamaliyeva, Turan Mirzaly Gasanly, Vagif Akber Gasymov and Mahammad Baba Babanly 227—236 Chemical, biochemical and environmental engineering Influence of Thermal and Bacterial Pretreatment of Microalgae on Biogas Production in Mesophilic and Thermophilic Conditions Beti Vidmar, Romana Marin{ek Logar, Mario Panji~ko and Lijana Fanedl Graphical Contents 237—247 Chemical, biochemical and environmental engineering Biosorption of 2,4 dichlorophenol Onto Turkish Sweetgum Bark in a Batch System: Equilibrium and Kinetic Study Dilek Ylldlz, Feyyaz Keskin and Ahmet Demirak 248-255 Analytical chemistry Separation/preconcentration of Cr(VI) with a Modified Single-drop Microextraction Device and Determination by GFAAS Sandor Kapitany, Erzsébet Soki, Jozsef Posta and Âron Béni Sample Crflll), Cr(V!) SOME — CrfVI) GFAAS , Cf(lll) SHORT COMMUNICATION 256-260 Physical chemistry About the Randi} Connectivity, Modify Randi} Connectivity and Sum-connectivity Indices of Titania Nanotubes TiO2(m,n) Wei Gao, Mohammad Reza Farahani and Muhammad Imran 261—265 Inorganic chemistry A Rarely Seen Phenolato and Azido-Bridged Polymeric Cadmium(II) Complex Derived from 2-Bromo-6-[(2-isopropylaminoethylimino)methyl]phe-nol Guo-Ping Cheng, Ling-Wei Xue and Cai-Xia Zhang Graphical Contents DOI: 10.17344/acsi.2016.2840 Acta Chim. Slov. 2017, 64, 1-14 ^creative tS/commons Review Theoretical Purge Factor Determination as a Control Strategy for Potential Mutagenic Impurities in the Synthesis of Drug Substances 1 V I 1 Nevenka Kragelj Lapanja,1* Renata Toplak Casar,1 Sabina Jurca1 and Bojan Doljak2 1 Lek Pharmaceuticals d.d., Verovškova 57, 1526 Ljubljana, Slovenia 2 Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: nevenka.lapanja@sandoz.com Tel: +386j 1 5803443 Received: 24-08-2016 Abstract Mutagenic impurities (MIs) are of serious concern for pharmaceutical industry, regulatory agencies and public health. The first guideline addressing the control of genotoxic impurities (GTIs) dates back to 2006. Since then there have been several updates and refinements, which eventually resulted in the guideline, published by the International Conference on Harmonisation (ICH) in June 2014. The ICH M7 guideline, compared to previous ones, offers greater flexibility in terms of control strategies for GTIs in drug substances. More specifically, it describes a control strategy that relies on process controls in lieu of analytical testing which is based on understanding the process chemistry and process parameters that impact the levels of GTIs. This principle is adopted in the theoretical purge factor determination tool proposed by Teas-dale et al. Several case studies applying the proposed theoretical purge factor determination tool were published in recent years. The results confirm the tool's good predictability of the extent to which the impurity is removed by the process. Hopefully, this approach will soon be released as an in-silico tool, generally accepted by the regulatory agencies. Keywords: Drug substance, mutagenic impurity, purge factors 1. Introduction The need to investigate the potential genotoxicity of drugs resulted from several incidents in the past and is nowadays a serious matter of concern for pharmaceutical industry. According to the definition given in the International Conference on Harmonisation (ICH) M7 guideline,1 genotoxicity refers to any deleterious change in the genetic material regardless of the mechanism by which the change is induced, whereas the term mutagen refers to a substance that induces mutation which is a heritable change in cells or organisms.2 It should be stressed that not all DNA damage results in mutation. However, many mutagens have the ability to induce cancer since there is a strong correlation between mutagenicity and carcinogeni-city.2 Without a doubt, mutagenicity and consequently potential carcinogenicity are strongly undesirable in relation to the use of medicines. However, in some cases, e.g., for treating life-threatening conditions, the use of drugs with higher risk may be acceptable. While a safe medicinal product is one with acceptable risk/benefit ratio, the same is not true for impurities found in drug substances and drug products; as impurities convey only risk with no associated benefit. Genotoxic impurities (GTIs) in drug substances are mainly the consequence of using electrophilic reagents for building up the molecular structure. If they don't react completely, they can persist in the reaction mixture and may be carried onward in the synthesis. Due to their high reactivity they can also react with the DNA and potentially induce genetic mutations. For this reason regulatory agencies established standards which assure that unavoidable impurities are limited to have no or acceptable levels of risk.3 Identification and control of potential mutagenic/genotoxic impurities in drug substances or drug products is still a challenging task for pharmaceutical companies. Hence, an overview of regulatory guide- Lapanja et al.: Theoretical Purge Factor Determination 2 Acta Chim. Slov. 2017, 64, 1-14 lines will be presented in this review article, together with identification and control strategies, especially the theoretical purge factor determination approach and its practical application. 2. Historical Background As already mentioned in the introduction, the risk related to the potential presence of GTIs emerged from various events in the past. In 2000 a first article regarding GTIs' related concern was published, i.e. an enquiry by the European Directorate for the Quality of Medicines and Healthcare (EDQM) on alkyl mesylate impurities in mesylate salts.4 This publication was the first that revealed the potential risk of formation of sulfonate esters during a salt formation process with sulfonic acids in alcoholic solutions and it is now considered as a milestone indicating a beginning of genotoxicity risk awareness.4,5 Two years later, in December 2002, the Committee for Proprietary Medicinal Products (CPMP) which was later renamed to Committee for Human Medicinal Products (CHMP), published a position paper on the limits of GTIs.6 The position paper was, after being significantly revised, released as a draft guideline in June 2004.7 The awareness of geno-toxic risk was significantly increased by the prominent incident of Viracept® in 2007. In June of that year excess levels of ethyl methane sulfonate (EMS) were detected in the nelfinavir mesylate active substance, manufactured by Roche Registration Ltd. EMS is a process-related impurity that was formed during manufacture of Viracept due to an inadvertent reaction between methane sulfonic acid used in the active pharmaceutical ingredient (API) salt formation and the solvent ethanol which was used to clean the acid storage tank. Since EMS is a potential human carcinogen, Roche had to recall the product from the European Union markets immediately.8 3. Regulatory Guidelines 3. 1. EMA Guideline on the Limits of Genotoxic Impurities The first guideline that addressed the control of GTIs in marketing applications for pharmaceuticals was the European Medicines Agency (EMA, formerly EMEA) guideline,9 finalized in 2006 (draft published in June 2004). Before its implementation, the issue of impurities with genotoxic potential was not specifically covered by the existing guidelines for qualification of impurities (ICH Q3A (R2)10/Q3B(R2)11/Q3C (R5)12/Q3D13). In the context of the EMA guideline,9 the term genotoxic impurity refers to positive findings in established in vitro or in vivo genotoxicity tests with the main focus on DNA reactive substances. GTIs may be classified as those with suf- ficient or those without sufficient (experimental) evidence for a threshold-related mechanism of genotoxicity. For compounds with clear evidence for threshold genotoxi-city, exposure levels that are without considerable risk of genotoxicity can be established based on calculation of a permitted daily exposure (PDE), which is derived from the no-observed-effect level (NOEL), or the lowest-observed-effect level (LOEL) in the most relevant animal study using uncertainty factors. For compounds without sufficient evidence for threshold-related mechanism the as low as reasonably practicable' (ALARP) principle should be followed, where avoiding is not possible. However, it is often impossible to define a safe exposure level for geno-toxic carcinogens without a threshold or completely eliminate GTIs from the drug substance. This has led to the need of a pragmatic approach that would recognize an acceptable risk exposure level. For this purpose a threshold of toxicological concern (TTC) has been developed. A TTC value of 1.5 ^g/person/day, corresponding to a 10-5 lifetime risk of cancer, defines a common exposure level for any unstudied chemical that will not pose a risk of significant carcinogenicity or other toxic effects.14,15 The limit was set based on the analysis of 343 carcinogens,16 expanded to more than 700 carcinogens from a carcinogenic potency database.1719 A simple linear extrapolation from 50 % tumor incidence (TD50) data for the most sensitive species and most sensitive site to a 1 in 106 incidence was used, which makes the principle very conservati-ve.14 Some high potency genotoxic carcinogens like afla-toxin-like-, N- nitroso-, and azoxy- compounds have to be excluded from the TTC approach.19 Compound-specific toxicity data is needed for the risk assessment of such compounds. A TTC value higher than 1.5 ^g/day may be acceptable for short term-exposure drugs, for treatment of life-threatening conditions, when life expectancy is less than 5 years, or where the impurity is a known substance and human exposure will be much greater from other sources, e.g. food. For the calculation of concentration limits in ppm of genotoxic impurity in drug substance the following equation is used, where dose applies to expected daily dose to the patient: concentration limit (ppm) : TTC [|ig/day] dose[g/day] (1) The guideline on the limits of GTIs 9 left certain concerns unaddressed. Besides that, industry struggled to fully understand how to interpret and apply it in its enti-rety.5 For this reason significant clarifications of several key topics have been issued in the Question and Answers (Q&A) on the 'Guideline on the limits of genotoxic impurities',20 published by the Safety Working Party (SWP) in September 2010. The Q&A document clarified that no ge-notoxicity testing or the ALARP principle application is needed when a potential GTI is controlled at the TTC level unless the impurity belongs to a class of very potent Lapanja et al.: Theoretical Purge Factor Determination 3 Acta Chim. Slov. 2017, 64, 1-14 genotoxic carcinogens, e.g., N-nitroso-, aflatoxin-like-and azoxy- compounds. It was also clarified that a negative bacterial mutagenicity test (Ames test) overrules a structural alert which means that no further studies are required providing the level remains below ICH Q3A10/Q3B11 limits. If the quantitative structure-activity relationship (QSAR) assessment gives no structural alerts it can be concluded that the impurity has no genotoxicity concern and no further qualification studies or justification will be required. It has also been clarified and confirmed that durational adjustments to the TTC limit are acceptable for investigational studies. The proposal of a staged TTC was first described by the Pharmaceutical Research and Manufacturers of America (PhRMA) cross-industry workgroup led by Mueller et al.21 However, the SWP incorporated a dose rate correction factor of 2 to account for deviations from the linear extrapolation model which gives slightly different values than those from the original PhRMA proposal. The acceptable limits for daily intake of GTI according to the SWP are 5, 10, 20 and 60 |g/day for duration of exposure of 6-12 months, 3-6 months, 1-3 months, and less than 1 month, respectively. For a single dose an intake of up to 120 |g is acceptable. With regards to the control of multiple GTIs, SWP stated that the TTC value of 1.5 |g/day can be applied to each individual impurity present in the drug substance only if the impurities are structurally unrelated. This is based on the assumption that the impurities act by the same genotoxic mode of action and have the same molecular target and thus might exert its effect in an additive manner. A limitation of the sum of the GTIs at 1.5 |g/day is recommended in such cases. The SWP document states that if a GTI is formed or introduced in a step before the final synthetic step, it is acceptable to not include the impurity in the drug substance specification if it is controlled to a suitable limit in a process intermediate. However, it has to be demonstrated by analysis results that the presence of this impurity does not exceed 30 % of the acceptable limit in the drug substance, otherwise it has to be included in the drug substance specification and the test has to be carried out on a routine basis. When a GTI is formed or introduced in the final synthesis step, it should be included in the specifications. However, skip testing can be applied if the level of the impurity does not exceed 30% of the acceptable limit in the drug substance. Data for at least 6 consecutive pilot scale or 3 consecutive production scale batches should be presented to support this approach. 3. 2. FDA Draft Guidance: Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches In December 2008, the Food and drug administration (FDA) published their draft guidance addressing the issue of GTIs.22 The guidance contained nonbinding recommendations to the pharmaceutical industry and never reached its finalization. FDA considers the approach taken in the EMA guideline9 for setting an exposure limit for ge-notoxic or carcinogenic impurities reasonable. However, the EMA guideline addresses the exposure limits only to products for marketing applications. Therefore, the FDA draft guidance provides recommendations on evaluation and acceptable exposure thresholds of genotoxic and carcinogenic impurities during clinical development as well as for marketing applications. According to the guidance, the potential lifetime cancer risk associated with genoto-xic and carcinogenic impurities can be reduced by changing the synthetic and/or purification route to minimize the formation and/or maximize the removal of the impurity of concern. Following the EMA guideline,9 a maximum daily exposure of 1.5 |g/day was proposed, allowing higher levels for products during clinical development.22 3. 3. ICH M7 Guideline: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk In June 2014 the ICH M7 guideline: Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk1 reached Step 4 of the ICH process, meaning that the final draft became recommended for adoption to the three regulatory bodies of the ICH: European Union, Japan and USA. Implementation of ICH M7 was encouraged after publication; however, its application was not expected prior to 18 months after the publication. The purpose of the ICH M7 guideline is to provide a practical framework that is applicable to the identification, categorization, qualification, and control of mutagenic impurities (MIs) to limit potential carcinogenic risk. It applies to new drug substances and new drug products during their clinical development and subsequent applications for marketing. It also applies to post-approval submissions of marketed products, and to new marketing applications for products with a drug substance that is present in an already approved product. This is only valid when (1) changes that result in new impurities are made or (2) increased limits for existing impurities are implemented or (3) when changes in indication or dosing regimen are made which significantly affect the acceptable cancer risk level. As previously already proposed by the EMA9 and FDA guideli-ne,22 the ICH M7 also finds it justified to use the TTC approach in the assessment of acceptable limits for any unstudied chemical. Higher acceptable intakes of impurities for less-than-lifetime (LTL) exposures are also allowed. Moreover, it is stressed that the TTC concept is a highly hypothetical concept that should not be regarded as a realistic indication of the actual risk and that exceeding the Lapanja et al.: Theoretical Purge Factor Determination 4 Acta Chim. Slov. 2017, 64, 1-14 TTC is not necessarily associated with an increased cancer risk. The impurity assessment according to the ICH M7 should include all actual and potential impurities that are likely to arise during the synthesis and storage of a drug substance, and during manufacturing and storage of a drug product. All these should then be evaluated for mu-tagenic potential by conducting database and literature searches for carcinogenicity and bacterial mutagenicity data. Based on the obtained data the impurities are classified as one of the following classes: Class 1: Impurities that are known mutagenic carcinogens. Class 2: Impurities that are known mutagens with unknown carcinogenic potential. Class 3: Impurities with alerting structure, unrelated to the structure of the drug substance; no mutagenicity data. Class 4: Impurities with alerting structure, same alert in drug substance or compounds related to the drug substance which have been tested and are non-mutagenic. Class 5: Impurities with no structural alerts, or alerting structure with sufficient data to demonstrate lack of mutagenicity or carcinogenicity. If data for carcinogenicity and bacterial mutagenicity are not available, a (Q)SAR assessment that focuses on bacterial mutagenicity predictions should be performed. Two (Q)SAR computational methodologies that complement each other are required according to the ICH M7. One methodology should be expert rule-based and the second one should be statistical-based. If none of the methods give structural alerts, it is sufficient to conclude that the impurity is non-mutagenic (Class 5). In case of an identified structural alert, a bacterial mutagenicity assay, e.g., Ames test, can be conducted. Negative result will overrule any structural alert, meaning that no further geno-toxicity assessment is needed (Class 5). In case of positive bacterial mutagenicity assay, a further assessment and/or control strategy is needed (Class 2). In vivo genotoxicity assays could also be performed, for example when levels of the impurity cannot be controlled at an acceptable limit and the relevance of the bacterial mutagenicity under in vivo conditions needs to be understood. If an impurity has the same structural alert as the drug substance or related compounds, this impurity can be considered as non-muta-genic if the bacterial mutagenicity assays of the drug substance or related compounds were negative. For class 1 impurities with positive carcinogenicity data a compound-specific acceptable intake calculated based on carcinogenic potency and linear extrapolation can be used. Other established risk assessment practices or already existing values used by regulatory bodies may also be applied. For impurities which are chemically similar to a known carcinogen compound class, class specific acceptable intakes can be applied when justified. For MIs with non-linear dose response or practical threshold a PDE can be calculated based on NOEL and uncertainty factors. When treatment duration is less than lifetime, the acceptable cumulative li- fetime dose is uniformly distributed over the total number of exposure days during treatment. Acceptable intakes for LTL to lifetime exposures for clinical development and marketing are presented in Table 1. The TTC-based acceptable intakes should be applied to each individual impurity. However, when there are three or more Class 2 or Class 3 impurities present in the drug substance, total mutagenic impurities should be limited as presented in the Table 1. Class 1 impurities with compound-specific or class-related acceptable intakes limits should be excluded from this total limits. Degradation impurities originating from drug products also need to be controlled individually. Table 1. Acceptable intakes for less-than-lifetime (LTL) to lifetime exposures for a) an individual impurity and b) for multiple impurities (based on ICH M71) Treatment duration Maximum daily dose [^g/day] a) b) < 1 month 120 120 > 1-12 months 20 60 > 1-10 years 10 30 > 10 years to lifetime 1.5 (TTC limit) 5 Besides the described acceptable intakes ICH M7 also lists some exceptions and flexibilities in approaches, e.g., higher acceptable intakes for impurities which are more abundant in other sources e.g., food, or products of endogenous metabolism (e.g., formaldehyde), than in pharmaceuticals. Exceptions can also be made in cases of severe disease, reduced life expectancy, late onset but chronic disease, or when there are limited therapeutic alternatives. Impurities with high carcinogenic potency (af-latoxin-like, N-nitroso, and alkyl-azoxy structures) need to be controlled with tighter limits, based on carcinogeni-city data. For classes 2 and 3 the TTC approach would usually be used. When an impurity has been identified as Class 1, 2 or 3, a control strategy needs to be developed; assuring that the level of this impurity in the drug substance and drug product is below the acceptable limit. ICH M7 lists 4 potential approaches for development of a control strategy for drug substance: Option 1: Test for the MI is included in the drug substance specification. Acceptance criterion is set at or below the acceptable limit using a suitable analytical method. When it can be shown that levels of the impurity in at least 6 consecutive pilot scale or 3 consecutive production scale batches of drug substance are less than 30 % of the acceptable limit, it is justified to apply periodic verification testing. Option 2: Test for the MI is included in the specification for raw material, starting material or intermediate, or as an in-process control. Acceptance criterion is set at or below the acceptable limit using a suitable analytical method. Option 3: Test for the MI is included in the specification for raw material, starting material or intermediate, or as an in-process control. Acceptance criterion is set Lapanja et al.: Theoretical Purge Factor Determination 5 Acta Chim. Slov. 2017, 64, 1-14 above the acceptable limit of the impurity in drug substance, using a suitable analytical method coupled with demonstrated understanding of fate and purge and associated process controls that assure the level in the drug substance is below the acceptable limit without the need for any additional testing later in the process. Option 3 can be justified when the level of the impurity will be less than 30 % of the acceptable limit by review of laboratory scale experiments data (e.g., spiking studies). Option 4: The MI does not need to be included on any specification when it can be demonstrated that the level of the impurity in the drug substance will be below the acceptable limit such that no analytical testing is required. Option 4 control strategy relies on understanding process chemistry and process parameters and their impact on residual impurity levels, including fate and purge knowledge. According to the ICH M7, justification of this control approach based on scientific principles alone is sufficient Table 2: A brief history of development of GTIs guidelines (based on Teasdale5 and Szekely et al.24). Year Issue Key points March 1995 ICH Q3A: Impurities in New Drug substances The term 'unusually toxic' is used to address GTIs. 2000 PharmEuropa Enquiry: Alkyl mesylate (methane sulfonate) impurities in mesylate salts The first article regarding the GTIs related concern published (potential risk of formation of sulfonate esters during a salt formation process). December 2002 CPMP: Position paper on the limits of genoto-xic impurities Wherever possible, alternative routes that avoid GTIs should be used. Otherwise they should be reduced to 'as low as technically feasible' level. Safety tests, including in vivo studies are required to determine a NOEL or to carry out a quantitative risk assessment. June 2004 CHMP: Guidelines on the limits of genotoxic impurities - Draft 'As low as technically feasible' terminology is replaced with the ALARP (As low as reasonably practical) principle. Requirement to introduce an alternative route is omitted. The need to provide justification of selected route remains. TTC concept is introduced. January 2006 PhRMA (Mueller) White paper A 'staged TTC' approach is introduced. A classification system, defining five separate classess of impurities, is defined. June 2006 CHMP: Guidelines on the limits of genotoxic impurities - Finalized The note that the guideline doesn't need to be applied retrospectively to authorised products unless there is specific cause for concern is added. Excipients are excluded from the finalized guideline. December 2008 FDA draft guidance: Genotoxic and carcinogenic impurities in drug substances and products: recommended approaches It is suggested to introduce lower limits for different patient populations (e.g. pediatric). Genotoxicity testing should be performed for any impurity above the ICH qualification threshold. Different staged TTC values for short term studies are proposed. September 2010 SWP: Questions and Answers on the CHMP Guideline on the limits of genotoxic impurities Durational adjustments to the TTC limit are acceptable for investigational studies. A 'cause of concern' terminology is explained. If a substance is controlled to an appropriate safety based limit, then no further actions are required. June 2014 ICH M7: Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk Two (Q)SAR computational methodologies that complement each other are required (one expert rule-based and the second one statistical-based). Four potential approaches to development of a control strategy for drug substance are proposed, including a control strategy that relies on understanding process chemistry and process parameters and their impact on residual impurity levels, including fate and purge knowledge. June 2015 ICH M7 Addendum: Application of the principles of the ICH M7 guideline to calculation of compound-specific acceptable intake Acceptable intakes have been derived for substances that are considered to be mutagens and carcinogens and are commonly used in the manufacture of drug substances. Lapanja et al.: Theoretical Purge Factor Determination 6 Acta Chim. Slov. 2017, 64, 1-14 in many cases. The scientific risk assessment used to justify this approach can be based on physicochemical properties and process factors that influence the fate and purge of an impurity. This includes chemical reactivity, solubility, volatility, ionizability and any physical process steps designed to remove impurities. The result of this risk assessment can be shown as an estimated purge factor for clearance of the impurity by the process. When justification based on scientific principles alone is not considered sufficient, analytical data to support the control approach is expected. If option 4 approach (and also option 3 approach) cannot be justified, a test for the impurity should be included on the specification of a drug substance, raw material, starting material, intermediate, or as an in-process control. ICH M7 guideline also clarifies that the application of ALARP principle is not necessary if the level of the MI is below acceptable limits. It is also not necessary to demonstrate that alternative routes of synthesis have been explored which was required by EMA guideline9 before the implementation of ICH M7. ICH M7 guideline addresses many issues that were left unclear in the previous guidelines. The guideline is still very complex and its application in the pharmaceutical industry and regulatory agencies is quite challenging. To complement the harmonized guideline finalized in June 2014, an Addendum to ICH M7 was proposed in June 2015 (Step 2): Application of the principles of the ICH M7 guideline to calculation of compound-specific acceptable intakes.23 The purpose of this document is to provide useful information regarding the acceptable limits of known mutagenic/carcinogenic impurities commonly found or used in drug synthesis and supporting monographs. The development of the guidelines toward the ICH M7 publication is presented in Table 2. Pharmaceutical industry can apply different approaches to mitigate the risk of GTIs in the synthesis of APIs. While the preferred approach (especially augmented by the regulatory agencies in early guidelines) is to avoid the use of genotoxic synthetic pathways by modifying the existing synthetic routes, this is not always possible since the use of highly reactive reagents is often required for the production of APIs.25 Therefore, a strategy based on elimination or reduction of GTI can be applied. This can be achieved by adjusting the process conditions (i.e., reaction time, pH, temperature, solvent matrix etc.). Furthermore, a Quality by Design (QbD) approach can also be applied to control GTI formation.26 Many purification steps (i.e. crystallization, solvent liquid-liquid extraction, precipitation, distillation, column chromatography, etc.) have the ability to remove GTIs along with other process impurities. Purging of impurities was previously addressed by Pierson et al.27 The risk of GTI carry over was defined considering the number of synthetic steps between the point of GTI appearance and final production step. If the GTI appears more than four steps before the final step, chemical rationale could be used to assess the need of GTI removal. The purging approach was later upgraded as it will be presented in the following section. 4. Theoretical Purge Factor Determination Approach Since publishing the guidelines covering the control of GTIs, regulatory authorities have requested evidence that any GTI is controlled in line with the acceptable limits. For this reason pharmaceutical companies had to present extensive analytical data. To avoid unnecessary analytical testing, Teasdale et al.28 took the challenge to develop an approach that would allow the likelihood of potential carryover of a GTI to be assessed ahead of performing analyses. In line with the ICH M7 Table 3. Physicochemical parameters and associated purge factors (adapted from Teasdale et al.8) Physicochemical Purge factors parameter 100 10 3 1 reactivity highly reactive moderately reactive - low reactivity/unreactive solubility - freely soluble moderately soluble sparingly soluble volatility - boiling point > 20 °C below that of the reaction/process solvent boiling point ± 10 °C that of the reaction/process solvent boiling point > 20 °C above that of the reaction/ process solvent ionizability ionization potential of GTI significantly different from that of the desired product (a specific purge factor is assigned where such an approach is specifically applied) physical processes -chromatography GTI elutes prior GTI elutes after desired to desired product product - - physical processes -recrystallization* freely soluble sparingly soluble * In the original approach the recrystallization process was described within the solubility term; however, based on the under-prediction of the purge factor tool in case of crystallization steps, it was proposed to describe it as an individual physical process with a scale from 1 to 100.29 Lapanja et al.: Theoretical Purge Factor Determination 7 Acta Chim. Slov. 2017, 64, 1-14 option 4 control strategy, the scientific approach proposed by Teasdale28 is based on physicochemical properties and process factors that influence the fate and purge of an impurity. In order to assess the carryover of potential GTIs into API, AstraZeneca developed a tool based on the assessment of key physicochemical properties of the agent of concern, relating them to the downstream processing conditions. A score is assigned for each of them to establish a 'purge factor'. The approach has been applied to various processes with available data. In order to assess the potential carry-over of a GTI, the following parameters are defined: reactivity, solubility, volatility, ionizability, and any physical process designed to remove impurities (e.g., chromatography). For each of the parameter a score is assigned as presented in Table 3. The scores are then multiplied together to give a purge factor for each stage of the process. Multiplying the purge factors for individual stages yields an overall purge factor. Teasdale et al.28 provided a case study, presenting both the outcome of the predictive purge factor and the real measured values. Theoretical purge factors were calculated for three potentially genotoxic impurities in the synthesis of AZD9056 (Scheme 1). Experimental purge factors were also determined for each of them by tracking the residual levels of impurities at successive stages. Results are summarized in Table 4. Scheme 1: Synthesis of AZD9056 (adapted from Teasdale et al.28). Lapanja et al.: Theoretical Purge Factor Determination 8 Acta Chim. Slov. 2017, 64, 1-14 Authors also noted that in the case of the impurity 1, the predicted purge factor in the isolated crude stage differed significantly from the experimental purge factor (10 versus 560, respectively). Based on this it could be argued that the scale for the solubility factor could be extended to 1-100 instead of 1-10. However, authors decided to retain the more conservative scale of 1-10 in order to compensate for any variance in processes such as uncontrolled crystallization, poor washing and/or inefficient deliquoring of the isolated product. Moreover, underprediction of the purge capacity of the process is preferable to an overpre-diction. Scheme 2: Synthesis of pazopanib hydrochloride (adapted from Elder et al. ). Lapanja et al.: Theoretical Purge Factor Determination 9 Acta Chim. Slov. 2017, 64, 1-14 Table 4. Summarized results of the case study for the synthesis of AZD9056 (based on Teasdale et al.28). Impurity of concern Theoretical purge factor Experimental purge factor Interpretation of the results Impurity 1 10 000 112 000 The calculated purge factor underpredicts the purge capacity of the process by a factor of 10. Even a conservatively calculated purge factors predicts that the risk of carryover of significant levels of this impurity into the API is low. Impurity 4 10 The calculated purge factor of 3 accurately predicts that the process has limited capacity of effectively removing this impurity. Impurity 5 10 000 38 500 The calculated purge factor accurately predicts the efficient removal of the impurity by the process. 3 Table 5. Summarized results of the case study for the synthesis of pazopanib hydrochloride (based on Elder et al.31). Impurity of concern Theoretical purge factor Experimental purge factor Interpretation of the results DMS 30 000 29 411 The tool very accurately predicts the purging capacity for DMS. Impurity II 8 100 30 044 The calculated purge factor underpredicts the purge capacity of the process by a factor of 3. Impurity 1 2 700 7 700 The calculated purge factor and experimental purge factor agree reasonably well. Impurity 3 52-174 Theoretical and experimental purge factor are in reasonable agreement, however a control strategy needs to be implemented due to a low factor. Impurity III 900 17 647 The calculated purge factor underpredicts the purge capacity of the process by a factor of 20. 9 In 2013 Teasdale et al.30 published further and more detailed information about the determination of theoretical purge factors, alongside various case studies. Instruc- tions are given on how to assign values for different physicochemical parameters, how to calculate the factors and how to evaluate the results. Lapanja et al.: Theoretical Purge Factor Determination 10 Acta Chim. Slov. 2017, 64, 1-14 Scheme 3: Synthesis of MK-8876 (adapted from McLaughlin et al.33). Another case study was described by Elder et al.31 in 2013, using the same approach to assess the ability to purge impurities in the synthesis of pazopanib hydroch-loride (Scheme 2). The theoretical purge factor assessment tool was applied to five mutagenic impurities (Table 5). The measured purge factor for each of the MI has been previously determined,32 therefore the authors were able to compare theoretical and experimental purge factors in order to assess the reliability of the proposed tool. Compared to the original approach, Elder et al.31 decided to include isolation steps within the physical process parameter, whereas a factor 3 was used if the isolation step was present and 1 if not. According to their results the tool very accurately predicted the purging capacity for the most reactive MIs. For less reactive MIs, measured and predicted values agreed reasonably well. In 2015 two additional practical applications of the proposed tool were published, i.e. by McLaughlin et al.33 and by Lapanja et al.29 McLaughlin et al.33 applied purge factor assessment tool to six MIs in the synthesis of a development compound MK-8876 (Scheme 3). Theoretical purge factors were compared with the analytically determined purge factors. Results are summarized in Table 6. It was emphasized that the proposed tool tends to underpre-dict the likely purge capacity of a process, thus staying on the safe / more conservative side. Lapanja et al.29 also used the same approach for assessing the presence of four potential MIs in the vortioxe-tine synthetic process (Scheme 4). Additionally, one minor modification regarding the physical process parameter was proposed, i.e. a recrystallization step was included within the physical process parameter, while according to Teasdale et al.28 recrystallization would be described within the solubility parameter. The theoretical purge factors were then compared with measured values and with the results of depletion studies. Results are summarized in Table 7. In conclusion it was noted that by assigning a va- Lapanja et al.: Theoretical Purge Factor Determination 11 Acta Chim. Slov. 2017, 64, 1-14 Table 6. Summarized results of the case study for the synthesis of MK-8876 (based on McLaughlin et al.33). Impurity of concern Theoretical purge factor Experimental purge factor Interpretation of the results EDC 1 110 > 50 000 The tool very accurately predicted the purging capacity for EDC. methyl iodide -1 1 000 000 100 000 The calculated purge factor overrpredicts the purge capacity of the process by a factor of 10. However, theoretical purge factor is in agreement with the actual analytical value of < 10 ppm of methyl iodide at intermediate stage. Chloroiodomethane 10 000 (crude) 100 000 (pure) 20 000 (crude) > 200 000 (pure) The calculated purge factor and experimental purge factors agree reasonably well. Arylboronic acid 10 000 (crude) 30 000 (pure) 143 000 (crude) > 1 000 000 (pure) Measured purge factors at the crude API stage and at the pure API stage are much higher than the theoretical purge factor. Bis boronic acid (BBA) 100 (crude) 1 000 (pure) > 3 333 (crude) > 250 000 (pure) Measured purge factors at the crude API stage and at the pure API stage are much higher than theoretical purge factor. Carbazole 100 > 375 The calculated and experimental purge factors agree reasonably well. CA3 Scheme 4: Synthesis of vortioxetine hydrochloride (adapted from Lapanja et al.29). lue of 3 for the recrystallization process the ability of the process to eliminate impurities was clearly underpredic-ted. However, Teasdale et al.28 suggested retaining a more conservative scale in order to compensate for any variance in processes. 5. Conclusion Several updates and refinements were done since the first guideline covering the issue of GTIs in pharmaceuticals was finalized by EMA in 2006. The ICH M7 guideli- Lapanja et al.: Theoretical Purge Factor Determination 12 _Acta Chim. Slov. 2017, 64, 1-14 Table 7. Summarized results of the case study for the synthesis of vortioxetine (based on Lapanja et al.29). Impurity of concern Theoretical purge factor Experimental purge factor Interpretation of the results (1) 8.1 x 106 4.9 x 101' The calculated purge factor underpredicts the purge capacity of the process by a factor of 6 000. Underprediction is especially significant in the case of recrystallization step (theoretical value of 9 versus 4 000). 8 100 Ames test for this compound was negative; however a theoretical purge factor has been calculated to assess the impact of reactivity parameter on the purge factor determination. The theoretical purge factor is clearly lower than the factor for compound I due to the different position of substituent and thus different reactivity. (5) hh 300 297 738 The experimental purge factor is approximately 1000-times higher than the theoretical purge factor. CI (4) 3 000 20 The calculated purge factor overpredicts the purge capacity of the process. ne which was released in June 2014 addressed many issues that were left unclear in the previous guidelines. Moreover, it offers greater flexibility in terms of mechanisms to demonstrate absence of MIs in drug substances. The use of theoretical purge factor determination tool which is in line with ICH M7 Option 4 control approach is very promising and allows avoiding analytical testing where not necessary. Many pharmaceutical companies have applied this semi quantitative approach using purge factors as described by Teasdale et al.28 and some of them published their results. Authors noted that the calculated purge factors agree very well or reasonably well with the experimental purge factors. In several cases it was noted that the purge factor tool tends to underpredict the purging capacity of the process. This underprediction was especially significant in the case of isolation steps during synthesis. While one could argue that the theoretically determined purge factors differ too much from the measured values, it must be emphasized that the underprediction is intentional in order to gain acceptance of the approach. When relating the theoretically determined purge factors to the required purge, it is expected that the theoretical purge would be preferably 100-times greater than the required purge. This makes the approach even more conservative and assures that we always stay on the safe side. Taking into account the conservatism of the approach, this tool should provide satisfactory evidence to the regulatory agencies for the absence of MIs above determined limits. It is to be hoped that this approach will become a regular practice benefiting the pharmaceutical industry, while not increasing any risk for the patients whatsoever. 6. Associated Content Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Lek Pharmaceuticals d.d., Verovškova 57, 1526 Ljubljana, Slovenia Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia Abbreviations ALARP, as low as reasonably practicable; API, active pharmaceutical ingredient; CHMP, Committee for Human Medicinal Products; CPMP, Committee for Proprietary Medicinal Products; DMSO, dimethyl sulphoxide; DNA, deoxyribonucleic acid; EDQM, European Directorate for the Quality of Medicines and Healthcare; EMA, European Medicines Agency; EMS, ethyl methane sulfo-nate; FDA, Food and Drug Administration; GTI, genoto-xic impurity; ICH, International Conference on Harmonisation; LOEL, lowest-observed effect level; LTL, Less than lifetime; MI, mutagenic impurity; NOEL, no-obser- Lapanja et al.: Theoretical Purge Factor Determination 13 Acta Chim. Slov. 2017, 64, 1-14 ved-effect level; PDE, permitted daily exposure; PhRMA, Pharmaceutical Research and Manufacturers of America; QbD, Quality by Design; Q&A, Questions and answers; QL, quantitation limit; (Q)SAR , (Quantitative) Structure-Activity Relationships; SWP, Safety Working Party; TTC, Threshold of Toxicological Concern. 7. References 1. ICH: Guidance for industry, M7 Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk, ICH, 2014. 2. R. W. Tennant, Mutagens and carcinogens, in AccessScience 2014 http://dx.doi.org/10.1036/1097-8542.441100 3. D. Jacobson-Kram, T. McGovern, Adv. Drug Delivery Rev. 2007, 59, 38-42. https://doi.org/10.1016Zj.addr.2006.10.007 4. European Directorate for the Quality of Medicines and Healthcare: Enquiry: Alkyl mesylate (methane sulfonate) impurities in mesylate salts, PharmEuropa 12:27, 2000. 5. A. Teasdale (Ed.): Genotoxic impurities: Strategies for identification and control, Wiley, New Jersey, United States, 2010, pp. 3-4. 6. EMEA/CPMP: Position paper on the limits of genotoxic impurities, EMEA, 2001. 7. EMEA/CHMP: Guidelines on the limits of genotoxic impurities, EMEA, 2004. 8. EMEA/CHMP: CHMP assessment report for Viracept, EMEA, 2007. 9. EMEA/CHMP: Guidelines on the limits of genotoxic impurities, EMEA, 2006. 10. ICH: Guidance for industry, Q3A (R2) Impurities in new drug substances, ICH, 2006. 11. ICH: Guidance for industry, Q3B (R2) Impurities in new drug products, ICH, 2006. 12. ICH: Guidance for industry, Q3C (R5) Guidelines for residual solvents, ICH, 2011. 13. ICH: Guidance for industry, Q3D Guideline for elemental impurities, ICH, 2014. 14. I. C. Munro, E. Kennepohl, R. Kroes, Food Chem. Toxicol. 1999, 57, 207-232. https://doi.org/10.1016/S0278-6915(98)00112-4 15. R. Kroes, G. Kozianowski, Toxicol. Lett. 2002, 127, 43-46. https://doi.org/10.1016/S0378-4274(01)00481-7 16. L. S. Gold, C. B. Sawyer, R. Magaw, G. M. Backman, M. de Veciana, R. Levinson, N. K. Hooper, W. R. Havender, L. Bernstein, R. Peto, M. C. Pike, B. N. Ames, Environ. Health Perspect. 1984, 58, 9-319. https://doi.org/10.1289/ehp.84589 17. I. C. Munro, Regul. Toxicol. Pharmacol. 1990, 12, 2-12. https://doi.org/10.1016/S0273-2300(05)80042-X 18. M. A. Cheeseman, E. J. Machuga, A. B. Bailey, Food Chem. Toxicol. 1999, 57, 387-412. https://doi.org/10.1016/S0278-6915(99)00024-1 19. R. Kroes, A. G. Renwick, M. Cheeseman, J. Kleiner, I. Mangelsdorf, A. Piersma, B. Schilter, J. Schlatter, F. van Schothorst, J. G. Vos, G. Würtzen, Food Chem. Toxicol. 2004, 42, 65-83. https://doi.org/10.1016Zj.fct.2003.08.006 20. EMEA/CHMP: Questions and Answers on the CHMP Guideline on the Limits of Genotoxic Impurities, EMEA, 2010. 21. L. Muller, R. J. Mauthe, C. M. Riley, M. M. Andino, D. D. Antonis, C. Beels, J. DeGeorge, A. G. De Knaep, D. Ellison, J. A. Fagerland, R. Frank, B. Fritschel, S. Galloway, E. Har-pur, C. D. Humfrey, A. S. Jacks, N. Jagota, J. Mackinnon, G. Mohan, D. K. Ness, M. R. O'Donovan, M. D. Smith, G. Vu-dathala, L. Yotti, Regul. Toxicol. Pharmacol. 2006, 44, 198-211. https://doi.org/10.1016/j.yrtph.2005.12.001 22. Guidance for industry: Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches (Draft), U.S. Food and Drug Administration (FDA), 2008. 23. ICH: Guidance for industry, M7 (R1) Addendum: Application of the principles of the ICH M7 guideline to calculation of compound-specific acceptable intake, ICH, 2015. 24. G. Szekely, M. C. Amores de Sousa, M. Gil, F. C. Ferreira, W. Heggie, Chem. Rev. 2015, 115, 8182-8229. https://doi.org/10.1021/cr300095f 25. N. V. V. S. S.Raman, A. V. S. S. Prasad, K. Ratnakar Reddy, J. Pharm. Biomed. Anal. 2011, 55, 662-667. https://doi.org/10.1016/jjpba.2010.11.039 26. Z. Cimarosti, F. Bravo, P. Stonestreet, F. Tinazzi, O. Vecchi, G. Camurri, Org. Process Res. Dev. 2010, 14, 993-998. https://doi.org/10.1021/op900242x 27. D. A. Pierson, B. A. Olsen, D. K. Robinson, K. M. DeVries, D. L. Varie, Org. Process Res. Dev. 2009, 13, 285-291. https://doi.org/10.1021/op8002129 28. A. Teasdale, S. Fenner, A. Ray, A. Ford, A. Phillips, Org. Process Res. Dev. 2010, 14, 943-945. https://doi.org/10.1021/op100071n 29. N. Lapanja, B. Zupančič, R. Toplak časar, D. Orkič, M. Uštar, A. Satler, S. Jurca, B. Doljak, Org. Process Res. Dev. 2015, 19, 1524-1530. https://doi.org/10.1021/acs.oprd.5b00061 30. A. Teasdale, D. Elder, S. J. Chang, S. Wang, R. Thompson, N. Benz, I. H. Sanchez Flores, Org. Process Res. Dev. 2013, 17, 221-230. https://doi.org/10.1021/op300268u 31. D. P. Elder, G. Okafo, M. McGuire, Org. Process Res. Dev. 2013,17, 1036-1041. https://doi.org/10.1021/op400139z 32. D. Q. Liu, T. Q. Chen, M. A. McGuire, A. S. J. Kord, J. Pharm. Biomed. Anal. 2009, 50, 144-150. https://doi.org/10.1016/jjpba.2009.04.002 33. M. McLaughlin, R. K. Dermenjian, Y. Jin, A. Klapars, M. V. Reddy, M. J. Williams, Org. Process Res. Dev. 2015, 19, 1531-1535. https://doi.org/10.1021/acs.oprd.5b00263 Lapanja et al.: Theoretical Purge Factor Determination 14 Acta Chim. Slov. 2017, 64, 1-14 Povzetek Mutagene necistote predstavljajo velik problem za farmacevtsko industrijo, regulatorne oblasti in javno zdravje. Prva regulatorna smernica, ki je obravnavala nadzor genotoksicnih necistot je bila izdana leta 2006, sledile pa so številne dopolnitve in izboljšave. Junija 2014 je bila s strani mednarodne konference o harmonizaciji zahtev izdana smernica ICH M7, ki v primerjavi s prvotnimi smernicami ponuja bolj pragmatične možnosti za nadzor genotoksicnih necistot v zdravilnih ucinkovinah. Poleg analitskega spremljanja genotoksicnih necistot ima sedaj farmacevtska industrija preko smernice ICH M7 možnost kontrolne strategije, ki sloni na razumevanju procesa sinteze in na oceni vpliva procesnih parametrov na nivo pridobljenih in nastalih necistot. Ta pristop je predlagal in prvi opisal A. Teasdale s sodelavci. Predlagani pristop izracuna teoreticnih faktorjev ocišcenja je bil v zadnjih letih uporabljen na številnih prakticnih primerih. Objavljeni rezultati kažejo na to, da lahko s tem pristopom precej dobro napovemo sposobnost ocišcenja necistot skozi proces. Upati velja, da bo omenjeni pristop kmalu na voljo v obliki racunalniškega orodja, ki bo splošno sprejemljiv s strani regulatornih oblasti. Lapanja et al.: Theoretical Purge Factor Determination DOI: 10.17344/acsi.2016.3134 Acta Chim. Slov. 2017, 64, 15-39 ^creative tS/commons Review The Lock is the Key: Development of Novel Drugs through Receptor Based Combinatorial Chemistry Nikola Marakovi} and Goran [inko* Institute for Medical Research and Occupational Health, Ksaverska cesta 2, p.p. 291, HR-10001 Zagreb, Croatia * Corresponding author: E-mail: gsinko@imi.hr Received: 16-12-2016 Abstract Modern drug discovery is mainly based on the de novo synthesis of a large number of compounds with a diversity of chemical functionalities. Though the introduction of combinatorial chemistry enabled the preparation of large libraries of compounds from so-called building blocks, the problem of successfully identifying leads remains. The introduction of a dynamic combinatorial chemistry method served as a step forward due to the involvement of biological macromo-lecular targets (receptors) in the synthesis of high affinity products. The major breakthrough was a synthetic method in which building blocks are irreversibly combined due to the presence of a receptor. Here we present various receptor-based combinatorial chemistry approaches. Huisgen's cycloaddition (1,3-dipolar cycloaddition of azides and alkynes) forms stabile 1,2,3-triazoles with very high receptor affinity that can reach femtomolar levels, as the case with acetylcholinesterase inhibitors shows. Huisgen's cycloaddition can be applied to various receptors including acetylcholinesterase, acetylcholine binding protein, carbonic anhydrase-II, serine/threonine-protein kinase and minor groove of DNA. Keywords: Drug design; Dynamic combinatorial chemistry; Huisgen's cycloaddition; in situ click-chemistry; Receptor-accelerated synthesis; Receptor-assisted combinatorial chemistry 1. Introduction The main focus of drug discovery is the identification of compounds that can modify molecular targets associated with certain diseases inducing a positive response. While natural products have inspired the design of most drugs in the past, the processes of lead discovery and optimization today rely on the preparation of large collections of new compounds, referred to as "libraries". Choosing large numbers of structurally diverse compounds is primarily governed by the complexity of natural products, which increases the difficulty, time, and cost of the preparation of such compounds. Also, as suggested by a computational study by Bohacek et al., the total number of "drug-like" compounds (< 30 non-hydrogen atoms, < 500 Daltons; only H, C, N, O, P, S, F, Cl and Br; stable in the presence of water and oxygen) is as large as 1063 indicating that the vast majority of "drug-like" compounds are yet to be discovered.1 The introduction of combinatorial chemistry seemed to resolve the problem of preparing large libraries by focusing on building libraries of more complex compounds from simple building blocks. Building blocks are combined in a maximum number of possible combinations through independent synthesis. In the final step, each compound is independently tested for activity. Independent testing of a large number of newly synthesized compounds significantly reduces the potential of conventional combinatorial methods. However, by the early 2000s, it became clear that conventional combinatorial chemistry turned out to be much less efficient than expected with only a few developed drugs reported and most industrial combinatorial chemistry libraries were disban- ded.2 In 1894, the German chemist Emil Fischer suggested a model of enzyme specificity by which an enzyme and its substrate possess specific complementary geometric shapes that fit exactly one into another like a lock and key. Although this model is more than 100 years old, E. Fischer's idea is still valid. Dixon and Villar showed that a protein can bind a set of structurally diverse molecules with similar affinities in the nanomolar range, whereas analogues closely related to one of the good binders show only weak affinities (> 2.5 mM).3 Chemists created an approach where novel potentially bioactive compounds are not synthesized by pure statistical reorganization of joi- Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs 16 Acta Chim. Slov. 2017, 64, 15-39 ning building blocks but forcing them in the right direction by including a macromolecular target (receptor) itself in this process. This was done through the introduction of a receptor-assisted combinatorial chemistry (RACC), sometimes also referred to as target-guided synthesis (TGS).4 In contrast to conventional combinatorial methods, in RACC the macromolecular target (protein or DNA) is directly involved in the choice of joining building blocks. The concept of RACC can be divided into dynamic combinatorial chemistry (DCC) and receptor-accelerated synthesis (RAS), also called kinetically controlled TGS. In DCC, the reaction that joins the building blocks is reversible, whereas RAS uses only reactive building blocks joined irreversibly. The subset of RAS called in situ click chemistry, which uses the Huisgen's 1,3-dipolar cycloaddition of azides and alkynes (Huisgen's cycloaddition) to irreversibly join the building blocks, will be covered with special interest.5,6 2. Dynamic Combinatorial Chemistry Method Dynamic combinatorial chemistry is a subset of RACC in which building blocks are joined through a reversible covalent reactions, generating a large equilibrium-controlled library of compounds referred to as a dynamic combinatorial library (DCL).7,8 The addition of biological targets during the generation of DCL stabilizes the library members with the highest affinity toward the biological target, moving the equilibrium toward high-affinity members. A comparison of the composition of the library with and without the biological target leads to the identification of a hit compound. Therefore, the synthesis and screening of library members are combined in one step, which speeds-up the process of hit identification. Moreover, hit identification is possible without any specific receptor assays used. Instead, increased amounts of the highest affinity library members are detected with established analytical methods like HPLC, mass spectrometry (MS), NMR spectroscopy or even X-ray crystallography.9,10 It may be more advantageous for the library to amplify many members with moderate affinities than just a few with high affinities. This behaviour reflects the complex nature of DCLs consisted of members interconnected through a set of equilibrium reactions.11 To address these problems numerous theoretical studies of DCLs have been done.12-16 The studies suggested that, unless excessive amounts of molecular target are used, good binders have a high probability of being significantly amplified. However, a major limitation for application of DCC in drug discovery is the limited number of reversible covalent reactions appropriate to be used to synthesize DCLs. Drug discovery applications of DCC require the following reaction conditions: (i) reaction at a biologically relevant tem- perature, (ii) compatibility with aqueous media, (iii) reaction at (close to) physiological pH and (iv) compatibility with the target functional groups.17,18 Compatibility with aqueous media is the most challenging condition as there are more reactions that have been developed in organic solvents than under aqueous conditions, thus preventing the use of a wider range of equilibration reactions. Additionally, the use of organic solvents in DCC is limited by the strong tendency of solvents to denature the target (enzyme, receptor, etc.). Examples of DCC applications for the discovery of high affinity ligands for biological receptors have been reported, including formation of DCLs of imines,19,20 hydrazones,21,22 oxime ethers,23 sulfides,24 disulfides25-28 and alkenes.29 2. 1. Reversible Imine Formation Huc and Lehn were the first to demonstrate the concept of DCC application in drug discovery by identifying inhibitors of carbonic anhydrase (CA) using a DCL of imines formed from amines and aldehydes.19 In addition to the fast and reversible nature of condensation between amines and aldehydes to imines, reversible imine formation is very convenient for drug discovery because it yields a Schiff base, a very common motive in metabolites and biologically active compounds.30,31 To detect products by HPLC, they "locked-in" the equilibrium by irreversible reduction of imines to corresponding amines using Na-BH3CN to fix the composition of the library prior to detection. Hochgurtel et al. created an imine library by condensing a diamine with more than fifty different ketones in the presence of neuraminidase from an influenza virus (Fig. 1).20 After reduction of imines, LC/MS analysis identified several hits (1-4). The negative control experiment included library synthesis in the presence of the bovine serum albumin (BSA). The second control experiment was carried out in the presence of the neuraminidase and Zanamivir, a potent competitive inhibitor of the neuraminidase. On both occasions, initial hit 4 was identified. The most abundant compound 3 lacked inhibitory potency, whereas the strongest inhibitor 2 was amplified three-fold less than 3. The authors suggested that this result could be explained by the lock-in reaction. Actual molecular species undergoing equilibration are imines and hemiaminals. The receptor amplifies the amount of these intermediates that are then reduced to fix the library composition. Reduced products have different structural and electronic properties and their interaction with the biological target may be worse, or better, than originating intermediates. This represents a major drawback for the application of reversible imine formation to the construction of DCLs in the presence of a biological target. Recent progress in analytical methods used for identification of binders from DCL had enabled access to larger libraries. For example, Guo et al. introduced a Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 17 Acta Chim. Slov. 2017, 64, 15-39 Figure 1. Formation of a library of potential neuraminidase inhibitors by condensing a diamine with several ketones. protocol for analysis of imine-based DCL using a suitable size-exclusion chromatography (SEC) column to retain all non-binders from DCL followed by denaturation of eluted protein-ligand complexes and MS analysis of binders.32 2. 2. Disulfide Interchange To demonstrate utility of a disulfide interchange for DCC approach, Ramström and Lehn designed a DCL of disulfides capable of binding to concavalin A (Con A), a member of lectins.25,33 DCL of disulfide carbohydrate di-mers (Table 1) was generated by incubating disulfide di-mers with an initiating reagent dithiothreitol (DTT) capable of reducing some disulfides to thiols. DTT is oxidized to a stable 6-membered cyclic disulfide that should not take part in the interconversion of the library disulfides. Upon initiation, interconversion between disulfides occurred with the rate dependent on pH. At pH 7.4, a reasonable rate of interconversion was obtained and receptor binding was not affected. Disulfide interchange could be stopped by lowering the pH (< 5) and final equilibrium distribution of DCL analyzed by HPLC. In the absence of any receptor, all expected ditopic combinations were generated in approximately equal amounts. When a receptor Con A was present during the interconversion, a significant amount of the bis-mannoside (Man/Man) and the mannose-containing heterodimers (Man/Gal, Man/Ara, Man/Xyl) was found to be bound to the receptor.25 Moreover, receptor-induced shifts in equilibrium resulted in the amplification of mannose-containing dimers, which is in accordance with concepts of the DCC approach. One of the major drawbacks of using DCL of disul-fides to identify potent inhibitors of protein targets is the labile nature of disulfide bond. However, once identified disulfide compounds can be replaced with their carbon Table 1. Structures of the disulfide-linked carbohydrate dimers.2 aß R2a R2e R4a R4e R5 n (Man/Man) a OH H H OH CH2OH 3 (Gal C2/Gal C2) ß H OH OH H ch2oh 2 (Gal C3/Gal C3) ß H OH OH H ch2oh 3 (Glc/Glc) ß H OH H OH CH22OH 2 (Ara/Ara) ß H OH OH H H 2 (Xyl/Xyl) ß H OH H OH H 2 Compounda 1 Man = D-mannose; Gal C2 = D-galactose, n = 2; Gal C3 = D-galactose, n = 3; Glc = D-glucose; Ara = L-arabinose; Xyl = D-xylose Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 18 Acta Chim. Slov. 2017, 64, 15-39 analogues, with bioisosteric thioether or amide linker instead of the disulfide bond. Using modified MS analysis that enables analysis of DCLs of thiols/disulfides under non-denaturing conditions, Schofield et al. have identified inhibitors to various protein targets by preparing carbon analogues of identified disulfide compounds.27,34 2. 3. Reversible Acylhydrazone Formation Ramström et al. developed DCLs of constituents potentially capable of binding to plant Con A using reversible hydrazidecarbonyl/acylhydrazone inter-conver-sion.21 Acylhydrazone libraries were generated from a series of oligohydrazide core building blocks a-i and a set of aldehyde counterparts 5-10 based on six common, naturally occurring carbohydrates, potentially capable of interacting with the binding site of Con A (Fig. 2). A set of initial 15 building blocks could give rise to a library containing at least 474 different species. Also, 15 sub-libraries were formed by mixing all building blocks except one specific hydrazide or aldehyde building block under the same conditions.21 Following equilibration libraries were subsequently subjected to the lectin assay in which the inhibitory potency of library constituents was monitored. The resulting inhibitory effects of the sub-libraries have been matched to the activity of the complete library. The largest effect was noticed on the removal of the mannose unit from complete DCL indicating that the mannose unit is necessary for inhibition. Similarly, triva-lent core building block G was the most active. The effect of the compound assembled from these two fragments was estimated in a binding assay, resulting in an IC50 value in the micromolar range (22 pM), indicating that the DCC approach using reversible hydrazidecar-bonyl/acylhydrazone interconversion enabled the identification of a novel tritopic mannoside showing potent binding to Con A (Fig. 3). However, the full potential of acylhydrazone-based DCLs in drug discovery is somewhat limited because of the requirement for acidic pH which is incompatible with most protein targets. Greaney et al. have managed to circumvent this obstacle by introducing nucleophilic catalysis of reversible acylhydrazone formation by using aniline as a nucleophilic catalyst at less acidic pH and thus identify acylhydrazone inhibitors of GST isozymes.35,36 Figure 2. A series of oligohydrazide A-I and aldehyde building blocks 5-10 generating an acylhydrazone dynamic combinatorial library of potential plant lectin Con A inhibitors.21 Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 19 Acta Chim. Slov. 2017, 64, 15-39 Figure 3. Compound 103-G identified as the best binder to Con A (IC50 = 22 ||M) from the acylhydrazone dynamic combinatorial library generated from a series of oligohydrazide and aldehyde building blocks.21 Figure 4. Dynamic combinatorial library composed of glutathione (GSH) conjugates potentially capable of binding to glutathione S-transferase (GST) generated from GSH, GSH analogues, and ethacrynic acid (EA).37 Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 20 Acta Chim. Slov. 2017, 64, 15-39 2. 4. Conjugate Addition of Thiols to Enones Shi and Greaney extended the number of reversible chemical reactions suitable for DCL generation by using conjugated addition of thiols to enones.24 Shi and Grea-ney designed a biased DCL generated using glutathione (GSH; 11), three GSH analogues 13-15, and the enone ethacrynic acid (EA; 12) (Fig. 4)37 Three analogues were expected to be misfits for the G site of glutathione S-transferase (GST) since the y-glutamyl residue is critical for binding,38 thus biasing the DCL equilibrium composition in the presence of GST toward the GSH adduct 16. EA is an inhibitor of GST and has provided a structural scaffold for development of GST inhibitors. Blank DCL, assembled in the absence of GST resulted in the distribution of four conjugates 16-19. Upon incubation with GST from Schistosoma japonica (SjGST), DCL reduced to the expected GS-EA adduct 16. Adduct 16 was increased from 35% of total conjugate concentration to 92% at equilibrium, due to large differences in binding affinity between 16 and peptides lacking the y-glutamyl residue. Control experiments with BSA instead of SjGST produced no changes to the blank DCL composition, confirming that the active site of SjGST is responsible in amplification of 16. Shi et al. used the thiol addition methodology to create new GST inhibitors from nonbiased DCLs. Since COjH N C02H Figure 5. A nonbiased DCL of potential GST inhibitors generated from glutathione (GSH) and 14 enone ethacrynic acid analogues.37 Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 21 Acta Chim. Slov. 2017, 64, 15-39 structural features of the H site change across different GST isozymes, the authors explored the H site of SjGST by constructing a DCL with reversed stoichiometry from that in biased DCL, whereby 14 EA analogues reacted with GSH to afford 14 GS-EA adducts (Fig. 5). MS analysis and deconvolution studies revealed that adducts 21a,m and n were amplified in the presence of SjGST, while ad-duct 21f was suppressed. To examine the inhibition potency of SjGST, 21a, 21n, non-amplified adduct 21b, and the suppressed adduct 21f were synthesized and their IC50 values measured. Results indicated that the extent of DCL amplification reflected the relative binding affinities of DCL components for the SjGST. Piperidine and leucine amides 21a (IC50 = 0.61 pM) and 21n (IC50 = 1.40 pM) were amplified from the library at the expense of the weaker binder lysine amide 21f (IC50 = 8.2 pM). Moreover, contrary to the proposed model structure of the SjGST/GS-EA Michaelis complex which identified a series of residues that could interact with the EA carboxylic acid group,39 amplified adducts 21a and 21n indicated that the carboxylic acid group of EA is not essential for binding in the H site and may be extended without change of inhibitory activity. 3. Receptor-Accelerated Synthesis Receptor-accelerated synthesis (RAS), also called kinetically controlled TGS, is a subset of RACC, which uses kinetic control to increase the relative amounts of the highest-affinity library members during library genera-tion.4,40 While the library members in the DCC approach are generated via reversible reactions, RAS uses building blocks which irreversibly combine into larger molecules. Process of hit identification and optimization takes advantage of combining synthesis and screening into one step (Fig. 6). Step 1 includes synthesis of reactive building blocks, while in step 2 these building blocks irreversibly combine due to the presence of a receptor. The hit identification consists of determining whether a formation of a product is significantly accelerated in the presence of a target molecule (receptor). The selectivity for one or more products over others arises from two factors, one related to the binding of building blocks to the receptor, and the other to the ability of a receptor to accelerate their irreversible joining. With regard to the binding of the starting building blocks to the receptor, simultaneous binding of highest-affinity building blocks in close proximity leads to rate acceleration. However, upon joining the starting building blocks to the product, the binding interactions of building blocks to the receptor may strengthen or weaken in accordance with the Fischer's lock and key model. Thus, highest-affinity building blocks might not form a product with the highest affinity for the receptor. As far as the ability of a given receptor to promote the coupling of reactive building blocks is concerned, it is important to note that receptors do not normally act as coupling catalysts. The demands for a reaction suitable for RAS are different from the DCC approach or from a conventional organic reaction. Ideally, complementary reactive groups should combine very slowly in solution generating a stable product with no or only minor side products. Kolb et al. identified Huisgen's cycloaddition as the one having the ideal reactivity profile for RAS.41,42 This methodology has been successfully applied in numerous examples known as in situ click chemistry.43 So far, RAC approaches have included C-N bond formation,44-46 C-S bond formation,47-49 C-C bond formation,50 and ami- Figure 6. Receptor-accelerated synthesis for hit discovery and optimization. Products are created from blocks properly stabilized within the receptor. Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 22 Acta Chim. Slov. 2017, 64, 15-39 de formation from thio acids and sulfonyl azides, also referred to as "sulfo-click reaction".51,52 Some of these approaches are described in more detail below. 3. 1. Substitution Reaction Using a Thiol as the Nucleophile Huc and Nguyen were the first to demonstrate the utility of a substitution reaction using a thiol as a nucleophile for the identification of an inhibitor via RAS approach.47 This reaction is widely used in organic chemistry since thiols are more reactive than alcohols. In initial study, they chose to target a zinc-containing metalloenzyme, bovine CA-II (EC 4.2.1.1).53 CA-II isozymes play a role in many important biological processes, including respiration, bone respiration, calcification, acid secretion, and pH control. The CA-II active site is a conical cleft with the Zn(II) ion located at its bottom with two secondary hydrophobic binding sites located in close proximity of this cleft. They tested the ability of CA-II to accelerate the formation of para-substituted aromatic sulfo-namide inhibitors 24a-e using competition assays optimized to limit side reactions, such as disulfide formation, alkyl chloride hydrolysis, and trialkyl sulfonium formation (Fig. 7).47 Thiol 22 was treated with two competing alkyl chlorides in buffered water at pH 6 for 48 h, first in the absence of CA-II, then in the presence of CA-II. HPLC analysis of the final thioether products confirmed that CA-II strongly favours formation of more potent inhibitors. For example, when chloride 23a competes with 23d, the yield of more potent inhibitor 24d changes from 50% in the absence of CA-II to 92% in its presence. On the contrary, when products have similar affinities for CA-II, their final yields are negligibly affected by the presence of CA-II. To confirm that CA-II serves as the reaction vessel, Huc and Nguyen conducted several control experiments, including varying CA-II concentration, replacing CA-II by BSA, replacing thiol 22 by a thiol that has no affinity for CA-II, and adding an inhibitor of CA-II, methazolamide.54 All of these experiments confirmed that the active site of CA-II templates product formation. Besides alkyl halides, thiols can also react with epo-xide rings in protein-templated irreversible formation of biologically active ligands. Okhanda et al. have utilized such epoxide ring opening to identify inhibitors of recombinant human 14-3-3 protein, involved in immunoglobulin class switching, via RAS approach.48 3. 2. Amide Formation Between Thio Acids and Sulfonyl Azides The choice of biological target for the RAS or the RACC is not limited to enzymes only. It has been shown that RAS can be utilized to discover small molecules that modulate or disrupt protein-protein interactions (PPIs) called protein-protein interaction modulators (PPIMs). PPIs are crucial for a large number of vital biological processes and interesting in the development of novel therapies for a variety of diseases.55 Among PPI targets for cancer treatment are also proteins of the Bcl-2 family. Some of the Bcl-2 proteins act as anti-apoptotic proteins (Bcl-2, Figure 7. The formation of para-substituted aromatic sulfonamide inhibitors 24a-e of CA-II.4 Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 23 Acta Chim. Slov. 2017, 64, 15-39 Figure 8. ALAcylsulfonamide compounds targeting Bcl-XL. Bcl-XL, and Mcl-1) and others as pro-apoptotic proteins. Pro-apoptotic proteins can be further classified into multidomain BH1-3 proteins (Bax and Bak) and BH3-only pro- teins (Bad, Bim, and Noxa).56 Bcl-2 proteins play an important role in the apoptosis. Most likely, apoptosis is initiated by binding the BH3 domain of BH3-only proteins SZ4TA2 K\ (BcI-Xl) = 19 nM Figure 9. PPIM identification via sulfo-click RAS approach.60 Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 24 Acta Chim. Slov. 2017, 64, 15-39 Figure 10. Screening of anti-apoptotic Bcl-XL via sulfo-click RAS approach for PPIM discovery. Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 25 Acta Chim. Slov. 2017, 64, 15-39 into a hydrophobic groove on the surface of anti-apoptotic proteins. Therefore, designing a molecule capable of mimicking the BH3 domain is a promising strategy for novel anticancer treatments. Thus, N-acylsulfonamides 25, ABT-737, and ABT-263, capable of disrupting Bcl-XL-Bad interaction, were prepared (Fig. 8).57-59 Hu et al. applied the RAS approach for the discovery of N-acylsulfonamide PPIMs.60 They designed building blocks structurally similar to ABT-737 and ABT-263, having a sulfonyl azide or a thio acid functional groups, and incubated these as binary mixture together with Bcl-XL for 6 h. LC/MS analysis revealed that, of all the 18 possible products, only N-acylsulfonamide SZ4TA2 was detected (Fig. 9). Control experiments involving incubation of reactive building blocks in the absence of Bcl-XL or in the presence of Bcl-XL and various BH3-containing peptides, confirmed that the surface of Bcl-XL protein acts as a template for the sulfo-click reaction. To generate new hit compounds, Kul-karni et al. designed two sublibraries, one with thio acids and the other with sulfonyl azides, among which were those with a structural resemblance to ABT-737 or ABT-263 and those that were randomly chosen.51 Eighty-one binary mixtures containing one thio acid (TA1-TA9) and one sulfonyl azide (SZ1-SZ9) were incubated with the protein Bcl-XL for 6 h at 37 °C (Fig. 10). LC/MS analysis of binary mixtures with or without Bcl-XL present during reaction resulted in elevated amounts of SZ4TA2, and three new products SZ7TA2, SZ9TA1, and SZ9TA6 in the presence of Bcl-XL. Control experiments with native and mutated pro-apoptotic Bim BH3 peptides and Bcl-XL proteins indicated that protein-templa-ted N-acylsulfonamide formation happened solely at the binding sites of Bcl-XL. In order to evaluate the IC50, all four hit compounds were subjected to dose-response studies and binding studies.60 All of the hit compounds show high to modest affinity for Bcl-XL protein and can modulate the interaction between Bcl-XL and BH3 peptide ligand. Nature of sulfo-click reaction and substrate scope challenge its applicability in the RAS approach. As thioa-cids are nucleophilic, readily dimerize, and present storage and stability issues, their preparation and handling is therefore very demanding.61 Namelikonda et al. optimized the one-pot deprotection/amidation variant of sulfo-click reaction in the presence and absence of Bcl-XL starting from the 9-fluorenylmethyl (Fm)-protected thioesters and sul- fonylazides.52 Optimal deprotection of Fm thioesters TA1'-TA3' prepared from thioacid building blocks TA1-TA3 was achieved in one minute at room temperature with 3.5% 1,8-diazabicycloundec-7-ene (DBU)/DMF. Resulting thioacids TA1-TA3 were immediately diluted with methanol and incubated with sulfonylazides SZ1-SZ6 as binary mixtures in the presence and absence of Bcl-XL. Product analysis failed to detect an increased amount of the previously reported hit compound SZ4TA2 in the presence of Bcl-XL, presumably due to the change in pH of the incubation sample probably due to the strong basicity of DBU. Experiments were repeated with a weaker base (5% piperidine/DMF), and the amount of SZ4TA2 was increased to the same level as before containing purified thioacid TA2. However, a side reaction producing pi-peridine amide was observed, but this unwanted byproduct did not interfere with Bcl-XL templated reaction. 4. In situ Click Chemistry So far, only a RAS approach using a combination of strong nucleophilic (basic) and electrophilic (acidic) building blocks has been discussed. However, a subset of receptor-accelerated synthesis, termed in situ click chemistry, has been developed utilizing the Huisgen's cycload-dition,5,6 a reaction independent to the acid-base reactivity paradigm, as shown in literature.62-67 4. 1. The Huisgen's 1,3-Dipolar Cycloaddition The Huisgen's 1,3-dipolar cycloaddition of azides and alkynes to form 1,2,3-triazoles is a model example among the reactions that meet the criteria of click chemistry (Fig. 11).41 Originally introduced by Barry Shar-pless in 1999, click chemistry refers to a group of reactions that generate carbon-heteroatom bonds. Click chemistry has been successfully applied in many areas, including organic synthesis,68-72 bioconjuga-tion,73-75 drug discovery,4,24,76,77 and polymer and material sciences.78-81 Huisgen's cycloaddition is preferred since azides and alkynes are easy to implement and are inert in the acidic/basic environments and under physiological conditions. However, spontaneous cycloaddition is very slow, since reaction proceeds only if azide and alkyne in- Figure 11. Huisgen's 1,3-dipolar cycloaddition of azides and alkynes. Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 26 Acta Chim. Slov. 2017, 64, 15-39 teract properly oriented. It was only after the discovery of dramatic rate acceleration of the azide-alkyne cycloaddition under copper(I) catalysis that it gained its popularity.82'83 This reaction exclusively forms 1,4-disubstituted 1'2,3-triazoles (anti-triazoles). The 1,5-disubstituted 1'2'3-triazoles (syn-triazoles) are prepared by using magnesium acetylides or ruthenium catalysis.84'85 Recently, efficient recyclable nanocatalysts have been developed for regioselective synthesis of 1'2,3-triazoles in water.86 Thermal reaction is extremely slow and gives a mixture of isomers which are chromatographically separable. In addition, 1'2,3-triazole moieties have some favourable physi-cochemical properties attractive for application to the drug discovery and biomedicine. They are very stable to both metabolic and chemical degradation' being inert to hydrolytic' oxidizing' and reducing conditions' even at higher temperatures.25 Due to resemblance with amide moiety in size, dipolar moment, and H-bond acceptor capacity, the 1,2,3-triazole ring can serve as its non-classic bioisostere.44'45'87'88 Since 1,2,3-triazoles are basic aromatic heterocyclic compounds, they are bioisosteres of aromatic rings and double bonds.6566 Additionally, the aforementioned physicochemical properties of 1,2,3-triazole moiety together with similarity to amide bond, make it a useful linker to generate "twin drugs",42 67 83 bidentate inhibitors,83-8589 linkers to immobilized fluorescent tags or small molecules,71 and anion receptors.90 4. 2. In situ Click Chemistry Using Acetylcholinesterase as a Template Inspired by a report by Mock et al. on dramatic rate acceleration of azide and alkyne cycloaddition by sequestering azide and alkyne moieties inside the cavity of cu- Figure 12. In situ click chemistry screening of binary mixtures of tacrine/phenylphenanthridinium-based building blocks for the discovery of bivalent inhibitors to AChE.9198 Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 27 Acta Chim. Slov. 2017, 64, 15-39 curbituril, a macrocycle made of glycouril,89 Lewis et al. were the first to investigate the potential of Huisgen's cycloaddition for application to target-guided synthesis.91 In their proof-of-concept study, they selected enzyme acetylcholinesterase (AChE; EC 3.1.1.7) which plays a vital role in neuro-transmission in central and peripheral nervous system.92'93 The active site of AChE is a narrow gorge with the catalytic binding site located at its bottom. The second binding site, known as peripheral site, is at the rim of the active site.94'95 Since reversible AChE inhi- BOH-A4 QH-A4 Figure 13. A library of acetylene building blocks for in situ click chemistry screening of AChE.106 Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 28 Acta Chim. Slov. 2017, 64, 15-39 bitors are used clinically to treat neurodegenerative disorders, such as Alzheimer's disease,96 various small-molecule ligands specific for each binding site have been developed, together with such which simultaneously bind to both sites and therefore possess higher affinity for AChE.97-99 Moreover, dimerization of an inactive fragment of a selective and potent reversible AChE inhibitor Huperzine A has shown that an inactive ligand can be transformed into highly potent inhibitors.100 To address the possibility of self-assembly of bivalent AChE inhibitors via Huisgen's cycloaddition, Lewis et al. used a library of known site-specific inhibitors based on tacrine (a catalytic site binder with Kd of 18 nM) and phenylphe-nanthridinium (a peripheral site binder with Kd of 1.1 pM) derivatized with alkyl chains bearing terminal azide and alkyne moieties (Fig. 12).99,100 Each of the binary mixtures was incubated with AChE at room temperature for 6 days. Upon examination of binary mixtures, it was established that only TZ2 + PA6 combination gave a detectable amount of the triazole product.101 Blocking the active site with reversible (tacri-ne) or irreversible (diisopropyl fluorophosphate) inhibitor blocked formation of the triazole product, confirming that the active site is a template for reaction. HPLC analysis revealed that the enzyme-templated product is exclusively a syn-izomer. A comparison of the dissociation constant of syn-TZ2PA6 (Kd is 77 fM) and anti-TZ2PA6 (Kd is 720 fM) showed that AChE templated the formation of a more potent inhibitor. Comparison of kinetic parameters and literature data for related non-covalent inhibitors of AChE, revealed that in situ generated syn-TZ2PA6 was the most potent non-covalent AChE inhibitor known at the time.99,102-104 Manetsch et al. revisited the AChE system to screen for additional in situ hits.105 LC/MS analysis revealed three new hit compounds - TZ2PA5, TA2PZ6, and TA2PZ5 - in addition to the TZ2PA6. All of the products were identified as syn-isomers with dissociation constants in femtomolar and picomolar range. Krasinski et al. substituted phenylphenanthridinium moeity with aromatic heterocycles that were not previously known to interact with AChE while tacrine building block TZ2 was chosen as an "anchor molecule" (Fig. 13).106 Analysis of binary TZ2/acetylene mixtures with AChE revealed that only phenyltetrahydro-isoquinolines PIQ-A5 and PIQ-A6 formed significant amounts of triazole products identified as syn-isomers. Incubation of a mixture of 10 acetylene building blocks with TZ2 and AChE gave only expected triazole products TZ2PIQ-A5 and TZ2PIQ-A6 demonstrating the feasibility of multi-component screening. With the equilibrium dissociation constant of only 33 fM, TZ2PIQ-A5 surpasses the inhibition potency of syn-TZ2PA6. Beside the development of potent reversible AChE inhibitors for treating Alzheimer's disease, another kind of medical treatment has preoccupied the attention of researc- hers in the field. Organophosphorus (OP) nerve agents acting as irreversible AChE inhibitors represent a constant threat to the general population because of their use as warfare agents in armed conflicts and terrorist attacks or as pest control agents.107,108 Thus, the current therapy in case of OP nerve agent poisonings includes an AChE reactiva-tor of the quaternary pyridinium oxime family.109,110 However, due to their permanent positive charge, these compounds do not readily cross the blood-brain barrier and thus cannot reactivate AChE in the central nervous sys-tem.111 Therefore, attempts have been made to develop centrally acting reactivators using click-chemistry approach.112,113 The AChE related enzyme butyrylcholineste-rase (BChE) is present in the plasma in high concentrations and differs in the amino acid composition.114,115 BCh-E is capable of hydrolyzing a variety of esters and plays an important role in the bioconversion of carbamates and other ester-based prodrugs.116-118 Both AChE and BChE display selectivity and stereoselectivity in interaction with reversible or irreversible inhibitors, various esters and carba-mates.119-123 The in situ click-chemistry approach may help in the development of novel chiral reactivators tailored by cholinesterase itself thus avoiding cumbersome synthetic procedures and/or enantiomer separation. 4. 3. In situ Click Chemistry Experiments with Acetylcholine Binding Protein Recently, Grimster et al. reported the preparation of ligands for nicotinic acetylcholine receptors (nACh-Rs) via in situ click chemistry thus expanding the tem-plation potential of this approach to more flexible inter-subunit binding sites.124 As a member of a superfamily of neurotransmitter ligand-gated ion channels, nAChRs have been investigated as therapeutic targets for medical treatment of central nervous system (CNS) disorders such as schizophrenia, nicotine addiction, and Alzheimer's disease.125-127 However, the development of novel and potent ligands for specific receptor subtypes using classical drug discovery approaches has been difficult because of the nAChR membrane disposition, receptor subtypes diversity, and the dynamic nature of the binding site. Grimster et al. turned their attention to the in situ click chemistry approach with the acetylcholine binding protein (AChBP) as a structural surrogate for n-AChRs.124 AChBPs are homologous to the ^-terminal 210 amino acids in the extracellular receptor domain with flexible subunit interface, thus imitating recognition properties of nAChRs. Initially, screening the triazole library synthesized under standard Cu-catalyzed azide alkyne cycloaddition reaction conditions against AChBPs from Lymnaea stagnalis (Ls), Aplysia californica (Ac), and the Y55W Aplysia californica mutant (AcY55W) revealed compound 26 as the strongest binder to all three nAChR surrogates, with the dissociation constant in the nanomolar range for Ls AChBP (Fig. 14). Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 29 Acta Chim. Slov. 2017, 64, 15-39 Figure 14. Compound 26 with high affinity to Lymnaea stagnalis, Aplysia californica, and the Y55W Aplysia californica mutant AChBPs and constituent alkyne 27 and azide 28 shown in retrosynthetic representation.124 Figure 15. In situ click chemistry screening of azide libraries 28a and 28b against alkyne 27.1* Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 30 Acta Chim. Slov. 2017, 64, 15-39 To confirm that flexible subunit interfaces in the AChBPs are capable to template the formation of 26, the constituent alkyne 27 and azide 28 were incubated in the presence of Ls, As, and AcY55W AChBPs in sodium phosphate buffer at room temperature for 3 days. Analysis of the reaction mixture by LC/MS-SIM method confirmed that Ls AChBP successfully catalyzed the formation of compound 26, while both Ac and AcY55W AChBPs gave the product but in much lower amount. Control reaction with Ls AChBP inhibited with a known competing ligand methyllycaconitine (MLA) gave a relatively low amount of product, thus confirming that the ACh binding site at flexible subunit interface indeed served as the template for the cycloaddition reaction. The search for new compounds with improved affinity and selectivity for closely related AChBPs continued using triazole 26 as a lead. Azide libraries 28a and 28b comprising building blocks with quaternary nitrogen centers, were incubated with alkyne 27 in the presence of Ls, As, and AcY55W AChBPs at room temperature for 3 days (Fig. 15). LC/MS-SIM analysis revealed that Ls AChBP catalyzed the formation of triazole products 26, 38, 39, 40, and 41 more efficiently than Ac or AcY55W AChBPs. It was also shown that the amount of in situ generated product is related to its affinity to the specific AChBP. For instance, the most amplified triazole 40 was shown to possess the highest affinity (Kd = 0.96 nM) to Ls AChBP. Next, the alkyne library with the previously tested quino-lone derivative 27 and diversely substituted aryl pro-pargyl ethers was incubated with azide 33 in the presence of Ls, Ac, and AcY55W AChBPs. LC/MS-SIM analysis revealed that all of the tested alkynes underwent AChBP-templated cycloaddition reactions with azide 33. However, the previously described triazole 40 was again formed in the highest amount with the highest affinity for all AChBPs. Finally, azides 28-37 were mixed with alkynes in the presence of Ls AChBP for 10 days. Analysis revealed that 40 was formed in the greatest amount, thus demonstrating that Ls AChBP can catalyze the formation of the highest affinity product from a bulk of various azides and alkynes present in the reaction mixture, analogously to the AChE system. All in situ click chemistry experiments with AChBPs included BSA control reaction which exhibited no product formation. Crystal structure of triazole 40 in complex with Ac AChBP confirmed a bound conformation, and a pose predicted from previously seen conformations of quaternary amines that bind to nAChRs through cation-quadrupole interactions involving n-electron-rich aromatic side chains (e.g., tryptophan).128 Triazole moiety forms a hydrogen bond with a neighbouring water molecule which again suggests that precursors in in situ click chemistry drive a conformation preferred by the triazole product rather than accommodating a conformation of the free protein, a fact previously reported for the AChE system. 4. 4. DNA Minor Groove Templation Role The templation potential of in situ click chemistry can be expanded to the minor groove of double-helical DNA, as shown by Poulien-Kerstien and Dervan129 and more recently by Imoto et al.130 In their pioneer work, Poulien-Kerstien and Dervan explored the Huisgen's cycloaddition to link two aromatic-substituted hairpin polyamides capable of sequence-specific binding to DNA in the DNA-templated reaction. Polyamides composed of three aromatic amino acids, N-metylpyrrole (Py), N-methylimidazole (Im), and N-methyl-3-hydroxypyrrole, distinguish four Watson-Crick base pairs by a set of pairing rules and represent a potential way to modulate transcription.131 Longer binding-site size is considered to be crucial for application in gene regulation since longer sequences should occur less frequently in genome leading to the development of various polyamide motifs for selective targeting.132,133 The most promising strategy came from chemical ligation of two hairpin polyamides to form di-mers.134,135 However, though having an excellent affinity and specificity to 10 base pair (bp) DNA sequences, hairpin dimers lack the cell and nuclear uptake properties of smaller hairpins, apparently due to size and shape.136 Six-ring hairpin polyamides with alkyne 42a and 42b or azide 43a and 43b moieties with different linker lengths were designed so that their matching sites are adjacent on the DNA, which allows the formation of hairpin dimers in situ (Fig. 16).137-140 Experiments were carried out at 37 °C at pH 7.0 with equimolar concentrations of one azide, one alkyne and DNA duplex A (1 pM). When any pair of hairpin polyamides (42a + 43a, 42a + 43b, 42b + 43a, 42b + 43b) was combined in solution, HPLC analysis of the reaction mixtures (verified using matrix-assisted laser desorp-tion/ionization-time of flight mass spectrometry) revealed significant acceleration of formation of hairpin dimers in the presence of DNA template with respect to the nontem-plated reaction between 42a and 43a. The rate of dimer formation from 42a and 43b was slower than the rate of formation from 42a and 43a, presumably due to the additional flexibility in the linker of 43b, which allows the reactants to more freely adopt nonproductive conformation. Also, the rate of product formation from pairings of 42b with 43a and 43b is decreased due to the differences in the reactivity between 42a, activated with an electron withdrawing group (EWG), and EWG-free alkyne 42b. Moreover, when the alkynyl reactant is substituted with an EWG, stereoelectronics of the reaction pathway favoured formation of 1,4-regioisomer.141 Thermal reaction between 42a and 43a or 43b afforded predominantly the 1,4-regioisomeric products, while DNA-templated reactions afforded them exclusively. When the EWG-free alkyne 42b was paired with either 43a or 43b, each thermal reaction produced two corresponding regioisomers in a ratio of 1:1, while DNA-templated reaction produced only a single isomer (42b + 43a) or a ratio of 3:1 (42b + 43b). Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 31 Acta Chim. Slov. 2017, 64, 15-39 Figure 16. DNA-templated dimerization of hairpin polyamides on DNA duplexes with hairpin binding sites separated with zero (A), one (B), or two (C) base pairs.130 DNA-templated cycloadditions were found to be sensitive upon separation of the hairpin-binding sites with additional bp. Thus, upon insertion of one bp between two adjacent five bp hairpin-binding sites for the hairpin polyamides 42a,b and 43a,b (DNA duplex B), the only product formed from 42b and 43b was detected with about 50% yield. When two intervening bp were inserted (DNA duplex C), no product was detected using various pairs of hairpin polyamides. DNA-templated cycloadditions were also found to be sensitive upon DNA sequence of the two hairpin-binding sites, as illustrated by the mismatch tolerance study of optimal pair 42a and 43a. When a single bp mismatch is present under azide hairpin polyamide-bin-ding or under each of the two harpin-binding sites, the rate of the hairpin dimer-forming cycloaddition is nearly halved or lowered over 2.5 fold, respectively. However, when the concentration of reacting hairpins 42a and 43a was varied from 1 pM to 0.5 pM, a threshold concentration that defined the ability of hairpins to distinguish between match site and double bp mismatch site was detected somewhere between 1 pM and 0.75 pM. The authors suggested that, at some lower concentration, an additional threshold exists that allows hairpins to distinguish the match site from a single bp mismatch site, rendering the possibility to increase the ratio of hairpin dimer formation on match over mismatch DNA and the overall hairpin di-mer yield. Recently, Di Antonio et al. have demonstrated the ability of the in situ click chemistry multicomponent approach to identify potent and selective small molecules binding a region of chromosomes formed by guanine-rich sequences of DNA called G-quadruplex (G4).142 In their study, they selected G4 formed by the human telomeric DNA (H-Telo).143 No adduct was formed when the reaction mixture was incubated in the absence of DNA, in the presence of double-stranded DNA, or in the presence of telomeric oligonucleotides pre-annealed to prevent G4 formation, thus confirming that H-Telo serves as a reaction pot. Moreover, adducts obtained from a reaction conducted in the presence of RNA G4-structure demonstrated selective RNA versus DNA G4 structure binding. More recently, Glassford et al. have expanded the templation potential of the in situ click chemistry to E. coli 70S ribo-somes or their 50S subunits and thus synthesized potent macrolide antibiotics that target bacterial ribosome.144 Also, the in situ click chemistry approach has been applied to explore the conformational space of the ligand binding site of a M. tuberculoisis transcriptional repressor EthR which regulates the transcription of monooxygenase EthA and thus controls the sensitivity of M. tuberculoisis to an- Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 32 Acta Chim. Slov. 2017, 64, 15-39 tibiotic ethionamide. The in situ formed inhibitor, displayed 10-fold higher activity than the starting azide, and induced a significant conformational change of the li-gand-binding domain of EthR.145 5. Iterative in situ Click Chemistry In addition to the development of coupled bivalent enzyme inhibitors targeting the active site, in situ click chemistry can produce multivalent ligands active on protein surface, such as allosteric, interfacial, or non-functional surface sites. Once a bivalent ligand has been formed via in situ approach from the corresponding azide and alkyne building blocks, that biligand can serve as an anchor ligand for the identification of a triligand, and so forth, in a so-called iterative in situ click chemistry approach. This approach has been successfully introduced by Agnew et al. to identify a triligand antibody-like capture agent against human or bovine CA-II (h(b)CA-II) (Fig. 17).146 Figure 17. Iterative in situ click chemistry approach for developing triligand capture agent for human or bovine carbonic anhydrase II (b(h)CA-II). Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 33 Acta Chim. Slov. 2017, 64, 15-39 Figure 18. In situ click chemistry approach for developing triligand capture agent/inhibitor for Aktl kinase.150 Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 34 Acta Chim. Slov. 2017, 64, 15-39 The first anchor ligand was identified by screening a comprehensive one-bead-one-compound (OBOC) peptide library consisting of short chain peptides, against fluores-cently labelled bCA-II.147,148 Analysis of the position-dependent frequency of amino acids identified the anchor ligand, a short heptapeptide comprised of non-natural D-amino acids and a terminal, acetylene-containing amino acid D-propargylglycine (D-Pra), showing an approximately 500 pM affinity for bCA-II. This anchor ligand was used in the second screen against the OBOC peptide library, in which peptides were modified with an azide linker, in the presence of bCA-II to identify the triazole product showing a 3 pM binding affinity for bCA-II. The screen was repeated with this terminal D-Pra-containing biligand as the new anchor unit to identify a triligand, which exhibited strong binding affinities against bCA-II (64 nM) and hCA-II (45 nM). However, no regioselecti-vity was observed for the two triazoles in the triazole capture agent. On-bead, protein-templated triligand formation was confirmed by an enzyme-linked colorimetric assay containing a biotin conjugate of the biligand anchor.149 The triligand was only formed in the presence of b(h)CA-II, and not when b(h)CA-II was absent or other proteins (transferrin, BSA) used instead. Similarly, on-bead, protein-templated formation was not observed when the incorrect biligand anchor was used. The triligand did not interfere with bCA-II intrinsic esterase activity, which indicated that it binds away from the active site. The strategy described was also applied to identify a high-specificity, triligand capture agent/inhibitor for Akt1 kinase.150 Akt1 kinase is responsible for signal transduc-tion from the plasma membrane to downstream effector molecules that control cell growth, apoptosis, and transla-tion.151 To ensure the development of an allosteric site inhibitor, Millward et al. carried out an initial screen against a large OBOC peptide library on a kinase preinhibited with an ATP-competitive inhibitor, Ac7.150 One of the N-terminal azido-amino acid-containing peptides generated in the initial screen showed almost 95% inhibition of the Akt1 kinase in the absence and presence of the conjugated small molecule inhibitor and was therefore employed as an anchor for biligand development (Fig. 18). The most promising candidate from biligand screens was modified with 5-hexynoic acid at the N-terminus and used as an anchor ligand for triligand development which finally resulted in the tertiary peptide containing two tria-zole moieties. An analytical assay based on immune-PCR152 revealed that the click reaction between the on-bead secondary peptide and the soluble anchor peptide was approximately 10-fold more efficient in the presence of Akt1 than in its absence, confirming the requirement for the target protein to template the click reaction. The biligand showed 100-fold improvement in its affinity for Akt relative to the anchor peptide, while the triligand showed 2-3 fold affinity gain for Akt1 (Kd = 200 nM). The specificity characterization of the anchor, biligand, and triligand for a panel of His-tagged protein kinases revealed that the anchor was very specific for the Aktl protein, with only modest cross-reactivity to GSK3P protein kinase. The biligand showed reduced specificity, with significant binding to GSK3p. For the triligand, binding to GSK3P was reduced to the level observed for the anchor peptide. These observations indicate that large improvements in affinity may come at the expense of reduced specificity, whereas increased specificity is not necessarily accompanied by increased affinity. This inverse correlation between affinity and selectivity is in accordance with previous studies on small molecule protein kinase inhibi-tors,153 antibody-small molecule interactions,154 DNA-protein interactions,155 and protein-protein interactions.156 Measuring Akt1 kinase activity under varying substrate and triligand concentrations eliminated the possibility of a competitive mode of Akt1 inhibition by the triligand with respect to ATP and peptide substrates.150 This confirmed that the triligand binds to a location away from the active site of the kinase and that inhibition occurs via an allosteric mechanism. Finally, the anchor, biligand, and triligand were tested for the ability to recognize Akt from the ovarian cancer cell line OVCAR3 in immunopre-cipitation (IP) experiments. IP experiments confirmed the increased affinity of the biligand relative to the anchor peptide in OVCAR3 cell lysates from both cells stimulated with a combination of epidermal growth factor (EGF) and insulin and from untreated control cells. The triligand showed somewhat increased IP of Akt relative to the biligand only in lysates from induced cells. However, an analysis of the total IP protein by SDS-PAGE electropho-resis showed low non-selective binding for all ligands. The authors observed IP of the protein that likely corresponds to the GSK3P kinase by the triligand, and to a lesser degree, by the anchor and the biligand.150 The underlying rationale for GSK3 binding to ligands is yet to be explained. However, IP experiments confirm the increase in capture efficiency of ligands, particularly in stimulated cells, as they are being translated from anchor to triligand with their affinity and selectivity criteria increased. 6. Conclusion Receptor-based combinatorial chemistry is a promising strategy developed for identifying possible leads in drug discovery whereby the biomolecular target of interest is used to "fish out" building blocks that couple into high affinity compounds. Theoretical studies have shown that, unless excessive amounts of a molecular target are used, high affinity compounds have a high probability of being significantly amplified over other possible combinations of building blocks. Also, any significantly amplified compound is guaranteed to be a high affinity compound. The examples listed in this review have illustrated the potential of various receptor-based combinatorial che- Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 35 Acta Chim. Slov. 2017, 64, 15-39 mistry approaches to identify high affinity compounds and, in some occasions, their potential to elucidate the binding modes of substrates to their biomolecular target. The in situ click chemistry approach combines building blocks through 1,3-dipolar cycloaddition of azides and alkynes (Huisgen's cycloaddition). This approach is predominantly used for the discovery of enzyme inhibitors targeting enzyme active sites as illustrated with examples from the AChE system, although the templation potential of this approach can be extended to more flexible intersubunit binding sites and even minor groove of double-helical DNA. Examples from AChE and AChBP systems have shown that in situ click chemistry allows one to freeze in-frame conformations that associate with high-affinity inhibitors and are normally not detected by conventional structural methods. 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Borrelli, M. Ventura, R. Pantano, G. Fu-magalli, M. S. Christodoulou, D. Monticelli, M. Luzzani, A. C. Fallacara, C. Tintori, M. Botta, D. Passarella, ACS Med. Chem. Lett., 2013, 4, 274-277. https://doi.org/10.1021/ml300394w 158. M. Mondal, N. Radeva, H. Köster, A. Park, C. Potamitis, M. Zervou, G. Klebe, A. K. H. Hirsch, Angew. Chem., Int. Ed., 2014, 53, 3259-3263. https://doi.org/10.1002/anie.201309682 Povzetek Sodobno odkrivanje zdravil v glavnem temelji na de novo sintezah velikega števila spojin z različnimi kemijskimi funkcionalnimi skupinami. Čeprav je kombinatorialna kemija omogočila pripravo velikih knjižnic spojin iz različnih gradnikov, še vedno ostaja težava identifikacije spojin vodnic. Odkritje dinamičnih metod kombinatorialne kemije predstavlja korak naprej, saj pri sami sintezi visoko afinitetnih produktov vključuje biološke makromolekularne tarče (receptorje). Glavni preboj predstavlja sintezna metoda pri kateri se gradniki ireverzibilno povežejo le ob prisotnosti receptorja. Predstavljamo različne pristope v kombinatorialni kemiji, ki temeljijo na prisotnosti receptorjev. Pri Huisgenovi cikloa-diciji (1,3-dipolarna cikloadicija azidov z alkini) nastanejo stabilni 1,2,3-triazoli; pogosto z zelo visokimi afinitetami do receptorja, ki lahko dosežejo celo femtomolarno območje, kot prikazuje primer z inhibitorji acetilholinesteraze. Huisgenovo cikloadicijo lahko uporabimo tudi pri različnih drugih receptorjih: acetilholinesterazi; proteinih, ki vežejo acetilholin; karboanhidrazi-II, serin/treonin-proteinski kinazi in pri vezavi na mali žleb DNA. Marakovic and Sinko: The Lock is the Key: Development of Novel Drugs ... 40 DOI: 10.17344/acsi.2016.2702 Acta Chim. Slov. 2017, 64, 40-44 ^creative ^commons Scientific paper Computational Investigation of the Dissociative Adsorption of Dichloroacetylene (C2Cl2) on N Functionalized Carbon and Carbon Germanium (CGe) Nanocone Sheets in the Gas Phase and Dimethyl Sulfoxide Meysam Najafi* Young Researchers and Elite Club, Kermanshah Branch, Islamic Azad University, Kermanshah, Iran * Corresponding author: E-mail: meysamnajafi2016@gmail.com Phone: +98-8337243181 Fax: +98-8337243181 Received: 25-06-2016 Abstract The possibility of dichloroacetylene-sensing on carbon nanocone sheet and carbon germanium nanocone sheet surfaces has been investigated. The effects of nitrogen functionalization and dimethyl sulfoxide on the adsorption of dichloroacetylene gas on carbon nanocone sheet and carbon germanium nanocone sheet surfaces were investigated. Results reveal that adsorption of dichloroacetylene on studied nanocone sheets were exothermic. Results show that, adsorption energy value of dichloroacetylene on carbon germanium nanocone sheet surface were more negative than corresponding values of carbon nanocone sheet. Results reveal that, N functionalization and dimethyl sulfoxide, increase and decrease the absolute adsorption energy value of dichloroacetylene on studied nanocone sheets, respectively. These results show that, there were good linearity dependencies between adsorption energy and orbital energy values of studied nano-cone sheets. Keywords: COSMO, DMSO, nanocone sheet, C2Cl2, sensor 1. Introduction Dichloroacetylene is an oily pyrophoric chemical compound with the chemical formula C2Cl2. The compound is volatile at standard temperature and pressure and explodes on contact with air. It is a toxic compound.1-3 It displays nephrotoxic effects to rats, but not to humans. It can be made from the compound trichloroethylene.1-3 The most common effect that the compound has on humans is the development of disorders.1-3 These disorders can persist for any amount of time between a number of days and a number of years. Exposure to the chemical can also cause a large range of other symptoms, including a headache, vomiting and nausea, jaw pain, cranial nerve palsy, appetite loss and acute lung edema. C2Cl2 level of carcinogenetic in humans is not classifiable, although there are small amounts of evidence that suggest that the chemical is carcinogenic in animals.4,5 Studies on male rats and rabbits have shown that inhalation of C2Cl2 can cause tubular necrosis, focal necro- sis, and other nephrotoxic effects.6'7 Additionally, the rabbits that were given C2Cl2 experienced hepatotoxic and neuropath logical effects. Inhalation of C2Cl2 also causes benign tumors of the livers and kidneys of rats. The chemical increase the incidences of lymphomas.9-10 In recent years, Carbon nanocone sheet (C-NCS) and their functionalized derivatives as gas toxic sensors have been used, widely. In addition to C-NCS, there are other nanocone sheets which are found experimentally such as carbon germanium nanocone sheets (CGe-NCS).11-19 In the current study, the interactions of C2Cl2 gas with C-NCS and CGe-NCS with disclination angles of 240° exploring its potential application as C2Cl2 gas sensor will be theoretically investigated. The N func-tionalization of nanostructures is very important and it can effectively change the electronic structures of nanostruc- tures.19,20 Ibrahim and et al.21 in previous study, polymerization of aniline by Cu (II) montmorillonite studied using attenuated total reflection Fourier-transform infrared Najafi: Computational Investigation of the Dissociative Adsorption ... Acta Chim. Slov. 2017, 64, 40-44 41 (ATR-FTIR) spectroscopy. Also experimental spectra were compared with that calculated by AMI, PM3, PM5, MINDO, Hartree-Fock, HF/6-31G(d), as well as Density Functional Theory, BLYP/DZVP and B3LYP/6-31g(d,p). Ibrahim and et al.22 used Density functional theory (DFT) to investigate both the structure and vibrational frequencies of acetate group. A model of B3LYP with four basis set was used to optimize and locate the energy minimum of the acetic acid molecule. Ibrahim and et al.23 studied molecular structure of gelatin by using Fourier transform infrared spectroscopy FTIR. The spectrum was subjected to deconvolution in order to elucidate the constituents of the molecular structure. Ibrahim and et al.24 promised na-nomaterials in the field of optical sensors due to their unique properties. Emeraldine base of polyaniline (Nano EB-PANI) was prepared, characterized and applied as an optical formaldehyde sensor. Figure 1. Complexes of C2Cl2 with C-NCS, CGe-NCS, N-C-NCS and N-CGe-NCS. In previous study, Warshel and et al.25 utilized computer simulations to elucidate the true molecular basis for the experimentally observed effect. They start by reproducing the trend in the measured change in catalysis upon mutations. They discuss the role of flexibility and conformational dynamics in catalysis, once again demonstrating that their role is negligible and that the largest contribution to catalysis arises from electrostatic preorganization. In previous studies, Warshel and et al.26,27 described a general approach for exploring the energetics of different feasible models of the action of CcO, using the observed protein structure, established simulation methods and a modified Marcus' formulation. They start by reviewing our methods for evaluation of the energy diagrams for different proton translocation paths and then present a systematic analysis of various constraints that should be imposed on any energy diagram for the pumping process. In previous study, Warshel and et al.28 considered the current state of simulations of electrostatic energies in macromolecules as well as the early developments of this field. They focused on the relationship between microscopic and macroscopic models, considering the convergence problems of the microscopic models and the fact that the dielectric 'constants' in semimacroscopic models depend on the definition and the specific treatment. In previous study, Warshel and et al.29 described application of the calculated geometry and vibrations to the analysis of vibronic structure. A preliminary account of the use of observed vibronic structure for determination of the geometry of excited electronic states was given. In previous study,30 it be observed that the predominant initiation reaction for oxidation of methane, propene, and o-xylene under fuel lean conditions involved hydrogen abstraction of the methyl hydrogen by molecular oxygen forming hydroperoxyl and hydrocarbon radical species. The study of adsorption of toxic gas on the solid surface of nanostructures in order to identify the suitable sensor to remove or reduce the toxic gas are important in environmental issue. C2Cl2 has a toxic effect on humans who are exposed to it. Therefore adsorption C2Cl2 by nano structures is important and fundamental objects of present paper are: (1) to investigate the C2Cl2 adsorption on C-NCS and CGe-NCS surfaces; (2) to compare the C2Cl2 adsorption ability of C-NCS and CGe-NCS; (3) to identify the effect of N functionalization of studied C-NCSs and CGe-NCSs on adsorption of C2Cl2; (4) to explore how the solvent alter the C2Cl2 adsorption on studied C-NCS and CGe-NCS surfaces; (5) To find the C-NCS and CGe-NCS with highly effective detection of C2Cl2. 2. Computational Details In this paper, structure of C-NCS (constructed of 108 C atoms) and CGe-NCS (constructed of 54 C and 54 Ge atoms) with disclination angles of 240° and their N functionalized derivatives were geometry optimized in the gas phase and solvent. Also the structure of complexes of studied C-NCSs and CGe-NCSs with C2Cl2 molecule were geometry optimized in gas phase and solvent (structures were shown in figure 1). In order to avoid boundary effects, atoms at the open ends of the studied C-NCSs and CGe-NCSs were saturated with hydrogen atoms.19 All the calculations were performed using the DFT/B3LYP method and 6-31G(d,p) basis set within the GAMESS package 19,31,32 Also, harmonic vibrational frequencies have been calculated, enabling us to confirm the real minima. Solva-tion effects were included through the use of the polarized continuum model (PCM).19,33 The B3LYP is a reliable and common used level of theory in the study of different na- Najafi: Computational Investigation of the Dissociative Adsorption 42 Acta Chim. Slov. 2017, 64, 40-44 nostructures.19'34-36 A dielectric constant of 46.7 was used corresponding to that for dimethyl sulfoxide (DMSO) as the solvent. The adsorption energy (Ead) of C2Cl2 molecule on the C-NCS and CGe-NCS is obtained using the following equation: Ead = E (nanocone sheet/C2Cl2) - E (nanocone sheet) - E (C2Cl2) + Ebsse (1) where E(nanocone sheet/C2Cl2) is the energy of C-NCS or CGe-NCS-C2Cl2 complex, and E(nanocone sheet) and E(C2Cl2) are referred to the energies of C-NCS or CGe-NCS and C2Cl2 molecule, respectively. The negative value of Ead indicates the exothermic specificity of the adsorption. The basis set superposition error (BSSE) has been corrected for all of the interactions.37 3. Results and Discussion wing order in gas phase and DMSO: C-NCS < N-C-NCS < CGe-NCS < N-CGe-NCS. In according to obtained Ead values of C2Cl2 on studied nanocone sheet surfaces in gas phase and DMSO' it can be concluded that N-CGe-NCS and C-NCS have higher and lower ability to adsorption of C2Cl2' respectively. These results in this section can be interpreted with a known fact that Ge atoms in studied CGe-NCS stabilize the CGe-NCS and their C2Cl2-CGe-NCS complexes; hence' these results in increased absolute Ead in comparison to studied C-NCS in gas phase, DMSO.19 Also results show that in compare to gas phase, DMSO attenuate the absolute Ead values of C2Cl2 on studied nanocone sheet surfaces ca 0.197 eV. Fundamental reason for decrease in absolute Ead values in DMSO, could be an unequal stabilization/destabilization of the studied nanocone sheets and their complexes with C2Cl2 in DMSO.19 Therefore results in this study show that, the N-CGe-NCS and C-NCS have the most and less absolute Ead values of C2Cl2 on studied nanocone sheet surfaces. 3. 1. The Ead values of C2Cl2 Gas on Studied Nanocone Sheet Surfaces in Gas Phase and DMSO The calculated Ead values of C2Cl2 gas on C-NCS and CGe-NCS and their N functionalized derivatives (N-C-NCS and N-CGe-NCS) in gas phase and DMSO were reported in the table 1. Results in table 1 show that, the Ead values of C2Cl2 on C-NCS and CGe-NCS in gas phase were -3.13 and -3.48 eV, respectively. Also the Ead values of C2Cl2 on C-NCS and CGe-NCS in DMSO aie -2.94 and -3.25 eV, respectively. Results in table 1 show that, the Ead values of C2Cl2 on N-C-NCS in gas phase and DMSO were -3.66 and -3.48 eV, respectively. Also the Ead values of C2Cl2 on N-CGe-NCS in gas phase and DMSO were -4.06 and -3.87 eV, respectively. Results reveal that, N functionalization of C-NCS increase the absolute Ead values of C2Cl2 in comparison to C-NCS ca 0.53 and 0.54- eV in gas phase and DMSO, respectively. Results indicated that, DMSO decrease the absolute Ead values of C2Cl2 on N-C-NCS and N-CGe-NCS in comparison to gas phase ca 0.18 and 0.19 eV, respectively. Results indicate that the absolute Ead values of the C2Cl2 on studied nanocone sheets decreased in the follo- Table 1. Calculated Ead (in eV) of C2Cl2 on C-NCS, CGe-NCS, N-C-NCS and N-CGe-NCS surfaces in gas phase and DMSO. DMSO Gas phase Nanostructure -2.94 -3.13 C-NCS -3.25 -3.48 CGe-NCS -3.48 -3.66 N-C-NCS -3.87 -4.06 N-CGe-NCS 3. 2. The Ehomo and ELUMO of Studied Nanocone Sheets In this work the EHOMO, ELUMO and EHLG values of C-NCS and CGe-NCS and their N functionalized derivatives were calculated and reported in table 2. In this section the dependencies of between Ead corresponding EHOMO, ELUMO and EHLG values of studied nanocone sheets were investigated. Results show that, calculated EH values of stu- died nanocone sheets range from -5.58 to -6.12 eV. Therefore obtained absolute EHOMO values of studied nanoco-ne sheets show that the N-CGe-NCS and C-NCS have higher and lower tendency to lose electron, respectively.19 Results reveal that, calculated ELUMO values of studied nanocone sheets range from -3.57 to -3.94 eV. Therefore obtained ELUMO values of studied nanocone sheets show that the N-CGe-NCS and C-NCS have higher and lower capacity to accept electrons, respectively. 19 Results indicated that, calculated EHLG values of studied nanocone sheets range from 1.64 to 2.55 eV. Therefore EHLG values of studied nanocone sheets show that the N-CGe-NCS have lower stability and higher reactivity and C-NCS have lower reactivity.19 In according to obtained results in table 2, it can be concluded that N functionalization of C-NCS and CGe-NCS increase the absolute ELUMO values and decrease the absolute EHOMO and EHLG values in comparison to C-NCS and CGe-NCS. The computed Ead values of C2Cl2 on studied nanocone sheet surfaces are corrected against corresponding calculated EHOMO, ELUMO and EHLG values of studied nanocone sheets. Equations obtained from the linear regression are as follows: Ead = - 1.71 X (Ehomo) - 13.54 (2) Najafi: Computational Investigation of the Dissociative Adsorption Acta Chim. Slov. 2017, 64, 40-44 43 Ead = 2.49 x (Ehomo) + 5.72 Ead= 1.03 x (EhLG) - 5.74 (3) (4) The correlation coefficients of equations 2, 3 and 4 reached ca 0.985, 0.992 and 0.990, respectively. These results show that, there are good linearity dependencies between Ead and orbital energy (EHOMO, ELUMO and EHLG) values of studied nanocone sheets. This can be useful in the selection of suitable nanocone sheets with enhanced C2Cl2 adsorption potential.19 As mentioned in tables 1 and 2, this can be concluded the calculated Ead and orbital energy scales have same trends for averment C2Cl2 adsorption potential of studied nanocone sheets. Therefore results in this study, reveal that N-CGe-NCS has highest and C-NCS has lowest C2Cl2 adsorption potential among studied nanocone sheets.19 Table 2. Calculated Ehomo (in eV) ELUMO (in eV) and Ehlg (in eV) of C-NCS, CGe-NCS, N-C-NCS and N-CGe-NCS. E HLG E LUMO E HOMO Nanostructure 2.55 -3.57 -6.12 C-NCS 2.16 -3.69 -5.85 CGe-NCS 2.03 -3.74 -5.77 N-C-NCS 1.64 -3.94 -5.58 N-CGe-NCS Finally higher absolute Ead and EL values and lo- wer Ehomo and EHLG values for studied nanocone sheets are appropriate benchmarks to approval the C2Cl2 adsorption potential. Therefore it can be concluded the Ead, Ehomo, Elumo and EHLG values of studied nanocone sheets can consider as important parameters to predicate and propose suitable nanocone sheets with enhanced C2Cl2 adsorption potential.19 4. Conclusion In this study the Ead values of C2Cl2 gas on C-NCS and CGe-NCS surfaces in gas phase were investigated using density functional theory calculations. The effects of N functionalization and DMSO on the adsorption of C2Cl2 gas on C-NCS and CGe-NCS surfaces were investigated. Results reveal that adsorptions of C2Cl2 on studied nanocone sheets were exothermic and experimentally possible from the energetic viewpoint. Results show that, Ead value of C2Cl2 on CGe-NCS surface are more negative than corresponding values of C-NCS. Results reveal that, N func-tionalization and DMSO causing an increase and decrease the absolute Ead values of C2Cl2 on studied nanocone sheets, respectively. Results show that, there are good linearity dependencies between Ead and orbital energy values of studied nanocone sheets. Therefore it can be concluded the E , and orbital energy values of studied nanocone sheets can consider as important parameters to propose suitable nano-cone sheets with enhanced C2Cl2 adsorption potential. 5. Acknowledgment Thank colleagues for their valuable discussion on the computational affairs. 6. 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Phys. 1970, 19, 553-566. https://doi.org/10.1080/00268977000101561 Povzetek S pomočjo funkcionalno gostotne teorije v plinski fazi smo proučevali možnost zaznavanja C2Cl2 na C-NCS in CGe-NCS površinah. Proučevali smo tudi učinke N funkcionalizacije in DMSO na adsorpcijo C2Cl2 na teh površinah. Rezultati kažejo, da je adsorpcija C2Cl2 na površini nanstožcev eksotermna in z energetskega vidika možna. Energija adsorp-cije, Ead, C2Cl2 na CGe-NCS površini je bolj negativna od Ead na C-NCS. Izkazalo se je, da N funkcionalizacija povzroči zvišanje in DMSO znižanje absolutne vrednosti Ead C2Cl2 na proučevane nanostožce. Dokazali smo tudi linearno zvezo med Ead in orbitalnimi energijami nanostožcev. Najafi: Computational Investigation of the Dissociative Adsorption DOI: 10.17344/acsi.2016.2772 Acta Chim. Slov. 2017, 64, 45-54 ^creative tS/commons Scientific paper Infrared Spectroscopy for Analysis of Co-processed Ibuprofen and Magnesium Trisilicate at Milling and Freeze Drying Manoj Acharya, Satyaki Mishra, Rudra N. Sahoo and Subrata Mallick* Faculty of Pharmaceutical Sciences, Siksha 'O' Anusandhan University, Kalinganagar, Khandagiri Square, Bhubaneswar, OR, India. * Corresponding author: E-mail: subratamallick@soauniversity.ac.in; s_mallickin @yahoo.com; profsmallick@ gmail.com Fax: +91-674-2386271; Tel: +91-674-2386209 Received: 28-07-2016 Abstract Assessment of interactions of ibuprofen and magnesium trisilicate after co-processing has been carried out by infrared spectroscopy. Dry-state ball-milling and, aqueous state kneading and freeze-drying were performed. FTIR spectroscopy of co-processed materials described acid-base reaction between the carboxylic acid containing ibuprofen to a significant extent. Increased absorbance of carboxylate peak accompanied by a consistently reduced absorbance of the car-bonyl acid peak was evident. Absorbance of carboxylate peak was more in freeze-dried sample compared to milled product. Intermolecular hydrogen bonding between ibuprofen and magnesium trisilicate in the co-processed material has been suggested. Inhibition of crystal morphology has been noticed in the photomicrographs of both the products. DSC report has shown absence or significantly decreased melting endotherm representing almost complete amorphization of ibuprofen. Release of drug increased greatly after co-processing in comparison to crystalline ibuprofen. Freeze-dried samples have improved drug release more significantly compared to ball-milled samples. Keywords: Infrared spectroscopy; co-milling; co-freeze drying; scanning electron microscopy; differential scanning calorimetry. 1. Introduction Infrared spectroscopy is a workhorse technique for pharmaceutical analysis in recent years. Infrared spectrum represents the molecular absorption and transmission, creating a molecular fingerprint of the sample. It corresponds to the frequencies of vibrations between the bonds of the atoms. Material is a unique combination of atoms and no two compounds produce the exactly same infrared spectrum. Changes in the frequency and shape of the bands of a drug could be utilized for the analysis of possible redistribution of electronic density in the structure of the molecule for the assessment of interactions. Ibuprofen, the most commonly prescribed NSAIDs1 [chemical formula: (CH3)2CHCH2C6H4CH(CH3)COOH] is known to induce injury of the gastrointestinal tract and cau- se changes in the permeability and structural properties of the membrane.2'3 Magnesium trisilicate is used therapeutically as an antacid in the treatment of peptic ulcers. Via a neutralization reaction it increases the pH of gastric juice. After precipitation colloidal silica can coat gastrointestinal mucosa which can confer further protection. Indigestion, heartburn, or gastroesophageal reflux can sometimes be symptoms of more serious conditions such as stomach ulcers or stomach cancer. Doctor consultation is necessary before taking magnesium trisilicate when an individual is taking a non-steroidal anti- inflammatory drug. Magnesium trisilicate interacts with a number of drugs and alter their absorption, thereby reducing their effecti-veness.4-8 Enteric coatings designed to prevent the dissolution in the stomach may also be damaged by magnesium trisilicate.9 Magnesium trisilicate is a compound of magnesium oxide and silicon dioxide with varying proportions of water (2MgO'3SiO2'XH2O) (USP 28). Magne- Acharya et al.: Infrared Spectroscopy for Analysis of Co-processed ... 46 Acta Chim. Slov. 2017, 64, 45-54 sium trisilicate is a solid adsorbent and could also be utilized to improve the dissolution of poorly soluble drugs.1011 Solid-dispersion granules of a poorly water-soluble drug containing microporous magnesium aluminosilicate (Neusilin) prepared by hot-melt granulation technique has shown improved dissolution of drug.1213 The solid dispersion granules of BAY 12-9566 containing Neusilin were successfully compressed into tablets and increased dissolution. The hydrogen-bonding potential of silanol groups on the surface of Neusilin brought about the increase in the drug release rate. In the present study assessment of interactions of ibuprofen and magnesium trisilicate has been undertaken by infrared spectroscopy after milling together in the dry-state and freeze-drying after aqueous state kneading. Ball milling is a powerful tool for particle size reduction and processing in the pharmaceutical industries.14 It is also a device for effecting chemical reactions by mechanical energy in dry-state and at ambient temperatu-res.1516 Ball milling presents a greener route for many processes compared to the use of microwave and ultrasound as energy sources. Impact and attrition during ball milling can bring about changes in the crystal structure of the drug and can induce amorphization17-22 and improve bioavailability.23 Freeze drying is a standard process used to stabilize and store the drug products in the pharmaceutical industries.24 FTIR spectroscopy was monitored to identify the mechanism of interaction25-27 of the carboxylic acid-containing drug ibuprofen with magnesium trisilicate. The interaction study has also been monitored by scanning electron microscopy and differential scanning calorimetry (DSC). Afterward, in-vitro drug release from the formulated co-processed powder was carried out to assure about the biological availability of the drug.28 The detailed infrared spectroscopy of this type of interaction after co-processing by dry-state milling, and aqueous state equilibration and freeze drying has rarely been reported earlier. Nokhodchi et al.,29 crystallized ibuprofen in presence of starch derivatives for improved pharmaceutical performance and found no significant change in FTIR spectroscopy and concluded that there is no change in molecular level of ibuprofen. Ibu-profen solid dispersions prepared using polyethylene glycol 4000 have shown no significant change in FT-IR spectra.27 2. Experimental 2. 1. Materials Ibuprofen was obtained from Tejani Life care, Cuttack, India and magnesium trisilicate (USP 28) was purchased from Burgoyne & Co, India (not less than 20% of magnesium oxide and not less than 45% of silicon dioxide; loss on ignition 17.0-34.0%). All other chemicals used were of analytical reagent grade. 2. 2. Co-processing of Ibuprofen and Magnesium Trisilicate Crystalline powder of ibuprofen and magnesium trisilicate powder were mixed for approximately 5 minutes by simple blending process using mortar and spatula at laboratory ambient condition in the dry-state (~30 °C; ~60% RH) without trituration. Ibuprofen and magnesium trisilicate (physical mixtures) weight ratios (3: 1, 2: 1, 1: 1 and 1: 2) were maintained as per formulation and left for immediate use in the co-process of dry-state ball-milling and, aqueous state kneading and freeze-drying. 2. 3. Dry-state Ball-milling The powder mixture of ibuprofen and magnesium trisilicate in the weight ratios was placed into a cylindrical vessel of ball mill (Swastik Electric and Scientific Work, India) and 1 h period of constant milling was performed in the dry-state at lab ambient condition of ~30 °C, ~60% RH. Significant increase in temperature of the milled material has not been detected at the end of the co-process. Ball charged in the vessel allowed smooth cascading motion, and significant attrition and impact during dry-state milling while operating the mill at 100 rpm for 1 h. 2. 4. Aqueous State Kneading and Freeze-drying Aqueous state kneading was performed by adding small amount of water in the physical powder mixtures of ibuprofen and magnesium trisilicate and left for a period of about 12 h at ambient conditions for equilibration. The kneaded samples were freeze-dried using a laboratory vacuum freeze dryer (4kg, 220 V) with attached vacuum (220V, 2.7A, 370W, 1400r/min, 50Hz) (Lark, Penguin Classic Plus, India) for 10-12 hours for effective drying. The pressure during freeze-drying was adjusted to 15-20 Pa while temperature maintained approximately at -40 °C. The freeze-dried samples were preserved in the desiccator till further analysis. The ball-milled and freeze-dried samples were left at ambient condition (~60% RH, ~30 °C) for few hours and dried in an incubator (Labotech, India) at 50 °C. The powder materials were passed through mesh 44 (opening ~350 pm) and assayed for drug content determination from the absorbance measured at 222 nm (^max) in the UV visible Spectrophotometer (Jasco-V630 UV Spectrophotometer Spectrometer, Software: Spectra Manager) using standard calibration curve of ibuprofen. 2. 5. Ibuprofen-magnesium Trisilicate Interaction Study FTIR spectra of pure crystalline ibuprofen and co-processed powder samples were performed for a compa- Acharya et al.: Infrared Spectroscopy for Analysis of Co-processed ... 47 Acta Chim. Slov. 2017, 64, 45-54 rative study between co-milling and co-freeze drying interaction. All the samples were mixed thoroughly with potassium bromide in the ratio of 1:100. KBr discs were prepared by compressing the powders at a pressure of 6 tonnes for 10 min in a Hydraulic pellet press (Techno-search Instruments, Maharashtra, India). FTIR spectrometer (FTIR-4100 type A, Jasco, Tokyo, Japan) was used for collecting all scans from 4000-400 cm1 of 80 accumulations at a resolution of 4 cm-1 and scanning speed of 2 mm/s. Spectral Manager for Windows software (Jasco, Tokyo, Japan) was used for data acquisition and holding. 2. 6. Surface Morphology and Thermal Analysis of the Particle Surface morphology and crystalline nature of the particulate samples were investigated using Scanning electron microscope (Instrument JSM-6390, Jeol, Tokyo, Japan). The powder samples were dried and sputtered with gold and scanned at room temperature using an accelerated voltage of 10 kV (Wd 19 and Spot_Size 48). Thermal behavior of the powder samples was characterized using a Differential scanning calorimeter (DSC, Universal V4.2E TA Instruments). Samples approximately 5-6 mg were weighed accurately and put into crimped aluminum pans with a pin hole in the lid. All samples were heated at a heating rate of 10 °C/min in an atmosphere of nitrogen gas purge at 50 ml/min from 30 and 300 °C. 2. 7. Drug release Studies Powdered samples containing 10 mg equivalent of ibuprofen were dispersed in 900 ml of distilled water and drug release was carried out using USP XXIV type II dissolution apparatus (Electrolab, dissolution tester USP TDT 06L, India) at a temperature of 37 ± 0.2 °C and paddle rotation set at 100 rpm. Ibuprofen concentration was determined by UV absorption at 222 nm. Aliquots were withdrawn at appropriate time intervals of 5, 10, 15, 30, 60, 90 and 120 min, and replaced with a fresh dissolu- tion medium. After proper rinsing of the cuvette and filtration of the aliquot through a 0.45 pm membrane filter, absorbance was recorded using the UV-Visible Spectro photometer. Standard calibration curve was used for calculating the respective concentration and the data were utilized to estimate cumulative percent drug release. Cumulative percent drug release was reported as the mean of not less than three determinations. 3. Results and Discussion The dry-state co-milling and aqueous state co-processing could be analogous to the commonly followed processes in the tablet granulation department of pharmaceutical industries. These processes are effective, simple and scalable for interaction study. Due to presence of varying amount of bound moisture in native magnesium trisilicate the co-milled materials became moisty in nature and needed drying. Instant character of the freeze-dried samples is to absorb moisture like a sponge when left at ambient condition of ~60% RH and 30 °C for few hours and drying in an incubator at 50 °C becomes necessary. The co-processed dried and equilibrated powder materials were passed through mesh of opening ~350 pm and assayed for actual drug content determination. Ibuprofen-magnesium trisilicate interaction study has been characterized by FTIR, and the usefulness of this powerful technique has been supported by scanning electron microscopy and differential scanning calorime-try as described below. Drug release from the formulated dosage form is important and ultimately related to the bi-oavailability of the drug. Dissolution of ibuprofen from the co-processed material has also been described below. Formulation detail and code of ibuprofen samples co-processed with magnesium trisilicate has been mentioned in Table 1. 3. 1. FTIR Analysis Spectral data of FTIR band assignments of ibupro-fen and co-processed samples are tabulated in Table 2. Table 1. Formulation code of ibuprofen samples co-processed with magnesium trisilicate (Ibuprofen = IB, Magnesium trisilicate = MTS). Formulation code Drug: MTS ratio Co-processing Ibuprofen assay (%) IB - - - IB1M1pm 1 : 1 Physical mixture without trituration - IB3M1B 3 : 1 Dry-state Ball-milling for one hour 71.61 ± 5.1 IB2M1B 2 : 1 Dry-state Ball-milling for one hour 68.65 ± 4.6 IB1M1B 1 : 1 Dry-state Ball-milling for one hour 46.09 ± 3.5 IB1M2B 1 : 2 Dry-state Ball-milling for one hour 37.54 ± 2.8 IB3M1F 3 : 1 Aqueous state equilibration and freeze-drying 74.11 ± 3.2 IB2M1F 2 : 1 Aqueous state equilibration and freeze-drying 68.55 ± 3.8 IB1M1F 1 : 1 Aqueous state equilibration and freeze-drying 48.65 ± 2.4 IB1M2F 1 : 2 Aqueous state equilibration and freeze-drying 30.54 ± 2.1 Acharya et al.: Infrared Spectroscopy for Analysis of Co-processed ... Table 2. Spectral data of FTIR of Ibuprofen and co-processed samples oo > 8- s» CS 2 s a. I -s S a ö n © 3 TO a. Band Tentative assignments Ibuprofen MTS IBJMJF 115,1X1,1 Wavenumber (cm x) 115,1X1,1 115,1X1,1 IBjMJB 115,1X1,15 115,1X1,15 II? ,1X1,15 1 OH stretching absent 3200- 3200- 3200- 3200- 3200- 3200- 3200- 3200- 3200- 3550 bb 3550 bb 3550 bb 3550 bb 3550 bb 3550 bb 3550 bb 3550 bb 3550 bb 2 CH2 asym str 3094 m - absent absent absent absent 3096 w 3096 w absent absent 3 CH3 asym str 2958 vs - 2955 vs 2955 vs 2954 vs 2954 vs 2955 vs 2955 vs 2954 vs 2953 vs 4 CH2 sym str 2868 m - 2868 m 2868 m 2868 m 2868 m 2869 m 2869 m 2869 m 2869 m 5 0-H...0 valance str combination 2729 m - 2729 aa 2729 aa 2729 aa 2729 aa 2730 w 2730 w 2730 vw Absent 6 0-H...0 valance str combination 2630 m - Absent Absent Absent Absent 2631 vw 2632 vw Absent Absent 7 C=0 str 1722 vs - 1720 vw 1720 vw 1720 vw Absent 1720 m 1720 m 1720 w Absent 8 carboxylate stretching mode Absent - 1600-1650 m 1600-1650 m 1600-1650 m 1600-1650 s 1600-1650w 1600-1650w 1600-1650 m 1600-1650 s 9 aromatic C=C str 1507 s — 1512 vw 1511 vw 1512 vvw 1512 vvw 1509 m 1508 m 1511vw 1511 vw 10 CH3 asym deformation, CH2 scissoring 1462 s - 1462 vw 1463 vw 1463 vw 1463 vw 1462 m 1462 m 1463 vw 1464 vw 11 CH-CO deformation 1420 s - 1421 vw 1421 vw 1421 vw 1415 vw 1420 m 1420 m 1421 vw 1421 vw 12 CH3 sym str 1380 s - 1383 vvw 1382 vvw 1382 vvw 1384 vvw 1380 w 1381 w 1380 vw 1381 vvw 13 OH in plane deformation 1321 s - 1322 vw 1321 vw 1322 vw 1322 vvw 1321 m 1321 w 1322 vw 1325 vvw 14 =C-H in plane deformation 1268 s - 1268 vw 1268 vw 1268 vw 1268 vvw 1263 m 1269 w 1269 vw 1268 vvw 15 C...C str 1230 vs - 1231 vw 1231 vw 1230 vw 1230 vvw 1231 m 1231 w 1232 vw 1232 vvw 16 C-O str 1183 s - 1183 vw 1184 vw 1183 vw 1183 vvw 1184 m 1184 w 1185 vw 1185 vvw 17 =C-H in plane deformation 1122 w - merger merger merger merger merger merger merger merger 18 =C-H in plane deformation 1067 m - merger merger merger merger merger merger merger merger 19 Si-O-Si asym str Absent 1027 bb -1027 mbb -1027 mbb -1027 bb -1027 bb -1027 mbb -1027 mbb -1027 bb -1027 bb 20 C-H in plane deformation 1008 m - merger merger merger merger merger merger merger merger 21 C-O-C str 970 m - 970 w 970 vw 970 vvw 970 vvw 970 w 970 w 970 vw 970 vvw 22 CH3 rocking vibration 935 s - 948 vw 948 vw 948 vvw absent 935 w 936 w 936 vw 936 vvw 23 C-H out of plane vibration 866 s - 866 vw 866 vw 866 vvw absent 866 w 866 w 866 vw 865 vw 24 CH2 rocking 779 s - 780 w 780 w 780 vw 780 vvw 779 w 779 w 780 vw 780 vvw 25 C=C ring str, C...C skeletal vibration 746 w - 746 vw 746 vw 746 vw 746 vvw 746 vw 746 vw 746 vvw 746 vvw 26 C-H out of plane deformation 668 s - 669 vvw 669 vvw 669 vvw 669 vvw 668 m 668 w 669 vw 669 vvw 27 C-H in plane ring deformation 636 w - 636 vvw 636 vvw 635 vvw 635 vvw 636 w 636 w 636 vw 636 vvw 28 C...C deformation 588 m - 588 vvw 588 vvw 588 vvw 588 vvw 588 m 588 w 588 vw 588 vvw 29 CH2 in plane rocking 522 m - 522 vvw 522 vvw 522 vvw 522 vvw 522 m 522 w 522 vvw 522 vvw 30 CH2/CH3 deformation vibration 479 vw - 472 vw 462 vw 464 vw 461 vw 461 vw 464 vw 462 vw 462 vw 31 O-Si-O bending Absent 471 bb 464 m 461 m 463 bb 463 bb 464 m 464 m 464 bb 464 bb 32 C=C-C ring asym bending 421 w - 420 aa 421 aa 421 aa 421 aa 420 vvw 421vvw 421vvw 421 vvw (s- strong; bb- broad band; mbb- medium broad band; w- weak; sym-symmetrical; asym-asymmetrical; str-stretching; m- medium; vs- very strong; vw - very weak; vvw - very very weak; aa- almost absent.) 49 Acta Chim. Slov. 2017, 64, 45-54 The very strong band at 2958 cm-1 in the FTIR spectrum of ibuprofen is assigned to CH3 asymmetric stretching.30 Ibuprofen has also shown the presence of free acid car-bonyl peak at 1722 cm-1 with high intensity,27'31 but became very weak when co-milled in the dry-state as well as co-freeze-dried after aqueous state kneading and equilibration with magnesium trisilicate (Fig. 1a,b). As the magnesium trisilicate (2MgO,3SiO2,xH2O) contains magnesium oxide (not less than 20% of magnesium oxide as per USP 28) and the acidic nature of the carboxylic acid group of ibuprofen, the possibility of an acid-base interaction between the drug and MgO of magnesium trisili-cate was explored. Also, very high intensity peak of ibu-profen at 1230 cm-1 was due to C-C stretching32 became gradually medium, weak, very weak and absent as the magnesium trisilicate amount increased in both the co-processed materials IB3M1F to IB1M2F and IB3M1B to IB1M2B. A strong band noticed at 779 cm-1 in ibuprofen was due to CH2 rocking vibration and the intensity observed to be weaker and weaker after co-processing.33,34 CH2 asymmetric stretching vibration (3094 cm-1 and 2868 cm" 1) and CH2 inplane rocking vibration (522 cm-1) were also detected in pure ibuprofen and found weaker and absent when co-milled and freeze dried after co-kneading. CH2 asymmetric stretching vibration (3094 cm-1 and 2868 cm-1), CH3 asymmetric deformation (1462 cm-1), CH3 rocking of strong intensity (935 cm-1), and CH2 inplane Figure 1. (contd.)Figure 1. FTIR spectroscopy of co-processed ibuprofen and magnesium trisilicate after dry-state ball-milling (a) MTS, IB, IB3M1F, IB2M1F, IB1M1F, and IB1M2F; and aqueous state equilibration and freeze-drying (b) IB, IB3M1B, IB2M1B, IB1M1B, and IB1M2B (abbreviations are explained in Table 1). Figure 1. (contd.) Acharya et al.: Infrared Spectroscopy for Analysis of Co-processed ... 50 Acta Chim. Slov. 2017, 64, 45-54 rocking vibration (522 cm-1) were also detected in pure ibuprofen. Poor band performance was perceived in the co-processed formulations. C-O stretching (1183 cm-1), CH2 scissoring vibration (1462 cm-1) and CH-CO deformation (1420 cm-1) contributed their presence strongly in ibuprofen alone and weakly in the co-processed powder. An acid-base reaction between the carboxylic acid containing ibuprofen and MgO containing MTS in presence of moisture can describe the changes in the FTIR spectra of co-processed formulations. The reaction has been facilita- ted in presence of water when co-freeze-dried after aqueous state kneading and equilibration with magnesium trisi-licate and also co-milled in the dry-state containing varying proportions of water in the MTS compound. Car-boxylate ion shows peak in the range of 1600-1650 cm-1 in the FTIR spectrum and this change was detected as a function of IB/MTS ratio. A reduction in absorbance of the carbonyl acid peak accompanied by a corresponding increase in the absorbance of carboxylate peak was prominent and the absorbance of carboxylate peak was relatively c) . j JttE* 10ÜV X500 .Styjj DOM) 194SSE! f) y 10kV X500 Stym 0000 19 48 SEI i) d) e) v r i ■ V ¿* Btr<* % , f A j iW'F - 4 m • 10kV X5000 Sum 0000 19 48 SEI lOkV^ 000p 19 4« SEI h) 7 , • ^floiV jL^K ' lOkV X5.000 Sym 0000 50 48 SEI J* [f^Jtl t ^ A 10kV X5.000 5|jm 0000 19 51 SEI Figure 2. (a) Ibuprofen pure, (b) IB1M1pm, (c) IB1M1B (X500), (d) IB1M1B (X5000), (e) IB1M2B (X500), (f) IB1M2B (X5000), (g) IB1M1F (X500), (h) IB1M1F (X5000), (i) IB1M2F (1:2)F(X500), and (j) IB1M2F (X5000) (abbreviations are explained in Table 1). Acharya et al.: Infrared Spectroscopy for Analysis of Co-processed ... 51 Acta Chim. Slov. 2017, 64, 45-54 more in freeze-dried product compared to milled product. A large broad band between 3550 to 3200 cm-1 ascribed to the presence of the O-H stretching frequency of silanol group bonded to the inorganic structure of MTS (containing SiO2), and also hydrogen bonds between adsorbed water and silanol.25'35 This large broad band is absent in ibuprofen pure drug but consistently maintained in all the co-processed materials could be due to intermolecular hydrogen bonding. The band related to the silanol (Si-O-Si) asymmetric stretching was found at 1027 cm-1 with high intensity in MTS and also in the co-processed formulations. Silanol asymmetric stretching intensity increased with the amount of MTS in the formulation. Another peak at 471 cm-1 in MTS due to O-Si-O bending36 prominently observed in the formulations. The small changes in the band intensity, band orientation and overlapping indicated only van der Waals or dipole-dipole interactions between ibuprofen and magnesium trisilicate molecules. 3. 2. Characterization by Scanning Electron Microscopy and Differential Scanning Calorimetry Scanning electron microscopy is a powerful tool to study the inhibition of crystal growth morphology. Fig. 2 shows distinctive plate like layers due to the crystalline nature in the initial samples of pure ibuprofen. Physical mixture of drug and magnesium trisilicate in 1:1 ratio (IB1M1pm) shows the presence of ibuprofen crystal geometry very clearly with slightly damaged morphology. Markedly reduced particle size has been noticed not only in the co-milled materials but also in the freeze-dried formulations after aqueous state kneading and co-processing. Crystal geometry of ibuprofen has been significantly disappeared in both the co-processed materials. Sub-micron and nano-crystalline agglomeration were observed particularly in the milled material whereas, freeze-dried materials have shown porous bed of irregular nanoparticles developing grain boundaries in the crystal structure indicating loss of crystal geometry. These grain boundaries supposed to disrupt the motion of dislocations and reduce the crystallite size of ibuprofen in the co-processed powder.37 Differential scanning calorimetry is frequently used in pharmaceutical research as an analytical tool for the identification and interaction study of active drug after coprocessing with other pharmaceutical compounds.38-43 It can explain the miscibility/incompatibility with its effects on thermal stability, yielding results promptly and effi-ciently.44 Thermograms after differential scanning calori-metry of pure ibuprofen and co-processed powder samples are presented in Fig 3. Ibuprofen has shown the melting endotherm at 76.7 °C which is approximately similar to the literature value.33 With the increase of MTS amount in the co-processed material melting temperature and enthalpy (data not mentioned) have been decreased markedly signifying the material is made up of a number of smaller crystals or crystallites, and paracrystalline phases. Melting endotherm of IBjM^ and IBjM2F has been disappeared indicating an almost amorphous structure where the atomic position is limited to short range order only. Amorphous phase of ibuprofen could be possible to pro- Figure 3. Differential scanning calorimetry of co-processed ibuprofen and magnesium trisilicate after dry-state ball-milling, and aqueous state equilibration and freeze-drying (abbreviations are explained in Table 1). Acharya et al.: Infrared Spectroscopy for Analysis of Co-processed ... 52 Acta Chim. Slov. 2017, 64, 45-54 duce by solid state co-milling with kaolin.31 The interaction between ibuprofen and the porous silica adsorbents indicated a significant loss of crystallinity of ibuprofen by the DSC studies.13 3. 3. In-vitro Release of Ibuprofen In-vitro drug release profiles of the co-processed material up to 120 min have been depicted in the Fig. 4a,b. The powder materials have shown significantly improved dissolution of drug after co-processing. Crystalline ibuprofen exhibited only 52.89% dissolution whereas, dry-state co-milling of ibuprofen and magnesium trisilica-te has improved dissolution to a great extent (77.98 to 85.84%). Formulated powder samples of aqueous state co-processing and freeze-drying of ibuprofen and magnesium trisilicate have presented relatively more improved drug release (84.87 to 100.29%). Percentage release of ibuprofen increased gradually with the gradual increase in magnesium trisilicate proportion in the freeze-dried samples. Mixtures of ibuprofen and magnesium trisilicate have presented substantially higher dissolution compared to the pure drug. Magnesium oxide (MgO) in magnesium trisilicate and carboxylic acid containing ibuprofen brought about the acid-base reaction and the hydrogen-bonding potential of silanol groups of SiO2 in the surface of magnesium trisilicate facilitated collectively the increase in the drug dissolution rate. Dissolution of nimesulide from pharmaceutical formulations exhibited better dissolution when the formulations contain micronized nimesu-lide crystals and medium become alkaline rather than aci-dic.28 Increased proportion of magnesium trisilicate in the mixture might have consumed the carboxylic acid containing ibuprofen and more of hydrogen-bonding potential of silanol groups can describe the increased release of ibuprofen of the co-processed formulations. Otsuka et al. have been able to transform the crystalline polymorphs of indomethacin to amorphous states during milling which had 60% higher dissolution than the crystalline state.20 The increased dissolution of drug from solid dispersions possibly be related to the decreased drug crystallinity or effective wetting of the reduced drug particles.45-48 Coprocessing of ibuprofen with magnesium trisilicate for enhanced dissolution possibly be a promising approach for improvement of ibuprofen bioavailability.47 4. Conclusions Detailed infrared spectroscopy has been utilized for the assessment of interactions of ibuprofen and magnesium trisilicate after dry-state ball-milling and, aqueous state kneading and freeze-drying. Changes in the frequency and shape of ibuprofen bands after co-processing have been detected for the analysis of redistribution of electronic density in the structure of ibuprofen molecule. Changes in the FTIR spectroscopy of co-processed formulations can describe acid-base reaction between the carboxylic acid containing ibuprofen and MgO of magnesium trisilicate (2MgO,3SiO2,xH2O). Varying proportions of water in the magnesium trisilicate facilitated the reaction in the dry-state milling rather gently while, aqueous state equilibration and freeze-drying brought about the reaction considerably. Reduced absorbance of the car-bonyl acid peak accompanied by a consistently increase in the absorbance of carboxylate peak was prominently visible and the absorbance of carboxylate peak was rather more in freeze-dried product compared to milled sample. i oo 40 60 80 Time (min) -ibsmib iuiyiih ■ -iiîim2iî ■ 40 60 80 Time (min) Figure 4. Dissolution profiles of co-processed ibuprofen and magnesium trisilicate: (a) dry-state ball-milling samples; (b) freeze-dried samples after aqueous state equilibration (abbreviations are explained in Table 1). Acharya et al.: Infrared Spectroscopy for Analysis of Co-processed ... 53 Acta Chim. Slov. 2017, 64, 45-54 O-H stretching frequency of silanol group due to the presence of SiO2 in the structure of MTS and the hydrogen bonds between adsorbed water and silanol attributed a large broad band between 3550 to 3200 cm-1 in all the co-processed materials and not in ibuprofen pure drug spectrum. That is the indication of intermolecular hydrogen bonding between ibuprofen and magnesium trisilicate in the co-processed material. Scanning electron microscopy revealed the inhibition of crystal growth morphology in both the co-processed materials. Milled material has shown sub-micron and nano-crystalline accumulation but, porous bed of irregular nanoparticles with developing grain boundaries was observed in the crystal structure of the freeze-dried samples. Missing of melting endotherm in the DSC report of IB1M2B and IB1M2F signified almost complete amorphization of ibuprofen. Significantly decreased melting temperature and enthalpy of ibuprofen in the other co-processed materials indicated inhibition of crystal growth to a great extent. Significantly increased dissolution of drug has been noticed after co-processing compared to crystalline ibuprofen alone. Freeze-dried process presented relatively more enhanced drug release compared to ball-milled samples. 5. Acknowledgments The authors are very much grateful to Prof. Manoj Ranjan Nayak, President, Siksha O Anusandhan University for his inspiration and facilities. Conflict of Interest None 6. References 1. R. Jones, Am. J. Med. 2001, 110, S4-S7. https://doi.org/10.1016/S0002-9343(00)00627-6 2. L. M. Lichtenberger, Y. Zhou, E. J. Dial, R. M. Raphael, J. Pharm. Pharmacol. 2006, 58, 1421-1428. https://doi.org/10.1211/jpp.58.10.0001 3. C. S. Levin, J. Kundu B. G. Janesko, G. E. Scuseria, R. M. Raphael, N. J. Halas, J. Phys. Chem. B. 2008, 112, 1416814175. https://doi.org/10.1021/jp804374e 4. D. M. Moss, M. 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Lin, Int J Pharm. 2011, 406, 106-110. https://doi.Org/10.1016/j.ijpharm.2011.01.009 40. R. Mohapatra, S. Mallick, A. Nanda, R. N. Sahoo, A. Prama-nik, A. Bose, D. Das, L. Pattnaik, RSC Adv. 2016, 6, 3197631987. 41. B. Tita, T. Jurca, G. Rusu, G. Bandur, D. Tita, Rev Chim (Bucharest). 2013, 64, 1089-1095. 42. B. Tita, E. Marian, G. Rusu, G. Bandur, D. Tita, Rev Chim (Bucharest). 2013, 64, 1390-1394. 43. B. Panda, A. S. Parihar, S. Mallick, Int J Biol Macromol. 2014, 67, 295-302. https://doi.org/10.1016/jijbiomac.2014.03.033 44. K. Klimova, J. Leitner, Thermochim Acta., 2012, 550, 59-64. https://doi.org/10.1016Zj.tca.2012.09.024 45. M. Newa, K. H. Bhandari, D. H. Oh, Y. R. Kim, J. H. Sung, J. O. Kim, J. S. Woo, H. G. Choi, C. S. Yong, Arch. Pharm. Res. 2008, 31, 1497-1507. https://doi.org/10.1007/s12272-001-2136-8 46. H. H. Baek, D. H. Kim, S. Y. Kwon, S. J. Rho, D. W. Kim, H. G. Choi, Y. R. Kim, C. S. Yong, Arch. Pharm. Res. 2012, 35, 683-689. https://doi.org/10.1007/s12272-012-0412-4 47. B. Karolewicz, M. Gajda, A. Owczarek, J. Pluta, A. Gorniak, Trop. J. Pharm. Res. 2014, 13, 1225-1232. https://doi.org/10.4314/tjpr.v13i8.5 48. B. Karolewicz, M. Gajda, A. Owczarek, J. Pluta, A. Gorniak, Pharmazie, 2014, 69, 589-594. Povzetek Zmes ibuprofena in magnezijevega trisilikata smo pripravili na dva načina: s suhim mletjem in z liofilizacijo vodne raztopine. Nastali zmesi smo preučevali s FTIR spektroskopijo. Opazili smo povečano absorpcijo kaboksilatne skupine povezane z zmanjšanjem absorbance karbonilne kisline, kar kaže na določeno reakcijo karboksilne kisline v ibuprofenu. Absorbanca karboksilne skupine je bila bolj izrazita v liofiliziranem vzorcu, kar kaže na možne intermolekularne vezi med ibuprofenom in magenezijevim trisilikatom v tem primeru priprave zmesi. Razliko smo opazili tudi na fotomikro-grafskih posnetkih in pri DSC meritvah tališča. Sproščanje ibuprofena iz liofiliziranega vzorca je hitrejše kot pa iz vzorca, pripravljenega s suhim mletjem. Acharya et al.: Infrared Spectroscopy for Analysis of Co-processed ... DPI: 10.17344/acsi.20l6.2780_Acta Chirn, Slov. 2017, 64, 55-62_©commons 55 Scientific paper MnO2 Submicroparticles from Chinese Brush and Their Application in Treatment of Methylene Blue Contaminated Wastewater Qi Wang,1* Chunlei Ma,1 Wanjun Li,1 Meng Fan,2 Songdong Li1 and Lihua Ma3* 1 Chemistry & Chemical Engineering Department, Taiyuan Institute of Technology, Taiyuan 030008, China. 2 Research Center for Eco-Environmental Sciences in Shanxi, Taiyuan 030009, China 3 Department of Chemistry, University of Houston at Clear Lake, Houston 77058, USA * Corresponding author: E-mail: wangqitit@163.com; mal@uhcl.edu. Received: 30-07-2016 Abstract Eggshell membrane (ESM) is selected as biotemplate to prepare MnO2 submicroparticles (SMPs) using Chinese Brush with sodium hydroxide solution. The size with average 710 nm of the obtained materials is in good consistency with the microsructured biotemplate. An efficient and convenient absorbent for methylene blue (MB) is developed. The removal efficiency could reach up to 93% in 35 min under room temperature without pH adjusting owing to the excellent adsorption from ESM itself and hydroxyl group formed on the surface of MnO2 crystal in the aqueous solution. Materials on the membrane can be separated from the wastewater simply to avoid the secondary pollution caused by the leak of material. This interesting approach to MnO2 SMPs and facile operation for MB adsorption could open a new path to the submicro-materials based wastewater treatment. Keywords: MnO2 particles, biotemplate, eggshell membrane, methylene blue 1. Introduction Synthesis of inorganic materials by biotemplating as a burgeoning technique has emerged for years in a wide variety of research fields.1 The use of biotemplate makes the synthetic procedure simple and product controllable taking advantage of the nature of their own. Biotemplates like organisms (butterfly wing,2 hair,3 wood fiber45 and pollen6), microorganisms (bacteria,7,8 fungus,9,10 and viruses11) and biological macromolecules (DNA,12-14 RNA,15 proteins,1619 and polysaccharides20) were reported to prepare inorganic materials. Among these templates, proteins have gained more popularity by researchers,21 ranging from ferritin,22-25 bovine serum albumin (BSA)26-31 to collagen32-34. However, proteins from natural extracted or artificial synthetic are difficult to obtain and thus cost a lot. This could be a pivotal limitation for the large-scale synthesis and practical application of the biotemplated materials. Eggshell membrane (ESM) is a kind of biomaterial with great imperative though it is generally considered as a domestic waste.35 This microscopic biopolymeric fibrous net is composed mainly of proteins (80-85%), 10% of which are collagens and 70-75% are other proteins and glycoproteins.36 Due to the unique structure and property, ESM has been utilized as a biotemplate for synthesis of inorganic materials. Novel metal materials such as gold nanoparticles, silver nanoparticles, macroporous silver network, Pt-Ag/polymers, have been constructed through ESM templating.37-40 On the other hand, sulfide,41 seleni-de,42 oxidide43,44 have been synthesized using ESM as a template. Besides, other kinds of material based on ESM have been studied.45-47 As a kind of inorganic nanomaterials, MnO2 have drawn much attention because of their flexible structures and unique properties and have been applied to catalysis, ion exchange, supercapacitors, molecule adsorption, biosensors and so on.48 One of the recent applications has fo- Wang et al.: MnO2 Submicroparticles from Chinese Brush 56 Acta Chim. Slov. 2017, 64, 55-62 cused on the MnO2 based micromotors.49-52 And micromotors containing manganese oxide and noble metal or graphene have also been studied.53-55 In this work, by consideration of its special microstructure, abundant component of protein, we choose ESM as the biotemplate to help synthesize MnO2. Most important of all, ESM could be obtained expediently and free of charge. Furthermore, on the basis of the interaction between protein and metal ions, a novel and interesting procedure with Chinese Brush to grow MnO2 submicro-particles (MnO2 SMPs) on ESM is developed. As reported by Furuichi et al, hydroxyl groups could be formed on the surface of MnO2 in aqueous solutions.56 Cao et al confirmed that the hydroxyl groups were involved in the adsorp-tion.48 Therefore, combining the adsorption capacities of both ESM itself57 and hydroxyl groups formed on the surface of MnO2 in the aqueous media, these accessible Mn-O2 SMPs are applied successfully to the treatment of methylene blue (MB) wastewater. 2. Experimental 2. 1. Reagents and Apparatus Deionized water with conductivity of 18.2 mQ cm-1 was used in this experiment from a water purification system (ULUPURE, Chengdu, China). Manganese acetate (MnAc2, Mw = 245.09, AR) and methylene blue (MB) were purchased from Kemiou Chemical Co. Ltd. (Tianjin, China). Sodium hydroxide (NaOH, AR) and all the other reagents were at least of analytical grade. Eggshell was obtained from Hongye student mess hall of Taiyuan Institute of Technology, and eggshell membrane was peeled off from the shell carefully. Diluents with different pH values were prepared by titrating with 0.1 mol L-1 sodium hydroxide or hydrochloric acid solution to the required pH values. Scanning electron microscopy (SEM) of ESM and MnO2 SMPs were carried out on a Quanta 200 FEG scanning electron microscope. The size distribution of as-prepared nanomaterial was performed at a laser particle sizer (Malvern Nano-ZS90). The X-ray photoelectron spectroscopy (XPS) was measured with an AXIS ULTRA DLD electron spectrometer (Kratos) using monochromatic Al Ka radiation for analysis of the surface composition and chemical states of the product. Thermogravimetry (TG) measurement was carried out in air at a heating rate of 10 °C min-1 on a Rigaku TG thermal analyzer (Rigaku Co., Japan). The UV-vis absorption spectra were recorded on a TU-1901 UV-vis spectrophotometer (Puxi, China). 2. 2. Synthesis of MnO2 SMPs MnO2 SMPs in this experiment were synthesized through a simple and interesting method. In a typical process, eggshell membrane (ESM) was firstly peeled off ca- refully from a fresh eggshell and cleaned 10 times with deionized water to remove residual egg white and then dried at room temperature. The clean ESM was cut into small pieces and soaked into 0.1 mol L-1 manganese acetate solution with a certain proportion (0.5 g to 100 mL). After 12 hours, the adsorbed ESM pieces were taken out and washed 5 times with deionized water and placed onto a watch glass to dry. At last, a Chinese Brush was dipped in 0.1 mol L-1 NaOH solution for 20 seconds. NaOH solution as ink was brushed evenly on the adsorbed ESM. Five minutes later, the color change of the membrane from white to light brown indicated that the MnO2 SMPs were synthesized successfully. The MnO2/ESM piece was washed and dried to preserve for characterization and practical use. 2. 3. Treatment of Methylene Blue Wastewater 15 mg MnO2/ESM materials and equal amounts of ESM, as a control experiment, were placed in the 4 mL MB solution with the concentration of 8 mg L-1 under stirring. After 35 min, materials and ESM were taken out to stop the adsorption. The UV-vis spectra of MB solutions after adsorption were recorded immediately at room temperature. All of the absorption intensity of MB measurement was set at wavelength 664 nm. The removal efficiency (R, %) and adsorption capacity (qe, mg g1) were calculated using the equations below: (1) (2) where C0 and Ce (mg L1) stand for the initial and final concentrations of MB in the treatment solutions, respectively, V is the volume of the mixture solution (L), and W is the mass of adsorbent used (g). 3. Results and Discussion 3. 1. Synthesis Mechanism Scheme 1 displays the schematic diagram of the synthesis process of submicro-structured MnO2 on ESM using Chinese Brush. As reported, eggshell membrane is composed of fibrous proteins with different kinds of acidic/basic amino acid residues like -OH, -COOH, -NH2, -SH, etc on the surface. When ESM pieces were soaked into the manganese acetate solution, Mn2+ showed a trend (from lone electron pair of heteroatom and unoccupied orbital in Mn atom) to adsorb onto the "active site" on the ESM, which resulted in a uniformly dispersive distribution of Mn2+ on the fibrous proteins. After washing and drying Wang et al.: MnO2 Submicroparticles from Chinese Brush ... 57 Acta Chim. Slov. 2017, 64, 55-62 at the room temperature, Chinese Brush with NaOH solution was brushed on the adsorbed ESM. This step caused a reaction in situ between Mn2+ and OH- around these "active site" and as a result MnO2 were obtained after 5 min.57 Owing to the uniformly dispersion of Mn2+ on the membrane, MnO2 particles were generated and grew along with the fibrous proteins to form a biomimetic material. Scheme 1. The schematic diagram of the synthesis process of Mn-O2 SMPs on ESM using Chinese Brush. 3. 2. Characterization of MnO2 SMPs 3. 2. 1. Scanning Electron Microscopy Morphologies of ESM before and after MnO2 preparation were investigated for comparison. Figure 1a displays the scanning electron microscopy (SEM) images of ESM, in which multilayer and overlapping fibrous proteins are observed. After the reaction with MnO2, by contrast, plenty of spherical particles array densely on the adsorbed membrane (Figure 1b and Figure S1a). Interestingly, particles arraying along with the original fiber-like protein is observed, and it is more obvious and straightforward in SEM image with smaller amplification factor (Figure S1a). To measure the particle size of synthesized material, a Nano Particle Analyzer testing was carried out. The results are shown in Figure S1b. And an average diameter of ~710 nm is obtained, which is a good consistency with the microstructured biotemplate. Figure 1. SEM images of (a) ESM and (b) MnO2 SMPs. Scale bar were 50 |im and 20 |im, respectively. 3. 2. 2. UV-Vis Spectroscopy and X-ray Photoelectron Spectroscopy The UV-Vis spectrum of as-prepared MnO2 SMPs is shown in Figure S2. A single absorption peak at 360 nm is found. To investigate the surface composition and elemental analysis for the resultant MnO2 SMPs, the X-ray photoelectron spectroscopy (XPS) was carried out. In the full scan spectrum (Figure S3), it shows that the synthesized material is composed of elements Mn 2p, O 1s, C 1s and N 1s. The elements C 1s, N 1s and partial O 1s come from proteins in ESM. To examine the details, XPS spectra of Mn 2p and O 1s were measured. As shown in Mn 2p spectrum (Figure 2a), two peaks are observed at 654.2 and 642.4 eV, which can be assigned to Mn 2p1/2 and Mn 2p3/2, respectively. Meanwhile, the O 1s spectrum (Figure 2b) can be resolved into three peaks. The strongest peak at Wang et al.: MnO2 Submicroparticles from Chinese Brush ... 58 Acta Chim. Slov. 2017, 64, 55-62 531.4 eV corresponds to the Mn-O-H, the other two small peaks (532.2 eV and 530.0 eV) adjacent reveal the existence of H-O-H and Mn-O-Mn, respectively. As a consequence, the aforementioned findings confirm that the as-prepared submicroparticles are MnO2. a) Mn 2p3/2 642,4 eV a Mn 2p1/2 A 654.2 eV b) 665 660 655 650 645 640 635 Binding Energy [eV] /\ Mn-O-H f L/ 531,4 eV H-O-H 532.2 eV // /\ Mn-O-Mn J J J \ \ 530.0 eV 536 534 532 530 528 526 Binding Energy [eV] Figure 2. (a) Mn2p and (b) O1s XPS spectra of as-prepared MnO2 SMPs. 200 400 600 Temperature [°C] Figure 3. The TG curves of ESM and as-prepared MnO2 SMPs. 3. 2. 3. Thermogravimetry Analysis Furthermore, a thermogravimetry (TG) analysis was carried out to illustrate the content of the composite (Figure 3). Blue and red curves indicate the mass changes of ESM only and synthesized MnO2/ESM material, respectively. It can be seen that ESM, as a kind of protein, is burnt out at about 600 °C and the quality is almost zero (blue curve in Figure 3). To study the relative amount of MnO2 SMPs coated on ESM, dotted portion in Figure 3 is zoomed in. It is vividly shown that the curves remain unchanged with the temperature rising afterwards. However, the horizontal part of MnO2/ESM is obviously higher than that of ESM only, which is attributed to the inorganic material existence. The difference of two horizontal curves stands for the relative amount of MnO2 SMPs in ESM, which is calculated to be 2.77%. 3. 3. Methylene Blue Wastewater Treatment The detailed characterization and measurement demonstrate that the synthesized material is ESM coated a) b) 2.5 2.0 3 ¿1.5 1.0H < 0.0- (1) (3) (2) -Original - ESM Only -MnOa/ESM 400 450 650 700 750 800 Wavelength [nm] Figure 4. (a) Photographs of ESM and MnO2/ESM before and after adsorption of MB [(1) ESM only; (2) ESM only after adsorption of MB; (3) MnO2/ESM SMPs; (4) MnO2/ESM after adsorption of MB.]. (b) The UV-vis absorption spectra of MB before and after adsorption by ESM only and MnO2/ESM. Wang et al.: MnO2 Submicroparticles from Chinese Brush ... 59 Acta Chim. Slov. 2017, 64, 55-62 MnO2 SMPs (MnO2/ESM). Owing to the handy operation of "put in" and "take out", these materials were further applied to removal of MB. Figure 4a displays the photographs of ESM and MnO2/ESM before and after adsorption of MB. Two sets of contrastive pictures show that ESM itself is capable of adsorbing for MB. Light pink ESM (1) turns into blue (2) after adsorption of a certain amount of MB. However the color change degree of MnO2/ESM before and after adsorption is bigger: brown MnO2/ESM (3) becomes dark green (4). The UV-Vis absorption spectra of MB before and after adsorption by ESM only and MnO2/ESM are recorded in Figure 4b. It is evidently indicated that the absorption intensity of MB at 664 nm after MnO2/ESM adsorption is significantly smaller than the one treated with ESM only. Figure S4a exhibits the equation of linear regression of MB solutions, by which the removal efficiencies of ESM and MnO2/ESM adsorption are calculated in Figure S4b. Inset photographs shows the color change of MB solution before and after adsorption: (5) is original MB solution; (6) and (7) are MB solution after ESM and MnO2/ESM adsorption, respectively. The color gap between (5) and (7) keeps pace with the removal efficiency of 93% by MnO2/ESM adsorption. 3. 4. Investigation of Time and pH for Adsorption Adsorption time for MB by MnO2/ESM adsorption was investigated by UV-Vis spectroscopy as shown in Figures 5a and S5a. Under different adsorption time the absorption intensity decreases gradually as a function of time and remains the same after 35 min, which represents the whole adsorption process. Figure S5a shows the time dependent removal efficiency curve for MB, it can be seen that the removal efficiency increases rapidly at first 10 min and flats out gradually afterwards. A maximal removal efficiency of 93% is obtained at 35 min. Moreover, Figure S5b demonstrates the effect of pH condition on the adsorption by MnO2/ESM. It turns out that the removal efficiency is kept in the range of 50%-62% under different pH values. The pH is not a factor to influence within the experimental error. It is worth noting that the removal efficiency under a certain pH condition is not as high as that in the distilled water solution. The additional adsorption for ions, which was used to adjust the acidity of the solution, took charge of this phenomenon. The desorption of MB was performed by placing the adsorbed Mn-O2/ESM into deionized water. Figure 5b shows the absorption spectra of MB by adsorption for 35 min and desorption for 24 h. It is seen obviously that the shape and position of absorption peak are the same before and after adsorption, which indicates that the molecule structure of MB keeps unchanged during the removal procedure. Therefore, the removal procedure is an adsorption-desorption equilibrium process. a) 2.5 -r-2.0 3 j> 1.5 < 1.0 0.5 0.0 400 -origina -5 min -10 min -15 min -20 min -25 min -30 min -35 min -40 min 450 500 550 600 650 Wavelength [nm] 700 750 b) JO ffl O < 300 400 500 600 Wavelength [nm] 700 Figure 5. (a) The UV-vis absorption spectra of MB under different time by MnO2/ESM adsorption. (b) The UV-vis absorption spectra of MB by adsorption for 35 min and desorption for 24 h. 3. 5. Study of Kinetics and Adsorption Isotherm In order to better understand the adsorption behavior of MB on MnO2/ESM, the adsorption capacities at different time (qt) were recorded. As shown in Figure 6a, the adsorption of MB increases gradually with the time prolonged and becomes balanced after 35 min. Based on this, experimental data are calculated and organized in Figure 6b to investigate the adsorption kinetics. Two kinetic models are generally used to evaluate the adsorption,59 and it can be concluded that the adsorption process of MB on MnO2/ESM is in accordance with the pseudo-second-order model (correlation coefficient of 0.99508 for pseudofirst-order model and 0.99915 for pseudo-second-order model). Moreover, the effect of initial MB concentration on equilibrium adsorption capacity (qe) is shown in Figure 7a, where the adsorption capacity steadily enhances with increasing the initial concentration of MB added. The adsorption behavior of MB on MnO2/ESM Wang et al.: MnO2 Submicroparticles from Chinese Brush ... 60 Acta Chim. Slov. 2017, 64, 55-62 a) b) ~3H -5- -64 Time [minj ->25 pseud ».first-Order -20 15 pseudosecondorder R:=0,99915 -10 10 20 Time [min] 30 -5 40 Figure 6. (a) Effect of adsorption time on adsorption capacity of MnO2/ESM in MB solutions. (b) Pseudo-first-order (red line) and pseudo-second-order (blue line) models for MB adsorption. a) b) ■2,0 . -1.B . -1,6 . -1.4 . -1.2 . -1,0 . -0,8 .-0.6 Figure 7. (a) Effect of initial concentrations of MB on equilibrium adsorption capacity of MnO2/ESM in MB solutions. (b) Langmuir (red line) and Freundlich (blue line) isotherms models for MB adsorption. was further studied through Langmuir and Freundlich isotherms models (Figure 7b), which were common adsorption isotherms models for evaluating the adsorption process.59 According to the data calculation and linear fitting, it is concluded that Langmuir model is able to interpret the MB adsorption process better (correlation coefficients are 0.9967 and 0.9787 for Langmuir isotherms and Freundlich isotherms models, respectively). 3. 6. Effect of Hydrogen Peroxide on Removal of MB The effect of H2O2 on the dye MB as a function of time monitored by uV-visible spectra was investigated in Figure S6a. We observed that the presence of H2O2 affected the absorbance of dye itself about 5% in 35 min and the shape of the peaks underwent no change. Then the effect of H2O2 with various concentrations on the removal efficiency of dye MB decontamination by MnO2 was examined. The results are showed in Figure S6b. It is straightforward that H2O2 decreases the removal efficiency of dye MB by MnO2. 4. Conclusions The MnO2 submicroparticles were prepared through an eggshell membrane based biotemplating method. The size of MnO2 SMPs kept correspondence with the diameter of the fibrous proteins, which indicated the bio-inspired growth of MnO2 SMPs. Taking advantages of macro-operability and adsorption performance stemmed from both protein membrane and hydroxyl on the surface of MnO2 in the aqueous solution, the ESM coated MnO2 SMPs was applied to the MB wastewater treatment. The adsorption process followed the pseudo-second-order kinetic model and Langmuir isotherms model, and the removal efficiency could reach up to 93% under room temperature without pH adjustment. This simple, green and interesting approach gives a facile concept of metal oxide materials synthesis, which is considered of great potential applications in wastewater treatment area. 5. Acknowledgements This work was supported by the Key Science Fund Project of Taiyuan Institute of Technology (2015LZ01) Wang et al.: MnO2 Submicroparticles from Chinese Brush ... 61 Acta Chim. Slov. 2017, 64, 55-62 and the Program for the (Reserved) Discipline Leaders of Taiyuan Institute of Technology. We thank Institute of Coal Chemistry, Chinese Academy of Science for the material characterization. 6. References 1. A. Kumar and V. Kumar, Chem. Rev. 2014, 14, 7044-7078. https://doi.org/10.1021/cr4007285 2. W. Zhang, D. Zhang, T. Fan, J. Ding, J. Gu, Q. Guo and H. Ogawa, Mater. Sci. Eng. 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Ma, L. Zhang, L. Wang and X. Yan, Chem. Lett. 2015, 44, 399-401. https://doi.org/10.1246/cl.140971 55. H. Wang, G. Zhao and M. Pumera, J. Am. Chem. Soc. 2014, 136, 2719-2722. https://doi.org/10.1021/ja411705d 56. H. Tamura, T. Oda, M. Nagayama and R. Furuichi, J. Elec-trochem. Soc. 1989, 136, 2782-2786. https://doi.org/10.1149/L2096286 57. T. Yang, M. L. Chen, X. W. Hu, Z. W. Wang, J. H. Wang and P. K. Dasgupta, Analyst 2011, 136, 83-89. https://doi.org/10.1039/C0AN00480D 58. X. Liu, Q. Wang, H. H. Zhao, L. C. Zhang, Y. Y. Su and Y. Lv, Analyst 2012, 137, 4552-4558. https://doi.org/10.1039/c2an35700c 59. L. Y. Hao, H. J. Song, L. C. Zhang, X. Y. Wan, Y. R. Tang and Y. Lv, J. Colliod Interface Sci. 2012, 369, 381-387. https://doi.org/10.1016/jjcis.2011.12.023 Povzetek Kot bio-predlogo (biotemplate) za pripravo submikronskih delcev MnO2 smo izbrali membrano jajčne lupine in uporabili kitajske čopiče pomočene v raztopino natrijevega hidroksida. Povprečna velikost tako pridobljenih delcev je bila 710 nm in je skladna z mikrostrukturo bio-predloge. Tako smo pripravili učinkovit in priročen absorbent za barvilo me-tilen modro. Učinkovitost odstranjevanja barvila lahko doseže do 93% v 35 minutah pri sobni temperaturi brez uravnavanje pH, tudi zaradi odličnega adsorpcije iz membrane jajčnih lupin in hidroksilnih skupin na površini kristalov MnO2 v vodni raztopini. Materiale na membrani lahko ločimo od odpadne vode, izogniti pa se moramo sekundarnemu onesnaženju. S tem zanimivim pristopom k sintezi submikronskih delcev MnO2 in učinkovitostjo odstranjevanja barvila metilen modro bi se lahko odprla nova pot priprave submikronskih materialov, pomembnih za čiščenje odpadnih voda. Wang et al.: MnO2 Submicroparticles from Chinese Brush ... DOI: 10.17344/acsi.2016.2821 Acta Chim. Slov. 2017, 64, 63-72 ^creative tS/commons Scientific paper Development of Chemistry Pre-Service Teachers During Practical Pedagogical Training: Self-Evaluation vs. Evaluation by School Mentors Vesna Ferk Savec* and Katarina S. Wissiak Grm Faculty of Education, University Ljubljana, Kardeljeva ploščad 16, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: vesna.ferk@pef.uni-lj.si Received: 16-08-2016 Abstract The research presented in this article deals with the self-evaluation of 4th year pre-service chemistry teachers' progress during their second year practical pedagogical training in chemistry teaching at primary schools (students' age 13-15 years) in comparison to the perception of their progress by their school mentors. The sample consisted of 21 pre-service teachers and 21 school mentors, in-service chemistry teachers, at primary schools. For the purpose of following to pre-service chemistry teachers' development, the pre-service teachers as well as their mentors completed the "Questionnaire for monitoring students' progress", focusing on eight characteristics of professional development during practical pedagogical training. The results reveal that student-teachers were stricter in their self-evaluation in comparison to their school mentors after their first chemistry lecture at school during the practical pedagogical training; however, after their last lecture, the evaluations were similar for most of the characteristics. The development of five randomly selected student-teachers is presented in detail from their own perspectives, as well as from their school mentors' perspectives. Keywords: Chemistry teacher education, practical pedagogical training, pre-service chemistry teachers, in-service chemistry teachers, school mentors 1. Introduction Within the framework of the education of pre-service teachers, practical pedagogical training is viewed as a crucial component in their professional development as teachers.1,2 Hascher and Hagenauer3 reviewed different terms referring to the various forms of practical training in teacher education, e.g. teaching practicum, student teaching, field experiences, teaching practice, clinical training, clinical teacher education, (guided) teaching experiences, internship, school practicum, school-based teacher education, and school placement. In this article, we use the term practical pedagogical training (PPT), which we define as a mandatory module in a pre-service teacher-education programme that takes place at school under the supervision of a school mentor, who is an in-service teacher of a specific school subject. PPT is aimed at providing pre-service teachers with an opportunity to gain experience in the classroom through their own teaching and/or co-teaching facilitated by continuous feedback about their teaching from their school mentor. PPT and their contribution to the learning of pre-service teachers have been an area of interest to researchers, teacher educators and teachers. Some studies have focused on pre-service teachers development, their beliefs, experiences, and expectations, as well as the challenges and their concerns relating to the PPT.4-8 Another group of studies focused on mentors and the mentoring provided by experienced teachers in schools.9-12 A third group of studies focused on the work of teacher educators in finding ways to support pre-service teachers in developing their teaching of specifics subject in school environ-ments.13-16 According to the literature review of Lawson et al.,17 a broad range of factors play roles in the PPT process for pre-service teachers. Among the outcomes in their review, the collaboration between student-teachers and mentors emerged as significant for the professional and individual development of pre-service teachers. It was pointed out that mentors' feedback is also a crucial aspect of the men-tor-pre-service teacher relationship, from the viewpoint of prospective teachers. Ferk Savec and Wissiak Grm: Development of Chemistry Pre-Service Teachers During 64 Acta Chim. Slov. 2017, 64, 63-72 Another viewpoint highlights pre-service teachers' individual differences and the effects of the characteristics of individual student-teachers on the processes during PPT and their outcomes.18-20 Hascher and Kittinger21 proposed students-teachers' learning and performance model to explain learning in PPT. Their model assumes that the quality of learning processes and learning outcomes during PPT is influenced by structural aspects (e.g. single or tandem placement, short-or long-term practicum), organizational aspects (e.g. university-school cooperation, school mentor professionali-zation), and social aspects (e.g. school social climate, teacher candidate's integration into the teaching staff). Their model also recognizes the role of individual factors of pre-service teachers such as cognition (e.g. pre-knowledge, attitudes, beliefs), motivation (e.g. interest, goal orientation), and emotions (e.g. enjoyment, anger) to contribute to the learning process. The model as well recognizes that factors at different levels (e.g. the culture of teacher education at the macro-level versus the teacher candidate-mentor interaction at the micro-level) co-determine the outcomes of teacher education.20,21 This article focuses on the self-evaluation of pre-service chemistry teachers' progress during their PPT in primary schools in comparison to the perception of their progress by their school mentors, who observed their teaching during PPT and provided feedback after each of the lessons. 2. The Context and the Purpose of the Study At the Faculty of Education of the University of Ljubljana, Slovenia, the PPT of pre-service chemistry teachers commences in the 3rd year of tertiary education and continues in the 4th year. PPT is organized in collaboration between teacher educators at the university and selected primary school mentors. It is conducted in primary schools in Slovenia. Within the framework of PPT, stu- dent-teachers prepare lesson plans and teach chemistry in the 8th and 9th years of Slovenian primary schools (the students are 14 to 15 years old). At selected primary schools, pre-service teachers have a school mentor (experienced in-service chemistry teacher). The role of the school mentor is to give directions prior to the commencement of PPT for successful inclusion in the current teaching plan, within the framework of which the student-teachers conduct and attend lessons during the time of PPT. The school mentor is also present during all of the lessons that the student-teacher conducts and, directly after each lesson, provides the student-teacher with feedback on the positive aspects of the individual performance, as well as on necessary improvements. In order to improve pre-service teachers' learning possibilities during PPT, we attempted to adjust PPT to pre-service teachers' suggestions based on previous re-search.7 Specifically, we have considered the following main proposals given by the pre-service chemistry teac-hers:7 (1) longer PPT, (2) independent choice of location and school for PPT, and (3) the possibility of doing PPT in several schools in cooperation with a number of different school mentors. The changes that have been introduced in PPT with regard to student-teachers' suggestions7 are presented in Table 1. This article deals with pre-service chemistry teachers', 4th-year student-teachers, development during their second-year experience with teaching during their PPT. The article focuses on the monitoring of pre-service chemistry teachers' first and the last lecture during their PPT based on their own and their school mentors' perceptions of eight characteristics of student-teachers' development measured by the "Questionnaire for monitoring students' progress .' The study addresses the following research question: How do pre-service chemistry teachers evaluate their development in comparison with their school mentors on their second-year experience with teaching during their PPT? Table 1. Changes that have been introduced in PPT with regard to student-teachers' suggestions Student-teachers' suggestions for optimization of PPT based on the evaluation of PPT7 State of PPT in the 2008/09 academic year7 - before optimization State of PPT in the 2014/15 academic year - after optimization (1) Student-teachers' suggestion for a longer PPT; • Five school days per academic year; • Ten school days per academic year; (2) Student-teachers' suggestion for an independent choice of location and school for PPT; • Seven primary schools • Within the Ljubljana Urban Municipality, Slovenia; • Schools chosen by the University; • 2-3 student-teachers conducted PPT simultaneously at the same school at the time; • Twenty-one primary schools (for 4th year student-teachers); • All Slovenian regions; • Schools chosen independently by each of the student-teachers; • One student conducted PPT at each of the schools; (3) Student-teachers' suggestion for the possibility of doing PPT in several schools in cooperation with a number of different school mentors; • Each of the student-teachers had the possibility to collaborate with one school mentor in the same academic year in the framework of PPT; • Each of the student-teachers had the possibility to collaborate with several school mentors in the same academic year in the framework of PPT; Ferk Savec and Wissiak Grm: Development of Chemistry Pre-Service Teachers During ... 65 Acta Chim. Slov. 2017, 64, 63-72 3. Method 3. 1. Instruments For the purpose of the investigation, the "Questionnaire for monitoring students' progress"7 was applied. The questionnaire showed appropriate internal consistency (Cronbach a = 0.89).7 The questionnaire enables reflection on pre-service teachers' development during PPT, in particular with regards to the following eight student-teacher characteristics: (1) the pre-service teacher's self-esteem while conducting the lessons -referred to as Self-esteem in this article, (2) the pre-service teacher's ability to establish discipline in class -referred to as Discipline in this article, (3) the suitability of the pre-service teacher's explanation of the chemistry topic taught -referred to as Explanation in this article, (4) the ability of the pre-service teacher to anticipate the appropriate amount of material to present during the lesson -referred to as The amount of contents in this article, (5) the pre-service teacher's experimental skills -referred to as Experimental skills in this article, (6) the pre-service teacher's expertise in providing an appropriate response to the students -referred to as Response in this article, (7) the pre-service teacher's ability to involve students actively - referred to as Active student's involvement in this article, and (8) the pre-service teacher's self-dependence in preparing for the lesson -referred to as Self-dependence in this article. Pre-service teachers and their school mentors evaluated pre-service teachers' development regarding each of the above-listed specific characteristics with a mark in the range 1-5, in which "1" represents the lowest student-teachers' competence and "5" the highest student-teachers' competence. 3. 2. Sample The sample consisted of student-teachers (N = 21) enrolled in the 2014/15 academic year in the 4th year of the undergraduate programmes "Chemistry and Biology" or "Chemistry and Physics" or "Chemistry and Home Economics" at the Faculty of Education, University of Ljubljana. The student-teachers involved were predominantly female (N = 20), and only one was male (N = 1); their average age was 23.91 years. Due to their future profession, they are referred to as pre-service teachers or student-teachers in this article. In addition to the pre-service teachers their school mentors, experienced in-service chemistry teachers (N = 21), from the twenty-one primary schools where PPT took place, were also involved in this study. All participating school mentors were female, and their average age was 46.20 years. In average, they had 20.81 years of experience in the teaching of the subject of chemistry in primary schools. In this study, the development of five 4th year pre-service chemistry teachers, who were chosen from the sample via random selection, is presented in detail from their own perspectives as well as from their school mentors' perspectives. Each student-teacher in PPT only had one mentor and visited only one school. In order to assure anonymity of student-teachers, their names - presented in the results of the article - are pseudonyms, i.e. Ina (female), Sara (female), Jan (male), Mara (female) and Ula (female). 3. 3. Data Collection The PPT for 4th year student-teachers was conducted in April 2015 at twenty-one primary schools throughout Slovenia. Every student spent two weeks (10 days) at an independently selected primary school, which was their second year experience of teaching chemistry. Each student-teacher monitored their own progress every day during PPT with the aid of the "Questionnaire for monitoring students' progress"7. The school mentors evaluated student-teachers' development by the use of "Questionnaire for monitoring students' progress"7 twice - after their first and last chemistry lecture during PPT. 3. 4. Data Analysis 3. 4. 1. Analysis of the "Questionnaire for Monitoring Students' Progress " The results collected from pre-service chemistry teachers and their school mentors in the "Questionnaire for monitoring students' progress"7 were entered into MS Excel, and appropriate calculations and figures were prepared. Further analysis was conducted using the Statistical Package for the Social Sciences (SPSS), version 21. The nonparametric test Wilcoxon Ranks Test (Z) was used to evaluate significant differences in perceptions of student-teachers' characteristics by pre-service teachers in comparison to their school mentors. Pre-service teachers' comments accompanying the numerical data were transcribed. 4. Results and Discussion At the pre-service teachers' first teaching of chemistry at school during their second year PPT, their school mentors evaluated the student-teachers' characteristics with higher values in comparison to pre-service teachers self-evaluation as can be seen from the mean values in Table 2. At the pre-service teachers' final teaching of chemistry at school during their second-year PPT, student-teachers' competences were again investigated. From the Ferk Savec and Wissiak Grm: Development of Chemistry Pre-Service Teachers During ... 66 _Acta Chim. Slov. 2017, 64, 63-72 Table 2. The mean values for eight characteristics measured by "Questionnaire for monitoring students' progress"7 from student-teachers' and school mentors' perspectives after their first and final presentation during PPT First presentation in PPT Final presentation in PPT Characteristic Pre-service teachers School mentors Pre-service teachers School mentors Mean SD Mean SD Mean SD Mean SD Self-esteem 3.39 0.70 4.39 0.61 4.92 0.26 4.72 0.46 Discipline 3.33 1.19 4.17 0.92 4.94 0.24 4.94 0.24 Explanation 3.78 0.73 3.94 0.64 4.83 0.38 4.72 0.46 The amount of contents 3.39 1.24 4.44 0.70 4.72 0.46 5.00 0.00 Experimental skills 3.72 1.13 4.00 0.69 4.72 0.46 4.56 0.51 Response 4.11 0.68 4.22 0.65 4.69 0.46 4.78 0.43 Active student's involvement 3.44 1.10 4.39 0.78 4.72 0.46 4.44 0.70 Self-dependence 3.89 1.23 4.33 0.69 4.83 0.38 4.50 0.62 mean values in Table 2, it can be determined that their perception of their own competence was closer to that of their school mentor's at that time. For the pre-service teachers' first teaching of chemistry, Wilcoxon Ranks Test showed significant differences in perception between pre-service teachers and their school mentors about the future teachers competence in four characteristics: Self-esteem (Z = -2.924, p = 0.003), Discipline (Z = -2.223, p = 0.026), The amount of contents (Z = -2.799, p = 0.005), Active student's involvement (Z = -2.315, p = 0.021). In contrast, no significant differences were found in the other four characteristics Explanation (Z = -0.566, p = 0.572), Experimental skills (Z = -0.366, p = 0.714), Response (Z = -0.540, p = 0.589), Self-dependen- Table 3: Ina's self-evaluation of her skills and knowledge in specific fields at her first and final presentation during their PPT in comparison with the evaluation of her school mentor Teacher's evaluation of Ina's skills and knowledge [Scale 1 to 5] Self-dependano T Self-esteem /: I : f : Active students' ' 1 involvment * T \ 1 \ I w .^Discipline fcjExpl a nation / fhe amount of w"" .,.■■ contents Experimental skills Ina's self-evaluation of her skills and knowledge [Scale 1 to 5] Self-dependance Active students" involvment Self-esteem ? 3 I dents J J lent ** Response 2-Q-" 1 0 ^¿Discipline ■-9 \ \ \ \ \ II \ 0 ^Explanation : / / :/ J The amount of conte nts Experimental skills ...g— First presentation —Final presentation To the question "How did you perceive the course of the lesson in the role of chemistry teacher?" Ina explained: After her first presentation: "After a year outside the school climate, I did not feel very self-confident, since I had not met students yet." After her final presentation: "During the practical pedagogical training, I had gained self-confidence, had a better feeling regarding explaining teaching topic and was better when came to establishing discipline in the class." •■■Q—First presentation — X— Final presentation To the question "How did you perceive the course of the lesson with Ina in the role of chemistry teacher?" Ina's school mentor explained: After her first presentation: "She presented the new chemistry topic thoroughly through the experimental work. Ina's explanation was clear and she was able to adapt to the students' rhythm of knowledge comprehension." After her final presentation: "During the lessons, she succeeded in applying all the teaching goals designed in advanced. Students were able to be actively involved in the process of presenting the new chemistry topic. Experimental work was carried out in a correct and appropriate manner. The content of the chemistry topic was properly introduced." Ferk Savec and Wissiak Grm: Development of Chemistry Pre-Service Teachers During ... 67 Acta Chim. Slov. 2017, 64, 63-72 ce (Z=-1.310, p = 0.190). Based on these results, it can be summarized that pre-service teachers are more realistic in estimating their competence for explanations of the chemistry topic taught, their experimental skills, their ability for providing an appropriate response to the students in the classrooms and their self-dependence in preparing for the lesson. However, pre-service teachers seem to be stricter in evaluation of their appearance of self-esteem while conducting the lessons and ability to establish discipline in class during lessons, also their ability to anticipate the appropriate amount of contents to present during the lesson and to actively involve students seem to be underestimated with regard to the perception of their school mentors. For the pre-service teachers' final teaching of chemistry, the Wilcoxon Ranks Test showed significant differences in perception between pre-service teachers and their school mentors about the future teachers' competence in only one of eight characteristics, in the amount of contents (Z = -2.236, p = 0.025). No significant differences were found in other seven characteristics: Self-esteem (Z = -1.536, p = 0.125), Discipline (Z = 0.000, p = 1.000), Explanation (Z = -0.707, p = 0.480), Experimental skills (Z = -0.832, p = 0.405), Response (Z = -1.342, p = 0.180), Active student's involvement (Z = -1.387, p = 0.166), Self-dependence (Z = -1.897, p = 0.058). Based on these results, it can be summarized that pre-service teachers gained more realistic estimation of their competences during the time of PPT in comparison to their school mentors' perceptions. To obtain insight into the situation of individual pre-service teachers', examples of the individual evaluations of eight characteristics are presented for five student-teachers in comparison with their development as seen by their school mentors. 4. 1. Example 1: 4th-year student-teacher Ina After her first lesson, it is clear from Table 3 that the student Ina had perceived herself as having very little self-confidence, which was in contradiction with her teacher mentor's comprehension of her behavior. In general, the teacher mentor Table 4. Sara's self-evaluation of her skills and knowledge in specific fields at her first and final presentation during their PPT in comparison with the evaluation of her school mentor Teacher's evaluation of Sara's skills and knowledge [Scale 1 to 5] Self-esteem S Self-d ependan cqo-.t.t ---^Jisciptin e Active students'^ . 'i,- . . . O 0 & .olanation involvment r^ \ Response N., À he amount of x --contents v Experimental skills Sara's self-evaluation of her skills and knowledge [Scale 1 to 5] Self-depend an cey ' J -X ' P-2-0-.....O \ Self-e steem § -'4 X 3 Discipline Acbve students' involvment i / 1 \ \ ^ O 0 Û ^Explanation Î V i l y ..■© ih „ L v „..-*Jthe amount of Responsaa-^ . . r ». O - contents Experimental skills "•©■•• First presentation —X— Final presentation To the question "How did you perceive the course of the lesson in the role of chemistry teacher?" Sara explained: After her first presentation: "I was satisfied with the way I was able to carry out the teaching lesson, since I was able to ask students enough different questions. The students were also very active, since they were involved in the teaching and learning process properly." After her final presentation: "I feel that my ability to establish discipline in the class has improved; students are listening to me and are willing to cooperate. I also feel that I am doing better when performing experiments in the class, I am no longer frightened when demonstrating the chemical experiment in front of the students in the class." -..©— FirS presentation —X— Final presentation To the question "How did you perceive the course of the lesson with Ina in the role of chemistry teacher?" Sara's school mentor explained: After her first presentation: "Sara was able to perform the lesson very well, involving students in the teaching and learning process. Her explanations were clear, and the lecture was designed appropriately due to the logical structure from the beginning to the end. She only could check the students intensively by reviewing their notes during the lesson." After her final presentation: "Sara is more confident and convincing when introducing new chemistry topics. She skillfully reacted when waiting for the experiment to occur, since the safety rules in the lab had been revised." Ferk Savec and Wissiak Grm: Development of Chemistry Pre-Service Teachers During ... 68 Acta Chim. Slov. 2017, 64, 63-72 had seen Ina's presence in the class as being very appropriate regarding all the significant characteristics observed. At Ina's final presentation (Table 3) in the class during PPT, her self-evaluation opinion had improved; she saw herself in a much better light also with regard to explanation of the topics taught and establishing discipline in the class. Ina's teacher mentor's opinion was consistent with Ina's self-evaluation, however the mentor described her improvement to the highest level in all areas described by influential characteristics. 4. 2. Example 2: 4th-year student-teacher Sara At her first presentation (Table 4) the student-teacher Sara had seen all of her influential characteristics through self-assessment as being quite low, except regarding the chemical experiment demonstration and the ability to anticipate the appropriate amount of matter to be presented during the lesson which is not coherent with her comment, where she stated that she is quite satisfied with the lesson. Also Sara's school mentor's opinion is very positive; except regarding Sara's self-esteem, she evaluated Sara with quite high marks regarding all other influential characteristics observed. From Table 4, it can be determined that the situation had changed significantly by Sara's last presentation during PPT. While conducting the final chemistry lesson, Sara had perceived herself to be very appropriate while grading all the influential characteristics; she only marked herself a bit lower regarding the successful involvement of Table 5: Jan's self-evaluation of her skills and knowledge in specific fields at his first and final presentation during their PPT in comparison with the evaluation of her school mentor Jan's self-evaluation of her skills and knowledge [Scale 1 to 5] Teacher's evaluation of Jan's skills and knowledge [Scale 1 to 5] Self-depend an zy;' Self-esteem -'4 3..-G... )i£xplan at I on '^liscipline / ,.0-'2 ~ .....V \ ! -f \ Actve students - ^ involvment i \ \L ReSD0nS(£---""0......am0Unt 0f Responses —----- contentE Experimental skills -■■©■■- First presentation —-X- Final presentation To the question "How did you perceive the course of the lesson in the role of chemistry teacher?" Jan explained: After his first presentation: "A bit frightened... it has been a year since I have been in front of the class performing the teaching lecture." After his final presentation: "My school mentor complimented me." •*•©■■■ First presentation —-x—■ Final presentation To the question "How did you perceive the course of the lesson with Ina in the role of chemistry teacher?" Jan's school mentor explained: After his first presentation: "Jan's teacher plan was correctly prepared in advance regarding the content and timetable. He was able to give brief and effective instructions to the students. Worksheets were appropriately prepared in advance; consequently, students were able to complete them independently, and then they were all checked at the end of the lesson. Therefore, students were active throughout the teaching process." After his final presentation: "Jan carried out the lesson independently. Prior to his lesson, he attended the observation of my class, and then he repeated the same topic. He carried out experimental group work successfully; the instructions were clearly and briefly delivered in advance. The results of the experiments were analysed with the students and therefore they successfully concluded the teaching lesson together." Ferk Savec and Wissiak Grm: Development of Chemistry Pre-Service Teachers During ... 69 Acta Chim. Slov. 2017, 64, 63-72 students in the lesson, thereby she described her improvement in various areas also in her comment. The opinion of Sara's teacher mentor was very similar; she gave her very good marks regarding almost all important characteristics observed and pointed out her improvement regarding confidence as well as the quality of teaching. 4. 3. Example 3: 4th-year student-teacher Jan It is clear from Table 5, that at his first presentation, the student-teacher Jan had evaluated all of his influential characteristics much more strictly than his school mentor did. Jan was not satisfied especially with his ability to clearly explain the topics thought; he commented to per- ceive himself as being frightened in the classroom after one-year pause since last practical pedagogical training in his third year of the study. It can be seen from Table 5, that the situation had changed during the time of practical pedagogical training, as at Jan's final presentation, he was very satisfied with his lesson. Jan and his mentor's opinions were quite consistent, except regarding Jan's experimental skills and the suitability of Jan's explanation of the topic taught. 4. 4. Example 4: 4th-year student-teacher Mara It is clear from Table 6 that Mara had seen her suitability of explanation of the topic taught to be extremely Table 6. Mara's self-evaluation of her skills and knowledge in specific fields at her first and final presentation during their PPT in comparison with the evaluation of her school mentor Mara self-evaluation of her skills and knowledge [Scale 1 to 5] Teacher's evaluation of Mara's skills and knowledge [Scale 1 to 5] Self-esteem ""A ^ Self-depend an c^-' * -^Discipline / / • O / \ / / 1 / V \ \ i to \ / n •i;-.. ¿{The amount of Response ...... V Experimental skills contents —©"•First presentation — X- Final presentation To the question "How did you perceive the course of the lesson in the role of chemistry teacher?" Mara explained: Self-depend an o Self-esteem Active students', involvment < "-^Zliscipline \ \ \\ \ V \ Ö.........g-...... Response^—— '^explanation i he amount of contents Experimental skills ■©•■• First presentation -X- Final presentation To the question "How did you perceive the course of the lesson with Ina in the role of chemistry teacher?" Mara's school mentor explained: After her first presentation: "My first lesson presentation after one year outside the school practice. I feel I am able to carry out the teaching lesson appropriately, but I do need my teacher mentor to supervise me and give me a proper advice where needed." After her final presentation: "My last day of practical pedagogical training. I am full of new impressions and experiences. I feel I am no longer so nervous, and I have gained self-confidence." After her first presentation: "In the future, Mara should work on step-by-step explanations of new chemical concepts introduced to the students during her lesson. For the whole image of the teaching lesson, it would be beneficial to add visual elements for better introducing and launching the new chemistry topics. There were some troubles with the time component of the teaching plan, which consequently was not appropriately carried out. She had quite a few problems with correct use of Slovenian language, as she spoke in a dialect." After her final presentation: "Mara should try to show a little bit more enthusiasm while teaching in the classroom. Consequently, the atmosphere in the classroom would be improved. The lesson should be more compact. In these terms, she should try to connect the parts of the lesson more tightly. However, she improved her teaching image in comparison to the last year of her PPT in our school. I recommend that she works on developing her natural body language when performing the teaching process." Ferk Savec and Wissiak Grm: Development of Chemistry Pre-Service Teachers During ... 70 Acta Chim. Slov. 2017, 64, 63-72 low at her first presentation, and she consequently also marked her self-esteem extremely low. Her school mentor did not evaluate her very well either, since she detected troubles with introducing and launching the new topics, as well as with the time component of the teaching plan and the discipline in the classroom. At her last presentation, both Mara and her teacher mentor changed their opinions as can be seen in Table 6. All the important characteristics were marked excellent by her teacher mentor, except in the case of Mara's experimental skills, where she can still improve. In the opinion of her school mentor, it is also important that in the future, she Mara shows more enthusiasm while teaching in the classroom. 4. 5.Example 5: 4th-year student-teacher Ula At her first presentation, it is clear from Table 7, that Ula was not satisfied with her teaching and that she had perceived all of her influential characteristics more strictly than her teacher mentor had. From her comment it is obvious, that she felt a relieve after her first lesson as she describes, that feels less nervous and to gain more control over the situation in the classroom. Table 7. Ula's self-evaluation of her skills and knowledge in specific fields at her first and final presentation during their PPT in comparison with the evaluation of her school mentor Ula's self-evaluation of her skills and knowledge [Scale 1 to 5] Teacher's evaluation of Ula's skills and knowledge [Scale 1 to 5] Seff-e steem 5 Self-dependance Active students' irivolv merit ■Q \ Discipline - 3 F ¿v- ^ Q 0 O J Explanation \>... ..A I The amount of contents Response Experimental skilis —O— First presentation —X- Final presentation To the question "How did you perceive the course of the lesson in the role of chemistry teacher?" Ula explained: After her first presentation: "The first teaching lessons have been successfully applied. I felt less nervous and have had more control over the situation in the classroom." After her final presentation: "The last day of my practical pedagogical training. I am full of new impressions; I am feeling much less nervous, and I have gained much self-confidence." Self-esteem 5 Self-dependance — ^Disciplin r t « v Active students', involvment Ö ^Explanation // 2 / 1 0 \ \ ; / M0""- 7 l ^ .........amount of contents Response Experimental skills ..-O " First presentation -X- Final presentation To the question "How did you perceive the course of the lesson with Ina in the role of chemistry teacher?" Ula's school mentor explained: After her first presentation: "Ula was able to prepare a compact teaching lesson plan, firstly, suitable for checking student's pre-knowledge and secondly for the introduction of new chemistry concepts, which has to be presented in the teaching lesson. Before this point, she had needed quite a lot of help, but after my careful review, she finally succeeded to prepare a good, complex and systematic teaching lesson plan. During carrying out the teaching lesson in the class, she had to face some problems, regarding the discipline, but it was an expected and understandable situation, since the class is, in general, a bit problematic." After her final presentation: "Ula was able to prepare an interesting lesson presenting the chemistry concepts in a way interesting for teaching and learning. Her appropriate teaching plan comprises several teaching methods; the students were actively engaged by discussion and question making; she was able to include context-based teaching goals were students enjoy real self-reflection regarding ecological problems presented during the teaching and learning process." Ferk Savec and Wissiak Grm: Development of Chemistry Pre-Service Teachers During ... 71 Acta Chim. Slov. 2017, 64, 63-72 Ula's teaching improved during the practical pedagogical training, as the situation had changed at Ula's final presentation (Table 7). Ula described, that she gained new ideas and valuable experience during practical pedagogical training. In the case of four evaluated characteristics the teacher mentor saw Ula's ability even better than Ula did. These four characteristics were the following: the pre-service teacher's ability to establish discipline in class, the suitability of the pre-service teacher's explanation of the topic taught, the ability of the pre-service teacher to anticipate the appropriate amount of contents to present during the lesson and the pre-service teacher's ability to involve students actively. The mentor especially pointed out that Ula's teaching plan involved different teaching methods and that the students were actively engaged by discussion and question making. Regarding the 4th-year student-teachers' PPT, it can be summarized from the overall results (Table 2), as well as from the analysis of individual teacher-students' reflections (Tables 3-7), that the school mentors were far less strict in evaluation of the student-teachers' performances in the class. All the teacher mentor's observations, especially regarding the students' first presentations, were far less demanding and much more indulgent regarding the student's behaviour in the class than the student-teachers' views of their selves were. However, regarding school mentor's and student-teachers' views of the last lesson in the class during their practical pedagogical training, there were far more matching reviews seen in comparison with their evaluations obtained at the first lessons in the class. The mentors' comment in the tables are in most cases also much longer than the comments of the student-teachers. The focus of student-teachers' comments, especially after their first lesson, is mostly about their self-esteem. Student-teachers report about their low confidence after one-year brake after the practical pedagogical training in the third year of their studies, they claim to be nervous, to be frighten during teaching, while in the case of the mentors, they are more specific and report about different skills by student-teachers, e.g. structure of chemistry lesson, students' active involvement, discipline in the classroom, student-teachers' enthusiasm during teaching, etc.. 5. Conclusion This investigation presents 4th-year pre-service chemistry teachers' development during their second-year experience with teaching during their PPT from their own perspective as well as from that of their school mentors. In particular, it focused on the monitoring of pre-service chemistry teachers' first and last chemistry lesson during their PPT based on their own and their school mentors' perceptions of eight characteristics of pre-service teachers' development measured by the "Questionnaire for monitoring students' progress".7 The results revealed that after their first chemistry lecture pre-service teachers and their school mentors estimated similar values of four of eight student-teacher characteristics, e.g. no statistically significant differences found for the explanation of the chemistry topic taught, their experimental skills, their ability for providing an appropriate response to the students in the classrooms and their self-dependence in preparing for the lesson. However, pre-service teachers seem to be stricter than their school mentors are; statistically significant differences found in the evaluation of their appearance of self-esteem while conducting the lessons and ability to establish discipline in class during lesson, as well as their ability to anticipate the appropriate amount of contents to present during the lesson and to involve students actively. According to the results following the last chemistry lecture during PPT, it can be concluded that pre-service teachers gained more realistic estimations of their knowledge and skills with regard to the eight observed characteristics, when compared to their school mentors' perception, as the statistically significant difference was observed only in their evaluation of their ability to anticipate the appropriate amount of contents to present during the lesson. From the content point of comments, it can be concluded, that mentors' comments in the tables are in most cases longer than the comments of the student-teachers. Student-teachers' comments, especially after their first lesson, are mostly about their self-esteem, while in the case of the mentors, they are more specific and report about different skills by student-teachers, e.g. structure of chemistry lesson, teaching methods, students' active involvement, discipline in the classroom, student-teachers' enthusiasm during teaching, etc. When focusing on specific characteristics, the results are in line with previous research findings,7 in which the lowest value by pre-service teacher was also ascribed to their ability to establish discipline in the classroom and higher grades were ascribed to their ability to involve students actively in the lesson, followed by their self-dependence in preparing for the lesson. Similarly, to previous studies,17 it can be concluded that school mentors' feedback to student-teachers is a very important part of PPT, especially because they observe student-teachers' progress from a broader, more holistic perspective of their future profession - chemistry teacher. Therefore, sustained efforts should be focused on productive school-university collaboration, but also to raising of the awareness for the need of in-service chemistry teachers' sustainable education in their subject area in relation to recent findings from chemical education research, e.g. studying the impact of different experimental methods in chemistry teaching on school practice,24,25 studying the impact of the online knowledge assessment system on students' knowledge,26 the development of concept maps as learning materials to foster students' meaningful learning of organic reactions.27 Ferk Savec and Wissiak Grm: Development of Chemistry Pre-Service Teachers During ... 72 Acta Chim. 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Postholm (Eds.): Teacher education research between national identity and global trends, Akademika, Trondheim, 2013, pp. 139-162. 19. T. Hascher, F. Hofmann, in: K.-H. Arnold, A. Gröschner, T. Hascher (Eds.): Pedagogical field experiences in teacher education, Waxmann, Münster, 2014, pp. 257-276. 20. T. Hascher, G. Hagenauer, Int. J. Educ. Res. 2016, 77, 15-25. https://doi.org/10.1016/jijer.2016.02.003 21. T. Hascher, C. Kittinger, in: K.-H. Arnold, A. Gröschner, T. Hascher (Eds.): Pedagogical field experiences in teacher education, Waxmann, Münster, 2014, pp. 221-235. 22. Z. Shechtman, M. Levy, J. Leichtentritt, J. Educ. Res. 2005, 98, 144-155. https://doi.org/10.3200/J0ER.98.3.144-155 23. I. Timostsuk, A. Ugaste, Teach. Teach. Educ. 2010, 26, 1563-1570. https://doi.org/10.1016/j.tate.2010.06.008 24. M. Vrtacnik, N. Gros, Acta Chim. Slov. 2013, 60, 209-220. 25. A. Logar, V. Ferk Savec, Acta Chim. Slov. 2011, 58, 866-875 26. B. Kralj, S. A. Glazar, Acta Chim. Slov. 2013, 60, 433-441. 27. B. Sket, S. A. Glazar, J. Vogrinc, Acta Chim. Slov. 2015, 62, 462-472. https://doi.org/10.17344/acsi.2014.1148. Povzetek V članku predstavljena raziskava se ukvarja s samo-evalvacijo napredka med praktičnim pedagoških usposabljanjem bodočih učiteljev kemije, študentov četrtega letnika, v primerjavi z mnenjem njihovih mentorjev na šoli. Vzorec sestavlja 21 bodočih učiteljev kemije in 21 njihovih mentorjev, izkušenih učiteljev kemije na osnovnih šolah. Za namen spremljanja razvoja bodočih učiteljev kemije med praktičnim pedagoških usposabljanjem so bodoči učitelji in njihovi mentorji izpolnjevali »Vprašalnik za spremljanje razvoja bodočih učiteljev kemije«, ki temelji na evalvaciji osmih karakteristik strokovnega razvoja učiteljev kemije. Rezultati kažejo, da so bili bodoči učitelji kemije v samo-evalvaciji bolj strogi od svojih mentorjev, še posebno po prvi izvedeni uri pouka kemije, medtem ko so bile ocene po zadnji izvedeni uri podobne z ocenami mentorjev glede večine ocenjevanih karakteristik. Podrobneje je predstavljen razvoj petih naključno izbranih bodočih učiteljev kemije iz njihovega osebnega vidika, kakor tudi iz perspektive njihovih mentorjev. Ferk Savec and Wissiak Grm: Development of Chemistry Pre-Service Teachers During ... DPI: 10.17344/acsi.20l6.2823_Acta Chirn. Slov. 2017, 64, 73-82_©commons 73 Scientific paper Preparation and Catalytic Study on a Novel Amino-functionalized Silica-coated Cobalt Oxide Nanocomposite for the Synthesis of Some Indazoles Mohammad Ali Ghasemzadeh,*'1 Bahar Molaei,2 Mohammad Hossein Abdollahi-Basir1 and Farzad Zamani3 1 Department of Chemistry, Qom Branch, Islamic Azad University, Qom, I. R. Iran 2 Young Researchers and Elite Club, Shahr-e-Qods Branch, Islamic Azad University, Tehran, Iran 3 School of Chemistry, University of Wollongong, New South Wales, 2522, Australia * Corresponding author: E-mail: GGhasemzadeh@qom-iau.ac.ir Received: 18-08-2016 Abstract In this research an efficient synthesis of a novel nanocomposite including SiO2 @ (3-aminopropyl)triethoxysilane-coated cobalt oxide (Co3O4) nanocomposite has been reported by three step method. The structure and magnetic characterization of Co3O4@SiO2@NH2 have been done by using various spectroscopic analyses which include FT-IR, X-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy and vibrating sample magnetometry. Amino-functionalized SiO2 coated Co3O4 nanocomposite exhibited superparamagnetic behavior and strong magnetization at room temperature. The average crystallite sizes of the Co3O4 are 23.7 nm. The obtained magnetic nanocomposite showed excellent catalytic activity as a new heterogeneous magnetic catalyst for the synthesis of some indazole derivatives under mild reaction conditions along with high level of reusability. Keywords: Co3O4@SiO2@NH2, heterogeneous catalyst, spectroscopic analysis, indazole derivatives, nanocomposite le under acidic conditions and inert to redox reactions, as compared with the organic coating materials, and hence functions like an ideal shell composite to protect the inner Co3O4 partciles. Silica-coated Co3O4 nanocomposite, i.e., Co3O4@SiO2, has recently been investigated for potential biomedical applications.810 Additionally, the SiO2 coating shell has an abundance of surface hydroxyl groups which can be easily coupled with organosilanes by formation of Si-O-Si covalent bonds. The importance of this field is highlighted by the use of bio molecules which control the self-assembly of nanodevices.11-13 This led to the idea of preparing an active catalyst, Co3O4@SiO2@NH2, through morphology-controlled synthesis which ensure that faces which are active specifically are exposed predominantly at the surface. As well as, to the best of our knowledge, no attempt has been made to synthesis of Co3O4@SiO2@NH2 nanostructures. In this study, a novel Co3O4 magnetic na-nocomposite was developed by grafting amino groups co-valently onto the surfaces of Co3O4@SiO2 nanocomposite. 1. Introduction Over the last decade, organic-inorganic magnetic na-nocomposites have become interesting as magnetic catalysts in both academic and industrial fields.1-3 The spinel cobalt oxide Co3O4 is a magnetic semiconductor and widely used catalyst for a variety of reactions.4-5 The use of this magnetic nanoparticle catalyst can address the isolation and recycling problem encountered in many heterogeneous and homogenous catalytic reactions. Most importantly, the magnetic-supported catalysts show not only high catalytic activity but also high degree of chemical stability. The Co3O4 surface has a strong affinity for silica, and the cobalt-oxide NPs were easily coated with silica via the sol-gel process.6 It has been exhibited that the formation of silica coating on the surface of Co3O4 NPs can hinder their aggregation and keep their chemical stability.7 In addition, the silanol (Si-OH) groups, which have often located in the terminal of silica coating surface, SiO2 is stab- Ghasemzadeh et al.: Preparation and Catalytic Study on a Novel 74 Acta Chim. Slov. 2017, 64, 73-82 The resulted nanocomposite was characterized by Fourier transform infrared (FTIR), transmission electron microscopy (TEM), X-ray powder diffraction (XRD), scanning electron microscopy (SEM) and vibrating sample magnetometer (VSM). This study on the synthesis of Co3O4@Si-O2@NH2 nanocomposite may open up new routes in the research for highly active catalysts. In continuing our efforts towards the development of efficient and environmentally benign heterogeneous ca-talysts,14-18 herein, Co3O4@SiO2@NH2 nanocomposite was prepared as a highly efficient magnetic catalyst by a simple method. The main goal of this catalytic synthesis was to introduce a novel and effective magnetic nanocom-posite to expand the use of these types of composites for organic reactions. In order to investigate the catalytic activity of this magnetic catalyst, synthesis of some indazole derivatives have been done via two-component reactions. 2. Experimental 2. 1. Chemicals and Apparatus Chemicals were purchased from the Sigma-Aldrich and Merck in high purity. All of the materials were of commercial reagent grade and have been used without further purification. The a,a-bis (substituted-arylidene) cycloalka-nones were synthesized via aldol condensation as described previously.19,20 All melting points are uncorrected and were determined in capillary tube on Boetius melting point microscope. The ultrasonic irradiation was used in reactions by a multi-wave ultrasonic generator (Sonicator 3200; Ban-delin, MS 73, Germany), equipped with a converter/transducer and titanium oscillator (horn), 12.5 mm in diameter, operating at 20 kHz with a maximum power output of 200 W. The ultrasonic generator automatically adjusted the power level. 1H NMR and 13C NMR spectra were obtained on Bruker 400 MHz spectrometer with CDCl3 as solvent using TMS as an internal standard. FT-IR spectrum was recorded on Magna-IR, spectrometer 550. The elemental analyses (C, H, N) were obtained from a Carlo ERBA Model EA 1108 analyzer. Powder X-ray diffraction (XRD) was carried out on a Philips diffractometer of X'pert Company with mono chromatized Cu Ka radiation (X = 1.5406 A). Microscopic morphology of products was visualized by SEM (LEO 1455VP). The mass spectra were recorded on a Joel D-30 instrument at an ionization potential of 70 eV. Transmission electron microscopy (TEM) was performed with a Jeol JEM-2100UHR, operated at 200 kV. Magnetic properties were obtained on a BHV-55 vibrating sample magnetometer (VSM) made by MDK-I.R.Iran. The compositional analysis was done by energy dispersive analysis of X-ray (EDX, Kevex, Delta Class I). 2. 2. Preparation of Co3O4 Nanoparticles Co3O4 MNPs were prepared according to previously reported procedure by Vela et. al with some modifica-tions.21 Firstly, cobalt nitrate hexahydrate (8.60 g) was dissolved in 100 ml of ethanol and the resulting mixture was stirred vigorously. Then, the mixture was heated up to 50 °C and kept for 30 min. Finally oxalic acid (2.14 g) was added quickly to the solution and the reaction mixture was stirred for 2 h at 50 °C. The formed precipitate which includes cobalt (II) oxalate was collected by centrifuges and then the prepared cobalt (II) oxalate powder was calcined at 400 °C for 2 h to produce Co3O4 nanopar-ticles. 2. 3. Preparation of Co3O4@SiO2 Nanoparticles Co3O4@SiO2 MNPs were prepared according to the slightly modified previously reported method by Vela et. al.21 Briefly, CTAB (2.2 g) was added to a solution of 0.5 g of Co3O4 nanoparticles in EtOH (350 mL), and then concentrated ammonia aqueous solution (40 mL, 28 wt %) was added dropwise to the reaction mixture under sonica-tion. After the treatment for 20 min which followed by the addition of tetraethylorthosilicate (TEOS) (0.4 mL in 10 mL of EtOH) to the mixture under ultrasound irradiation, then solution was stirred for 20 h at room temperature. Co3O4 nanoparticles coated with porous SiO2 shell were collected by centrifugation and washed three times with deionised water and then were calcined at 600 °C for 6 h. 2. 4. Preparation of Co3O4@SiO4@NH2 Nanocomposite Co3O4@SiO2 nanoparticles (0.5 g) were added to the three-necked flask and ultrasonically dispersed for 15 min in dry toluene (25 mL). Afterwards, 1 mililiter (4.27 mmol) of 3-aminopropyltriethoxysilane (APTES) was added into the flask, and the reaction mixture was refluxed at 110 °C with continuous stirring for 10 h under nitrogen atmosphere. After completion of the reaction, the resulting amine-functionalized Co3O4@SiO2 was gathered by centrifugation and washedwith water and ethanol for several times. Finally, it was dried at 50 °C under vacuum conditions for 10 h (Scheme1). Nitrogen content of the amine-grafted sample was estimated by back titration using NaOH (0.1 mol/L).22-24 First, the known amount of the catalyst was stirred in HCl (0.5 mol/L) for 30 min. Then, the mixture was filtrated and titrated with NaOH (0.1 mol/L). Nitrogen content of the catalyst was 5.86 mmol/g using 8.54 mmol/g trimet-hoxysilylpropylamine. 2. 5 .General Procedure for Synthesis of Some Indazole Derivatives In a typical procedure, a mixture of a,cx-bis (substi-tuted-arylidene) cycloalkanone (1 mmol), phenyl hydra- Ghasemzadeh et al.: Preparation and Catalytic Study on a Novel ... 75 Acta Chim. Slov. 2017, 64, 73-82 Scheme 1. Preparation steps for fabricating Co3O4@SiO2@NH2 nanocomposite zine (2 mmol), and Co3O4@SiO2@NH2 (0.003 g) were placed in a round-bottom flask. The suspension was stirred under solvent-free conditions at 80 °C. Completion of the reaction was monitored by Thin Layer Chromatography (TLC). After termination of the reaction, the catalyst was separated from the solid crude product by using an external magnet. The precipitated solid was then collected and recrystallized from ethanol to afford the pure product. The products were identified with *HNMR, 13CNMR and FT-IR spectroscopic techniques. 3. Results and Discussion 3. 1. Catalyst Characterization The synthesis strategy of Co3O4/SiO2/NH2 MNPs involves three steps. Figure 1 shows the XRD patterns of prepared Co3O4, Co3O4@SiO2 and Co3O4@SiO2@NH2. All the XRD patterns show raising background which is attributed to X-ray fluorescence since Cu-Ka has been used as the X-ray source during the measurements.25 The reflections of XRD pattern of Co3O4 in Fig. 1a confirm the synthesis of cubic normal spinel Co3O4 (JCPDS file no. 42-1467). Fig. 1b shows the SiO2 coating of Co3O4 by the presence of the new broad peak at 2e approximately 22-25°. As shown in Figure 1, the characteristic peaks of Co3O4 are also observed for Co3O4@SiO2 and Co3O4@SiO2@NH2, which represent the stability of the crystalline phase of Co3O4 nanoparticles during silica coating and surface amino-functionalization. Although these characteristic diffraction peaks are weakened in Co3O4@SiO2 and Co3O4@SiO2@NH2, because of the silica coating and surface amino-functionalization. The average crystallite sizes of the Co3O4 in Figure 1 (a, b and c) which have been estimated by using the Scherrer equation were 23.5, 24.2 and 26.0 nm respectively. Further information about the chemical structure of Co3O4, Co3O4 @ SiO2 and Co3O4 @ SiO2 @ NH2 nanocom-posites have been obtained from FT-IR spectroscopy Ghasemzadeh et al.: Preparation and Catalytic Study on a Novel ... 76 Acta Chim. Slov. 2017, 64, 73-82 Figure 1. X-ray diffraction of Co3O4 (a), Co3O4@SiO2 (b) and Co3O4 @ SiO2-NH2 (c) MNPs. shown in Figure 2. For all three nanoparticles, the analyses indicated two strong absorption bands at 565 and 662 cm-1 which correspond to the vibrations of Co-O in Co3O4. The peaks at 460 and 1070 cm-1 are attributed to the Si-O-Si bond stretching of Co3O4@SiO2 and Co3O4@ SiO2@NH2. The weak intensity band at 830 cm-1 can be Si-O-Si V V CoU) ,n——i—>—--1—1 1 1 i 11—i—1—1—1—i—1—1—1—i—1 1 1—i—1—1—1—i 1 1—1—r—i—..........iii 1200 3900 3600 3300 3000 2700 2400 2100 1800 1500 1200 900 6C0 Vtovenurrtoers / (qm-1) Figure 2. Comparative FT-IR spectra of Co3O4 (a), Co3O4@SiO2 (b) and Co3O4 @ SiO2-NH2 (c) MNPs. Ghasemzadeh et al.: Preparation and Catalytic Study on a Novel ... 77 Acta Chim. Slov. 2017, 64, 73-82 10- c) N Co b) Co 0 Co 1 L J L L 1 J J i 8 IS Co —i—■—i—1—'—i—i— ( 1 i t i Figure 3. EDX spectra of Co3O4 (a), Co3O4@SiO2 (b) and Co3O4 @ SiO2-NH2 (c) MNPs. ascribed to the stretching of non-bridging oxygen atom in Si-OH bond. Therefore the silica coating on the surface of Co3O4 nanoparticles were confirmed by these absorption bands (Figure 2b and 2c). As indicated in Figure 2c, the peaks of Co3O4@SiO2@NH2 are located at 1480 cm-1 (C-H bending), 2880 cm-1 (C-H stretching), 1645 cm-1 (N-H bending), and 3360 cm-1 (N-H stretching). These peaks indicated that APTES has been bonded with the surface of Co3O4@SiO2. The characteristic peaks of C-H stretching and N-H bending for the synthesized Co3O4@SiO2@NH2 are too weak to be observed clearly. Therefore, another analytical method, EDX, was employed to prove that the amine group has been bonded on the surface of Co3O4@ SiO2.26-28 Figure 3 shows the EDX data for Co3O4, Co3O4@SiO2, Co3O4@SiO2@NH2 MNPs. In Figure 3 c, the weight ratio for C: N: O: Si: Co was calculated to be 12: 3.5: 36: 6.5: 42. These data demonstrate formation of Co3O4@SiO2@NH2 nanocomposite. Figure 4 represents the room-temperature magnetization curves of the Co3O4, Co3O4@SiO2 and Co3O4@ SiO2-NH2 MNPs which have been obtained using a VSM. As it can be observed, there are no hysteresis, coercivity and remanence in the three synthesized nanoparticles which indicate their typical superparamagnetic property. The plots which have been shown in Figure 4 exhibited a change in saturation magnetization (Ms) of the particles Figure 4. VSM magnetization curves of the Co3O4 (a), Co3O4@ SiO2 (b) and Co3O4 @ SiO2-NH2 (c) MNPs. Ghasemzadeh et al.: Preparation and Catalytic Study on a Novel ... 78 Acta Chim. Slov. 2017, 64, 73-82 after incorporation of a NH2/SiO2 shell. The Ms values were measured to be 47.1, 36.9 and 33.8 emu/g respectively. It is clear that saturation magnetization of silica-coated Co3O4 nanoparticles is lower than that of pristine Co3O4 nanoparticles, and saturation magnetization of Co3O4@SiO2-NH2 is lower than Co3O4@SiO2. This reduction in saturation magnetization can be attributed to the surface effects such as magnetically inactive layer which contains spins that are not collinear with the magnetic field.29 Because the silica coating is a nonmagnetic mass, and this decrease was ascribed to the contribution of the nonmagnetic NH2/SiO2 shell to the total mass of the particles. Figure 5 shows TEM image of amino-functionalized SiO2 coated Co3O4 nanoparticles. Typical size of the structure has been measured about 50 nm, and the aggregation of the nanoparticles can be observed clearly. Therefore, the TEM observation confirmed the formation of an amino-functionalized SiO2 around the Co3O4 nanoparticles with typical nanostructure. The scanning electron microscopy (FE-SEM) of the Co3O4@ SiO2@NH2 MNPs shows the morphology and structure of the as-prepared samples (Figure 6). The Co3O4 nanoparticles are irregular sheets (non-spherical) in shape and hard aggregated powders with diameters ranging from 35 to 80 nm as seen in Figure 6a. The irregular Bullet-shaped Co3O4@SiO2 nanoparticles with diameters ranging from 95 to 220 nm are shown in Figure 6b. This illustrated that SiO2 has been successfully coated on the Co3O4 nanoparticles. The micrograph of Figure 5. TEM images of Co3O4@SiO2@NH2 MNPs Ghasemzadeh et al.: Preparation and Catalytic Study on a Novel ... 79 Acta Chim. Slov. 2017, 64, 73-82 Figure 6. SEM images of Co3O4 (a), Co3O4@SiO2 (b) and Co3O4 @ SiO2-NH2 (c) MNPs. Co3O4 @ SiO2 @ NH2 MNPs represents a cloudy network of particles with spherical shape, as indicated in TEM image. This network is the result of self-poly condensation of aminopropylsilane groups. 3. 2. Catalyst Testing for the Synthesis of Some 7-benzylidene-2,3-diphenyl-3,3a,4,5,6,7-hexahydro-2H-indazole Derivatives In order to optimize the reaction conditions and to obtain the best catalytic activity, the synthesis of 7-benzy-lidene-2,3-diphenyl-3,3a,4,5,6,7-hexahydro-2H-indazole derivatives was chosen as a model reaction. The reactions were conducted under solvent-free conditions at 80 °C (Scheme 2). The synthesis of 7-benzylidene-2,3-diphenyl-3,3a,4,5,6,7-hexahydro-2H-indazoles with different amounts of the Co3O4@SiO2@NH2 MNPs has been considered. It was observed that while the amount of catalyst increased from 0 to 0.003 g, the product yield raised from 0% to 98% significantly. No reaction yield without using the catalyst corroborates that the Co3O4@SiO2@NH2 MNP catalyst plays a pivotal role in the synthesis of 7-benzylidene-2,3-diphenyl-3,3a,4,5,6,7-hexahydro-2H-indazole derivatives. In the respect of industrial aims, reusability of the catalyst was examined by repeating the model reaction under the optimizedreaction conditions (Table 1). In order to reuse the catalyst after each cycle, it was separated by a magnet, washed several times with deionized water and chloroform. Then, it was dried in oven at 60 °C and reused in the next run. According to the results, the Co3O4@ SiO2@NH2 MNPs can be reused six times without any significant loss of activity in this organic reaction. Moreover, nitrogen content of the catalyst was estimated by back titrationafter sixth cycle (5.72 mmol/g), which indicates low NH2 leaching during the reaction. Table 1. Reusability of the Co3O4 @ SiO2-NH2 nanocomposite First Second Third Yield (%) Fourth Fifth Sixth 98 96 95 92 91 87 In order to evaluate scope of this research, we tried to prepare a range of 7-benzylidene-2, 3-diphenyl-3,3a,4,5,6,7-hexahydro-2H-indazole derivatives under the same reaction conditions. The results are presented in (Table 2).30 Scheme 2. The model reaction for the synthesis of 7-benzylidene-2,3-diphenyl-3,3a,4,5,6,7-hexahydro-2H-indazole Ghasemzadeh et al.: Preparation and Catalytic Study on a Novel ... 80 Acta Chim. Slov. 2017, 64, 73-82 Table 2. Co3O4@5 m02-NH2 catalyzed synthesis of some indazolesa Entry Productb Time (min) Yield (%)c Lit. M.p.°C M.p.°C 3a 17 94 136-1383 136-136 3b 20 91 141-1433 142-144 3c 15 93 156-158 30 155-157 3d 12 96 174-1763 174-176 3e 10 98 202-2043 200-202 a Reaction conditions: phenyl hydrazine (1 mmol), a,a-bis (substituted-arylidene) cycloalkanone (1 mmol), catalyst (0.003 g, Co3O4 @ SiO2-NH2), under solvent-free conditions at 80 °C b Products were characterized by FT-IR, 1H NMR and 13C NMR analysis c Isolated yield. 1 2 3 4 5 4. Conclusions In this research, Co3O4 nanoparticles were coated with amino-functionalized SiO2 as organic shell via three step method. The average crystallite size of the Co3O4 was calculated 23.7 nm, by using the Scherrer equation. The synthesized nanocomposite exhibited super paramagnetic behaviour at room temperature because of the magneti- cally inactive layer of SiO2@NH2. The saturation magnetization of Co3O4@SiO2@NH2 MNPs is less than that of pure Co3O4 nanoparticles. This new magnetic nanocomposite showed the following advantages: (a) simple preparation; (b) recoverability and easy separation by an external magnet, c) highly effective for chemical transformations as a heterogeneous catalyst. These unique results open new perspectives for application of these types of Ghasemzadeh et al.: Preparation and Catalytic Study on a Novel ... 81 Acta Chim. Slov. 2017, 64, 73-82 magnetic nanocomposites in many reactions. Moreover, we have developed a facile, convenient and environmentally benign synthesis of some 7-benzylidene-2,3-dip-henyl-3,3a,4,5,6,7-hexahydro-2H-indazole derivatives by utilizing novel nano-scale materials including Co3O4@ SiO2@NH2 nanocomposite. 5. Acknowledgements This work is funded by the Research Affairs Office of the Islamic Azad University, Qom Branch, Qom, I. R. Iran grant number 2014-13929]. 6. References 1. K. K. Senapati, S. Roy, C. Borgohain, P. Phukan, J. Mol. Ca-tal. 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Slov. 2017, 64, 73-82 Povzetek V tej raziskavi poročamo o učinkoviti sintezni poti v treh stopnjah s katero smo pripravili nov nanokompozit kobaltove-ga oksida (Co3O4) prevlečen s SiO2 @ (3-aminopropil)-trietoksisilanom. Strukturne in magnetne lastnosti kompozita Co3O4 @ SiO2 @ NH2 smo določili s pomočjo različnih metod: infrardečo spektroskopijo (FT-IR), rentgensko praškov-no difrakcijo, vrstično elektronsko mikroskopijo (SEM), presevno elektronsko mikroskopijo (TEM), energijsko disper-zijsko spektroskopijo (EDX) in magnetometrijo z vibrirajočim vzorcem (VSM). V nanokompozitu Co3O4, ki je prevlečen z amino funkcionaliziranim SiO2 je opaziti superparamagnetne lastnosti in močno magnetizacijo pri sobni temperaturi. Povprečne velikosti kristalitov Co3O4 so 23,7 nm. Dobljeni magnetni nanokompozit je pokazal odlično katalit-sko aktivnost kot novi heterogeni magnetni katalizator za sintezo nekaterih derivatov indazola pri blagih reakcijskih pogojih in visoko stopnjo ponovne uporabe. Ghasemzadeh et al.: Preparation and Catalytic Study on a Novel ... DPI: 10.17344/acsi.20l6.2852_Acta Cliim, Slov. 2017, 64, 83-94_©commons 83 Scientific paper Forward Osmosis in Wastewater Treatment Processes Jasmina Korenak,1 Subhankar Basu,2 Malini Balakrishnan,2 Claus Helix-Nielsen1'3 and Irena Petrinic1 1 University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova ulica 17, SI-2000 Maribor, Slovenia 2 The Energy and Resources Institute (TERI), Darbari Seth Block, IHC Complex, Lodhi Road, New Delhi 110003, India 3 Technical University of Denmark, Department of Environmental Engineering, Bygningstorvet 115, DK2800 Kgs. Lyngby, Denmark. * Corresponding author: E-mail: jasmina.korenak@um.si Tel: +386 2 2294 474 Fax: +386 2 2527 774 Received: 30-08-2016 Abstract In recent years, membrane technology has been widely used in wastewater treatment and water purification. Membrane technology is simple to operate and produces very high quality water for human consumption and industrial purposes. One of the promising technologies for water and wastewater treatment is the application of forward osmosis. Essentially, forward osmosis is a process in which water is driven through a semipermeable membrane from a feed solution to a draw solution due to the osmotic pressure gradient across the membrane. The immediate advantage over existing pressure driven membrane technologies is that the forward osmosis process per se eliminates the need for operation with high hydraulic pressure and forward osmosis has low fouling tendency. Hence, it provides an opportunity for saving energy and membrane replacement cost. However, there are many limitations that still need to be addressed. Here we briefly review some of the applications within water purification and new developments in forward osmosis membrane fabrication. Keywords: Biomimetic membranes, Desalination, Draw solutions, Forward osmosis, Wastewater treatment 1. Introduction The last decade has witnessed extensive research and technological achievements in water production and wastewater treatment processes. Also, it is being realized that water, energy and food are inter-connected - often expressed as the water-energy-food nexus. This necessitates further developments to establish more energy efficient solutions. Therefore, a growing number of academic and industrial research groups around the world are conducting work on water treatment and reuse - in particular, within membrane-based water treatment. Forward Osmosis (FO) is one example of a promising membrane process and potentially a sustainable alternative/supplement to reverse osmosis (RO) process for wastewater reclamation and sea/brackish water desalination. FO has shown good performance in a variety of applications, such as desalination, concentration of waste-water and resource recovery, wastewater treatment and it is also attracting attention as a potential technology to augment water supplies using seawater and wastewater.1-3 However, Van der Bruggen et al (2015) stated that FO as stand-alone process is usually not viable for water treatment purposes.4 Nevertheless, membrane fouling limits its large-scale applications. To reduce the membrane fouling in FO, many improvements has been attempted, e.g. synthesis of different membrane materials, fabrication of membrane modules, membrane coatings etc. Further, there have been improvements in the productivity and decrease in the cost of synthetic membranes used for water and wastewater applications. One of the novelties in membrane development research field is application of biomimetic membranes in separation processes including FO.5 Biomimetics is defined as the study of the structure and function of biological systems and processes as models or inspiration for the sustainable design and engineering of materials and machines. In particular the use of aquaporins (AQPs) - biological water channel proteins6 which are highly selective and effective has prompted considerable interest in recent years.7 Korenak et al.: Forward Osmosis in Wastewater Treatment Processes 84 Acta Chim. Slov. 2017, 64, 83-94 In this paper, we review, (i) the membrane process based on osmotic pressure, principles and transport of water molecules, (ii) applications of FO in water purification, and (iii) recent developments in FO membrane fabrication. 2. Osmotically Driven Membrane Processes FO is a membrane process in which no hydrostatic pressure is applied. The transport of water molecules across a semi-permeable membrane occurs due to the osmotic pressure difference of solutions on either side of the membrane. The natural flow of water is from the low solu- te concentration side to the high solute concentration side across a semi-permeable membrane to equilibrate the osmotic pressure difference. PRO is an osmosis process in which there is a hydraulic pressure applied to the high concentration solution, but the osmotic pressure difference is higher, so the water flux is still opposite to the flux in RO process. PRO possesses characteristics intermediate between FO and RO, where water from a low osmotic pressure feed solution (FS) diffuses through a membrane into a pressurized high osmotic pressure draw solution (DS). In order for water transport to occur, the osmotic pressure difference between the FS and DS should exceed the hydrostatic pressure on the DS side. The classical PRO application is electrical power generation which can be achieved by de- Pressure (AP > An) Pressure (0 < AP < An) ÛP = 0 Pressure (AP<0) - An An RO PRO FO AFO Figure 1. Osmotic processes in membrane filtration. AP is applied hydraulic pressure; Arc is osmotic pressure difference between the two solutions; Jw is water flux; Js is salt reverse flux Figure 2. Relationship between water flux and applied pressure in RO, PRO, FO, and AFO. Korenak et al.: Forward Osmosis in Wastewater Treatment Processes ... 85 Acta Chim. Slov. 2017, 64, 83-94 pressurizing the diluted seawater through a hydro-turbine or generator set.8 Pressure-assisted forward osmosis (AFO) has been proposed that applies the pressure at the feed side to further enhance the performance of the FO process to increase water flux. AFO adds a medium pressure pump to a conventional FO system. The system takes advantage of additional hydraulic pressure that results in water transport in both mechanisms: flux driven by hydraulic pressure (RO mechanism) and that by osmotic pressure (FO mechanism). Figure 1 describes the flux directions of the permeating water in the RO, PRO, FO and AFO processes respectively. The theoretical water flux across the membrane (Jw) is calculated using a variation of Darcy's law: J = Aw x (oAn- AP) (1) ring the process and the DS is diluted. Thus, FO offers several advantages; (i) high rejection of a wide range of contaminants, (ii) reduction in energy consumption, (iii) lower brine discharge, and (iv) lower membrane fouling propensity compared to pressure-driven membrane pro- 2 9 cesses.2" 9 The main challenges in the FO process are related to: - Development of high performance, such as higher water flux and lower salt reverse flux of FO membranes. - Reduction in concentration polarisation of membranes. - Ensuring low DS reverse solute flux across the membrane. - Economical reuse and regeneration of the DS. 4. Types of DS where, Aw is the pure water permeability coefficient of the membrane, AP is the applied hydrostatic pressure, An is the differential osmotic pressure, and o is the reflection coefficient which represents the rejection capability of a membrane. A perfect semipermeable membrane has o = 1. Fig. 2 presents the relation between water flux and applied pressure. In RO, solutes diffuse from the feed into permeate. However, in FO, solutes diffuse in two directions: from the feed into the DS (i.e., forward diffusion) and simultaneously from the DS into the feed (i.e., reverse diffusion). Reverse permeation of solutes from the DS into the FS decreases the osmotic driving force and consequently this reduces the water transport. In a FO system, this could dramatically increase the costs of the process. The flux of a solute (Js) through semipermeable membranes is governed by chemical potential gradients and is commonly described by Fick's law: 1 = B(Ci - Cm) (2) where B is the solute permeability coefficient and Ct and CFm represent the solute concentration at the membrane-solution interface on the DS side and FS side, respectively. 3. The Forward Osmosis Process In FO process, the water molecules are drawn from the FS through a semi-permeable membrane to the DS side (from a lower osmotic pressure to a higher osmotic pressure side). The driving force of the process is an osmotic pressure generated by the concentrated DS. The process ends when the hydraulic difference between the two solutions equals the osmotic pressure difference. The semi-permeable membranes used in FO has comparable rejection range in size of pollutants (1nm and below) as RO membranes. Purified water is produced du- In the FO process, the concentrated solution is commonly known as the DS although different terms can be found in the open literature. The DS plays an important role in the efficiency and performance of the process, and the selection of appropriate DS is crucial. The driving force involved in FO is shown in Fig. 3; where Cs, Cd, as, ad and ^s, ^d are the solute concentrations, water activities and water chemical potentials in the feed (s) and draw (d) solution, respectively. Figure 3. Schematic representation of the driving force involved in FO in an ideal system where only water (H2O) is transported across the membrane (i.e. 100% solute rejection by the membrane).10 In this process it is the ability of the draw solution to generate the relevant osmotic pressure level that is para-mount.11 The osmotic pressure of solution is affected by adding a second solute that can influence the solute-solvent interaction. Solutes disturb the solvent structure. In the case of water as the solvent, the presence of solute affects the structure of liquid water. In pure liquid water, the molecules are heavily hydrogen bonded in an ordered structure. The presence of ions disturb such structures by creating strong electric fields, the water dipoles are then arranged in an orderly manner and strongly bound, thus Korenak et al.: Forward Osmosis in Wastewater Treatment Processes ... 86 Acta Chim. Slov. 2017, 64, 83-94 affecting the freedom of water molecules and influencing their hydrogen bond system.12 Osmotic pressure of a solution n can be expressed by the Morse equation (applies to solutions with dilute concentrations, i.e. <0.5M), as follows: where i is the van't Hoff factor, M is the molarity of the solute which is equal to the ratio of the number of solute moles (n) to the volume of the solution (V), R is the gas constant of 8.3145 J K1 mol1, and T is the absolute temperature. The right side of the equation includes the chemical potential of water which allows for calculating the activity of water aw where Vw is the molar volume of water. Hence, to achieve a high osmotic pressure, a good solubility of the draw solute in water is required to get a high n or M value. In addition, an ionic compound which is able to fully dissociate to produce more ionic species is preferred because it may result in a high i value. This indicates that multivalent ionic solutes are the most favourable. Therefore, compounds with high water solubility and a high degree of dissociation are potential candidates as draw solutes. Different DS and their physio-chemical properties are presented in Table 1. Since FO is an osmotic-driven process, a higher osmotic potential of DS than the feed solution is essential to induce a water flux. In addition, it must exhibit minimum reverse transport from the DS side to the feed side, be easily separated and re-used upon water extraction or be readily available if regeneration is not required. Further to these characteristics, a desirable DS should be non-toxic, highly soluble, of neutral pH, inert and causing a minimum chemical or physical impact on the membrane, low molecular weight and low viscosity to reduce the concentration polarisation, be relatively low cost, and stable. Many studies have been performed to identify appropriate draw solutes over the past few decades.22 Based on the available literature, NaCl appears to be the most promising DS (approximately 40% of experiments), due to its high solubility, low cost and relatively high osmotic potential. It has been used as a DS in concentrations from 0.3 M to 6 M, but is often used at 0.5 M simulating the osmotic pressure of seawater and prompting the use of real seawater or RO brine as a DS.3 Nevertheless, the type of wastewater (feed solution) and the required product purity have influence on the DS selection also. Some studies have used magnetic and/or hydrophilic nanoparticles as a DS.23,24 However, it seems that there are only few that can be selected as a perfect draw solute, since the regeneration step has to be included for draw solution. As such, the benefits of the process have to be larger than the costs of DS and the additional regeneration step. 4 4. 1. Fouling in Osmotically Driven Membrane Processes Fouling is due to the deposition of retained matter (particles, colloids, macromolecules, salts, etc.) on the membrane surface or inside the membrane pores. The interaction (chemical and hydrodynamic) between the foulants and the membrane surface reduces the membrane water flux either temporarily or permanently.25 There are Table 1. Overview of draw solutes used in FO processes. Draw Conc. Osmotic Feed Js Jw Ref. solute(s) pressure (bar) solution (g/m2h) (L/m2h) EDTA-2Naa 0,61 M 60 Raw wastewater 0.1 3.3 13 NaOAc 1,49 M 60 0.4 5.4 NaCl 1,27 M 60 2.4 5.5 EDTA-2Na and NP7b 0.1M and 15 mM 7.31 DI water 0,067 2.65 14 EDTA-2Na and NP9c 0.1M and 15 mM 7.4 DI water 0.092 PUFd/hydrogel 50 to 89 wt% DI water 3.9 to 17.9 15 composites of hydrogel PSSPe 20 wt% 20.85 DI water 0.14 14.50 16 PAspNaf 0.3 g/mL 51.5 atm DI water 4.9 31.8 17 Sucrose 1 26.7 DI water 12.9 18 PAA-Nag 0.72 g/mL 44 DI water 0.18 22 19 HCOONah 0.68 28 DI water 2.73 9.4 20 Sodium hexa- carboxylatophenoxy 0.067 None DI water 7 21 phosphazene a Ethylenediaminetetraacetic acid disodium salt b Nonylphenol ethoxylate surfactants, Tergitol NP7 c Nonylphenol ethoxylate surfactants, Tergitol NP9 d Polyurethane foam eOligomeric poly(tetrabutylphosphonium styrenesulfonate)s f Poly (aspartic acid sodium salt) g Polyacrylic acid sodium salts h Sodium formate Korenak et al.: Forward Osmosis in Wastewater Treatment Processes ... 87 Acta Chim. Slov. 2017, 64, 83-94 four major types of fouling: (1) organic fouling, which is caused by macromolecular organic compounds such as alginate, protein, and natural organic matters; (2) inorganic fouling, which is due to crystallization of sparingly soluble mineral salts when the salt concentration exceeds saturation; (3) biofouling, which involves bacteria deposition, attachment, and subsequent growth to form biofilm; and (4) colloidal fouling, which results from the deposition of colloidal particles.26 Depending on its severity, fouling can have varied degree of adverse impact on membrane performance, such as decreasing water flux, deteriorating product water quality, and increasing maintenance bur-den.27 Furthermore, the foulants might also chemically degrade the membrane material.28 Fouling is a considerable problem that occurs in most liquid membrane processes and consequently influences the economics of the operation. Hence, a lot of research has been done to reduce the impacts of fouling in pressure driven membrane processes. The problem can be addressed by changing operational conditions, cleaning, membrane surface modification, and membrane material choices. However, fouling in osmotically driven membrane processes is different from fouling in pressure driven membrane processes (Figure 4). Depending on the membrane orientation, the deposition of foulants occurs on different membrane surfaces. In FO process, foulant deposition occurs on the relatively smooth active layer. In PRO and other pressure driven processes, the foulant deposition takes place on the rough support layer side, or even within the support layer.25 Recent studies have demonstrated that membrane fouling in FO process is relatively low compared to the pressure driven processes. The reversible fouling can be minimized by optimizing the hydrodynamics, and a variety of contaminants can be effectively removed by physical cleaning.2,30-33 In FO process, fouling due to organic materials is more severe than inorganic material.34 Alginate as a model foulant was studied in FO and RO.30 NaCl was used as draw solute in FO and feed solute in RO, severe flux decline in FO was observed than in RO. However, when dextrose was used as draw solute in FO, the flux decline was almost identical to RO. This indicates a cake formation from reverse salt flux. Humic acid filtration shows higher flux decline in FO than in RO. This also occurs in colloidal fouling with silica particles.35 The flux decline is attributed to intermolecular bridging of humic acid molecules by the salt ions. A strong correlation between intermolecular adhesion and fouling in FO was observed. Strong foulant-foulant interactions, such as adhesion, causes faster accumulation of foulant on the membrane surface.36 It was further concluded that Ca binding, permeation and hydrodynamic shear force are some of the major factors that influences the rate of membrane fouling. The combined effect of organic and inorganic fouling using alginate and gypsum (CaSO4) as model foulants was found to have a synergistic effect between the two foulants; the coexistence of the two foulants displayed a severe flux decline than the individual foulants.37 Alginate fouling and gypsum scaling on the membrane surface could be removed by physical cleaning. However, this observation is true when cellulose acetate membrane is used in FO process. The water flux recovery after physical cleaning of gypsum was less than with a polyamide thin film composite membrane.32 These findings demonstrate that membrane surface modification and material choices should be an effective strategy to mitigate FO membrane fouling. Motsa et al (2014) reported that membrane orientation had an impact on fouling behaviour since the membrane fouled more easy when operated in PRO mode than in FO mode. There was severe permeate flux decline in PRO mode mainly due to the calcium-alginate complexes blocking the pores in the support layer.38 Yong Ng and Parid, focused on the impact of lower organic loads (10, 30, 50 ppm) in secondary effluents with calcium inclusion on Figure 4. Illustration of the fouling mechanisms in membrane processes a) fouling in RO and osmotically driven membrane processes (b) fouling in PRO mode; (c) fouling in FO mode.29 Korenak et al.: Forward Osmosis in Wastewater Treatment Processes ... 88 Acta Chim. Slov. 2017, 64, 83-94 the fouling characteristics of FO membranes both in the FO and PRO modes.39 In their work, they demonstrated that the FO mode had lower fouling compared to the PRO mode, which was also seen by other authors.31,40 This was attributed to the denser, smoother and tighter structure of the membrane active layer which prevented the adhesion and accumulation of foulants on the membrane surface, while the porous support layer, being a looser structure, allowed the accumulation and deposition of the foulants on its surface and inside the membrane, by the mechanisms of direct interception and subsequent pore plugging. Thus it is clear that the nature of fouling in osmoti-cally driven membrane process is different from fouling in pressure driven membrane processes. Further investigations of the mechanism of FO fouling are required to fully understand the differences. The mechanism of fouling is complex and depends on many factors such as water quality, temperature, system design, membrane cleaning, water flow, membrane surface etc. To mitigate fouling, these factors need to be considered in the process design and development. Figure 5. Applications of FO in the water industry, desalination (left) and water reuse (right).11 5. Forward Osmosis Applications FO has a potential benefit as it requires a low hydraulic pressure compared to the pressure-driven process (RO). FO has low energy consumption therefore it involves lower costs, and with appropriate draw solutes and its regeneration methods, the process could be developed to be economically feasible and technically sound. While FO has been investigated in a wide range of applications, including power generation, seawater/brac-kish water desalination, wastewater treatment and food processing, this review focuses mainly on wastewater treatment. In general, there are two clusters of applications concerning FO in the water production and water treat- ment industry (Figure 5), (i) desalination and (ii) water reuse.11 5. 1. Desalination In early 1970s, the FO process was proposed as pre-treatment step to the RO process.41 However, the advent of commercial FO cellulose triacetate (CTA) membranes prompted applications within seawater/brackish water desalination. With the FO desalination process, fresh water can be obtained directly (Figure 6) obtained from saline water (seawater or brackish water) at low (or no) pressure. This can be obtained by using an osmotic reagent based on volatile salts such as NH4HCO3 as the DS3,22. A DS recovery process is needed to separate the draw solute from the solution.42 and in this case raising the DS temperature abo- Oraw Solute Recovery Unit Figure 6. FO process for desalination of seawater/brackish water. Korenak et al.: Forward Osmosis in Wastewater Treatment Processes ... 89 Acta Chim. Slov. 2017, 64, 83-94 ve 60 °C will produce CO2 and NH3 which can then be reused to produce NH4HCO3 in the next cycle43. Also, polymer hydrogels and modified magnetic particles have been suggested as DS in FO desalination with no pressure required. Indirect FO desalination uses a high salinity water (e.g. seawater/brackish water) as the DS and a poor-quality water source such as wastewater effluent or urban storm water runoff as FS.44,45 The diluted seawater/brac-kish water DS can then be desalinated using low pressure reverse osmosis (LPRO). The FO-LPRO hybrid process reduces the cost of the total desalination process compared to pure RO33. This is due to the fact that desalination occurs with a lower salinity and can run at 50% recovery 46. Nicoll (2013) compared three different desalination systems: i) conventional pre-treatment with a dual media filter (DMF), cartridge filtration and SWRO; ii) UF based pre-treatment with SWRO; and iii) conventional pre-treat-ment feeding a FO/RO plant. The summary calculations showed that the DMF/FO/RO configuration has the lowest energy consumption.46 Many studies were focused on DS and their recovery for FO desalination. Different draw solutes (i.e. Na-Cl, KCl, CaCl2, MgCl2, MgSO4, Na2SO4 and C6H12O6) were investigated for seawater desalination using a hybrid FO-NF process.47 Nanoparticles (superparamagnetic) were also tested as a DS in FO desalination, where the nano-particles could be regenerated by UF.24 5. 2. Wastewater Treatment Most FO approaches for poor quality water treatment and reuse are similar to the direct seawater desalination method, where poor-quality water is used as feed, while a DS is used to reduce the volume of the feed. The DS is further treated by other post-treatment process for the recovery of the salt (e.g. RO, membrane distillation). In general, wastewater has lower osmotic pressure and higher fouling propensity. FO integrated with membrane distillation (MD) process was studied for treatment of municipal wastewater, where stable water flux was attained in a continuous operation at the recovery rate up to 80%.48 The FO showed a moderate to high rejection of most organic contaminants while MD rejected the residual contaminants to achieve a near complete rejection in the hybrid process. To recover clean water from secondary wa-stewater effluent, a photovoltaic powered FO - electro-dialysis (FO-ED) process was tested. The process resulted in high removal of total organic carbon (TOC) from the feed wastewater and production of fresh water.49 By using FO and ED through solar energy, this process has been able to supply potable water in isolated and remote areas and islands. In addition, FO process showed several benefits for space missions, including high wastewater recovery, low energy cost and minimized resupply. Further, natural steroid hormones were removed from wastewater by FO membrane contactors. FO has also been used for other wa- stewater such as oily wastewater, industrial and municipal wastewater, nuclear wastewater, landfill leachate, oil-water separation.50 Additionally, application of FO for wastewater treatment was performed in membrane bioreactor (Figure 7), called osmotic membrane bioreactor (OsMBR). Submerged membrane bioreactors (MBRs) involve biodegradation and membrane filtration in a single reactor. It has become one of the most commonly applied WASTEWATER IN BIOREACTOR t PURIFIED WATER Pump SLUDGE OUT Figure 7. Schematic representation of an OsMBR.2 technologies in the treatment of different types of waste-water. FO process replaces the pressure driven membrane process (microfiltration, ultrafiltration) used in MBR. Integration of FO in MBR provides lower fouling propensity, no applied hydraulic pressure, and equally good quality effluent. Unlike the conventional MBR, FO-MBR does not involve high pressure diffused air for reducing the cake layer formation on the membrane surface and pump for collecting the effluent. In addition, FO provides a more sustainable flux and reliable removal of contaminants. The study of novel FO-MBR or osmotic MBR (OsMBR) has been initiated in the last five years.40,51 A salt accumulation model to investigate FO performance in Os-MBR shows that the ratio of the membrane salt permeability (B) to the water permeability (A) (i.e. B/A) and the ratio of hydraulic retention time (HRT) to sludge retention time (SRT) (i.e. HRT/SRT) are two important parameters for the optimization of OsMBR operation.52 To minimize the flux decline caused by salt accumulation, these two ratios should be low. 6. Recent FO Membrane Developments The ideal FO membrane exhibits high water permeability and solute rejection, minimal external and internal concentration polarization (ICP) as well as high chemical and mechanical stability. These features are somewhat contradictory. For example, a low ICP requires a low Korenak et al.: Forward Osmosis in Wastewater Treatment Processes ... 90 Acta Chim. Slov. 2017, 64, 83-94 Table 2. An overview of various FO application in last five years Feed Process / FO membrane material / DS / module: Objective Remarks Synthetic wastewater with sludge Submerged OsMBR / CTA / NaCl (aq.) / Flat-sheet: Water reclamation from wastewater The bioinspired surface modification improved the antifouling ability of the CTA FO membrane.53 Polyvinyl chloride (PVC) latex FO / CTA / NaCl (aq.) / Flat-sheet: Condensation of PVC latex with FO as a pretreatment step The apparent TOC rejection in the FO process is slightly higher than that in RO.54 Boiler feed water (BFW) FO / PA-TFC / NaCl (aq.) / Flat-sheet: treatment of BFW of steam assisted gravity drainage (SAGD) process Reducing the temperature (during fabrication) of the organic solution down to -20 °C effectively reduced the thickness of the PA selective layer.55 High-nutrient sludge FO-MD / TFC / Na3PO4 (aq.) / Flat-sheet: concentrating high-nutrient sludge in an FO-MD hybrid system At pH 9, the Na3PO4 was providing a high water flux and mitigating salt leakage resulting from the formation of the high charge of phosphate and complexion.56 Wastewater with sludge OsMBR / - / Fertilizer / - /: anaerobic fertilizer-drawn forward osmosis membrane bioreactor (AnFDFOMBR) for biogas production Mono-ammonium phosphate (MAP) showed the highest biogas production while other fertilizers exhibited an inhibition effect on anaerobic activity with solute accumulation.57 Raw sewage FO-MD / CTA / NaCl (aq.) / Flat-sheet: Direct sewer mining Trace organic contaminants (TrOC) transport through the FO membrane is governed by "solute-membrane" interaction, whereas that through the MD membrane is strongly correlated to TrOC volatility.48 Secondary wastewater effluent FO-ED / CTA / NaCl (aq.) / Flat-sheet, parallel plate-and-frame module: Potable water production, utilization of natural energy for water treatment and reuse In the ED unit, the diluted draw solution was desalted and high-quality water was produced; the concentrate was recycled to the FO unit and reused as the draw solution.49 Synthetic wastewater FO / CTA / NaCl (aq.) / Flat-sheet: Tetracycline recoverable separation from antibiotic wastewater An effective treatment of tetracycline antibiotic wastewater as well as the recovery of antibiotics from the wastewater.58 Synthetic surfactant wastewater FO / CTA / NaCl (aq) / Flat-sheet: Dehydrate and treat Olive Mill Wastewater (OMWW) Complete decolorization of permeate, and more than 98% rejection to OMWW components, including biophenols and ions.59 Synthetic dye wastewater FO-CF(coagulation/flocculation) / TFC / Poly(acrylic acid) NaCl (aq) / Flat-sheet: treatment and reuse of textile wastewater Remarkable reverse fouling behaviour has been observed where the Jw of the fouled membrane was fully restored to the initial value by physical flushing without using any chemicals.60 Wastewater containing heavy metals FO / cellulose acetate butyrate (CAB) / NaCl (aq.) / Hollow fiber: Water reclamation from emulsified oily wastewater through FO under the PRO mode Water flux declines slightly by 10% after a 12 h oil/water test under the PRO mode and water flux of the fouled membrane can be restored to 97% by simple water rinse.61 S-value (structural parameter) which in turn requires a low thickness and high porosity. Thus, providing sufficient mechanical stability to a thin highly porous membrane is one of the key outstanding problems in FO membrane development. The membrane structural parameter S is defined as: £ where D is the diffusion coefficient of the draw solute, ts is the support layer thickness, t the tortuosity, and e the porosity.62 Several materials have been investigated for FO membrane fabrication. These include materials based on cellulose, polyamide (and other polymers), and polyelectrolytes. Also so-called mixed matrix membranes have been investigated. These membranes typically consist of 'fillers' or inclusions (e.g. zeolites) embedded in a polymeric matrix. A special case is the concept of biomimetic FO membranes where aquaporin proteins are incorporated in the membrane enhancing water flux while preserving high solute rejection. Cellulose acetate (CA) and cellulose triacetate (CTA) have been used in RO membrane fabrication since the 1960s so it is perhaps not surprising that FO membra- Korenak et al.: Forward Osmosis in Wastewater Treatment Processes ... 91 Acta Chim. Slov. 2017, 64, 83-94 Table 3. List of commercial producers and developers of FO membranes Company Membrane Type Aquaporin A/S Oasys Water Fluid Technology Solutions, Inc. Nitto Denko Woongjin Chemical Co., Ltd. Porifera nes based on CTA were amongst the first to be commercially available from Hydration Technologies Incorporated (HTI).63 In recent years there have been significant developments in CA and CTA based FO membrane both in flat sheet and hollow fibre geometries. Generally, these membranes are fabricated in a phase inversion process where a polymer is transformed in a controlled way from a solution state to a solid state. Thus, when a polymer solution (polymer plus solvent) is cast on a suitable support and immersed in a coagulation bath containing a non-solvent precipitation occurs because of the exchange of solvent and non-solvent. The procedure allows for making membranes with very low S-values (of the order of 50 pm) which makes them potentially good FO membranes. The general trend is that CA membranes have acceptable water fluxes but tend to have lower rejection (and thus higher reverse solute fluxes) whereas the opposite trend is the case for CTA based FO membranes.64 The cellulose hydroxyl can be reacted with reagents to generate cellulose esters beyond CA and CTA. These include materials such as cellulose propionate (CP), cellulose acetate butyrate (CAB) or cellulose acetate propionate (CAP). Dual layer FO hollow fibres made from CA and CAP show superior performance compared to CA-based flat sheet or hollow fibre membranes. However, the limited stability to temperature and pH generally restricts the use of cellulose-based materials.64 Cellulose-based membranes were dominant throughout the 1960s until the advent of thin film composite (TFC) membranes in the 1970s. Most TFC membranes are made with a porous, highly permeable support such as polysulfone, which is coated with a cross-linked aromatic polyamide thin film.65 The coating - also sometimes referred to as the active layer - provides the solute rejection properties while the support provides the mechanical stability. The typical coating is made by interfacial polymerization to create a 100-200 nm thick polyamide coating exemplified by the reaction between m-phenyl diamine and trimesoyl chloride monomers. A good polyamide layer requires optimization of the exact monomer composition, reaction time, temperature and ambient humidity. In FO membranes, addition of the detergent sodium dodecyl sulfate (SDS) can enhance solute rejection without major impact on the water flux, and post treatment using SDS/glycerol followed by thermal Configuration Status Hollow fiber and flat sheet Commercial Flat sheet Commercial Flat sheet Commercial - Development - Development Flat sheet Commercial annealing facilitates removal of unreacted monomers resulting in increased free volume and reduced thickness leading to improved flux without detrimental effects on rejection.66'67 The presence of cetyltrimethylammonium chloride (CTAC) which can react with the m-Phenylene diamine (MPD) can decrease water flux while increasing the solute rejection. Thus, there are a number of possibilities for fine-tuning FO membrane active layers. A good support for a TFC membrane shows a low ICP and typically supports are based on polysulfone or polyethersulfone.62 Also bucky papers made from Carbon Nanotubes (CNTs) and nanofiber mats formed from elec-trospun fibres have been suggested as good FO membrane support due to high porosity and tensile strength.68'69 Structurally it has been argued that an open 'finger' like structure of the support is to be favoured over a more dense 'sponge' like structure.70 However a more open structure is also mechanically weaker and a more dense structure also may have a higher ICP. An obvious compromise would be to have an anisotropic support with a sponge structure interfacing the active layer supported by a finger like structure below.62'71 But the structural features are not the only determinants for FO membrane performance. A sponge like support structure may in fact give rise to a higher water flux than a finger like structure provided that hydrophilicity and thickness are well controlled.72-74 This illustrates the complexity behind ICP where many specific physico-chemical factors give rise to a phenomenological effect. Polyelectrolytes have attracted considerable attention over the last decade as an alternative to the TFC approach. The typical polyelectrolyte membrane consists of a layer-by-layer (LbL) deposition of alternating cationic and anionic electrolyte-films onto a suitable support where hydrolysed (and thus negatively charged) polyacryloni-trile is an exemplary material. Large scale production of LbL assembled membranes has proved to be difficult; nevertheless' the approach offers the potential of fabricating membranes with good rejection combined with good solvent resistance and thermal stability.75'76 One of the latest design approaches for FO (and RO) membranes is based on the concept of membrane biomime-tics where technological developments take cues from natu-re.77'78 The basic concept is based on the fact that biological membranes have excellent water transport characteristics. Biomimetic aquaporin Thin film composite Cellulose tri-acetate Composite semipermeable membrane Composite membrane Thin film composite Korenak et al.: Forward Osmosis in Wastewater Treatment Processes ... 92 Acta Chim. Slov. 2017, 64, 83-94 They employ natural proteins known as aquaporins (AQPs) to regulate the flow of water, providing increased permeability and near-perfect solute rejection.79 Thus by using reconstituted AQPs as building blocks one can create membranes with unique flux and rejection properties.80 AQP membrane design approaches have been recently revie-wed.7 According to membrane structural design, AQPs incorporated biomimetic membranes can be classified into two basic types, (1) AQPs containing vesicle encapsulated membranes (VEMs), where AQPs containing vesicles (pro-teoliposomes or proteo-polymersomes) are immobilized in a dense polymer layer and (2) AQP containing supported (lipid or polymer) membrane layers (SMLs). AQP-based membranes are currently being produced and commercialised by the Danish company Aquapo-rin A/S, its Singaporean affiliate, Aquaporin Asia Pte. Ltd., and its Joint Ventures AquaPoten Limited in China and Aquaporin Space Alliance in Denmark in flat sheet and hollow fibre geometries. The membranes are currently tested in several processes including pesticide removal, CO2 capture, and water reuse in space and textile wastewater treatment.81-84 7. Conclusions The FO process used in wastewater treatment and water purification shows promising results, and has many advantages in comparison to the conventional water/wa-stewater treatment processes. The studies are focused on improving the FO process by developing new membranes, membrane surface modifications, different DSs and their compatibility with various wastewaters. However, there are other issues (e.g. membrane fouling, raw water characteristics) in FO process that needs to be studied. FO processes are highly compatible with other treatment processes therefore, the whole treatment process could become more cost effective by incorporating FO process. As it is seen from the literature, many studies and improvements were done on the membrane materials and their surface, and new technologies were implemented, such as membranes with biological materials (aquaporins). Higher quality water is in demand due to the imposition of new and ever-changing water quality standards. Therefore, interest in FO technology is growing as a potential, cost- competitive and reliable alternative. 8. Acknowledgements The authors would like to acknowledge financial support from the Slovenian Research Agency (Javna Agencija za Raziskovalno Dejavnost RS) for their Project No. 1000 - 14 - 0552) and Department of Science and Technology (DST), Government of India for Grant No. INT/Slovenia/P-15/2014. CHN also acknowledges support from the Innovation Fund Denmark via the IBISS and MENENTO projects. 9. References 1. E. M. Garcia-Castello, J. R. Mccutcheon, M. Elimelech, J. Membr. Sci., 2009, 338, 61-66. https://doi.org/10.1016/j.memsci.2009.04.011 2. A. 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Ye, J. Ling, H. T. Madsen, E. G. S0gaard, C. Hélix-Niel-sen, P. Luis, B. Van Der Bruggen, J. Membr. Sci, 2015, 498, 75-85. https://doi.org/10.1016/j.memsci.2015.09.010 83. T. Hill, B. W. Taylor, International Conference on Environmental Systems (ICES). San Diego, CA; United States, 2012 84. I. Petrinic, C. Hélix-Nielsen, Tekstil, 2015, 63, 252-260. Povzetek V zadnjih letih se membranska tehnologija vse pogosteje uporablja v procesih čiščenja odpadne vode in vode za proizvodnjo. Procesi membranske filtracije so enostavni za izvajanje in dajejo kakovostni produkt/filtrat za nadaljnjo uporabo tako v industrijske namene kot tudi za proizvodnjo pitne vode. Ena od obetavnih tehnologij za proizvodnjo vode in obdelavo odpadnih voda je proces osmoze. Princip delovanja osmoze predstavlja metodo čiščenja vode, ki deluje brez hidravličnega tlaka, kar zagotavlja trajnostno (nizkoenergetsko) tehnologijo obdelave vode. Gonilna sila je razlika v kemijskem potencialu med vhodno in gonilno raztopino, ki sta ločeni z membrano, prepustno samo za vodo. Prednost osmoze pred obstoječimi visokotlačnimi membranskimi procesi je ravno delovanje brez dodatnega visokega tlaka, kar vodi tudi k manj pogostemu mašenju membran. Torej, omogoča delovanje z nižjo porabo energije ter podaljša življenjsko dobo membran. Vendar pa še vedno obstajajo nekatere pomembne tehnološke pomanjkljivosti procesa. V prispevku je predstavljena uporabnost tehnologije osmoze pri različnih sistemih čiščenja ter razvoj proizvodnje osmoznih membran. Korenak et al.: Forward Osmosis in Wastewater Treatment Processes ... DOI: 10.17344/acsi.2016.2894 Acta Chim. Slov. 2017, 64, 95-101 ^creative tS/commons Scientific paper A Novel High-performance Electrospun Thermoplastic Polyurethane/Poly(vinylidene fluoride)/Polystyrene Gel Polymer Electrolyte for Lithium Batteries Yuanyuan Deng, Zeyue He, Qi Cao*, Bo Jing, Xianyou Wang and Xiuxiang Peng Key Laboratory of Environmentally Friendly Chemistry and Applications of Minister of Education, College of Chemistry, Xiangtan University, Xiangtan 411105, China * Corresponding author: E-mail: wjcaoqi@163.com Received: 06-09-2016 Abstract A novel high-performance gel polymer electrolyte (GPE) based on poly(vinylidene fluoride) (PVDF), thermoplastic polyurethane (TPU) and polystyrene (PS) has been prepared. Its characteristics are investigated by scanning electron microscopy (SEM), thermal analysis (DSC), universal testing machines (UTM), galvanostatic charge-discharge and electrochemical impedance spectroscopy. The GPE based on TPU/PVDF/PS (10 wt.%) show a high ionic conductivity of 5.28 x 10-3 S cm-1 with the electrochemical stability window of 5.0 V. In addition, its first charge-discharge capacity reached to 169.5 mAh g-1, high mechanical strength and stability to allow safe operation in rechargeable lithium ion polymer batteries. Keywords: Gel polymer electrolytes; Electrospinning; Poly (vinylidene fluoride); Polystyrene; Thermoplastic polyurethane 1. Introduction Polymer-based nanocomposites have attracted considerable academic and industrial attention over the years.1'2 Various combinations of polymer matrices and nanofillers have been investigated. It is known to us that superior performance of lithium ion battery is determined by active electrode materials and excellent electrolytes. Among them, gel polymer electrolytes (GPEs) have been reported with high ionic conductivity at room temperature, stable and well compatibility with lithium electrodes,3-5 and good mechanical stability. There are many ways to produce GPEs such as phase inversion method, /-ray irradiation method, solvent casting technique, thermally induced phase separation technique, and electros-pinning technique.6-8 In these methods, electrospinning technique which made the solution of polymer into lots of uniform and slender nanofibers under high voltage is a simple, controllable and efficient approach. Thermoplastic polyurethane (TPU) contains two-phase microstructure which are soft segments and hard segments.9-11 The hard sections are incompatible with the soft section in thermodynamics, while these two phases are interconnec- ted throughout each other. The whole system benefits from these two phases since that the hard parts afford spatial stability and the soft phases are conducive to good ionic conductivity owing to the soft segments don't form ionic cluster after being dissolved alkali metal salt. Many investigations were devoted to copolymerizing TPU with other polymers for processing GPEs. Some articles based on coaggregant like thermoplastic polyurethane (TPU)/li-near poly (ethylene oxide) (PEO) (TPU-PEO), thermoplastic polyurethane (TPU)/polyacrylonitrile (PAN) (TPU-PAN) and polyurethane/poly (vinylidene fluoride) (PU-PVDF) as GPEs for rechargeable lithium batteries have been reported lately.12-14 Different concentrations of thermoplastic polyurethanes/poly(vinylidene fluoride-co- he-xafluoro propylene) (TPU/PVDF-HFP) derived from some researchers including our study group member Xiuxiang Peng having done related research.15 Poly (vinylidene fluoride) (PVDF) is a semi-crystalline polymer.16 With low water absorption, high mechanical properties and interfacial stability with lithium metal,17-19 PVDF has been adopted as polymer electrolyte in lithium ion polymer bat-teries.20 Polystyrene (PS) polymers possess excellent mechanical properties: high strength, fatigue resistance Yuanyuan et al.: A Novel High-performance Electrospun Thermoplastic ... 96 Acta Chim. Slov. 2017, 64, 95-101 and dimension stability. Besides, it also has high glass transition temperature and high dielectric breakdown field. From the properties of the three kinds of materials, each of these three kinds of materies is very appropriate as a gel polymer matrix. Our group have done some research, which was the first trial of making TPU/PVDF/PS fiber membranes.21 In comparison to PU/PVDF, TPU/PS and PVDF/PS films, the TPU/PVDF/PS films show more noticeable electrochemical characteristic and mechanical performance. We would like to continue our efforts to develop TPU/PVDF/ PS porous fibrous films by electrospinning using different concentration polymer solutions. In order to investigate the influence of various polymer concentration stresses on the TPU/PVDF/PS fiber membranes, membrane morphology, charge and discharge capacity, ionic conductivity, and mechanical properties will be examined systemically. In this study, we expect to provide a deep investigation and insight on the preparation of TPU/PVDF/PS microporous fiber membranes with prominent electrochemical and mechanical performance. Primary results showed that it is very suitable for application in lithium ion batteries. 2. Experimental 2. 1. Materials Thermoplastic polyurethane (TPU, yantaiwanhua, 1190A), polystyrene (PS,yangzishihua) and poly( vinyli-dene fluoride) (PVDF, Alfa Aesar) were dried under vacuum at 80 °C for 24 h. LiClO4 ■ 3H2O (AR, Sinopharm Chemical Reagent Co., Ltd.) was dehydrated in vacuum oven at 120 °C for 72 h. 1.0 M Liquid electrolyte was made by dissolving a certain quality of LiClO4 in ethylene carbonate (EC, Shenzhen capchem technology Co., Ltd.)/propylene carbonate (PC, Shenzhen capchem Technology Co. Ltd.) (1/1, v/v). N, N-dimethylforamide (DMF) and acetone were analytical purity and used as received without further treatment. rous fibrous films were finally dried under vacuum at 80 °C for 12 h. 2. 3. Preparation of Gel Polymer Electrolytes The thickness of the TPU/PVDF/PS nonwoven films used was about 100 pm. At room temperature, the dried TPU/PVDF/PS nonwoven films were activated by 1 M LiClO4-EC/PC liquid electrolyte solutions for 1 h in a glove box filled with argon. Wipe the surface of swelled membranes by filter paper and then get the gel polymer electrolytes. 2. 4. Membrane Characterization Scanning electron microscope (SEM, Hitachi S-3500 N, Japan) was used to examine the morphology of the films. The thermal stability of the films was monitored using thermogravimetric analysis (model TQAQ 50, TA Company, USA). DSC measurements were carried out under the temperature range from 20-200 ° at a scan rate of 10 °/min. The mechanical strength of the gel polymer electrolyte films was measured by universal testing machines (UTM, Instron Instruments). There are some difficulties in surveying the "wet" films (with electrolyte), therefore the test was measured the mechanical properties of the "dry" membrane (without electrolyte). The extension rate was kept at 5 mm min-1. The dimensions of the sheet used were 2 cm x 5 cm x 150-250 pm (width x length x thickness). The porosity was investigated by immersing the membranes into n-butanol for 1 h and then calculated by using the following relation: W - W p = >Lz-]00 0/o P„Ve (1) Ww and Wd are the mass of the wet and dry membrane, respectively, nb is the density of n-butanol, and Vp is the volume of the dry membrane. The electrolyte uptake was determined by measuring the weight increase and calculated according to Eq: 2. 2. Preparation of TPU/PVDF/PS fibrous Membrane In the first place, a certain amount of dried PVDF, TPU and PS (6:6:1, wt/wt/wt) were dissolved in the mixture of acetone/N, N-dimethylacetamide (1:3, wt/wt) forming a 9 wt.% solution, then they were stirred by mechanical stirring for 12 h at room temperature. Then 10 wt.%, 11 wt.%, 12 wt.% TPU/PVDF/PS solutions were made by the same way. After being stayed for 10 minutes to remove air bubbles, the viscous blending polymer solution was put into the needle injection pump. The tip of the needle was connected to high voltage source (24.5kV) and elec-trospined at ambient atmosphere. Porous fibrous films were obtained on the collector plate. The electrospun po- W -W i/ptake(%) = ----ü-x 100% ^i (2) W0 is the weight of dried films and W is the weight of swelled films. The ionic conductivity of the composite film was measured with SS/PE/SS blocking cell by AC impedance measurement using Zahner Zennium electrochemical analyzer with a frequency range of 0.1-1 MHz. The thin films were prepared about 100pm in thickness and 1.96 cm2 in area for impedance measurement. Thus, the ionic conductivity could be calculated from the following equation: (3) Yuanyuan et al.: A Novel High-performance Electrospun Thermoplastic 97 Acta Chim. Slov. 2017, 64, 95-101 In Eq. (3), o is the ionic conductivity, Rb is the bulk resistance, h and S are the thickness and area of the films, respectively. 2. 5. Cell Assembly and Performance Characteristics Electrochemical stability was measured by a linear sweep voltammetry (LSV) of a Li/PE/SS cell using Zahner Zennium electrochemical analyzer at a scan rate of 5 mV s-1, with voltage from 2 V to 6 V. For charge-discharge cycling tests, the Li/PE/LiFePO4 cell was assembled. The cell was subjected to electrochemical performance tests using an automatic charge-discharge unit, Neware battery testing system (model BTS-51, ShenZhen, China), between 2.5 and 4.2 V at 25 °C, at different current densities. 3. Results and Discussion 3. 1. Morphology and Structure Fig. 1 shows the SEM images of the membranes prepared by electrospinning of different concentrations of 9 to 12 wt.% TPU/PVDF/PS polymer solution. All of these four membranes show a microporous structure, but we can see that the fibers of TPU/PVDF/PS (Fig.1(b)10 wt.%) are relatively uniform and slender, with the diameter distribution about 1pm. While the fibers of TPU/PVDF/PS (Fig.1 (a)9 wt.%) are cross linked unevenly in the middle part of it. Both of the fibers of TPU/PVDF/PS (Fig.1(c)11 wt.%) and (Fig.1(d)12 wt.%) diameter distribution values are thicker than the fibers of TPU/PVDF/PS (Fig.1(b)10 wt.%), so do the fiber smoothness. From the principle of electrospinning we know there are many factors that can affect fiber membranes' morphology. The parameters influencing the morphology of electrospun fiber membranes contain the distance between the nozzle of the syringe and the collector, the applied voltage, dielectric constant of the solution and the concentration of the polymer solution. In this work, the only difference is the concentration of the polymer solution. Finally, we found that TPU/PVDF/PS polymer solution of 10 wt.% is the best for electrospinning. After blending, there is interface between different materials. The interfacial interation force which has a great influence on the morphology of electrospinning film, the greater the force, the poorer the performance of membrane. The interface force is the minimum when the mass fraction is 10%, which is why the membranes of TPU/PVDF/PS (10 wt.%) is smooth and slender. a) f 5c JtkV WD! lad 55-10 llO.MO 1|im 5:1 MkV WD! (mm 5S40 S 10.000 l»ni c) Jfifim iijfcafii™ d) S = l 30kV WD! mil SS25 110.000 1|tm Fig. 1. SEM images of TPU/PVDF/PS electrospun membranes (a) 9 wt.% (b) 10 wt.% (c) 11 wt.% (d) 12 wt.% Yuanyuan et al.: A Novel High-performance Electrospun Thermoplastic ... 98 Acta Chim. Slov. 2017, 64, 95-101 3. 2. DSC Analysis Typical DSC curves of the nanofibrous membranes varied with the relative weight of PVDF/TPU/ PS, which are presented in Fig.2. From the Table 1, the crystallinity of TPU/PVDF/PS (9 wt.%) is 20.43%; the crystallinity of TPU/PVDF/PS (10 wt.%) is 13.64%; the crystallinity of TPU/PVDF/PS (11 wt.%) is 21.37%; the crystallinity of TPU/PVDF/PS (12 wt.%) is 26.65%. We can find that the crystallinity decreased when concentration increased from 9 wt.% to 10 wt.%. However, with the concentration continuing to increase, the crystallinity gets enlargement. So we can get a conclusion that 10 wt.% concentration has the lowest degree of crystallinity. __iJwty. —~ 11 Wt,% - 9 wt.% _ 10 wt.% _ i i 1,1:1 100 120 M0 160 180 TeinpciHlui e( ) Fig. 2. DSC thermograms of different concentration of TPU/ VDF/PS Table 1. Thermodynamic properties of different concentration of TPU/PVDF/PS Sample AHf (J/g) Crystallinity X c (%) TPU/PVDF/PS (9 wt.%) 8.91 20.43 TPU/PVDF/PS (10 wt.%) 6.61 13.64 TPU/PVDF/PS (11 wt.%) 11.39 21.37 TPU/PVDF/PS (12 wt.%) 15.5 26.65 3. 3. Electrolyte Uptake and Ionic Conductivity Fig.3 shows the uptake behaviors of the electrospun fibrous membranes. The percentage of electrolyte uptake can be calculated according to Eq(A). The TPU/PVDF/PS (9 wt.%) fibrous film shows an electrolyte uptake of about 310% within 2 min, The TPU/PVDF/PS(10 wt.%) fibrous film is 331%, The TPU/PVDF/PS (11 wt.%) fibrous film is 296%, The TPU/PVDF/PS (12 wt.%) fibrous film is 274% after 15 min, it is found that the electrolyte uptake of these four membranes become stabile. The uptake of the electrolyte solution reaches up to 320% (9 wt.%), 341% (10 wt.%), 305% (11 wt.%), 298% (12 wt.%), respectively. The absorption of large quantities of liquid electrolyte by the composite membranes results from the high porosity of the membranes and the high amorphous content of the polymer. The fully interconnected pore structure makes fast penetration of the liquid into the membrane possible, and hence the uptake process is stabile within the initial 15 min. TPU/PVDF/PS (10 wt.%) membrane owns the highest porosity, so it also has the highest electrolyte uptake percentage. Furthermore, the TPU/ PVDF/PS (10 wt.%) membrane's average fiber diameter is minimal that leads to the increasing in the absorption ratio of the electrolyte solution. Because the porosity and the surface area of the pore wall of the film will increasing with the average fiber diameter decreasing. The increasing of surface area of the pore wall and more pores result in a higher uptake of the liquid electrolyte, which means more Li+ in the same volume.22 _§_# r ! 1 ■ C X î ï I £~" t $ —9 wt.% t • 10 wt.% ! 1 wt.% T 12 Wt.% • 0 5 10 15 20 Tïmi^min) Fig. 3. The uptake behavior of the TPU/PVDF/PS electrospun fibrous films Fig.4 shows the impedance spectra of TPU/PVDF/ PS based fibrous polymer electrolyte. It is typical AC impedance for gel polymer electrolyte. The self-resistance (R) is the major contribution to the total resistance and ionic conductivity is calculated according to Eq.(3). The ionic conductivity of TPU/PVDF/PS (10 wt.%) membrane was 5.28 x 10-3 mS cm1 at room temperature. From table 2, we know that the ionic conductivity of TPU/PVDF/PS (10 wt.%) membrane is maximal , and the body resistance of TPU/PVDF/PS (10 wt.%) membrane is the smallest. The solution crystallinity, porosity and absorption rate have relationships with the self-resistance, from the previous experimental results we can know why the ionic conductivity of TPU/PVDF/PS (10 wt.%) membrane is the biggest. Yuanyuan et al.: A Novel High-performance Electrospun Thermoplastic ... 99 Acta Chim. Slov. 2017, 64, 95-101 Table 2. Different concentration of TPU/PVDF/PS membranes' parameters and ionic conductivity Materials Rb(Q) H(cm) S(cm-2) o(10-3S cm1) TPU/PVDF/PS (9 wt.%) 292 0.0014 202 2.37 TPU/PVDF/PS (10 wt.%) 1.12 0.0012 2.03 5.28 TPU/PVDF/PS (11 wt.%) 4.67 0.0014 1.98 1.51 TPU/PVDF/PS (12 wt.%) 5.2 0.0012 2.05 1.13 Fig. 4. Impedance spectra of gel polymer electrolytes 3. 4. Evaluation in Li/LiFePO4 Cell Fig.5 shows the first charge-discharge capacity curves of the cells with GPEs of TPU/PVDF/PS. The GPEs of TPU/PVDF/PS (10 wt.%) delivers a charge capacity of 169.81 mAh g1 and discharge capacity of 169.5 mAh g-1, which is about 99% of the theoretical capacity. The GPEs of TPU/PVDF/PS (9 wt.%; 11 wt.%; 12 wt.%) deliver a charge capacity of 161.79 mAh g-1; 159.49 mAh g-1; Fig. 5. first Charge-discharge capacity of different concentration of GPEs based on electrospun TPU/PVDF/PS membrane Fig. 6. The cycle performance (discharge capacitie) of different concentrations of GPE based on electrospun TPU/PVDF/PS membranes 151.82 mAh g 1and discharge capacity of 160.65 mAh g 1; 156.32 mAh g1; 151.74 mAh g1. The Li cells with GPEs have been evaluated for cycle ability property under the 0.1 C rate at 25 oC and the results are shown in Fig. 6. The cell with GPE (10 wt.%) has a highest discharge capacities in the whole 50 cycles. From the above data, we can know that the GPEs of TPU/PVDF/PS (10 wt.%) owns the best charge-discharge capacity and cycle ability property. 3. 5. Mechanical Property The stress-strain curves of different concentrations of electrospun PVDF/TPU/PS membranes are presented in Fig. 7, and their mechanical properties are summarized in Table 3. Because no phase separation of the nanofi-brous membranes was observed from SEM, the nanofi-brous membranes presented acceptable mechanical properties to be applied into practice.23 It can be found that PVDF/TPU/PS (10 wt.%) membrane owns the longest elongation of 98.2% and can bear the tensile strength below 12.9 MPa. Both the tensile strength and elongation are better than others. Because electrospun membranes are constituted by three kinds of polymer, all of three kinds of polymer are dissolved in the mixture of aceto-ne/N,N-dimethylacetamide (1:3, wt/wt) solution, and there is interfacial force between each others. As we know that the PVDF/TPU/PS (10 wt.%) membrane's interfacial Yuanyuan et al.: A Novel High-performance Electrospun Thermoplastic ... 100 Acta Chim. Slov. 2017, 64, 95-101 Fig. 7. Stress strain curves of different concentration of electrospun PVDF/TPU /PS membranes 2 3 4 5 Ê Vo!tage(V VS.Li/Li ) Fig. 8. Linear sweep voltammograms of the gel polymer electroly- tes Table 3. Mechanical properties of different concentration of electrospun PVDF/TPU /PS Samples Stress (Mpa) Strain (%) TPU/PVDF/PS (9 wt.%) 9.9 ± 0.2 85.4 ± 0.2 TPU/PVDF/PS (10 wt.%) 11.9 ± 0.2 94.2 ± 0.2 TPU/PVDF/PS (11 wt.%) 9.14 ± 0.2 63.2 ± 0.2 TPU/PVDF/PS (12 wt.%) 6.16 ± 0.2 62.8 ± 0.2 force is the smallest, so do the crystallinity of PVDF/TPU/PS (10 wt.%) membrane. The greater the degree of crystallinity, the worse of the toughness. So the PVDF/TPU/PS (10 wt.%) membrane has the best mechanical properties. 3. 6. Electrochemical Stability The results of electrochemical stability tests of the gel polymer electrolytes by LSV are shown in Fig.8. From Fig.8, the electrochemical stability of the gel polymer electrolyte with PVDF/TPU/PS (10 wt.%) membrane is 4.9 V. And their electrochemical stability follows the order: TPU/PVDF/PS (10 wt.%) 4.9 V> TPU/PVDF/PS (11 wt.%) 4.3 V> TPU/PVDF/PS (9 wt.%) 4.0 V> TPU/ PVDF/PS (12 wt.%) 3.6 V. It is clearly that the gel polymer electrolyte of TPU/PVDF/PS (10 wt.%) shows the best electrochemical stability, which may due to better compatibility with liquid electrolyte and nanofibrous membranes with less leakage of liquid electrolytes. In addition, the electrochemical stability was also influenced by the large and fully interconnected pores, high porosity, higher specific surface area, uniform morphology of membranes and the AFD. From the SEM images of TPU/PVDF/PS electrospun membranes you can know that the gel polymer electrolyte of TPU/PVDF/PS (10 wt.%) possesses high porosity and surface area. Therefore the gel polymer electrolyte of TPU/PVDF/PS (10 wt.%) is best for applications in lithium-ion. 4. Conclusions GPEs based on fibrous TPU/PVDF/PS blend membranes were prepared by electrospinning the polymer solution in DMF/acetone (3:1, w/w) at room temperature. It has been observed that the optimum proportion of a novel high-performance gel polymer electrolyte is TPU/PVDF/ PS (10 wt.%). It has a high ionic conductivity of 5.28 x 10-3 mS cm1 with electrochemical stability up to 5.0 V versus Li+/Li at room temperature. The first charge-discharge capacity of gel polymer electrolyte lithium battery based on PVDF/TPU/PS (10 wt.%) is about 169.5 mAh g-1 at 25 The PDVF/TPU/PS (10 wt.%) mixed film owns the longest elongation of 98.2%, and it can bear the tensile strength below 12.9 MPa. Both the tensile strength and elongation are excellent. The PVDF/TPU/PS (10 wt.%) based gel polymer electrolyte is the optimum proportion of a novel high-performance gel polymer electrolyte for rechargeable lithium batteries. 5. Acknowledgements The workers gratefully appreciate the financial supports from the Youth Project of National Nature Science Foundation of China (No. 51203131). 6. References 1. Zhu YS, Wang FX, Liu LL, Xiao SY, Chang Z, Wu YP, Energy Environ. 2013, 6, 618-624. Yuanyuan et al.: A Novel High-performance Electrospun Thermoplastic ... Acta Chim. Slov. 2017, 64, 95-101 101 https://doi.org/10.1039/C2EE23564A 2. Theron SA, Zussman E, Yarin AL, J. polymer. 2004, 45, 2017-2030. 3. J. J. Xu, H. Ye, Electrochem. Commun. 2005, 7, 829-835. https://doi.Org/10.1016/j.elecom.2005.04.034 4. S.-I. Kim, H.-S. Kim, S.-H. Na, S.-I. Moon, Y.-J. Kim, N.-J. Jo, Electrochim. Acta. 2004, 50, 317-321. https://doi.org/10.1016Zj.electacta.2003.12.068 5. N. Wu, B. Jing, Q. Cao, X. Wang, H. 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Singh, Solide State Ionics. 2002, 169, 152-153. 21. X. L. Tang, Q. Cao, X. Y. Wang, X. X. Peng, J Zeng, RSC Adv., 2015, 5, 58655- 58662 22. L. Zhou, Q. Cao, B. Jing, X. Y. Wang, X. L. Tang, N. Wu, J. Power Sources. 2014, 263, 118-124. https://doi.org/10.1016/jjpowsour.2014.03.140 23. W. L. Li, Y. H. Wu, J. W. Wang, D. Huang, L. Z. Chen, European Polymer Journal. 2015, 67, 365-372. https://doi.org/10.1016/j.eurpolymj.2015.04.014 Povzetek Pripravili smo visoko učinkovit gel polimerni elektrolit (GPE), ki temelji na polivinilidenfluoridu (PVDF), termopla-stičnem poliuretanu (TPU) in polistirenu (PS). Njegove lastnosti smo preučevali z naslednjimi tehnikami: vrstično elektronsko mikroskopijo (SEM), termično analizo (DSC), meritvami mehanskih lastnosti (UTM) in elektrokemijsko impe-dančno spektroskopijo. Gel polimerni elektroliti (GPE), ki temeljijo na TPU/PVDS/PS (10 wt.%) imajo visoko ionsko prevodnost 5.28 x 10-3 S cm-1 in elektrokemijsko okno stabilnosti 5.0 V. Poleg tega pa prva kapaciteta polnjenja in praznjenja doseže 169.5 mAh g-1. Zaradi dobrih mehanskih lastnosti in stabilnosti bi bili ti materiali lahko uporabni v litij ionskih polimernih baterijah. Yuanyuan et al.: A Novel High-performance Electrospun Thermoplastic 102 DOI: 10.17344/acsi.2016.2901 Acta Chim. Slov. 2017, 64, 102-116 ^creative ^commons Scientific paper Synthesis, Characterization and Cytotoxicity of Substituted [1]Benzothieno[3,2-e][1,2,4]triazolo [4,3-a]pyrimidines Samir Botros,1 Omneya M. Khalil,1 Mona M. Kamel1 and Yara S. El-Dash1* 1 Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, P.O. Box 11562, Cairo, Egypt * Corresponding author: E-mail: yara.el-dash@pharma.cu.edu.eg Tel: +202 27043364 / Fax: +202 3635140 Received: 10-09-2016 Abstract A new series of 4-benzyl-6,7,8,9-tetrahydro[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidines was synthesized motivated by the widely reported anticancer activity of thieno[2,3-d]pyrimidines and triazolothienopyrimidines. The in vitro cytotoxic activity of some selected compounds was evaluated against two human cell lines: prostate cancer (PC-3) and colon cancer (HCT-116). A preliminary study of the structure-activity relationship of the target compounds was discussed. Most of the synthesized compounds showed remarkable activity on the tested cell lines, while compound 16c had the highest potency against the PC-3 cell line with an IC50 of 5.48 |M compared to Doxorubicin (IC50 = 7.7 |M), the reference standard used in this study. On the other hand, 6c and 18c were the most active against HCT-116 (IC50 = 6.12 and 6.56 |M, respectively) relative to IC50 = 15.82 |M of the standard. Thus, some of the synthesized thienopyri-midine derivatives, specially 6c, 16c and 18c, have the potential to be developed into potent anticancer agents. Keywords: Thienopyrimidines; 1,2,4-Triazoles; Anticancer activity; PC-3; HCT-116 1. Introduction Despite decades of research that have resulted in an enormous leap in cancer therapy, cancer remains a major cause of death worldwide thus there is a continuous need for the discovery and development of new anticancer agents.1'2 It is worth mentioning that 60% of world's total new annual cases occur in Africa, Asia and Central and South America.3 Thiophenes have been reported to possess interesting biological activities particularly as anticancer agents.45 Many research groups reported the synthesis of biologically active thiophene derivatives through the well-known Gewald reaction.67 As an example, Mohareb et al.s synthesized some thiophene derivatives and investigated their antitumor activity. The prepared compounds exhibited GI50 ranging from 0.02 to 0.08 |M against MCF-7, NCI-H450 and SF-268 cell lines compared to Doxorubicin. Meanwhile, thieno[2,3-d]pyrimidines represent an important class of bioactive heterocycles attracting much attention due to their wide range of biological and pharmaceutical activities.910 The presence of pyrimidine ring in the basic building scaffolds of DNA and RNA modules (thymine, cyto-sine and uracil) is probably the reason of their diverse biological activities.11 In addition, the tricyclic system, cycloalkylthieno[2,3-d]pyrimidine, which is considered to be a bioisostere of quinazoline, has been used as a core for the mechanism-based design and synthesis of a variety of compounds for anticancer therapy.12-16 On the other hand, the 1,2,4-triazole heterocycle is of great value as a building block in the structure of several anticancer drug candidates.11,17,18 Letrozole, Anastro-zole and Ribavirin are representative examples of commercially available anticancer drugs containing triazole scaffolds (Fig. 1).19-21 Among these heterocycles, the mer-capto substituted 1,2,4-triazole ring systems have been well studied and so far a variety of biological activities have reported for them.17,22,23 Recently, 4-amino-1,2,4-triazol-3-thione was used as an intermediate for the synthesis of several biologically active fused heterocyclic compounds where the amino and mercapto groups are appropriate nucleophile centers for many chemical modifications.24 Further, many alkylated Botros et al.: Synthesis, Characterization and Cytotoxicity Acta Chim. Slov. 2017, 64, 102-116 103 Figure 1. Chemical structures of anticancer drugs containing triazole moiety available on the market. Figure 2. Structures of some reported pyrimidines, thieno[2,3-d]pyrimidines and triazole derivatives with cytotoxic activity showing the possible chemical optimization to obtain target compounds A and B Botros et al.: Synthesis, Characterization and Cytotoxicity ... 104 Acta Chim. Slov. 2017, 64, 102-116 mercapto 1,2,4-triazoles linked to various aromatic ring systems either through amide or ester linkages have been reported to exhibit significant antitumor activities.25-27 In the last few years, many research groups investigated thienopyrimidine derivatives fused to 1,2,4-triazole moiety as potential cytotoxic agents.28-30 For example, the fusion of a triazole ring to cycloalkylthieno[2,3-d]pyrimi-dine (VII) showed significant in vitro cytotoxic activity against human colorectal cancer cells (HCT-116) (IC50 = 2.8 |^g/mL) compared to the reference drug Doxorubicin (Fig. 2).31 In our search for new classes of potential anticancer agents, the aforementioned findings prompted us to synthesize a series of 4-benzyl[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-a] pyrimidines with varying the substitution at position 1 (Target compound A) in order to investigate the effect of combining these bioactive moieties on the anticancer activity. Moreover, we aimed in this work to prepare a series of 4-benzyl[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-a] pyrimidines bearing various S-(substituted amino alkyl) moieties at position 1 (Target compound B) to act as cytotoxic agents. In this series, different alkyl linkers and different aliphatic and aromatic amines were used to study the effect of these variations on the cytotoxic activity. Some selected compounds were tested for possible anti-cancer activity against two cell lines (PC-3 and HCT-116). 2. Experimental 2. 1. Chemistry All melting points were determined with Stuart SMP10 apparatus and the values given are uncorrected. IR spectra (KBr, cm-1) were determined on Shimadzu IR 8400s spectrophotometer (Faculty of Pharmacy, Cairo University, Egypt). 1H-NMR and 13C-NMR spectra were recorded on Mercury 300-BB 300 MHz (Microanalytical Center, Faculty of Science, Cairo University, Egypt) and Bruker 400-BB 400 MHz spectrometers (Microanalytical Unit, Faculty of Pharmacy, Cairo University, Egypt) using TMS as the internal standard. Chemical shift values are given in ppm on 5 scale. Mass spectra were recorded on Hewlett Packard 5988 spectrophotometer (Microanalyti-cal Center, Faculty of Science, Cairo University, Egypt). Elemental analyses were carried out at the Regional center for Mycology and Biotechnology, Faculty of Pharmacy, Al Azhar University, Egypt; values found were within ±0.35% of the theoretical ones. Progress of the reactions was monitored by TLC using aluminum sheets precoated with UV fluorescent silica gel (Merck 60F 254) and visualized using UV lamp. The solvent system used was chloroform : benzene : methanol [9:5:2]. The starting compounds, ethyl 2-amino-4,5,6,7-te-trahydro[1]benzothiophene-3-carboxylate (1),32 3-benzyl-2-sulphanyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyri- midin-4(3#)-one (2)33 and the a- and P-chloroamides (13a-d, 14a-d, 15a-d)34-40 were prepared according to reported procedures. 2. 1. 1. 3-Benzyl-2-hydrazino-5,6,7,8-tetrahydro [1]benzothieno[2,3-rf]pyrimidin-4(3H)- one (3) A mixture of 3-benzyl-2-sulphanyl-5,6,7,8-tetrahy-dro[ 1 ]benzothieno[2,3-d]pyrimidin-4(3#)-one (2) (1.64 g, 50 mmol) and hydrazine hydrate 99-100% (7 mL, 140 mmol) in dry pyridine (25 mL) was heated under reflux for 25 h. The mixture was evaporated under reduced pressure and the residue was treated with ethanol. The solid product was collected by filtration, washed with ethanol, dried and crystallized from ethyl acetate. Yield: 50%; mp: 226-228 °C; IR (KBr, cm-1): 3248-3211 (NH, NH2), 3061-3035 (CH aromatic), 2916, 2848 (CH aliphatic), 1666 (C=O), 1624, 1568, 1529 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.75-1.78 (m, 4H, 2 x CH2 at C-6, C-7), 2.62-2.764 (m, 2H, CH2 at C-5), 2.79-2.81(m, 2H, CH2 at C-8), 5.21 (s, 2H, NCH2C6H5), 6.98 (s, 1H, NH, D2O exchangeable), 7.15-7.34 (m, 5H, Ar-H); EI-MS m/z 326 (M+, 26.29%); Anal. Calcd for C17H18N4OS (326.42): C, 62.55; H, 5.56; N, 17.16. Found: C, 62.74; H, 5.64; N, 17.38. 2. 1. 2. 4-Benzyl-6,7,8,9-tetrahydro[1]benzothieno [3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5 (4#)-one (4a) A mixture of 3-benzyl-2-hydrazino-5,6,7,8-tetrahy-dro[ 1 ]benzothieno[2,3-d]pyrimidin-4(3#)-one (3) (0.32 g, 1 mmol) and formic acid (5 mL, 130 mmol) was heated under reflux for 4 h. The white precipitate formed upon cooling was collected by filtration, washed with water, dried and crystallized from ethyl acetate. Yield: 78%; mp: 198-200 °C; IR (KBr, cm-1): 3115 (CH aromatic), 2922, 2850 (CH aliphatic), 1670 (C=O), 1595, 1552, 1517 (C=C aromatic); 1H-NMR (CDCl3-d6) 5: 1.81-1.92 (m, 4H, 2 x CH2 at C-7, C-8), 2.76-2.79 (m, 2H, CH2 at C-6), 3.02-3.06 (m, 2H, CH2 at C-9), 5.48 (s, 2H, NCH2C6H5), 7.26-7.67 (m, 5H, Ar-H), 8.37 (s, 1H, aromatic CH); EI-MS m/z 336 (M+, 35.04), 91 ([C7H7]+, 100%); Anal. Calcd for C18H16N4OS (336.4): C, 64.26; H, 4.79; N, 16.65. Found: C, 64.-42; H, 4.86; N, 16.90. 2. 1. 3. 4-Benzyl-1-methyl-6,7,8,9-tetrahydro [1]benzothieno[3,2-e][1,2,4]triazolo[4,3-a] pyrimidin-5(4.ff)-one (4b) A mixture of 3 (0.32 g, 1 mmol) and acetic acid (10 mL, 70 mmol) was heated under reflux for 6 h. The reaction mixture was poured onto ice cold water (25 mL). The white precipitate formed was collected by filtration, washed with water, dried and crystallized from acetonitrile. Botros et al.: Synthesis, Characterization and Cytotoxicity ... Acta Chim. Slov. 2017, 64, 102-116 105 Yield: 85%; mp: 242-244 °C; IR (KBr, cm-1): 3061, 3043 (CH aromatic), 2937, 2870 (CH aliphatic), 1664 (C=O), 1593, 1558, 1541 (C=C aromatic); 1H-NMR (CDCl3-d6) 5: 1.83-1.91 (m, 4H, 2 x CH2 at C-7, C-8), 2.75 (s, 3H, CH3), 2.77-2.78 (m, 2H, CH2 at C-6), 3.04-3.07 (m, 2H, CH2 at C-9), 5.44 (s, 2H, NCH2C6H5), 7.22-7.66 (m, 5H, Ar-H); 13C-NMR (CDCl3-d6) 52 12.07, 22.06, 22.97, 24.92, 25.63, 45.94, 118.29, 128.15, 128.63, 129.73, 130.91, 134.06, 136.12, 138.67, 144.07, 149.09, 156.29; EI-MS m/z 350 (M+, 64.90), 91 ([C7H7]+, 100%); Anal. Calcd for C19H18N4OS (350.42): C, 65.12; H, 5.18; N, 15.99. Found: C, 65.38; H, 5.29; N, 16.31. 2. 1. 4. 1-Chloromethyl-4-benzyl-6,7,8,9-tetrahy-dro[1]benzothieno[3,2-e][1,2,4]triazolo [4,3-a] pyrimidin-5(4H)-one (5) To a solution of 3 (1 g, 3 mmol) in dry DMF (10 mL), chloroacetyl chloride (1.5 mL, 20 mmol) was added dropwise with cooling. The solution was then heated under reflux in a boiling water bath for 9 h. After cooling, the reaction mixture was poured onto ice-cold water and the suspension formed was stirred at room temperature for 2 h. The separated solid was collected by filtration, washed with cold water, dried and crystallized from methanol. Yield: 88%; mp: 188-190 °C; IR (KBr, cm-1): 3080, 3040 (CH aromatic), 2939, 2852 (CH aliphatic), 1681 (C=O), 1622, 1591, 1550 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.74-1.80 (m, 4H, 2 x CH2 at C-7, C-8), 2.76-2.80 (m, 2H, CH2 at C-6), 2.82-2.87 (m, 2H, CH2 at C-9), 5.11 (s, 2H, CH2Cl), 5.29 (s, 2H, NCH2C6H5), 7.15-7.34 (m, 5H, Ar-H); EI-MS m/z 386 (M+2, 3.9); 384 (M+, 16.95%); Anal. Calcd for C19H17ClN4OS (384.87): C, 59.29; H, 4.45; N, 14.56. Found: C, 59.41; H, 4.52; N, 14.71. 2. 1. 5. General procedure for the preparation of compounds 6a-d A mixture of 5 (0.25 g, 0.6 mmol) and the appropriate N-substituted piperazine (4 mmol) in absolute etha-nol (30 mL) was heated under reflux for 6 h. The product obtained was collected by filtration, washed with water and crystallized from the suitable solvent. 4-Benzyl-1-[[4-methylpiperazin-1-yl]methyl]-6,7,8,9-tetrahydro[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4ff)-one (6a). Crystallized from aqueous ethanol; yield: 36%; mp: 172-174 °C; IR (KBr, cm-1): 3040, 3020 (CH aromatic), 2922, 2850 (CH aliphatic), 1677 (C=O), 1591 (C=C aromatic); 1H-NMR (CDCl3-d6) 5: 1.86-1.92 (m, 4H, 2 x CH2 at C-7, C-8), 2.27 (s, 3H, CH3), 2.43-2.50 (m, 4H, 2 x CH2 piperazine), 2.63-2.70 (m,3 2H, CH2 at C-6), 2.78-2.812 (m, 2H, CH2 at C-9), 3.06-3.10 (m, 4H, 2 x CH2piperazine), 3.88 (s, 2H, CH2), 5.46 (s, 2H, NCH2C6H5), 7.28-7.69 (m, 5H, Ar-H); EI- MS m/z 448 (M+, 0.57), 91 ([C7H7]+, 100%); Anal. Calcd for C24H28N6OS (448.56): C, 64.26; H, 6.29; N, 18.73. Found: C, 646.38; H, 6.37; N, 18.56. 4-Benzyl-1-[[4-phenylpiperazin-1-yl]methyl]-6,7,8,9-tetrahydro[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4ff)-one (6b). Crystallized from ethyl acetate; yield: 48%; mp: 228-230 °C; IR (KBr, cm-1): 3057, 3032 (CH aromatic), 2941, 2918, 2848, 2821 (CH aliphatic), 1672 (C=O), 1587, 1558, 1539 (C=C aromatic); 1H-NMR (CDCl3-d6) 5: 1.85-1.87 (m, 4H, 2 x CH2 at C-7, C-8), 2.77-2.80 (m, 6H, CH2 at C-6 and 2 x CH2 piperazine), 3.05-3.10 (m, 2H, CH2 at C-9), 3.18-3.19 (m, 4H, 2 x CH2 piperazine), 3.95 (s, 2H, CH2), 5.47 (s, 2H, N-CH2-C6H5), 6.83-7.70 (m, 10H, Ar-H); EI-MS m/z 511 (M+1, 4.13), 510 (M+, 6.27%); Anal. Calcd for C29H30N6OS (510.63): C, 68.21; H, 5.92; N, 16.46. Found: C, 68.44; H, 5.98; N, 16.82. 4-Benzyl-1- [[4-(4-chlorophenyl)piperazin-1-yl]methyl] -6,7,8,9-tetrahydro[1]benzothieno[3,2-e][1,2,4]triazo-lo[4,3-a]pyrimidin-5(4ff)-one (6c). Crystallized from ethyl acetate; yield: 51%; mp: 254-256 °C; IR (KBr, cm-1): 3100, 3040 (CH aromatic), 2929, 2819 (CH aliphatic), 1670 (C=O), 1581, 1550, 1510 (C=C aromatic); 1H-NMR (CDCl3-d6) 5: 1.86-1.88 (m, 4H, 2 x CH2 at C-7, C-8), 2.77-2.80 (m, 6H, CH2 at C-6 and 2 x CH2 piperazine), 3.05-3.10 (m, 2H, CH2 at C-9), 3.12-3.13 (m, 4H, 2 x CH2 piperazine), 3.96 (s, 2H, CH2) 5.47 (s, 2H, N-CH2-C6H5), 6.87-7.70 (m, 9H, Ar-H); EI-MS m/z 546 (M+2, 32.02), 544 (M+, 37.08%); Anal. Calcd for C29H29ClN6OS (545.08): C, 63.90; H, 5.36; N, 15.42. Found2 C, 654.07; H, 5.44; N, 15.67. 4-Benzyl-1-[[4-(4-methoxymphenyl)piperazin-1-yl] methyl]-6,7,8,9-tetrahydro[1]benzothieno[3,2-e][1,2,4] triazolo[4,3-a]pyrimidin-5(4ff)-one (6d). Crystallized from acetonitrile; yield: 37%; mp: 238-240 °C; IR (KBr, cm-1): 3040, 3000 (CH aromatic), 2926, 2808 (CH aliphatic), 1681 (C=O), 1591, 1556, 1535 (C=C aromatic); 1H-NMR (CDCl3-d6) 5: 1.85-1.87 (m, 4H, 2 x CH2 at C-7, C-8), 2.75-23.786 (m, 6H, CH2 at C-6 and 2 x CH2 piperazine), 3.05-3.09 (m, 6H, CH2 at C-9 and 2 x CH2 pi-perazine), 3.75 (s, 3H, OCH3), 3.294 (s, 2H, CH2) 5.472 (s, 2H, N-CH2-C6H5), 6.83-7.70 (m, 9H, Ar-H); EI-MS m/z 541 (M+1," 5.84), 540 (M+, 15.74), 91 ([C7H7]+, 100%); Anal. Calcd for C30H32N6O2S (540.68): C, 66.674; H, 5.97; N, 15.54. Found: C, 66.88; H, 6.05; N, 15.66. 2. 1. 6. 4-Benzyl-6,7,8,9-tetrahydro[1]benzothie-no[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-1,5(2H,4#)-dione (7) A mixture of 3 (0.64 g, 2 mmol) and N,N-car-bonyldiimidazole (CDI) (0.7 g, 4.3 mmol) in dry benzene (30 mL) was heated under reflux for 15 h. After cooling, Botros et al.: Synthesis, Characterization and Cytotoxicity ... 106 Acta Chim. Slov. 2017, 64, 102-116 the solvent was evaporated under reduced pressure and the residue was triturated with cold water. The solid product was collected by filtration, dried and crystallized from acetonitrile. Yield: 81%; mp: 306-308 °C; IR (KBr, cm-1): 3170 (NH), 3055, 3034 (CH aromatic), 2933, 2852 (CH aliphatic), 1720, 1683 (2 x C=O), 1610, 1560, 1523 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.78-1.80 (m, 4H, 2 x CH2 at C-7, C-8), 2.73-26.80 (m, 2H, CH2 at C-6), 2.81-2.84 (m, 2H, CH2 at C-9), 5.05 (s, 2H, NCH2C6H5), 7.28-7.35 (m, 5H, Ar-H), 12.0 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 22.03, 22.86, 24.47, 25.22, 43.86, 115.40, 127.84, 128.14, 128.80, 130.05, 131.47, 136.45, 138.97, 141.21, 149.17, 156.63; EI-MS m/z 352 (M+, 32.62), 91 ([C7H7]+, 100%); Anal. Calcd for C18H16N4O2S (352.41): C, 61.35; H, 4.58; N, 15.90. Found: C, 61.54; H, 4.65; N, 15.88. 2. 1. 7. General procedure for the preparation of compounds 8a-e A mixture of 3 (0.32 g, 1 mmol) and the appropriate isothiocyanate (2 mmol) in absolute ethanol (30 mL) was heated under reflux for 8 h. The precipitated product was collected by filtration, dried and crystallized from etha-nol/CHCl3 (2:1). 4-Benzyl-1-methylamino-6,7,8,9-tetrahydro[1]benzot-hieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4_ff)-one (8a). Yield: 43%; mp: 206-208 °C; IR (KBr, cm-1): 3370, 3196 (NH), 2944, 2880 (CH aliphatic), 1681 (C=O), 1575, 1537, 1506 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.74-1.80 (m, 4H, 2 x CH2 at C-7, C-8), 2.62-2.65 (m, 2H, CH2 at C-6), 2.78-2.81 (m2, 2H, CH2 at C-9), 2.81 (s, 3H, CH3), 5.22 (s, 2H, NCH2C6H5), 7.19-7.38 (m, 5H, Ar-H), 9.293 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 22.34, 23.10, 24.76, 25.67, 31.21, 43.31, 115.03, 127.09, 127.56, 127.66, 128.86, 130.82, 136.52, 138, 151.17, 158.15, 164.13; EI-MS m/z 365 (M+, 68.95), 91 ([C7H7]+, 97.00%); Anal. Calcd for C19H19N5OS (365.45): C, (52.44; H, 5.24; N, 19.16. Found: C, 62.61; H, 5.30; N, 19.34. 4-Benzyl-1-ethylamino-6,7,8,9-tetrahydro[1]benzot-hieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4_ff)-one (8b). Yield: 46%; mp: 200-202 °C; IR (KBr, cm-1): 3358, 3257, 3169 (NH), 2972, 2848 (CH aliphatic), 1681 (C=O), 1571, 1535, 1506 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 0.96 (t, J = 7.2 Hz, 3H, CH3), 1.76-1.79 (m, 4H, 2 x CH2 at C-7, C-8), 2.65-2.70 (m, 2H, CH2 at C-6), 2.80-2.85 (m, 2H, CH2 at C-9), 3.39 (q, J = 7.2 Hz, 2H, CH2-CH3), 5.26 (s, 2H, NCH2C6H5), 7.19-7.36 (m, 5H, Ar2H), 9.30 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 14.28, 21.80, 22.57, 24.24, 25.14, 38.20, 42.64, 126.56, 127.03, 128.35, 130, 130.32, 133.61, 136, 150.52, 157.64, 163.8; EI-MS m/z 379 (M+, 100), 91 ([C7H7]+, 97.00%). 4-Benzyl-1-butylamino-6,7,8,9-tetrahydro[1]benzot-hieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4_ff)-one (8c). Yield: 30%; mp: 172-174 °C; IR (KBr, cm-1): 3360, 3178 (NH), 2924, 2850 (CH aliphatic), 1685 (C=O), 1535, 1454 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 0.76 (t, J = 14.7 Hz, 3H, CH3), 1.18-1.23 (m, 2H, CH2-CH3), 1.31-1.36 (m, 2H, C^-CH^CHA 1.75-1.80 (m, 4H, 2 x CH2 at C-7, C-8), 2.61-2.65 (m, 2H, CH2 at C-6), 2.752-2.77 (m, 2H, CH2 at C-9), 3.36 (t, J = 12.6 Hz, 2H, NH-CH2), 5.19 (s, 2H, NCH2C6H5), 7.18-7.38 (m, 5H, Ar-H), 99.20 (s, 1H, NH, D2O"exchangeable); EI-MS m/z 407 (M+, 1.32), 91 ([C7H7]+, 100%); Anal. Calcd for C^H^NjOS (407.53): C, 64.84; H, 6.18; N, 17.18. Found: C, 65.01; H, 6.22; N, 17.39. 4-Benzyl-1-allylamino-6,7,8,9-tetrahydro[1]benzothie-no[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4_ff)-one (8d). Yield: 38%; mp: 184-186 °C; IR (KBr, cm-1): 3360, 3167 (NH), 2924, 2850 (CH aliphatic), 1685 (C=O), 1651 (C=N), 1531, 1454 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.75-1.80 (m, 4H, 2 x CH2 at C-7, C-8), 2.64-2.70 (m, 2H, CH2 at C-6), 2.79-2.282 (m, 2H, CH2 at C-9), 4.00-4.028 (m, 2H, CH2 allylic), 4.98-5.012 (m, 1H, CH2=CH), 5.11-5.14 (m, 1H, CH2=CH), 5.24 (s, 2H, NCH2C6H5), 5.69-5.74 (m,1H, CH2=CH), 7.18-7.33 (m, 5H, Ar-H), 9.40 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 21.82, 22.59, 24.23, 25.16, 42.73, 45.50, 115, 1176, 126.59, 127, 128.31, 130.32, 131, 134.54, 136.4, 138.6, 151, 158.2, 164.1; EI-MS m/z 391 (M+, 2.27), 91 ([C7H7]+, 100%); Anal. Calcd for C^H^NjOS (391.49): C, 64.43; H, 5.41; N, 17.89. Found: C, 64.67; H, 5.48; N, 18.04. 4-Benzyl-1-[(4-methoxyphenyl)amino]-6,7,8,9-te-trahydro[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-a] pyrim idin-5(4tf)-one (8e). Yield: 65%; mp: 244-246 °C; IR (KBr, cm-1): 3215 (NH), 3111, 3070 (CH aromatic), 2941, 2835 (CH aliphatic), 1683 (C=O), 1618 (C=N), 1543, 1512, 1487 (C= C aromatic); 1H-NMR (DMSO-d6) 5: 1.77-1.79 (m, 4H, 2 x CH2 at C-7, C-8), 2.72-2.79 (m, 2H, CH2 at C-6), 2.80-2.86 (m, 2H, CH2 at C-9), 3.74 (s, 3H, OCH3), 5.14 (s, 2H, NCH2C6H5), 6^7-7.37 (m, 9H, Ar-H), 9.339 (s, 1H, NH, D2O exchangeable); EI-MS m/z 457 (M+, 0.75), 91 ([C7H7]+, 100%); Anal. Calcd for C^H^N^S (457.55): C, 65.63; H, 5.07; N, 15.31. Found: C, 65.79; H, 5.12; N, 15.47. 2. 1. 8. Ethyl(4-benzyl-5-oxo-4,5-dihydro-6,7,8,9-tetrahydro[1jbenzothieno[3,2-e][1,2,4] triazolo[4,3-a] pyrimidin-1-yl) acetate (9) A mixture of 3 (0.32 g, 1 mmol) and diethyl malona-te (2 mL, 13 mmol) was refluxed for 9 h. The reaction was allowed to cool, the formed residue was triturated with et-hanol, collected by filtration, dried and crystallized from isopropanol to yield the title compound 9. Botros et al.: Synthesis, Characterization and Cytotoxicity ... Acta Chim. Slov. 2017, 64, 102-116 107 Yield: 41%; mp: 220-222 °C; IR (KBr, cm-1): 3035, 3055 (CH aromatic), 2935, 2854 (CH aliphatic), 1732, 1678 (2 x C=O), 1635 (C=N), 1589, 1539, 1504 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.18 (t, J = 7.2 Hz, 3H, CH3), 1.78-1.82 (m, 4H, 2 x CH2 at C-7, C-8), 2.63 (s, 2H, CH2-CO), 2.77-2.81 (m, 2H, CH2 at C-6), 2.91-2.98 (m, 2H, CH2 at C-9), 4.16 (q, J = 7.2 Hz, 2H, CH2-CH3), 5.28 (s, 2H, NCH2C6H5), 7.28-7.37 (m, 5H, Ar-H); 13C-NMR (DMSO-d6) 5: 14.45, 21.92, 22.77, 24.55, 25.52, 32.31, 45.51, 62.0, 117.61, 127.99, 128.39, 128.86, 131.32, 132.78, 136.59, 138.98, 142.06, 149.17, 155.17, 168.17; EI-MS m/z 422 (M+, 60.13), 91 ([C7H7]+, 100%); Anal. Calcd for C22H22N4O3S (422.46): C, (52.54; H, 5.25; N, 13.26. Found: C, 62.771; H, 5.34; N, 13.48. 2. 1. 9. 4-Benzoyl-1-(3-benzyl-5,6,7,8-tetrahydro-4-oxo-3,4-dihydro[1]benzothieno[2,3-d] pyrimidin-2-yl)thiosemicarbazide (10) To an ice cold solution of ammonium thiocyanate (0.17 g, 2 mmol) in dry acetone (5 mL), a solution of benzoyl chloride (0.3 mL, 2 mmol) in acetone (5 mL) was added dropwise. An ice-cold suspension of 3 (0.34 g, 1 mmol) in acetone (15 mL) was added to the previous mixture. The reaction mixture was heated on a water-bath for 15 h. The reaction mixture was cooled and filtered. The filtrate was evaporated and the obtained product was crystallized from ethanol/CHCl3 (2:1). Yield: 33%; mp: 114-116 °C; IR (KBr, cm-1): 3346, 3159 (NH), 3057, 3030 (CH aromatic), 2927, 2856 (CH aliphatic), 1687, 1674 (2 x C=O), 1622 (C=N), 1598, 1581, 1539 (C=C aromatic); 1H-NMR (DMSO-d6) 5: I.76-1.80 (m, 4H, 2 x CH2 at C-7, C-8), 2.65-2.73 (m, 2H, CH2 at C-6), 2.81-2.84 (m, 2H, CH2 at C-9), 5.31 (s, 2H, NCH2C6H5), 7.27-7.91 (m, 10H, Ar-H), 9.79, 11.71, 12.49 (s, 3H, NH, D2O exchangeable); EI-MS m/z 489 (M+, 2.60), 91 ([C7H7]+, 100%); Anal. Calcd for C25H23N5O2S2 (489.61): C, 61.33; H, 4.73; N, 14.30. Found: C, 61.49; H, 4.79; N, 14.51. 2. 1. 10. 4-Benzyl-1-sulphanyl-6,7,8,9-tetrahydro [1]benzothieno[3,2-e][1,2,4]triazolo[4,3-a] pyrimidin-5(4.ff)-one (11) A mixture of 3 (1.4 g, 4.3 mmol), KOH (0.42 g, 7.5 mmol) and CS2 (4.5 mL, 7.5 mmol) in absolute ethanol (70 mL) was heated under reflux for 25 h. The solvent was evaporated under reduced pressure. The obtained residue was dissolved in H2O (20 mL) followed by acidification with dilute HCl (1 mL). The precipitated product was collected by filtration, dried and crystallized from methanol. Yield: 50%; mp: 274-276 °C; IR (KBr, cm-1): 3446 (NH), 3182, 3134 (CH aromatic), 2947, 2852 (CH aliphatic), 1662 (C=O), 1618 (C=N), 1585, 1516, 1489 (C=C aromatic), 1159 (C=S); 1H-NMR (DMSO-d6) 5: 1.77-1.82 (m, 4H, 2 x CH2 at C-7, C-8), 2.77-2.80 (m6, 2H, CH2 at C-6), 2.89-2.92 (m, 2H, CH2 at C-9), 5.14 (s, 2H, NCH2C6H5), 7.26-7.38 (m, 5H, Ar-H), 14.06 (s, 1H, SH, D2O exchangeable); EI-MS m/z 368 (M+, 30.43), 91 ([C^f, 100%); Anal. Calcd for C18H16N4OS2 (368.48): C, 58.67; H, 4.38; N, 15.21. Found: C, 58.92; H, 4.41; N, 15.42. 2. 1. ii.General procedure for the alkylation of the thienotriazolopyrimidine 11 yielding 12a,b, 16a-d, 17a-d, 18a-d A mixture of the triazolo derivative 11 (0.36 g, 1 mmol) and the appropriate alkyl iodide or a- and P-chlo-roamides (13a-d, 14a-d, 15a-d) (1.5 mmol) in the presence of anhydrous sodium acetate (5 mmol) in absolute ethanol (70 mL) was heated under reflux till TLC indicated completion of the reaction. The product precipitated was collected by filtration, dried and crystallized from the appropriate solvent. 4-Benzyl-1-methylsulphanyl-6,7,8,9-tetrahydro[1]ben-zothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4_ff)-one (12a). Reaction time: 12 h, crystallized from ethanol, yield: 43%; mp: 246-248 °C; IR (KBr, cm-1): 2916, 2846 (CH aliphatic), 1674 (C=O), 1585, 1546, 1508 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.76-1.83 (m, 4H, 2 x CH2 at C-7, C-8), 2.62 (s, 3H, CH3), 2.79-2.80 (m, 2H, CH22 at C-6), 2.92-2.93 (m, 2H, CH32 at C-9), 5.31 (s, 2H, NCH2C6H5), 7.24-7.42 (m, 5H, Ar-H); EI-MS m/z 382 (M+, "24.5), 91 ([C7H7]+, 81.15%); Anal. Calcd for C19H18N4OS2 (382.5): C, 59.66; H, 4.74; N, 14.65. Found: C, 59.89; H, 4.79; N, 14.91. 4-Benzyl-1-ethylsulphanyl-6,7,8,9-tetrahydro[1]ben-zothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4_ff)-one (12b). Reaction time: 15 h, crystallized from ethanol, yield: 46%; mp: 210-212 °C; IR (KBr, cm-1): 2935, 2854 (CH aliphatic), 1670 (C=O), 1585, 1550, 1508 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.27 (t, 3H, CH3), 1.75-1.82 (m, 4H, 2 x CH2 at C-7, C-8), 2.77-2.81 (m, 2H, CH2 at C-6), 2.90-2.94 (m, 2H, CH2 at C-9), 3.05 (q, 2H, CH2-CH3), 5.31 (s, 2H, NCH2C6H5), 7.26-7.43 (m, 5H, Ar-H); EI-MS m/z 396 (M+, 39.74), 91 ([C7H7]+, 100%); Anal. Calcd for C50H50N4OS5 (396.53): C, (50.58; H, 5.08; N, 14.13. Found: C, 60.85; H, 5.14; N, 14.28. N-(4-chlorophenyl)-2-[(4-benzyl-6,7,8,9-tetrahydro-5-oxo-4,5-dihydro[1]benzothieno[3,2-e][1,2,4]triazolo [4,3-a]pyrimidin-1-yl)sulfanyl]acetamide (16a). Reaction time: 9.30 h, crystallized from ethyl acetate/ethanol, yield: 57%; mp: 238-240 °C; IR (KBr, cm-1): 3259 (NH), 3190, 3064 (CH aromatic), 2941, 2858 (CH aliphatic), 1685 (br. 2 x C=O), 1591, 1548, 1506 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.76-1.80 (m, 4H, 2 x CH2 at C-7, C-8), 2.65-2.70 6(m, 2H, CH2 at C-6), 2.88-2.920 (m, 2H, CH2 at C-9), 3.84 (s, 2H, SCH2), 5.32 (s, 2H, NCI LC I L), 7.28-7.42 (m, 9H, Ar-H), 10.15 (s, 1H, NH, Botros et al.: Synthesis, Characterization and Cytotoxicity ... 108 Acta Chim. Slov. 2017, 64, 102-116 D2O exchangeable); EI-MS m/z 537 (M+2, 11.44), 535 (M+, 25.2), 91 ([C7H7]+, 100%); Anal. Calcd for C26H22Cl-N5O2S2 (536.07): C, 58.25; H, 4.14; N, 13.06. Found: C, 585.44; H, 4.11; N, 13.21. 4-Benzyl-1-{[2-morpholino-2-oxoethyl]sulphanyl}-6,7,8,9-tetrahydro[1]benzothieno[3,2-e][1,2,4]triazolo [4,3-a]pyrimidin-5(4ff)-one (16b). Reaction time: 5.30 h, crystallized from acetonitrile, yield: 73%; mp: 256-258 °C; IR (KBr, cm-1): 3020, 3000 (CH aromatic), 2966, 2870 (CH aliphatic), 1670, 1633 (2 x C=O), 1585, 1550, 1508 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.78-1.80 (m, 4H, 2 x CH2 at C-7, C-8), 2.79-2.81 (m, 2H, CH2 at C-6), 2.92-2.98 (m, 2H, CH2 at C-9), 3.39 (t, J = 9.9 Hz, 4H, CH2-N), 3.53 (t, J = 9.9 Hz, 4H, CH2-O), 4.18 (s, 2H, SCH2), 5.32 (s, 2H, NCIKC.IL), 7.26-7.42 (m, 5H, Ar-H); EI-MS m/z 495 (M+, 5725), 91 ([C7H7]+, 100%); Anal. Calcd for C24H25N5O3S2 (495.62): C, 58.16; H, 5.08; N, 14.13. Found: C, 58.42; H, 5.17; N, 14.29. 4-Benzyl-1-{[2-(4-phenylpiperazin-1-yl)-2-oxoethyl] sulphanyl}-6,7,8,9-tetrahydro[1]benzothieno[3,2-e] [1,2,4]triazolo[4,3-a]pyrimidin-5(4ff)-one (16c). Reaction time: 5.30 h, crystallized from acetonitrile, yield: 65%; mp: 224-226 °C; IR (KBr, cm-1): 3040, 3000 (CH aromatic), 2918, 2812 (CH aliphatic), 1672, 1635 (C=O), 1585, 1550, 1508 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.78-1.81 (m, 4H, 2 x CH2 at C-7, C-8), 2.76-2.80 (m, 2H, CH2 at C-6), 2.91-2.98 (m, 2H, CH2 at C-9), 3.09-3.14 (m, 4H, 2 x CH2 piperazine), 3.55-3.60 (m, 4H, 2 x CH2 piperazine), 4.23 (s, 2H, SCH2), 5.32 (s, 2H, NCH2C6H5), 6.80-7.42 (m, 10H, Ar-H); "EI-MS m/z 570 (M+, 21.19), 91 ([C7H7]+, 100%); Anal. Calcd for C30H30N6O2S2 (570.73): C, 63.13; H, 5.30; N, 14.73. Found: C, 63.40; H, 5.36; N, 14.89. 4-Benzyl-1-{[2-[4-(4-methoxyphenyl)piperazin-1-yl]-2-oxoethyl]sulphanyl}-6,7,8,9-tetrahydro[1]benzothie-no[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4_ff)-one (16d). Reaction time: 6.30 h, crystallized from ethyl acetate, yield: 83%; mp: 230-232 °C; IR (KBr, cm-1): 3040, 3000 (CH aromatic), 2941, 2818 (CH aliphatic), 1672, 1635 (2 x C=O), 1585, 1548, 1510 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.77-1.79 (m, 4H, 2 x CH2 at C-7, C-8), 2.76-2.80 (m, 2H, CH2 at C-6), 2.91-2.99 (m, 6H, CH2 at C-9 and 2 x CH2 pip2erazine), 3.53-3.59 (m, 4H, 2 x CH2 piperazine), 3.68 (s, 3H, OCH3), 4.22 (s, 2H, SCH2), 5.322 (s, 2H, NCH^Hj), 6.80-7.42 (m, 9H, Ar-H); EI-MS m/z 600 (M+,~2.79), 232 (M-C18H16N4OS2, 100%); Anal. Calcd for C31H32N6O3S2 (600.76): C, 61.98; H, 5.37; N, 13.99. Found: C, (52.17; H, 5.46; N, 14.12. N-(4-chlorophenyl)-2-methyl-2-[(4-benzyl-6,7,8,9-te-trahydro-5-oxo-4,5-dihydro[1]benzothieno[3,2-e] [1,2,4]triazolo[4,3-a]pyrimidin-1-yl)sulphanyl]aceta-mide (17a). Reaction time: 9.30 h, crystallized from chlo- roform, yield: 60%; mp: 264-266 °C; IR (KBr, cm-1): 3305, 3261, 3194 (NH), 3066 (CH aromatic), 2945, 2858 (CH aliphatic), 1681 (br. 2 x C=O), 1610, 1589, 1548 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.48 (d, J = 6.6 Hz, 3H, CH3), 1.71-1.79 (m, 4H, 2 x CH2 at C-7, C-8), 2.60-2.62 (m, 2H, CH2 at C-6), 2.83-2.85 (m, 2H, CH2 at C-9), 4.16 (q, J = 6.6 Hz, 1H, CH), 5.34 (s, 2H, NCHXUU, 7.23-7.42 (m, 9H, Ar-H), 10.03 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 17.09, 21.43, 22.14, 23.8, 24.68, 44.89, 47.15, 1177.43, 120.46, 126.83, 127.44, 127.84, 128.23, 128.31, 131.1, 131.97, 135.97, 137.67, 137.98, 138.53, 149.62, 155.47, 168.49; EI-MS m/z 551(M+2, 14.86), 549 (M+, 19.21), 91 ([C7H7]+, 100%); Anal. Calcd for C27H24ClN5O2S2 (550.10): C, 58.95; H, 4.40; N, 12.73. Found: C, 59.17; H, 4.48; N, 12.85. 4-Benzyl-1-{[2-morpholino-1-methyl-2-oxoethyl]sulp-hanyl}-6,7,8,9-tetrahydro[1 ]benzothieno[3,2-e ] [1,2,4]triazolo[4,3-a]pyrimidin-5(4fl)-one (17b). Reaction time: 5 h, crystallized from ethyl acetate, yield: 65%; mp: 260-262 °C; IR (KBr, cm-1): 2943, 2860 (CH aliphatic), 1670, 1635 (2 x C=O), 1587, 1550, 1508 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.45 (d, J = 6 Hz, 3H, CH3), 1.80-1.85 (m, 4H, 2 x CH2 at C-7, C-8), 2.78-2.79 (m, 2H, CH2 at C-6), 2.91-2.95 (m, 2H, CH2 at C-9), 3.46 (t, J = 11 Hz, 4H, CH2-N), 3.52 (t, J = 11 Hz, 4H, CH2-O), 4.56 (q, J = 62 Hz, 1H, CH), 5.33 (s, 2H, NCH^CgHj), 7.26-7.43 (m, 5H, Ar-H); EI-MS m/z 509 (M+, ^^X 91 ([C7H7]+, 100%); Anal. Calcd for C25H27N5O3S2 (509.65): C, 58.92; H, 5.34; N, 13.74. Found: C, 59.13; H, 5.41; N, 13.87. 4-Benzyl-1-{[2-(4-phenylpiperazin-1-yl)-1-methyl-2-oxoethyl]sulphanyl}-6,7,8,9-tetrahydro[1]benzothieno [3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4ff)-one (17c). Reaction time: 7 h, crystallized from acetonitrile, yield: 63%; mp: 240-242 °C; IR (KBr, cm-1): 3020, 3000 (CH aromatic), 2931, 2820 (CH aliphatic), 1670, 1629 (2 x C=O), 1598, 1583, 1548 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.48 (d, J = 6.6 Hz, 3H, CH3), 1.73-1.78 (m, 4H, 2 6x CH2 at C-7, C-8), 2.89-2.92 (m3, 2H, CH2 at C-6), 3.02-3.08 (m, 2H, CH2 at C-9), 3.14-3.20 (m, 4H, 2 x CH2 piperazine), 3.59-3.70 (m, 4H, 2 x CH2 piperazine), 4.61 (q, J = 6.6 Hz, 1H, CH), 5.33 (s, 2H, NCUXMl-), 6.78-7.43 (m, 10H, Ar-H); EI-MS m/z 584 (M+, 2.10), 216 (M-C18H16N4OS2, 100), 91 ([C7H7]+, 72.31%); Anal. Calcd for C31H32N6O2S2 (584.765): C, 63.67; H, 5.52; N, 14.37. Found: C, 63.81; H, 5.58; N, 14.59. 4-Benzyl-1-{[2-[4-(4-methoxyphenyl)piperazin-1-yl]-1-methyl-2-oxoethyl]sulphanyl}6,7,8,9-tetrahydro [1]benzothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4ff)-one (17d). Reaction time: 7 h, crystallized from acetonitrile, yield: 83%; mp: 236-238 °C; IR (KBr, cm-1): Botros et al.: Synthesis, Characterization and Cytotoxicity ... Acta Chim. Slov. 2017, 64, 102-116 109 2933, 2816 (CH aliphatic), 1670, 1629 (2 x C=O), 1585, 1548, 1510 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.47 (d, J = 7.2 Hz, 3H, CH3), 1.74-1.79 (m, 4H, 2 x CH2 at C-7, C-8), 2.65-2.67 (m, 4H, 2 x CH2 piperazine), 2.89-2.91 (m, 2H, CH2 at C-6), 2.97-3.01 (m, 2H, CH2 at C-9), 3.49-3.58 (m, 4H, 2 x CH2 piperazine), 3.68 (s, 3H, OCH3), 4.60 (q, J = 7.2 Hz, 1H, CH), 5.33 (s, 2H, NCH2C6H5), 6.80-7.43 (m, 9H, Ar-H); 13C-NMR (DM-SO-d6) 5: 19.35, 21.45, 22.18, 23.96, 24.78, 38.66, 45.27, 49.60, 50.04, 55.14, 114.22, 117.0, 120.50, 127.45, 127.95, 128.31, 131.50, 131.88, 135.97, 138.0, 144.86, 149.0, 153.28, 155.56, 168.54; EI-MS m/z 614 (M+, 2.22), 246 (M-C18H16N4OS2, 100), 91 ([C7H7]+, 48.71%); Anal. Calcd for C^H^NgO^ (614.78): C, 62.52; H, 5.57; N, 13.67. Found: C, 62.74; H, 5.66; N, 13.89. N-(4-chlorophenyl)-3-[(4-benzyl-6,7,8,9-tetrahydro-5-oxo-4,5-dihydro[1]benzothieno[3,2-e][1,2,4]triazolo [4,3-a]pyrimidin-1-yl)sulphanyl]propanamide (18a). Reaction time: 34 h, crystallized from chloroform, yield: 68%; mp: 228-230 °C; IR (KBr, cm-1): 3309, 3275 (NH), 3100, 3000 (CH aromatic), 2931, 2840 (CH aliphatic), 1681 (br. 2 x C=O), 1589, 1546 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.74-1.79 (m, 4H, 2 x CH2 at C-7, C-8), 2.62-2.65 (m, 2H, CH2 at C-6), 2.72 (t, J = 15 Hz, 2H, CH^CH^), 2.83-2.85 (m, 2H, CH2 at C-9), 3.31(t, J = 15 Hz, 2H, CH^CH^), 5.30 (s, 2H, NCH2C6H5), 7.22-7.45 (m, 9H, Ar-H), 9.99 (s, 1H, NH, D2O exchangeable); EI-MS m/z 551.8 (M+2, 0.65), 549.8 (M+, 1.27), 91 ([C7H7]+, 100%); Anal. Calcd for C^H^ClNjO^ (550.10): C, 58.95; H, 4.40; N, 12.73. Found: C, 59.12; H, 4.47; N, 12.91. 4-Benzyl-1-{[2-morpholino-3-oxopropyl]sulphanyl}-6,7,8,9-tetrahydro[1]benzothieno[3,2-e][1,2,4]triazolo [4,3-a]pyrimidin-5(4ff)-one (18b). Reaction time: 30 h, crystallized from chloroform, yield: 51%; mp: 198-200 °C; IR (KBr, cm-1): 2947, 2862 (CH aliphatic), 1674, 1639 (2 x C=O), 1589, 1554, 1508 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.74-1.80 (m, 4H, 2 x CH2 at C-7, C-8), 2.75-2.77 (m, 2H, CH2 at C-6), 2.77 (t, J = 12.6 Hz, 2H, CH^CH^), 2.91-2.95 (m, 2H, CH2 at C-9), 3.22 (t, J = 12.6 Hz, 2H, CH^CH^), 3.43-3.44 (t, 4H, CH2-N), 3.49-3.51 (t, 4H, CH2-O), 5.30 (s, 2H, NCH^Hj), 7.25-7.41 (m, 5H, Ar-H); EI-MS m/z 509 (M+, 0.30), 91 ([C7H7]+, 100%); Anal. Calcd for C^H^Np^ (509.65): C, 578.92; H, 5.34; N, 13.74. Found: C, 59.21; H, 5.36; N, 13.89. 4-Benzyl-1-{[ 3-(4-phenylpiperazin-1-yl)-3-oxo-propyl]sulphanyl}-6,7,8,9-tetrahydro[1]benzothieno [3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4#)-one (18c). Reaction time: 26 h, crystallized from acetonitrile, yield: 40%; mp: 214-216 °C; IR (KBr, cm-1): 2937, 2852 (CH aliphatic), 1676, 1643 (2 x C=O), 1585, 1552, 1508 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.70-1.71 (m, 4H, 2 x CH2 at C-7, C-8), 2.70-2.81 (m, 4H, CH2 piperazine), 2.842 (t, J = 6 Hz, 2H, CH^CH^), 2.97-2.99 (m, 2H, CH2 at C-6), 3.04-3.12 (m, 2H, CH2 at C-9), 3.23 (t, J = 6 Hz, 2H, CH2-CH2S), 3.46-3.50 (m, 4H, CH2piperazine), 5.30 (s, 2H, NCH2C6H5), 6.79-7.42 (m, 10H, Ar-H); 13C-NMR (DMSO-d6) 5: 21.39, 22.16, 23.98, 24.79, 31.18, 32.72, 44.83, 47.98, 48.35, 115.63, 119.17, 120.50, 127.37, 127.78, 128.26, 128.88, 131.60, 131.71, 136.02, 138.0, 140.66, 148.0, 150.60, 155.58, 168.22; EI-MS m/z 584 (M+, 4.13), 91 ([C7H7]+, 100%); Anal. Calcd for C^H^NgO^ (584.76): C, 63.67; H, 5.52; N, 14.37. Found: C, 63.84; H, 5.63; N, 14.61. 4-Benzyl-1-{[3-[4-(4-methoxyphenyl)piperazin-1-yl]-3-oxopropyl]sulphanyl}-6,7,8,9-tetrahydro[1]benzot-hieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4_ff)-one (18d). Reaction time: 29 h, crystallized from chloroform, yield: 42%; mp: 222-224 °C; IR (KBr, cm-1): 3055, 3001 (CH aromatic), 2949, 2833 (CH aliphatic), 1674, 1641 (2 x C=O), 1587, 1554, 1510 (C=C aromatic); 1H-NMR (DMSO-d6) 5: 1.71-1.73 (m, 4H, 2 x CH2 at C-7, C-8), 2.71-2.78 (m, 4H, 2 x CH2 piperazine), 2.80 (t, J = 6.6 Hz, 2H, CH^CH^), 2.83-2.92 (m, 4H, 2 x CH2 at C-6 and C-9), 3.25 (t, J = 6.6 Hz, 2H, CH^CH^), 3.45-3.55 (m, 4H, 2 x CH2 piperazine), 3.68 (s, 3H, OCH3), 5.31 (s, 2H, NCH2C6H5), 6.79-7.42 (m, 9H, Ar-H); EI-MS m/z 616 (M+2, 0.95), 614 (M+, 4.66), 91 ([C7H7]+, 100%); Anal. Calcd for C^H^NgO^ (614.78): C, 62.52; H, 5.57; N, 13.67. Found: C, (52.70; H, 5.54; N, 13.84. 2. 2. In vitro Anticancer Screening 2. 2. 1. Materials and Methods The prostate tumor cell line (PC-3) and the colon tumor cell line (HCT-116) were obtained frozen in liquid nitrogen (-180 °C) from the American Type Culture Collection (ATCC) and were maintained in the National Cancer Institute, Cairo, Egypt, by serial sub-culturing. All chemicals used in this study were of high analytical grade. They were obtained from either Sigma-Aldrich or Bio-Rad. 2. 2. 2. Measurement of Potential Cytotoxicity The cytotoxic activity of some selected compounds was measured in vitro against human prostate cancer cell line (PC-3) and colon cancer cell line (HCT-116) at five different doses (0, 5.0, 12.5, 25.0 and 50.0 pg/mL). The screening was carried out at the Pharmacology Unit, Cancer Biology Department, National Cancer Institute, Cairo University using Sulforhodamine-B (SRB) assay, applying the method of Skehan et al.41 as follows. Cells were plated in 96 multi-well plate (104 cells/well) for 24 h before treatment with the tested compound to allow attachment to the wall of the plate. Different concentrations of the compounds (0, 5.0, 12.5, 25.0 and 50.0 pg/mL) were added to the cell monolayer in tri- Botros et al.: Synthesis, Characterization and Cytotoxicity ... 110 Acta Chim. Slov. 2017, 64, 102-116 plicate and wells were prepared for each individual dose. Monolayer cells were incubated with the compounds for 48 h at 37 °C in atmosphere of 5% CO2. After 48 h, cells were fixed, washed and stained with Sulforhodamine-B stain. Excess stain was washed with acetic acid and the attached stain was recovered with Tris EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration was plotted to get the survival curve of each tumor cell line. IC50 values (the concentration required for 50% inhibition of cell viability) were calculated using sigmodial dose response curve-fitting models (GraphPad, Prizm software incorporated), each concentration was repeated three times. The results are given in Table 1 and represented graphically in Fig. 3. 3. Results and Discussion 3. 1. Chemistry The synthetic strategies adopted for the synthesis of the intermediate and final compounds are illustrated in Schemes 1 and 2. In Scheme 1, the starting compound ethyl 2-amino-4,5,6,7-tetrahydro[ 1 ]benzothiophene-3-carboxylate (1) was prepared according to the well-known Gewald procedure.32 Reacting 1 with benzyl isothiocya-nate in acetonitrile afforded the corresponding 3-benzyl-2-sulfanylthienopyrimidine derivative 2. The 2 formed was treated with 99% hydrazine hydrate in dry pyridine to give the 2-hydrazino derivative 3. Structural elucidation of 3 was based on IR and 1H-NMR spectroscopy. Reacting the key intermediate 3 with formic acid or acetic acid induced cyclization to the corresponding triazolo derivatives 4a and 4b. IR and 1H-NMR spectra confirmed the cyclization through the disappearance of NH and NH2 signals. The presence of a signal at 5 12.07 ppm in 13C-NMR verified the presence of the CH3 group in 4b. Compound 5 was obtained upon treatment of 3 with chloroacetyl chloride in dry DMF. The notable feature in the 1H-NMR spectrum was the appearance of a singlet peak at 5 5.18 ppm indicating CH2Cl group. The successful formation of the intermediate 5 prompted us to investigate the nucleop-hilic replacement of the active chlorine atom with different amines. Compound 5 underwent nucleophilic substitution with various substituted piperazines to afford 6a-d. The 1H-NMR spectra of the products 6a-d showed the appearance of the protons of the piperazine moiety in the range of 5 2.43-3.19 ppm. Moreover, a singlet signal at 5 3.94-3.96 ppm characteristic to the CH2 linking the tria-zole ring and the piperazine ring confirmed the successful incorporation of piperazine moieties. Compound 7 was obtained in good yield by heating the key intermediate 3 with N,N-carbonyldiimidazole in dry benzene. IR spectrum of 7 showed absorption bands at v 1720 and 1632 cm-1 indicating the presence of two C=O groups of the triazole ring and the pyrimidinone ring, res- pectively. Furthermore, the 1H-NMR spectrum displayed an exchangeable singlet signal at 5 12.0 ppm corresponding to the NH proton of the triazole ring. 13C-NMR spectrum of 7 showed two signals at 5 149.17 and 156.63 ppm confirming the presence of two carbonyl moieties. Furthermore, the reaction of 3 with various isothiocyanates yielded the corresponding 1-substituted aminotriazolo derivatives 8a-e. 1H-NMR spectra of 8a-e showed D2O exchangeable signals in the range of 5 9.29-9.39 ppm assignable to the NH. On the other hand, reacting 3 with diethyl malonate in acetic acid afforded the unexpected product 1-methyltriazolo derivative 4b. The formation of 4b may be explained by the hydrolysis and decarboxylation of the ester group in the intermediate compound 9 in acidic medium. However, the direct interaction of 3 with excess di-ethyl malonate in the absence of solvent at the refluxing temperature afforded the expected product 9. The IR spectrum showed the presence of two C=O moieties at v 1740 and 1666 cm-1 while the 1H-NMR spectrum confirmed the presence of the ethyl ester group. Further evidence was obtained from the 13C-NMR spectrum of 9 which confirmed the presence of ethyl ester group through signals at 5 14.45 and 62.0 ppm in addition to a signal at 5 32.31 ppm corresponding to the -CH2- flanked between the thienop-yrimidin-2-ylsulphanyl group and carbonyl function. Furthermore, the reaction of 3 with benzoyl chloride and ammonium thiocyanate in dry acetone afforded the ben-zoyl thiourea derivative 10. 1H-NMR spectrum of compound 10 showed the presence of three D2O exchangeable signals assignable to three NH moieties at 5 9.79, 11.71 and 12.49 ppm. In Scheme 2, the reaction of 3 with carbon disulfide in ethanolic potassium hydroxide followed by acidification with hydrochloric acid yielded the thiol (11) / thione (11a) tautomers. One of the objectives of this work was to prepare a series of S-alkylated triazolopyrimidine derivatives with varying the linker skeleton as well as varying the bioactive amine to test their cytotoxicity. Herein, a series of alkylated mercapto 1,2,4-triazo-les was synthesized via the reaction of the key intermediate 11 with various alkyl halides or a- and P-chloroamides (13a-d, 14a-d, 15a-d) in absolute ethanol in the presence of anhydrous sodium acetate to afford the corresponding S-alkyl derivatives (12a,b, 16a-d, 17a-d, 18a-d). The success of alkylation was confirmed by the absence of SH or NH signals in 1H-NMR spectra of 12a and 12b together with the appearance of peaks characteristic to methyl and ethyl moieties in each compound, respectively. Moreover, the mass spectrum of 12a,b showed their corresponding molecular ion peaks at m/z 382 and 396, respectively. The structure of the mercapto alkylated derivatives 16a-d, 17a-d and 18a-d linked to different secondary amines with different linkages was supported by elemental analyses and spectral data. IR spectra of all target Botros et al.: Synthesis, Characterization and Cytotoxicity ... Acta Chim. Slov. 2017, 64, 102-116 111 compounds indicated the appearance of new amide C=O absorption band at v 1629-1643 cm-1. Moreover, 1H-NMR spectra of compounds 16a-d, 17a-d and 18a-d showed the disappearance of the SH signal at 5 14.06 ppm. Besides, the alkyl protons in the linker between the triazolop-yrimidine ring and the amine appeared as follows; )-SH ^—NHNH2 R—CH3, C2H5, Ch^Ch^^Ch^, ch2=ch-ch2_, 4-och3-c6h4 cooc2h6 Scheme 1. Synthesis of triazolo derivatives 4a,b, 5, 6a-d, 7, 8a-d, 9 and 10: (i) PhCH2NCS / K2CO3 / acetonitrile, followed by acidification; (ii) NH2-NH2-H2O / pyridine, reflux; (iii) RCOOH, reflux; (iv) ClCH2COCl / DMF, 100 °C; (v) piperazines / EtOH, reflux; (vi) CDI / benzene, reflux; (vii) RNCS / EtOH, reflux; (viii) diethyl malonate, reflux; (ix) benzoyl chloride / NH4SCN / acetone, reflux. Botros et al.: Synthesis, Characterization and Cytotoxicity ... 112 Acta Chim. Slov. 2017, 64, 102-116 1H-NMR spectra of 16a-d showed a characteristic singlet in the range of 5 3.84-4.23 ppm assigned for SCH2 protons, while the spectra of 17a-d revealed doublet signals in the range of 5 1.44-1.51 ppm assignable to CH3 moiety and quartet signals assignable to CH protons in the range of 5 3.93-4.61 ppm. The presence of ethylene fragment (CH2-CH2) in compounds 18a-d was revealed by two triplet signals in the range of 5 2.72-2.87 ppm and 5 3.20-3.40 ppm in :H-NMR spectra. Further proof for these compounds was obtained using 13C-NMR spectroscopy where the spectrum of compound 18c showed signals at 5 31.18 and 32.72 ppm assignable to the SCH2 and Scheme 2. Synthesis of triazolo derivatives 11, 12a,b, 16a-d, 17a-d, 18a-d: (i) CS2 / KOH / EtOH, reflux; (ii) diluted HCl; (iii) anhydrous sodium acetate / EtOH, reflux. Botros et al.: Synthesis, Characterization and Cytotoxicity ... Acta Chim. Slov. 2017, 64, 102-116 113 CH2-CO moieties, respectively. In addition, 1H-NMR spectra of all the target products 16a-d, 17a-d and 18a-d displayed the expected signals of the morpholino, 4-chlo-roanilino and substituted piperazine moieties. 3. 2. In vitro Cytotoxicity The in vitro cytotoxic activity of 24 selected compounds was evaluated against two human cancer cell lines including cells derived from human prostate cancer (PC-3) and human colon cancer (HCT-116) according to the standard protocol for IC50 determination. Doxorubicin (DOX), being one of the most effective anticancer agents, was chosen as the reference standard anticancer drug.42 The IC50 values in pM are listed in Table 1 and the results are represented graphically in Fig. 3. From the results in Table 1 it is evident that most of the tested compounds displayed moderate to potent cancer cell growth inhibition. Generally, all the tested compounds tended to be more active against HCT-116 than against PC-3. Examining the IC50 of the tested compounds against PC-3 cell line revealed that compounds 10, 12b, 17b and 18c exhibited significant anticancer activities with lower IC50 values compared to DOX, with compound 16c being the most potent with an IC50 of 5.48 pM. Meanwhile, compound 12a showed equipotent activity to DOX, while compounds 6a, 6c, 8a, 17c and 18a exhibited IC50 values (ranging from 8.25-8.97 pM) very close to DOX (IC50 = 7.7 pM) against PC-3. As for the HCT-116 cell line, compounds 6c and 18c were the most active (IC50 = 6.56 and 6.12 pM, respectively) in contrast to 15.82 pM for the standard on the same cell line. In addition, compounds 4a, 5, 6a, 8a, 8b, 8d, 10, 12b, 17b, 17c, and 18a displayed more potent cytotoxic activity compared to the standard with IC50 values ranging from 7.4 to 14.77 pM. Table 1. Results of in vitro cytotoxic activity of some selected compounds against human prostate cancer cell line (PC-3) and colon cancer cell line (HCT-116). (Results in bold represent compounds with better activity than DOX.) Compound no. IC50 in ^M* PC-3 HCT-116 4a 13.58 10.64 4b 11.41 >100 5 10.13 12.47 6a 8.91 14.77 6c 8.25 6.56 7 11.35 29.51 8a 8.97 12 8b 15 11 8d 10.7 12 8e 10.05 17.15 10 6.53 8.86 11 >100 >100 12a 7.8 20.1 12b 7 7.57 16a >100 >100 16c 5.48 17.52 17a >100 60.53 17b 6.4 7.63 17c 8.5 7.4 17d >100 20.33 18a 8.5 8.18 18b 16.01 >100 18c 7.5 6.12 18d 36 27.32 Doxorubicin 7.7 15.82 * The values given are means of three experiments. Referring to the IC50 values listed in Table 1, the following SAR can be deduced: among the triazolo derivatives 4a and 4b, the unsubstituted derivative 4a showed Figure 3. Cytotoxicity of some selected compounds against human prostate cancer cell line (PC-3) and colon cancer cell line (HCT-116) Botros et al.: Synthesis, Characterization and Cytotoxicity ... 114 Acta Chim. Slov. 2017, 64, 102-116 good activity against HCT-116. Concerning the piperazi-ne derivatives 6a-d, compounds 6a and 6c displayed good activity against both cell lines whereas the 4-chlorophenyl piperazine derivative 6c showed 2.4 fold higher activity than DOX against HCT-116 cell line in agreement with the reported anticancer activity of derivatives incorporating piperazine scaffolds and halogen atoms.43'44 Upon analyzing the results of the substituted amino triazoles 8a-e, compounds 8a, 8b and 8d exhibited higher activity than DOX against HCT-116 but it was difficult to reach conclusions regarding the effect of varying the substituent since the cytotoxicity of 8a-e was almost the same. The N-methyl derivative 8a was the only potent analogue against PC-3 cell line. Interestingly, compound 10 displayed potent cytotoxic activity against both cell lines in accordance with the reported antitumor activity of thiose-micarbazide derivatives.45 Among the 1,2,4-triazole derivatives, the mercapto substituted 1,2,4-triazole ring systems have been studied and so far a variety of antitumor properties have been reported for a large number of these compounds.25-27 Based on the above findings, we investigated herein in Scheme 2, the structure-activity relationship of ^-alkylated series of compounds 12a,b, 16a-d, 17a-d and 18a-d, focusing in particular on the effect of the linker skeleton as well as varying the bioactive amine on the cytotoxic activity, the following was observed: - The incorporation of ethyl substituent in 12b resulted in a more potent derivative than 12a against both cell lines. - Among compounds 16a-d with CH2 linker, the 4-phenyl piperazine analogue 16c showed selective high activity against PC-3. - The cytotoxic activity of compounds 17a-d with branched alkyl linker (-CHCH3) showed that the incorporation of morpholine ring (17b) and phenyl piperazine moiety (17c) resulted in compounds with potent activity against both cell lines. - The phenyl piperazine derivatives (16c and 18c) afforded better cytotoxic activity compared to other amines against PC-3 and HCT-116, respectively. -Extending the side chain caused pronounced change in the activity of the 4-chloroaniline derivative 18a against both cell lines compared to 16a with acetamide linkage and 17a with branched linker which were devoid of activity. 4. Conclusion A series of substituted 4-benzyl[1]benzothieno[3,2-e][1'2'4]triazolo[4'3-a]pyrimidines was designed, synthesized, and screened for their anticancer activity against PC-3 and HCT-116 cell lines. Many of the newly synthesized compounds showed remarkable activity on the tested cell lines with higher sensitivity towards the HCT-116 cell line. Compounds 10,12b, 17b and 18c sho- wed higher cytotoxic activity against both PC-3 and HCT-116 cell lines compared to DOX. Incorporation of a 4-phenylpiperazine moiety resulted in higher activity against both cell lines where compound 16c was the most active against PC-3 with 1.4 fold higher activity than DOX, while 18c showed 2.5 fold higher anticancer activity against HCT-116. The obtained results suggest that thienopyrimidines containing 1,2,4-triazole scaffold might be suitable candidates for further chemical modifications in order to obtain more potent and selective anticancer agents. 5. References 1. 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Slov. 2017, 64, 102-116 Povzetek Številna poročila o proti rakastem delovanju različnih tieno[2,3-d]pirimidinov in triazolotienopirimidinov so nas spodbudila k pripravi nove serije 4-benzil-6,7,8,9-tetrahidro[1]benzotieno[3,2-e][1,2,4]triazolo[4,3-a]pmmidinov. Raziskali smo in vitro citotoksično aktivnost izbranih spojin proti dvema človeškima celičnima linijama: raka prostate (PC-3) ter raka debelega črevesa in danke (HCT-116). Izvedli smo tudi začetno študijo odvisnosti med aktivnostjo tarčnih spojin in njihovo strukturo. Večina pripravljenih spojin je izkazala precejšnjo aktivnost proti testiranima celičnima linijama, zlasti obetavna je bila aktivnost spojine 16c proti celični liniji PC-3 z IC50 vrednostjo 5.48 |M, kar je zelo ugodno v primerjavi z vrednostjo za doksorubicin (IC50 = 7.7 |M), referenčnim standardom uporabljenim v tej raziskavi. Po drugi strani pa sta se spojini 6c in 18c izkazali kot najbolj aktivni proti celični liniji HCT-116 (IC50 = 6.12 in 6.56 |M), kar je tudi ugodno v primerjavi z vrednostjo za standard (IC50 = 15.82 |M). Zato lahko zaključimo, da bi nekateri izmed sin-tetiziranih tienopirimidinskih derivatov, zlasti 6c, 16c in 18c, lahko predstavljali potencialno zanimive spojine za nadaljnji razvoj v učinkovita zdravila proti raku. Botros et al.: Synthesis, Characterization and Cytotoxicity ... DPI: 10.17344/acsi.20l6.2920_Acta Chirn. Slov. 2017,64, 117-128_©commons n? Scientific paper Synthesis, Cytotoxic and Anti-proliferative Activity of Novel Thiophene, Thieno[2,3-#]pyridine and Pyran Derivatives Derived from 4,5,6,7-tetrahydrobenzo[#]thiophene Derivative Rafat Milad Mohareb,1'* Nadia Youssef Megally Abdo2 and Fatma Omar Al-farouk3 1 Department of Chemistry, Faculty of Science, Cairo University, Giza, A. R. Egypt 2 Chemistry Department, Faculty of Education, Alexandria University, 21526 Alexandria, Egypt 3 Department of Chemistry, Faculty of Science, American University in Cairo, 5th Settlement, A.R., Egypt * Corresponding author: E-mail: raafat_mohareb@yahoo.com Received: 15-09-2016 Abstract Novel tetrahydrobenzo[b]thienopyrole derivatives are synthesized from 2-amino-3-cyano-4,5,6,7-tetrahydroben-zo[b]thiophene (1) through its reaction with a-chloroacetone to give the corresponding N-alkyl derivative 3. Compound 3 undergoes ready cyclization in sodium ethoxide solution to give the tetrahydrobenzo[b]thienopyrrole 4. The latter compound 4 is used as the key starting material for the synthesis of thiophene, thieno[2,3-b]pyridine and pyran derivatives. The cytotoxicity of the synthesized products towards the human cancer cell lines namely gastric cancer (NUGC), colon cancer (DLD-1), liver cancer (HA22T and HEPG-2), breast cancer (MCF-7), nasopharyngeal carcinoma (HONE-1) and normal fibroblast (WI-38) cell lines are measured. Compounds 4, 7a, 7b, 8a, 8b, 10c, 10d, 10f, 12a, 12b, 14b and 15b exhibit the optimal cytotoxic effect against cancer cell lines. Compounds 7b and 14b show the maximum inhibitory effect and these are much higher than the reference CHS-828 (pyridyl cyanoguanidine). On the other hand, the anti-proliferati-ve evaluations of these compounds with high potency against the cancer cell lines L1210, Molt4/C8, CEM, K562, K562/4 and HCT116 show that compounds 7b and 8b give IC50's against Molt4/C8 and CEM cell lines higher than that of the reference, doxorubicin. Keywords: Tetrahydrobenzo [b] thiophene, pyran, thiophene, cytotoxicity, anti-proliferative activity 1. Introduction Sulfur containing heterocycles paved way for the active research in the pharmaceutical Chemistry. Nowadays benzothiophene derivatives in combination with other ring systems have been used extensively in pharmaceutical applications.1-3 A large number of compounds containing thiophene system have been investigated because of their broad spectrum of biological activities which include anal-gesic,4 antibacterial,5 antifungal,6 antiparasitic,7 antiviral,8 anti-inflammatory,9 anticonvulsant,10 anti-nociceptive,11 DNA cleavage,12 herbicidal,13 antitubercular,14 protein kinase inhibition,15 respiratory syndrome protease inactiva-tion,16 an active ester in the peptide synthesis and agonists of peroxisome proliferator activated receptors.17 In addition to these considerable biological applications, tetrahydro-benzo[ft]thiophenes are important intermediates, protecting groups and final products in organic synthesis. Recently, our research group was involved through comprehensive program aiming for the synthesis of 4,5,6,7-tetrahydroben-zo[b]thiophene derivatives followed by their antitumor eva-luations.18,19 Moreover, we reported the multi-component reactions with 3-(a-bromoacetyl)coumarin to give pyan and pyrididine derivatives.20 In continuation of this program we are demonstrating the use of 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[ft]thiophene for the synthesis of te-trahydrobenzo[b]thienopyrrole derivatives followed by their cytotoxic and the anti-proliferative evaluations.21,22 Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel 118 Acta Chim. Slov. 2017, 64, 117-128 2. Results and Discussion The reaction of the 2-amino-3-cyano-4,5,6,7-te-trahydrobenzo[ft]thiophene (1) with a-chloroacetone in the presence potassium carbonate afforded the 2-((2-oxo-propyl)amino)-4,5,6,7-tetrahydrobenzo[ft]thiophene-3-carbonitrile (3). Compound 3 was characterized by 1H-NMR and 13C-NMR. Thus, the 1H-NMR spectrum display the presence of beside the expected tetrahydro-benzene moiety, a singlet at 5 5.20 ppm indicating the presence of the N-CH2 group, a singlet at 5 2.88 ppm assigned to the CH3 group and a broad singlet at 5 8.30 ppm due to the NH group. Moreover, the 13C-NMR spectrum showed 5: 19.6 (CH3), 20.3, 22.0, 25.7 and 34.6 (4 CH2), 55.6 (CH2), 116.8 (CN), 124.1, 124.9, 128.7 and 139.5 (thiophene C), 164.8 (C=O). Compound 3 under- Shema 1. Synthesis of compounds 3 and 4. Shema 2. Synthesis of compounds 6a,b and 7a,b. Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ... Acta Chim. Slov. 2017, 64, 117-128 119 went ready cyclization when heated in sodium ethoxide solution in a boiling water bath to yield the 1-(3-amino-4,5,6,7-tetrahydro-1H-benzo[4,5]thieno[2,3-fc]pyrrol-2-yl)ethanone (4) (Scheme 1). Compound 4 showed interesting reactivity towards different reagents, thus, it reacted with either malononitri-le (5a) or ethyl cyanoacetate (5b) in the presence of ammonium acetate in an oil bath at 120 °C afforded the Knoevenagel condensated products 6a and 6b, respectively. The latter products underwent ready cyclization in sodium ethoxide solution to give the annulated products 7a and 7b, respectively (Scheme 2). The structures of the latter products were established on the basis of the analytical and spectral data. Thus, the 1H-NMR spectrum of 7a showed the presence of 5 2.89 ppm assigned to the CH3 group, a singlet at 5 4.89 ppm indicating the NH2 group and a singlet at 5 8.33 ppm confirming the presence of the NH group. Moreover, the 13C-NMR spectrum showed 5 19.8 (CH3), 20.1, 22.7, 25.2 and 34.6 (4 CH2), 116.8 (CN), 12(3.1, 122.6, 123.8, 124.2, 125.3, 127.2, 135.6, 142.3 (thiophene, pyrrole, pyridine C) and 168.2 (C=N). Compound 4 was studied to produce thiophene derivatives through the Gewald's reaction23-26 as many thiophe-nes were used as anticancer drugs. Thus, the reaction of compound 4 with either of malononitrile or ethyl cyanoace-tate and elemental sulphur gave the thiophene derivatives 8a and 8b, respectively. On the other hand, the one pot reaction of compound 4 with either malononitrile or ethyl cya-noacetate and any of benzaldehyde, 4-chlorobenzaldehyde or 4-methoxybenzaldehyde gave the pyran derivatives 10a-f, respectively. The 1H-NMR and 13C-NMR spectra 10a-f were consistent with their respective structures. Further confirmations for the structure of compounds 10a-f were obtained through their synthesis via another synthetic root. Thus, the reaction of compound 4 with the cinnamonitrile derivatives 11a-f in the presence of a catalytic amount of Shema 3. Synthesis of compounds 8a,b and 10a-f. Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ... 120 Acta Chim. Slov. 2017, 64, 117-128 triethylamine gave the same products 10a-f, respectively (m.p., mixed m.p. and fingerprint IR) (Scheme 3). Moreover, the reaction of either of compound 8a or 8b with ethyl cyanoacetate in refluxing dimethylforma-mide afforded the 2-amido derivatives 12a and 12b, respectively. Formation of the latter products was explained on the condensation of ethyl cyanoacetate with the 2-aminothiophene moiety not to the 3-aminopyrrol moiety on the basis of the :H-NMR spectra of such products. Thus, the :H-NMR spectrum of either 12a or 12b displayed the missing of the NH2 group that attached to thiop-hene ring which is expected to appear within the range 5 5.10-5.24 ppm while that of the 3-aminopyrrole moiety existing at 5 4.81-4.83 ppm. Similar acylation of the 2-aminothiophene was reported before in literature.27 The high yield of compound 12a, encouraged us to make furt- her work. Thus, the reaction of 12a with either of the aryl diazonium salts 13a-d gave the aryl hydrazo derivatives 14a-d, respectively. Moreover, compounds 12a,b underwent ready cyclization in sodium ethoxide to produce the thieno[2,3-fc]pyridine derivatives 15a and 15b, respectively (Scheme 4). 2. 2. Anti-tumor Cell Activity 2. 2. 1. Chemicals and Cell cultures Fetal bovine serum (FBS) and L-glutamine, were purchased from Gibco Invitrogen Co. (Scotland, UK). RPMI-1640 medium was purchased from Cambrex (New Jersey, USA). Dimethyl sulfoxide (DMSO), doxorubicin, CHS-828, penicillin, streptomycin and sulforhodamine B (SRB) were purchased from Sigma Chemical Co. (Saint Shema 4. Synthesis of compounds 12a,b-15a,b. Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ... Acta Chim. Slov. 2017, 64, 117-128 121 Louis, USA). The cell cultures was obtained from the European Collection of cell Cultures (ECACC, Salisbury, UK) and human gastric cancer (NUGC), human colon cancer (DLD-1), human liver cancer (HA22T and HEPG-2), human breast cancer (MCF-7), nasopharyngeal carcinoma (HONE-1) and normal fibroblast cells (WI-38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). They grow as monolayer and routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 lg/mL), at 37 °C in a humidified atmosphere containing 5% CO2. Exponentially growing cells were obtained by plating 1.5 x 105 cells/mL for the six human cancer cell lines including cells derived from 0.75 x 104 cells/mL followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay. 2. 2. 2. In vitro Cytotoxicity Assay The heterocyclic compounds, prepared in this study, were evaluated according to standard protocols28,29 for their in vitro cytotoxicity against the six human cancer cell lines including cells derived from human gastric cancer (NUGC), human colon cancer (DLD-1), human liver cancer (HA22T and HEPG-2), human breast cancer (MCF-7), nasopharyngeal carcinoma (HONE-1) and a normal fibroblast cells (WI-38). All of IC50 values were listed in Table 1. Some heterocyclic compounds were observed with significant cytotoxicity against most of the cancer cell lines tested (IC50=10-1000 nM). Normal fibroblasts cells (WI-38) were affected to a much lesser extent (IC50>10,000 nM). The reference compound used was the CHS-828 which is the pyridyl cyanoguanidine anti-tumor agent.30 It is a new che-motherapeutic drug in addition it has low toxicity and lacks known patterns of multidrug resistance.31 2. 2. 3. Structure-activity Relationship From Table 1 it is clear that the thiophene moiety was found to be crucial for the cytotoxic effect of the cyclic compounds 3 -15a,b. Compounds 4, 7a, 7b, 8a, 8b, 10c, 10d, 10f, 12a, 12b, 14b and 15b exhibited optimal cytotoxic effect against cancer cell lines, with IC50's in the nM range. Comparing the cytotoxicity of the tetrahy-drobenzothiophene 3 and the cyclized product 4, it is obvious that the cytotoxicity of compound 4 is higher than that of compound 3. The presence of the pyrrol ring through the tetrahydrobenzo[fc]thiophene in compound 4 is responsible for its high potency. The condensation reac- Table 1. Cytotoxicity of the newly synthesized products against a variety of cancer cell lines [IC50a (nM)] Compound Cytotoxicity (IC50 in nM) No. UGCb DLD-1b HA22Tb HEPG-2b HONE-1b MCF-7b WI-38b 3 2142 1222 1340 1028 1828 2246 NA 4 86 45 313 128 212 310 NA 6a 2101 2380 3258 2266 2380 3330 NA 6b 1335 1140 1072 1154 1064 1258 NA 7a 218 146 220 337 241 380 NA 7b 48 92 260 46 74 32 NA 8a 320 240 230 165 1281 265 NA 8b 48 35 53 170 49 78 NA 10a 1220 1033 2250 1275 2126 2372 NA 10b 1165 1322 2350 2221 2152 1322 NA 10c 330 532 822 442 1529 1224 NA 10d 30 62 74 39 1330 88 NA 10e 1135 2160 2160 814 780 296 NA 10f 149 2220 3210 550 2451 1286 120 12a 69 74 190 448 2871 2690 NA 12b 26 65 38 220 440 57 NA 14a 1350 1160 2290 2120 1126 2230 NA 14b 83 59 80 64 87 48 1330 14c 1480 1156 1346 1226 1275 1240 NA 14d 1245 2160 2180 2220 1869 1765 NA 15a 1845 1210 1218 1076 1270 436 NA 15b 1220 2063 377 740 253 2210 NA CHS-828 25 2315 2067 1245 15 18 NA a Drug concentration required to inhibit tumor cell proliferation by 50% after continuous exposure of 48 h. b NUGC, gastric cancer; DLD-1, colon cancer; HA22T, liver cancer; HEPG-2, liver cancer; HONE-1, nasopharyngeal carcinoma; MCF-7, breast cancer; WI-38, normal fibroblast cells. NA: Not Active. Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ... 122 Acta Chim. Slov. 2017, 64, 117-128 tion of compound 4 with either malononitrile or ethyl cya-noacetate to produce compounds 5a and 5b, respectively showed a decrease of cytotoxicity. On the other hand, the cyclization of compounds 6a and 6b to the ben-zo[4',5']thieno[3',2':4,5]pyrrolo[3,2-fc]pyridine derivatives 7a and 7b showed remarkable increase of the cytotoxicity. Moreover, it is clear that compound 7b showed more cytotoxicity than 7a, this is attributed to the presence of the oxygen rich COOE-t group. The introduction of the second thiophene moiety to compound 4 that gives both of compounds 8a and 8b showed high potency-especially-in case of compounds 8b which was attributed due to the presence of the COOEt. Considering the pyran derivatives 10a-f, the cytotoxicity of compounds 10c and 10d showed the highest values among the six compounds. However, compound 10c showed high cytotoxicity against the four cancer cell lines HUGC, DLD-1, HA22T and HEPG-2, but it is of great value to notice that compound 10d showed high cytotoxicity against five cancer cell lines and such cytotoxicity is higher than that of compound 10c. The high cytotoxicity of compound 10d is attributed to the presence of the OH and the Cl group as well. The thiophene derivatives 12a and 12b showed high cytotoxicity similarl to that of compounds 8a,b. Moreover, compound 12b with the COOE-t showed high potency than that of compound 12a. The coupling of the diazonium salts 13a-d with compound 12a afforded the arylhydrazone derivatives 14a-d. Compound 14b with the Cl group showed the maximum cytotoxicity among the arylhydrazone derivatives 14a-d. Finally, considering the thieno[2,3-fc]pyridi-ne derivatives 15a,b where the presence of the OH in compound 15b conserved an interesting cytotoxicity against the cancer cell lines HA22T, HEPG-2 and HONE-1 with the IC50's 377, 740, 253 nM, respectively. It is of great value to notice that compounds 7b, 8b and 12b showed the maximum cytotoxicity among the tested compounds. 2. 2. 4. Anti-proliferative Cell Activity Against Cancer Cell Lines We used a panel of tumor cell lines to test the cytoto-xicity of the new compounds, especially those showed high potency against the six cancer cell lines through Table 2. Importantly, this panel included the cell lines and their iso-genic sub-lines with the determinants of drug resistance: murine leukemia L1210, T-lymphocyte cell lines Molt4/C8 and CEM, human leukemia R562 and its MDR subline K562/4 that over expressed P-glycoprotein, and the colon carcinoma HCT116. The above determinants alter the response of cells to many anticancer drugs including doxoru-bicin. Data on cytotoxic (anti-proliferative) activity are presented in Table 2 in which IC50 values represent the concentrations that inhibit cell proliferation by 50%. It is clear from Table 2 that tested compounds 4, 7a, 7b, 8a, 8b, 10c, 10d, 10f, 12a, 12b, 14b and 15b showed high potency against the cell lines. The benzo[4',5']thieno[3',2':4,5]pyr-rolo[3,2-b]pyridine derivative 7b and the benzo[4,5]thieno-[2,3-b]pyrrol-2-yl)-thiophene derivative 8b showed high potency against Molt4/C8 and CEM cell lines and their IC50's are higher than that of the reference doxorubicin. It is clear from Table 2 that the twelve tested compounds showed high IC50 against K562/4 cell line than doxorubicin. 3. Experimental 3. 1. General All melting points were determined on an electrothermal apparatus (Buchi 535, Switzerland) in an open capillary tube and are uncorrected. 13C-NMR and 1H-NMR spectra were recorded on Bruker DPX200 instrument in DMSO with TMS as internal standard for protons and solvent signals as internal standard for carbon spectra. Chemical shift values are mentioned in a (ppm). Mass spectra Table 2. Anti-proliferative activity (IC50) of selected compounds against variety of cell lines Compound Cytotoxicity (IC50 in nM) No. L1210 Molt4/C8 CEM K562 K562/4 HCT116 4 1.5 ± 0.5 1.1 ± 0.03 0.3 ± 0.01 0.4 ± 0.08 0.9 ± 0.02 0.8 ± 0.05 7a 0.4 ± 0.1 0.8 ± 0.04 2.0 ± 0.4 1.8 ± 0.03 0.9 ± 0.06 1.3 ± 0.02 7b 0.3 ± 0.08 0.4 ± 0.04 0.9 ± 0.05 1.30 ± 0.08 1.1 ± 0.07 2.4 ± 0.09 8a 1.2 ± 0.09 0.8 ± 0.02 0.6 ± 0.01 0.2 ± 0.01 0.9 ± 0.08 1.4 ± 0.2 8b 1.1 ± 0.06 0.02 ± 0.002 0.7 ± 0.03 0.9 ± 0.06 1.6 ± 0.07 0.8 ± 0.02 10c 0.8 ± 0.05 0.4 ± 0.02 1.3 ± 0.05 0.6 ± 0.02 0.02 ± 0.01 1.2 ± 0.08 10d 0.6 ± 0.02 1.5 ± 0.07 2.5 ± 0.05 1.7 ± 0.02 2.5 ± 0.02 2.8 ± 0.07 10f 1.4 ± 0.05 0.8 ± 0.03 2.6 ± 0.09 0.02 ± 0.01 2.8 ± 0.06 0.4 ± 0.08 12a 2.1 ± 0.05 0.6 ± 0.02 0.5 ± 0.01 0.3 ± 0.01 0.4 ± 0.06 2.4 ± 0.07 12b 1.8 ± 0.09 0.9 ± 0.04 1.8 ± 0.6 0.7 ± 0.06 0.8 ± 0.06 0.9 ± 0.08 14b 0.5 ± 0.03 0.3 ± 0.05 2.6 ± 0.06 0.5 ± 0.07 0.6 ± 0.02 0.1 ± 0.01 15b 0.9 ± 0.02 0.3 ± 0.01 0.6 ± 0.05 2.1 ± 0.07 2.7 ± 1.03 0.3 ± 0.04 Dox. 0.37 ± 0.07 0.20 ± 0.02 0.06 ± 0.02 0.14 ± 0.03 7.2 ± 0.9 1.4 ± 0.1 Doxorubicin (Dox.) was used as the reference drug Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ... Acta Chim. Slov. 2017, 64, 117-128 123 were recorded on EIMS (Shimadzu) and ESI-esquire 3000 Bruker Daltonics instrument. Elemental analyses were carried out by the Microanalytical Data Unit Lud-wig-Maximilians-Universitat-Munchen, Germany. The progress of all reactions was monitored by TLC on 2 x 5 cm pre-coated silica gel 60 F254 plates of thickness of 0.25 mm (Merck). 3. 1.1. Synthesis of 2-((2-Oxopropyl)amino)-4,5,6,7-tetrahydrobenzo[6]thiophene-3-carbonitrile (3) To a solution of compound 1 (1.78 g, 0.01 mol) in I,4-dioxane (40 mL) containing sodium carbonate (1.00 g) a-chloroacetone (0.94 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 2 h then poured onto ice/water and the formed solid product was collected by filtration and crystallized from ethanol. White crystals; yield: 2.01 g (86%); mp: 182-183 °C; IR (KBr, cm-1): 3465-3328 (NH), 2220 (CN), 1705 (C=O), 1615 (C=C); 1H-NMR (dimethyl sulfoxide (DMSO)-d6) 8:1.80-1.85 (m, 4H, 2CH2), 2.22-2.26 (m, 4H, 2CH2), 2.88 (s, 3H, CH3), 5.20 (s, 2H, CH2), 8.30 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 8 : 19.6, 20.3, 22.0, 25.7, 34.6, 55.6, 116.8, 124.1, 124.9, 128.7, 139.5, 164.8; MS electron impact (EI): m/z (%) 234 (M+). Anal. Calcd for C12H14N2OS: C, 61.51; H, 6.02; N, 11.96; S, 13.68. Found: C, 61.82; H, 6.22; N, II.77; S, 13.73. Synthesis of 1-(3-Amino-4,5,6,7-tetrahydro-1ff-ben-zo[4,5]thieno[2,3-£]pyrrol-2-yl)ethanone (4) A suspension of compound 3 (2.34 g, 0.01 mol) in sodium ethoxide (0.02 mol) [prepared by dissolving metallic sodium (0.46 g, 0.02 g) in absolute ethanol (20 mL] was heated in a boiling water bath for 6 h then poured onto ice/water containing few drops of hydrochloric acid. The formed solid product was collected by filtration and crystallized from 1,4-dioxane. White crystals; yield: 1.80 g (77%); mp: >300 °C; IR (KBr, cm-1): 3479-3348 (NH, NH2), 1715 (C=O), 1618 (C=C); 1H-NMR (DMSO-d6) 8: 1.78-1.83 (m, 4H, 2CH2), 2.20-2.27 (m, 4H, 2CH2),62.91 (s, 3H, CH3), 4.83 (s, 2H, NH2, D2O exchangeable), 8.27 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 8: 19.8, 20.2, 22.0, 25.6, 34.8, 124.0, 124.9, 128.5, 139.6, 165.6; MS (EI): m/z (%) 234 (M+). Anal. Calcd for C12H14N2OS: C, 61.51; H, 6.02; N, 11.96; S, 13.68. Found: C, (51.68; H, 5.89; N, 12.20; S, 13.83. 3. 1. 2. General Procedure for the Synthesis of Thieno[2,3-6]pyrrol Derivatives 6a and 6b To the dry solid of compound 4 (2.34 g, 0.01 mol) either malononitrile (0.66 g, 0.01mol) or ethyl cyanoace- tate (1.13 g, 0.01 mol) was added followed by ammonium acetate (0.50 g, 0.01 mol).The whole reaction mixture was heated in an oil bath at 120 °C for 1h then left to cool. The solidified product was boiled with ethanol then left to cool. The formed solid product was collected by filtration and crystallized from acetic acid. 2-(1-(3-Amino-4,5,6,7-tetrahydro-1_ff-benzo[4,5]thie-no[2,3-£]pyrrol-2-yl)ethylidene)-malononitrile (6a) Yellow crystals; yield: 1.92 g (68%); mp: 167-168 °C; IR (KBr, cm-1): 3488-3334 (NH, NH2), 3054 (CH aromatic), 2227, 2222 (2CN), 1620 (C=C); 1H-NMR (DMSO-d6) 8: 1.79-1.86 (m, 4H, 2CH2), (m, 4H, 2CH2), 2.69 (s, 3H, CH3), 4.86 (s, 2H, NH2, D2O exchangeable;), 8.29 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 8: 19.4, 20.3, 22.2, 25.6, 34.5, 116.3, 116.9, 122.3, 123.8, 124.0, 124.9, 127.2, 135.2; MS (EI): m/z (%) 282 (M+). Anal. Calcd for C15H14N4S: C, 63.80; H, 5.00; N, 19.84; S, 11.36. Found: C, 63.72; H, 4.93; N, 20.05; S, 11.59. Ethyl 3-(3-amino-4,5,6,7-tetrahydro-1ff-benzo[4,5]thie-no[2,3-£]pyrrol-2-yl)-2-cyanobut-2-enoate (6b) Yellow crystals; yield: 2.46 g (75%); mp: 121-122oC; IR (KBr, cm-1): 3473-3330 (NH, NH2), 3054 (CH aromatic), 2222 (CN), 1640 (C=C); 1H-NMR (DMSO-d6) 8: 1.13 (t, 3H, J = 7.26 Hz, CH3), 1.80-1.86 (m, 4H, 2CH2), 2.22-2.27 (m, 4H, 2CH2), 2.66 (s, 3H, CH3), 4.22 (q, 2H, J = 7.26 Hz, CH2), 4.88 (s, 2H, NH2, D2O exchangeable), 8.27 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 8: 16.3, 19.6, 20.2, 22.5, 25.6, 34.8, 116.6, 122.0, 123.5, 124.6, 124.7, 127.2, 134.8, 166.1; MS (EI): m/z (%) 329 (M+). Anal. Calcd for C17H19N3O2S: C, 61.98; H, 5.81; N, 12.76; S, 9.73. Found: C, 62.08; H, 6.07; N, 12.59; S, 9.88. 3. 1. 3. General Procedure for the Synthesis of the Benzo[4',5']thieno[3',2':4,5]-pyrrolo [3,2-#]pyridine Derivatives 7a and 7b Method (A): A suspension of either compound 6a (2.28 g, 0.01 mol) or 6b (3.29 g, 0.01 mol) in sodium et-hoxide (0.02 mol) [prepared by dissolving metallic sodium (0.46 g, 0.02 mol) in absolute ethanol (20 mL) was heated in a boiling water bath for 8 h then poured onto ice/water containing few drops of hydrochloric acid. The formed solid product was collected by filtration and crystallized from acetic acid. Method (B): To a solution of compound 4 (2.34 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethyla-mine (0.50 mL) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added. The whole reaction mixture, in each case, was heated under reflux for 4 h then poured onto ice/water containing few drops of hydrochloric acid. The formed solid product was collected by filtration and crystallized from acetic acid. Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ... 124 Acta Chim. Slov. 2017, 64, 117-128 2-Amino-4-methyl-7,8,9,10-tetrahydro-5_ff-benzo [4',5']thieno[3',2':4,5]pyrrolo[3,2-£]pyridine-3-carbo-nitrile (7a) Yellow crystals; yield: 2.27 g (80%); mp: 232-233 °C; IR (KBr, cm-1): 3474-3314 (NH, NH2), 3056 (CH aromatic), 2220 (CN), 1626 (C=C); 1H-NMR (DMSO-d6) 5: 1.76-1.84 (m, 4H, 2CH2), 2.21-2.26 (m, 4H, 2CH2), 2.89 (s, 3H, CH3), 4.89 (s, 2H, NH2, D2O exchangeable;), 8.33 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 19.8, 20.1, 22.7, 25.2, 34.6, 116.8, 120.1, 122.6, 123.8, 124.2, 125.3, 127.2, 135.6, 142.3, 168.2; MS (EI): m/z (%) 282 (M+). Anal. Calcd for C15H14N4S: C, 63.80; H, 5.00; N, 19.84; S, 11.36. Found: C, (53.66; H, 4.83; N, 20.25; S, 11.37. Ethyl 2-amino-4-methyl-7,8,9,10-tetrahydro-5_ff-benzo [4',5']thieno[3',2':4,5]pyrrolo[3,2-£]pyridine-3-car-boxylate (7b) Yellow crystals; yield: 2.24 g (68%), mp: 195-196 °C; IR (KBr, cm-1): 3466-3327 (NH, NH2), 3056 (CH aromatic), 1640 (C=C); 1H-NMR (DMSO-d6) 5: 1.14 (t, 3H, J = 7.07 Hz, CH3), 1.82-1.86 (m, 4H, 2CH2), 2.20-2.27 (m, 4H, 2CH2), 2.88 (s, 3H, CH3), 4.24 (q, 2H, J = 7.07 Hz, CH2), 4.84 (s, 2H, NH2, D2O exchangeable), 8.32 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 16.2, 19.8, 20.3, 22.5, 25.6, 34.5, 55.6, 120.3, 122A, 123.8, 124.6, 124.7, 127.6, 133.9, 143.2, 164.4, 168.9; MS (EI): m/z (%) 329 (M+). Anal. Calcd for C17H19N3O2S: C, 61.98; H, 5.81; N, 12.76; S, 9.73. Found: C, 651.68; H, 5.94; N, 12.63; S, 9.90. 3. 1. 4. General Procedure for the Synthesis of [4,5]thieno[2,3-6]pyrrol-2-yl)thiophene Derivatives 8a and 8b To a solution of compound 4 (2.34 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL) and elemental sulfur (0.32 g,0.01 mol) either malononitri-le (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added. The reaction mixture, in each case was heated under reflux for 2 h then was left to cool and the formed solid product, in each case, was collected by filtration and crystallized from ethanol. 2-Amino-4-(3-amino-4,5,6,7-tetrahydro-1_ff-benzo [4,5]thieno[2,3-£]pyrrol-2-yl)thiophene-3-carbonitrile (8a) Orange crystals; yield: 2.42 g (77%), mp: 141-142 °C; IR (KBr, cm-1): 3462-3354 (NH, NH2), 3053 (CH aromatic), 2221 (CN), 1628 (C=C); 1H-NMR (DMSO-d6) 5: 1.78-1.84 (m, 4H, 2CH2), 2.23-2.28 (m, 4H, 2CH2), 4.80, 5.25 (2s, 4H, 2NH2, D^ exchangeable), 6.11 (s, 1H, thiophene H-5), 8.26 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO—d6) 5: 20.4, 22.9, 25.0, 34.6, 116.6, 120.3, 123.1, 123.8, 124.2, 125.3, 127.2, 139.3, 140.6, 142.3; MS (EI): m/z (%) 314 (M+). Anal. Calcd for C15H14N4S2: C, 57.30; H, 4.49; N, 17.82; S, 20.40. Found: C, 57.44; H, 4.39; N, 18.04; S, 20.28. Ethyl 2-amino-4-(3-amino-4,5,6,7-tetrahydro-1_ff-ben-zo[4,5]thieno[2,3-£]pyrrol-2-yl)-thiophene-3-carboxy-late (8b) Orange crystals; yield: 2.60 g (74%), mp: 131-132 °C. IR (KBr, cm-1): 3479-3331 (NH2), 3053 (CH aromatic), 1690 (C=O), 1632 (C=C); 1H-NNMR (DMSO-d6) 5: 1.13 (t, 3H, J = 6.83 Hz, CH3), 1.81-1.87 (m, 4H, 2CH2), 2.22-2.25 (m, 4H, 2CH2), 4.23 (q, 2H, J = 6.83 Hz, CH2), 4.81, 5.03 (2s, 4H, 2NH2, D2O exchangeable), 6.13 (s, 1H, thiophene H-5), 8.30 (s, 1H, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 16.0, 20.(2, 22.7, 25.6, 34.5, 55.6, 120.8, 122.7, 123.8, 124.6, 124.9, 127.6, 133.9, 143.5, 164.2; MS (EI): m/z (%) 361 (M+). Anal. Calcd for C17H19N3O2S2: C, 56.48; H, 5.30; N, 11.62; S, 17.74. Found: C, 56.71; H, 5.55; N, 11.42; S, 17.49. 3. 1. 5. General Procedure for the Synthesis of Pyran Derivatives 10a-f Method (A): General procedure: To a solution of compound 4 (2.34 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.5 mL), either of malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) and either of benzaldehyde (1.06 g, 0.1 mol), 4-chloro-benzaldehyde (1.40 g, 0.01 mol) or 4-methoxybenzal-dehyde (1.36 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 1 h and the formed solid product produced from the hot solution was collected by filtration and crystallized from ethanol. Thin layer chro-matography revealed just a single spot which proved the presence of a single product. Method (B): To a solution of compound 4 (2.34 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.5 mL), either of the cinnamonitrile derivatives 11a-f (0.01 mol) were added. The reaction mixture was heated under reflux for 2 h and the formed solid product produced from the hot solution was collected by filtration and crystallized from ethanol. Thin layer chromatography revealed just a single spot which proved the presence of a single product. 2-Amino-6-(3-amino-4,5,6,7-tetrahydro-1_ff-benzo [4,5]thieno[2,3-£]pyrrol-2-yl)-4- phenyl-4_ff-pyran-3-carbonitrile (10a) Pale yellow crystals; yield: 3.10 g (80%); mp: 167-168oC; IR (KBr, cm-1): 3489-3321 (NH, NH2), 3056 (CH aromatic), 2220 (CN), 1630 (C=C); 1H-NM2R (DMSO-d6) 5: 1.76-1.85 (m, 4H, 2CH2), 2.21-2.27 (m, 4H, 2CH2), 4.83, 5.41 (2s, 4H, 2NH2, D2O exchangeable), 5.66-5.90 (2d, 2H, pyran H-4, H-5), 7.28-7.38 (m, 5H, C6H5), 8.24 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 20.6, 22.9, 25.3, 34.8, 39.3, 116.9, 120.6, 122.8, 123.8, 123.9, 125.3, 126.9, 127.2, 129.4, 130.8, Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ... Acta Chim. Slov. 2017, 64, 117-128 125 139.3, 140.6, 141.8, 142.3; MS (EI): m/z (%) 388 (M+). Anal. Calcd for C22H20N4OS: C, 68.02; H, 5.19; N, 14.42; S, 8.25. Found: C, 67.93; H, 5.32; N, 14.60; S, 8.44. 6-(3-Amino-4,5,6,7-tetrahydro-1_ff-benzo[4,5]thieno [2,3-£]pyrrol-2-yl)-2-hydroxy-4-phenyl-4_ff-pyran-3-carbonitrile (10b) Pale yellow crystals; yield: 2.57 g (66%), mp: 264-265 °C; IR (KBr, cm1): 3520-3341 (NH, NH2, OH), 3055 (CH aromatic), 2222 (CN), 1632 (C=C); 1^-NMR (DMSO-d6) 5: 1.77-1.86 (m, 4H, 2CH2), 2.20-2.27 (m, 4H, 2CH2), 4.86 (s, 2H, NH2, D2O exchangeable), 5.68-5.87 (2d, 2H, pyran H-4, H-5), 7.30-7.41 (m, 5H, C6H5), 8.22 (s, 1H, NH, D2O exchangeable), 10.30 (s, 1H, OH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 20.4, 22.7, 25.4, 34.8, 39.9, 116.7, 120.8, 122.8, 123.3, 123.9, 125.7, 126.9, 127.0, 130.4, 133.6, 139.3, 140.8, 142.0, 142.7; MS (EI): m/z (%) 389 (M+). Anal. Calcd for C22H19N3O2S: C, 67.84; H, 4.92; N, 10.79; S, 8.23. Found: C 67.60; H, 4.69; N, 10.99; S, 8.40. 2-Amino-6-(3-amino-4,5,6,7-tetrahydro-1_ff-benzo [4,5]thieno[2,3-£]pyrrol-2-yl)-4-(4-chlorophenyl-4_ff-pyran-3-carbonitrile (10c) Pale yellow crystals; yield: 2.87 g (68%); mp: 274-275 °C; IR (KBr, cm-1): 3474-3330 (NH, NH2), 3055 (CH aromatic), 2220 (CN), 1633 (C=C); 1H-NMR (DMSO-d6) 5: 1.78-1.85 (m, 4H, 2CH2), 2.18-2.25 (m, 4H, 2CH2), 4.86, 5.40 (2s, 4H, 2NH2, D2O exchangeable), 5.68-5.73 (2d, 2H, pyran H-4, H-5), 7.30-7.38 (m, 4H, C6H4), 8.26 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 20.3, 22.8, 25.5, 34.8, 39.7, 116.7, 120.4, 122.6, 123.9, 124.3, 125.3, 126.9, 128.8, 130.6, 139.0, 140.9, 142.8, 144.3; MS (EI): m/z (%) 423 (M+). Anal. Calcd for C22H19ClN4OS: C, 62.48; H, 4.53; N, 13.25; S, 7.58. Found: C, 62.22; H, 4.72; N, 13.51; S, 7.28. 6-(3-Amino-4,5,6,7-tetrahydro-1_ff-benzo[4,5]thieno [2,3-£]pyrrol-2-yl)-4-(4-chlorophenyl)-2-hydroxy-4_ff-pyran-3-carbonitrile (10d) Yellow crystals; yield: 3.21 g (76%), mp: 222-223 °C; IR (KBr, cm-1): 3541-3333 (NH, NH2), 3055 (CH aromatic), 2220 (CN), 1626 (C=C); 1H-NMR (DMSO-d6) 5: 1.78-1.87 (m, 4H, 2CH2), 2.21-2.28 (m, 4H, 2CH2), 4.83 (s, 2H, NH2, D2O exchangeable), 5.65-5.72 (2d, 2H, pyran H-4, H-5), 7.30-7.41 (m, 4H, C6H4), 8.24 (s, 1H, NH, D2O exchangeable), 10.28 (s, 1H, OH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 20.2, 22.6, 25.8, 34.3, 39.8, 116.5, 120.2, 122.6, 123.7, 123.9, 125.7, 126.9, 127.4, 130.2, 139.3, 141.3, 142.0, 142.8; MS (EI): m/z (%) 424 (M+). Anal. Calcd for C22H18ClN3O2S: C, 62.33; H, 4.28; N, 9.91; S, 7.56. Found: C, 62.09; H, 4.46; N, 9.75; S, 7.39. 2-Amino-6-(3-amino-4,5,6,7-tetrahydro-1_ff-benzo [4,5]thieno[2,3-£]pyrrol-2-yl)-4-(4-methoxyphenyl-4_ff-pyran-3-carbonitrile (10e) Orange crystals; yield: 3.01 g (72%), mp: 167-168 °C; IR (KBr, cm-1): 3531-3312 (NH, NH2), 3058 (CH aromatic), 2223 (CN), 1628 (C=C); 1H-N!VIR (DMSO-d6) 5: 1.74-1.86 (m, 4H, 2CH2), 2.20-2.28 (m, 4H, 2CH2), 3.01 (s, 3H, OCH3), 4.8(5, 5.22 (2s, 4H, 2NH2, D2O exchangeable), 5.67-5,74 (2d, 2H, pyran H-4, H-5), 7.32-7.38 (m, 4H, C6H4), 8.25 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMISO-dg) 5: 20.0, 22.8, 25.8, 34.8, 30.8, 39.6, 116.9, 120.6, 122.6, 123.4, 123.9, 125.7, 126.9, 127.6, 130.4, 139.4, 141.7, 142.3, 143.6; MS (EI): m/z (%) 418 (M+). Anal. Calcd for C23H22N4O2S: C, 66.01; H, 5.30; N, 13.39; S, 7.66. Found: C, 66.24; H, 5.48; N, 13.19; S, 7.80. 6-(3-Amino-4,5,6,7-tetrahydro-1_ff-benzo[4,5]thieno [2,3-£]pyrrol-2-yl)-2-hydroxy-4-(4-methoxyphenyl)-4_ff-pyran-3-carbonitrile (10f) Orange crystals; yield: 3.01 g (70%), mp: 229-230 °C; IR (KBr, cm-1): 3566-3332 (NH, NH2, OH), 3056 (CH aromatic), 2220 (CN), 1626 (C=C); 1H-NMR (DMSO-d6) 5: 1.74-1.86 (m, 4H, 2CH2), 2.22-2.29 (m, 4H, 2CH2), 3.08 (s, 3H, OCH3), 4.83 (s, 2H, NH2, D2O exchangeable), 5.64, 5.71 (2d, 2H, pyran H-4, H-5), 7.30-7.44 (m, 4H, C6H4), 8.23 (s, 1H, NH, D2O exchangeable), 10.32 (s, 1H, D2O exchangeable, OH); 13C-NMR (DMSO-d6) 5: 20.5, 22.8, 25.3, 34.5, 30.8, 39.1, 116.9, 120.6, 122.6, 123.4, 123.9, 125.7, 126.9, 127.6, 130.6, 139.4, 141.7, 142.3, 143.9; MS (EI): m/z (%) 419 (M+). Anal. Calcd for C23H21N3O3S: C, 65.85; H, 5.05; N, 10.02; S, 7.64. Found: C, 66.19; H, 5.17; N, 10.22; S, 7.59. 3. 1. 7. General Procedure for the Synthesis of Benzo[4,5]thieno-[2,3-£]pyrrol-2-yl)-2-(2-Cyanoacetamido)thiophene Derivatives 12a and 12b To a solution of either compound 8a (3.14 g, 0.01 mol) or 8b (3.61 g, 0.01 mol) in dimethylformamide (40 mL) ethyl cyanoacetate was added. The reaction mixture was heated under reflux for 2 h then poured onto ice/water. The formed solid product was collected by filtration and crystallized from ethanol. N-(4-(3-Amino-4,5,6,7-tetrahydro-1fl-benzo[4,5]thie-no [2,3-£]pyrrol-2-yl)-3-cyano-thiophen-2-yl)-1-cya-noacetamide (12a) Yellow crystals; yield: 3.43 g (90%), mp: 184-185 °C; IR (KBr, cm-1): 3482-3323 (NH, NH2), 3055 (CH aromatic), 2225, 2220 (2CN), 1705 (C=O), 1630 (C=C); 1H-NMR (DMSO-d6) 5: 1.79-1.83 (m, 4H, 2CH2), 2.25-2.26 (m, 4H, 2C6H2), 4.83 (s, 2H, NH2, D2O excha2n-geable), 5.20 (s, 2H, CH2), 6.20 (s, 1H, thiophene H-5), 8.28, 8.32 (2s, 2H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 20.3, 22.9, 25.4, 34.7, 52.7, 116.9, 117.2, 120.3, 123.1, 124.1, 124.6, 125.3, 127.2, 138.8, 141.2, Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ... 126 Acta Chim. Slov. 2017, 64, 117-128 142.6, 168.2; MS (EI): m/z (%) 381 (M+). Anal. Calcd for C18H15N5OS2: C, 56.67; H, 3.96; N, 18.36; S, 16.81. Found: C, 56.88; H, 3.58; N, 18.56; S, 16.93. Ethyl 4-(3-amino-4,5,6,7-tetrahydro-1_ff-benzo[4,5] thieno-[2,3-£ ]pyrrol-2-yl)-2-(2-cyano-acetamido) thiophene-3-carboxylate (12b) Orange crystals; yield: 2.99 g (70%); mp: 194-195 °C; IR (KBr, cm-1): 3453-3320 (NH, NH2), 3056 (CH aromatic), 2223, 1702, 1688 (2C=O), 1632 (C=C); 1H-NMR (DMSO-d6) S: 1.13 (t, 3H, J = 6.83 Hz, CH3), 1.81-1.87 (m, 4H, 26CH2), 2.22-2.25 (m, 4H, 2CH2), 4.233 (q, 2H, J = 6.83 Hz, CH2), 4.81 (s, 2H, NH2, D2O exchangeable), 5.23 (s, 2H, CH2), 6.23 (s, 1H, tliiophene H-5), 8.30, 8.34 (s, 2H, 2NH, D2O exchangeable); 13C-NMR (DMSO-d6) S: 16.0, 20.3, 22.2, 25.6, 34.8, 47.1, 51.4, 116.5, 120.4, 122.7, 123.8, 124.3, 124.9, 127.6, 133.9, 143.8, 164.3, 170.2; MS (EI): m/z (%) 428 (M+). Anal. Calcd for C20H20N4O3S2: C, 56.06; H, 4.70; N, 13.07; S, 14.97. Found: C, 56.22; H, 4.53; N, 13.31; S, 15.07. 3. 1. 8. General Procedure for the Synthesis of Hydrazoacetamide Derivatives 14a-d To a cold solution (0-5 °C) of compound 12a (3.81 g, 0.01 mol) in ethanol (50 mL) containing sodium acetate (3.50 g, 0.50 mol) either benzenediazonium chloride (0.01 mol), 4-chlorobenzene-diazonium chloride (0.01 mol), 4-methoxybenzenediazonium chloride (0.01 mol) or 4-methylaniline (0.01 mol) [prepared by adding a cold solution of sodium nitrite (0.70 g, in water (10 mL)) to a cold solution (0-5 °C) of either aniline oil (0.93 g, 0.01 mol), 4-chloroaniline (1.27 g, 0.01 mol) 4-methoxybenzenedia-zonium chloride (1.24 g, 0.01 mol) or 4-methylaniline (1.07 g, 0.01 mol) in concentrated hydrochloric acid (12 mL) with continuous stirring] was added with continuous stirring. The whole reaction mixture was left at room temperature for 1 h then the formed solid product was collected by filtration and crystallized from acetic acid. 2-((4-(3-amino-4,5,6,7-tetrahydro-1ff-benzo[4,5]thie-no[2,3-£]pyrrol-2-yl)-3-cyanothiophen-2-yl)amino)-2-oxo-W-phenylacetohydrazonoyl cyanide (14a) Red crystals; yield: 3.78 g (78%), mp: 223-224 °C; IR (KBr, cm-1): 3475-3320 (NH), 3053 (CH aromatic), 2223, 2220 (2CN), 1708 (C=O), 1630 (C=C); 1H-NMR (DMSO-d6) S: 1.77-1.85 (m, 4H, 2CH2), 2.25-2.28 (m, 4H, 2CH2), 4.80 (s, 2H, NH2, D2O exchangeable), 6.15 (s, 1H, thiophene H-5), 7.25-7.41 (m, 5H, C6H5), 8.25, 8.30, 8.56 (3s, 3H, 3NH, D2O exchangeable); 163C5-NMR (DM-SO-d6) S: 20.5, 22.9. 25.8, 34.7, 116.7, 117.0, 120.2, 121.7, 123.1, 124.0, 124.1, 124.6, 125.3, 126.9, 127.2, 129.3, 133.1, 138.8, 141.2, 142.8, 164.2, 168.7; MS (EI): m/z (%) 485 (M+). Anal. Calcd for C24H19N7OS2: C, 59.36; H, 3.94; N, 20.19; S, 13.21. Found: C, 59.42; H, 3.72; N, 20.53; S, 13.08. 2-((4-(3-Amino-4,5,6,7-tetrahydro-1H-benzo[4,5]thie-no[2,3-£]pyrrol-2-yl)-3-cyanothiophen-2-yl)amino)-W-(4-chlorophenyl)-2-oxoacetohydrazonoyl cyanide (14b) Red crystals; yield: 4.41 g (85%), mp: 194-195 °C; IR (KBr, cm-1): 3488-3315 (NH, NH2), 3056 (CH aromatic), 2225, 2220 (2CN), 1710 (C=O), 1628 (C=C); 1H-NMR (DMSO-d6) S: 1.79-1.85 (m, 4H, 2CH2), 2.23-2.27 (m, 4H, 2CH2), 4.863 (s, 2H, NH2, D2O exchangeable), 6.12 (s, 1H, thiophene H-5), 7.28-7.39 (m, 4H, C6H4), 8.23, 8.32, 8.42 (3s, 3H, 3NH, D2O exchangeable); 13C-NMR (DMSO-d6) S: 20.6, 22.4, 25.8, 34.9, 116.8, 117.3, 120.0, 121.4, 123.1, 124.0, 124.1, 124.8, 125.3, 127.2, 138.8, 140.4, 141.2, 143.4, 164.8, 168.6; MS (EI): m/z (%) 520 (M+). Anal. Calcd for C24H18ClN7OS2: C, 55.43; H, 3.49; N, 18.85; S, 12.33. Found: C, 55.70; H, 3.62; N, 18.59; S, 12.48. 2-((4-(3-Amino-4,5,6,7-tetrahydro-1H-benzo[4,5]thie-no[2,3-£]pyrrol-2-yl)-3-cyanothiophen-2-yl)amino)-W-(4-methoxyphenyl)-2-oxoacetohydrazonoyl cyanide (14c) Reddish brown crystals; yield: 4.63 g (90%); mp: 168-169 °C; IR (KBr, cm-1): 3462-3335 (NH, NH2), 3053 (CH aromatic), 2227, 2221 (2CN), 1720 (C=O), 1638 (C=C); 1H-NMR (DMSO-d6) S: 1.74-1.82 (m, 4H, 2CH2), 2.21-2.28 (m, 4H, 2CH2), 3.38 (s, 3H, OCH3), 4.88 (s, 2H, NH2, D2O exchangeable), 6.13 (s, 1H, thiophene H-5), 7.321-7.42 (m, 4H, C6H4), 8.21, 8.32, 8.45 (3s, 3H, 3NH, D2O exchangeable); 63C-NMR (DMSO-d6) S: 20.8, 22.7, 25.8, 34.3, 55.3, 116.3, 117.0, 120.3, 121.4, 123.8, 124.0, 124.0, 124.8, 125.9, 127.0, 133.2, 138.2, 140.8, 141.9, 164.9, 168.6; MS (EI): m/z (%) 516 (M+). Anal. Calcd for C25H21N7O2S2: C, 58.24; H, 4.11; N, 19.02; S, 12.44. Found: C, 58.40; H, 4.26; N, 19.11; S, 12.29. 2-((4-(3-Amino-4,5,6,7-tetrahydro-1H-benzo[4,5]thie-no[2,3-£]pyrrol-2-yl)-3-cyanothiophen-2-yl)amino)-2-oxo-W-(p-tolyl)acetohydrazonoyl cyanide (14d) Reddish brown crystals; yield: 3.44 g (69%); mp: 129-130 °C; IR (KBr, cm-1): 3482-3318 (NH, NH2), 3057 (CH aromatic), 2227, 2220 (2CN), 1712 (C=O), 1630 (C=C); 1H-NMR (DMSO-d6) S: 1.76-1.83 (m, 4H, 2CH2), 2.23-2.28 (m, 4H, 2CH2), 26.65 (s, 3H, CH3), 4.86 (s, 2H2 , NH2, D2O exchangeable), 6.11 (s, 1H, thiophene H-5), 7.302-7.329 (m, 4H, C6H4), 8.23, 8.30, 8.48 (3s, 3H, 3NH, D2O exchangeable); 613C4-NMR (DMSO-d6) S: 20.4, 22.9, 23.3, 25.8, 34.6, 116.4, 117.3, 120.6, 122.8, 123.8, 124.0, 124.3, 124.8, 125.2, 126.4, 138.8, 140.6, 141.7, 143.9, 164.6, 168.7; MS (EI): m/z (%) 500 (M+). Anal. Calcd for C25H21N7OS2: C, 60.10; H, 4.24; N, 19.62; S, 12.84. Found: C, 60.32; H, 4.52; N, 19.48; S, 12.64. 3. 1. 9. General Procedure for the Synthesis of Thieno[2,3-#]pyridine Derivatives 15a and 15b A suspension of either compound 12a (3.81 g, 0.01 mol) or 12b (4.28 g,0.01 mol) in sodium ethoxide (0.02 Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ... Acta Chim. Slov. 2017, 64, 117-128 127 mol) [prepared by dissolving metallic sodium (0.46 g, 0.02 mol) in absolute ethanol (20 mL] was heated in a boiling water bath for 12 h then poured onto ice/water containing few drops of hydrochloric acid. The formed solid product was collected by filtration and crystallized from 1,4-dioxane. 4-Amino-3-(3-amino-4,5,6,7-tetrahydro-1_ff-benzo[4,5] thieno[2,3-é]pyrrol-2-yl)-6-hydroxy-thieno[2,3-é]pyri dine-5-carbonitrile (15a) Yellow crystals; yield: 2.29 g (60%); mp: > 300 °C; IR (KBr, cm 1): 3593-3355 (NH, NH2, OH), 3056 (CH aromatic), 2224 (CN), 1635 (C=C); 1H-NMR (DMSO-d6) 5: 1.75-1.85 (m, 4H, 2CH2), 2.23-2.27 (m, 4H, 2CH2), 4.68, 5.09 (2s, 4H, 2NH2, D2O exchangeable), 6.16 (s, 1H, thiophene H-5), 8.28 (s, 1H, NH, D2O exchangeable), 9.90 (s, 1H, OH, D2O exchangeable); 13C-NMR (DMSO-d6) 5: 20.8, 22.9, 25.8, 34.7, 116.7, 120.2, 121.7, 123.1, 124.1, 124.6, 125.3, 126.5, 127.0, 129.6, 138.8, 142.8, 144.5, 162.8; MS (EI): m/z (%) 381 (M+). Anal. Calcd for C18H15N5OS2: C, 56.67; H, 3.96; N, 18.36; S, 16.81. Found: C, 56.93; H, 3.65; N, 18.48; S, 17.09. 3-(3-Amino-4,5,6,7-tetrahydro-1_ff-benzo-4,5]thieno [2,3-è]pyrrol-2-yl)-4,6-dihydroxy-thieno[2,3-è]pyridi-ne-5-carbonitrile (15b) Yellow crystals; yield: 2.79 g (73%) g); mp: 289-290 °C; IR (KBr, cm-1): 3578-3345 (NH, NH2, OH), 3056 (CH aromatic), 2222 (CN), 1628 (C=C); 1^-NMR (DMSO-d6) 5: 1.79-1.85 (m, 4H, 2CH2), 2.23-2.27 (m, 4H, 2CH2), 4.86 (s, 2H, NH2, D2O exchangeable), 6.17 (s, 1H, thiophene H-5), 8.26 (s, 1H, NH, D2O exchangeable), 10.29, 10.34 (2s, 2H, D2O exchangeable, 2OH); 13C-NMR (DMSO-d6) 5: 20.3, 22.8, 25.8, 34.7, 116.6, 120.2, 121.6, 123.1, 124.7, 124.1, 124.8, 125.3, 126.8, 127.5, 133.2, 140.8, 143.8, 144.2, 162.9; MS (EI): m/z (%) 382 (M+). Anal. Calcd for C18H14N4O2S2: C, 56.53; H, 3.69; N, 14.65; S, 16.77. Found: C, 56.72; H, 3.46; N, 14.80; S, 16.37. 4. Conclusions Novel 4,5,6,7-tetrahydro-1#-benzo[4,5]thieno[2,3-ft]pyrrol-derivatives were synthesized in good yields. Some compounds were used to produce annulated products. The cytotoxicity of the newly synthesized compounds indicate that compounds 4, 7a, 7b, 8a, 8b, 10c, 10d, 10f, 12a, 12b, 14b and 15b showed the highest potency among the tested compounds. In addition, the anti- proliferative evaluations of these twelve compounds indicated that the benzo[4',5']thieno[3',2':4,5]pyrrolo[3,2-è]pyridine derivative 7b and the benzo[4,5]thieno-[2,3-ft]pyrrol-2-yl)-thiophene derivative 8b showed high potency against Molt4/C8 and CEM cell lines and their IC50's are higher than the reference drug "doxorubicin". 5. Acknowledgments R. M. Mohareb would like to thank the Alexander von Humboldt for affording him regular fellowships in Germany for doing research and completing this work. 6. References 1. S. Xue, H. Guo, M. Liu, J. Jin, D. Ju, Z. Liu, Z. Li, Eur. J. Med. Chem. 2015, 26, 151-161. http://dx.doi.org/10.1016/j.ejmech.2015.04.016 2. L. Ye, J. He, Z. Hu, Q. Dong, H. Wang, F. Fu, J. Tian, Food Chem. 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Cito-toksičnost sintetiziranih spojin smo preverili na rakavih celicah želodčnega (NUGC), črevesnega (DLD-1), jetrnega (HA22T in HEPG-2) ter nazofaringealnega karcinoma (HONE-1), raka dojk (MCF-7) in na normalnih fibroblastnih celicah (WI-38). Izkazalo se je, da imajo spojine 4, 7a, 7b, 8a, 8b, 10c, 10d, 10f, 12a, 12b, 14b in 15b optimalni citotok-sični učinek na rakave celice. Spojini 7b in 14b kažeta maksimalni inhibicijski efekt, ki je precej večji od efekta referenčne spojine CHS-828 (piridil cianogvanidina). Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ... DPI: 10.17344/acsi.20l6.2956_Acta Cliim, Slov. 2017,64, 129-143_©commons 129 Scientific paper Green Biosynthesis of Spherical Silver Nanoparticles by Using Date Palm (Phoenix Dactylifera) Fruit Extract and Study of Their Antibacterial and Catalytic Activities Saeed Farhadi,1* Bahram Ajerloo1 and Abdelnassar Mohammadi2 1 Department of Chemistry, Lorestan University, Khoramabad 68151-44316, Iran 2 Department of Biology, Lorestan University, Khoramabad 68151-44316, Iran * Corresponding author: E-mail: sfarhadi1348@yahoo.com Tel.: +98-6633120618, fax: +98-6633120611 Received: 30-09-2016 Abstract In this work, we have synthesized spherical silver nanoparticles (Ag NPs) by a low-cost, rapid, simple and ecofriendly approach using Date palm fruit extract as a novel natural reducing and stabilizing agent. The product was characterized by UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), energy-dispersive X-ray (EDX) spectroscopy and Zeta potential measurements. The reaction conditions including time, content of reducing agent and silver nitrate, temperature and pH were investigated. The optimum yield of Ag NPs was obtained when 10 mM of silver nitrate was reacted with Date fruit extract at pH 11 and heated it to 55 °C within 10 minutes. The elemental and crystalline nature of Ag NPs were confirmed from EDX and XRD analysis. SEM and TEM images showed that the Ag NPs were spherical and with sizes in the range of 25-60 nm. On the base of FT-IR analysis, it can be stated that the functional groups present in bio-molecules of Date fruits are responsible for the reduction and stabilization of Ag NPs, respectively. The Ag NPs showed good antibacterial activity against a few human pathogenic bacteria. The catalytic activity of the Ag NPs for rapid and efficient reduction of toxic nitro compounds into less toxic corresponding amines by using NaBH4 was also investigated. Keywords: Biosynthesis, Silver nanoparticles, Date palm fruit extract, Antibacterial activity, Nitro reduction, Catalyst 1. Introduction Among various transition metal nanoparticles, silver nanoparticles (Ag NPs) have attracted considerable attention in nanoscience and nanotechnology due to their excellent optical and electronic properties as well as their wide applications in various fields such as catalysis,1 surface enhanced Raman scattering,2 degradation of environmental pollutants,3 biosensors4 cancer therapy5 and antibacterial effects.6 Several synthetic strategies have been developed for the synthesis of Ag NPs including photoc-hemical,7 sonochemical,8 sovothermal9 and spin coating methods.10 Among these, chemical reduction of a silver ions (Ag+) in presence of a stabilizer is the most frequently applied method for the preparation of Ag NPs as stable colloidal dispersions in water or organic solvents.11 The major drawback of chemical method is that the high- ly reactive chemical reductants as well as the stabilizers such as synthetic polymers, surfactants and dendrimers used in this method cause chemical toxicity and serious environmental problems, thus limiting their utility. In recent years, biosynthesis of metal nanoparticles has received considerable attention due to the growing need to develop clean and nontoxic chemicals, environmentally friendly solvents and renewable materials.12 The selection of a non-toxic reducing agent, a cost-effective and easily renewable stabilizing agent and an environmentally benign solvent system are the three main criteria for a greener metal nanoparticles synthesis. In this regard, a great deal of effort has been devoted toward the biosynthesis of silver nanoparticles using bac-teria,13-17 fungi,18-20 actinomycetes,21-23 yeast24 and viruses25-27 but the rate of nanoparticle synthesis is faster using fruits and plants extracts than microbes, and the pro- Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles 130 Acta Chim. Slov. 2017, 64, 129-143 duced nanoparticles are more stable.28 In recent regards, the synthesis of Ag NPs has been reported by using the natural extract of leaves, seeds and or roots of plants such as Nelumbo nucifera,29 Anisochilus carnosus,30 Mi-musops elengi,31 marine macroalga Chaetomorpha li-num,32 Bunium persicum,33 Olea europaea,34 Hamamelis virginiana,35 Justicia adhatoda,36 Suaeda acuminata,37 Mentha piperita,38 Phlomis,39 Pennyroyal,40 Murraya keenigii,41 Mangifera indica,42 Nicotiana tobaccum,43 Bunium persicum,44 Hamamelis virginiana.45 However, the reaction time of Ag+ ions for complete reduction in these works was very long. To enable the biosynthesis methods of Ag NPs to compete with the chemical methods, there is a need to achieve faster synthesis rates with high monodispersion. The use of fruit extracts of plants is an appropriate candidate for this purposes. Several papers on the synthesis of Ag NPs using the extract of fruits such as Terminalia chebula46 Solanum triloba-tum41 Dillenia indica,48Solanum lycopersicums49 Tana-cetum vulgare,50 Crataegus douglasii,51 Emblica Offici-nalis,52 and Kiwifruit 53 have been reported in the literatures. The Date palm tree (Phoenix dactylifera), a tropical and subtropical tree, is one of mankind,s oldest culti- vated plants, and it has played an important role in the day-to-day life of the people for the last 7000 years.54 Dates are produced in 35 countries worldwide and cultivated on about 2.9 million acres of land. The world production of date fruit estimate to be more than 7000000 metric tons, and Iran (14% of world production) is the second major producer after Egypt (11% of world production). Figure 1 shows the photographs of Date palm trees ant their fruits. The Date fruit is considered to be an inexpensive and easily available important fruit in Iran.55 The Date palm fruits are an important source of nutrition, especially in the arid regions where due to the extreme conditions, very few plants can grow. Date fruit also shows some functional properties in the food industry, such as water-holding, oil-holding, emulsifying and gel formation. Indeed, Date fruit can be incorporated in food products to modify textural properties, avoid synthesis and stabilize high fat food and emulsions.56 The study by Abdelhak has shown that different varieties of ripe Date fruits contained mainly p-coumaric, ferulic and sinapic acids and some cinnamic acid derivatives.51 The in vitro study by Vayalil reported that the aqueous extract of Date fruits has antioxidative and antimutagenic proper-ties.58 On the other hand, the study by Bilgari had shown Figure 1(a)-(d) Photographs of Date palm trees and their fruits. Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles Acta Chim. Slov. 2017, 64, 129-131 143 a strong correlation between the antioxidant activity and the total phenolic and total flavonoids of palm dates.59 The Date fruit is rich in phytochemicals like carbohydrates and sugars, phenolics, sterols, carotenoids, anthocya-nins, procyanidins, and flavonoids.60 Most of the biomo-lecules can act as reducing and capping agent in the reactions. Then, the Date fruits extract that are inherently rich in these phytochemicals could be used as a novel reducing agent for synthesizing Ag NPs in large-scale production. In this paper, we report on rapid, simple and low-cost synthesis of Ag NPs by the reduction of aqueous Ag+ solution using Date fruit extract. To our knowledge, this is the first report on the use of Date fruit for the rapid synthesis of Ag NPs. The nearly monodisperse Ag were formed under mild conditions, without any additive protecting nano-particles. The formation of Ag NPs was recorded by the UV-visible spectra. Additionally, the obtained Ag NPs were analyzed by Fourier transform infrared (FT-IR) spectra, and X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray (EDX) spectroscopy. The rapid approach using Date fruit extract would be suitable for developing a biological process for large-scale production. Various parameters (e.g. concentration of the reactants, reaction temperature, pH and time) were optimized that would increase the yield of nanoparticle synthesis. The antibacterial and catalytic activities of the biologically synthesized Ag NPs were also investigated. 2. Experimental 2. 1. Materials Silver nitrate (AgNO3), NaBH4, 4-nitrophenol, and 4-nitroanilin were obtained from Merck and were of analytical grade. Double distilled de-ionized water was used for the experiments. All glass wares were properly washed with distilled water and dried in oven. 2. 2. Preparation of Date Palm Fruit Extract Date Palm fruit extract was used as a reducing and stabilizing agent for the synthesis of Ag NPs. Date palm fruits were purchased from local supermarket in Iran and used for the synthesis of silver nanoparticles. The fresh fruits of Date washed repeatedly with distilled water to remove the dust and organic impurities present in it. About 15 g of fruit were crushed into fine pieces with sterilized knife. The fruit of Date Palm were taken into the 250 ml beaker containing 100 mL double distilled de-ionized water and then the solution was stirred for 30 min and filtered through Whatman No.1 filter paper twice. The obtained light yellow extract was stored in refrigerator at 4 °C. The extract is used as reducing agent as well as stabilizing agent. 2. 3. Synthesis of Ag Nanoparticles In a typical experiment, Ag NPs were prepared by using Date fruit extract as follows: in a 50 mL round-bottom flask equipped with a magnet bar, 3 ml of aqueous solution of Date fruit extract was mixed with 20 ml of 10 mM aqueous silver nitrate solution. The mixture was then heated at 55 °C under constant stirring for an appropriate time (e.g. 10 min) in an oil bath. The formation process and the optical properties of the silver nanoparticles were identified from both the color change and UV-Vis spectra of the solution. In order to remove the Ag NPs product, the solution was centrifuged at 5500 rpm for 20 min. The supernatant was decanted and the precipitate was re-dispersed in double distilled water for another round of cen-trifugation. The precipitate was then washed with deioni-zed water for three times to remove any impurities if any. Finally, the washed precipitate was dried in an oven maintained at 60 °C for 2 h and finally ground into powder for characterization. In a similar manner described above, a series of experiments were conducted to investigate the effect of various parameters including reaction time, Ag+ ion concentration, the Date fruit extract amount, pH and temperature on the reaction. The reaction mixtures were monitored by a UV-Vis spectrophotometer at different time intervals and the Ag NPs were characterized further. The effect of pH on the Ag NPs synthesis was determined by adjusting the pH of the reaction mixtures (10 mM silver nitrate, 3 mL date extract) to 3, 5, 7, 9, 11 or 13 by using 0.1 M HCl or NaOH aqueous solutions. The effect of the silver salt was determined by varying the concentration of silver nitrate (0.1, 1, 10 and 100 mM). The Date fruit extract content was varied to 1, 3, 5, 7, 9 mL, while keeping the silver nitrate concentration at a level of 10 mM. To study the effect of temperature on nanoparticle synthesis, reaction mixtures containing 3 mL Date extract, and 10 mM Ag-NO3 at pH 11 were incubated at 25, 40, 55 or 70 °C. 2. 4. Methods of Characterization The UV-visible absorption spectra of Ag NPs colloidal solutions were recorded on a double beam UV-visible spectrometer (Cary 100, VARIAN) operated at a resolution of 2 nm with quartz cells with path length of 1 cm in 300-800 nm range. Blanks were prepared with deionized (DI) water. Infrared spectra were obtained using a FT-IR 160 Schimadzu Fourier transform infrared spectrophotometer using KBr pellets. The XRD pattern of the silver nanoparticles was obtained on an X-ray diffractomer (PANalytical/X'Pert Pro MPD) using Cu Ka (1.54059 A) radiation. The particle size and shape was confirmed using a scanning electron microscope (MIRA3 TESCAN) equipped with EDX attachment. Transmission electron microscopy (TEM) observations were conducted on a Philips CM120 microscope at the accelerating voltage of 200 kV. AFM images were recorded on a multi-mode ato- Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles ... 132 Acta Chim. Slov. 2017, 64, 129-143 mic force microscopy (ARA-AFM, model Full Plus, ARA Research Co., Iran). The surface charge of samples was measured with Zeta potential measurements in water (NICOMP 380ZLS Zeta potential/Particle sizer). Magnetic measurements were carried out at room temperature using a vibrating sample magnetometer (VSM, Magnetic Daneshpajoh Kashan Co., Iran) with a maximum magnetic field of 10 kOe. 2. 5. Antibacterial Tests Antibacterial activity of the biosynthesized Ag NPs was evaluated against strains of Gram-positive bacteria: Bacillus cereus (PTCC 1015), Staphylococcus aureus (1431) and Staphylococcus epidermidis (PTCC 1114), Gram-negative bacteria: Escherichia coli (PTCC 1330) and Klebsiella pneumonia (PTCC 1290) by modified Kirby-Bauer disk diffusion method [66]. Bacteria were cultured for 18 h at 37 °C in Nutrient agar medium and then adjusted with sterile saline to a concentration of 2 x 106 cfu/mL. Bacterial suspension in Petri dishes (8 cm) containing sterile Mueller-Hinton agar (MA) were cultured using a sterile cotton swab. The compounds were dissolved in water and sterile paper discs of 6 mm thickness were saturated with 30 |l of silver nanoparticles and then placed onto agar plates which had previously been inoculated with the tested microorganisms. Amikacin (30 |g/disk) for gram negative and penicillin for gram positive (10 |g/disk) was used as positive controls. After incubation at 37 °C for 24 h, the diameter of inhibition zone was measured. The diameter of such zones of inhibition was measured using a meter ruler, and the mean value for each organism was recorded and expressed in millimetres. 2. 6. Catalytic Tests In order to study the catalytic performance of the biosynthesized Ag NPs, the reduction of 4-nitrophenol (4-NP) to 4-amiophenol (4-AP) by excess sodium borohydri-de (NaBH4) in aqueous solution was used as the model reaction. In a typical catalytic reaction, 3 mL of aqueous solution of 4-NP (0.1 mM) and 0.5 mL of aqueous NaBH4 (10 mM) solution were mixed together in a standard quartz cell, having 1 cm path length and then 1 mL of aqueous Ag suspensions (0.5 mg mL-1) was added to the reaction mixture under constant magnetic stirring. Immediately after that, the solution was transferred to a standard quartz cell, and the concentration of p-nitrophenol in the reaction mixture was monitored by the UV-visible absorption spectra recorded with a time interval of 2 min in a scanning range of 200-800 nm at ambient temperature. For recycling experiment, after completion of the reaction the catalyst was recovered by centrifugation. The precipitate was washed repeatedly with deionized water in consecutive washing cycles. Ultrasonic treatment was used in every cycle in order to re-disperse the catalyst and remove adsorbed impurities. After washing, the catalyst was used directly for recycling test. After each recycle, the centrifuge supernatant was collected and detected by Atomic absorption spectroscopy to determine the content of Ag metal. The reduction 4-nitroaniline was also investigated under the same conditions. 3. Results and Discussion 3. 1. Phytoreduction of Silver Ions A study on phytosynthesis of Ag NPs by the aqueous fruit extract of date was carried out in this work. During the visual observation, silver nitrate treated with date fruit extract showed a color change from yellow to brown within 20 min whereas no color change could be observed in silver nitrate solution without date extract (Figure 2). The appearance of yellowish brown color in fruit extract treated flask is a clear indication for the formation of Ag NPs. This color arises due to excitation of surface pla-smon resonance (SPR) vibrations in Ag nanoparticles. Figure 2. Photographs of: (a) aqueous extract of date fruits, (b) 10 mM of aqueous AgNO3 solutions, and (c) Colloidal aqueous Ag NPs solution formed by reduction of AgNO3 with Date fruit extract. 3. 2. UV-Visible Absorption Studies UV-Vis spectroscopy is a powerful tool to study the formation of Ag NPs. The reaction mixtures containing silver salt and Date fruit extract were, therefore characterized by UV-Visible spectroscopy. Based on UV-Vis spectroscopy various chemical and physico-pa-rameters (concentration of the fruit extract and silver salt, pH, temperature and reaction time) were optimized for the reduction Ag+ ions to Ag NPs using Date fruit extract. To optimize the reaction time, a time variation study was carried out using the concentration of AgNO3 (10 mM) and aqueous date extract (3 mL). Figure 3(a) shows the UV-Vis absorption spectra of Ag NPs synthesized at Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles Acta Chim. Slov. 2017, 64, 129-133 143 different time durations. It is observed that the intensity of SPR bands increases as the reaction time progresses and within 10 min a considerable intensity of the SPR bands is achieved. However, these values were hardly changed after 10 min. It suggested that the reduction time of Ag+ was almost completed within 10 min in the presence of date extract. Therefore, the optimal reaction time for the reduction Ag+ ions to Ag NPs using Date fruit extract is 10 min. As shown in the inset of Fig. 3(a), after the reaction between Ag+ and date extract, the color was changed from clear yellow to dark brown and it shows the formation of Ag NPs. Next, various concentrations of silver nitrate solution (0.1-100 mM) were reacted with 3 mL fruit extract. Figure 3(b), shows the UV-Vis absorption spectra of Ag NPs obtained at different concentrations of AgNO3 (0.1, 1, 10 and 100 mM). At 0.1 mM concentration, an observable SPR band was not appeared, indicating very low yield of Ag NPs formed (Figure 3(b), curve i), but with increasing concentration of AgNO3 to 1 mM, the SPR of Ag NPs appears at 395 nm and remarkably increases with the increase of AgNO3 concentration to 10 mM with increasing in the peak wavelength to 410 nm (Figure 3(b), curves i and ii, respectively). High intensity of the 410 nm SPR band indicates increasing concentration of nanoparticle. However, further increasing the concentration of AgNO3 from 10 to 100 mM did not increase the SPR band further-in contrast, it give a broad SPR band with decreased intensity and shifted to longer wavelength region (~425 nm). This phenomenon may be due to the fast growth of the particles at high concentration. The appearance of red shifted band at higher concentration of AgNO3 suggests the formation of larger particles. The yield of Ag NPs increased with the increase in silver nitrate concentration (0.1-10 mM) and maximum yield was obtained with 10 mM, and this concentration was selected for further studies. Additionally, the effect of the date extract amount on the synthesis of Ag NPs was investigated under the provided reaction conditions, and the results are shown in Figure 3(c). As observed, with increasing the date extract quantity from 1 to 3 mL in 20 mL of 10 mM Ag+ ion solution, the intensity of characteristic SPR absorption bands for Ag NPs increases (Figure 3(c), curves i and ii) and then decreases when the date extract increases further (Figure 3(c), curves iii-v). The maximum absorption was found at a concentration of 3 mL fruit extract. From the UV-Vis absorption spectrum in Figure 3(c), it was observed that there is a shift in wavelength from 400 to 412 nm indicating a redshift with increase in date extract concentration from 1 to 3 mL. Accordingly, it can be concluded that with the increase in Date extract amount, the size of Ag nanoparticles increases. The temperature also affected the process of silver reduction. The effect of reaction temperature was also evaluated with varying reaction temperatures from 25 to 75 °C (Figure 3(d)). As shown in Figure 3(d) (curves i and ii), the reaction mixtures incubated at room temperature (25 °C) and 40 °C showed less pronounced SPR peaks during a long time of 50 min while by heating the reaction mixtures at 55 and 70 °C the reduction process was faster and the intense peaks were developed within a short time of 10 min (Figure 3(d), curves iii and iv). This indicates that higher temperature facilitates the formation of Ag NPs due to the increase in the reaction rate. The maximum SPR peak intensity was detected at 70 °C. However, a slight increase in SPR band intensity occurs at 75 °C when compared with the temperature of 55 °C. Then, the temperature of 55 °C is preferred for further study. It is noteworthy to mention that with the increase in reaction temperature, UV-Vis spectra show sharp narrow peaks at lower wavelength regions (~412 nm at 55 and 70 °C), which indicate the formation of smaller nanoparticles, whereas, at lower reaction temperature, the peaks observed at higher wavelength region (425 nm at 25 °C) which clearly indicates increase in silver nanoparticles size. It is a well-known fact that when the temperature is increased, the reactants are consumed rapidly leading to the formation of smaller nanoparticles [61, 62]. Among the various parameters, the initial pH of solution plays a significant role in the synthesis of metal na-noparticle. Thus, in the present study, the effect of pH on the synthesis of Ag NPs was studied at acidic, natural and basic values using 3 ml Date fruit extract and 10 mM Ag-NO3. As can be seen in Figure 3(e) (curves i and ii) the formation of Ag nanoparticles was not observed at all at acidic pHs 3 and 5. Under the acidic conditions, biomole-cules are likely to be inactivated. This suggests that acidic pH is not favorable for the Ag NPs synthesis. At pH 7, the Ag NPs formation was observed at relatively low concentration, as confirmed by the appearance of a weak absor-bance band at about 425 nm (Figure 3(e), curve iii). However, Ag NPs were readily obtained at pH higher than 7, as evidenced through progressive evolution of the characteristic SPR band in the spectral region from 400 to 415 nm. As can be seen in Figure 3(e) (curves iv-vi), the intensity of the SPR band of these Ag NPs increased significantly upon increasing the pH to 9, 11 and then 13, indicating that correspondingly higher yields of Ag NPs were obtained, probably due to the presence of a considerable number of reactive functional groups to bind with silver ions. In addition, a slight red shift of the SPR band of the Ag NPs (from 400 to 415 nm) occurred upon increasing the pH. These results suggest that larger-diameter Ag NPs were obtained at higher pHs. The optimal pH for nanopar-ticle synthesis was chosen to be pH 11, which is in good agreement with the reported literature.63 The differences in the amount of Ag NPs obtained over the range of pH could be ascribed to a variation in the dissociation constants (pKa) of functional groups (OH and COOH) on the biomolecules that are involved.64 Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles ... 134 Acta Chim. Slov. 2017, 64, 129-143 a) CO (H) (m) (¡V) m ■■V Curve [Ag+]mH -1-1-1- 300 400 500 600 700 Wavelength (nm) 800 300 —i-r 400 500 600 700 Wavelength (nm) c) _ Extract Lun/e amounts(mL) BOO 300 400 S00 600 700 Wavelength (nm) 800 d) -i-1-1-1300 400 500 600 700 Wavelength (nm) BOO 300 800 Wavelength (nm) Figure 3. Effect of various parameters on the synthesis of Ag NPs: (a) The effect of reaction time; The inset photo shows the color change of solution with time of reaction, (b) The effect of Ag+ Concentration; The inset photo shows the color change of solution at different concentrations of AgNO3, (c) The effect of different amounts of Date fruit extract, (d) the effect of different temperatures and (e) the effect of pH. 3. 3. XRD Analysis Figure 4 shows the XRD pattern of Ag NPs synthesized using Date fruit extract after the complete reduction of Ag+ to Ag under the optimized conditions (10 mM Ag-NO3, 3 mL Date extract, pH 11 at 55 °C for 10 min). As observed in the XRD pattern, the four characteristic diffraction peaks at 29 values of 38.10°, 44.15°, 64.67°, and 77.54° can be indexed to the (111), (200), (220), and (311) reflection planes of faced center cubic (fcc) structure of silver (JCPDS card no 04.0784). The considerable broadening of the diffraction peaks demonstrates the nanometer nature of the Ag particles. The average crystallite size of the Ag product is approximately 39.5 nm as estimated by the Debye-Scherrer equation: Dxrd = 0.9X/(3cos9), where Dxrd is the average crystallite size, X is the wavelength of Cu Ka radiation, ¡3 is the corrected full-width at half-maximum of the main diffraction peak of (111), and 9 is the Bragg angle. The XRD pattern obtained is consistent with earlier reports.65,66 Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles Acta Chim. Slov. 2017, 64, 129-135 143 Figure 4. XRD pattern of Ag NPs synthesized by Date fruit extract. 3. 4. SEM, TEM and EDX Analysis The size and morphology of the Ag NPs were determined via SEM, TEM and AFM images. Figure 5 shows the SEM images of the as-prepared Ag NPs. From the SEM images in different magnifications (Figure 5(a)-(c)), it is clearly evident that the product consists of extremely fine particles with sphere-like morphologies that appreciably aggregated as clusters due to the extremely small dimensions and high surface energy of the obtained nanoparticles. We also can find from the images that the morphology of the particles is almost homogeneous. The resulting images show the presence of large number of spherical nanoparticles with an average particle size of 42.5 nm. The EDX was used to further characterize the composition of the sample. Figure 5(d) shows the EDX spectrum of the Ag NPs prepared by using Date Figure 5. (a-c ) SEM images of the as-prepared Ag NPs, (d) EDX elemental spectrum of the Ag NPs. The inset of Figure 5(d) shows EDX elemental mapping for Ag NPs. Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles ... 136 Acta Chim. Slov. 2017, 64, 129-143 fruit extract as reducing agent. The intense peaks around 3.40 keV and 3.45 keV are correspond to the binding energies of Ag KLa and Ag KLj3, respectively, while the peaks situated blow 0.5 keV corresponding of N, C and O from Date fruit extract. Further, the EDX elemental mapping of the product in the inset of Figure 5(d) displays the uniform distribution of the Ag element. The results further indicate that the Ag NPs have been successfully prepared in this work. The TEM image and size distribution of the Ag NPs are shown in Figure 6. The TEM sample was prepared by dispersing the powder in ethanol by ultrasonic vibration. It can be seen from Figure 6 that the nanoparticles show approximately sphere-like morphologies with a uniform size. Because of the small dimensions and high surface energy of the particles, it is easy for them to aggregate. We also can find from this figure that the morphology of the particles is almost homogeneous. To investigate the size distribution of the Ag NPs, the particle size histogram was also determined from the TEM image. The inset of Figure 6 shows the size distribution of the Ag particles. It is clear that the diameter sizes of the Ag NPs are in the range of 25 to 60 nm with a narrow size distribution. The average particle size is approximately 40 nm, which is in agreement with the result calculated for the half-width of diffraction peaks using the Scherrer's formula, allowing for experimental error. AFM is a beneficial tool for studying various morphological features and parameters. Since, it has the advantage of probing in deep insights of surface topography qualitatively due to its both lateral and vertical nanometer scale spatial resolution. The AFM images in Figure 7 display the surface morphology of the Ag-NPs formed by Date fruit extract. As observed in Figure 7(a), AFM image Figure 6. TEM image of the Ag NPs. The inset shows the size distribution of the Ag NPs. reveals the appearance of spherical nanoparticles and their respective particle size and morphology clearly were close to those determined by the SEM and TEM images. As can be seen from Figure 7(b), the surface of Ag NPs sho- 53.43 r ■39,95 1 Figure 7. (a) and (b) AFM images of the Ag NPs. Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles a) Acta Chim. Slov. 2017, 64, 129-137 143 wed a dense and uniform packed structure. Thus, the Ag NPs could provide a biocompatible and rough surface for biological uses, e.g., cell immobilization. 3. 5. Zeta Potential Measurements Zeta potential provides the information about the stability of nanoparticles and surface charge. Zeta potential is an essential parameter for characterization of stabi- lity in aqueous colloidal Ag-NPs suspensions. Zeta potential of the synthesized Ag NPs is pictured in Figure 8. The zeta potential value was measured to be about -35 mV which confirms the good stability of the colloidal Ag NPs aqueous suspension formed by reduction of AgNO3 with Date fruit extract.67 The high negative values illustrate the repulsion between the particles and thereby attainment of better stability of Ag NPs formation avoiding agglomeration in aqueous solutions. Figure 8. Zeta potential analysis of colloidal Ag NPs solution prepared with Date fruit extract. 3. 6. FT-IR Chemical Analysis The identification of the possible biomolecules responsible for the reduction and the stabilization of biosynthesized Ag NPs can be achieved by the FTIR studies. It has been reported that the Date palm fruit is rich in phytochemicals like carbohydrates (mainly glucose, sucrose and fructose), phenolic acids, sterols, carote-noids, anthocyanins, procyanidins and flavonoids.60 Figure 9(A) shows the structures of some phytochemicals present in Date fruits As can be seen, these components are containing carboxyl (-COOH), phenolic -OH and carbonyl (C=O) functional groups. Figure 9(B) shows FT-IR spectra recorded for the Date fruit extract and the Ag NPs synthesized with the Date extract before and after washing. The FT-IR spectrum of Date extract in Figure 9(B) (spectrum a) shows phenolic O-H, C=O, and a) i i i i i i i 401)0 .«00 «00 2800 2100 2 WW ¡6(1 u ¡200 800 400 Wave number (cm-1) Figure 9. (A) The structure of some of phytochemicals present in Date fruits: (a)-(c) Phenolic acids, (d) a Flavonoid, (e) a Procyanidin, (f) a Sterol, (g) a Carotenoid. (B) FT-IR spectra of: (a) Date palm fruit extract, (b) Ag NPs capped with Date fruit extract solution and (c) Ag NPs after washing with deionized water Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles ... 138 Acta Chim. Slov. 2017, 64, 129-143 C-OH stretching bands, corresponding to a number of bands at 3475, 1639, and 1039 cm-1, respectively. The absorption bands at 2918, 1420, and 1352 cm-1 are related to the C-H stretching bands in Date fruit. As shown in Figure 9(B), (spectrum b), after the reduction of Ag-NO3 the decreases in intensity of bands at 3450 and 1039 cm-1 and redshift of these bands signify the involvement of the OH groups in the reduction process. On the hand the shift of the band from 1639 cm-1 to 1630 cm-1 is attributed to the binding of C=O groups with Ag NPs. On the base of FT-IR analysis, it can be stated that the hydroxyl, carboxyl and carbonyl functional groups present in carbohydrates, flavonoids, tannins and phenolic acids of Date fruit extract may be accountable for the reduction of the Ag+ ions and stabilization of Ag NPs. In an experiment, the Ag NPs capped with Date extract were washed with deionized water for three times and the FT-IR spectrum of the dried precipitate was again taken for the purity of the sample. As can be clearly seen in Figure 9(B), (spectrum c), the intensity of the characteristic bands of biomolecules markedly decreases after washing the product, confirming the removal of biomolecules on the surface of Ag NPs. From the FTIR analysis and previously reported mechanisms,68-70 it can be stated that the hydroxyl and carbonyl groups present in carbohydrates, flavonoids, procyanidin and phenolic compounds are powerful reducing agents and they may be accountable for the bioreduc-tion of Ag+ ions leading to Ag0 nanoparticle synthesis. FTIR study confirms that the carbonyl groups of biomole-cules have a strong ability to bind metal ions and they may be encapsulated around the Ag NPs forming a protective coat-like membrane to avoid the agglomeration and thus results in nanoparticle stabilization in the medium. Thus, the Date fruit extract components act as bioreductants and surfactants too. The plausible mechanism of the formation of Ag NPs by using a Flavonoid biomolecule as a typical reducing agent is shown in Figure 10. In this pursuit, proteins and all secondary metabolites of extract play a critical role in both reducing and capping mechanism for na-noparticle formation. 3. 7. Antibacterial Activity of Ag Nanoparticles The antibacterial activity of Ag NPs were analyzed against five bacteria: Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumonia, and Escherichia coli by disk diffusion method. The results of the antibacterial activity of silver nanoparticles were showed in Figure 11. The Figure shows that Ag NPs have good antibacterial activity; bacteria cells have been killed at the concentration of 30 ^g/ml. Table 1 represented the inhibition zone of these bacteria. Highest activity of Ag NPs was obtained against epidermidis, while lowest activity were observed against B. cereus and E. coli. Biosynt-hesized Ag NPs exhibit more antimicrobial activity on Figure 11. Images of antibacterial activities of Discs 30 ng/mL Ag NPs on (a) E. Coli, (b) K. Pneumonia, (c) S. Epidermidis, (d) B. Cereus. (e) S. Aureus. Figure 10. The plausible mechanism of the formation of Ag NPs using Date fruit extract Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles Acta Chim. Slov. 1011, 64, 129-143 139 Table 1. Average of inhibition zones synthesized silver nanoparticles with Date fruit extract. Entry Bacteria Type Inhibition zone diameter (mm) Silver nanoparticle Disc standard E. Coli K. Pneumonia S. Epidermidis B. Cereus S. Aureus Gram-negative Gram-negative Gram-positive Gram-positive Gram-positive 11 11 17 12 13 13 13 14 11 41 gram-positive microorganism than gram-negative. The potential antimicrobial activities showed by Ag NPs have made them encouraging candidates as novel generation antimicrobials. 3. 8. Catalytic Activity of Ag Nanoparticles To evaluate the catalytic activity of the Ag NPs prepared in this work by using Date fruit extract, the reduction of 4-nitrophenol (4-NP) and 4-nitroaniline (4-NA) in aqueous solution by excess NaBH4 was used as the model systems. The catalytic process was monitored by UV-Vis spectroscopy as shown in Figure 12. From Figure 12(a), it was seen that an absorption peak of 4-NP undergoes a red shift from 317 to 400 nm immediately upon the addition of aqueous solution of NaBH4, corresponding to a significant change in solution color from light yellow to yellow-green due to formation of 4-nitrophenolate ion. In the absence of Ag NPs catalyst (0.5 mg), the absorption peak at 400 nm remained unaltered for a long duration, indicating that the NaBH4 itself cannot reduce 4-nitrophenolate ion without a catalyst. In the presence of Ag NPs catalyst and NaBH4 the 4-NP was reduced, and the intensity of the absorption peak at 400 nm decreased gradually with time and after about 24 min it fully disappeared (Figure 12(a)). In the meantime, a new absorption peak appeared at about 295 nm and increased progressively in intensity. This new peak is attributed to the typical absorption of 4-aminophe-nol (4-AP). This result suggests that the catalytic reduction of 4-NP exclusively yielded 4-AP, without any other side products. In the reduction process, the overall concentration of NaBH4 was 10 mM and 4-NP was 0.1 mM. Considering the much higher concentration of NaBH4 compared to that of 4-NP, it is reasonable to assume that the concentration of BH4- remains constant during the reaction. In this context, pseudo-first-order kinetics could be used to evaluate the kinetic reaction rate of the current catalytic reaction, together with the UV-Vis absorption data in Figure 12(a). The absorbance of 4-NP is proportional to its concentration in solution; the absorbance at time t (At) and time t = 0 (A0) are equivalent to the concentration at time t (Ct) and time t = 0 (C0). The rate constant (k) could be determined from the linear plot of ln(C/C0) versus reduction time in minutes. As expected, a good linear correlation of ln(Ct/C0) versus time was obtained as shown in the inset of Figure 12(a), whereby a kinetic reac- tion rate constant k is estimated to be 1.34 x 10 min . Figure 12(b) shows the UV-Vis absorption spectra of the reduction of 4-nitroaniline by NaBH4 at various reaction times in the presence of Ag NPs. The observed peak at 385 nm for the 4-nitroaniline shows a gradual decrease in intensity with time and a new peak appeared at 295 nm indicating the formation of p-phenylenediamine (1,4-PD). As shown in Figure 12(b), it took 24 min for the complete reduction of 4-NA in the presence of Ag NPs (0.5 mg). a) b) soo T 400 500 600 Wavelength (nm) 800 Figure 12. UV-Vis spectra of (a) 0.1 mM 4-nitrophenol (4-NP) with 10 mM NaBH4 and (b) 0.1 mM 4-nitroaniline (4-NA) with 10 mM NaBH4 in the presence of Ag NPs as catalyst. The insets show the plots of ln(Ct/C0) against the reaction time for pseudo-first-order reduction kinetics of 4-NP and 4-NA in the presence of excess NaBH4 (10 mM) in aqueous solutions. Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles 140 Acta Chim. Slov. 2017, 64, 129-143 The corresponding k value was 7.38 x 10-2 min-1 (see the inset in Figure 12(b)). The results indicated that Ag NPs exhibited considerably high activity for the reduction of nitroarenes with NaBH4 as the hydrogen donor. The reusability of catalysts is a very important parameter to assess the catalyst practicability. Therefore, the recovery and reusability of the Ag catalyst was investigated for the reduction of 4-NP under the present reaction conditions. After the completion of reaction, Ag NPs were separated from the reaction mixture by centrifugation. The catalyst was washed with water and ethanol several times, dried and employed for the next reaction. The activity of the four consecutive runs (98, 98, 97 and 95%) revealed the practical recyclability of the applied catalyst. No significant loss in activity was observed for up to four catalytic cycles, thereby indicating that the as-prepared catalyst is stable and efficient in the reduction of nitro-compounds. As shown in Fig. 13(a) and (b), XRD and SEM image of the recycled catalyst did not show significant change after the fourth run in comparison with the fresh catalyst (see Figures 4 and 5). This observation confirmed that the Ag NPs are stable under the reaction conditions and are not affected by the reactants. Moreover, we have compared the obtained results in the reduction of 4-NP with NaBH4 catalyzed by Ag NPs prepared in this work with some reported catalysts in the literature (Table 2). It is clear that with respect to the reaction conditions and/or reaction times, the present method Table 2. Comparison of the result obtained for the reduction of 4-NP in the present work with those obtained by some reported catalysts. Entry Catalyst Conditions Time Ref. 1 Ni-PVA/SBA-15 H2O, NaBH4, r.t. 85 min [71] 2 Hierarchical Au/CuO NPs H2O, NaBH,,, r.t 80 min [72] 3 Cu NPs THF/H2O, NaBH4, 50 °C 2 h [73] 4 PdCu/graphene EtOH/H2O, NaBH4, 50 °C 1.5 h [74] 5 Au-GO H2O, Na2BH4, r.t. 4 30 min [75] 6 CoFe2O4 NPs H2O, NaBH4, r.t. 50 min [76] 7 FeNi2 nano-alloy H2O, NaBH,,, r.t 60 min [77] 8 NiCo2 nano-alloy H2O, NaBH4, r.t. 30 min [78] 9 CdS/GO H2O, NaBH4, r.t. 30 min [79] 10 dumbbell-like CuO NPs H2O, NaBH4, r.t. 32 min [80] 11 Ni NPs H2O, NaBH4, r.t. 16 min [81] 12 CuFe2O4 NPs H2O, NaBH4, r.t. 14 min [82] 13 Au NPs H2O, NaBH4, r.t. 4 min [83] 14 Pd/RGO/Fe3O4 NPs H2O, NaBH4, r.t. 1 min [84] 15 Cu/Fe3O4 N3Ps4 H2O, NaBH4, r.t. 55 sec [85] 16 Cu NPs/perlite H2O, NaBH4, r.t. 2.5 min [86] 17 Ag NPs H2O, NaBH4, r.t. 24 min This work Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles Acta Chim. Slov. 2017, 64, 129-141 143 is more suitable and/or superior (Table 2, entries 1-10). It is clear that reaction in the presence of most reported catalysts required longer reaction times. However, compared with some these reports, the present catalyst also presented close or lower catalytic activity for the reduction of 4-NP (Table 2, entries 11-16). Furthermore, compared with the other catalysts, the Ag NPs can be easily prepared using Date fruit extract without the use of harsh, toxic and expensive chemicals which is very important in practical applications. 4. Conclusions In the present work, Date fruit extract was used as an effective reducing as well as capping agent for the biosynthesis Ag NPs in aqueous solution. The synthesis of Ag NPs was affected by the variation in reaction conditions such as time, temperature, concentration of extract and silver solution and pH. The synthesized Ag NPs were spherical, 25-60 nm in size, crystal in nature and showed absorption spectrum at -400-420 nm. The formed Ag NP-s were quite stable, showed good antimicrobial activity and were utilized as a catalyst for the reduction of several aromatic nitro-compounds into their corresponding amino derivatives. Thus Date extract can be effectively used for the synthesis of Ag NPs. Further experiments for the synthesis other metal nanoparticles such as Au, Pd, and Cu, using Date fruit extract are in progress in our laboratory. Synthesis of metallic nanoparticles using green resources like Date fruit extract is a challenging alternative to chemical synthesis, since this novel green synthesis is cost effective, pollutant free and eco-friendly synthetic route. 5. Acknowledgements The authors gratefully acknowledge the Lorestan University Research Council and Iran Nanotechnology Initiative Council (INIC) for their financial supports. 6. References 1. K. Nishioka, T. Sueto, N. Saito, Appl. Surf. Sci. 2009, 255, 9504-9507. https://doi.org/10.1016/j.apsusc.2009.07.079 2. Y. W. C. Cao, R. C. Jin, C. 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Optimalni reakcijski pogoji sinteze srebrovih nanodelcev (Ag NPs) so bili doseženi v primeru reakcije 10 mM raztopine srebrovega nitrata s sadnim izvlečkom datljeve palme pri pH 11 in temperaturi do 55 ° C v 10 minutah. Elementarno in kristalinično naravo nanodelcev srebra (Ag NPS) smo potrdili z EDX in XRD analizama. SEM in TEM slike so pokazale, da so nanodelci srebra (Ag NPs) sferični, z velikostjo v območju od 25-60 nm. Na osnovi FT-IR analize, lahko rečemo, da so funkcionalne skupine prisotne v bioloških molekulah sadnega izvlečka datljevih palm odgovorne za redukcijo in stabilizacijo nanodelcev srebra (Ag NPs). Dokazali smo njihovo učinkovito antibakterijsko delovanje proti nekaterim patogenim bakterijam. Preučevali smo tudi katalitsko aktivnost nanodelcev srebra (Ag NPs) za hitro in učinkovito zmanjšanje strupenih nitro spojin v manj strupene amine z uporabo NaBH4 Farhadi et al.: Green Biosynthesis of Spherical Silver Nanoparticles ... 144 DOI: 10.17344/acsi.2016.2983 Acta Chim. Slov. 2017, 64, 144-158 ^creative ^commons Scientific paper Utilization of Corn Cob and TiO2 Photocatalyst Thin Films for Dyes Removal Hui-Yee Gan,1 Li-Eau Leow1 and Siew-Teng Ong1'2* 1,2 Faculty of Science, Centre for Biodiversity Research, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900 Kampar, Perak, Malaysia * Corresponding author: E-mail: ongst@utar.edu.my; ongst_utar@yahoo.com Tel: 605-4688888; Fax: 605-4661676 Received: 11-10-2016 Abstract The effectiveness of using TiO2 and corn cob films to remove Malachite Green oxalate (MG) and Acid Yellow 17 (AY 17) from binary dye solution was studied. The immobilization method in this study can avoid the filtration step which is not suited for practical applications. Batch studies were performed under different experimental conditions and the parameters studied involved initial pH of dye solution, initial dye concentration and contact time and reusability. The equilibrium data of MG and AY 17 conform to Freundlich and Langmuir isotherm model, respectively. The percentage removal of MG remained high after four sorption cycles, however for AY 17, a greater reduction was observed. The removal of both dyes were optimized and modeled via Plackett- Burman design (PB) and Response Surface Methodology (RSM). IR spectrum and surface conditions analyses were carried out using fourier-transform infrared spectrophotometer (FTIR), scanning electron microscope (SEM) and atomic force microscope (AFM), respectively. Keywords: Malachite Green; Acid Yellow 17; Immoblization; Plackett Burman; Response Surface Methodology l.Introduction Dye is a common coloring agent used in textile, paper, ink, food and leather industries. The usage of these dyes has continuously increased and it has been reported that there are more than 100,000 commercial dyes with a rough estimated production of 7 x 105 to 1 x 106 tons per year.1,2 Although this colored pollutant imparts only a small fraction of the total organic load in wastewater, it is easily recognizable and damages the aesthetic nature of the environment. Many dyes used in these industries are difficult to degrade, as they are generally stable to light and oxidizing agents, as well as resistant to aerobic digestion. Therefore, conventional effluent treatment methods based on oxidation and/or aerobic digestion may not be effective. Malachite Green (MG) is a water soluble cationic dye that is widely used in aquaculture as an effective fungicide. However, scientific evidence indicated that MG and its metabolites, leucomalachite green (LMG) is environmentally persistent. This dye causes a serious public health hazards as both clinical and experimental observations reveal that MG is a multi-organ toxin.3-5 As MG belongs to the same group of triphenylmethane dyes as cry- stal violet, in which carcinogenic effects have been demonstrated, therefore based on this group classification, a carcinogenic effect can be assumed.4 Acid Yellow 17 (AY 17) is a mono-azo acid dye, widely used in the textile, leather, cosmetic and paper industry. It is also a common additive in household products such as shampoo, detergent, soap and shower gel.6 This dye synergizes dermatitis to sensitive skin and causes irritation to eyes. Besides, its thermal decomposition emits toxic fumes of CO, CO2 and NO.7 Due to these severe problems, water contamination originated from the dyeing and finishing in textile industry has become a major concern. The most common physical method utilized by textile industry for waste water treatment is adsorption. Amongst all, activated carbon is one of the most popular adsorbents and it has also demonstrated its efficiency in the removal of various pollutants. However, this type of adsorbent remains as a costly material and it is difficult to regenerate. Thus, there is a need to continue exploring other economical feasible treatment method for dyes removal. Maize also known as corn, is one of the major feed grains in the world. However, after the removal of corns, the abundant agriculture residues such as corn cob, corn husk, Gan et al.: Utilization of Corn Cob and TiO2 Photocatalyst Acta Chim. Slov. 2017, 64, 144-158 145 corn leaf and corn stalk are often burnt without utilization.8'9 But corn cob can actually serves as an attractive low cost adsorbent as it possesses some fairly amazing properties. It contains approximately 39.1% cellulose, 42.1% he-micellulose, 9.1% lignin, 1.7% protein and 1.2% ash.10 Apart from adsorption technique, photocatalytic oxidation is also one of the emerging technologies for the elimination of organic pollutants. From the literature, photocatalysis have demonstrated different degrees of applicability for the removal of organic pollutants from aqueous solutions and often, this is viewed as a promising method because it requires no addition of chemi-cals.11-15 The basic principle involve can be depicted as follows: once excited by light with energy higher than the band gap energy of photocatalyst, pairs of holes (h+) and electrons (e) generate and migrate to the surface to react with adsorbed reactants. The holes, together with other oxidizing species such as hydroxyl radicals resulting from certain photochemical reactions, oxidize the organic pollutants to carbon dioxide, water and some simple mineral acids.16 The main drawback for these two wastewater treatment processes was low economical feasible. Often, extra energy or equipment is required for the post-filtration, centrifugation and sedimentation process. Therefore, in this current work, attempt has been made to immobilize both corn cob and TiO2 onto a thin film to overcome the problem associated with separation of fine particles mentioned earlier. In order to further enhance the usefulness and efficiency of the proposed treatment method, the percentage uptake for both MG and AY 17 were optimized and modeled via Plackett- Burman design (PB) and Response Surface Methodology (RSM). 2. Materials and Methods 2. 1. Adsorbent Corn cob was collected from Kampar night market and cut into small pieces, approximately 2 cm/ piece. It was then washed several times with distilled water and consequently boiled for 3 hours to remove the adhering dirt and residues. The clean corn cob was then dried in oven at 60 °C for 24 hours. Dried sorbent was subsequently grinded into powder form and passed through 1 mm sieve before stored into the air tight container for further experimental use. 2. 2. Immobilization of TiO2 and Corn Cob Chitosan solution was prepared by dissolving 5.05 g of chitosan powder (coarse ground flakes and powder, Sigma-Aldrich Pte. Ltd) in 500 mL of 1% (v/v) acetic acid solution under continuous stirring for a night at room temperature to ensure all the chitosan powder was well dissolved and the solution was bubble free. TiO2 Degussa P25 (mainly in anatase form, mean particle size of 30 nm, BET surface area of 50 m2/g) was dispersed well and free from agglomeration into chitosan solution via the combination of mechanical stirring and sonica-tion methods with slight modicfication.17 Both corn cob film (1.0 g of corn cob / 63 g chitosan solution) and TiO2 film (0.25 g of TiO2 / 63 g chitosan solution) were prepared with evaporative casting method onto a 10.16 x 10.16 cm of polymer plate and dried in oven at 45 °C for 24 hours to evaporate all the moisture. The dried films were then neutralized by soaking it in 0.5 M of NaOH solution for 4 hours. Thereafter, the films were washed till neutral pH and subjected for further drying in oven at 35 °C for 24 hours. 2. 3. Adsorbates Binary dye solution was selected for this study and it involved the mixing of Acid Yellow 17, AY 17 (C.I.= 18965) and Malachite Green crystal, MG ( C.I. = 40000). Both dyes were purchased from Sigma-Aldrich Pte. Ltd and were used as received without further purification. The prepared binary dye solution was kept in dark for prevent degradation from light. 2. 4. Instrumental and Characterization Analysis The functional groups that present on corn cob film before and after dye removal process were determined using Perkin Elmer FTIR, Spectrum RX1 at the wavenum-ber range of 400-4000 cm-1 with the number of 4 scans per sample and resolution of 4.0 cm-1. The surface morphology of corn cob and TiO2 film was studied by using field emission scanning electron microscope (JEOL FES-EM JSM 6701F), operated at emission current of 3.0 kV with working distance of 4.6 mm. Besides, atomic force microscope was also employed (AFM, Park System, XE-70) to observe the surface topography of film before and after the dye removal process by using the contact mode on a 15 x 15 pm2 area. 2. 5. Batch Study Batch study was performed under the exposure of sunlight continuously for 4 hours. Light intensity was recorded at every 1 hour interval with UVA/B light meter. Based on the results from our previous studies in the laboratory, the amount of dyes adsorbed by TiO2 in dark was negligible. Both TiO2 thin film and corn cob film were immersed in 500 mL of binary dye solution (10.0 mg/L of MG and 40.0 mg/L of AY 17) in the aquarium tank. Aeration was provided by an air pump. At predetermined time intervals, a known volume of dye solution was withdrawn from the tank and analyzed for its dye content using UV-visible spectrophotometer to determine the % of dye removal. The same experimental conditions were employed Gan et al.: Utilization of Corn Cob and TiO2 Photocatalyst ... 146 Acta Chim. Slov. 2017, 64, 144-158 throughout the study unless otherwise stated. The percentage uptake of dye was calculated based on Equation 1. Percentage removal (%) = x 100 % (1) where, Co = Initial concentration of dye, mg/L Ce = Concentration of dye in equilibrium, mg/L 2. 5. 1. Effect of pH The removal of dyes at different initial pH was investigated in range of pH 4.45 ± 0.50 (natural pH of the binary dye solution) to 7. Dilute sodium hydroxide (NaOH) solution was added dropwise to adjust the pH to the desire pH, prior to the experiment. 2. 5. 2. Effect of Initial Dye Concentrations and Contact Time The effect of initial dye concentrations and contact time on the percentage uptake of MG and AY 17 was studied by using the dye concentrations of 20, 40 and 80 mg/L. Dye solution was collected at various time intervals, 5, 10, 15, 30, 60, 120, 180, 240 and 300 minutes and the concentration was determined. 2. 5. 3. Sorption Isotherm Sorption isotherms were obtained by varying the initial dye concentrations of MG from 10.0 mg/L to 50.0 mg/L and 40.0 mg/L to 80.0 mg/L for AY 17. The experiment was carried out by adding 0.1 g of corn cob film into 20 mL of binary dye solutions. This sorption mixture was then shaken at 150 rpm in a centrifuge tube at room temperature for 4 hours. 2. 5. 4. Reusability of TiO2 Film and Corn Cob Film The possibility of repetitive usage of films was studied in this parameter. The same TiO2 and corn cob films were reused for multiple sorption cycles (up to 4 cycles). Before the films were subjected for the next cycle of sorption process, the previously sorbed dyes were removed from the films by soaking it in 0.5 M NaOH solution for desorption process. This was followed by several washings until neutral and the films were air-dry. 2. 6. Statistical Approach 2. 6. 1. Evaluation of Factors Affecting the Percentage Uptake of Dyes The effect of various factors that influence the percentage uptake of MG and AY 17 were investigated with Plackett-Burman design. The validity of 3 factors including initial dye concentrations, contact time and initial pH of binary dye solution were screened by Design Expert Version 7.1.3 software to generate 12 experimental designs. 2. 6. 2. Optimization Study The factors resulted from Plackett-Burman study was continued with central composite design (CCD) model in Response Surface Methodology (RSM) by using Design Expert Version 7.1.3 software. The correlation of factors and percentage uptake for binary dye was described with modified cubic model. 3. Results and Discussion 3. 1. Instrumental Analysis 3. 1. 1. Fourier Transform Infrared Spectroscopy (FTIR) Figure 1 shows the FTIR spectra of native chitosan film and corn cob film before and after adsorption in the wavenumber range from 4000 to 400 cm-1. From the spectrum, the peak observed at 3436 and 3437 cm-1 corresponded to the amine stretching N-H and confirmed the presence of amine group in the chitosan structure. The peaks appeared at 2920 cm-1 indicated that the stretching vibration of C-H bond of methylene and methane group, whereas 2844 cm-1 shows C-H stretching for sp3 carbon atom. As for peak observed at 1632, 1638 and 1642 cm-1, this would suggest the presence of N-H bending amine groups. A weak intensity of C=C stretching bands for aromatic rings were assigned at 1425cm-1. It was noticed that the FTIR spectra of corn cob film (before and after adsorption) are very similar to each other. Apart from the limitations in the sensitivity of the instrument, this could also be due to the nature of the process. As it has been postulated that the dye removal process mainly involved adsorption, which is a surface chemistry process, therefore the FTIR spectra before and after the process would shown not much difference. Similar results were reported in the removal of Methylene Blue by using nitrilotriacetic acid modified banana pith.2 3. 1. 2. Surface Characterization The surface morphology involving shape and porosity of the films was studied using SEM. The SEM micrographs that showed the surface texture of TiO2 film and corn cob film before and after the dyes removal process was presented in Figures 2 and 4. The analysis was performed under the magnification of 10,000x. From these SEM micrographs, it is apparent that before the dyes removal process, TiO2 powders has been Gan et al.: Utilization of Corn Cob and TiO2Photocatalyst ... Acta Chim. Slov. 2017, 64, 144-158 147 evenly disperse onto the chitosan matrix. This can be observed from the homogeneity shown by the film (Figure 2a). The energy dispersive X-ray (EDX) analysis was performed on the white spots shown in Figure 2a. The Ti peaks in the spectrum (Figure 3) confirm the presence of TiO2 in the film. As for corn cob film, it is clear that it is a non-porous type of materials (Figure 4a). Significant dif- ference was observed on film morphology after it undergoes dye removal process. Both of the film's surfaces displayed less uniformity than before dyes removal. It is suggested that the rough and uneven surfaces shown in these films is due to the adhesion of dye molecules. Besides SEM, color mapping using contact mode, atomic force microscope (AFM) was also employed to Figure 2. SEM micrographs of TiO2 film before (a) and after (b) dyes removal process Gan et al.: Utilization of Corn Cob and TiO2 Photocatalyst ... 148 Acta Chim. Slov. 2017, 64, 144-158 Figure 3. EDX analysis spectrum of the white spot in TiO2 film Figure 4. SEM micrographs of corn cob film before (a) and after (b) dyes removal process define the saturation of film's surface. This is one of the usual methods used for displaying data whereby high features or high topography is illustrated by lighter color and vice versa. From the images obtained (Figures 5-6), films after the dyes removal process exhibited lighter color and rougher surface. This is most probably caused by the agglomeration of dyes. During the removal process, with the introduction of dye molecules on the surface of the films, these films become more intense and this ex- plains the higher topography shown after the removal process. 3. 2. Effect of Initial pH of Dye Solution Figure 7 shows the percentage uptake of MG and AY 17 from natural pH of binary dye solution (4.54) to 7 after 4 hours of contact time. The pH of dye solution is a crucial controlling parameter as it is going to influence the Gan et al.: Utilization of Corn Cob and TiO2Photocatalyst ... Acta Chim. Slov. 2017, 64, 144-158 149 Figure 5. AFM image of TiO2 film before (a) and after (b) dyes removal process Figure 6. AFM image of corn cob film before (a) and after (b) dyes removal process Figure 7. Effect of pH in the removal of MG and AY 17 aqueous chemistry as well as the surface binding sites of adsorbent.18 Generally, the removal of MG should be increased as the pH of the solution increased whereas for AY 17, higher removal will be facilitated at low pH. However, the current results obtained indicated that the removal of both dyes was more favorable in acidic condition and this agreed well with some of the previously reported works.19-21 The high affinity shown by the films in acidic pH can be attributed to the usage of chitosan as the supporting matrix in this study. At lower pH, amino groups of chito-san can be easily protonated to form -NH3+. With decreasing pH, there will be more protons available to protonate amino groups of chitosan and this enhance the attraction of negatively charged dye (AY 17) towards the cationic amines.22,23 However, as chitosan is a type of pH sensitive cellulose biopolymer which will dissolve and formed hydrogel under extreme acidic condition, therefore the effect of pH was not carried out beyond pH 4. And since by using the natural pH of the binary dye solution, an appreciate amount of both dyes could be removed simultane- Gan et al.: Utilization of Corn Cob and TiO2 Photocatalyst ... 150 Acta Chim. Slov. 2017, 64, 144-158 ously, therefore no pH adjustment was carried out in subsequent experiments. 3. 3. Effect of Initial Dye Concentration and Contact Time The influence of the contact time was studied in order to identify the equilibrium time for maximum adsorption. Figure 8 indicates the rates of adsorption of MG and AY 17 at various concentrations. The uptake for three different concentrations which were 20, 40 and 80 mg/L for both MG and AY 17 showed the similar adsorption trend. From the results, it can be noticed that the adsorption of dyes was rapid at beginning, followed by a gradual process. This fast uptake at the beginning may be attributed to the large amount of available vacant binding sites of sor-bent whereas a subsequent slower adsorption could be related to intraparticle diffusion. The current uptake pattern followed essentially the same trend in most of the reported works dealing with the adsorption studies whereby it can be customarily classified into rapid formation of an equilibrium interfacial concentration, followed by slow diffusion into the adsorbent.24 With increasing contact time, the percentage uptake of dye removal rate decreased due to limited vacant adsorption sites as the binding sites of sorbent become saturated with dye molecules.23 Hence, this has turned into a limiting factor for dye uptake. Similar observations were reported in the removal of colored textile wastewater using chitosan and the authors explai- Figure 8. Effect of initial dye concentration and contact time in the removal of MG and AY 17 ned that these were due to the competition for active adsorption sites. 20,25 At higher dye concentration, the number of available adsorption sites becomes fewer and many dye molecules competed strongly to the limited adsorption sites. Consequently, large number of dye molecules was not being adsorbed successfully onto the sorbent. 3. 4. Kinetics Studies Sorption kinetic studies were explored as it can provide some important insight about the mechanism of adsorption processes as well as describe the reaction pathways. The modeling of the kinetic studies of MG and AY 17 onto the sorbent was examined individually by applying two different kinetic models, namely pseudo-first-order26 and pseudo-second-order.27 The applicability of the model was chosen based on their respective linear regression correlation coefficient, R2 values. 3. 4. 1. Pseudo-first Order Kinetic Model For pseudo-first order kinetic model, it assumes that the rate of the solute change is directly proportional to the amount of solid uptake with time. The linear equation of pseudo-first order equation is expressed as follows: log(q-qt) = logqe- ¿t (2) where, qe = amount of dyes adsorbed at equilibrium, mg/g qt = amount of dyes adsorbed at time t, mg/g K1 = rate constant of pseudo-first order, 1/min t = time, min A linear graph of log (qe-qt) versus time for the adsorption of MG and AY 17 onto the corn cob films at the concentration of 20, 40 and 80 mg/L was plotted (Figure not shown). The experimental, qe(expt) and theoretical, qe(cal) adsorption capacities of dye at equilibrium and the firstorder rate constant, K1 with the correlation coefficient, R2 for each dye concentration of was tabulated in Table 1. The qe(expt) and K1 were determined from the intercept and gradient of the kinetic plot, respectively. Based on the re- Table 1. Adsorption capacities, kinetic model parameters and correlation coefficients based on pseudo-first and pseudo-second order kinetic models Dye Initial dye concentration qe (expt) (mg/L) Pseudo-first order kinetic model qe (cai) Kx (1/min) R2 (mg/g) mg/g) Pseudo-second order kinetic model qe, cal K h R2 (mg g 1) (1/min) (mg/g.min) MG 20 5.4106 1.4983 0.00253 0.2583 5.0556 0.0286 0.7301 0.9837 40 9.8819 5.4425 0.01474 0.5810 11.0375 0.0031 0.3831 0.9960 80 14.8624 10.6856 0.01036 0.8083 18.6220 0.0009 0.2971 0.9830 AY 17 20 2.2865 1.7939 0.006909 0.5023 3.0544 0.0040 0.0370 0.9926 40 3.3817 2.1857 0.005297 0.6686 3.5727 0.0074 0.0947 0.9758 80 3.2961 1.6749 0.005758 0.4756 3.3852 0.0137 0.1570 0.9887 Gan et al.: Utilization of Corn Cob and TiO2Photocatalyst ... Acta Chim. Slov. 2017, 64, 144-158 151 suits, for both MG and AY 17, the R2 values were relatively low and the qe(cal) values gave unreasonable values compared to those determined experimentally. Besides, it was found that the pseudo-first order kinetic equation does not fit well for the whole range of the adsorption process. This clearly indicates the non-applicability of pseudo-first order kinetic model for the studied dyes and implies more than one parameter could be involved in the adsorption process. From the literature, the reviews of experimental works also reveal that (in most cases) the pseudofirst order equation is unable to correlate the measured kinetics well. 28-30 3. 4. 2. Pseudo-second Order Kinetic Model The adsorption kinetic data was further studied by using pseudo-second order model. Pseudo-second order model assume that rate limiting step may be chemisorp-tion involving the valence forces transferring through electron sharing or exchanging between sorbent and sorbate as covalent forces, and ion exchange.29,31 The linear equation of the model was shown: t 1 1 ~= T+ 1, unfavorable; Rl = , linear; 0 < RL < 1, favorable; RL = 0, ir-whereas R2 for AY 17 was 0.9684. This result indicates reversible). The calculated RL value lies between 0.0207 that monolayer adsorption of AY 17 on the surface of corn to 0.4534 and this indicates that the adsorption process A linear graph of Ce/qe against Ce for the adsorption of MG and AY 17 onto corn cob films was plotted and shown in Figures 11 and 12, respectively. The correlation coefficient, R2 value was 0.9406 for the linear plot of MG, C o Gan et al.: Utilization of Corn Cob and TiO2Photocatalyst ... Acta Chim. Slov. 2017, 64, 144-158 153 is favorable and corn cob thin films is a potential adsorbent for the removal of MG and AY 17 from aqueous solution. The Freundlich isotherm assumes a physiochemical multilayer adsorption process on heterogeneous surfaces energy system. This isotherm is more towards a non-ideal adsorption that is more flexible and does not assume adsorption limit. The exponential Freundlich isotherm model equation is expressed as: (12) where KF = Freundlich isotherm constant for adsorption and n = Freundlich constant for intensity of adsorption. By taking the logarithm, the equation will therefore be in a linearized form and appeared as below: Figure 14. Freundlich isotherm of AY 17 The graphs of log qe against log Ce for MG and AY 17 were plotted and shown in Figures 13 and 14, respectively. The linear regression line on the plot could be used to determine the value of 1/n and KF from gradient and y-intercept, respectively. The coefficients for the linearized forms of the isotherm models for the adsorption of both dyes are listed in Table 2. The results implied that adsorption of MG on the corn cob films was more towards the heterogeneous surface and belong to multilayer adsorption system. The values of n for MG and AY 17 were 1.484 and -0.618 whereas the intensities of Freundlich 1A 1 ■a as - ot a "3 a6 ■ M O - 0.4 0.2 - 0 -I-r--,-r-T--i--T- -06 -05 -0.4 -03 -0.2 -0.1 0 0 1 log C'.(mg'L) Figure 13. Freundlich isotherm of MG constant were 16.881 and 333.657, respectively. Adsorption system will be termed as favorable process when the n value is in the range of 1 < n < 10. Based on the n value obtained, the adsorption of MG was termed as favorable. As for AY 17, Langmuir model appears to provide a more reasonable fitting and therefore this explains why a lower n value was obtained. 3. 6. Reusability of TiO2 Film and Corn Cob Film 2 Reusability is a major concern as this is one of key steps to make this type of economical dyes removal method applicable for practical usage. Therefore, a study on the repetitive usage and recycle of the thin films was performed. Figure 15 shows the effect of repetitive usage of TiO2 and corn cob films on the percentage removal of MG and AY 17. The percentage removal of MG was maintained around 90 % whereas percentage removal of AY 17 decreased from cycle 1 to 4. This can be attributed by the non-negligible adsorbed dye molecules on the films. Although the film was subjected to regeneration process by using NaOH before the next cycle of usage, some of the AY 17 dye molecules might still be strongly bind to the films and this condition hinders other AY 17 dye molecules from reaching to the active site and subsequently, a lower uptake was observed. As for MG, the recycling method adopted shown that this is a suitable method to desorb the previously attached MG dye and as a result, a high removal efficiency was maintained throughout the process. Table 2. Langmuir and Freundlich isotherm parameters Dye q,m mg/g Langmuir Ka, L/mg R2 kf Freundlich n R2 MG AY 17 35.336 0.241 0.882 0.039 0.9406 0.9684 16.881 333.657 1.484 -0.618 0.9633 0.9081 Gan et al.: Utilization of Corn Cob and TiO2 Photocatalyst ... 154 Acta Chim. Slov. 2017, 64, 144-158 3. 7. Statistical Experimental Design-Plackett-Burman (PB) and Response Surface Methodology (RSM) Statistical approach was employed to determine the important factors and to optimize the experimental condition for the removal of MG and AY 17 in binary dye solution. Design-Expert version 7.1.3 was used to validate the model through function of desirability. Significant factors that affect the dyes removal through combination of photodegradation and adsorption were screened through Plac-kett- Burman (PB) design. Figure 15. Effect of reusability in the removal of MG and AY 17 A total of three assigned parameters which were initial dye concentrations, contact time and pH were screened in total 12 experimental runs. For both MG and AY 17, the generated experimental condition and the differences of % removal between observed and predicted values were calculated and shown in Tables 3 and 4, respectively. It was observed that the largest and smallest differences between the observed and predicted removal for MG were 1.97% and 13.57%, respectively. As for AY 17, the percentage of differences was recorded in the range of 0.21% to 18.61%. The differences shown between the experimental and predicted percentage of removal is most probably due to the involvement of insignificant variables in the analysis. In some of the previously reported works, the researchers also noticed that there'll some differences in terms of the observed and predicted response and they attributed this kind of deviation to the non-negligible effect of insignificant variables in the design.2,32 Table 5 shows the analysis of variance (ANOVA) of both MG and AY 17 in binary dye solution. The studied variables were identified as significant Prob > F was less than 0.05. Based on the value, the studied model was found to be significant. For both MG and AY 17, the significant factors in affecting the removal process were contact time and initial pH of binary dye solution. The effect of contact time was termed as significant and this is closely related to the involvement of various stages in the process of adsorption. As for the effect of pH, this suggests that the degree of ionization of the adsorbate and the surface properties of the adsorbent play an important role in determining the efficiency of the process. The influential factors identified through PB were further studied and optimized using response surface methodology (RSM). A total of 13 experimental runs were conducted and Table 6 shows the combination of the generated contact time and initial pH. Besides, the observed and predicted response was also presented in the same table. The modified cubic model was employed to describe the correlation between these two important factors and the percentage removal was shown as follows in terms of coded form: MG in binary dye solution: % uptake of MG = +94.16 + 16.95 A -- 10.04 B -9.18 AB - 15.96 A2 - 11.36 B2 AY 17 in binary dye solution: % uptake of AY 17 = +101.87 + 35.05 A- 10.06B -12.41 AB - 50.89 A2 - 6.53 B2 Where A = contact time and B = initial pH (14) (15) Table 3. Plackett-Burman design and results for the percentage removal of MG in binary dye solution Experiment Contact time, mins vanaoie Initial concentration, mg/L PH Observed response, % Predicted response, % Differences, % 1 240.00 10.00 7.00 54.05 59.51 -5.46 2 240.00 10.00 7.00 54.05 59.51 -5.46 3 240.00 10.00 4.54 96.74 83.17 13.57 4 240.00 20.00 4.54 93.22 88.43 4.79 5 5.00 20.00 7.00 42.54 44.52 1.97 6 240.00 20.00 7.00 52.54 64.77 -12.23 7 5.00 20.00 4.54 47.08 58.94 -11.86 8 5.00 20.00 7.00 42.54 44.52 1.97 9 5.00 10.00 7.00 38.68 44.52 1.97 10 5.00 10.00 4.54 48.04 53.69 -5.65 11 5.00 10.00 4.54 48.04 53.69 -5.65 12 240.00 20.00 4.54 93.22 88.43 4.79 Gan et al.: Utilization of Corn Cob and TiO2Photocatalyst ... Acta Chim. Slov. 2017, 64, 144-158_ 155 Tables 7 and 8 were the ANOVA results and from and AY 17, respectively. The relatively high R2 values in these tables, both models were found to be significant (P < MG and AY 17 models indicated that there were good 0.0001) with model F-value of 102.21 and 36.37 for MG agreements between the experimental and predicted va- Table 4. Plackett-Burman design and results for the percentage removal of AY17 in binary dye solution Experiment Contact time, mins varia me Initial concentration, mg/L pH Observed response, % Predicted response, % Differences, % 1 5.00 40.00 4.54 11.90 20.60 -8.70 2 5.00 40.00 4.54 11.90 20.60 -8.70 3 240.00 60.00 4.54 100.00 91.89 8.11 4 240.00 40.00 7.00 47.26 53.98 -6.72 5 5.00 60.00 4.54 13.68 28.33 -14.65 6 240.00 60.00 7.00 43.10 61.71 -18.61 7 240.00 60.00 4.54 100.00 91.89 8.11 8 240.00 40.00 7.00 47.28 53.98 -6.70 9 5.00 60.00 7.00 6.67 7.31 -0.64 10 5.00 60.00 7.00 6.67 7.31 -0.64 11 5.00 40.00 7.00 5.43 5.22 0.21 12 240.00 40.00 4.54 100.00 84.16 15.84 Table 5. Regression analysis (ANOVA) of Placktt-Burman of MG and AY 17 in binary dye solution Dye Source Degree of freedom Sum of squares Mean square F-value Prob > F Description MG Model 3 4369.61 1456.54 14.14 0.0015 Significant Contact time 1 2607.80 2607.80 25.32 0.0010 Significant Initial MG concentration 1 82.90 82.90 0.80 0.3958 Not significant Initial pH 1 1678.91 1678.91 16.30 0.0037 Significant Residual 8 823.92 102.99 - - - AY 17 Model 3 15032.24 5010.75 25.77 0.0002 Significant Contact time 1 12120.26 12120.26 62.33 0.0001 Significant Initial AY 17 concentration 1 179.18 179.18 0.92 0.3652 Not significant Initial pH 1 2732.80 2732.80 14.05 0.0056 Significant Residual 8 1555.58 194.45 - - - Total 11 16587.82 - - - Table 6. Central composite design (CCD) matrix for two independent variables and the observed respond on MG and AY 17 in binary dye solution variable Respond Experiment Contact Initial Experimental % Predicted % Differences, Experimental Predicted Differences, time pH uptake of MG uptake of MG % % uptake of AY 17 % uptake of AY 17 % 1 122.50 5.77 93.1 93.28 -0.18 100 100 0.00 2 240.00 7.00 62.75 64.58 -1.83 45.02 57.03 -12.01 3 122.50 4.54 96.7 93.28 3.42 100 100 0.00 4 5.00 7.00 49.39 49.03 0.36 9.05 11.75 -2.70 5 122.50 5.77 93.1 93.28 -0.18 100 100 0.00 6 122.50 5.77 93.1 93.28 -0.18 100 100 0.00 7 240.00 5.77 100 95.16 4.84 100 86.03 13.97 8 122.50 5.77 93.1 93.28 -0.18 100 100 0.00 9 122.50 5.77 93.1 93.28 -0.18 100 100 0.00 10 240.00 4.54 100 93.28 6.72 100 100 0.00 11 5.00 4.54 49.93 50.76 -0.83 14.4 7.05 7.35 12 5.00 5.77 61.73 61.26 0.47 11.28 15.93 -4.65 13 122.50 7.00 74.23 72.76 1.47 100 85.28 14.72 Gan et al.: Utilization of Corn Cob and TiO2 Photocatalyst 156 Acta Chim. Slov. 2017, 64, 144-158 Table 7. Regression analysis (ANOVA) of RSM of MG Source Degree of freedom Sum of squares Mean square F-value p-value (Prob>F) Description Model 5 4351.68 870.34 102.21 <0.0001 Significant A 1 1723.81 1723.81 202.44 <0.0001 Significant B 1 605.21 605.21 71.08 <0.0001 Significant AB 1 336.91 336.91 39.57 0.0004 Significant A2 1 703.36 703.36 82.60 <0.0001 Significant B2 1 356.31 356.31 41.85 0.0003 Significant Residual 7 59.61 8.52 - - - R2: 0.9865, Adjusted R2: 0.9768, Predicted R2: 0.8937, Adequate precision: 27.233 and C.V.: 3.58 % Table 8. Regression analysis (ANOVA) of RSM of AY 17 Source Degree of freedom Sum of squares Mean square F-value p-value (Prob>F) Description Model 5 17914.53 3582.91 36.37 <0.0001 Significant A 1 7370.31 7370.31 74.82 <0.0001 Significant B 1 606.62 606.62 6.16 0.0421 Significant AB 1 615.78 615.78 6.25 0.0410 Significant A2 1 7152.23 7152.23 72.61 <0.0001 Significant B2 1 117.70 117.70 1.19 0.3105 Not significant Residual 7 689.54 98.51 - - - R2: 0.9629, Adjusted R2: 0.9365, Predicted R2: 0.6454, Adequate precision: 14.585 and C.V.: 13.17 % lues. The R2 that is close to unity signified a stronger model and it would be able to provide a better response.39 The signal to noise ratio is represented by adequate precision and a ratio that is greater than 4 is desirable.40' 41 From this study, the adequate precision for MG and AY 17 models were 27.233 and 14.585, respectively and this shown an adequate signal. The coefficient of variance (C.V.) of MG model was recorded as 3.58% wheraeas for AY 17 model was 13.17%. A low value of C.V. is preferred as this represents a greater precision and reliability of the experiments carried out.40 As both models have shown an adequate signal, therefore they were used to navigate the design space. Figure 16. 3D surface plot of MG as a function of initial pH and contact time (54 !B Figure 17. 3D surface plot of AY 17 as a function of initial pH and contact time Figures 16 and 17 showed the 3D surface plot of MG and AY 17, respectively for the interaction between contact time and initial pH. For the removal of both dyes, a more favourable condition was observed when the contact time was at the maximum point while initial pH was at the minimum point within the studied range. This is because by prolonging the contact time, it leads to more diffusion time and therefore a greater amount of dye molecules can be adsorbed onto the sorbent sites. As for the effect of initial pH, again, this is related to the surface charge and the usage of chitosan as the immobilizing agent. Gan et al.: Utilization of Corn Cob and TiO2Photocatalyst ... Acta Chim. Slov. 2017, 64, 144-158 157 4. Conclusion The results from this study have shown the effectiveness of TiO2 and corn cob films in the removal of MG and AY 17 from aqueous solution. The kinetics of dyes adsorption revealed that dye adsorption was more appropriately described by pseudo-second order model which is a kind of chemisorption process, involving valency forces through the sharing or exchange of electrons between the adsorbent and adsorbate as covalent forces, and ion exchange. The equilibrium data obtained was best conformed to Freundlich isotherm for MG and Langmuir isotherm for AY 17. This indicated that the adsorption of both dyes followed their respective heterogeneous and homogeneous adsorption pattern. The maximum adsorption capacity of MG and AY 17 was 35.336 and 0.241 mg/g, respectively. It is interesting to note that the efficiency of the films remained high after being repeated used for 2 cycles. From the statistical experimental design, it was shown that both models were highly significant with relatively high R2 values. Within the studied range, the crucial factors in affecting the percentage of removal for both dyes were identified to be contact time and initial pH. 5. Acknowledgements The authors are thankful for the financial support provided by the Malaysia-Toray Science Foundation through the research grant: 4417/002. The research facilities provided by Universiti Tunku Abdul Rahman (UTAR) are acknowledged. 6. References 1. V. K. Gupta and Suhas, J. Environ. Manage., 2009, 90, 2313-2342. https://doi.org/10.1016/jjenvman.2008.11.017 2. S. L. Lee, S. W. Liew and S. T. Ong, Acta Chim. Slov., 2016, 63, 144-153. https://doi.org/10.17344/acsi.2015.2068 3. S. Srivastava, R. 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Wang, Nazih Shammas (Eds); World Scientific Publishing Co.: Singapore, 2012; 929-978. https://doi.org/10.1142/9789814327701_0021 25. R. D. C. Soltani, A. R. Khataee, M. Safari and S. W. Joo, Int. Biodeterior. Biodegradation, 2013, 85, 383-391. https://doi.org/10.1016/jibiod.2013.09.004 26. S. Lagergren and B. K. Svenska, Veternskapsakad Handlin-gar, 1898, 24, 1-39. 27. Y. S. Ho and G. McKay, Process Biochem., 1999, 34, 451465. https://doi.org/10.1016/S0032-9592(98)00112-5 28. W. Plazinski, Adv. Colloid Interface Sci., 2013, 197-198, 58-67. https://doi.org/10.1016/j.cis.2013.04.002 Gan et al.: Utilization of Corn Cob and TiO2 Photocatalyst ... 158 Acta Chim. Slov. 2017, 64, 144-158 29. Y.S. Ho, J Hazard Mater, 2006, B136, 681-689. https://doi.org/10.1016/jjhazmat.2005.12.043 30. J. Febrianto, A.N. Kosasih, J. Sunarso, Y. H. Ju, N. Indraswa-ti, N. and S. Ismadji, J Hazard Mater, 2009, 162, 616-645. https://doi.org/10.1016/jjhazmat.2008.06.042 31. Ho, Y. S. and G. McKay, Water Res., 2000, 34, 735-742. https://doi.org/10.1016/S0043-1354(99)00232-8 32. S. T. Ong and C. K. Seou, Desalin. Water Treat., 2014, 52, 7673-7684. https://doi.org/10.1080/19443994.2013.830684 33. P. Saha, S. Chowdhury, S. Gupta, I. Kumar and R. Kumar, Clean Soil, Air and Water, 2000, 38, 437-445. https://doi.org/10.1002/clen.200900234 34. T. Santi and S. Manonmani, Malachite Green Removal from aqueous solution by the peel of Cucumis Sativa fruit. Clean Soil, Air and Water, 2011, 39, 162-170. https://doi.org/10.1002/clen.201000077 35. K. T. Karthikeyan and Jothivenkatachalam, J. Environ. Na-notechnol., 2014, 3, 69-80. https://doi.org/10.13074/jent.2014.03.142070 36. I. Langmuir, J. Am. Chem. Soc., 1918, 40, 1361-1403. https://doi.org/10.1021/ja02242a004 37. H. Freundlich, Phys. Chem. Soc., 1906, 40, 1361-1368 38. T. W. Weber and R. K. Chakkravorti, Am. Ins. Chem. Eng. J., 1974, 20, 228-238. https://doi.org/10.1002/aic.690200204 39. K. Chauhan, U. Trivedi, and K. C. Patel, J. Microbial Bio-technol., 2006, 16, 1410-1415. 40. J. K. Kim, B. R. Oh, H. Shin, C. Eom and S. W. Kim, Process Biochem., 2008, 43, 1308-1312. https://doi.org/10.1016Zj.procbio.2008.07.007 41. E. C. Khoo, S. T. Ong, Y. T. Hung and S. T. Ha, Desalin. Water Treat., 2013, 51, 7109-7119. https://doi.org/10.1080/19443994.2013.791774 Povzetek Proučevana je bila učinkovitost uporabe filmov z vsebnostjo TiO2 oziroma mikrodelcev koruznih storžev za odstranjevanje barvil malahitno-zeleno (MG) in kislo-rumeno 17 (AY 17) iz raztopine. Uporabljena metoda imobilizacije se lahko izogne filtraciji, ki v praksi ni primerna. V šaržnih eksperimentih so bili pročevani začetni pH raztopine, začetna koncentracija barvila, kontaktni čas in ponovna uporaba adsorbenta. Ravnotežni podatki za MG in AY 17 sledijo Freundlic-hovi in Langmuirjevi izotermi. Odstotek odstranjenega MG je ostal visok po štirih sorpcijskih ciklih, vendar je bila za AY 17 dosežena višja redukcija. Odstranjevanje obeh barvil je bilo modelirano in optimirano s pomočjo metode po Plackett-Burmanu (PB) in metode odzivne površine (RSM). Pogoji na površini so bili analizirani s pomočjo infrardeče spektroskopije (IR), fourierjeve transformacijske infrardeče spektroskopije (FTIR), elektronske vrstične mikroskopije (SEM) in mikroskopije na atomsko silo (AFM). Gan et al.: Utilization of Corn Cob and TiO2Photocatalyst ... DPI: 10.17344/acsi.20l6.2986_Acta Chirn, Slov. 2017,64, 159-169_©commons 159 Scientific paper Synthesis of Some Unique Carbamate Derivatives bearing 2-Furoyl-1-piperazine as a Valuable Therapeutic Agents Muhammad Athar Abbasi,1* Ghulam Hussain,1 Aziz-ur-Rehman,1 Sabahat Zahra Siddiqui,1 Syed Adnan Ali Shah,2'3 Muhammad Arif Lodhi,4 Farman Ali Khan,4 Muhammad Ashraf,5 Qurat-ul-Ain,5 Irshad Ahmad,6 Rabia Malik,6 Muhammad Shahid7 and Zahid Mushtaq7 1 Department of Chemistry, Government College University, Lahore-54000, Pakistan 2 Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia 3 Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Level 9, FF3, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia 4 Department of Biochemistry, Abdul Wali Khan University, Mardan-23200, Pakistan 5 Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan 6 Department of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan 7 Department of Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan * Corresponding author: E-mail: atrabbasi@yahoo.com; abbasi@gcu.edu.pk tel: (+92)-42-111000010, ext. 266. Received: 12-10-2016 Abstract The aim of the research work was to synthesize different biologically active carbamate derivatives bearing 2-furoyl-1-piperazine and having modest toxicity. The synthesis was completed as a multiple sequence. The structural confirmation of all the synthesized compounds was obtained by EI-MS, IR and 1H-NMR spectral data. The enzyme inhibition and antibacterial potential of the synthesized compounds was evaluated. To find the utility of the prepared compounds as possible therapeutic agents their cytotoxicity was also checked. All the compounds were active against acetylcholinesterase enzyme, especially 12 and 14 showed very good inhibitory potential relative to Eserine, a reference standard. Almost all the compounds showed good activities against both Gram-positive and Gram-negative bacterial strains. Keywords: 2-Furoyl-1-piperazine; 1H-NMR; Acetylcholinesterase; Antimicrobial activity; Hemolytic activity 1. Introduction Heterocyclic compounds are cyclic compounds having hetero atoms e.g, N, O or S, having diverse medicinal importance.1 Piperazine is a medicinally important hete-rocyclic nucleus which consists of a six membered ring containing two nitrogen atoms at the positions 1 and 4 in the ring. The piperazine has been classified as a privileged structure and is frequently found in biologically active compounds across a number of different therapeutic areas2 which encompass anti-microbial, anti-tubercular, anti-psychotic, anti-convulsant, anti-depressant, anti-inflammatory, cytotoxic, anti-malarial, anti-arrhythmic, anti-oxidant and anti-viral activities.3,4 Abbasi et al.: Synthesis of Some Unique Carbamate Derivatives 160 Acta Chim. Slov. 2017, 64, 159-169 Carbamates are derivatives of carbamic acid (NH2COOH). A carbamate group, carbamate group, carbamate ester and carbamic acids functional groups are unified structurally and often are interconverted chemically. Carbamate esters are also called urethanes. Although most of the literature is concerned with organic car-bamates, yet, the inorganic salt ammonium carbamate is produced on a large scale from ammonia and carbon dioxide. The amino groups of the lysine residues in urease and phosphotriesterase also attribute carbamate. The carbamate resulting from aminoimidazole is an intermediate in the biosynthesis of . Carbamoyl phosphate is generated from carboxyphosphate rather than CO2.5 The carbamate insecticides featuring the carbamate ester functional group, e.g, Aldicarb, Carbofuran, Carbaryl (Fig. 1) etc., encompass this group. O Fig. 1. Carbaryl (insecticide carbamate) The organophosphate pesticides also hinder this enzyme, although irreversibly, and originate a more severe form of cholinergic poisoning.6 Iodopropynyl butylcar-bamate is a wood and paint preservative and used in the cosmetics7. Urethane (ethylcarbamate) was once produced commercially in the United States as an anti-neoplas-tic agent and for other medicinal purposes. It was found toxic and largely ineffective and is now seldom used as a veterinary medicine.8 a-Glucosidase comprises class of hydrolase enzymes, located in the brush border surface membrane of small intestinal cells.9 The vital function of a-glucosidase is to hydrolyze the 1,4 glycosidic linkage from the non reducing end of the a-glucosides, substrates to produce aD-glucose and other monosaccharide which are operated as carbon and energy source.10 For oral anti-diabetic drugs for patients with type-2 Diabetes mellitus a-glucosidase inhibitors compounds are used. Postprandial hyperglyce-mia has a role in the growth of type-2 diabetes and problems associated with disease such as nephropathy and ma-croangipathy etc.11 The inhibitors of enzyme can hinder the release of D-glucose of oligosaccharides and disacc-harides beginning dietary complex carbohydrates and holdup glucose absorption, resulting in compact postprandial hyperglycemia.12 Acetyl and butyrylcholinesterases (AChE/BChE) comprise a family of serine hydrolases. The different spe- cificities for substrates and inhibitors for these enzymes are caused by the differences in amino acid residues of the active sites of AChE and BChE. The enzyme scheme is liable for the termination of acetylcholine at cholinergic synapses. These are main components of cholinergic brain synapses and neuromuscular junctions. The chief function of AChE and BChE is to catalyze the hydrolysis of the neurotransmitter acetylcholine.13,14 It has been found that BChE is present in appreciably higher quantities in Alzheimer's plaques in the normal age related to non dementia of brains. Cholinesterase inhibitors increase the amount of acetylcholine offered for neuromuscular and neuronal transmission through their reversible or irreversible inhibitory activity.15 Hence, the search for new cholinesterase inhibitors is consider an important strategy to introduce new drug candidates for the treatment of Alzheimer's disease and other related diseases.16 Different microbes have been found to be involved in many diseases17-22 and some of them are included in the current study. So, in continuation of our previous work on carbamates,23-25 hereby we report the synthesis of some unique carbamates having amalgamation with 2-furoylpiperazine moiety, which might find their utility as potential and safe thereapeutic agents. 2. Results and Discussion The aim of the present research work was to synthesize new biologically active compounds with low toxicity. Indeed, the current need is to introduce pharmacologically active drugs to help in pharmacy against the increasing resistance of microorganisms. 2. 1. Chemistry In the present research work, different carbamate derivatives bearing 2-furoyl-1-piperazine were synthesized in a series of steps by a reported method26 as shown in Scheme 1 and then all the derivatives were screened for enzyme inhibition, antimicrobial and hemolytic activities. The structural analysis of one of the compounds is discussed here in detail for the benefit of the reader. The molecule 16 was synthesized as an off-white amorphous solid having melting point 80-92 °C and molecular formula C19H20Br3N3O4, which was confirmed by EI-MS having [M]+ peak at m/z 591 and by the number of protons in its 1H-NMR spectrum. The CHN analysis data of this molecule also supported the assignement of its molecular formula. Its structure was corroborated by the distinct ion peak at m/z 93 related to N-furoyl group and another at m/z 332 for O-(2,6-dibromophenyl)-N-(allyl)carbamate part. The suggested mass fragmentation pattern is given in Fig. 2. In IR spectrum of 16, characteristic peaks appeared at u 3406 (N-H), 3086 (Ar C-H), 2882 (R C-H), 1657 (C=O), 1582 (Ar C=C), 1497 (N=O), 1197 (C-O-C), 1110 (C-N-C), 847 (C-N) and 548 cm-1 (C-Br) which al- Abbasi et al.: Synthesis of Some Unique Carbamate Derivatives Acta Chim. Slov. 2017, 64, 159-161 169 together confirmed the presence of the carbamate group and 2-furoyl-1-piperazine ring. In the aromatic region of 1H-NMR spectrum, a two-proton singlet appeared at 8 7.67 (s, 2H, H-3 and H-5) which is typical for a 2,4,6-tribromophenyl moiety attached via an oxygen atom. The other three peaks in the aromatic region at 87.49 (brs, 1H, H-5'''), 7.04 (d, J = 4.1 Hz, 1H, H-3''') and 6.49 (dd, J = 4.0, 2.0 Hz, 1H, H-4''') are characteristic for a 2-furyl ring. Moreover, a singlet 8 5.19 represents NH of the carbamate group, while the 1,4-disubstituted piperazine ring was deduced through two broad singlets in aliphatic region, and each broad singlet representing two symmetrical methylene groups. These two singlets resonated at 8 3.94 (4H) and 8 3.47 (4H). The former was assigned to symmetrical CH2-3'' and CH2-5'' while latter was assigned to symmetrical CH2-2'' and CH2-6'' in the piperazine entity. Similary, the presence of a central 1,3-disubstitued propyl group was ascertained by the signals resonating at 8 4.12 (t, J = 6.6 Hz, 2H, CH2-1'), 3.61-3.57 (m, 2H, CH2-3') and 2.10 (quintet, J = 6.8 Hz, 2H, CH2-2'). The 1H-NMR spectrum of this molecule is shown in Fig. 3. So, on the basis of aforementioned spectral evidences, the structure of 16 was confirmed as 2,4,6-tribromophenyl 3-[4-(2-furoyl)-1-piperazinyl]propylcarbamate. All the synthesized carbamate derivatives bearing 2-furoyl-1-pi-perazine were characterized by IR, 1H-NMR and EI-MS spectral analysis in a similar way. 2. 2. Biological Activities (in vitro) 2. 2. 1. Enzyme Inhibition Activity The synthesized compounds exhibited variable inhibitory potentials against a-glucosidase, acetylcholineste-rase and butyrylcholinesterase as evident from their IC50 values presented in Table 1. Only two compounds, 2,4,6-tribromophenyl 2-[4-(2-furoyl)-1-piperazinyl]ethylcarba-mate (9) and 2,4,6-tribromophenyl 3-[4-(2-furoyl)-1-pipe-razinyl]propylcarbamate (16) showed weak inhibitory po- Fig. 2. Suggested mass fragmentation pattern of 2,4,6-tribromophenyl 3-[4-(2-furoyl)-1-piperazinyl]propylcarbamate (16). Abbasi et al.: Synthesis of Some Unique Carbamate Derivatives ... 162 Acta Chim. Slov. 2017, 64, 159-169 Fig. 3. 1H-NMR spectrum of 2,4,6-tribromophenyl-3-[4-(2-furoyl)-1-piperazinyl]propylcarbamate (16) Table 1. Bioactivity studies of different carbamate derivatives bearing 2-furoyl-1-piperazine Compound O-Glucosidase % Inhibition IC50 (^M) % Inhibition AchE IC50 (^M) % Inhibition BChE IC50 (^M) 5 25.45 ± 0.31 - 78.59 ± 0.23 34.62 ± 0.19 28.35 ± 0.16 - 7 - - 81.35 ± 0.96 456.45 ± 0.29 - - 9 94.32 ± 0.28 345.16 ± 0.60 77.15 ± 0.18 32.51 ± 0.03 48.16 ± 0.27 - 12 32.34 ± 0.26 - 81.63 ± 0.15 18.91 ± 0.04 55.71 ± 0.24 361.27 ± 0.13 14 27.67 ± 0.45 - 82.45 ± 0.11 23.22 ± 0.05 33.26 ± 0.25 - 16 85.54 ± 0.32 422.61 ± 0.30 81.76 ± 0.12 24.71 ± 0.07 43.45 ± 0.21 - Control 92.23 ± 0.14" 38.25 ± 0.12" 91.27 ± 1.17* 0.04 ± 0.0001* 82.82 ± 1.09* 0.85 ± 0.0001* NOTE: All compounds were dissolved in methanol and experiments were performed in triplicate (mean±SEM, n = 3). a = Acarbose, b = Eserine, AChE = Acetylcholinesterase, BChE = Butyrylcholinesterase tential against a-glucosidase, having IC50 value of 345.16 ± 0.16 pM and 422.61 ± 0.13 pM, respectively, relative to Acarbose, used as a reference standard having IC50 value of 38.25 ± 0.12 pM. This inhibitory potential might be attributed to the presence of 2,4,6-tribromophenyl group in both molecules. All the molecules were active against acetylcholinesterase enzyme but among all molecules phenyl 3-[4-(2-furoyl)-1-piperazinyl]propylcarbamate (12) exhibited the best inhibitory potential against this enzyme with IC50 value of 18.91 ± 0.04 pM, relative to Eserine, a reference standard having IC50 value of 0.04 ± 0.0001 pM. This enhanced inhibitory potential of 12 can be a result of its unique skeleton as a whole. Moreover, this was the only molecule which showed some inhibitory tendency against butyrylcholinesterase enzyme with IC50 value of 361.27 ± 0.13 pM. In the future some modified derivatives of this molecule are suggested to show much closer IC5„ value to the standard Eserine. 2. 2. 2. Antibacterial Activity All the synthesized molecules were screened against Gram-positive and Gram-negative bacteria, and were found to be excellent-to-good antibacterial agents. The re- Abbasi et al.: Synthesis of Some Unique Carbamate Derivatives Acta Chim. Slov. 2017, 64, 159-163 169 suits are shown as MIC values in Table 2. Among the synthesized carbamates, 16 showed the lowest MIC value (8.96 ± 0.49 pM) against S. typhi credibly because of the presence of 2,4,6-tribromophenyl group. In the case of E. coli, carbamate 14 showed the lowest MIC value (9.95 ± 0.48 pM) probably due to the presence of 2,4,6-trinitrop-henyl group. Against P. aeruginosa and B. subtilis, 16 and 9 exhibited excellent antibacterial potential with MIC values 9.27 ± 0.16 pM and 9.43 ± 0.85 pM, respectively, predominantly because of the presence of 2,4,6-trinitrop-henyl group and 2,4,6-tribromophenyl group, respectively, in these molecules. Similarly, the carbamate 16 also rendered a great antibacterial activity against S. aureus with MIC value 16.87 ± 0.41 pM. Amongst the synthesized compounds, 5 and 16 showed MIC values against all the bacterial strains while compound 16 showed the excellent MIC value in the following order towards all bacterial strains: S. typhi > P. aeroginosa > B. subtilis > E. coli > S. aureus, probably due to the presence of 2,4,6-tribro-mophenyl moiety. In general, we can say that most of the carbamates possessed very good antibacterial activities against both Gram-positive and Gram-negative bacterial strains and hence these molecules might lead to the discovery of very potent antibacterial agents in future. 2. 2. 3. Hemolytic Activity Most of the molecules exhibited very modest cytotoxicity values, except 14 (72.38%), yet it was lower than the positive control (Triton-X-100). The lowest activity was shown by the molecule 16 (9.20%), although it was a little higher than the negative controls (PBS). So it can be concluded that these molecules might be further tested for their therapeutic applications in the drug designing program because of their moderate toxicity, as shown in Table 2. 2. 2. 4. Computational Docking In order to get an insight about the validity of accuracy, the co-crystallized ligands of the following enzymes Table 2. Antibacterial activity (MIC) and hemolytic activity of different carbamate derivatives bearing 2-furoyl-1-piperazine Compound S. typhi (-) E. coli (-) MIC (^M) P.aeroginosa (-) B. subtilis (+) S. aureus (+) Hemolytic activity % 5 9.08 ± 0.50 17.43 ± 0.61 19.87 ± 0.51 10.85 ± 0.14 19.98 ± 0.58 24.26 7 9.14 ± 0.15 16.98 ± 0.75 18.34 ± 0.92 10.78 ± 0.93 - 15.94 9 9.89 ± 0.17 14.43 ± 0.05 9.87 ± 0.43 9.43 ± 0.85 - 12.10 12 9.88 ± 0.75 15.64 ± 0.32 17.67 ± 0.34 11.76 ± 0.54 - 53.13 14 9.78 ± 0.90 9.95 ± 0.48 17.78 ± 0.33 16.49 ± 0.27 - 72.38 16 8.96 ± 0.49 10.64 ± 0.58 9.27 ± 0.16 9.78 ± 0.62 16.87 ± 0.41 9.20 Ciprofloxacin 7.45 ± 0.58 7.16 ± 0.58 7.14 ± 0.18 7.29 ± 0.90 7.80 ± 0.19 PBS Triton 0.09 100 Abbasi et al.: Synthesis of Some Unique Carbamate Derivatives ... 164 Acta Chim. Slov. 2017, 64, 159-169 Fig. 5. 3D interacted image of phenyl 3-[4-(2-furoyl)-1-pipera-zinyljpropylcarbamate (12) against acetylcholinestrase. were extracted and then re-docked into the binding pockets of the receptors. In all these cases, RMSD values bet-w. een docked and co-crystallized ligands were less than 2 A which indicates the reliability of docking method and thus showing that our protocol can be used for further studies. Almost all the synthesized derivatives were computationally docked against a-glucosidase, AChE and BChE to explore the binding modes of all the ligands. The carbamate 12 was docked against acetylcholinesterase. There were observed four prominent interactions between 12 and active residues of the protein. First strongest side chain donor interaction was found between TyrA130 and carbonyl oxygen giving a distance of 1.77 A, second between SerA122 and another carbonyl oxygen with the distance of 3.67 A. Third strong back bone donor interaction was established between GlyA117 and amide proton of the ligand with a distance of 2.09 A. Similarly the last hydrophobic interaction of a distance of 3.10 A was found between TrpA81 and phenyl ring of the ligand as shown in Fig. 4 and 5. From the same compound 12 protein docked complex of butyrylcholinesterase, this interacted weakly Abbasi et al.: Synthesis of Some Unique Carbamate Derivatives Acta Chim. Slov. 2017, 64, 159-165 169 with Tyr332 and His438 amino acid residues. Tyr332 displayed arene-arene interaction with furoyl ring with the distance of 3.36 A, while His438 displayed arene cation interaction with phenyl ring with a distance of 3.74 A as it is shown in Fig. 6 and 7. 3. Experimental 3. 1. General Chemicals and solvents of analytical grade were purchased from Sigma Aldrich and Alfa Aesar (Germany). By using an open capillary tube method, melting points were determined on Griffin and George apparatus and are uncorrected. By using thin layer chromatography (TLC) in various percentages of ethyl acetate and n-hexane as mobile phase, initial purity of compounds was detected at 254 nm. IR peaks were recorded on a Jasco-320-A spectrometer by using a KBr pellet method. 1H-NMR signals were recorded at 500 MHz in CDCl3 using Bruker spectrometers. EI-MS signals were recorded by utilizing a JMS-HX-110 spectrometer. 3. 2. Synthesis of Phenyl (N-substituted) carbamates 3 and 11 2-Chloroethylamine (2-chloro-1-ethanamine; 2; 0.1 mol) and 3-bromopropylamine (10; 0.1 mol) were taken separately in two iodine flasks, each containing 10 mL distilled water. The pH of the solution was maintained at 9-10 by 10% aqueous Na2CO3 followed by the addition of phenyl carbonochloridic acid (phenylchloroformate; 1; 0.1 mol,) in equimolar ratios in each flask along with vigorous shaking. The reaction mixture in each case was stirred at room temperature for 3-4 h. Progress of the reaction was confirmed by TLC (n-hexane : EtOAc; 70:30), visualized by UV lamp. Phenyl 2-chloroethylcar-bamate (3) and phenyl 3-bromopropylcarbamate (11) were collected as white precipitates by filtration. These were washed with distilled water and dried to acquire pure compounds. 3. 3. Nitration of Phenyl (^-substituted) carbamates Yielding 6 and 13 Phenyl 2-chloroethylcarbamate (3; 0.1 mol) and phenyl 3-bromopropylcarbamate (11; 0.1 mol) were taken separately in two 50 mL round bottom flasks. 5-10 mL concentrated H2SO4 was added in each flask to dissolve the respective compound. Each mixture was stirred for 15-20 min at room temperature and then equimolar amount of nitric acid was added to each mixture dropwise at 10 °C. Then each reaction mixture was stirred for 4 h and monitored by TLC. On reaction completion, ice cold water was added to the reaction flasks to produce the precipitates which were collected by filtration, washed with distilled water and dried to afford the nitrated compounds 6 and 13, separately. 3. 4. Bromination of Phenyl (^-substituted) carbamates Yielding 8 and 15 Phenyl 2-chloroethylcarbamate (3; 0.1 mol) and phenyl 3-bromopropylcarbamate (11; 0.1 mol) were taken separately in two 50 ml round bottomed flasks and were dissolved in glacial acetic acid (5-10 mL). Liquid bromine was added slowly in equimolar amount to eack flask. The reaction mixture in each case was stirred at room temperature and monitored with TLC for the completion of the reaction. Distilled water was added to each reaction flask to quench the reaction. Precipitated products were filtered, washed with distilled water and dried to obtain pure brominated compounds 8 and 15, separately. 3. 5. Synthesis of Different Carbamate Derivatives Bearing 2-Furoyl-1 -piperazine Moiety 2-Furoyl-1-piperazine (4; 4.5 mmol) dissolved in 20-30 mL acetonitrile was taken in a 100 mL round bottom flask, solid K2CO3 (13.5 mmol) was added and the reaction mixture was refluxed for half an hour followed by the addition of respective carbamates (3, 6, 8,11, 13 or 15; one in each case) in equimolar ratio. The mixture was refluxed for 4-5 h. TLC was carried out to check the reaction completion (20% ethyl acetate: 80% n-hexane). Distilled water was added to the reaction mixture to acquire the respective precipitates. On completion, 1-2 drops of aqueous NaOH were added to the reaction mixture. Precipitates were filtered, washed and dried to obtain the respective carbamates 5, 7, 9, 12, 14 or 16 (one in each case) bearing 2-furoyl-1-piperazine. 3. 5. 1. Phenyl 2-[4-(2-furoyl)-1-piperazinyl] ethylcarbamate (5) Sticky brown liquid; Yield: 90%; Mol. F.: C18H21N3O4; Mol. Mass.: 343 g/mol; IR (KBr, cm-1) vmax: 3406 (N-H), 3086 (Ar C-H), 2882 (R C-H), 1657 (C=O), 1582 (Ar C=C), 1498 (N=O), 1197 (C-O-C), 1110 (C-N-C), 853 (C-N); 1H-NMR (500 MHz, CDCl3, ppm): ¿7.49 (brs, 1H, H-5'''), 7.45 (brt, J = 7.2 Hz, 2H, H-3 and H-5), 7.15 (brt, J = 7.3 Hz, 1H, H-4), 7.07 (brd, J = 7.7 Hz, 2H, H-2 and H-6), 7.01 (d, J = 4.1 Hz, 1H, H-3'''), 6.50 (dd, J = 1.9, 3.2 Hz, 1H, H-4'''), 5.19 (s, 1H, NH), 3.84 (brs, 4H, CH2-3'' and CH2-5''), 3.39 (t, J = 6.2 Hz, 2H, CH2-1'), 2.56 (brt, J = 6.0 Hz, 4H, CH2-2'' and CH2-6''), 2.42 (t, J = 6.8 Hz, 2H, CH2-2'); EI-MS m/z 343 [M]+, 207 [C11H15N2O2]+, 165 [C9HnNO2r+, 163 [C9H9NO2n, 95 [C5H3O2]+, 94 [C6H5O]+, 93 [C5HO2]+. Anal. Calc. for C18H21N3O4 (343.15): C, 62.96; H, 6.16; N, 12.24. Found: C, 62.84; H, 6.25; N, 12.37. Abbasi et al.: Synthesis of Some Unique Carbamate Derivatives ... 166 Acta Chim. Slov. 2017, 64, 159-169 Phenyl Scheme 1. Outline for the synthesis of different carbamate derivatives bearing 2-furoyl-1-piperazine. Reagents and conditions: (I) 10% aq. Na2CO3 soln./pH 9-10/stirring at RT for 3-4 h. (II) 2-Furoyl-1-piperazine (4)/CH3CN/K2CO3/reflux for 4-5 h. (III) Conc. HNO3/conc. H2SO4/stirring ait RT for 3-4 h. (IV) Br2/CH3COOH/stirring at RT for 3-4 h. 3. 5. 2. 2,4,6-Trinitrophenyl 2-[4-(2-furoyl)-1-piperazinyl]ethylcarbamate (7) Sticky brown liquid: 82%; Mol. F.: C18H18N6O10; Mol. Mass.: 478 g/mol; IR (KBr, cm-1) vmax: 3406 8N-H), 3086 (Ar C-H), 2882 (R C-H), 1657 (C=O), 1582 (Ar C=C), 1490 (N=O), 1197 (C-O-C), 1110 (C-N-C), 848 (C-N); 1H-NMR (500 MHz, CDCl3, ppm): S 8.15 (s, 2H, H-3 and H-5), 7.49 (brs, 1H, H-5'''), 6.99 (d, J = 4.1 Hz, 1H, H-3'''), 6.49 (dd, J = 1.9, 3.2 Hz, 1H, H-4'''), 5.19 (s, 1H, NH), 3.84 (brs, 4H, CH2-3'' and CH2-5''), 3.39 (t, J = 6.2 Hz, 2H, CH2-1'), 2.56 (brt, J = 6.0 Hz, 4H, CH2-2'' and CH2-6''), 2.422 (t, J = 6.8 Hz, 2H, CH2-2'); EI-MS m/z 478 [M]+, 300 [C.H^O.r, 298 [C9H6N4O8n, 254 [C9H6N3O6]+, 229 [C6H2N3O7]+, 207 [C11H15N2O2]+, 95 [C5H3O2]+, 93 [C6HO2]+. Anal. Calc. for C18H18N6O10 (478.11): C, 45.19; H, 3.79; N, 17.57. Found: C, 45.26; H, 3.85; N, 17.66. 3. 5. 3. 2,4,6-Tribromophenyl 2-[4-(2-furoyl)-1 -piperazinyl]ethylcarbamate (9) Sticky brown liquid; Yield: 85%; Mol. F.: C18H18Br3N3O4; Mol. Mass.: 577 g/mol; IR (KBr, cm-1) vmax: 3406 (N-H), 3086 (Ar C-H), 2882 (R C-H), 1657 (C=O), 1582 (Ar C=C), 1491 (N=O), 1197 (C-O-C), 1110 (C-N-C), 853 (C-N), 545 (C-Br); 1H-NMR (500 MHz, CDCl3, ppm): S 7.75 (s, 2H, H-3 and H-5), 7.47 (brs, 1H, H-5'''), 7.02 (d, J = 4.1 Hz, 1H, H-3'''), 6.47 (dd, J = 1.9, 3.3 Hz, 1H, H-4'''), 5.18 (s, 1H, NH), 3.83 (brs, 4H, CH2-3'' and CH2-5''), 3.36 (t, J = 6.1 Hz, 2H, CH2-1'), 2.58 (brt, J = 6.1 Hz2 4H, CH2-2'' and CH2-6''), 2.45 (t, J = 6.8 Hz, 2H, CH2-2'); EI-MS m/z 577 [M]+, 399 [C9H8Br3NO2n, 397 [C8H6Br3NO2]^+, 318 [C9H8Br2NO2]+, 327 [C6H2Br3O]+, 207 [CnH15N2O2]+, 95 [C5H3O2]+, 93 [C5HO2]+. Anal. Calc. for C18H18Br3N3O4 (576.88): C, 37.27; H, 3.13; N, 7.24. Found1 C, 37.34; H, 3.21; N, 7.33. 3. 5. 4. Phenyl 3-[4-(2-furoyl)-1-piperazinyl] propylcarbamate (12) Sticky brown liquid; Yield: 87%; Mol. F.: C19H23N3O4; Mol. Mass.: 357 g/mol; IR (KBr, cm-1) vmax: 34106 (N-H), 3086 (Ar C-H), 2882 (R C-H), 1657 (C=O), 1582 (Ar C=C), 1496 (N=O), 1197 (C-O-C), 1110 (C-N-C), 850 (C-N); 1H-NMR (500 MHz, CDCl3, ppm): 87.49 (brs, 1H, H-5'''), 7.42 (brt, J = 7.1 Hz, 2H, H-3 and Abbasi et al.: Synthesis of Some Unique Carbamate Derivatives Acta Chim. Slov. 2017, 64, 159-167 169 H-5), 7.16 (brt, J = 7.4 Hz, 1H, H-4), 7.09 (brd, J = 7.8 Hz, 2H, H-2 and H-6), 7.05 (d, J = 4.0 Hz, 1H, H-3'''), 6.48 (dd, J = 1.9, 4.0 Hz, 1H, H-4'''), 5.15 (s, 1H, NH), 4.10 (t, J = 6.7 Hz, 2H, CH2-1'), 3.90 (brs, 4H, CH2-3'' and CH2-5''), 3.64-3.56 (m, 2H, CH2-3'), 3.46 (brs, 4H, CH2-2'' and CH2-6''), 2.19 (quintet, J = 6.7 Hz, 2H, CH2-2'); EI-MS m/z 357 [M]+, 221 [C12H17N2O2]+, 179 [C10H13NO2n, 177 [C10H11NO2]^+, 95 [C5H3O2]+, 93 [C6H50]+, 93 [C5HO2]+. Anal. Calc. for C19H23N3O4 (3567.17): C, 63.85; H, (5.49; N, 11.76. Found: C, 63.92; H, 6.56; N, 11.82. 3. 5. 5. 2,4,6-Trinitrophenyl 3-[4-(2-furoyl)-1-piperazinyl]propylcarbamate (14) Sticky brown liquid; Yield: 86%; Mol. F.: C19H20N6O10; Mol. Mass.: 492 g/mol; IR (KBr, cm-1) vmax: 34106 (N-6H), 3086 (Ar C-H), 2882 (R C-H), 1657 (C=O), 1582 (Ar C=C), 1492 (N=O), 1197 (C-O-C), 1110 (C-N-C), 855 (C-N); 1H-NMR (500 MHz, CDCl3, ppm): ¿7.96 (s, 2H, H-3 and H-5), 7.48 (brs, 1H, H-5'''), 7.02 (d, J = 4.0 Hz, 1H, H-3'''), 6.46 (dd, J = 2.0, 4.0 Hz, 1H, H-4'''), 5.16 (s, 1H, NH), 4.10 (t, J = 6.7 Hz, 2H, CH2-1'), 3.95 (brs, 4H, CH2-3'' and CH2-5''), 3.60-3.57 (m, 2H, CH2-3'), 3.48 (brs, 4H, CH2-2'' and CH2-6''), 2.14 (quintet, J = 6.8 Hz, 2H, CH2-2'); EI-MS m/z 492 [M]+, 314 [C10H10N4O8r, 312 [C^H^r, 266 [C^H^f, 228 [C6H2N3O7]+, 221 [C12H17N2O2]+, 95 [C5H3O2]+, 93 [C5HO2]+. Anal. Calc. for C19H20N6O10 (492.12): C, 46.35; H, 4.09; N, 17.07. Found: C, 46.44; H, 4.17; N, 17.19. 3. 5. 6. 2,4,6-Tribromophenyl 3-[4-(2-furoyl)-1 -piperazinyl]propylcarbamate (16) Off-white amorphous solid; Yield: 90%; m.p.: 80-92 oC; Mol. F.: C19H20Br3N3O4; Mol. Mass.: 591 g/mol; IR (KBr, cm-1) vmax: 3406 (N-H), 3086 (Ar C-H), 2882 (R C-H), 1657 (C=O), 1582 (Ar C=C), 1497 (N=O), 1197 (C-O-C), 1110 (C-N-C), 847 (C-N), 548 (C-Br); 1H-NMR (500 MHz, CDCl3, ppm): 87.67 (s, 2H, H-3 and H-5), 7.49 (brs, 1H, H-5'''), 7.04 (d, J = 4.1 Hz, 1H, H-3'''), 6.49 (dd, J = 2.0, 4.0 Hz, 1H, H-4'''), 5.19 (brs, 1H, NH), 4.12 (t, J = 6.6 Hz, 2H, CH2-1'), 3.94 (brs, 4H, CH2-3'' and CH2-5''), 3.61-3.57 (m, 2H, CH2-3'), 3.47 (brs, 4H, CH2-2" and CH2-6''), 2.10 (quintet, J = 6.8 Hz, 2H, CH2-2'); EI-MS m/z 591 [M]+, 413 [C10H10Br3NO2]^+, 411 [C10H8Br3NO2r+, 332 [C10H10Br2NO2]+, 328 [C6H3Br3O]•+, 221 [C12H17N2O2]+, 95 [C5H3O2]+, 93 [C5HO2]+. Anal. Calc. for C19H20Br3N3O4 (590.90): C, 3841; H, 3.39; N, 7.07. Found: C, 38.49; H, 3.44; N, 7.18. 3. 6. Biological Activity Assays (in vitro) 3. 6. 1. a-Glucosidase Assay The a-glucosidase inhibition activity was performed in accordance with the slightly modified method of Pierre et al.27 Total volume of the reaction mixture of 100 pL contained 70 pL 50 mM phosphate buffer saline, pH 6.8, 10 pL (0.5 mM) test compound, subsequently the addition of 10 pL (0.057 units) enzyme. The contents were mixed, preincubated for 10 min at 37 °C and pre-read at 400 nm. The reaction was initiated by the addition of 10 pL of 0.5 mM substrate (p-nitrophenylglucopyranoside). Acarbose was used as positive control. After 30 min of incubation at 37 °C, absorbance was measured at 400 nm using Synergy HT microplate reader. All experiments were carried out in duplicates. The percent inhibition was calculated by the following equation: Inhibition (%) = x 100 (l) IC50 values (concentration at which there is 50% in enzyme catalyzed reaction) compounds were calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA). 3. 6. 2. Cholinesterase Inhibition Assay The AChE and BChE inhibition activities were performed according to the method of Ellman et al., with minor modifications.28 Total volume of the reaction mixture was 100 pL. It contained 60 pL Na2HPO4 buffer with concentration of 50 mM and pH 7.7. Ten pL test compound (0.5 mM per well) was added, followed by the addition of 10 pL (0.005 unit per well) enzyme. The contents were mixed and pre read at 405 nm. Then contents were pre incubated for 10 mins at 37 °C. The reaction was initiated by the addition of 10 pL of 0.5 mM per well substrate (acetylthiocholine iodide / butyrylthiocholine iodide), after that the addition of 10 pL DTNB (0.5 mM per well). After 15 mins of incubation at 37 °C absorbance was measured at 405 nm. All experiments were carried out with their individual controls in triplicate. Eserine (0.5 mM per well) was used as a positive control. The inhibition (%) and IC50 were calculated by the same method as described in a-glucosidase assay. 3. 6. 3. Antibacterial Activity The antibacterial activity was evaluated in sterile 96-wells microplates under aseptic conditions. The method is rooted in the principle that microbial cell number increases as the microbial growth proceeds in a log phase of growth which results in increased absorbance of broth medium.29,30 Three gram-negative (Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa) and two gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) were included in the study. The organisms were maintained on stock culture agar medium. The test samples with suitable solvents and dilutions were pipette into wells (20 pg per well). Overnight maintained fresh bacte- Abbasi et al.: Synthesis of Some Unique Carbamate Derivatives ... 168 Acta Chim. Slov. 2017, 64, 159-169 rial culture after suitable dilution with fresh nutrient broth was poured into wells (180 pL). The initial absorbance of the culture was strictly maintained between 0.12-0.19 at 540 nm. The total volume in each well was kept to 200 pL. The incubation was done at 37 °C for 16-24 hours with lid on the microplate. The absorbance was measured, before and after incubation and the difference was noted as an index of bacterial growth at 540 nm by using microplate reader. The percent inhibition was calculated by using the formula: Inhibition (%) = x 100 (2) where X is absorbance in the control with bacterial culture and Y is absorbance in the test sample. Results are mean of triplicate (n = 3, ± SEM). Ciprofloxacin was used as the standard. Minimum inhibitory concentration (MIC) was measured with suitable dilutions (5-30 pg per well) and results were calculated using EZ-Fit 5 Perrella Scientific Inc. Amherst USA software, and data expressed as MIC. 3. 6. 4. Statistical Analysis The results are written as mean ± SEM after performance in three-folds and statistical analysis by Microsoft Excel 2010. Minimum inhibitory concentration (MIC) was calculated by using different dilutions (ranging 5-30 pg per well) and EZFit Perrella Scientific Inc. Amherst USA software. 3. 6. 5. Hemolytic Activity Hemolytic activity was done by a reported method.31,32 Bovine blood was obtained from the Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan. After centrifugation, separation and washing, the % RBCs lysis was computed by noting the absorbance. 3. 6. 6. Molecular Docking Methodology For the prediction of bioactive conformations, various synthesized compounds were docked into the active pockets of the following chosen proteins/enzymes by using the default parameters of MOE-Dock program. Earlier to dock the ligands into enzyme molecules, Builder of MOE 2009-10 was implemented to sketch the structures of synthesized compounds. Energy minimization was carried out up to 0.05 gradients by using MMFF94x force field through the default parameter of the MOE energy minimization algorithm. Then the energy minimized molecules were saved in the mdb file format as an input database for molecular docking in the subsequent step. The enzyme molecules of a-glucosidase (PDB ID code: 3NO4), acetylcholinesterase (PDB ID code: 1GQR) and butyrylcholinesterase (PDB ID code: 1POP) were retrieved from Protein Data Bank having the possible resolutions of 2.02 A, 1.69 A and 2.30 A respectively. Then all the water molecules were extracted from the receptor enzymes and 3D protonation was carried out through Pro-tonate 3D Option. Energies of protein molecules were minimized by using the default parameters of MOE 2009-10 energy minimization algorithm (gradient: 0.05, Force Field: MMFF94X). Then all the ligands were docked into the binding pockets (selective residues/amino acids) of the above enzymes using default parameters of MOE-Dock Program. To increase the validity of docking protocol, re-docking was also applied.33 LigPlot which is implemented in MOE (Molecular Operating Environment) was used to determine the interactions between enzymes and ligands. 4. Conclusion The structures of synthesized unique carbamate derivatives bearing 2-furoyl-1-piperazine moieties were elucidated by spectral techniques. All the derivatives showed decent inhibitory potential against acetylcholinestrase enzyme and almost all the derivatives were active against the studied strains of Gram-positive and Gram-negative bacteria. The results of cytotoxicity studies were used to evaluate their cytotoxicity profile as these molecules exhibited modest toxicity. Hence, these molecules may be considered as suitable therapeutic entrants for the drug designing programs leading to some life saving medication. 5. Acknowledgement The authors are highly thankful to Higher Education Commission (HEC) of Pakistan for providing financial support in this study. 6. References 1. R. Kharb, P. C. Sharma, A. Bhandari, M. Shaharyar, Der. Pharmacia. Lett. 2012, 4, 652-657. 2. J. Faist, W. Seebacher, R. Saf, R. Brun, M. Kaiser, R. Weis, Eur. J. Med. Chem. 2012, 47, 510-519. https://doi.org/10.1016/j.ejmech.2011.11.022 3. K. Kulig, J. Sapa, D. Maciag, B. Filipek, B. Malawska, Arch. 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Pharm. 1961, 7, 88-95. https://doi.org/10.1016/0006-2952(61)90145-9 29. M. Kaspady, V. K. Narayanaswamy, M. Raju, G. K. Rao, Lett. Drug Des. Discov. 2009, 6, 21-28. https://doi.org/10.2174/157018009787158481 30. C.-R. Yang, Y. Zang, M. R. Jacob, S. I. Khan, Y.-J. Zhang, X.-C. Li, Antimicrob. Agents. Chemother. 2006, 50, 1710-1714. https://doi.org/10.1128/AAC.50.5.1710-1714.2006 31. P. Sharma, J. D. Sharma, J. Ethnopharmacol. 2001, 74, 239-243. https://doi.org/10.1016/S0378-8741(00)00370-6 32. W. A. Powell, C. M. Catranis, C. A. Maynard, Lett. Appl. Microbiol. 2000, 31, 163-168. https://doi.org/10.1046/j.1365-2672.2000.00782.x 33. M. J. Bostro, J. R. Greenwood, J. Gottfries, Mol. Graph. Model. 2003, 21, 449-462. https://doi.org/10.1016/S1093-3263(02)00204-8 Povzetek Namen predstavljenega raziskovalnega dela je bila sinteza različnih biološko aktivnih karbamatnih derivatov, ki bi vsebovali 2-furoil-1-piperazinski fragment in bi bili le malo strupeni. Sintezo smo izvedli kot večstopenjsko sekvenco. Strukturno potrditev pripravljenih spojin smo izvedli s pomočjo EI-MS, IR in 1H-NMR spektroskopskih metod. Za pripravljene spojine smo določili tudi sposobnost inhibicije encimov in njihovo antibakterijsko delovanje. Da bi ugotovili potencialno uporabnost dobljenih spojin kot terapevtskih učinkovin, smo določili tudi njihovo citotoksičnost. Vse spojine so se izkazale kot aktivne proti acetilholinesterazi; spojini 12 in 14 sta izkazovali še posebej dobro inhibitorno aktivnost v primerjavi z referenčnim standardom ezerinom. Skoraj vse spojine so se pokazale kot učinkovite tudi proti Gram-pozitivnim in Gram-negativnim bakterijskim sevom. Abbasi et al.: Synthesis of Some Unique Carbamate Derivatives ... 170 DOI: 10.17344/acsi.2016.2995 Acta Chim. Slov. 2017, 64, 170-178 recreative ^/commons Scientific paper Nitrogen Doped Graphene Nickel Ferrite Magnetic Photocatalyst for the Visible Light Degradation of Methylene Blue Rajinder Singh, Jigmet Ladol, Heena Khajuria and Haq Nawaz Sheikh* Department of Chemistry, University of Jammu, Jammu Tawi,180 006 India * Corresponding author: E-mail: hnsheikh@rediffmail.com Received: 14-10-2016 Abstract A facile approach has been devised for the preparation of magnetic NiFe2O4 photocatalyst (NiFe2O4-NG) supported on nitrogen doped graphene (NG). The NiFe2O4-NG composite was synthesized by one step hydrothermal method. The na-nocomposite catalyst was characterized by Powder X-ray diffraction (PXRD), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), Ultraviolet-visible spectroscopy (UV-Vis) and Vibrating sample magnetometry (VSM). It is found that the combination of NiFe2O4 nanopartic-les with nitrogen-doped graphene sheets converts NiFe2O4 into a good catalyst for methylene blue (MB) dye degradation by irradiation of visible light. The catalytic activity under visible light irradiation is assigned to extensive movement of photogenerated electron from NiFe2O4 to the conduction band of the reduced NG, effectively blocking direct recombination of electrons and holes. The NiFe2O4 nanoparticles alone have efficient magnetic property, so can be used for magnetic separation in the solution without additional magnetic support. Keywords: Nanostructures, photodegradation, nickel ferrite, catalysts, absorption, UV/Vis spectroscopy. 1. Introduction Photocatalysis especially by TiO2 has been widely used for the purification of waste water. The energy band gap of 3.2 eV is required for the excitation of electron by light in TiO2 catalyst so UV light can only be used in the process of photodegradation. The development of visible light sensitive photocatalysts by band gap modifications and external surface changing for waste water treatment and degradation of organic dye is an active area in photo-catalysis.1-7 Graphene has attracted the attention due to various applications.8-11 Graphene has sp2 hybridized carbon and one atom thick (2-D) sheet of conjugated system and extraordinary physical and chemical properties.12-16 There has been so much focus to develop graphene-metal oxide photocatalysts such as TiO2-graphene and ZnO-graphene for the photodegradation of organic dye by the irradiation of visible light.17-22 The heterogeneous systems are mostly used to perform the photodegradation reactions. The repeated use of photocatalysts after degradation is of great importance for sustainable use of the catalyst. The magnetic nanoparticles anchored on solid sup- port serve as heterogenous catalyst allowing facile separation of catalyst from reaction products.23 Superparamagnetic copper ferrite-graphene nanocomposite prepared via hydrothermal method acts as excellent catalyst for the reduction of nitroarenes. The big advantage of the catalyst is that it can be easily recovered and retains the catalytic activity even after five catalytic cycles.24 Copper-cobalt ferrites prepared by hydrothermal method from co-precipa-ted precursor serve as efficient catalyst in the decomposition of methanol to CO and H2.25 The various metal ferrites have been used as catalysts in phenols decomposition, detoxification of CO gas from automobile exhaust, anodic material for lithium ion batteries.26-29 Nickel ferrite (Ni-Fe2O4) has the inverse spinel structure. The ferrimagne-tism arises due to antiparallel spin of Fe3+ ions present at tetrahedral sites and Ni2+ occupying octahedral sites.30 The Nickel ferrite is considered as the efficient magnetic material which has good electrical resistivity, high-Curie temperature and chemical stability. Magnetic nanopartic-les of nickel ferrite have been used to manufacture titania-coated nickel ferrite, which can act as magnetically separable photocatalyst.31 The TiO2 doped NiFe2O4 nanopar- Singh et al.: Nitrogen Doped Graphene Nickel Ferrite Magnetic ... Acta Chim. Slov. 2017, 64, 170-178 171 ticles possess band gap of 2.19 eV and have displayed enhanced photocatalytic activity as compared to TiO2 for degradation of Rhodamine B dye in aqueous solution under visible light irradiation.32 Pure nickel ferrite is photo-ca-talytically inactive but its composite with another semiconductor (e.g., graphene sheets) can find an effective mechanism for separation of charges leading to increased photocatalytic performance. One such example is Zn-Fe2O4-graphene photocatalyst and its great performance in the photocatalytic degradation of MB under visible light irradiation.33 Carbon material doped with a heteroatom, such as B, N or S, can increase the pseudo capacitance by manipulating its electronic properties and chemical reactivity leading to increased performance of doped grapheme.34-37 Nitrogen-doped graphene (NG) has great utility because of its higher specific capacitance matched to the pristine graphene and good durability, therefore, enabling its use as electrode materials for supercapacitors and applications in photocatalysis.38 In this paper, we report the development of one step method to design magnetically separable nitrogen doped graphene-based photocatalyst having excellent catalytic activity. The approach is designed to deposit NiFe2O4 na-nocrystals on nitrogen doped graphene sheets via a one-step hydrothermal method. Interestingly, in the presence of nitrogen doped graphene, the inert nanocrystals of Ni-Fe2O4 have been converted into a highly efficient catalyst for the methylene blue (MB) degradation under visible light irradiation. In addition, NiFe2O4 nanoparticles themselves have a magnetic property, which makes the Ni-Fe2O4-NG composite magnetically separable in liquid medium. 2. Experimental 2. 1. Materials Iron(III) nitrate nonahydrate Fe(NO3)3 ■ 9H2O, Nic-kel(II) nitrate hexahydrate Ni(NO3)2 ■ 6H2O, graphite powder flakes, phosphoric acid and hydrogen peroxide were purchased from Alfa Aesar. All chemicals were used as received without further purification. Ethanol, urea, sodium hydroxide and sulphuric acid were purchased from Sigma Aldrich. Deionized water was used throughout. 2 .2. Synthesis of Magnetic NiFe2O4-Nitrogen Doped Graphene Composite Photocatalyst Purified natural graphite was used for the synthesis of graphene oxide (GO) by the well known method given by Hummers and Offeman.39 The graphene oxide (GO) (0.08 g) was dispersed in 20 ml of absolute ethanol and sonicated for 45 min. In a separate beaker 0.28 g of Ni(NO3)2 ■ 6H2O and 0.78g of Fe(NO3)3 ■ 9H2O mixture was added to 10 ml absolute ethanol with constant stirring for 30 min forming homogenous solution. The two solutions were mixed and pH of the mixture solution was kept 10.0 using 6 M NaOH solution and then 1 g urea was added into it. The resulting mixture was put into a 50 mL Teflon-lined stainless steel autoclave and heated to 180 °C for 18 h in an oven. After cooling the reaction mixture to room temperature and the precipitates were filtered, washed with distilled water and dried in oven at 70 °C for 12 h. The product was named as NiFe2O4-NG. Same method was applied to synthesize pure NiFe2O4 with the modification that GO and urea were excluded. Sulfur was estimated as BaSO4 by gravimetric method and Chloride was estimated as AgCl by Volhard's method.40 2. 3. Spectroscopic and Microscopic Measurements The phase and size of the as-prepared samples were determined from powder X-ray diffraction (PXRD) using D8 X-ray diffractometer (Bruker) at a scanning rate of 12° min1 in the 28 range from 10° to 70°, with Cu Ka radiation (X = 0.15405 nm). Scanning electron microscopy (SEM) micrographs of the samples were recorded on FEI Nova Nano SEM 450. High Resolution Transmission Electron Microscopy (HRTEM) was recorded on Tecnai G2 20 S-TWIN Transmission Electron Microscope with a field emission gun operating at 200 kV. The samples for TEM measurements were prepared by evaporating a drop of the colloid onto a carbon coated copper grid. The infrared spectra were recorded on Shimadzu Fourier Transform Infrared Spectrometer (FT-IR) over the range of wave number 4000-400 cm1 and the standard KBr pellet technique was employed. The magnetic moment as a function of applied field was recorded using Vibrating Sample Magnetometer (VSM), Lakeshore 7410. All the measurements were performed at room temperature. 2. 4. Photocatalytic Activity Measurement The catalytic activity of the as synthesized sample was performed by degradation of organic dye MB under the irradiation of visible light. For the Photo irradiation 500 W xenon lamp was used fitted with UV cut-off filters (JB450) in order to completely remove any radiation below 420 nm ensuring the exposure to only visible light. The whole procedure was performed at 25 °C. A 100 mL of MB dye solution was prepared (20 mg/L concentration) and 0.025 g of photocatalyst was mixed with dye solution. The resulting mixture was stirred for 60 min before illumination in order to establish the adsorption - desorption equilibrium between MB and catalyst surface. At same instant of time 5 mL of dye-catalyst mixture was taken out and concentration of the residual dye was determined with the help of UV-vis spectroscopy by measuring the absorption at 664 nm. The absorbance of dye at 664 nm was monitored with time after fixed intervals of time. The absor- Singh et al.: Nitrogen Doped Graphene Nickel Ferrite Magnetic ... 172 Acta Chim. Slov. 2017, 64, 170-178 bance of dye with time without catalyst was also recorded for reference. 3. Results and Discussion 3. 1. PXRD Measurements The structural characterization of the nanoparticles has been carried out by Powder X-ray diffraction technique using CuKa radiation. Figure 1(a-b) show the differences of phase composition between GO and NG. The doping of nitrogen in GO can be clarified easily by PXRD spectrum. The PXRD pattern of GO exhibits a characteristic (002) peak of graphite emerging at 24.2°. Compared with GO, it is found that the (002) peak of NG appears at 26.3° which indicates that nitrogen atoms have entered into the crystal lattice of graphite and caused the increased distance between the graphite layers. This confirms the formation of nitrogen-doped graphene by urea assisted hydrothermal reaction. Figures 1c, d show the PXRD diffraction patterns of the pure NiFe2O4 and as prepared Ni-Fe2O4-NG. The diffraction peaks a4 30.9°, 35.7°, 43.4°, 53.7°, 57.2° and 63.2° corresponding to the planes (220), (311), (400), (422), (511) and (440) are allocated to spinel-type NiFe2O4 (JCPDS No. 54-0964).41 Similar diffraction patterns are observed for NiFe2O4-NG. The nitrogen doped graphene oxide can be reduced by the alcohol under hydrothermal conditions and no peak at (002) is observed in the composite. It can also be related to well exfoliation of the NG sheets in the resulting composite material. So the diffraction pattern of NG disappears in the XRD pattern of NiFe2O4-NG. The average crystallite size of these nanoparticles was calculated according to the Scherrer's equation. (1) where, L (nm) is the crystallite size, X (nm) is the wavelength of the Cu Ka radiant, X = 0.15405 nm, P(°) is the full-width at half-maximum (FWHM) of the diffraction peak, 8 is the diffraction angle and K is the Scherrer constant equal to 0.89. All the major peaks were used to calculate the average crystallite size of the NiFe2O4 and Ni-Fe2O4-NG nanoparticles. The estimated average crystallite sizes of nanoparticles are in the range of 80-120 nm. 3. 2. SEM and TEM Analysis Figure 2a shows representative scanning electron microscopy and transmission electron microscopy images of the prepared GO. From the SEM image, morphology and structure of as-prepared graphene oxide sample was investigated. GO sheets were cast on a gold coated (100 nm) Si/SiO2 substrate. It has been found that the graphene flakes have wrinkled surfaces. Furthermore, in the TEM image (Figure 3a) GO shows layer-by-layer stacked structure and has wrinkled paper like morphology. Such morphological changes can be attributed to the increased formation of phenolic and epoxy functional groups on the basal plane of GO. The curled and overlapped nanosheets can be clearly observed. The SEM image (Figure 2b) and TEM image (Figure 3b) reveal that nitrogen-doped grape-ne nanosheets exhibit a typical wrinkled structure, which results from stable bending thermodynamically.42,43 Figures 2(c-d) show SEM images of the NiFe2O4 and NiFe2O4-NG samples where as Figures 3(c-d) show TEM images of the NiFe2O4 and NiFe2O4-NG samples. In Figure 2c and Figure 3c, NiFe2O4 nanoparticles are clear- Figure 1. PXRD patterns of (a) GO, (b) NG, (c) NiFe2O4 and (d) NiFe2O4-NG. Singh et al.: Nitrogen Doped Graphene Nickel Ferrite Magnetic Acta Chim. Slov. 2017, 64, 170-178 173 ly visible in the SEM and TEM images. The NiFe2O4 na-noparticles distributed on NG to form nanoparticles bound on the surface of NG sheets is seen in the Figure 2d and Figure 3d. Measurements showed that the average diameter of NiFe2O4-NG particles is approximately 80 nm. The particle size data obtained from TEM data are in very close agreement to the size calculated from the Deb-ye-Scherrer method. 3. 3. FT-IR Characterization Figure 4(a-d) shows the FTIR spectra of GO, NG, NiFe2O4 and NiFe2O4-NG. There are many O-containing groups that exist on GO sheets, such as hydroxyl, epoxy, and carboxyl groups. Majority of the O-containing groups will disappear after reduction. FTIR bands at 1050, 1220, 1405 and 1730 cm-1 were observed for GO. These bands correspond to C-O stretching, C-O-C stretching, O-H deformation vibration and C=O car-bonyl stretching.44 FTIR bands at 1400 cm-1 due to C=C stretching is observed in NG and the vC=O band at 1730 cm-1 completely disappeared due to reduction. The bands located at 1180 and 1565 cm-1 in Figure 4b are assigned to the v C-N and v C=C respectively. The FTIR spectra suggest N doping of GO. Figure 4 (c-d) shows the FT-IR bands of NiFe2O4 and NiFe2O4-NG. The bands observed in the range of 620-650 cm-1 corresponds to the intrinsic stretching vibrations of the M-O in the tetrahedral site. The second band around 3400-3500 cm-1 corresponds to O-H stretching vibrations.45 Furthermore, it is observed that almost all the characteristic bands of oxygen containing functional groups (C=O, O-H, C-OH and C-O-C) disappeared in the FT-IR spectrum of NiFe2O4-NG depicting the change in the surface morpholgy of NG-NiFe2O4 composite. These findings show that NiFe2O4 nanoparticles are bonded to the NG. The results above show the heteroatom N was entered in the graphene structure and the NiFe2O4-NG composites was prepared favourably. 3. 4. Photocatalytic Measurements The adsorption of light by the photocatalysts is the key feature of photocatalysis method. Figure 5a show the Singh et al.: Nitrogen Doped Graphene Nickel Ferrite Magnetic ... 174 Acta Chim. Slov. 2017, 64, 170-178 Figure 4. FT-IR spectra of (a) GO (b) NG (c) NiFe2O4 (d) NiFe2O4-NG UV Spectrum of NiFe2O4-NG. The photocatalytic activities of the as-obtained NiFe2O4-NG nanocomposite pho-tocatalysts were evaluated by monitoring the degradation of methylene blue (MB) under visible-light irradiation at 25 °C. Figure 5a shows the changes in the absorbance pro- files of MB solution (concentration of MB, C = 0.075 M and path length, l = 1cm) in the presence of NiFe2O4-NG photocatalyst under visible-light irradiated at 25 °C recorded at different time intervals. The adsorption-desorption equilibriated solution of MB and NiFe2O4-NG was used Singh et al.: Nitrogen Doped Graphene Nickel Ferrite Magnetic Acta Chim. Slov. 2017, 64, 170-178 175 0.7 0.6 0.5 0-S 0.2 0.1 0.0 Odin 30 min ........ JJ/XÜ........ ........... A SO min J///-A 90 min 6~~y// \\t 120 min ..................>1.................... i MÄ I M /V/ ™ ISO min ISO mill Wavelength (ddi) Figure 5a. Absorption spectra of the MB solution (C = 0.075 M and l = 1 cm) taken at different photocatalytic degradation times using Ni- Irradiarion time (ruin) Figure 5b. Kinetics of photodegradation of (a) Pure MB and (b) the pure MB solution. The catalyst acts as magnetic material which gives good performance in magnetic separation for the NiFe2O4-NG photocatalysts using an external magnet. 3. 4. 1. Mechanism of Photocatalytic Activity Measurements The photocatalytic activity for MB degradation can be best explained by the following mechanism. The notable increase in photocatalytic activity under visible light exposure can be attributed to exceptional synergistic effect between NiFe2O4 and the nitrogen-doped graphene sheets causing the effective separation of carriers generated by the light exposure in the NiFe2O4-NG composite system. A plausible mechanism for enhancement in pho-tocatalysis process is shown as follows: When the visible-light is irradiated on the surface of NiFejO^e) + N-doped graphene NiFe^O,) + N-doped graphene (e) NiFe204(h) + OH + 02"+ CI6H15C1N3S(MB) NiFe204 + CO, + H,0 + S042" + NO," + CI" + Nfl4+ (2) (3) (4) (5) (6) as starting solution. In Figure 5b C/C0 was plotted versus time where C0 is initial concentration of methylene blue (0.075 M at time t = 0 min. and C is concentration at time t min.). It can be clearly seen that almost all the MB in the solution is decomposed after 180 min in presence of the NiFe2O4-NG while there is least photodegradation in NiFe2O4, the electron-hole pairs are formed (Eq. 2). Then by the percolation mechanism, the electrons generated by the photogeneration process are instantly transfer onto NG sheets (Eq. 3). Superoxide anion radical is produced from oxygen dissolved and activated through nitrogen doped graphene carrying negative charge (Eq. 4). The adsor- Fe2O4-NG. Singh et al.: Nitrogen Doped Graphene Nickel Ferrite Magnetic ... 176 Acta Chim. Slov. 2017, 64, 170-178 bed water can react with holes to produce hydroxyl radical (Eq. 5). At the end superoxide anion, and hydroxyl radical cause the oxidation of MB dye adsorbed on the surface of NiFe2O4-NG composite by electrostatic interaction and n-n interaction between aromatic rings of methylene blue and graphene layer (Eq. 6). In the photocatalytic degradation process, the electrons of the photocatalyst i,e Ni-Fe2O4-NG nanocomposite are excited from the valence band (VB) to the conduction band (CB) by the visible light irradiation. The photogenerated holes in the VB are scavenged by OH- of water forming OH radicals which are responsible for the MB degradation process afterwards. The N-graphene performs two functions; (a) it acts as charge carrier to trap the delocalised electrons thereby restricting the (h-e) recombination. (b) Secondly, it increases the adsorption of MB dye on the catalyst surface thereby increasing the n-n interaction between aromatic rings of methylene blue and graphene layer.46 3. 5. Magnetic Characterization Magnetization hysteresis loops of the as-prepared NiFe2O4 and NiFe2O4-NG samples at room temperature were measured using vibrating sample magnetometer as shown in Figure 6(a-b). The magnetic properties of the NiFe2O4 having inverse spinel structure can be described in terms of cations distribution. The magnetization originates from the Fe3+ ions at both tetrahedral and octahedral sites and Ni2+ is present only in octahedral sites.47,48 Coer-civity and saturation magnetization of NiFe2O4-NG are 47.4 G and 10.1 emu/g respectively, whereas that of Ni-Fe2O4 are 33.5 G and 9.2 emu/g respectively. The values observed for NiFe2O4-NG are larger than those for Ni-Fe2O4 which shows that NiFe2O4-NG is more easily separable than NiFe2O4. The increase in the saturation magne- m— J I I 1 15 -10 -5 0 5 10 15 Applied Field (kOe) Figure 6. Magnetic hysteresis loop measured at 300 K for (a) Ni-Fe2O4 (b) NiFe2O4-NG tization was possibly attributed to the increasing crystalli-nity and particle size of the nanoparticles. 4. Conclusions In the outcome, a magnetic NiFe2O4-NG photoca-talyst has been fabricated through hydrothermal route. The SEM and TEM images show that nitrogen-doped graphene sheets are flaked and furnished with NiFe2O4 nanoparticles having an average diameter of 80 nm. The photocatalytic activity measurements confirm that the Ni-Fe2O4 nanoparticles combined with nitrogen-doped grap-hene sheets lead to exciting conversion of the inactive Ni-Fe2O4 into very good catalyst for the degradation of methylene blue (MB) under visible light irradiation. The notable increase in photoactivity can be ascribed to the superior conductivity of the reduced NG sheets leading to favourable and efficient separation of photogenerated carriers (hole-electron) in the NiFe2O4-NG system. Subsequently, there is very large and useful change in photoca-talytic activity after coupling nickel ferrite with nitrogen-doped graphene sheets. 5. Acknowledgements We would like to acknowledge SAIF, Panjab University, Chandigarh and Indian Institute of Technology Guwahati for their technical support. 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Int. 2016, 42, 15235-15241. https://doi.org/10.1016/j.ceramint.2016.06.160 47. H. Nathani, S. Gubbala, R. D. K. Misra, Mater. Sci. Eng. B 2005, 121, 126-136. https://doi.org/10.1016/j.mseb.2005.03.016 48. A. B. Nawale, N. S. Kanhe, K. R. Patil, S. V. Bhoraskar, V. L. Mathe, A. K. Das, J Alloys Compd. 2011, 509, 4404-4413. https://doi.org/10.1016/jjallcom.2011.01.057 Singh et al.: Nitrogen Doped Graphene Nickel Ferrite Magnetic ... 178 Acta Chim. Slov. 2017, 64, 170-178 Povzetek Preprost sintezni način smo uporabili za pripravo magnetnega fotokatalizatorja NiFe2O4 na grafenu, dopiranem z dušikom (NG). Kompozit NiFe2O4-NG smo pripravili z enostopenjsko hidrotermalno sintezo. Nanokompozitni katalizator smo karakterizirali z naslednjimi metodami: rentgensko praškovno difrakcijo (XRD), vrstično elektronsko mikroskopijo (SEM), presevno elektronsko mikroskopijo (TEM), infrardečo spektroskopijo (FT-IR), UV-Vis spektroskopijo in magnetometrijo z vibrirajočim vzorcem (VSM). Kombinacija nanodelcev NiFe2O4 in grafena, dopiranega z dušikom pretvori NiFe2O4 v dober katalizator za fotokatalitični razpad barvila metilen modro (MB). Fotokatalitično aktivnost pod vplivom vidne svetlobe lahko pripišemo obsežnemu premiku vzbujenih elektronov iz NiFe2O4 v prevodni pas redu-ciranega grafena (NG). Že sami nanodelci NiFe2O4 imajo takšne magnetne lastnosti, da jih lahko uporabimo za magnetno separacijo v raztopini brez dodatne uporabe magneta. Singh et al.: Nitrogen Doped Graphene Nickel Ferrite Magnetic DPI: I0.l7344/acsi.20l6.30l9_Acta Chirn, Slov. 2017,64, 179-185_©commons m Scientific paper Synthesis, Structures, and Antimicrobial Activities of Two Cobalt(II) Complexes [Co(L1)2(OH2)2] and [Co(L2)2] Yong-Jun Han, Li Wang, Qing-Bin Li and Ling-Wei Xue* College of Chemistry and Chemical Engineering, Pingdingshan University, Pingdingshan Henan 467000, P.R. China * Corresponding author: E-mail: pdsuchemistry@ 163.com Received: 27-10-2016 Abstract A new cobalt(II) complex, [Co(L1)2(OH2)2] (1), was prepared by the reaction of 3-bromo-5-chlorosalicylaldehyde (HL1) with cobalt nitrate in methanol. Reaction of 1 with cyclopropylamine in methanol afforded the Schiff base cobalt(II) complex, [Co(L2)2] (2), where L2 is the deprotonated form of 2-bromo-4-chloro-6-(cyclopropyliminomethyl)phenol (HL2). The complexes have been characterized by elemental analyses, IR spectroscopy, and single-crystal X-ray diffraction. The L1 ligand coordinates to the Co atom through the phenolate O and carbonyl O atoms, while the L2 ligand coordinates to the Co atom through the phenolate O and imino N atoms. The Co atom in complex 1 adopts octahedral coordination and that in complex 2 adopts tetrahedral coordination. The effect of the free ligands and the cobalt complexes on the antimicrobial activities against Staphylococcus aureus, Escherichia coli, and Candida albicans was studied. Keywords: Synthesis; Crystal structure; Antimicrobial; Schiff base; Cobalt complex 1. Introduction Schiff bases are a kind of versatile ligands in coordination chemistry.1-6 In recent years, metal complexes of Schiff bases have attracted considerable attention due to their remarkable biological activity, such as antifungal, antibacterial and antitumor property.7-9 It has been shown that the Schiff base complexes derived from salicylaldehyde and its derivatives with primary amines, bearing the N2O, N2S, NO2 or NSO donor sets, have interesting biological activity.9-12 Furthermore, cobalt complexes in its varied oxidation states have become a central theme of current research because of their potentially useful properties in the realm of relevant scientific and technological fields. Recently, we have reported some Schiff base complexes and their application in biological area.13-15 In the present work, two new cobalt(II) complexes, [Co(L1)2(OH2)2] (1) and [Co(L2)2] (2), where L1 and L2 are the deprotonated forms of 3-bromo-5-chlorosa-licylaldehyde (HL1) and 2-bromo-4-chloro-6-(cyclopropyli-minomethyl)phenol (HL2), respectively, are reported. 2. Experimental 2. 1. Material and Methods 3-Bromo-5-chlorosalicylaldehyde, cyclopropylami-ne, and cobalt nitrate were purchased from Fluka. Other reagents and solvents were analytical grade and used without further purification. Elemental (C, H and N) analyses were made on a Perkin-Elmer Model 240B automatic analyzer. Cobalt analysis was carried out by EDTA titration. Infrared (IR) spectra were recorded on an IR-408 Shimadzu 568 spectrophotometer. 2. 2. Preparation of [Co(L1)2(OH2)2] (1) HL1 (0.23g g, 1.0 mmol) was dissolved in methanol (20 mL), then a methanol solution (10 mL) of Co(NO3)2 ■ 6H2O (0.29 g, 1.0 mmol) was added while stirring. The mixture was stirred for 1 h at ambient temperature to give a red solution. Red block-shaped single crystals suitable for X-ray diffraction were formed by slow evaporation of the solution in air for about a week. Yield: 45%. D.p. 173 °C. Elemental analysis found: C, 29.63; H, 1.92; Co, 10.67%. C14H10Br2Cl2CoO6 calcd: C, 29.82; H, 1.79; Co, 10.45%. IR data (KBr, cm-1): 3433 (br, m), 1647 (vs), 1505 (m), 1443 (s), 1413 (s), 1313 (w), 1205 (m), 1139 (s), 1080 (s), 989 (m), 926 (w), 864 (m), 747 (s), 693 (w), 543 (m), 409 (w). 2. 3. Preparation of [Co(L2)2] (2) To the methanolic solution (10 mL) of complex 1 (56.4 mg, 0.100 mmol) was added a methanolic solution Han et al.: Synthesis, Structures, and Antimicrobial Activities 180 Acta Chim. Slov. 2017, 64, 179-185 (10 mL) of cyclopropylamine (11.5 mg, 0.200 mmol). The mixture was stirred for 1 h at ambient temperature to give a red solution. Red block-shaped single crystals suitable for X-ray diffraction were formed by slow evaporation of the solution in air for three days. Yield: 61%. D.p. 232 °C. Elemental analysis found: C, 39.77; H, 2.58; N, 4.72; Co, 9.9%. C20H16Br2Cl2CoN2O2 calcd: C, 39.64; H, 2.66; N, 4.62; Co, 9.7%. IR data (KBr, cm1): 1622 (m), 1438 (m), 1360 (m), 1160 (s), 1072 (s), 951 (s), 860 (s), 543 (m), 518 (m), 464 (w). 2. 4. X-ray Diffraction Data were collected from selected crystals mounted on glass fibers. The diffraction data were collected on a Bruker SMART 1000 CCD with Mo Ka radiation (X = 0.71073 Â) at 298(2) K. The data for the two complexes were processed with SAINT16 and corrected for absorption using SADABS.17 Semi-empirical absorption corrections were applied with y-scans.18 The structures were solved by direct methods using SHELXS-97, and refined by full-matrix least-squares techniques on F2 using anisotropic displacement parameters.19 The water hydrogen atoms were located from a difference Fourier map and refined isotropically, with O-H and H—H distances restrained to 0.85(1) and 1.37(2) Â, respectively. The remaining hydrogen atoms were placed at the calculated positions. Idealized H atoms were refined with isotropic displacement parameters set to 1.2 times the equivalent isotropic U values of the parent atoms. The low bond precision on C-C bonds of 0.01614  for 1 was caused by the poor quality of the crystal diffraction. The 18 restraints of 1 we- re generated by the O-H and H—H distances restraints, and the isotropic behavior restraint of C14. The cyclopropane group C18-C19-C20 of 2 was disordered over two sites, with occupancies of 0.352(5) and 0.648(5), respectively. The crystallographic data for the complexes are listed in Table 1. 3. Results and Discussion 3. 1. Chemistry A new cobalt(II) complex with L1 as ligand has been prepared. Reaction of this complex with cyclo-propylamine afforded a new cobalt(II) complex bearing Schiff base ligand, L2 (Scheme 1). The results of the elemental analyses are in accord with the calculated composition of these complexes. The air-stable cobalt complexes are soluble in DMF, methanol, ethanol, chloroform, and acetonitrile. 3. 2. Infrared Spectra The infrared spectrum of complex 1 exhibits strong band at 1647 cm-1, which can be assigned to the C=O stretching frequency of L1 ligands. When the carbonyl groups form azomethine groups with cyclopropylamine, the band is absent in the spectrum of complex 2. Instead, a new band indicative of C=N bond is observed at 1622 cm-1.20,21 When compared with the spectrum of the free Schiff base HL2, it can be seen that the band is shifted to the lower frequency. This indicates the coordination of the imino N atom to the cobalt center. The medium and broad Table 1. Crystal and structure refinement data for 1 and 2 1 2 Empirical formula CMH1oBr2Cl2CoO6 C2()H16Br2Cl2CoN2O2 Formula weight 563.9 606.0 Temperature (K) 298(2) 298(2) Crystal system Monoclinic Monoclinic Space group P21/c P21/c Unit cell dimensions a (À) 7.590(2) 12.319(2) b (À) 27.685(2) 22.916(2) c (À) 8.639(2) 7.952(1) ß(°) 101.451(2) 108.83(3) y (À3) 1779.3(5) 2124.6(5) Z 4 4 Density (g cm-3) 2.105 1.895 Absorption coefficient (mm-1) 5.784 4.841 Reflections collected 10619 8098 Independent reflections 2778 2979 Data/parameters 1734/238 1478/290 Restraints 18 52 Final R indices [I > 2a(I)] 0.0777, 0.1509 0.0355, 0.0500 R indices (all data) 0.1411, 0.1737 0.1024, 0.0615 Goodness-of-fit on F2 1.054 0.913 Han et al.: Synthesis, Structures, and Antimicrobial Activities Acta Chim. Slov. 2017, 64, 179-181 185 Scheme 1. The synthetic procedure of the complexes band centered at 3433 cm-1 for the spectrum of complex 1 can be attributed to the O-H vibrations of the water li-gands. The bands in the region 550-400 cm-1 are assigned to the Co-N and Co-O vibrations.22 3. 3. Crystal Structure Description of the Complex 1 The molecular structure of the complex 1 is shown in Figure 1. The Co atom has an octahedral geometry and coordinated by two deprotonated 3-bromo-5-chloroben-zaldehyde ligands, and two water molecules. The aldehyde ligands act as bidentate ligands and coordinate to the Co atom through the phenolate O and carbonyl O atoms. For the octahedral coordination, the three trans angles are in the range 170.3(3)-177.2(3)°, and the other angles are in the range 84.9(3)-95.5(3)°, indicating a slightly distorted octahedral geometry (Table 2). The distances of the Co-O and Co-N bonds are comparable to the values observed in other cobalt(II) complexes with similar coordination.23,24 The dihedral angle between the two benzene rings of the ligands is 2.7(3)°. In the crystal structure, the molecules are connected by intermolecular hydrogen bonds O-H-O and O-H-Br, forming a 3D network, as Table 2. Coordinate bond distances (A) and angles (°) for 1 and 2 1 Co1-O1 Co1-O3 Co1-O5 O4-Co1-O1 O1-Co1-O2 01-Co1-O3 O4-Co1-O5 02-Co1-O5 04-Co1-O6 O2-Co1-O6 05-Co1-O6 2 Co1-O1 Co1-N1 O2-Co1-O1 O1-Co1-N2 O1-Co1-N1 shown by Figure 2. The corresponding hydrogen bonding parameters are listed in Table 3. In addition, there are n---n stacking interactions (Table 4) among the adjacent benze- 2.062(7) Co1-O2 2.069(6) 2.087(7) Co1-O4 2.062(7) 2.096(7) Co1-O6 2.109(7) 176.6(3) O4-Co1 O2 95.5(3) 87.1(3) O4-Co1 O3 86.7(3) 90.7(3) O2-Co1 O3 177.2(3) 92.8(3) O1-Co1- O5 84.9(3) 94.4(3) O3-Co1 O5 87.3(3) 94.5(3) O1-Co1- O6 87.5(3) 91.2(3) O3-Co1 O6 86.8(3) 170.3(3) 1.904(3) Co1-O2 1.890(3) 1.995(4) Co1-N2 1.956(5) 116.89(14) O2-Co1 N2 95.83(16) 115.96(15) O2-Co1 N1 121.64(14) 95.18(15) N2-Co1- N1 112.84(16) ne rings 25 Table 3. Hydrogen bonding parameters for 1 and 2 D-H-A d(D-H) (A) d(H-A) (A) d(D-A) (A) Z(D-H-A) (°) 1 O6-H6B O6-H6A O6-H6A O5-H5B O5-H5B O5-H5A 2 C19-H19-Br1 C17-H17-Br1 ••O4a ••Br1a ••O2a ••Br2b ••O4b ••O2b 0.85(1) 0.85(1) 0.85(1) 0.85(1) 0.85(1) 0.85(1) 0.97 0.93 1.96(5) 2.72(5) 2.26(9) 2.78(5) 2.40(10) 1.93(5) 2.92(3) 2.92(3) 2.766(9) 3.486(7) 2.911(11) 3.583(7) 3.018(10) 2.729(9) 3.660(5) 3.824(5) 156(11) 151(9) 133(11) 158(11) 130(11) 156(12) 134(6) 163(6) Symmetry codes: (a) 1 - x, - y, - z; (b) - x, - y, - z; (c) x, y, 1 + z; (d) 1 - x, - y, 1 - z. c Han et al.: Synthesis, Structures, and Antimicrobial Activities ... 182 Acta Chim. Slov. 2017, 64, 179-185 Table 4. Parameters between the planes for 1 and 2 Cg Cg Cg 'Cg Dihedral 8 8 distance (A) angle (°) Perpendi cular distance of Cg(I) on Cg(J) (A) P(°) Y (°) Perpendicular distance of Cg(J) on Cg(I) (A) 1 Cg(1)-Cg(2)b 3.827 2.28 3.562 19.24 Cg(1)-Cg(2)a 3.961 2.28 3.660 21.42 Cg(1) and Cg(2) are the centroids of the C1-C6 and C8-C13 benzene rings, respectively. 2 Cg(3)-Cg(3)b 3.660 0.00 3.384 22.39 Cg(3) is the centroid of the C1-C6 benzene ring. 21.45 22.48 22.39 3.613 3.687 3.384 Figure 1. Perspective view of the complex 1 with 30% probability thermal ellipsoids. Figure 2. Molecular packing of the complex 1 along the b axis. Han et al.: Synthesis, Structures, and Antimicrobial Activities Acta Chim. Slov. 2017, 64, 179-183 185 3. 4. Crystal Structure Description of the Complex 2 The molecular structure of the complex 2 is shown in Figure 3. The Co atom has a tetrahedral geometry and is coordinated by two deprotonated Schiff base ligands 2-bromo-4-chloro-6-(cyclopropyliminomethyl)phenol. The Schiff base ligands act as bidentate ligands and coordinate to the Co atom through the phenolate O and imino N atoms. For the tetrahedral coordination, the angles are in the range 95.18(15)-121.64(14)°, indicating a slightly distorted tetrahedral geometry (Table 2). The distances of the Co-O and Co-N bonds are comparable to the values Figure 3. Perspective view of the complex 2 with 30% probability thermal ellipsoids. Only the major component of the disordered cyclohexane group is shown. Figure 4. Molecular packing of the complex 2 along the a axis. Han et al.: Synthesis, Structures, and Antimicrobial Activities ... 184 Acta Chim. Slov. 2017, 64, 179-185 observed in other cobalt(II) complexes with similar coordination.26'27 The dihedral angle between the two benzene rings of the ligands is 97.0(3)°. In the crystal structure, the molecules are connected by intermolecular hydrogen bonds C-H—Br' forming 2D layers parallel to the ac plane, as shown by Figure 4. The corresponding hydrogen bonding parameters are listed in Table 3. In addition, there are n—n stacking interactions (Table 4) among the adjacent benzene rings.25 3. 5. Antimicrobial Activity Qualitative determination of antimicrobial activity was done using the disk diffusion method.28'29 The results are summarized in Table 4. A comparative study of minimum inhibitory concentration (MIC) values of the free li-gands and the complexes indicate that the cobalt complexes have better activity than the free ligands. Generally, this is caused by the greater lipophilic nature of the complexes than the ligands. Such increased activity of the metal chelates can be explained on the basis of chelating the-ory.30 On chelation, the polarity of the metal atoms will be reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of positive charge of the metal atoms with donor atoms. Further, it increases the delocali-zation of n-electrons over the whole chelate ring and enhances the lipophilicity of the complexes. This increased lipophilicity enhances the penetration of the complexes into lipid membrane and blocks the metal binding sites on enzymes of microorganisms. From Table 5, it is obvious that the cobalt complexes show greater antimicrobial and antifungi activities against Staphylococcus aureus, Escherichia coli, and Candida albicans when compared to HL1 and HL2. The complex with Schiff base ligand seems to be more active than that with aldehyde ligand. The activity of complex 2 is stronger than 1. For Staphylococcus aureus and Escherichia coli, even though the activities of the cobalt complexes are stronger than those of the free ligands, it is still less than the control drug tetracycline. But for Candida albicans, both complexes show stronger activity than the free li-gands and tetracycline. This trend is in accordance with those reported in literature, that cobalt complexes have stronger activities than the free Schiff bases in the antibacterial fields.31,32 Table 5. MIC values (ig/mL) for the antimicrobial activities of the tested compounds Staphylococcus Escherichia Candida aureus coli albicans HL1 256 128 > 1024 HL2 64 64 > 1024 1 16 8.0 256 2 1.0 4.0 128 Tetracycline 0.32 2.12 > 1024 4. Conclusion Two new cobalt(II) complexes with 3-bromo-5-chlorosalicylaldehyde or 2-bromo-4-chloro-6-(cyclo-propyliminomethyl)phenol as ligands have been prepared and characterized. The crystal structures of both complexes were confirmed by X-ray single crystal diffraction. The Co atom in complex 1 is in an octahedral coordination, while in complex 2, it gives a tetrahedral coordination. The antimicrobial tests show that both complexes have potential activity against Staphylococcus aureus, Escherichia coli and Candida albicans. 5. Acknowledgements This research was supported by the National Sciences Foundation of China (No. 20676057 and 20877036) and Top-class foundation of Pingdingshan University (No. 2008010). 6. Supplementary Material The crystallographic data of the structures described in this paper were deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-1489222 (1) and 1489223 (2). Copies of these data are available free of charge from http://www.ccdc.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44)1223-336-033; or email: deposit@ccdc.cam.ac.uk. 7. References 1. M. He, Q.-Z. Jiao, X.-F. Chen, J. Li, J. Chen, G.-H. Sheng, Z.-L. You, Chinese J. Inorg. Chem. 2016, 31, 1590-1596. 2. D. Qu, F. Niu, X. Zhao, K.-X. Yan, Y.-T. Ye, J. Wang, M. Zhang, Z. You, Bioorg. Med. Chem. 2015, 23, 1944-1949. https://doi.org/10.1016/j.bmc.2015.03.036 3. Y. Zhu, C.-F. Wang, K. Yan, K.-D. Zhao, G.-H. Sheng, Q. Hu, L. Zhang, Z. You, J. Coord. Chem. 2016, 69, 2493-2499. https://doi.org/10.1080/00958972.2016.1186801 4. J. Qin, Q. Yin, S.-S. Zhao, J.-Z. Wang, S.-S. Qian, Acta Chim. Slov. 2016, 63, 55-61. https://doi.org/10.17344/acsi.2015.1918 5. D. Barut, N. Korkmaz, S. T. Astley, M. Aygun, Acta Chim. Slov. 2015, 62, 88-94. https://doi.org/10.17344/acsi.2014.734 6. F.-M. Wang, Acta Chim. Slov. 2016, 63, 406-410. https://doi.org/10.17344/acsi.2016.2520 7. Z.-C. Liu, B.-D. Wang, Z.-Y. Yang, Y. Li, D.-D. Qin, T.-R. Li, Eur. J. Med. Chem. 2009, 44, 4477-4484. https://doi.org/10.1016Zj.ejmech.2009.06.009 Han et al.: Synthesis, Structures, and Antimicrobial Activities Acta Chim. Slov. 2017, 64, 179-185 185 8. D.-D. Qin, Z.-Y. Yang, G.-F. Qi, T.-R. Li, Transition Met. Chem. 2009, 34, 499-505. 9. Y.-Y. Yu, H.-D. Xian, J.-F. Liu, G.-L. Zhao, Molecules 2009, 14, 1747-1754. https://doi.org/10.3390/molecules14051747 10. Z. You, M. Liu, C. Wang, G. Sheng, X. Zhao, D. Qu, F. Niu, RSC Advances 2016, 6, 16679-16690. https://doi.org/10.1039/C6RA00500D 11. Y.-T. Ye, F. Niu, Y. Sun, D. Qu, X.-L. Zhao, J. Wang, D.-M. Xian, H. Jurg, Z.-L. You, Chinese J. Inorg. Chem. 2015, 31, 1019-1026. 12. C. Jing, C. Wang, K. Yan, K. Zhao, G. Sheng, D. Qu, F. Niu, H. Zhu, Z. You, Bioorg. Med. Chem. 2016, 24, 270-276. https://doi.org/10.1016/j.bmc.2015.12.013 13. L. Wang, Y.-J. Han, Q.-B. Li, L.-W. Xue, Acta Chim. Slov. 2016, 63, 822-826. https://doi.org/10.17344/acsi.2016.2699 14. G. P. Cheng, L. W. Xue, C. X. Zhang, Russ. J. Coord. Chem. 2014, 40, 284-288. https://doi.org/10.1134/S1070328414040022 15. L.-W. Xue, Y.-X. Feng, C.-X. Zhang, Synth. React. Inorg. Met.-Org. Nano-Met. Chem. 2014, 44, 1541-1544. https://doi.org/10.1080/15533174.2013.802340 16. Bruker, SMART and SAINT. Area Detector Control and Integration Software, Bruker Analytical X-ray Instruments Inc., Madison, WI, USA, 1997. 17. G. M. Sheldrick, SADABS. Program for Empirical Absorption Correction of Area Detector Data. University of Gottingen, Gottingen, Germany, 1997. 18. A. C. T. North, D. C. Phillips, F. S. Mathews, Acta Crystallo-gr. 1968, A24, 351-359. https://doi.org/10.1107/S0567739468000707 19. G. M. Sheldrick, SHELXL-97. Program for the Refinement of Crystal Structures, University of Gottingen, Gottingen, Germany, 1997. 20. L. Pan, C. Wang, K. Yan, K. Zhao, G. Sheng, H. Zhu, X. Zhao, D. Qu, F. Niu, Z. You, J. Inorg. Biochem. 2016, 159, 22-28. https://doi.Org/10.1016/j.jinorgbio.2016.02.017 21. F. Niu, K.-X. Yan, L. Pang, D. Qu, X. Zhao, Z. You, Inorg. Chim. Acta 2015, 435, 299-304. https://doi.org/10.1016/jica.2015.07.014 22. S. Chandra, U. Kumar, Spectrochim. Acta Part A 2005, 61, 219-224. https://doi.org/10.1016Zj.saa.2004.03.036 23. R. L. De, K. Samanta, K. Maiti, E. Keller, Inorg. Chim. Acta 2001, 316, 113-116. https://doi.org/10.1016/S0020-1693(01)00369-3 24. Y. Li, Q. Wu, L. Lecren, R. Clerac, J. Mol. Struct. 2008, 890, 339-345. https://doi.org/10.1016/j.molstruc.2008.05.044 25. A. L. Spek, Acta Crystallogr. 2009, D65, 148-155. 26. Z.-L. You, S.-Y. Niu, Synth. React. Inorg. Met.-Org. Nano-Met. Chem. 2007, 37, 29-33. https://doi.org/10.1080/15533170601172393 27. C. Jing, C. Wang, K. Yan, K. Zhao, G. Sheng, D. Qu, F. Niu, H. Zhu, Z. You, Bioorg. Med. Chem. 2016, 24, 270-276. https://doi.org/10.1016/j.bmc.2015.12.013 28. A. Barry, Procedures and theoretical considerations for testing antimicrobial agents in agar media. in: Lorian (Ed.), Antibiotics in Laboratory Medicine, 5th ed. Williams and Wilkins, Baltimore, 1991. 29. T. Rosu, M. Negoiu, S. Pasculescu, E. Pahontu, D. Poirier, A. Gulea, Eur. J. Med. Chem. 2010, 45, 774-781. https://doi.org/10.1016/j.ejmech.2009.10.034 30. J. W. Searl, R. C. Smith, S. Wyard, J. Proc. Phys. Soc. 1961, 78, 1174-1176. https://doi.org/10.1088/0370-1328/78/6/311 31. T. Yang, F. Niu, L. X. Li, Z. N. Xia, Y. Zhang, Z. L. You, Russ. J. Coord. Chem. 2016, 42, 402-409. https://doi.org/10.1134/S1070328416050109 32. X. M. Hu, L. W. Xue, G. Q. Zhao, W. C. Yang, Russ. J. Coord. Chem. 2015, 41, 197-201. https://doi.org/10.1134/S1070328415030045 Povzetek Pripravili smo nov kobaltov(II) kompleks, [Co(L1)2(OH2)2] (1), z reakcijo 3-bromo-5-klorosalicilaldehida (HL1) s ko-baltovim nitratom v metanolu. Pri reakciji 1 s ciklopropilaminom v metanolu nastane kobaltov(II) kompleks s Schiffovo bazo, [Co(L2)2] (2), kjer je L2 deprotonirana oblika 2-bromo-4-kloro-6-(ciklopropiliminometil)phenola (HL2). Kompleksa sta bila okaratkerizirana z elementno analizo, IR spektroskopijo in monokristalno rentgensko difrakcijo. Ligand L1 se koordinira na Co atom preko fenolatnega O atoma in karbonilnega O atoma, medtem ko se ligand L2 koordinira na Co atom preko fenolatnega O atoma in imino N atoma. Co atom v kompleksu 1 ima oktaedrično koordinacijo, v kompleksu 2 pa tetraedrično koordinacijo. Določena je bila tudi antimikrobna aktivnost prostih ligandov in kobaltovih kompleksov na Staphylococcus aureus, Escherichia coli in Candida albicans. Han et al.: Synthesis, Structures, and Antimicrobial Activities ... 186 DOI: 10.17344/acsi.2016.3054 Acta Chim. Slov. 2017, 64, 186-192 ^creative ^commons Scientific paper Monodispersed Gold Nanoparticles as a Probe for the Detection of Hg2+ Ions in Water Bindhu Muthunadar Rajam,1 Parimaladevi Ramasamy2 and Umadevi Mahalingam2* 1 Department of Physics, Nanjil Catholic College of Arts and Science, Nedumcode, Kaliyakavilai-695502, Tamil Nadu, India 2 Department of Physics, Mother Teresa Women's University, Kodaikanal-624101, Tamil Nadu, India * Corresponding author: E-mail: ums10@yahoo.com Tele: 04542241685 (UM and PR) Received: 08-11-2016 Abstract Gold nanoparticles were synthesized using Ananas comosus as reducing agent. UV-visible spectra show the surface pla-smon resonance peak at 544 nm. TEM measurement shows that the formation of monodispersed spherical nanoparticles with average size of 7 nm. Crystalline nature of the nanoparticles was evident from TEM images and peaks in the XRD pattern. FTIR analysis provides the presence of biomolecules responsible for the reduction and capping of the prepared gold nanoparticles. A selective and sensitive method is proposed for detecting mercury based on the SPR change of gold nanoparticles. This mercury sensor based on surface plasmon optical sensor can be used in water analysis. Keywords: Ananas comosus. gold nanoparticles, mercury, optical sensor 1. Introduction The determination of heavy metal ions in water is of great importance because of their role in the physiological functions of biological systems.1 Among the heavy metal ions, mercury is an most dangerous metal ions for environment and has most commonly toxic risks for human contacting areas as a result of natural processes, because it is widely distributed in air, water and soil and it is a toxic element that exists in metallic, inorganic, and organic forms.2 Mercuric ion (Hg2+), exists mostly in surface water due to its high water solubility and it can cause several developmental delays and health problems that can damage the brain, nervous system, kidneys, and endocrine sys-tem.3,4 Therefore, the analysis and measurement of detecting mercury in aqueous media is important. A variety of methods have been developed for quantification of Hg2+ concentrations such as atomic absorption spectros-copy, inductive coupled plasma mass spectroscopy, electrochemical impedance spectroscopy, voltammetry and polarography. But, these methods are expensive, complicated sample treatment and mostly take a long measuring period. The selective optical sensor is an alternative method and has been attracted due to the excellent sensitivity, rapid response, the ability to do the detection in a non-destructive manner and cost-effective. Metal nanoparticles have been received much attention due to their unique optical, electrical and catalytic properties. The size, shape and surface morphology of the particles were crucial in tuning these properties of nanosi-zed metal particles. This was mostly significant for noble metals having strong surface plasmon resonance (SPR) oscillations. There were many synthetic methods have been developed to prepare nanoparticles, including chemical, physical and biological methods, among which green synthesis of metal nanoparticles remains the simplest and environment friendly method. All these synthetic methods vary generally in the way the electrons required for the reduction were provided. Green synthesis of nanoparticles using D.carota, S.lycopersicums, Beetroot, H.Cannabinus leaf, Moringa oliefera flower, Avena sativa and Hibiscus cannabinus stem has been reported.5-11 Among the different metallic nanoparticles, gold nanopar-ticles have diverse activities and exhibit novel properties such as high surface and variation in electronic and optoelectronic properties; have made them more appropriate for therapeutic use and broad applications in nanobiotechno-logy. The chemical inertness and resistance to surface oxi- Rajam et al.: Monodispersed Gold Nanoparticles as a Probe ... Acta Chim. Slov. 2017, 64, 186-192 187 dation make gold an important material for use in nano-scale technologies and devices. This property is crucial when particle size approaches the nanostructure and the dominance of surface atoms results in an enhanced chemical reactivity. In this work, gold nanoparticles were synthesized using Ananas comosus fruit extract as reducing agent. Since Ananas comosus is a readily available fruit and it is a good source of water, carbohydrates, sugars, vitamins A, C and carotene, beta.12 It contains low amounts of protein, fat, ash and fibre. It is a good source of citric acid, malic acid and ascorbic acid12,13 and also contain three types of amino acids. Along with this, it also contains bromelain, a protein-digesting enzyme that reduces inflammation. Modified pineapple peel fibre was used to remove heavy metal ions in water through the reaction with succinic acid anhydride.14,15 Bhosale et al. reported the synthesis of na-noparticles using Ananas comosus extract as reducing agent with kanamycin A and neomycin as stabilizing agents.16 They prepared larger nanoparticles with agglomeration. In the present study, the synthesis and characterization of monodispersed small gold nanoparticles using fruit extract of Ananas comosus has been described. Here the size and aggregation of the nanoparticles were controlled without any additional stabilizing agents. The sensing activity of gold nanoparticles obtained by this method has been also described. 2. Experimental Techniques 2. 1. Materials and Methods Ananas comosus fruit was collected from local supermarket in Kodaikanal, Tamilnadu, India. Chloroauric acid and various heavy metals were obtained from Sigma Aldrich Chemicals. All glasswares were properly washed with distilled water and dried in hot air oven before use. 2. 2. Preparation of Ananas Comosus Extract Fully riped Ananas comosus fruit weighing 100 g cut into fine pieces and were crushed into 100 ml distilled water in a mixer grinder for extraction. The extract was then separated by centrifugation at 1000 rpm for 10 min to remove insoluble fractions and macromolecules. Then the extract obtained was filtered and finally a light yellow extract was collected for further experiments. 2. 3. Synthesis of Gold Nanoparticles For the synthesis of gold nanoparticles, 5ml of Ananas comosus extract was added to aqueous solution of HAuCl4 (3 mM) and stirred continuously for 5min at room temperature. Upon addition of fruit extract, the color of the solution gradually changes from light pink to charac- teristic dark ruby red upon completion of reaction of the gold colloid (g1). Similarly by adding 10 and 15 ml of fruit extract two more set of samples henceforth called (g2) and (g3) respectively were prepared. UV-visible spectra of these solutions were recorded. Then the solutions were dried. The dried powders were characterized by X- ray diffraction (XRD), Fourier Transform Infrared Radiation (FTIR), Transmission Electron Microscope (TEM) and Energy Dispersive X-ray Spectroscopy (EDX). 2. 4. Characterization Methods and Instruments The absorption spectra of the prepared nanoparticles were measured using a Shimadzu spectrophotometer (UV 1700) in 300-800 nm range. X- Ray Diffraction analysis of the prepared nanoparticles was done using PANalytical X'pert - PRO diffractometer with Cu Ka radiation operated at 40 kV/30 mA. FTIR measurements were obtained on a Nexus 670 FTIR instrument with the sample as KBr pellets. Transmission Electron Microscopic (TEM) analysis was done using a JEOL JEM 2100 High Resolution Transmission Electron Microscope equipped with an EDX attachment, operating at 200kV. 3. Results and Discussion 3. 1. UV-visible Studies Noble metals are known to exhibit unique optical properties due to the property of SPR which is the collective oscillation of the conduction electrons in resonance with the wavelength of irradiated light. In the present 300 400 500 600 700 800 Wavelength (nm) Figure 1. Optical absorption spectra of AuNPs at different concentration of A.comosus fruit extract (inset: colour changes of the prepared AuNPs) (a, b and c vs 5, 10 and 15 ml respectively). Rajam et al.: Monodispersed Gold Nanoparticles as a Probe ... 188 Acta Chim. Slov. 2017, 64, 186-192 study the formation of gold nanoparticles was initially conformed using UV-Visible spectroscopy by measuring Surface Plasmon Resonance (SPR) peaks. Gold nanoparticles exhibit plasmon absorption bands that depend on their size and shape. Fig. 1 shows the absorption spectra obtained for gold nanoparticles with different concentration of fruit extract. The colour variation of the obtained gold nanoparticles for different concentration of Ananas comosus fruit extract has been shown in Fig. 1(inset). These characteristic color variations are due to the excitation of the surface plasmon resonance in the metal nano-particles. As the concentration of fruit extract increases, an fwhm value decreased from 105 nm to 94 nm and blue shift observed from 550 to 544 nm in the reaction medium, indicating the formation of small nanoparticles. As the particles decrease in size, the absorption peak usually shifts toward the blue wavelengths caused by the donation of electrons to the particles. It has been well established that the maximum wavelength of nanoparticles strongly depends on size, shape, state of aggregation and the dielectric environment. This directly corresponds to a shift of the absorption peak, whereby small gold particle sizes will cause an absorption peak shift to smaller wavelengths, higher frequency and energies.17 The observed symmetric nature of the SPR indicates the formation of spherical nanoparticles. As the concentration of the extract increases more number of citric, malic and ascorbic acids are available to reduce gold ion and forms large number of very small nanoparticles gives rise to sharp, intense and blue shifted SPR. It was further confirmed by the TEM images shown in Fig. 4 and 5. The symmetric nature of the SPR and the absence of peaks in the longer wavelength region indicate the absence of nanoparticle aggregation. As-corbate, malate and citrate ions in the fruit extract introduce the negative charge onto the particle surface and thus preventing the particles from aggregation. Thus from the results it can be concluded that the concentration of fruit extract plays an important role in the formation of gold na-noparticles. The obtained nanoparticles were stabilized by physical adsorption of excess negatively charged citrate, malate and ascorbate ions in the solution medium, and thus a repulsive force worked along particles electrostatically and preventing them from aggregation. 3. 2. XRD Studies The crystalline structure and phase purity of the prepared gold nanoparticles were confirmed with X-ray diffraction (XRD) analysis. Fig. 2(a) shows the XRD pattern for the dried powder of Ananas comosus. Three diffraction peaks were observed at 28.5°, 40.8° and 50.9° signify the presence of ascorbic acid (JCPDS 22-1560), citric acid (JCPDS 22-1568) and malic acid (JCPDS 23-1631) in the Ananas comosus extract. Fig. 2(b) shows the XRD pattern for g1 and g3. The broad diffraction peaks were observed at 38.2°, 44.1°, Rajam et al.: Monodispersed Gold Nanoparticles as a Probe ... a) ■ A. Comosus ex t ract _i_I_I_I_I_I_i_I_I_I_i_ 20 30 40 50 60 70 80 2'Kdegree) b) 44,f (200) (iï) JK ,1 (111) JCPDS. 04-0784 (220) (311) (i> 20 30 — 40 50 —i—■ 60 —i—■ 70 80 20(deg reei Figure 2. X-ray diffraction pattern of (a) A.comosus fruit extract and (b) AuNPs (i) g1 and (ii) g3. 64.8° and 77.6° in the 28 range and they corresponding to (111), (200), (220) and (311) Bragg's reflections based on the FCC structure of gold nanoparticles with space group of Fm-3m (JCPDS: 04-0784). No peaks of crystal-lographic impurities in the sample have been found. Generally, the breadth of a specific phase of material is directly proportional to the mean crystallite size of that material. The obtained broader peaks with increasing fruit extract concentration indicating smaller particle size. The XRD line width can be used to estimate the size of the particle by using the Debye-Scherrer formula as D = kX/p cos8 where D is the particle size (nm), k is a constant equal to 0.94, X is the wavelength of X-ray radiation (1.5406 A), p is the full-width at half maximum (FWHM) of the peak (in radians) and 28 is the Bragg angle (degree). The average particle size, lattice constant, cell volume, surface area to volume (SA: V) ratio, specific surface area (SSA) and Crystallinity index were calculated and tabulated Table.1. The calculated average particle size for both g1 and g3 indicates that the particle size decreased with the concentration of the fruit extract increased. The calculated lattice constant values are very close to the standard data Acta Chim. Slov. 2017, 64, 186-192 189 Table.1. The average particle size, lattice constant, cell volume, surface area to volume (SA: V) ratio, specific surface area (SSA) and crystallinity index of the prepared nanoparticles. Prepared Particle Size Lattice constant Cell volume SSA SA:V Crystallinity AgNPs (nm) ( A) ( A3) (m2/g) ratio index Icry gl 16 4.0529 66.57 18.46 0.35 -1.0625 g3 7 4.0815 67.99 40.64 0.78 -0.714 (JCPDS File no. 04-0784) and the sample exhibit smaller cell volumes that of bulk. As shown in Table. 1, the observed values of both specific surface area (SSA) and SA:V ratios were increased with decreasing particle size. The SSA has a particular importance in reactivity. It gives the rate at which the reaction will proceed. Because of the large number of atoms available in the reaction medium (g3) makes the reaction faster and hence make them more suitable for broad kind of applications. Crystallinity was evaluated by comparing the crystalline size obtained by XRD to TEM particle size determination. The calculated values of crystallinity index were close to one which indicates the monocrystalline nature of g1 and g3. 3. 3. FTIR Studies FTIR analysis was carried out to identify the chemical change of the functional groups involved in bioreduc-tion. Fig.3(a) shows the FTIR spectrum of the Ananas co-mosus fruit extract, shows prominent bands at 3417, 2924, 1640, 1019 and 801 cm1 in the 4000 -500 cm1 region. These peaks are assigned to O-H stretching, CH stretching, C=C ring stretching, C-O-C stretching and C-C ring stretching of ascorbic acid, respectively.18 Fig. 3(b) shows that the FTIR spectrum of g3. The peak at 3417 cm-1 was also due to the OH stretching of citric and malic acid.19,20 —i—|—i—|—i—|—i—i—i—i—i—\—i— 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber {cm ) Figure 3. FTIR spectra of (a) A.comosus fruit extract and (b) g3. An interesting peak observed at 2369 cm-1 in the spectrum of extract was assigned to NH- stretching of amines. This vibrational mode was completely reduced in the spectrum of g3. It may the presence of bromelain in the extract. Bromelain is a protein which functions as an enzyme known as proteolytic enzymes. These enzymes have the ability to separate all important peptide bonds. This possibly leads to the absence of this vibrational mode during the synthesis of gold nanoparticles (g3). The interesting peak at 1640 cm-1in the spectrum of extract was assigned to C=C ring stretching of vitamin C, OCO asymmetric stretching of malic acid and C=O stretching of citric acid, was appeared at a sharp peak at 1601 cm-1 in the spectrum of g3. Another interesting broad peak observed at 1414 cm-1 in the spectrum of extract was show at a symmetric peak at 1390 cm-1 in the spectrum of g3, was due to OCO symmetric stretching of malic acid, COH deformation of citric acid and CH2 wagging of ascorbic acid. Similarly, the symmetric peak observed at 1115 cm-1 was due to C-O-C stretching of ascorbic acid and C-C stretching of malic and citric acid. This indicates that the carboxylic acid groups present in the Ananas Comosus fruit extract was responsible for reduction of AuNPs. 3. 4. TEM Studies The TEM images of the g1 and g3 were shown in Fig. 4 and 5 respectively. The prepared nanoparticles exhibit size dependent morphology. At the TEM image of g1, monodispersed and spherical nanoparticles of average size of 17 nm with diameter ranging from 13 nm to 26 nm (Fig.4). The TEM image of g3, synthesized by higher fruit extract concentration showing the presence of monodis-persed spherical nanoparticles of average size of 7 nm ranging from 3 to 15 nm size (Fig. 5). Here, most of the particles observed in the range of 4 nm to 8 nm. As the concentration of fruit extract increases large number of citrate, malate and ascorbate ions are available to reduce gold ion and forms small nanoparticles. The smaller size of g3 was also due to their high specific surface area and its monocrystalline nature. More number of nanoparticles observed in TEM images of g3 in comparison to g1. In both cases, the observed nanoparticles were spherical and homogeneous distribution, which was confirmed from the symmetric nature of SPR shown in Fig. 1(a). Strong interaction between biomolecules in the fruit extract and sur- Rajam et al.: Monodispersed Gold Nanoparticles as a Probe ... 190 Acta Chim. Slov. 2017, 64, 186-192 face of nanoparticles was sufficient to the formation of spherical nanoparticles preventing them from sintering. At lower concentration of fruit extract the citric, ascorbic and malic acid present in fruit extract was insufficient to reduce gold ion, indicating larger size particle. The twined particles observed in Fig. 4(c), 4(d), 5(c) and 5(d) were identified by showing brightness in part of the particles as compared to the other parts. Generally, twinning, the planar defect is observed for face-centered cubic (fcc) structured metallic nanocrystals. Sharing of a common crystallographic plane by two subgrains gives rise to twinning. Face-centered cubic (fcc) structured metallic nanostructures have a tendency to nucleate and grow into twinned particles with their surfaces bounded by lowest energy facets (111)21. The formation of gold was further confirmed by the analysis of the energy dispersive spec-troscopy shown in Fig. 6. 3. 5. Sensing Activity Sensing is one of the important applications of na-noparticles. Nanoparticle-based optical surface sensors have received much attention due to their faster response and better resolutions. The interaction between natural biomolecules and the surface of the inorganic nanopartic-les paves the way for development of sensing system. The interaction of prepared AuNPs with various alkali metal (Li+, K+, Fe3+) and transition metal ions (Ni2+, Mn2+, Cu4+, Figure 6. EDX graph of g3. Rajam et al.: Monodispersed Gold Nanoparticles as a Probe ... Acta Chim. Slov. 2017, 64, 186-192 191 Zn2+, Hg2+, Cd2+) was examined by adding 1ml of (3mM) salts of these metals into the 2 ml of AuNPs by drop by drop and stirred for 5 min. UV-vis spectra (Fig.6 (a)) of AuNPs were taken immediately after addition of metal ions, after 5 min of interaction. It was observed that except Hg2+ no other metal ions exhibited a colour change. UV-vis spectra of these heavy metals interacted with Au-NPs were shown in Fig.7 (a). It was observed that the intensity of the SPR bands get reduced for all metal ions as compared to that of the AuNPs. Only mercury got almost quenching of the SPR peak among all the metals, including alkali metal (Li+, K+, Fe3+) and transition metal ions (Ni2+, Mn2+, Cu4+, Zn2+, Hg2+, Cd2+). It was also observed that for Hg2+ gave fading of pale pink colour, indicating the prepared AuNPs were sensitive and selective towards Hg2+. The sensitivity of this method was measured by adding various concentration of aqueous solution of Hg2+ ions to the aqueous AuNPs (5 ml) at room temperature. With the increase of Hg2+ ions, the color sequentially changed from purple to colorless. The addition of 0.188 mM to 0.653mM Hg2+ to the AuNPs solution causes color changes from light purple to colorless were observed shown in Fig. 7 (b) (inset). The UV-vis spectrum correspondingly recorded and shown in Fig. 7(b). With increasing the concentration of Hg2+ ion to the AuNPs causes immediate reduction in the intensity of surface plasmon peak at 544 nm. This could be accounted for the slight blue shift of the SPR band of gold nanoparticles. It shows absorbance strength decreases gradually by increasing the concentration of Hg2+ ion. With increasing Hg2+ ion concentration, blue shift of the SPR peak was also obtained. When Hg2+ ion added to the prepared nanoparticles, Hg2+ ions interact with the biomolecules (carboxylic acid groups) in the Ananas comosus fruit extract on the surface of the nanoparticles form bonds among nanoparticles with Hg2+ ions performing as link for binding sites of biomole-cules and eliminating it away from the surface of the na-noparticle surface, in that way aggregation of nanopartic-les had taken place. This could be accounted for the slight blue shift of the SPR band of gold nanoparticles. There was no SPR peak was observed after the addition of 0.653 mM Hg2+, suggesting the concentration of Hg2+ was limited to 0.653 mM. So The linear variation of absorbance (AA) changes and the concentration of Hg2+ over the range from 0.188 mM to 0.653mM shown in Fig. 7(c). This plot can be fit by a linear equation y = 1.527x-1.0633, R2 = 0.9889. The sensitivity of the system towards analyte concentration was found to be 1.5273/mM is measured from the plot of absorbance (AA) versus concentration of Hg2+. The limit of detection was estimated by defined as the following formula of CL = 3SB/m, where CL SB andm are the limit of detection, standard deviation of the sample, and the slope of the calibration curve, respectively. It was found to be 0.1198 mM. Applications of nanoparticle sensors by the aggregation of small particles were useful Rajam et al.: Monodispersed Gold Nanoparticles as a Probe ... 500 600 Wavelength (nm) AuNPs O.lSmM BJOmM 0J7mM 0.43 m M 0.47m M 0.53 m M 0,55m M 0,56m M D.58mM 0.59 m M 0.611 m M 0.61 m M 0.62 m M 0.62 5m M 0.63m M 0.638 m M 0.643m M 0.643 in M 0.652 m M C) 0.9 0.8 0.7 0.6< y=-1,5273x+1,0633 R'=0.9B89 [HOjiM Figure. 7. (a) UV-vis absorption spectrum and photographs (inset) of AuNPs with different heavy metal ions, (b) UV-vis absorption spectrum of AuNPs solution upon addition of Hg2+ ions (0.188 mM to 0.653mM) and (c) plot of absorbance (AA) intensity at 544 nm versus Hg2+ ions concentration. because aggregates with multiple particles yield large enhancements due to the enormous electromagnetic field that coherently interfere at the junction site between the 192 Acta Chim. Slov. 2017, 64, 186-192 particles. This mercury sensor based on surface plasmon optical sensor can be used in environmental monitoring especially in water purification. 4. Conclusion The present simple study was designed to slow reduction of chloroauric acid using fruit extract of Ananas comosus as reducing agent. This green synthesis method has formed monodispersed spherical gold nanoparticles with average size of 7 nm. The prepared nanoparticles were characterized by UV-visible, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS) technique to identify the size, shape of nanoparticles and biomo-lecules act as reducing agents. FTIR measurements show that carboxylic acid groups present in Ananas comosus fruit extract was used as reducing agent. The prepared gold nanoparticles were stable for one month without aggregation. The surface plasmon resonance of prepared gold nanoparticles was confirmed by UV-visible spectral analysis. As the concentration of Ananas comosus fruit extract increases, absorption spectra shows blue shift with decreasing particle size. The prepared AuNPs were sensitive and selective towards Hg2+. This mercury sensor based on surface plasmon optical sensor can be used in water analysis by detecting the concentration of Hg2+ ions. 5. Acknowledgements The authors are thankful to DST-CURIE New Delhi, UGC-DAE CSR Indore for financial assistance. 6. References 1. M. A. Anderson, F. M. M. Morel, Limnol. Oceanogr. 1978, 25, 283-295. https://doi.org/10.4319/lo.1978.23.2.0283 2. F. A. Cotton, G. Wilkinson, C. A. Murillo, M. Bochmann, Advanced Inorganic Chemistry, 6th ed., John Wiley & Sons, New York, 1999. 3. T. W. Clarkson, L. Magos, G. J. Myers, N. Engl. J. Med. 2003, 349, 1731-1737. https://doi.org/10.1056/NEJMra022471 4. Y. Wang, F. Yang, X. Yang, Biosens. Bioelectron. 2010, 25, 1994-1998. https://doi.Org/10.1016/j.bios.2010.01.014 5. M. Umadevi, S. Shalini, M. R. Bindhu, Adv. Nat. Sci. Nanos-ci. Nanotechnol. 2012, 3 025008, 1-6. 6. M. Umadevi, M. R. Bindhu, V. Sathe J. Mater. Sci. Technol. 2013, 29, 317-322. https://doi.Org/10.1016/j.jmst.2013.02.002 7. M. R. Bindhu, M. Umadevi, Spectrochim. Acta A, 2015, 135, 373-378. https://doi.Org/10.1016/j.saa.2014.07.045 8. M. R. Bindhu, M. Umadevi, Spectrochim. Acta A, 2013, 101, 184-190. https://doi.org/10.1016Zj.saa.2012.09.031 9. M. R. Bindhu, V. G. Sathe, M. Umadevi, Spectrochim Acta A, 2013, 11, 5409-415 10. V. Armendariz, I. Herrera, J. R. Peralta-Videa, M. Jose-Yaca-man, H. Troiani, P. Santiago, J. L. Gardea-Torresdey, J. Na-nopart. Res. 2004, 6, 377-382. https://doi.org/10.1007/s11051-004-0741-4 11. M.R. Bindhu, P.Vijaya Rekha, T. Umamaheswari, M.Uma-devi, Mater. Lett. 2014, 131, 194-197. https://doi.org/10.1016/j.matlet.2014.05.172 12. J. L. Collins, The Pineapple, Interscience Publishers Inc New York. (1960) 250. 13. E. K. Nelson, J. Am. Chem. Soc. 1925,47, 1177-1179. https://doi.org/10.1021/ja01681a039 14. X. Hu, M. Zhao, H. Huang, Water Environ. Res. 2010, 8, 2733-741. 15. X. Hu, M. Zhao, G. Song, H. Huang, Environ Technol. 32, 2011, 739-746. https://doi.org/10.1080/09593330.2010.510853 16. V. Santosh Nalage, V. Sidhanath Bhosale, V. Sheshanath Bhosale, ONJ 2011 5, 78-82. 17. S. L. Smitha, K. M. Nissamudeen, D. Philip, K. G. Gopchan-dran, Spectrochim. Acta A 2008 71, 186-190. https://doi.org/10.1016/j.saa.2007.12.002 18. C. Y. Panicker, H. T. Varghese, D. Philip, Spectrochim. Acta A, 2006 65, 802-804. https://doi.org/10.1016/j.saa.2005.12.044 19. P. Tarakeshwar, S. Manogaran, Spectrochim. Acta A 1994 50, 2327-2343. https://doi.org/10.1016/0584-8539(94)E0017-5 20. J. L. Castro, M. R. Lopez-Ramyfrez, J. F. Arenas, J. C. Otero, Vib. Spectrosc. 2005 39, 240-243. https://doi.org/10.1016/j.vibspec.2005.04.007 21. J. G. Allpress, J. V. Sanders, Surf. Sci. 1967, 7,1-25. https://doi.org/10.1016/0039-6028(67)90062-3 Povzetek Nanodelce zlata smo pripravili z uporabo Ananas comosus kot reducirnega reagenta. UV-Vis spektri kažejo površinsko resonančni plazmonski (SPR) vrh pri 544 nm. Z meritvami s presevnim elektronskim mikroskopom (TEM) pa smo prikazali sintezo monodispergiranih sferičnih nanodelcev s povprečno velikostjo 7 nm. Kristalna narava nanodelcev je razvidna iz TEM slik in vrhov, določenih z rentgensko praškovno difrakcijo (XRD). FTIR spektroskopija kaže na prisotnost biomolekul, ki so odgovorne za redukcijo in ločevanje pripravljenih nanodelcev zlata. Za določanje živega srebra predlagamo selektivno in občutljivo metodo, ki temelji na osnovi SPR sprememb nanodelcev zlata. Takšen senzor bi lahko uporabljali pri analizi voda Rajam et al.: Monodispersed Gold Nanoparticles as a Probe ... DPI: 10.17344/acsi.20l6.3097_Acta Chirn, Slov. 2017,64, 193-201_©cernons 193 Scientific paper Poly-Dianix Blue/Multi-Walled Carbon Nanotube Modified Electrode for Detection of Levodopa in the Presence of High Concentrations of Ascorbic and Uric Acids Abdolhamid Hatefi-Mehrjardi,1'2'* Mohammad Ali Karimi,1 Azam Barani2 and Mahdiyeh Soleymanzadeh2 1 Department of Chemistry, Payame Noor University, 19395-3697, Tehran, Iran 2 Department of Chemistry & Nanoscience and Nanotechnology Research Laboratory (NNRL), Payame Noor University (PNU), Sirjan, Iran * Corresponding author: E-mail: hhatefy@pnu.ac.ir or hhatefy@Yahoo.com Tel: +98-34-423-335-41; Fax: +98-34-423-335-40 Received: 24-11-2016 Abstract A selective and sensitive electrochemical sensor was studied for determination of levodopa (LD) in the presence of uric acid (UA) and ascorbic acid (AA) using poly-dianix blue and multi-walled carbon nanotubes (PDB/MWCNTs) modified glassy carbon electrode. Cyclic voltammetry, differential pulse voltammetry, and chronoamperometry methods were applied to investigate the electrocatalytic oxidation of LD, UA and AA in aqueous solutions. By DPV technique, LD, UA and AA give oxidation peaks at 0.380, 0.520 and 0.180 V, respectively. Under the optimized experimental conditions LD, UA and AA give a linear response in the range of 0.09-75 |mol L-1, 0.3-110 |mol L-1 and 10-160 |mol L-1, respectively. Accordingly, the obtained detection limits were 0.003, 0.002 and 0.023 |mol L-1. The method provides a simple electrochemical sensor for successful determination of LD in human blood serum samples. Keywords: Dianix Blue; Carbon Nanotubes; Modified Electrode; Levodopa; Uric Acid; Ascorbic Acid. 1. Introduction Parkinson's disease (PD) is a progressive neurologic disorder that leads to a slowly increasing asthenia in movement. It is caused by a lack of dopamine, a natural substance usually found in the brain. Dopamine cannot be administered directly because it does not cross the blood-brain barrier easily. Levodopa (LD) is one of central nervous system drugs and passes into the brain and is then converted to dopamine by decarboxylase. Then, LD is utilized to increase dopamine levels in the brain.1 Clearly, the process of LD detection and its concentration determination is an important property in pharmaceutical and clinical procedures. Different analytical methods have been developed in order to measure LD levels in various sample matrices, such as spectrophotometric,2 high-performance liquid chromatography,3 and capillary zone elec- trophoresis.4,5 All these methods involve complicated techniques and expensive instruments. Compared to other choices, electrochemical methods provide useful alternatives that are faster, cheaper and highly sensitive.610 Ascorbic acid (AA) is commonly known as vitamin-C.11 AA plays an important role in several enzymatic reactions and in the defense against oxidative stress.12 According to these properties, it is utilized for the prevention and treatment of infertility, Alzheimer's disease, atherosclerosis, cancer13,14 and AIDS.15,16 However, at higher concentration levels, AA contributes to the formation of kidney stones. Uric acid (UA) is a nitrogenous compound and the primary major product of purine catabolism.17 Continuous monitoring of UA in the body fluid is vital since its abnormal concentration levels result in different diseases, such as hyperuricaemia and gout.18 Several methods for the de- Hatefi-Mehrjardi et al.: Poly-Dianix Blue/Multi-Walled Carbon Nanotube 194 Acta Chim. Slov. 2017, 64, 193-201 tection of UA have been explained in papers including enzymatic-spectrophotometry19 and chemiluminescen-ce.20 However, most of these methods are complicated because they need derivatization of compound with variety detection methods. Therefore, it is favorable to have a simple, sensitive and fast method for monitoring the concentration of UA in biological fluids such as electrochemical techniques.21,22 Whereas LD, UA and AA play the main role in the human body and often coexist in biological fluids, the selective detection of these three compounds has always been the subject of many types of research.15 As LD, UA and AA are all electroactive, electrochemical methods are often utilized to the determination of these three spe-cies.23,24 However, the direct redox reactions of these species at the bare electrodes take place at very similar potentials25-28 and often suffer from a pronounced fouling effect, which results in a poor selectivity and reproducibi-lity.29,30 Also, the voltammetric sensing of neurotransmit-ter metabolites usually suffers from the interference of AA, which usually coexists in vivo as anion at high concentrations and possesses an oxidation potential close to that of neurotransmitter metabolites at the unmodified electrode.31 Moreover, one promising approach for minimizing overvoltage effects and facilitating the determination is through the use of an electrocatalytic process at chemically modified electrodes. The most commonly used electrode material is carbon particularly glassy carbon (GC),32 accordingly the chemical modifications of the inert substrate of glassy carbon electrode with redox active thin films offer significant advantages in the design and development of electrochemical sensors.33 Modification of GC electrodes can be achieved by numerous ways, and the electropolymerization method has been widely explored.34 Compared with the conventionally adsorbed layer, the electropolymerized conductive sensing film is more uniform and the thickness is easily controlled by controlling the number of potential sweep cycles. More importantly, the polymeric sensing films on the electrode surface can yield a three-dimensional reaction zone which can provide more active sites for anodic oxidation of LD, UA and AA and greatly increase the sensitivity of the resulting sensor.35 Carbon nanotubes (CNTs) are considered to be good supports for polymer-modified GC electrodes, because of their good electric conductivity, small dimensions, high mechanical strength,36 electric37,38 and thermal behavior,39,40 and the property of being polymer carriers.41,42 In the previous work, the poly-(Alizarin Red S)-mo-dified glassy carbon electrode was successfully fabricated and used for the electrochemical detection of LD, homo-vanillic acid, and AA in the presence of the each other.43 However, modification with new nanocomposite materials offers advanced properties. In this study, PDB/MWCNTs-modified GC electrode was electrochemically prepared and used as an elec- trochemical sensor for determination of LD, UA and AA in the presence of the each other. The results have been compared with the bare GCE and PDB/GCE based on electrocatalytic oxidation, and some parameters influencing the performances of this electrode in the determination of the three species are discussed. In fact, the redox active sites shuttle electrons between the analytes and the electrode shows a significant reduction in activation overpotentials. 2. Experimental 2. 1. Chemicals and Solutions LD, UA and AA were obtained from Alfa Aesar, Fluka (Switzerland) and Merck (Germany), respectively. Dianix blue (4,8-diamino-1,5-dihydroxy-2-(4-hydroxyp-henyl)-4a,9a-dihydroanthracene-9,10-dione) with the molecular mass of 362.34 g mol-1, the structural formula of C20H14N2O5 and the following molecular formula (Scheme 1) was purchased from Dy Star. Scheme 1. The structural formula of Dianix blue. MWCNTs with purity more than 95% were purchased from Research Institute of Petroleum Industry (Iran). MWCNT purification was performed as given in the literature:44 0.150 g of MWCNTs were stirred in 12 mL of concentrated HNO3/H2SO4 mixture 3:1 for 24 h. The solid product was filtered using a membrane filter with a pore size of 0.2 m, washed with double distilled water until neutral pH was reached. The filtrate was dried at 80 °C in an oven for 24 h. Other reagents were of analytical grade purchased from Merck and used without further purification. Electrolyte solutions were prepared using Smalley method.45 The initial pH of the solution 0.10 mol L-1 KCl + 0.01 mol L-1 H3PO4 was ca. 2.1. The higher pHs were adjusted by the addition of 0.11 mol L-1 NaOH. Ionic strength was constant over the entire range of pH. All electrochemical experiments were carried out in 0.11 mol L-1 PBS at pH 3.0. Freshly prepared LD, UA and AA solutions were used for each experiment. All aqueous solutions were made with double-distilled water. 2. 2. Apparatus A conventional cell with three electrodes including bare GCE or modified GCE with PDB or PDB/MWCNTs Hatefi-Mehrjardi et al.: Poly-Dianix Blue/Multi-Walled Carbon Nanotube ... Acta Chim. Slov. 2017, 64, 193-195 201 as working electrode, Ag/AgCl (3.0 mol L-1 KCl, Metrohm) as reference electrode and platinum bar (Metrohm) as auxiliary electrode, was employed for electrochemical experiments. The cyclic voltammetry and differential pulse voltammetry and chronoamperometry experiments were carried out using an Autolab P/GSTAT 12 (Eco Chemie, The Netherlands) interfaced with a computer and controlled by GPES 4.9 software. The topological imaging of the electrodes was performed by AFM using Na-nosurf Easy Scan 2 AFM (Nanosurf AG, Switzerland) and Field Emission Scanning Electron Microscope (FESEM, MIRA, TESCAN, USA). AFM images were taken in the air in the contact/tapping mode and were obtained at least in three different sites in given samples. 2. 3. Electrode Modification Before electrode modification, the GCE (nominal area of 0.0314 cm2, Azar electrode Co., Urmia, Iran) was polished using aqueous slurries of alumina (0.05 pm) on polishing cloth. Then it was rinsed with double-distilled water, and sonicated in water/ethanol/water each for 3 min respectively. The suspension of DB/MWCNTs was prepared from at least 2 h ultra-sonication of DB (0.1 mmol L-1) and MWCNTs (1 wt% DB) in PBS.46 The cleaned electrode was immersed in the suspension of DB/MWCNTs and conditioned out by cyclic potential sweeping between -0.2 to +1.8 V at 0.100 V s-1 for 40 scans. After electropolymerization, the modified electrode was rinsed with distilled water and utilized for electrochemical measurements. 3. Results and Discussion 3. 1. Fabrication and Characterization of PDB/MWCNTs Modified GCE The non-conducting polymer films devoted to developing sensors and biosensors have a very thin thickness (10-100 nm) due to their self-limited growing.47 The nonconducting films also have favorable perm-selective properties which could be used to reduce possible electrochemical interferences in samples. Therefore, fast response time and high selectivity could also be expected for nonconducting polymers modified GCE. Based on non-cova-lent interactions such as n-n stacking, van der Waals interaction and strong adsorption, they interact with MWCNTs, increasing the solubility of MWCNT in water and therefore stabilizing the DB/MWCNTs solution. Cyclic voltammetry was used to form electro-polymerized film and the redox behavior of DB in the presence of MWCNTs was investigated between -0.2 and 1.8 V at the clean glassy carbon electrode. The consecutive cyclic vol-tammograms (the first 10 cycles) are plotted in Figure 1. As the number of cycles increases, the anodic currents in- Figure 1. Successive cyclic voltammograms of GCE in 0.11 M PBS (pH 3) containing the suspension of DB/MWCNTs for first 10 cycles. The scan rate was 0.100 V s-1. crease until a steady state after about 7 cycles. It is an evidence that a polymeric product with the anthraquinone basis formed on the electrode surface. The morphological characteristics of the modified electrodes were studied by SEM and AFM. Fig. 2 represents the topography SEM and AFM images acquired from the surface of bare GC, PDB/GC and PDB/MWC-NT-GC electrodes. The SEM images of smooth and homogeneous surface correspond to the unmodified (a) and modified GCE with PDB (b). While the PDB/MWCNTs modified GCE (c) reveal different patterns, this obviously shows that the electrode surface is covered electrochemically by PDB/MWCNTs in three dimensions. The AFM images indicate that the modified electrode surface with PDB/MWCNTs film is throughout rough and in comparison to PDB/GC and bare GC electrode, increases its microscopic area significantly and the resulting currents in voltammetric measurements. 3. 2. Electrochemical Behavior of LD, UA and AA in a Mixture at Modified GCE In order to study the selectivity of the PDB/MWCNTs-GCE, the cyclic voltammograms of LD, UA and AA in PBS, pH 3, were recorded at the bare and Hatefi-Mehrjardi et al.: Poly-Dianix Blue/Multi-Walled Carbon Nanotube 196 Acta Chim. Slov. 2017, 64, 193-201 Figure 2. SEM (top) and AFM (down) images of bare GC (a), PDB/GC (b) and PDB/MWCNTs-GC (c) electrodes -0.3 0.0 0,3 0.6 EN (vs. Ag/AgCl) 0.« -0.3 0.0 0.3 0.6 E/V (vs. Ag.'AgC I) 4 < a — o ■A -0.3 0.0 0.3 0.6 E/V (vi. Ag/AgCJ) 0.9 -0.3 0,0 0.3 0.6 E/V (VS. Ag^AgCl) Figure 3. Cyclic voltammograms of blank solution in the absence of any analyte (red dotted lines) and 2 ^mol L-1 LD (A), 60 ^mol L-1 UA (B), 100 nmol L-1 AA (C) and the mixture of the three analytes (D) obtained on the surface of bare GC (green short dashed lines), modified PDB/GC (blue long dashed lines), and PDB/MWCNTs-GC electrodes (solid black lines). The potential scan rate was 0.100 V s-1 and supporting electrolyte was 0.11 mol L-1 PBS, pH 3.0. Hatefi-Mehrjardi et al.: Poly-Dianix Blue/Multi-Walled Carbon Nanotube ... Acta Chim. Slov. 2017, 64, 193-197 201 modified electrodes (Fig. 3). It can be shown that the anodic peak potentials for the LD (A), UA (B), and AA (C) oxidation at the bare GC electrode are about 0.432, 0.554, and 0.268 V, respectively, whereas the respective potentials at the surface of the PDB/MWCNTs modified GC electrode are about 0.411, 0.573, and 0.182 V. Fig. 3 (D) shows cyclic voltammograms for a mixture of 2 |mol L-1, 60 |imol L-1 and 100 |imol L-1of LD, UA and AA, respectively in 0.11 mol L-1 PBS solution (pH 3.0) at bare GCE, PDB/GCE and PDB/MWCNTs-GCE. As can be seen, at bare GCE the oxidation peaks for LD, UA and AA are overlapped together with low currents and this shows slow electron transfer kinetics. At the PDB/MWCNTs modified GCE, three well-defined oxidation peaks appear at 0.450, 0.607 and 0.255 V for LD, UA and AA, respectively. The oxidation responses of LD, UA and AA show a great enhancement in the peak currents at PDB/MWCNTs-GCE in comparison with PDB-GCE and bare GCE. Also, when we compare the oxidation peak potentials of LD, UA and AA, there is an enhancement of the anodic peak separation at the PDB/MWCNTs-GCE relative to the values specified at the PDB/GCE and bare GCE. So, the LD, UA and AA peaks potential separations are large enough for the determination of these compounds in the presence of each other at PDB/MWCNTs-GCE. The enhancement in the LD, UA and AA oxidation peak current is mainly attributed to the considerable increment in the electroactive area of the electrode due to the presence of MWCNTs. This phenomenon makes possible the determination of all of these compounds with satisfactory separation between their oxidation peak potentials in voltam-metry. 3. 3. Effect of pH on the Oxidation of LD In order to find the optimum pH for determination of LD, the effect of supporting electrolyte pH was studied. In this case, cyclic voltammetry studies were carried out in the pH range of 2.0-9.0 (PBS, 0.11 mol L-1) at the surface of PDB/MWCNTs-GCE. Fig. 4 shows cyclic voltammo-grams obtained for oxidation of LD at the surface of PDB/MWCNTs-GCE at different pH values. The maximum peak current can be observed at pH 3.0. In addition, all the peak potentials for the oxidation of LD shifted towards negative direction with increasing pH. Therefore, pH 3.0 was selected for further experiments. According to the linear plots of Epa vs. pH concerning the observed slope of -0.057 V/pH for LD (above of the Fig. 4), which is very close to the expected Nernstian value of 0.059 V at 25 °C, where np (number of protons) = ne (number of electrons). 3. 4. Chronoamperometry Studies The catalytic electro-oxidation of LD at the surface of the PDB/MWCNTs-GCE was studied by short time BN[vs Ag/AgCI) Figure 4. Cyclic voltammograms of 60 |M LD at the PDB/MWCNTs-GCE in 0.11 mol L-1 PBS at pH (a) 2.0, (b) 3.0, (c) 4.0, (d) 5.0, (e) 6.0, (f) 7.0, (g) 8.0 and (h) 9.0. The scan rate is 0.100 V s-1. Also, the plots of the extracted I and E0' vs. pH are shown above. chronoamperometry technique. Fig. 5A indicates the chronoamperograms of the different concentrations of LD in PBS (pH 3.0) obtained on PDB/MWCNTs-GC modified electrode by setting the working electrode potentials to 0.5 V vs. Ag/AgCl (KCl 3 mol L-1). Hatefi-Mehrjardi et al.: Poly-Dianix Blue/Multi-Walled Carbon Nanotube 198 Acta Chim. Slov. 2017, 64, 193-201 Figure 5. Chronoamperograms of (a) 5.0; (b) 30.0; (c) 50.0; (d) 80.0; (e) 120.0 |imol L-1 of LD in PBS (0.11 mol L-1, pH 3.0) obtained on PDB/MWCNTs-GCE, at the initial potential of 0.0 V and step potential of 0.5 V vs. Ag/AgCl (KCl 3 mol L-1) (A). The inset shows I as a function of t-1/2 (B). The inset shows the slope of lines B as a function of the concentrations of LD (C). The diffusion coefficient (D) for oxidation of LD at the surface of the modified electrode can be evaluated using Cottrell's equation: I = nFAD1/2 C n-1/2t-1/2 (1) Where D and Cb are the diffusion coefficient (cm2 s-1) and the bulk concentration (mol cm-3), respectively. Under diffusion control conditions, the plots of selected currents versus t-1/2 would be linear. The value of D could be evaluated from the slope of these plots, according to the Cottrell equation. Fig. 5B indicates the experimental plots for different concentrations of LD in the range of 5-120 |imol L-1. The mean value of the diffusion coefficients for LD was calculated to be 6.23 x 10-5 cm2 s-1 using the slopes of the resulting straight lines plotted versus the LD concentrations (Fig. 5C). 3. 5. Differential Pulse Voltammetric Determination of LD, UA and AA Since differential pulse voltammetry (DPV) has a much higher current sensitivity and better resolution than cyclic voltammetry, it was applied for study of LD, UA and AA concentration at PDB/MWCNTs-GCE. Under the optimized solution conditions (0.11 mol L-1 PBS, pH 3), the DPVs of various concentrations of LD, UA and AA were separately recorded (Fig. 6). The respective calibration curves of the anodic peak currents for solutions containing different amounts of each analyte were plotted (Fig. 6, inset) and the linear ranges of 0.09-75 |imol L-1, 0.3-110 |imol L-1and 10-160 |imol L-1were obtained for LD, UA, and AA, respectively. The limits of detection (3 a) for determination of LD, UA, and AA on the modified electrode surface, were found to be 3, 2, and 23 nmol L-1, respectively. Also, the modified electrode presented good repeatability. The relative standard deviations (RSDs) for LD at 0.5 |imol L-1, UA at 3 |imol L-1, and AA at 15 |imol L-1 were 0.25%, 0.61%, and 2.1%, respectively, for 6 measurements which reveal that the sensor had good repeatability. 3. 6. Simultaneous Determination of LD, UA and AA in the Mixture The ability of the PDB/MWCNTs modified GC electrode for simultaneous determination of each analyte was 0.25 0.30 0,35 040 0.45 0.50 0.45 0.50 0,55 0.60 0.0 0.1 0.2 0.3 EN {vs. Ag/AgCl} EN ( vs. Ag/AgCl) EA/ ( vs. Ag/AgCl) Figure 6. Differential pulse voltammograms of LD (A), UA (B), and AA (C) at PDB/MWCNTs-GCE in 0.11 mol L-1 PBS (pH 3). LD concentrations: (a) 0.09, (b) 0.4, (c) 3, (d) 8, (e) 11, (f) 20, (g) 32, (h) 43, (i) 54, (j) 62, (k) 75|imol L-1; UA concentrations: (a) 0.3, (b) 2.5, (c) 7, (d) 12.5, (e) 18, (f) 26.5, (g) 33.5, (h) 50, (i) 63, (j) 82, (k) 110 |imol L-1and AA concentrations: (a) 10, (b) 30, (c) 50, (d) 80, (e) 120, (f) 140, (g) 160 |imol L-1. Insets show the calibration lines from the DPVs shown in (A), (B) and (C). Hatefi-Mehrjardi et al.: Poly-Dianix Blue/Multi-Walled Carbon Nanotube ... Acta Chim. Slov. 2017, 64, 193-199 201 examined by addition of various concentrations of the species in the presence of the constant concentration of the others (Fig. 7). Under the optimal conditions, by increasing of various concentrations of LD, UA and AA, three separated peaks appeared at the potential of about 0.380, 0.520 and 0.180 V, respectively. By increasing the concentration of LD in the presence of 50 pmol L-1 UA and 200 pmol L-1 AA (Fig. 7A), the peak current of LD increased linearly with increasing LD concentration in the range of 0.8-72 pmol L-1 and the related regression calibration is I/pA = 0.10 C/pmol L-1+0.58 (Fig. 7A, inset). It is observable that the oxidation peaks related to UA and AA are approximately constant. Furthermore, different concentrations of UA in the presence of 1.7 pmol L-1 LD and 220 pmol L-1 AA illustrate excellent DPVs responses (Fig. 7B); the peak current of UA grows linearly by increasing UA concentration in the range of 0.3-110 pmol L-1 and the related regression calibration is I/pA = 0.008 C/pmol L-1 + 0.11 (Fig. 7B, inset) which shows simultaneous determination of UA in the presence of LD and AA on the surface of PDB/MWCNTs-modified GCE. We also observed oxidation peaks of various amounts of AA in the presence of a constant concentration of LD (2 pmol L-1) and UA (20 pmol L-1) (Fig. 7C). There is no serious variation observed in the peak current of LD and UA, but the peak current of AA in the concentration range of 1-160 pmol L-1 increased linearly with calibration regression equation of I/pA = 0.013 C/pmol L-1 + 1.0 (Fig. 7C, inset). These results indicate that the electrochemical determination of three analytes in the presence of each other on the PDB/MWCNTs- modified GCE surface is possible independently. of 5.5 pmol L-1 LD was investigated. The tolerance limit was taken as the maximum concentration of the foreign compound which caused an approximately ±5% relative error in the determination of the analyte. The experimental results show that neither 500-fold excess concentration of Ni2+, Fe3+, Cu2+, Co2+, Mn2+, Na+, K+, Mg2+, Ca2+, Al3+, Pb2+, Cl-, NO3-, SO42-, PO43-, CO32-, HCO3- nor 300-fold excess of glucose, lactose, sucrose, fructose, glycine, L-lysine, and riboflavin did not interfere, but practically equal molar concentrations of dopamine, DOPAC, homo-vanillic acid, epinephrine, and norepinephrine showed interference on determination of LD. 3. 8. Real Samples Analysis In order to evaluate the analytical applicability of the proposed sensor, direct determination of LD, UA and AA were applied for two physiological samples (human blood serum). The human blood plasma samples were collected from clinical laboratory and diluted 4 times by 0.11 mol L-1 PBS solution (pH 3) without any treatment. The recoveries of these three analytes in blood serum were determined by the standard addition method (Table 1) and satisfactory results were obtained. These results show that the PDB/MWCNTs-GC modified electrode is an excellent sensitive tool for simultaneous determination of the analytes in physiological samples. 4. Conclusions 3. 7. Interference Studies Under the optimal experimental conditions, the influence of various interfering species on the determination In the present work, it was shown that poly-DB/MWCNTs film on the GCE can be considered as a sensitive and selective sensing element in the simultaneous voltammetric determination of LD, UA and AA. The E/V < vs. Ag/AgCI) E/V( vs. Ag/AgCI) E/V ( vs. Ag/AgC!) Figure 7. Differential pulse voltammograms of PDB/MWCNTs-GCE in PBS 0.11 mol L-1 (pH 3), containing (A) LD concentrations: (a) 0.8, (b) 4.5, (c) 6, (d) 12, (e) 18, (f) 27, (g) 35, (h) 46, (i) 65, (j) 72 pmol L-1in the presence of 50 pmol L-1 UA and 200 pmol L-1 AA; (B) UA concentrations: (a) 0.3, (b) 1, (c) 10, (d) 22, (e) 53, (f) 82, (g) 90, (h) 110 pmol L-1in the presence of 1.7 pmol L-1 LD and 220 pmol L-1 AA; (C) AA concentrations: (a) 1, (b) 3, (c) 4.5, (d) 6, (e) 10, (f) 30, (g) 66, (h) 97, (i) 130, (j) 160 pmol L-1in the presence of 2 pmol L-1 LD and 20 pmol L-1 UA. Insets: The related calibration plots from the DPVs are shown in (A), (B) and (C). Hatefi-Mehrjardi et al.: Poly-Dianix Blue/Multi-Walled Carbon Nanotube 200 Acta Chim. Slov. 2017, 64, 193-201 Table 1. Determination and recovery tests of LD, UA and AA in real samples obtained using PDB/MWCNTs-GC modified electrode. Analyte Sample Added(^mol L1) Found(^mol L 1) Recovery(%) LD Serum 1 0 0.112 - 10 9.909 98.00 Serum 2 0 0.130 - 16 16.21 100.5 Serum 1 a 0 0.154 - 10 10.50 103.5 Serum 2 a 0 0.093 - 4 3.958 96.70 AA Serum 1 0 0.297 - 25 24.60 97.26 Serum 2 0 0.221 - 20 20.62 102 UA Serum 1 0 0.168 - 20 20.00 99.20 Serum 2 0 0.087 - 10 10.52 104.3 The recovery tests of LD were performed in the presence of 35 nmol L-1 AA and 12 ^mol L-1 UA in real samples modified electrode showed an effective electrocatalytic activity toward the anodic oxidation of LD, UA and AA, which leads to a significant increase in the peak currents and a decrease in peak over-potentials. 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Eng. R 2005, 49, 89-112. https://doi.org/10.1016/j.mser.2005.04.002 47. M. Yuqing, C. Jianrong, W. Xiaohua, Trends Biotechnol. 2004, 22, 227-231. https://doi.org/10.1016/j.tibtech.2004.03.004 Povzetek Preučevali smo selektiven in občutljiv elektrokemijski senzor na osnovi s poli-dianiks modrim in večstenskimi ogljikovimi nanocevkami (PDB/MWCNT) modificirano elektrodo iz steklastega ogljika za določanje levodope (LD) v prisotnosti sečne kisline (UA) in askorbinske kisline (AA). Za raziskave elektrokatalitske oksidacije LD, UA in AA v vodnih raztopinah smo uporabili metode ciklične voltametrije, diferencialne pulzne voltametrije in kronoamperometrije. Pri tehniki DPV so LD, UA in AA dali oksidacijske vrhove pri 0,380 V, 0,520V in 0,180 V. Pri optimiziranih eksperimentalnih pogojih je bil linearen odgovor za LD v območju 0,09-75 |mol L-1, za UA 0,3-110 |mol L-1 in za AA v območju 10-160 |mol L-1. V skladu s tem so bile meje zaznave 0,003, 0,002 in 0,023 |mol L-1. Metoda predstavlja preprost elektrokemijski senzor za uspešno določitev LD v serumskih vzorcih iz humane krvi. Hatefi-Mehrjardi et al.: Poly-Dianix Blue/Multi-Walled Carbon Nanotube 202 DOI: 10.17344/acsi.2016.3109 Acta Chim. Slov. 2017, 64, 202-207 recreative ^commons Scientific paper Mn(II), Zn(II) and Cd(II) Complexes Based on Oxadiazole Backbone Containing Carboxyl Ligand: Synthesis, Crystal Structure, and Photoluminescent Study Li-Na Wang, Lin Fu, Jia-Wei Zhu, Yu Xu, Meng Zhang, Qi You, Peng Wang and Jie Qin* School of Life Sciences, Shandong University of Technology, Zibo 255049, P. R. China * Corresponding author: E-mail: qinjietutu@ 163.com Tel.: 0086-533-2780271; Fax: 0086-533-2781329 Received: 26-11-2016 Abstract Three coordination polymers, [Cd(L)2(H2O)2]n (1), [Zn(L)2(H2O)2]n (2) and [Mn(L)2]n (3) were prepared by reacting 5-(3-pyridyl)-1,3,4-oxadiazole-2-thioacetic acid (HL) with corresponding metal acetate in DMF/CH3CN medium under solvothermal condition. The isolated complexes were characterized by elemental analysis and infrared spectroscopy. The X-ray crystallographic analysis revealed double strand structure of 1 and 2, and 3D framework of 3. The different structures of these complexes indicate that the configuration of the ligand and the reaction condition play a key role in self-assemble of complexes 1-3. Furthermore, photoluminescent properties of 1 and 2 were also studied in the solid state. Keywords: Oxadiazole ligand; solvent thermal synthesis; crystal structure; photoluminescent property 1. Introduction Nowadays, more and more attention has been paid to coordination compounds with various of topological structures and potential promising applications ranging from functional material to therapeutic agent.1-6 Many factors need to be considered during the self-assembly process of coordination compound, including the nature of the metal ion, the well-designed organic ligand, the auxiliary ligand, the solvent medium, the pH value, the temperature, and so on.7 Therefore the rational design and precise crystal engineering of coordination compounds with desired structures and specific properties still remain a challenge.8 According to previously reported work, the choice of organic ligand has been verified as a decisive role in the construction of the overall architectures of coordination polymers, as the organic spacer serves to link metal nodes and to propagate the structural information.9,10 Rigid linear organic ligands such as 4,4'-bipyridine and its derivatives are well adopted in generating polymers bearing linear chain,11 honeycomb-like,12 square-li-ke,13 or brick-wall-like structures.14,15 While the bent organic ligands can offer the possibility of constructing novel polymer network owing to their variable conforma- tion.15 1,3,4-Oxadiazole is an intensively investigated class of bent organic bridging moiety due to its convenient synthesis as well as the versatile coordination mode.9,1618 Coordination polymers, with structures like helical chain,19 zeolite-like net,16 and 3-fold interpenetrated 3D framework,15 have been reported by Dong group. They are based on symmetric 2,5-diaryl-1,3,4-oxadiazole containing pyridyl, aminophenyl or cyanophenyl groups as terminal coordination sites. Herein we focus on the coordination behavior of 5-(3-pyridyl)-1,3,4-oxadiazole-2-thioacetate (L), which is mostly based upon the following considerations. (i) L is an unsymmetric ligand bearing both pyridine and carboxyl groups bridged by the oxadiazole backbone. Hence L can show diverse coordination modes. Especially the carboxyl group can feature unidentate, chelate or bridging fashions.20 (ii) L is a bent ligand, which can adopt either gauche- or anti-configuration in the self-assembly reaction (Scheme 1).8,20 (iii) Heteroatoms such as N, O, and S of L could be considered as potential hydrogen bond acceptors to expand polymeric frameworks via hydrogen bonding interactions.16 Coordination polymers based on L and its isomer 5-(4-pyridyl)-1,3,4-oxadiazole-2-thioacetate (4-pyoa) were first reported by Du et al. under the layer separation diffusion condition.20 Wang et al.: Mn(II), Zn(II) and Cd(II) Complexes Based Acta Chim. Slov. 2017, 64, 202-207 203 Reaction of HL and 4-pyoa with metal salts afforded 1D coordination polymers of {[M2(4-pyoa)4(H2O)2](H2O)2}n (M = Co, Zn), anatase type network of {[Pb(4-pyoa)2] (H2O)}n, 2D layer of {[Cu(L)2(H2O)](H2O)2}n, and 3,6-connected 3D net of [Cd(L)2]n.20 Indeed, this demonstrates that HL is well-tailored in constructing new polymers with attractive properties. The aim of the presented work is the construction of complexes derived from HL under solvent thermal condition. The reactions of HL and M(CH3COO)2 (M = Cd, Zn, and Mn) in DMF/CH3CN at 110 °C afford three polymers, [Cd(L)2(H2O)Jn (1), [Zn(L)2(H2O)Jn (2) and [Mn(L)2]n (3). Herein, the preparation, and crystallograp-hic analyses of these complexes are described. Moreover, luminescent properties of 1 and 2 were investigated in the solid state. gauche- Scheme 1. Two possible configurations of L. 2. Experimental 2. 1. Physical Measurements and Materials Reagents and solvents were purchased commercially from Aladdin Industrial Corporation (China) and used without further purification. The starting com- pound HL was synthesized according to the literature method.20'21 The IR spectra were taken on a Vector22 Bru-ker spectrophotometer (400-4000 cm1) prepared as KBr pellets. Elemental analyses were performed on a Perkin-Elmer model 2400 analyzer. Fluorescence spectra were recorded on Cary Eclipse spectrofluorimeter (Varian, Australia) at room temperature. 2. 2. General Procedure for the Synthesis of Complexes 1-3 HL (0.1 mmol) and metal acetate salts (0.2 mmol) in 10 mL mixed solvent of DMF/CH3CN (v/v = 1:1) were sealed in a 25 mL Teflon cup. The mixture was heated at 110 °C for 3 days and cooled to room temperature at a rate of 5°C/h. Yellow crystals were obtained. [Cd(L)2(H2O)2]n (1) Yield: 15.8 mg (51% on the basis of HL). The IR (KBr, cm-1): 3446, 3098, 3033, 1607, 1577, 1474, 1462, 1438, 1393, 1222, 1198, 1091, 1049, 1031, 999, 960, 821, 715, 701, 684, 640, 443. Anal. Calcd. for C18H16CdN6O8S2: C, 34.82; H, 2.60; N, 13.53. Found: C, 34.92; H, 2.59; N, 13.57%. [Zn(L)2(H2O)2]n (2) Yield: 13.8 mg (48% on the basis of HL). IR (KBr, cm-1): 3468, 3085, 3067, 2985, 1646, 1614, 1459, 1417, 1364, 1326, 1191, 1087, 1052, 1004, 959, 820, 712, 698, 650, 537. Anal. Calcd. for C18H16ZnN6O8S2: C, 37.67; H, 2.81; N, 14.64. Found: C, 37.82; H, 2.80; N, 14.69%. [Mn(L)2]n (3) Yield: 8.4 mg (32% on the basis of HL). IR (KBr, cm-1): 3033, 1574, 1462, 1393, 1196, 1088, 1046, 996, 921, 819, 679, 639, 441. Anal. Calcd. for C18H12MnN6O6S2: C, 40.99; H, 2.93; N:15.93. Found: C, 4L12; H, 2.91; N, 15.99%. Table 1. Crystallographic data for 1-3. 1 2 3 Empirical formula C18H16CdN6O8S2 C18H16ZnN608S2 C18H12MnN6°6S: Mr 620.89 573.86 527.40 Crystal System triclinic triclinic monoclinic Space group P-1 P-1 C2/c a (A) 7.4145(11) 7.3585(6) 25.302(3) b (A) 7.6738(11) 7.4216(7) 10.6816(11) c (A) 10.6697(15) 10.7069(9) 7.1462(7) a(°) 88.064(4) 88.979(3) 90.00 m 82.611(4) 82.757(2) 95.747(3) r(°) 74.497(4) 73.709(3) 90.00 v (A3) 580.13(14) 556.67(8) 1921.7(3) Z 1 1 4 pc (g cm-3) 1.777 1.712 1.823 F(000) 310 292 1068 T / K 298(2) 298(2) 298(2) l(Mo-Ka)/ mm-1 1.179 1.351 0.960 GOF (F2) 1.131 1.084 1.107 Data / restraints / parameters 2614/0/160 2531 / 0 / 160 2207 / 0 / 150 R1a, wR2b (I>2a(I)) 0.0205, 0.0540 0.0245, 0.0611 0.0248, 0.0662 a R1 = EjjFoj - |Fc||/E|Fo|. b wR2 = [Ew(Fo2 - Fc2)2/Ew(Fo2)]1 Wang et al.: Mn(II), Zn(II) and Cd(II) Complexes Based ... 204 Acta Chim. Slov. 2017, 64, 202-207 2. 3 Determination of Crystal Structures X-ray intensity data for crystals 1-3 were collected on a Bruker SMART APEX CCD-based diffractometer (Mo Ka radiation, X = 0.71073 Â) at 298 K. The raw frame data were integrated into SHELX format reflection files and corrected for Lorentz and polarization effects using SAINT.22 Multi-scan absorption corrections were applied by SADABS.23 All the structures were solved by direct methods and refined by full-matrix least-square methods applying SHELXL program package.24 Anisotropic thermal parameters were used to refine all non-hydrogen atoms. H atoms of C-H were geometrically generated and refined with isotropic thermal parameters riding on the parent atoms. The H atoms of water molecules were fixed by difference Fourier maps with O-H = 0.85(2) Â, H-H = 1.44(2)  and Uiso(H) = 1.5Ueq(O). Details of crystallo-graphic parameters, data collection, and refinements are summarized in Table 1. Relevant bond distances and bond angles are given in Tables 2, 3 and S1. 3. Results and Discussion 3. 1. Synthesis and General Characterization The mixing of metal salts and carboxylic ligand solution resulted in precipitation in traditional aqueous reaction system therefore solvothermal synthesis was adopted. By performing parallel experiments, it was found that using M(NO3)2 or M(ClO4)2 (M = Cd, Zn, Mn) as the source of metal salts could also isolate these complexes, which indicates that the complexes are independent of the coun-ter-anions of the metal salts. The acetate salts were found to achieve products in a somewhat higher crystal quality and yield. 3. 2. IR Spectra The IR spectra of complexes 1-3 (see Figure S1, Supporting Information) exhibiting the absence of characteristic absorption bands of the carboxyl group (1718 cm-1 in HL) reveals the complete deprotonation. As a consequence, the antisymmetric (vas(COO-)) and symmetric (vs(COO-)) stretching vibrations of carboxylate groups appear. The separation value Av between vas(COO-) and vs(COO-) can be used to identify the coordination mode of the carboxylate ligand.25'26 The Av value is 214 cm-1 for 1, 229 cm-1 for 2, indicating a monodentate coordination mode of carboxylate group. While the Av value for 3 is 181 cm-1 indicative of bidentate carboxylate coordination. These IR results are in agreement with the crystal structural analyses. 3. 3. Crystal Structures X-ray single-crystal diffraction reveals that complexes 1 and 2 are isostructural and crystallize in the same Table 2. Selected bond distances (Â) and angles (°) for complex 1. Cd1-O4 2.3030(13) Cd1-O3iii 2.2584(14) Cd1-N1 2.3703(14) S1-C7 1.7275(18) S1-C8 1.8042(19) O2-C9 1.242(2) O3-C9 1.251(2) N1-Cd1-N1i 180.000(1) O3iii-Cd1-N1 90.24(6) O4i-Cd1-N1i 91.59(5) O3iii-Cd1-O4 91.31(5) O3ii-Cd1-O4 88.69(5) O4-Cd1-O4i 180.0 O3ii-Cd1-N1 89.76(6) O4i-Cd1-N1 88.41(5) C7-S1-C8 98.83(9) Symmetry codes: (i) -x, -y + 1, -z + 2; (ii) -x + 1, - -y + 1, -z + 1; (iii) x - 1, y, z + 1 Table 3. Selected bond distances (Â) and angles (°) for complex 3 Mn1-O2 2.1024(10) Mn1-O3ii 2.1878(10) Mn1-N1™ 2.3479(11) S1-C7 1.7235(14) S1-C8 1.7969(14) O2-C9 1.2453(17) O3-C9 1.2485(17) N1iii-Mn1-N1i 94.98(6) O2-Mn1-O2iv 93.00(6) O2-Mn1-O3v 103.46(4) O2i-Mn1-O3v 92.20(4) O3#v-Mn1-O3#ii 157.28(6) O2-Mn1-N1iii 176.41(4) O2iv-Mn1-N1iii 86.11(4) O3v-Mn1-N1iii 80.06(4) O3ii-Mn1-N1iii 84.63(4) C7-S1-C8 98.76(6) Symmetry codes: (i) -x + 3/2, y - /, -z + /2; (ii) x, -y + 1, z - '/2; (iii) x - /2, y - /2, z; (iv) -x + 1, y, -z + /2; (v) -x + 1, -y + 1, -z + 1 NÎ Figure 1. Coordination environment of Cd11 in 1. Symmetry codes: (i) -x, -y + 1, -z + 2; (ii) -x + 1, -y + 1, -z + 1; (iii) x - 1, y, z + 1. triclinic PI space group with similar cell parameters. Therefore only the structure of 1 is described here in detail as a representative example. The ORTEP plots of complexes 1 and 2 with atomic numbering scheme are shown in Figures 1 and S2 As drawn in Figure 1, the Cd11 ion is located at the inversion center, and the asymmetric unit of compound 1 is composed of one Cd11 ion with the occupancy of 0.5, one L1- ligand, and one coordinated water molecule. The central Cd11 is six coordinate with two N and two O atoms from four crystallographically independent L1-, and two water O atoms. The coordination geometry of the {CdN2O4} can be described as an almost perfect octahe- Wang et al.: Mn(II), Zn(II) and Cd(II) Complexes Based Acta Chim. Slov. 2017, 64, 202-207 205 dron, which is reflected by the axial N1-Cd-NT 180.0°, and the sum of the equatorial bond angels being 360.0°. The gauche style of the ligand is observed in complex 1, which was confirmed by the value of the torsion angle of C7-S1-C8-C9 being -70.94(15)°. Figure 2. 1D coordination framework of 1. Du and coworkers have prepared the complex [Cd(L)2]n (1A) in CH3OH/H2O-NaOH mixed solvent system at room temperature using Cd(NO3)2 and HL.20 In 1A, the octahedral coordination sphere of Cd11 is provided by four carboxylate O and two pyridyl N atoms coming from six separated ligands. The authors ascribed this coordination geometry to the metal-ligand synergistic effect that the CdII ion with larger radii is capable of holding six ligands around it. In 1A, the ligand serves as a 3-connected node resulting in the 3D rutile framework. While in complex 1, the pyridyl and the car-boxylic groups both adopted the monodentate coordination mode acting as 2-connected node. The Cd11 ions are bridged by paired L ligands. As a consequence, 1-D double-strand coordination array of 1 is formed running along [1 0 -1] direction with Cd -Cd separation of 12.1848(14) A (Figure 2). The Cd-Npyridyl bond length (2.3703(14) A) is comparable to that in 1A (2.373(2) A), while the Cd-Ocarboxylic bond length in 1 being 2.2584(14) A is longer than that in 1A 2.291(2) A. The intra-chain hydrogen bond interactions were found between the uncoordinated carboxylic O atoms and the coordinated water molecules (O4-H4B-O2111, symmetric code: (iii) x - 1, y, z + 1). Analysis of the crystal packing of 1 reveals the existence of two types of inter-chain hydrogen bonds, including O4-H4A-O2iv and O4-H4A-S1iv (symmetric co- Figure 3. View of the 2D hydrogen-bonding supramolecular layer in 1. Wang et al.: Mn(II), Zn(II) de: (iv): -x, -y + 1, z + 1) between the water ligands and the uncoordinated carboxylic O atoms as well as S atoms. Therefore, these 1D chains are connected through these hydrogen bonds, forming a two-dimensional su-pramolecular layer along the [001] plane, as depicted in Figure 3. Figure 4. The coordination environment of Mn(II) in 3 at 50% probability displacement. Symmetry codes: (i) -x + 3/2, y - /2, -z + /2; (ii) x, -y + 1, z - /2; (iii) x - /2, y - /2, z; (iv) -x + 1, y, -z + /2; (v) -x + 1, -y + 1, -z + 1. Polymer 3 crystallized in monoclinic C2/c space group. As illustrated in Figure. 4, the independent unit of 3 is composed of half Mn(II) cation and one deprotonated ligand L1-. The L1- serves as a ^-bridging ligand in 3, which is identical to that in complex 1A. The Mn center is hexa-coordinated in distorted octahedron coordination geometry and is bonded by four carboxyl oxygen atoms from four L1- anions [Mn1-O2 = 2.1024(10) A; Mn1-O3 = 2.1878(10) A], and two pyridine nitrogen atoms from the other two ligands with Mn-N bond lengths of 2.3479(11) A. The N1', O3u, O2iv and O3v are located in the equatorial plane, while the O2 and N1m atoms occupy the axial positions. The difference of coordination geometry between Mnn in 3 and Cdn in 1A lies in that the pyridine N atoms are in the axial positions in 1A.20 The Mn-O and Mn-N bond distances are close to other manganese complexes derived from (4-pyridylthio)acetic acid (PTA), such as [(Mn-salen)PTA] and [Mn(PTA)2(H2O)]n.27,28 Compared with complexes 1 and 2, the ligand adopted anti-configuration in 3 as evidenced by the torsion angle of C7-S1-C8-C9 being -167.9°. Figure 5. ThelD chain structure in 3. and Cd(II) Complexes Based ... 206 Acta Chim. Slov. 2017, 64, 202-207 In 3 the adjacent Mn11 centers are doubly bridged by the carboxyl groups of L1-, forming an infinite chain structure along the c direction, with a Mn—Mn distance of 4.567(4) A (Figure 5). In each L1- ligand, the mean plane of the carboxylate group and the plane of the pyridine group are inclined to each other with a dihedral angle of 19.2°. Such 1D chains are aligned side by side in the ab plane, and are further linked together by Mn1-N1 linkages, eventually forming the three dimensional network of 3 (Figure 6). Figure 6. The 3D network of 3. 3. 4. Photoluminescent Properties Taking into account that coordination compounds based on d10 metal centers are promising candidates for photoactive materials with potential applications,29,30 the ambient temperature photoluminescent properties of 1 and 2 as well as the free ligand HL were measured in the solid state. As depicted in Figure 7, upon excitation at 290 nm, the free compound HL has an emission band maxima at 325 nm. The emission of HL can be assigned to the n* to n and n* to n intraligand transitions.31,32 As Cd2+ or Zn2+ ions are 300 400 soo Wavelength (nm) Figure 7. Solid state emission spectra of compounds HL, 1 and 2 at room temperature. difficult to oxidize or reduce owning to their d10 configuration,33 the emission spectra of complexes 1 and 2 are similar with that of HL. Hence the luminescent emissions of 1 and 2 are attributed to the intraligand transition. Moreover, approximately a three-time increase in the luminescence intensity was observed for complexes compared with the free li-gand. The red shifts (10 nm for 1 and 17 nm for 2) and enhancement of luminescence intensity of the complexes may be ascribed to the deprotonation and coordination to metal ions, which can effectively enhance the rigidity of HL and further reduce the loss of energy by radiationless decay of the intraligand emission excited state.34 4. Conclusions To sum up, three Cd(II), Zn(II) and Mn(II) coordination polymers based on semirigid asymmetric ligand 5-(3-pyridyl)-1,3,4-oxadiazole-2-thioacetate were successfully prepared. Complexes 1 and 2 are double-strand structures, and two-dimensional supramolecular networks were further observed through O-H-O and O-H—S hydrogen bonding interactions. While 3 features three-dimensional framework. The structural diversity reveals that the configuration of the ligand and the reaction condition play an important role during the self-assembly process of complexes 1-3. In addition, complexes 1 and 2 exhibit intense blue fluorescent emission indicating promising candidates for functional inorganic-organic photoactive materials. 5. Supplementary Material Crystallographic data (excluding structure factors) for the structural analysis have been deposited with the Cambridge Crystallographic Data Center as supplementary publication Nos. CCDC 1518672 (1), 1518673 (2), and 1518674 (3). Copies of the data can be obtained free of charge via www.ccdc.ac.uk/conts/retrieving.html (or from The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, Fax: +44-1223-336-033. E-mail: deposit-@ ccdc.cam.ac.uk). 6. Acknowledgment This work was supported by the National College Students' Innovative and Entrepreneurial Training Plan of China (201510433068) and Natural Science Foundation of Shandong Province (ZR2011HM065). 7. References 1. W. J. Gee, L. K. Cadman, H. A. Hamzah, M. F. Mahon, P. R. Raithby, A. D. Burrows, Inorg. Chem. 2016, 55, 10839- Wang et al.: Mn(II), Zn(II) and Cd(II) Complexes Based Acta Chim. Slov. 2017, 64, 202- 207 207 10842. https://doi.org/10.1021/acs.inorgchem.6b01917 2. J. Qin, N. Qin, C. H. Geng, J. P. Ma, Q. K. Liu, D. Wu, C. W. Zhao, Y. B. Dong, CrystEngComm. 2012, 14, 8499-8508. https://doi.org/10.1039/c2ce25561h 3. R. Vafazadeh, A. C. Willis, Acta Chim. Slov. 2016, 63, 186192. https://doi.org/10.17344/acsi.2016.2263 4. J. Qin, S. S. Zhao, Y. P. Liu, Z. W. Man, P. Wang, L. N. Wang, Y. Xu, H. L. Zhu, Bioorg. Med. Chem. Lett. 2016, 26, 4925-4929. https://doi.org/10.1016/j.bmcl.2016.09.015 5. J. Qin, Q. Yin, S. S. Zhao, J. Z. 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Chem. 2015, 641, 2657-2663. https://doi.org/10.1002/zaac.201500613 Povzetek Pripravili smo tri koordinacijske polimere [Cd(L)2(H2O)2]n (1), [Zn(L)2(H2O)2]n (2) in [Mn(L)2]n (3) z reakcijo med 5-(3-piridil)-1,3,4-oksadiazol-2-tioocetno kislino (HL) z ustreznim kovinskim acetatom v mešanici DMF/CH3CN pri sol-votermalnih pogojih. Izolirane komplekse smo okarakterizirali z elementno analizo in infrardečo spektroskopijo. Rentgenska strukturna analiza razkrije strukturo dvojne vijačnice pri spojinah 1 in 2 ter 3D mrežo pri 3. Različne strukture teh kompleksov kažejo, da imajo konfiguracija liganda in reakcijski pogoji ključno vlogo pri zlaganju v kristalno strukturo. Proučili smo tudi fotoluminiscenčne lastnosti 1 in 2 v trdnem stanju. Wang et al.: Mn(II), Zn(II) and Cd(II) Complexes Based ... 208 DOI: 10.17344/acsi.2016.3116 Acta Chim. Slov. 2017, 64, 208-214 ^creative ^commons Scientific paper Synthesis and Structure of [Cu(Hapn)]NO3]NO3, [Cu(Hapn)(H2O)2]SiF6, [Cu(Hapn)(H2O)BF4]BF4 • H2O and [Cu(Hapn)(NH2SO3)2] n-complexes (apn = 3-(prop-2-en-1-ylamino)propanenitrile) Mykhailo Luk'yanov,1* Evgeny Goreshnik,2 Vasyl Kinzhybalo,3 and Marian Mys'kiv1 1 Department of Inorganic Chemistry, Ivan Franko National University of Lviv, Kyryla i Mefodiya Str., 6, 79005, Lviv, Ukraine 2 Department of Inorganic Chemistry and Technology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia 3 Institute of Low Temperature and Structure Research, Okolna 2, Wroclaw, 50-422, Poland * Corresponding author: E-mail: mishalukianov@gmail.com; Tel.: +380 32 23 94 506 Received: 29-11-2016 Abstract Four copper(I) n-complexes: [Cu(Hapn)NO3]NO3 (1), [Cu(Hapn)(H2O)2]SiF6 (2), [Cu(Hapn)(H2O)BF4]BF4-H2O (3) and [Cu(Hapn)(NH2SO3)2] (4) were prepared using alternating-current electrochemical technique, starting from alcohol solutions of 3-(prop-2-en-1-ylamino)propanenitrile (apn) titrated with appropriate acid and copper(II) salts (Cu(NO3)2 • 3H2O, CuSiF6 • 4H2O, Cu(BF4)2 • 6H2O or Cu(NH2SO3)2 • xH2O, respectively). Obtained compounds were characterized by single-crystal X-ray diffraction and partially by IR spectroscopy. In the structures of complexes 1, 2 and 4 Cu(I) cation possesses a tetrahedral environment formed by the C=C bond of one organic cation Hapn, the N atom of cyano group from another Hapn moiety, and two O atoms (from NO3- anions in 1, from H2O molecules in 2) or N atoms (NH2SO3- anions in 4). In compound 3 strongly pronounced trigonal-pyramidal coordination environment of Cu(I) is formed by a mid-point of C=C-bond of one Hapn cation, nitrogen atom (of cyano group) of another Hapn unit, O atom of H2O molecule in the basal plane, and F atom of BF4- anion at the apical position. Keywords: Copper(I); n-complex; aminonitrile derivative; crystal structure; coordination polymer 1. Introduction For almost two centuries the attention of scientists within different branches has been paid to aminonitriles, ranging from a-aminonitriles discovered by A. Strecker as far as in 1850,1 to various P-, y-, o-, ra- aminonitriles obtained in our days.2 Representatives of this class are well-known not only as versatile intermediates in organic synthesis and in many other reactions,3,4 but also as reagents for synthesis of heterocyclic compounds,5 inhibitors of enzymes,6 precursors of peptides,7 amino-acids,8 which, in turn, exhibit antibiotic,9 antifungal,10 and other important biological and pharmacological properties.11,12 The coordination behaviour of aminonitriles in the complexation reactions with Cu(I) salts can be characteri- zed on the basis of only several related,13,14 or closely related,15 compounds, though the matter under discussion is still relevant. It has been noticed that atoms of Cl or Br compete for space in coordination polyhedron with allyl groups and cyano group in the halide complexes of Cu(I) with diallylaminopropanenitrile (the tertiary amine N-atom is protonated).16,17 Still one of the two olefin bonds and ha-lide atoms have a priority, and CN-group (as well as the second C=C-bond) does not coordinate to the metal ion. Generally speaking, there are few ways for apn-mo-iety to coordinate with Cu ions. Depending on the status (cation or molecular) of 3-(prop-2-en-1-ylamino)propanenitrile the number of active groups for coordination changes, which, in turn, influences the composition of coordination polyhedron of the Cu ion (other ligand moieties, such as sol- Luk'yanov et al.: Synthesis and Structure of [Cu(Hapn)]NO3]NO Acta Chim. Slov. 2017, 64, 208-214 209 vent molecules or anions, occupy usually the apical position of the coordination polyhedron) and complexity of the arisen inorganic component in a compound: from (CuCl)2 to (Cu2Cl3)nn-.16 Thus, being in molecular state, apn is coordinated to Cu with allyl- and amino- group and Cl- in the following sequence (C=C > NH >...), whereas C^N-group is not coordinated. Cationic form of apn provides these groups the same chance to be coordinated with the metal atom: C=C > C=N > Hal. In order to study a coordination ability of C=C-bond or C^N-group to the copper atom, the compounds with ionic copper salts have been studied. Therefore, we have undertaken the synthesis and crystal structure determination of copper(I)-n-complexes with 3-(prop-2-en-1-ylamino)propanenitrile. 2. Experimental 2. 1. Synthesis of 3-(prop-2-en-1-ylamino) propanenitrile (apn) A mixture of 0.15 mol allylamine (11.2 mL) and 0.10 mol acrylonitrile (6.8 mL) was continuously stirred and cooled (5 h, 20 °C) preventing the temperature rising higher than 30 °C,18 then it was heated for 1 h in a water bath with a reflux condenser at 60 °C. The product (orange liquid) was purified by distillation in a vacuum of a water-jet pump (85 °C /40 mm Hg). The yield of apn was 88% (15 mL). IR (KBr) n 3315(w), 3077(m), 2977(m) 2912(s), 2837(s), 2247(s), 1642 (m), 1528(wv), 1465(s), 1419(s), 1118(s), 996(s), 922(vs) cm-1. 2. 2. Preparation of Complexes Four crystalline copper(I) compounds with 3-(prop-2-en-1-ylamino)propanenitrile were prepared using alternating-current electrochemical syntheses.19 The density of crystals of 1-4 was determined by the flotation method in a chloroform-bromoform mixture (Table 1). 2. 2. 1. Preparation of [Cu(Hapn)NO3]NO3 (1) The apn (4.8 mmol) in 2 mL of ethanol titrated by HNO3 to pH 5.5 was mixed with Cu(NO3)2 ■ 3H2O (4.3 mmol) in 2 mL of ethanol. The solution was placed into a small test-tube and copper-wire electrodes in cork were inserted. After applying U = 0.50 V of alternating-current tension (frequency 50 Hz, /init = 0.5 mA) for 16 h a starting coloured solution was discoloured and good quality colourless crystals of 1 appeared on the copper electrodes. Yield of complex 1 was 70%. 2. 2. 2. Preparation of [Cu(Hapn)(H2O)2]SiF6 (2) The same synthesis (frequency 50 Hz, U = 0.55 V, Iinit = 0.54 mA), starting from CuSiF6 ■ 4H2O (3.8 mmol) and 4 mL of methanolic solution of the apn (4.2 mmol), previously titrated with an aqueous solution of 19% H2Si-F6 to pH 3, resulted in a formation of good quality crystals of 2 in 12 h. Yield of the complex was 95%. IR (Nujol) n 3434(vs), 2953(vs), 2846(vs), 2260(w), 1642 (s), 1458(s), 1376(m), 1019(s), 953(w), 728(s) cm-1. 2. 2. 3. Preparation of [Cu(Hapn)(H2O)BF4]BF4 • H2O (3) Good quality crystals of complex 3 were obtained in a similar way (alternating-current, U = 0.65 V, Iinit = 0.5 mA) starting from 4 mL of propanol solution of the 4 mmol of apn (titrated with HBF4 to pH = 4) and Cu(BF4)2 ■ 6H2O (4 mmol). Colourless prismatic crystals of compound 3 appeared on copper wire electrodes after 120 h. The yield was 60%. 2. 2. 4. Preparation of [Cu(Hapn)(NH2SO3)2] (4) Colourless needle-like crystals of complex 4 appeared from a methanol solution (4 mL) of Cu(NH2SO3)2 ■ xH2O (4.1 mmol) and the apn (4.1 mmol) previously titrated with water solution of 50% NH2SO3H to pH 6.5 under conditions of the alternating-current electrochemical technique (U = 0.6 V, /init = 0.4 mA) during 7 days. A yield of 4 was 65%. IR (KBr) n 3787(vw), 3264(w), 2923(m), 2361(s), 1662(w), 1249(vs), 1203(vs), 1055(m), 787(w), 643(vw), 593(vw), 560(vw) cm-1. 2. 3. Crystallography The experimental details, crystallographic parameters and summaries of the data collection for 1-4 are presented in Table 1. Single crystals of 1-4 were preliminarily studied by the photo-method and then diffraction data were collected on a Rigaku AFC7R (for 1-2) or KU-MA-KM4/CCD (for 3-4) diffractometers with graphite monochromated Mo^a radiation (X = 0.71073 Â). Corrections to the Lorentz and polarization factors were applied to reflection intensities. The X-ray experimental data were processed using the Rigaku Crystal Clear pro-gram,20 for compounds 1 and 2. The CrysAlisRED program was used for processing the X-ray data for complexes 3 and 4.21 An absorption correction was applied by the analytical method.22 Structures 1-4 were solved using direct methods and light atoms were revealed from the difference Fourier syntheses using the SHELX program package.23 Full-matrix least-squares refinements based on F2 were carried out for the positional and thermal parameters of all non-hydrogen atoms. Four fluorine atoms of SiF62- anion in 2 are split with roughly 50% s.o.f. The hydrogen atoms in structures 1-4 were revealed from the difference Fourier syntheses and refined in the riding model along with the non-hydrogen atoms (fixed C-H distances and with Uiso(H) equal to 1.2Ueq(C)). Hydrogen Luk'yanov et al.: Synthesis and Structure of [Cu(Hapn)]NO3]NO 210 Acta Chim. Slov. 2017, 64, 208-214 Table 1. Crystallographic data and experimental details for structures 1—4 Compound 2 3 Empirical formula Formula weight Temperature (K) Space group Unit cell dimensions (A, °) a b c a P Y Volume (A3), Z Dc (g cm-3) Dm (g cm-3) Absorption coefficient (mm ') F(000) Measured reflections Independent reflections Observed reflections [I > 2o(I)] Goodness-of-fit on F2 Parameters refined Final R indices [I > 2o(/)] C6HnCuN4O6 298.73 200(2) P21/n 8.2341(4) 7.9905(3) 17.4008(8) 90 98.959(2) 90 1130.91(9), 4 1.755 1.73 1.96 608 4673 2552 2163 1.09 155 R1 = 0.049, wR2 = 0.134 C6H15CuN2O2F6Si 352.83 200(2) P21/c 8.5929(9) 9.7426(8) 15.2109(16) 90 103.448(4) 90 1238.5(2), 4 1.892 1.88 1.93 712 5091 2795 2542 1.12 204 R1 = 0.052, wR2 = 0.145 C6H15CuN2O2B2F8 384.36 100(2) P21/n 12.351(4) 12.351(4) 13.497(4) 90 97.98(3) 90 1420.9(7), 4 1.797 1.80 1.63 768 14002 4825 3266 0.99 226 R1 = 0.036, wR2 = 0.087 C12H30Cu2N8O12S4 733.76 100(2) PI 8.217(2) 9.018(3) 17.439(5) 91.93(3) 92.52(3) 90.21(3) 1290.3(6), 2 1.889 1.88 2.05 752 11271 8361 6055 1.00 343 R1 = 0.032, wR2 = 0.073 1 4 atoms of amino group and water were refined freely. The figures were prepared using DIAMOND 3.1 software.24 3. Results and Discussion Analysis of the obtained new Cu(I) n-complexes proves that the type of anion influences strongly on a structure formation in these complexes.25'26 3. 1. Crystal Structure of [Cu(Hapn)NO3]NO3 Complex (1) Complex [Cu(Hapn)NO3]NO3 (1) is formed with anion NO3— which is structurally related to halogenide ones. In this compound due to bridged functions of both Hapn and NO3— units the known [Cu(NO3)]2 inorganic fragments27-30 are interconnected with organic cations Hapn forming goffer chains in the direction [111] (Fig. 1). The Figure 1. Infinite chains and hydrogen bonding in complex 1. Symmetry operations: (i) x + /2, -y + /2, z + '/2; (ii) —x + 2, -y, -z + 2. Luk'yanov et al.: Synthesis and Structure of [Cu(Hapn)]NO3]NO3 Acta Chim. Slov. 2017, 64, 208-214 211 angle between planes passing through two neighbouring inorganic linkers [Cu(NO3)]2 of the polymer is 67°. For comparison, the analogous angle between planes passing through two neighbouring inorganic units [CuCl]2 in the halide complex is 65°.16 The metal atom possesses a tetrahedral surrounding consisting of the middle (further m) of double C(5)=C(6)-bond, N (C^N-group) and 2 oxygen atoms from two NO3- anions. Lengths of the bonds are Cu-m 1.933(3), Cu-N 1.939(3), Cu-O(1) 2.102(2) and Cu-O(1)" 2.202(2) A (Table 2). The angle formed by three Cu atoms in the chain [Cu(Hapn)2+]n equals to 152°. The chain is not straight because of an influence of the non-coordinated NO3- anion, which forms N-H—O hydrogen bonds (Table 3).31 Table 2. Selected bond distances and angles for 1-4. Distance (A) Angle (°) 1a Cu-N1 1.939(3) Cu-mi 1.933(3) Cu-O1 2.102(2) Cu-O1ii 2.202(2) N1-C1 1.129(4) C5-C6 1.361(4) 2b Cu-N1i 1.987(3) Cu-O1w 2.003(3) Cu-m 1.936(3) Cu-O2w 2.239(3) C5-C6 1.363(5) N1-C1 1.132(5) 3 Cu-N1 1.946(2) Cu-O1w 1.992(2) Cu-mi 1.893(5) N1-C1 1.132(2) C5-C6 1.364(3) 4 Cu1-N11i 1.990(2) N11i-Cu1-N1 106.2(7) Cu1-N1 2.061(2) N11i-Cu1-N2 92.2(7) Cu1-m1 1.977(7) N1-Cu1-N2 99.2(7) Cu1-N2 2.275(9) N11i-Cu1-m1 117.4(6) C16-C15 1.352(3) N2-Cu1-m1 110.3(5) C11-N11 1.134(3) C11-N11-Cu1i 176.1(8) Cu2-N3 2.039(8) N3-Cu2-N4 104.6(7) Cu2-N4 2.143(9) N3-Cu2-N21ii 96.6(7) Cu2-m2 1.971(7) N21ii-Cu2-N4 93.9(8) Cu2-N21ii 2.130(2) N4-Cu2-m2 111.1(5) C26-C25 1.352(3) N3-Cu2-m2 130.5(6) C21-N21 1.138(3) C21-N21-Cu2ii 165.8(8) m - middle point of C5=C6 (in 4: C15=C16 and C25=C26) double bond. Symmetry codes: " (i) x + /, -y + /, z + /; (ii) -x + 2, -y, -z + 2; b (i) -x + 1, y - /, -z - /; (ii) -x + 1, y + /, -z - /; c (i) -x + /, y - /, -z + /; (iii) - x + 1.5, y - /, -z + /; d (i) -x + 1, -y + 1, -z; (ii) -x + 2, -y, -z + 1. Table 3. Geometry of selected hydrogen bonds in 1-4. Atoms involved Distances, A Angle, deg D-H-A D-H H-A D-A D-H-A 1a N2-H1N-O5i 0.90 2.48 3.116(4) 128 N2-H1N-O6i 0.90 1.94 2.798(2) 159 N2-H2N-O5 0.90 1.94 2.807(4) 161 2b N2-H1N-F1 0.90 1.89 2.784(8) 174 O1w-H2w1-F2i 0.97 1.70 2.661(1) 170 N2-H2N-F2ii 0.90 1.95 2.812(5) 160 O2w-H2w2-F1 0.96 2.39 3.259(6) 151 3c O1w-H1w1-F4i N2-H1N-O2w" O1w-H2w1-F5iii N2-H2N-F1 O2w-H1w2-F7iv O2w-H2w2-F8 4d N1-H1B-O2T 0.92 2.02 2.919(1) 165 N2-H2A-O31 0.92 1.93 2.820(8) 162 N2-H2B-O12" 0.92 2.07 2.992(1) 176 N3-H3B-O43iii 0.92 2.01 2.925(2) 173 N22-H2N2-O42iv 0.92 1.81 2.730(3) 178 N4-H4B-O32ii 0.92 2.07 2.992(6) 177 Symmetry codes: a (i) /2 - x, y - /2, /2 - z; b (i) —x, 1 - y, —z; (ii) 1 -x, y - /, -z; c (i) -x, -y, 1 - z; (ii) x - /, / - y, z- /; (iii) x - 1, y, z; (iv) 1.5 - x, / + y, / - z; d (i) 2 - x, -y, -z; (ii) x - 1, y, z; (iii) 1 -x, 1 - y, 1 - z; (iv) -x, 1 - y, 1 - z. 3. 2. Crystal Structure of [Cu(Hapn)(H2O)2] SiF6 Complex (2) In following two complexes 2 and 3 water molecules act as co-ligands. The structure of the compound 2 consists of infinite metal-organic spiral-like ribbons of [Cu(Hapn)(H2O)2]2+ composition. The angle between three neighbouring copper atoms is 63°. Located between mentioned ribbons SiF62- anions are bound to metal-organic fragment via O-H -F and N-H -F hydrogen bonds (Fig. 2). Despite the existence of Cu1 complexes with hexaflourosilicate-anion with the direct CuI-F-Si-F5 bond,32 SiF62--anion does not enter the internal coordination sphere of the metal. Tetrahedral coordination polyhedron of copper(I) ion is formed by a mid-point of C(5) = C(6) bond, one nitrogen (C=N) and two O (H2O molecules) atoms. Respective bond lengths are Cu-m 1.936(3), Cu-N(1)' 1.987(3), Cu-O(1w) 2.003(3) and Cu-O(2w) 2.239(3)  (Table 2). A system of hydrogen bonds is much more developed in the given complex (table 2) in comparison with 1. This promotes relatively dense packing of metal-organic chains and the inorganic anions. C6i-Cu-C5i 38.8(2) N1-Cu-O1 104.8(2) N1-C1-C2 177.1(4) N1-Cu-O1ii 101.0(2) O1-Cu-O1ii 71.9(1) mi-Cu-N1 132.6(1) N1i-Cu-O1w 107.1(2) N1i-Cu-m 118.5(9) N1i-Cu-O2w 95.9(2) O1w-Cu-O2w 93.7(1) C1-N1-Cuii 162.9(3) N1-C1-C2 178.4(4) N1-Cu-O1w 99.7(7) O1w-Cu-mi 129.1(5) C1-N1-Cu 169.7(8) N1-C1-C2 177.7(2) O1w-Cu-F8iii 89.1(6) 0.73 1.99 2.710(6) 169 0.86 1.88 2.725(2) 168 0.72 2.01 2.716(1) 169 0.96 1.96 2.755(1) 139 0.92 1.95 2.834(9) 160 0.84 2.09 2.827(3) 146 Luk'yanov et al.: Synthesis and Structure of [Cu(Hapn)]NO3]NO 212 Acta Chim. Slov. 2017, 64, 208-214 3. 3. Crystal Structure of [Cu(Hapn)(H2O) BF4]BF4 • H2O Complex (3) In the complex 3 water molecules and BF4- anions (apart from the active centers of Hapn) are included in the internal coordination sphere of Cu. The presence of BF4- anions promotes transformation of coordination polyhedron of the metal from tetrahedron to trigonal pyramid formed by m of (C=C)-bond, N (C^N-group) and O (H2O) atoms in the basal plane. Fluorine atom Figure 3. Cu(I) coordination in 3. Structure fragment of complex 3. Symmetry operations: (i) —x + /2, y - '/2, -z + '/2; (ii) -x+ 1, - y + 1, -z; (iii) - x + 1.5, y - /2, -z + /2. (80% probability displacement ellipsoids). Luk'yanov et al.: Synthesis and Structure of [Cu(Hapn)]NO3]NO3 Acta Chim. Slov. 2017, 64, 208-214 213 from BF4- anion occupies the apical position (Cu-F(8)"' 2.640(2) A) of coordination polyhedron. Atom of Cu is somewhat (A = 0.03 A) removed from the (m, N, O) plane. Another crystallographically independent H2O molecule and BF4-anion are not coordinated to copper® and fixed in a crystal space by relatively strong hydrogen bonds. As one can see from Figure 3, the structure 3 is similar to 2, but separate fragments of coordination polymer due to the Hapn flexibility demonstrate bulbous chain structure (the angle between three atoms of Cu [Cu(Hapn)2+]n equals to 59°). Since one distance of Cu-O(1w) equals to 1.992(2) A, and the other opposite Cu-O(1w)" is equal to 2.900(2) A, one may regard from a certain distance (Cu(H2O))2 moiety as dimeric fragment and Cu(I) polyhedron as a trigonal bipyramid. 4. Conclusions Flexibility of Hapn allows using it as a convenient tool in a construction of coordination compounds. In all the above-mentioned compounds Hapn totally realizes its coordination abilities attaching to the metal atom with (C=C)-bond of allyl- and N atom of cyano-group. The protonated N-amine atom being deprived of its donor ability participates actively in a formation of strong N-H-X hydrogen bonds (Table 2). On the other hand, the combination of Hapn with ionic copper(I) salts (CuNO3, Cu2Si-F6, CuBF4, CuSO3NH2) promotes an effective interaction of both n- and o-ligands with the central atom, which serve to a formation of stable frameworks. 5. Supplementary Material 3. 4. Crystal Structure of [Cu(Hapn)(NH2SO3)2] Complex (4) As in complexes 1 and 2, in the compound 4 coordination polyhedra for both independent Cu(I) ions possess tetrahedral shape. The Cu(1) environment comprises of the mid-point of C(15)=C(16) bond (m1), N(11) atom from CN-group and two nitrogen atoms from two NH2SO3- anions. The Cu(2) polyhedron involves m2 (C(25) =2 C(326)), N(21) (C=N) and N(3) and N(4) (NH2SO3-) centers. Bonds lengths: Cu(1)-m1 1.977(7), Cu(1)-N(11y 1.990(2), Cu(1)-N(1) 2.061(8) and Cu1-N(2) 2.275(9) A; Cu(2)-m2 1.971(7), Cu(2)-N(21)" 2.130(2), Cu(2)-N(3) 2.039(8) and Cu(2)-N(4) 2.143(9) A. The main structural feature of the complex 4 is the appearance of [Cu(Hapn)]2 rings (Fig. 4). Two closest rings are tilted by 72° and linked with H-bonds among inorganic anions and organic cations (N(2)-H(2A)-O(31) 1.93 A etc. Table. 3). CCDC 913397 (1), 913398 (2), 913399 (3) and 913400 (4) contain the supplementary crystallographic data for this article. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam. ac.uk). 6. References 1. A. Strecker, Ann. Chem. Pharm. 1850, 75, 27-45. https://doi.org/10.1002/jlac.18500750103 2. V. Chhiba, M. L. Bode, K. Mathiba, W. Kwezi, D. Brady, J. Mol. Catal. B: Enz. 2012, 76, 68-74. https://doi.org/10.1016/j.molcatb.2011.12.005 3. D. Enders, J. P. Shilvock, Chem. Soc. Rev. 2000, 29, 359373. https://doi.org/10.1039/a908290e 4. E. Rafiee, A. Azad, M. Joshaghani, Lett. Org. Chem. 2007, 4, Figure 4. Copper(I) coordination in 4 and [Cu(Hapn)]2. Symmetry operation: (i) -x + 1, -y + 1, -z; (80% probability displacement ellipsoids). Luk'yanov et al.: Synthesis and Structure of [Cu(Hapn)]NO3]NO 214 Acta Chim. Slov. 2017, 64, 208-214 60-63. https://doi.org/10.2174/157017807780037478 5. E. C. Taylor, J. G. Berger, J. Org. Chem. 1967, 32, 23762378. https://doi.org/10.1021/jo01283a003 6. S.-S. Tang, P. C. Trackman, H. M. Kagang, J. Biol. Chem. 1983, 258, 4331-4338. 7. G. Cardillo, C. Tomasini, Chem. Soc. Rev. 1996, 25, 117128. https://doi.org/10.1039/CS9962500117 8. M. Preiml, K. Hillmayer, N. Klempier, Tetrahedron Lett. 2003, 44, 5057-5059. https://doi.org/10.1016/S0040-4039(03)01136-5 9. S. G. Davies, O. Ichihara, I. Lenoir, I. A. S. Walters, J. Chem. Soc. Perkin Trans. 1 1994, 11, 1411-1415. https://doi.org/10.1039/P19940001411 10. F. Theil, S. Ballschuh, Tetrahedron Asymmetry 1996, 7, 3565-3572. https://doi.org/10.1016/S0957-4166(96)00465-X 11. D. Steinhuebel, Y. Sun, K. Matsumura, N. Sayo, T. Saito, J. Am. Chem. 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Version 1.171.31.8, Oxford: Oxford Diffraction Ltd. (2007). 22. R. C. Clark, J. S. Reid, Acta Crystallogr, Sect. A: Found. Crystallogr 1995, 51, 887-897. https://doi.org/10.1107/S0108767395007367 23. G. M. Sheldrick, Acta Cryst., Sect. A: Found. Crystallogr. 2008, A64, 112-122. https://doi.org/10.1107/S0108767307043930 24. DIAMOND v3.1. Crystal Impact GbR, Bonn, Germany (2004-2005). 25. E. Goreshnik, M. Mys'kiv, J. Chem. Crystallogr. 2010, 40, 381-383.https://doi.org/10.1007/s10870-009-9666-1 26. M.Y. Luk'yanov, A.V. Pavlyuk, E. A. Goreshnik, M. G. Mys'kiv, Russ. J. Coord. Chem. 2012, 38, 639-645. (Koord. Khimiya 2012, 38, 663-670). 27. S. P. Neo, Z.-Y. Zhou, T. C. W. Mak, T. S. A. Hor, J. Chem. Soc., Dalton Trans. 1994, 23, 3451-3458. https://doi.org/10.1039/DT9940003451 28. R. D. Hart, G. A. Bowmaker, J. D. Kildea, E. N. de Silva, B. W. Skelton, A. H. White, Aust. J. Chem. 1997,50, 604-620. https://doi.org/10.1071/C96041 29. E. A. Goreshnik, M. G. Mys'kiv, Acta. Chim. Slov. 2011, 58, 772-775. 30. Y. Slyvka, E. Goreshnik, N. Pokhodylo, O. Pavlyuk, M. Mys'kiv, Acta. Chim. Slov. 2016, 63, 399-405. https://doi.org/10.17344/acsi.2016.2486 31. G. R. Desiraju, Angew. Chem. Int. Ed. 2011, 50, 52-59. https://doi.org/10.1002/anie.201002960 32. E. A. Goreshnik, Y. I. Slyvka, M. G. Mys'kiv, Inorg. Chim. Acta 2011, 377, 177-180. https://doi.org/10.1016/jica.2011.08.008 Povzetek Pripravili smo štiri bakrove(I) n-komplekse: [Cu(Hapn)NO3]NO3 (1), [Cu(Hapn)(H2O)2]SiF6 (2), [Cu(Hapn) (H2O)BF4]BF4-H2O (3) in [Cu(Hapn)(NH2SO3)2] (4) z uporabo elektrokemijske tehnike z izmenično napetostjo iz alkoholnih raztopin 3-(prop-2-en-1-ilamino)propannitrila (apn) titriranega z ustrezno kislino ter z bakrovo(II) soljo (Cu(NO3)2 • 3H2O, CuSiF6 • 4H2O, Cu(BF4)2 • 6H2O ali Cu(NH2SO3)2 • xH2O). Pripravljene spojine smo okarakterizira-li z monokristalno rentgensko difrakcijo in delno z IR spektroskopijo. Pri strukturah 1, 2 in 4 ima Cu(I) kation te-traedrično razporeditev ligandov, ki nastane z C=C vezjo enega organskega kationa Hapn, N atoma ciano skupine iz drugega Hapn liganda ter dveh O atomom (iz NO3- aniona pri 1, iz H2O molekule pri 2) oziroma N atoma (anion NH2SO3- pri 4). Pri spojini 3 je prisotna trigonala-piramidalna koordinacija Cu(I) s sredinsko točko C=C-vezi enega Hapn kationa, N atoma (iz ciano skupine) drugega Hapn liganda in O atoma molekule H2O v osnovni ravnini ter s F atomom iz BF4- aniona v navpični legi. Luk'yanov et al.: Synthesis and Structure of [Cu(Hapn)]NO3]NO3 DPI: I0.l7344/acsi.20l6.3l32_Acta Chirn. Slov. 2017,64, 215-220_©commons 215 Scientific paper Three 1D cyanide-bridged M(Ni, Pd, Pt)-Mn(II) Coordination Polymer: Synthesis, Crystal Structure and Magnetic Properties Jingwen Shi,1 Chongchong Xue,1 Lingqian Kong2 and Daopeng Zhang1* 1 College of Chemical Engineering, Shandong University of Technology, Zibo 255049, China 2 Dongchang College, Liaocheng University, Liaocheng 252059, P.R. China * Corresponding author: E-mail: dpzhang73@ 126.com Received: 13-12-2016 Abstract Abstract: Three tetracyanide-containing building blocks K2[M(CN)4] (M = Ni, Pd, Pt) and one semi-closed macrocycle seven-coordinated manganese(II) compound have been employed to assemble cyanide-bridged heterometallic complexes, resulting in three cyanide-bridged Mn-Mnn complexes: [Mn(L)][Ni(CN)4] • 2H2O (1) [Mn(L)][Pd(CN)4] (2) and [Mn(L)][Pt(CN)4] (3) (L = 2,6-bis[1-(2-(N-methylamino)ethylimino)ethyl]pyridine). Single-crystal X-ray diffraction analysis shows their similar one-dimensional structure consisting of the alternating [Mn(L)]2+ species and [M(CN)4]2-building blocks, generating a cyanide-bridged neutral polymeric chain. In all three isostructural complexes the coordination geometry of manganese ion is a slightly distorted pentagonal-bipyramidal with the two cyanide nitrogen atoms at the trans positions and N5 coordinating mode at the equatorial plane from ligand L. Investigation over magnetic properties of these complexes reveals very weak antiferromagnetic interaction between neighboring Mn(II) ions bridged by the long NC-M-CN unit. A best-fit to the magnetic susceptibility of complexes 1-3 leads to the magnetic coupling constant of J = -0.081, -0.103 and -0.14 cm-1, respectively. Keywords: Cyanide-bridged, heterometallic complex, crystal structure, magnetic property 1. Introduction In the past several decades, the ultimate goal of crystal engineering is to directional design and construction of molecular crystals with new structures, properties and functions. During which, many effective strategies have been developed to rationally designing and controlling assembly of metal complexes with diversified topological structures and interesting properties. Among the various transition metal coordination systems, the rational design of the cyanide-bridged heterometallic complexes with target structure types have been given intense attention because not only the structures and the nature of the magnetic, optic and electric properties of corresponding complexes can be readily controlled and anticipated, but also the excellent stabilizing ability of cyanide group for many transition metal centers and oxidation states with or without the peripheral ligands.1-23 As has been known, except the several factors from the cyanide precursor such as the number and position of cyanide group, number and nature of charge of cyanide-containing building block, and steric effect of reactants that can be used to tune the structure of the cyanide-bridged complexes formed, the ancillary ligands attached to the counterpart assembling cations also play a crucial role for constructing cyanide-bridged complexes with different structures. The polyaza macrocyclic ligands with some rigid character obtained by condensation of 2,6-dia-cetylpyridine and polyamine, which are usually coordinated to the equatorial plane of metal ions with only two trans replaceable sites weakly bonded to other ligands, have proved to be good ancillary ligands to assemble low-dimensional structural cyanide-bridged complexes. 24-31 Interested also in these types of ligands, we have reported many cyanide-bridged bimetallic complexes by using cyanide precursors containing different cyanide groups.32-36 Here, we investigated the reactions the Mn(II) compound based-on a semi-closed macrocyclic ligand L (L = 2,6-bis[1-(2-(N-methylamino)ethylimi-no)ethyl]pyridine) with three tetra-cyanometallates Shi et al.: Three 1D cyanide-bridged M(Ni, Pd, Pt)-Mn(II) 216 Acta Chim. Slov. 2017, 64, 215-220 Scheme 1. The semi-closed macrocycle ligand and the cyanide precursors used to synthesize the complexes 1-3. (Scheme 1), and obtained three one-dimensional cyanide-bridged heterobimetallic complexes with the formula [Mn(L)][Ni(CN)4] 2H2O (1) [Mn(L)][Pd(CN)4] (2) and [Mn(L)][Pt(CN)4] (3). It should be mentioned that current complexes are the first one-dimensional example assembled from the semi-closed macrocyclic manganese compound. The synthesis, crystal structure and magnetic properties of all the three complexes are described in this paper. solved in 5 mL of water was laid in the bottom of a tube, upon which a mixture solvent of water and methanol with a ratio of 1:1 was carefully added. Then, a solution of [Mn(L)]Cl2 (0.10 mmol, 40.1 g) in 5 mL of methanol was carefully added to the top of the mixture solvent layer above formed. About two weeks later, single yellow crystals suitable for X-ray diffraction were obtained from the interface, collected by filtration and dried in air. Yield: 35.5 mg, 67.1%. Anal. Calcd. for C19H29MnN9NiO2: C, 43.13; H, 5.52; N, 23.82. Found: C, 43.01; H, 5.45; N, 24.01. Main IR bands (cm 1): 3255(s), 2915(m), 2845(m), 2153(s), 2125(s), 1650(m), 1594(m), 1455(m), 1370(m), 1190(m), 965(m). Complex 2: Yield: 30.8 mg, 57.1%. Anal. Calcd. for C19H25MnN9Pd: C, 42.20; H, 4.66; N, 23.31. Found: C, 42J0; H, 4.61; N, 23.15. Main IR bands (cm1): 3257(s), 2910(m), 2850(m), 2150(s), 2120(s), 1652(m), 1590(m), 1458(m), 1377(m), 1197(m), 961(m). Complex 3: Yield: 40.1 mg, 63.6%. Anal. Calcd. for C19H25MnN9Pt: C, 36.25; H, 4.00; N, 20.02. Found: C, 3(5-14; H, 3.85; N, 19.81. Main IR bands (cm1): 3260(s), 2900(m), 2853(m), 2155 (s), 2123(s), 1658(m), 1597(m), 1452(m), 1375(m), 1191(m), 959(m). 2. Experimental Section 2. 1. Instruments Elemental analyses of carbon, hydrogen, and nitrogen were carried out with an Elementary Vario El. The infrared spectroscopy on KBr pellets was performed on a Magna-IR 750 spectrophotometer in the 4000-400 cm1 region. Variable-temperature magnetic susceptibility and field dependence magnetization measurements were performed on a Quantum Design MPMS SQUID magnetometer. The experimental susceptibilities were corrected for the diamagnetism of the constituent atoms (Pascal's tables). 2. 2. General Procedures and Materials All the reactions were carried out under an air atmosphere and all chemicals and solvents used were reagent grade without further purification. The [Mn(L)(H2O)2]Cl2 were prepared by using the reported method for similar manganese macrocycle complex.33 Caution! The cyanide compounds are hypertoxic and hazardous and they should be handled in small quantities with care. 2. 4. X-ray Data Collection and Structure Refinement Single crystals of all complexes for X-ray diffraction analysis with suitable dimensions were mounted on the glass rod and the crystal data were collected on a Bruker SMART CCD diffractometer with a MoKa sealed tube (X = 0.71073 Â) at 293 K using a œ scan mode. The structures were solved by direct method and expanded using Fourier difference techniques with the SHELXTL-97 program package. All the non-hydrogen atoms were refined with anisotropic displacement coefficients. Hydrogen atoms were assigned isotropic displacement coefficients U(H) = 1.2U(C) or 1.5U(C) and their coordinates were allowed to ride on their respective carbons using SHELXL97 except some of the H atoms of the solvent molecules that were refined isotro-pically with fixed U values and the DFIX command was used to rationalize the bond parameter. CCDC 1521398-1521400 for these three complexes contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via . Details of the crystal parameters, data collection, and refinement are summarized in Table 1. 2. 3. Preparation of Complexes 1-3 These three complexes were prepared using one similar three layers diffusion procedure, therefore only the synthesis of 1 is reported as a typical representative. A solution containing K2[Ni(CN)4] (0.10 mmol, 24.1 mg) dis- 3. Results and Discussion 3. 1. Synthesis and General Characterization As has been known, the closed macrocycle ligand 3,6-diazaoctane-1,8-diamine and 3,6-dioxaoctano-1,8- Shi et al.: Three 1D cyanide-bridged M(Ni, Pd, Pt)-Mn(II) ... Acta Chim. Slov. 2017, 64, 215-220 217 Table 1. Crystallographic data for complexes 1-3. 1 2 3 Formula C19H29MnN9NiO2 C19H25MnN9Pd C19H25MnN9Pt M 5929.16 540.82 19 62259.51 9 Crystal system Monoclinic Monoclinic Monoclinic Space group P2(1)/c C2/c C2/c a/A 18.1273(5) 11.3312(7) 11.3015(6) b/A 16.7829(5) 11.2415(6) 11.2472(6) c/A 7.7459(2) 17.4718(9) 17.4485(8) a/° 90 90 90 !b/° 93.440(3) 91.187(6) 91.305(4) Y° 90 90 90 v/A3 2352.28(11) 2225.1(2) 2217.3(2) Z 4 4 4 F(000) 1100 1092 1220 GOF 1.038 1.049 0.995 R1[I>2a(I)] 0.0350 0.0303 0.0332 wR2 (all data) 0.0834 0.0751 0.0701 diamine are good auxiliary ligands for assembling cyanide-bridged magnetic complexes by incorporating some paramagnetic metal ions such as Mn(II), Fe(II) and Co(II), etc.24-31 With comparison to the above two ma-crocyclic ligands, the semi-closed pentadentate macrocycles ligand used here (Scheme 1) may have more flexibility due to its semi-open nature and the two pendulous methyl groups, which is maybe beneficial to produce single axial magnetic anisotropy for paramagnetic metal ions. As has been known, the Mn(II) ion in some complexes based-on aliphatic amines ligands can be easily oxidized to Mn(III) ion. However, the seven-coordinated Mn(II) species obtained by incorporating Mn(II) ion into these types of macrocyclic ligands are very robust and can be handled in air and in aqueous solution without being oxidized. Furthermore, the large equatorial steric effect from the macrocyclic ligand can effectively lower the dimensionality of the complexes formed, thus far more favoring of constructing functional complexes with low dimensional structure through replacing the two weakly bonded and replaceable ligands at the two trans positions. The reactions between the manganese(II) compound with the semi-closed macrocycle acting as auxiliary ligand and three tetra-cyanidemetallates result in three isostructural one-dimensional cyanide-bridged complexes. In the IR spectra of complexes 1-3 two sharp peaks due to the cyanide-stretching vibration were observed at about 2120 and 2150 cm-1, respectively, indicating the presence of bridging and nonbridging cyanide ligands in these complexes. 3. 2. Crystal Structures of Complexes 1-3. Some important structural parameters for complexes 1 -3 are collected in Table 2. The neutral binuclear independent unit, one-dimensional structure and the cell pac- king diagram of compound 1 are shown in Figures 1-3, respectively, and the other compounds show similar structures. The calculated and measured partner of XPRD data for these three complexes is given in Figures S1-S3 (Supporting Information), respectively. As can be found, complexes 1-3 possess similar one dimensional neutral single chain structure comprising of repeating [-NC-M(CN)2-CN-Mn(L)-] (M = Ni, Pd, Pt) unit. In these three complexes, each [M(CN)4]2- unit, acting as a bidentate ligand through it's a pair of trans cyanide groups, connects the Mn(II) ion of two independent semi-closed macrocyclic manganese units. The structure of these three complexes is very similar to the reported 1D linear chain complex {[Mn(L1)][Fe(1-MeIm)(CN)5]}n, but different from {[Mn(L1)(H2O)][Mn(L1)][Fe(CN)6]}n ■ n(CH4O)3.5nH2O and {[]Mn(L1)(H2O)][Mn(L1)][iM' (CN)8]}n ■ 4nH2O,36 for the latter which can be structurally characterized as one-dimensional zig-zag chain structure. (L1 = 2,13-dimethyl-3,6,9,12,18-pentaazabicyclo[12.3.1] octadeca-1(18),2,12,14,16-pentaene), M' = Mo, W). The M-Cbridged-CN bond lengths and the M-C=Nbridge bond angles are almost equal to those corresponding parameters found in other non-bridged cyanide groups, demonstrating that the coordination or non-coordination of the N atom to the metal atom has no obvious influence on the geometry of the cyanide precursor. The Mn(II) ion in complexes 1-3 is seven-coordinated forming a slightly distorted pentagonal-bipyramidal coordination geometry in which the five equatorial positions are occupied by N5 unit coming from the semi-closed macrocyclic ligand and the two axial ones coordinated by two N atoms of cyanide groups. The distances between Mn ion and the equatorial N atoms in complexes 1-3 are almost equal to each other within the very narrow range 2.322(5)-2.383(2)  (Table 2). The average Mn-Ncyanide bond lengths in all these complexes are 2.257(2), 2.236(3) Shi et al.: Three 1D cyanide-bridged M(Ni, Pd, Pt)-Mn(II) ... 218 Acta Chim. Slov. 2017, 64, 215-220 Figure 1. The representative neutral binuclear independent unit of complex 1. All hydrogen atoms and solvent molecules have been omitted for clarity. and 2.225(5) Â, respectively, slightly shorter than the Mn-Nequatorial bond lengths. As tabulated in Table 2, the bond angle of N1-Mn1-N2 are 176.74(9), 177.42(16) and 177.6(3)°, respectively, indicating the good linear configuration of these three atoms. However, the Mn-C^N bond angle is somewhat bent with the values about 155°. The intramolecular Mn-Mn separation through the dia-magnetic bridging cyanide precursor in 1-3 is 9.926, 10.476 and 10.450 Â, respectively. 3. 3. Magnetic Properties of Complexes 1-3. The temperature dependence of magnetic susceptibility for complex 1 measured in the range of 2-300 K under the external magnetic field of 2000 Oe is showed in Fig. 4. For complexes 2 and 3 their temperature dependen- Figure 2. The representative 1D structure of complex 1. All hydrogen atoms and solvent molecules have been omitted for clarity. Figure 3. The cell packing diagram along b for complex 1. All the non-solvent hydrogen atoms have been omitted for clarity. Shi et al.: Three 1D cyanide-bridged M(Ni, Pd, Pt)-Mn(II) ... Acta Chim. Slov. 2017, 64, 215-220 219 Table 2. Selected bond lengths (Â) and angles (°) for 1-3. 1 (M = Ni) 2 (M = Pd) 3 (M = Pt) Mn(1)—N(1) 2.257(2) 2.236(3) 2.225(5) Mn(1)—N(2) 2.359(2) 2.322(5) 2.367(5) Mn(1)—N(3) 2.366(2) 2.338(3) 2.342(6) Mn(1)—N(4) 2.383(2) 2.378(3) 2.325(8) M(1)—C(1) 1.871(3) 1.989(4) 2.007(6) M(1)—C(2) 1.858(3) 1.996(5) 1.999(8) C(1)—N(1)—Mn(1) 156.4(2) 155.2(3) 154.6(5) N(1)—C(1)—M(1) 178.0(3) 177.6(3) 178.3(6) N(1)i—Mn(1)—N(1) 176.74(9) 177.42(16) 177.6(3) Symmetry code: (i) —x + 3/2, —y + 3/2, -z + 1. ce of magnetic susceptibilities is given in Figure S4 (Supporting Information). The changing tendency of xmT for these three complexes is comparatively similar. The xmT value at room temperature is 4.31, 4.30 and 4.29 emu K mol-1 for complexes 1-3, respectively, slightly lower than the spin only value of 4.375 emu K mol-1 for the isolated high spin Mn(II) (S = 5/2). With the temperature decreasing, the xmT value is with no obvious change from 300 to about 50 K. Below this temperature the xmT begins to decrease rapidly and reaches their lowest value of 1.73 for 1, 1.85 for 2 and 2.72 for 3 at 2 K, respectively. The magnetic susceptibility for these three complexes conforms well to Curie-Weiss law in a range of 2-300 K (the inset of Fig. 4) and gives the negative Weiss constant 6 = -3.38 K and Curie constant C = 4.17 emu K mol1 for 1, 6= -4.75, C = 4.20 emu K mol-1 for 2 and 6 = -1.16, C = 3.99 emu K mol-1 for 3. These results primarily show the antiferro-magnetic magnetic coupling between the two Mn(II) centers bridged by [-NC-M-CN-] unit in these three complexes. The magnetic data are analyzed by using the Hamil-tonian: Hit = -2£/SjSi+1. The temperature dependence of the magnetic susceptibility is given by the equation:37,38 Figure 4. Temperature dependences of XmT-T (the solid line represents the best fit based on the parameters discussed in the text) for complex 1. Inset: Temperature dependences X„-1-T (the solid line was calculated from the Curie-Weiss law). found in the reported complexes assembled from the closed macrocyclic manganese compounds and other dia- magnetic cyanometallates. chain AM Ng-pz{S„AS- + 0/3*7-} if + A)} (1) 4. Conclusion (Fisher's infinite chain model) with: /.i = coth[JSH&' +1)/ KT]-[KTf JSu,(Su,,+1)] (2) The least-squares fit to the data leads to J = -0.081 cm-1, g = 1.99, R = 1.19 ■ 10-5 for 1, J = -0.103 cm-1, g = 1.99, R = 1.23 ■ 10-5 for 2 and J = -0.14 cm-1, g = 1.98, R = 2.12 ■ 10-5 for 3, respectively. These results reveal also the antiferromagnetic coupling between adjacent manganese ion bridged by the cyanide precursor and the small J value can be attributed to the long distance separated by the diamagnetic bridging unit. Both of the thermal magnetic behavior and the theoretical simulation results of the above three complexes are basically consistent with those In summary, three new cyanide-bridged M(II)-Mn(II) (M = Ni, Pd, Pt) complexes structurally characterized as one-dimensional single chain have been synthesized with tetracyanide-containing precursor K2[M(CN)4] as building blocks and semi-closed macrocycle ligand based manganese(II) compound as assemble segment. The magnetic studies demonstrate the weak antiferromagnetic interaction between the Mn(II) ions through [-NC-M-CN-] unit in all the three complexes. The current results and those reported recently28,29 indicate that the semi-closed macrocycle mangaese(II) compound employed here is good candidate for assembling cyanide-bridged heterometallic complexes with low dimensional structures and sometime interesting magnetic properties. Shi et al.: Three 1D cyanide-bridged M(Ni, Pd, Pt)-Mn(II) ... 220 Acta Chim. Slov. 2017, 64, 215-220 5. Acknowledgement This work was supported by the Natural Science Foundation of China (21171107 and 21671121). 6. References: 1. S. Ferlay, T. R. Mallah, P. Ouahès, M. V. Veillet, Nature 1995, 378, 701-703. 2. W. R. Entley, G. S. Girolami, Science 1995, 268, 397-400. 3. J. N. Rebilly, T. Mallah, Struct. Bond. 2006, 122, 103-131. 4. R. Lescouezec, L. M. Toma, J. Vaissermann, M. Verdaguer, F. S. Delgado, C. Ruiz-Perez, F. Lloret, M. Julve, Coord. Chem. Rev. 2005, 249, 2691-2729. 5. H. Miyasaka, A. Saitoh, S. Abe, Coord. Chem. Rev. 2007, 251, 2622-2664 and references therein. 6. O. Sato, T. Kawakami, M. Kimura, S. Hishiya, S. Kubo, Y. Einaga, J. Am. Chem. Soc. 2004, 126, 13176-13177. 7. L. M. Toma, R. Lescouëzec, L. D. Toma, F. Lloret, M. Julve, J. Vaissermann, M. J. Andruh, J. Chem. Soc., Dalton Trans. 2002, 3171-3176. 8. L. M. C. Beltran, J. R. Long, Acc. Chem. Res. 2005, 38, 325-334. 9. S. S. Kaye, J. R. Long, J. Am. Chem. Soc. 2005, 127, 65066507. 10. K. W. Chapman, P. D. Southon, C. L. 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Ababei, C. Pichon, O. Roubeau, Y. G. Li, N. Breifuel, L. Buisson, P. Guionneau, C. Mathoniežre, R. Cleirac, J. Am. Chem. Soc. 2013, 135, 14840-14853. 22. D. P. Zhang, S. P. Zhuo, H. Y. Zhang, P. Wang, J. Z. Jiang, Dalton Trans. 2015, 44, 4655-4664. 23. D. P. Zhang, L. Q. Kong, H. Y. Zhang, Acta. Chim. Slov. 2015, 62, 219-224. 24. M. Mousavi, V. Beireau, C. Desplanches, C. Duhayonab, J. P. Sutter, Chem. Commun. 2010, 46, 7519-7521. 25. T. S. Venkatakrishnan, S. Sahoo, N. Breifuel, C. Duhayon, C. Paulsen, A. L. Barra, S. Ramasesha, J. P. Sutter, J. Am. Chem. Soc. 2010, 132, 6047-6056. 26. C. Paraschiv, M. Andruh, Y. Journaux, Z. ZaCk, N. Kyritsa-kasd, L. Ricard, J. Mater. Chem. 2006, 16, 2660-2668. 27. G. Rombaut, S. Golhen, L. Ouahab, C. Mathonière, O. Kahn, J. Chem. Soc, Dalton Trans. 2000, 3609-3614. 28. K. Qian, X. C. Huang, C. Zhou, X. Z. You, X. Y. Wang, K. R. Dunbar, J. Am. Chem. Soc. 2013, 135, 13302-13305. 29. S. L. Zhang, X. H. Zhao, X. Y. 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Povzetek Tri strukturne motive s štirimi ciano skupinami K2[M(CN)4] (M = Ni, Pd, Pt) in manganovo(II) spojino s koordinacijskim številom sedem, ki vsebuje polzaprti makrociklični ligand, smo uporabili za pripravo mostovnih ciano heteroko-vinskih kompleksov in tako pripravili tri Mn-Mnn komplekse s mostovno ciano skupino: [Mn(L)][Ni(CN)4] • 2H2O (1) [Mn(L)][Pd(CN)4] (2) in [Mn(L)][Pt(CN)4] (3) (L = 2,6-bis[1-(2-(N-metilamino)etilimino)etil]piridin). Monokristalna rentgenska strukturna analiza razkrije podobno enodimenzionalno strukturo pri vseh treh spojinah zgrajeno iz izmeničnih [Mn(L)]2+ in [M(CN)4]2- strukturnih motivov, ki so povezani preko ciano mostov. Pri vseh treh izostrukturnih kompleksih je koordinacijska geometrija manganovega iona v obliki rahlo popačene pentagonalne bipiramide z dvema ciano dušikovima atomoma v trans položaju in z N5 koordinacijo liganda L v ekvatorialni legi. Raziskave magnetnih lastnosti teh kompleksov so razkrile zelo šibko antiferomagnetno interakcijo med sosednjimi Mn(II) ioni, ki so povezani preko daljših NC-M-CN enot. Na podlagi magnetne susceptibilnosti smo določili magnetne sklopitvene konstante za komplekse 1-3, ki so J = -0.081, -0.103 in -0.14 cm-1. Shi et al.: Three 1D cyanide-bridged M(Ni, Pd, Pt)-Mn(II) ... DPI: 10.l7344/acsi.20l7.3207_Acta Chirn. Slov. 2017,64, 221-226_©commons 22\ Scientific paper Phase Equilibria and some Properties of Solid Solutions in The Tl5Te3-Tl9SbTe6-Tl9GdTe6 System Samira Zakir Imamaliyeva,*'1 Turan Mirzaly Gasanly,2 Vagif Akber Gasymov1 and Mahammad Baba Babanly1 1 Institute of Catalysis and Inorganic Chemistry named after acad.M.Nagiyev, Azerbaijan National Academy of Sciences, H.Javid ave., 131, Az-1143, Baku, Azerbaijan 2 Baku State University, Z.Khalilov str., 23, Az-1148, Baku, Azerbaijan * Corresponding author: E-mail: samira9597a@gmail.com Received: 15-01-2017 Abstract Phase equilibria in the Tl5Te3-Tl9SbTe6-Tl9GdTe6 system were experimentally studied by thermal analysis, X-ray diffraction and microhardness measurements applied to equilibrated alloys. Some isopleth sections, isothermal section at 760 K, and also projections of the liquidus and solidus surfaces, were constructed. A continuous series of solid solutions was found in this system. Solid solutions crystallize in the tetragonal Tl5Te3 structure type. Keywords: Thallium-antimony tellurides; thallium-gadolinium tellurides; phase equilibria; projections of the liquidus and solidus; solid solutions; crystal structure 1. Introduction A number of works have illustrated the continuing interests in new multinary chalcogenides of heavy p-ele-ments, including rare earth elements. Due to their important functional properties, they find applications in a wide range of devices such as ion-selective sensors, microbatteries, modern day solar cells, and thermoelectric energy conversion.1-3 Moreover; some of them have attracted interest as topological insulators.4'5 Thallium subtelluride, Tl5Te3, thanks to features of crystal structure (Sp.gr.I4/mcm, a = 8.930; c = 12.598 Â) has a number of ternary derivatives of Tl4AIVTe3 and Tl9BVTe6-type (AIV-Sn, Pb; BV-Sb, Bi).6-9 These compounds exhibit good thermoelectric properties, and Tl9Bi-Te6 has reported a ZT ~1.2 at 500 K.10-12 Furthermore, the Dirac-like surface states were observed in [Tl4]TlTe3 (Tl5Te3) and its tin-doped derivative [Tl4](Tl1-xSnx)Te3.13 The new ternary compounds of Tl9LnTe6- type (Ln-Ce, Nd, Sm, Gd, Tm, Tb) which are a new of substitution derivatives of Tl5Te3 were reported in some wo-krs.14-16 Later, H.Kleinke and co-workers have reported the crystal structure as well as magnetic and thermoelectric properties for a number of Tl9LnTe6-type com-pounds.17-19 Further studies of phase equilibria in the systems including the Tl5Te3 compound or its structural analogs showed that these systems are characterized by the formation of unlimited solid solutions.20-22 This study reports a detailed investigation of phase equilibria in the Tl5Te3-Tl9SbTe6-Tl9GdTe6 system. Tl5Te3 and Tl9SbTe6 melt congruently at 723 and 790 K while Tl9GdTe6 melts with decomposition by the peri- tectic reaction at 800 K.7 The lattice parameters of Tl9SbTe6 and Tl9GdTe6 are following: a = 8.829, c = 13.001 Â, z = 2; a = 8.870; c = 13.027 Â, z = 2.24, 25 The Tl5Te3-Tl9SbTe6 system is characterized by the formation of continuous solid solutions areas based on Tl5Te3.7 2. Experimental 2. 1. Materials and Syntheses For the synthesis, we used the high purity thallium, antimony, gadolinium, and tellurium (the purity of the ingredient was better than 99.99 mass. %). The surface of thallium was coated by a thin oxide film, which was removed before use. Imamaliyeva et al.: Phase Equilibria and some Properties of Solid Solutions 222 Acta Chim. Slov. 2016, 64, 221-226 It should be noted that, thallium and its compounds are extremely toxic, and should be handled with great care. Thallium is readily absorbed through the skin and care should be taken to avoid this route of exposure. Therefore, we used protective gloves at all times when working with thallium. However, no respiratory tract covers are required since thallium is not volatile. The elements were weighed to be about 10 g in total according to the molar ratio of the corresponding binary and ternary compound, were placed in silica tubes of about 20 cm in length and then were sealed under a vacuum of 10-2 Pa. Taking into account the congruent melting of Tl5Te3 and Tl9SbTe6, their synthesis was carried out by heating of elements in one zone electric furnace at the 750 and 830 K, respectively followed by cooling in the switched-off furnace. The obtained intermediate ingot of Tl9GdTe6 was carefully ground in an agate mortar, pressed into the circular pellet of about 10 mm diameter and annealed at 770 K within ~1000 h as it was done in previous work.24 The weight losses during the pellet preparation were less than 0.5 mass. %. In order to prevent a reaction between the gadolinium and the quartz during high temperature reactions, quartz tubes coated internally with a thin layer of carbon were used. The purity of the synthesized compounds was checked by the X-ray diffraction (XRD) and differential thermal analysis (DTA). Only one thermal effect was observed for Tl5Te3 (723 K) and Tl9SbTe6 (790 K); whereas two peaks for Tl9GdTe6 which were relevant the peritectic reaction at 800 K and its liquidus at 1190 K. These data are in good agreement with the literature references.7,23,24 XRD confirmed that synthesized compounds were phase-pure. Powder XRD pattern of the Tl9SbTe6 and Tl9GdTe6 were similar to that of Tl5Te3. The unit cell parameters were practically equal to literature data (Table 1).24,25 Synthesized binary and ternary compounds were used for the fabrication of the alloys of the Tl5Te3-Tl9Sb-Te6-Tl9GdTe6 system. The alloys weighing 1 g were synthesized in quartz tube evacuated to 10-2 Pa. Taking into account the fact that an equilibrium state could not be obtained even after the long-time (1000 h) annealing, after synthesis the samples containing more than 60 mol% Tl9GdTe6 were powdered, mixed, pressed into circular pellets of about 10 mm diameter and annealed at 700 K for 1 month. 2. 2. Methods X-ray powder diffraction (XRD), differential thermal analysis (DTA) and also microhardness measurements were employed to check the purity of the synthesized starting compounds and analyze the samples in order to plot the phase diagrams. DTA was performed using a NETZSCH 404 F1 Pegasus differential scanning calorimeter within room temperature and ~1400 K depending on the composition of the alloys at a heating rate of 10 K min-1 and accuracy about ±3°. Temperatures of thermal effects were taken mainly from the heating curves. The XRD measurements of the powdered specimen were recorded using a Bruker D8 diffractometer utilizing CuKaradiation within 28 = 10 - 70°. The unit cell parameters were calculated by indexing of powder patterns using Topas V3.0 software. An accuracy of the crystal lattice parameters is shown in parentheses (Table). Microhardness measurements were done with a mi-crohardnesmeter PMT-3, the typical loading being 20 g and accuracy about 20 MPa. 3. Results and Discussion The combined analysis of obtained experimental and literature data [7, 24, 25] allowed us to construct the diagram of the phase equilibria in the Tl5Te3-Tl9SbTe6-Tl9GdTe6 system (Table, Fig.1-6). The 2Tl5Te3-Tl9SbTe6 system is quasi-binary and characterized by the formation of unlimited solid solutions (5) with Tl5Te3-structure.7 The 2Tl5Te3-Tl9GdTe6 and Tl9SbTe6-Tl9GdTe6 systems (Table 1, Figs. 1a, 2a) are characterized by the formation of continuous solid solutions (5) with Tl5Te3-structure. However, they are non-quasi-binary sections of the Tl-Gd-Te ternary and Tl-Sb-Gd-Te quaternary systems due to the peritectic melting of the Tl9GdTe6 compound. This leads to crystallization infusible X phase in a wide composition interval and formation two-phase L + X and three-phase L + X + 5 areas. These areas are not experimentally fixed due to narrow temperature interval and shown by dotted line. We have assumed that the X phase has a composition TlGdTe2. This assumption is confirmed by the presence of the most intense reflection peaks of TlGdTe2 on diffractograms of the as-cast alloys from the region more than 63 mol% Tl9GdTe6.26 It should be noted that regardless a very close melting temperature of Tl9SbTe6 (790K) and peritectic decomposition of Tl9GdTe6 (800 K) compounds, the liqui-dus and solidus curves have not extremum points and temperature interval of the crystallization of the 5-phase is less than 3 K. Such phenomenon is realized when the enthalpy of mixing during the formation of solid and liquid solutions from starting compounds is practically equal to zero. In other words, in the studied system the Sb ^ Gd replacement in the solid and liquid states are not accompanied by a significant thermal effect. This fact allows us to characterize the 5-solid solutions as quasi-ideal solution. Imamaliyeva et al.: Phase Equilibria and some Properties of Solid Solutions ... Acta Chim. Slov. 2017, 64, 221-226 223 Table 1. Some properties of phases in the Tl5Te3-Tl9SbTe6-Tl9GdTe6 system. Thermal effects, Microhardness, Parameters of Phase K MPa, tetragonal lattice, A (accuracy ±3°) (accuracy ±20 MPa) a c Tl5Te3 723 1130 8.9303(3) 12.5987(8) Tl9,8Gd0,2Te6 730-744 1180 8.9184(4) 12.6848(9) Tl9.6Gd0.4Te6 740-763 1160 8.9064(4) 12.7707(9) Tl9.5Gd0.5Te6 750-770 - - - Tl9.4Gd0.6Te6 760-773 1150 8.8953(4) 12.8558(8) Tl9.2Gd0.8Te6 775-788; 1100 1150 8.8824(3) 12.9417(8) Tl9.1Gd0.9Te6 785-793; 1150 - - - Tl9GdTe6 800; 1190 1100 8,8705(4) 13,0277(7) Tl9Sb0,2Gd0,8Te6 798; 1100 1150 8.8616(5) 13.0218(8) Tl9Sb0,4Gd0,6Te6 795 1130 8.8536(5) 13.0167(9) Tl9Sb0,5Gd0,5Te6 794 - - - Tl9Sb0,6Gd0,4Te6 793 1120 8.8454(4) 13.0115(8) Tl9Sb0,8Gd0,2Te6 792 1050 8.8373(3) 13.0066(7) Tl9SbTe6 790 1000 8.8315(4) 13.0017(7) a,A 8.93 H., MPii T,K c) o -C b) a) 2TlsTe, 20 c,A 13,0 12.8 12.6 ! 1[>0 800 40 60 mol % Tl,GdTe„ SO TI.GdTc,, Fig. 1. Polythermal section (a), concentration relations of micro-hardnesses (b), and lattice parameters (c) for the system 2Tl5Te3- 780 Tl.,GdTe,. 20 40 60 mol%Tl,SbTe, 80 TlsSbTe„ Fig. 2. Polythermal section (a), concentration relations of micro-hardnesses (b), and lattice parameters (c) for the system Tl9GdTe6- Tl,GdTe6 n„Sb'i'e6 The curves of microhardness dependencies have a flat maximum, which is typical for systems with continuous solid solutions (Fig. 1b and 2b). The XRD patterns obtained are presented in Fig. 3. Powder diffraction patterns of Tl5Te3, Tl9SbTe6 and Tl9GdTe6, and intermediate alloys were very similar to that of Tl5Te3 with slight reflections displacement from one compound to another. The lattice parameters of solid solutions depend linearly on the composition, i.e. obey the Vegard's rule. Projections of the liquidus and solidus surfaces of the Tl5Te3-Tl9SbTe6-Tl9GdTe6 system. Liquidus of the Tl5Te3-Tl9SbTe6-Tl9GdTe6 system consists of two fields of the primary crystallization of X-phase and 5- solid solutions, limited by the ab curve corresponds to the monovariant peritectic L + X o 5 equilibrium (Fig. 4). Imamaliyeva et al.: Phase Equilibria and some Properties of Solid Solutions 224 Acta Chim. Slov. 2016, 64, 221-226 0 —1—r-î—T—~—■—.......—FTTH.........................................t—---1—■ ■ ■ -—......................................... If 30. ao 40 or 60 2-Theta - Scale Fig. 3. XRD patterns for different compositions in the Tl5Te3-Tl9GdTe6 (patterns 1-3) and Tl9GdTe6-Tl9SbTe6 (patterns 3-5) systems. 1- Tl5Te3; Tl9GdTe6 system, where A, B and C are equimolar compositions of the boundary systems as shown in Fig. 4. According to the phase diagram of the Tl9GdTe6-[B] cut, the primary crystallization of the 5-phase occurs from the liquid phase in the composition area < 60 mol% Tl9GdTe6. In the Tl9GdTe6- rich alloys the X-phase first crystallizes, then a monovariant peritectic equilibrium L + X o 5 takes place. As can be seen, over the entire compositions area of the Tl9SbTe6-[A] and Tl5Te3-[C] cuts only 5-phase crystallizes from the melt. Comparison between isopleth sections (Fig. 5) with the isothermal section (Fig. 6) shows, that tie-lines positions in two-phase area L + 5 do not correspond to the cross section planes and continuously change with temperature. The tie-lines positions at 760 K are shown in Fig. 6. 4. Conclusion A T-x-y diagram of the Tl5Te3-Tl9SbTe6-Tl9GdTe6 system, including the phase diagrams of boundary systems Tl5Te3-Tl9TbTe6 and Tl9SbTe6-Tl9TbTe6, isothermal section at 760 K, some isopleth sections and also the liqui-dus and solidus surfaces projections, were constructed. Imamaliyeva et al.: Phase Equilibria and some Properties of Solid Solutions ... Fig.4. Projections of the liquidus and solidus (dashed lines) surfaces of the Tl5Te3-Tl9GdTe6-Tl9SbTe6 system. Dash-dot lines show the investigated sections. Primary crystallization phases: 1-8; 2-X phase. Isopleth sections of the Tl5Te3-Tl9SbTe6-Tl9GdTe6 system (Fig.5). Figs. 5a-c show the isopleth sections 2Tl5Te3-[C], Tl9SbTe6-[A] and Tl9GdTe6-[B] of the Tl5Te3-Tl9SbTe6- 2-50 mol % Tl9GdTe6; 3-Tl9GdTe6; 4-50 mol % Tl9GdTe6; 5-Tl9SbTe6 Acta Chim. Slov. 2017, 64, 221-226 225 T,K 780 760 740 [AJ 20 40 60 80 Tl,SbTet mol%TlgSbTefi Tl„,Te, L_ L__,.!.. _ J_ _1___1 TyMTej 20 40 60 SO Tl»SbTe„ mo I % Tl vSbTe„ Fig.6. The isothermal section of the phase diagram at 760 K of the Tl5Te3-Tl9GdTe6-Tl9SbTe6 system. mol %Tl,GdTe„ Fig. 5. Polythermal sections Tl10Te6-[C], Tl9SbTe6-[A], and Tl9GdTe6-[B] of the phase diagram of the Tl5Te3 -Tl9SbTe6-Tl9GdTe6 system. Components of the system display unlimited solubility in the solid state. Obtained experimental data can be used for choice the composition of solution-melt and for determining of temperature conditions for growing crystals of 5-phase with a given composition. 5. Acknowledgment This work was done in the international joint research laboratory between Institute of Catalysis and Inorganic Chemistry of ANAS (Azerbaijan) and Donostia International Physics Center (Basque Country, Spain). 6. References 1. Applications of Chalcogenides: S, Se, and Te, ed. by Gurin-der Kaur Ahluwalia, Springer, 2016. 2. A. R. Jha, Rare Earth Materials: Properties and Applications, CRC Press, United States, 2014. https://doi.org/10.1201/b17045 3. CRC Handbook of Thermoelectrics, ed. by D. M. Rowe, CRC Press, New York, 1995. 4. B. Yan, H-J.Zhang, C-X.Liu, X-L. Qi, T. Frauenheim and S-C. Zhang, Phys. Rev. B. 2010, 82, 161108(R)-7 5. N. Singh and U. Schwingenschlogl, Phys. Status Solidi RRL. 2014, 8, 805-808. https://doi.org/10.1002/pssr.201409110 6. I. Schewe, P. Böttcher, H. G. Schnering, Z. Kristallogr. 1989, Bd188, 287-298. https://doi.org/10.1524/zkri.1989.188.3-4.287 7. M. B. Babanly, A. Azizulla, A. A. Kuliev, Russ. J. Inorg. Chem. 1985, 30, 1051-1059. 8. M. B. Babanly, A. Azizulla, A. A. Kuliev, Russ. J. Inorg. Chem. 1985, 30, 2356-2359. 9. A. A. Gotuk, M. B.Babanly, A. A. Kuliev, Inorg. Mater. 1979, 15, 1062-1067. 10. K. Kurosaki, H. Uneda, H. Muta and S. Yamanaka, J. Alloys Compd. 2004, 376, 43-48. https://doi.org/10.1016/jjallcom.2004.01.018 11. Q. Guo, A. Assoud, H. Kleinke. Adv.Energy Mater. 2014, 4, 1400348/1-8. 12. B. Wolfing, C. Kloc, J. Teubner, E. Bucher, Phys. Rev. Let. 2001, 36, 4350-4353. https://doi.org/10.1103/PhysRevLett.86.4350 13. K. E. Arpino, D. C.Wallace, Y. F. Nie, T. Birol, P. D. C. King, S. Chatterjee, M. Uchida, S. M. Koohpayeh, J.-J. Wen, K. Page, C. J. Fennie, K. M.Shen, and T. M. McQueen, Phys. Rev. Lett. (PRL). 2014, 112, 017002-5. https://doi.org/10.1103/PhysRevLett.112.017002 Imamaliyeva et al.: Phase Equilibria and some Properties of Solid Solutions 226 Acta Chim. Slov. 2016, 64, 221-226 14. S. Z. Imamalieva, F. M. Sadygov, M. B. Babanly, Inorg. Mater. 2008, 44, 935-938. https://doi.org/10.1134/S0020168508090070 15. M. B. Babanly, S. Z. Imamaliyeva, D. M. Babanly, Azerb. Chem. J. 2009, 2, 121-125. 16. M. B. Babanly, S. Z. Imamaliyeva, F. M. Sadygov, News of BSU. Nat. Sci. Ser. 2009, 4, 5-10. 17. S. Bangarigadu-Sanasy, C. R. Sankar, P. Sohlender, H. Kleinke, J. Alloys Compd. 2013, 549, 126-134. https://doi.org/10.1016/jjallcom.2012.09.023 18. S. Bangarigadu-Sanasy, C. R. Sankar, P. A. Dube, J. E. Gree-dan, H. Kleinke, J. Alloys. Compd. 2014, 589, 389-392. https://doi.org/10.1016/jjallcom.2013.11.229 19. Q. Guo, H. Kleinke, J. Alloys. Compd. 2015, 630, 37-42. https://doi.org/10.1016/jjallcom.2015.01.025 20. M. B. Babanly, J.-C. Tedenac, S. Z. Imamalieva, F. N. Gu-seynov, G. B. Dashdieva, J. Alloys Compd. 2010, 491, 230236. https://doi.Org/10.1016/j.jallcom.2009.08.157 21. S. Z. Imamaliyeva, F. N. Guseynov, M. B. Babanly, J. Chem. Probl. 2008, 4, 640-646. 22. S. Z. Imamaliyeva, F. N. Guseynov, M. B. Babanly, Azerb. Chem.J. 2009, 1, 49-53. 23. M. M. Asadov, M. B. Babanly, A. A. Kuliev, Inorg. Mater. 1977, 13, 1407-1410. 24. S. Z. Imamaliyeva, T. M. Gasanly, I. R. Amiraslanov, M. B. Babanly, Austr. J. Basic Appl. Sci. 2015, 9, 541. 25. K. Wacker, Kristallogr. Supple. 1991, 3, 281. 26. C. R. Sankar, S. Bangarigadu-Sanasy, H. Kleinke, J. El. Mater. 2012, 41, 1662-1266. Povzetek V sistemu Tl5Te3-Tl9SbTe6-Tl9GdTe6 smo preučevali fazna ravnotežja s termično analizo, rentgensko praškovno difrak-cijo in meritvami mikrotrdote. Pripravili smo nekatere izopletne in izotermične krivulje pri 760 K ter projekcije tekočinsko trdnih površin. V tem sistemu smo našli serijo kontinuirnih trdnih raztopin. Trdne raztopine kristalizirajo v tetrago-nalnem Tl5Te3 kristalnem sistemu. Imamaliyeva et al.: Phase Equilibria and some Properties of Solid Solutions DPI: 10.17344/acsi.20l6.3095_Acta Chirn, Slov. 2017,64, 227-236_©commons 227 Scientific paper Influence of Thermal and Bacterial Pretreatment of Microalgae on Biogas Production in Mesophilic and Thermophilic Conditions Beti Vidmar,1 Romana Marin{ek Logar,1 Mario Panji~ko2 and Lijana Fanedl1* 1 Chair of Microbiology and Microbial Biotechnology, Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domzale, Slovenia 2 Sustainable Technologies Development Centre Ltd (CROTEH), Dragutina Golika 63, HR-10020 Zagreb, Croatia * Corresponding author: E-mail: Lijana.Fanedl@bf.uni-lj.si Tel: 00386 1 3203 835 Received: 23-11-2016 Abstract Microalgae biomass has a great potential in search for new alternative energy sources. They can be used as a substrate for the biogas production in anaerobic digestion. When using microalgae, the efficiency of this process is hampered due to the resistant cell wall. In order to accelerate the hydrolysis of cell wall and increase the efficiency of biogas production we applied two different pretreatments - biological and thermal under mesophilic and thermophilic conditions. During biological pretreatment we incubated microalgae with anaerobic hydrolytic bacteria Pseudobutyrivibrio xylanivo-rans Mz5T. In thermal pretreatment we incubated microalgae at 90 °C. We also tested a combined thermal and biological pretreatment in which we incubated P. xylanivorans Mz5T with thermally pretreated microalgae. Thermal pretreatment in mesophilic and thermophilic process has increased methane production by 21% and 6%, respectively. Biological pretreatment of microalgae has increased methane production by 13%, but only under thermophilic conditions (pretreatment under mesophilic conditions showed no effect on methane production). Thermal-biological pretreatment increased methane production by 12% under thermophilic conditions and by 6% under mesophilic conditions. Keywords: biogas production; anaerobic digestion; microalgae; biological pretreatment; thermal pretreatment; Pseudo-butyrivibrio xylanivorans Mz5T 1. Introduction Global human population growth, rapid technological development, climate changes and depletion of fossil fuels have led to an accelerated search for new renewable energy sources. Renewable energy sources are rapidly evolving area with positive effects on the environment (with little or zero carbon dioxide emissions and substrate low sulfur content) and promising economic aspect.1 Given alternative energy sources provoked a lot of controversy, despite initial positive expectations. Renewable energy sources are classified into groups; first generation biofuels (derived exclusively from crops of cultivated plants) and second generation biofuels (derived from lignocellulosic biomass)2-4 have serious flaws, including a great need for arable land and large amount of consumed water. They are also creating a lot of pressure on agriculture and have a low productivity, since produced biomass cannot cover global demand.5 In recent decades we are witnessing increase in interest of exploitation of the algae energy potential. Algae biomass represents the substrate for rapidly developing group of third generation biofuels. This generation offers the perfect solution for solving the above-mentioned drawbacks.6 The main advantages of using algae are low water consumption (they can be grown in salty, waste and non-potable water), possible production on uncultivated areas with high carbon dioxide concentrations, theoretical high photosynthetic efficiency and high productivity.7,8 For a long time technology focused mainly on obtaining biodiesel from algae biomass, which proved to be energy consuming and unbalanced process. More simple process for supplying renewable energy is anaerobic digestion (AD).9,10 Biogas from AD is an alternative, but Vidmar et al.: Influence of Thermal and Bacterial Pretreatment 228 Acta Chim. Slov. 2017, 64, 227-236 much more economically and energetically-favourable process.8 Microbial anaerobic methanogenic process is applied for the multistep decomposition of organic substrates into biogas. Biogas consists of different gases - methane (~65%), carbon dioxide (~35%) and others (nitrogen, nitrogen oxides, hydrogen, ammonia and hydrogen sulfide).11 Other products in AD, such as heat and digestate can be used in other processes or as a soil conditioner.8 Efficacy of AD is influenced by various factors such as composition of substrates, carbon and nitrogen ratio (C:N) of digester contents, composition of microbial community, degree of mixing, pH and temperature. It has been shown that among technological parameters temperature and pH have the biggest impact on speed of the biogas produc-tion.12-15 The process of anaerobic degradation can run under psychrophilic (<20 °C), mesophilic (25-40 °C) or thermophilic (50-65 °C) conditions.16,17 Technically speaking, the industry is only interested in mesophilic and thermophilic process,18 since the decomposition at lower temperatures is very slow.19 When speaking about AD of the same substrates the mesophilic and thermophilic processes are distinguished mainly by their composition of microbial community, resulting in biogas production differences from the same substrate.20,21,22 There are some important microbiological characteristics associated with thermophilic anaerobes, which may affect the biogas production. These characteristics include slow bacterial growth, high cell death, lower bacteria variety, which show an effect on relatively high fatty acids concentration (more than 1 g l-1), reduced substrate degradation etc.21 Since AD is a multi-step process, it is depending on interactions among bacterial and archaeal microbial communities and their substrate and product specificities. Knowledge about the dynamics of microbial community structure and activity is essential for successful planning of the biogas process, monitoring its parameters and for reaching main goal: process stability and maximum yield.23 The link between community structure and performance is still not completely clear and more studies are nee-ded.24,25 Mesophilic conditions represent the optimum temperature range for larger group of microorganisms (anaerobic bacteria and archaea), as thermophilic conditions. Nevertheless the most important fact is to maintain a stable temperature, irrespective of applied process.20 Biochemical reactions at higher temperatures are faster therefore the degradation is faster too. Generally, but not always, thermophilic AD is up to 8-times faster and up to 4-times more productive than mesophilic. It allows better organic matter decomposition and increased biogas production (up to 36%), although the actual methane yield in ther-mophilic AD is dependent on substrate composition and its C:N ratio. Higher temperature also enables thermal destruction of pathogenic bacteria, which is considered as a big advantage over other processes. Disadvantages of thermophilic AD are instability, higher energy inputs and in comparison to the mesophilic process higher temperatures can cause reduced CO2 solubility, which leads to higher proportion of free ammonium and increase in pH.20 Microalgae represent a promising substrate for AD, because they are rich in nutrients, such as carbon, nitrogen and phosphorus, which are essential for the anaerobic microorganisms. Microalgae cells contain a lot of water (78-90%),26,27 many species have high content of carbohydrates (up to 64% of their dry matter) and lipids (2-75% of their dry matter).28,29 Carbohydrates occur in the form of starch, cellulose and various sugars,30 so the substrate is suitable for microbial fermentation. Freshwater microalgae species can contain up to 31% free fatty acids (FFA), but the composition of FFA and lipids is heavily depending on growth conditions (light, temperature, nitrogen level, growth stage at which they are harve-sted).31 In comparison to carbohydrates and proteins, li-pids have higher theoretical potential for methane production. Nevertheless, when the buffer capacity of the system is low, higher lipid content can result in formation of intermediate products (long chain fatty acids) during AD and consequently process inhibition.32 Some species of microalgae may contain lignin (<2%),33 a high level of cellulose (7,1%) and hemicellulose (16,3%).34 High ash contents are typical for winter months and in early spring. The C:N ratio is around 10:1.34 35 Despite the positive aspects of microalgae as substrate for biogas production, we may encounter several problems that also limit their use for anaerobic decomposition. Problems may occur due to low concentration of biodegradable substrate, cell walls resistant to biodegradation, low C:N and sometimes higher lipids concentra- tions.32 Some green algae are covered by multiple layers of intricately sculpted scales while others have crystalline glycoprotein coverings or thick multilaminate fibrillar cell walls. A few taxa though have cell walls with remarkable structural and biochemical similarity to cell walls found in land plants.36 As an example we can take a known representative of the genus Scenedesmus, wherein the rigid cell wall is composed of glucose, mannose and galactose. Individual sugars are otherwise well biodegradable, but in the cell wall they are linked together and form cellulose, hemicellulose and some other polymers (e.g. sporopolle-nin). These molecules form a strong cell wall, highly resistant to bacterial degradation.10 One of the possible solutions to enhance the AD of microalgae biomass are different types of pretreatments, which we use in order to make substrate more susceptible to biodegradation.37 Pretreatments can be divided into four groups - thermal, mechanical, chemical and biological. Most studied area is thermal pretreatment of microal-gae biomass, which shows favourable results and certain industrial processes already run continuously. Mechanical pretreatment generally requires more energy input in Vidmar et al.: Influence of Thermal andBacterial Pretreatment ... Acta Chim. Slov. 2017, 64, 227-236 229 comparison to the chemical, thermal or biological treatments. Chemical pretreatment has proved successful, especially in combination with thermal, but the main disadvantage is contamination and complexity of downstream processes. Biological pretreatment of biomass is also very promising, mainly due to low energy consumption.38 In the presented research work the biodegradability of untreated and pretreated microalgae was examined in anaerobic digestion. In order to accelerate the hydrolysis and increase the efficiency of biogas production two different pretreatments were applied - biological (bacterial) and thermal. A combined thermal-biological pretreatment was tested, too. Biogas production was measured in biochemical methane potential assay under mesophilic and thermophilic conditions. 2. Experimental 2. 1. Substrate for Biogas Production Microalgae biomass was obtained from the open photobioreactor of company Koto d.o.o. Microalgae are produced in digestate (liquid part of the effluent after separation to liquid and solid part) of thermophilic biogas reactor, which converges into 26 m3 big pool. Microalgae biomass was pumped out of the pool with a peristaltic pump and stored in larger containers, later divided into smaller volumes (up to 1 l) and frozen at -20 °C. Chemical composition of the dry microalgae biomass is shown in Table 1. Table 1. Chemical composition of the dry microalgae biomass. Legend: TVS (total volatile solids), TOC (total organic carbon), TN (total nitrogen) (Determined by Koto d.o.o.). Parameters Content (g kg 1) TVS 796,8 TN 70,7 Ash 203,2 Protein 441,3 TOC 404,8 C:N ratio 5,7 2. 2. Microbial Inoculum for Biogas Production Two different microbial inoculums were used to test the differences between mesophilic and thermophilic process of biogas production. Mesophilic microbial inoculum was taken from an active CSTR (continuous stir-red-tank reactor) operating at 37 °C (biogas plant Petrol d.d., Slovenia). Before the experiment, the microbial inoculum was pre-incubated for eight days at 37 °C. Thermophilic microbial inoculum was taken from CSTR operating at 55 °C (biogas plant Koto d.o.o., Slovenia) and was pre-incubated for eight days at 55 °C. 2. 3. Pretreatment of Microalgae Biomass The temperature of 90 °C was applied for thermal treatment of microalgae in this experiment. The selected temperature based on previous research reports.10 Microalgae were first thawed, thoroughly mixed and distributed into glass bottles of 250 ml. The bottles were closed with gas-tight rubber and aluminium stoppers. Thermal pretreatment of microalgae was conducted in water bath for three hours at 90 °C. Occasionally the bottles were mixed and vented. Bacterial strain Pseudobutyrivibrio xylanivorans Mz5T (DSM 14809) originates from the microbial collection of the Department of Microbiology and Microbial Biotechnology at Biotechnical Faculty and was used for biological pretreatment of microalgae biomass. P. xylani-vorans Mz5T holds excellent cellulolytic, xylanolytic, amylolytic and pectinolytic activity.39,40 Due to these characteristics, P. xylanivorans Mz5T was selected for biological pretreatment of microalgae. The bacterium was cultured in DSMZ medium M330 (50 ml) and incubated overnight (~20 h) at 37 °C. When the culture reached optical density (X = 600 nm) 0,5 ± 0,05, it was centrifuged and the precipitate was anaero-bically transferred into 1 l batch reactors to pretreat mi-croalgae biomass. Pretreatment was carried out for 24 hours at 37 °C (120 rpm), then microbial inoculum was added to the substrate. 2. 4. Experimental Setup of Biochemical Methane Potential (BMP) Assay BMP assay was conducted to examine and determine the effect of different microalgae pretreatments on biogas and methane production. On the first day biological and thermal pretreatments were performed, but the BMP assay started the second day. Experimental setup was the same for both processes (mesophilic and thermophilic), as seen on Figure 1. For biological pretreatment we incubated P. xylani-vorans Mz5T together with untreated microalgae (as described in chapter 2.3), for thermal pretreatment only thermally pretreated microalgae (as described in chapter 2.3) were added and for thermal-biological pretreatment we incubated P. xylanivorans Mz5T with thermally pretreated microalgae. All pretreatments lasted for 24 hours, after which methanogenic microbial inoculum was added to the experimental bottles to start the anaerobic digestion. Before experiments the appropriate loading of the bioreactors was determined by measuring TTS (total solids) and TVS (total volatile solids) for both microbial inoculums and chemical oxygen demand (COD) for mi-croalgae.41 The microbial inoculum concentration for both experiments was 4 g TVS l1 and microalgae loading was 1,228 g TVS (144 ml). Phosphate buffer (20 ml) and anoxic tap water were added to all experimental mixtures. Working volume for Vidmar et al.: Influence of Thermal and Bacterial Pretreatment 230 Acta Chim. Slov. 2017, 64, 227-236 all mixtures was 500 ml. Sole microbial inoculum served as a negative control for residual methanogenic activity. For positive control (standard), which represents the internal control for BMP assay, glucose was added as a substrate. Loading for standard mixtures was 0,748 g l-1 (0,2 g CODglucose). While mixing all ingredients, anaerobic conditions were maintained by sparging gaseous nitrogen.42 Mixtures with autoclaved culture of P. xylanivorans Mz5T were tested to measure the medium's nutrients and dead cell COD effect (negative control to experimental mixtures with live culture of MZ5) on biogas production. Both experiments were conducted in laboratory bioreac-tors (1 l) at 37 °C for mesophilic conditions and at 55 °C for thermophilic conditions. The bioreactors were kept in dark at 120 rpm for 46 days (thermophilic process) and 55 days (mesophilic process) with three replicate experimental mixtures. In order to gain information on the cumulative biogas production in each mixture, after each measurement the volume of produced biogas was added to the sum of previous measurements. In presentation of the final results of biogas production the amount of generated biogas in negative control was also taken into account. To calculate the net quantity of the produced biogas (how much biogas was generated at the expense of the added substrate), the average amount of biogas produced in ne- gative controls was subtracted from the production of the test mixtures. The same was done for cumulative methane production. The resulting methane yields were normalized to standard conditions as described by Hansen et. al (2004).43 2. 5. Analytical Methods TTS and TVS of experimental mixtures were determined at the beginning (t0) and the end (t46 for thermophi-lic and t55 for mesophilic process) of each experiment according to standard methods.41 COD was also performed, using closed reflux method.41 The pH-values of mixtures were measured at the beginning (t0) and the end (t46 for thermophilic and t55 for mesophilic process) of each experiment. The quantity and composition of produced biogas was determined 12 times during both processes. Short-chain fatty acids (SCFAs) were monitored 4 times during both processes. The amount of produced biogas was measured manually with a pressure gauge and water column.43 The proportion of methane, carbon dioxide and nitrogen was monitored by Shimadzu 14A gas chromatograph (GC) equipped with thermal conductivity detector (TCD). The separation of gases was carried out on a steel column (diameter 1/8'') filled with PORAPAK Q (Agilent). He- Figure 1. Experimental set-up for BMP assays. NC - negative control (microbial inoculum), ST - standard respectively positive control, A - untreated microalgae, TA - thermally pretreated microalgae, P-MZ5 - biologically pretreated microalgae (Mz5 culture added), P-MZ5a - negative control for biologically pretreated microalgae (autoclaved Mz5 culture added), P-MZ5-T - thermally and biologically pretreated microalgae (Mz5 culture added), P-MZ5a-T - negative control for thermally and biologically pretreated microalgae (autoclaved Mz5 culture added). Vidmar et al.: Influence of Thermal andBacterial Pretreatment ... Acta Chim. Slov. 2011, 64, 227-236 231 lium with flow rate of 25 ml min-1 was used as a gas carrier. For analysis, we used the following program: injector temperature was 50 °C, column temperature 30 °C, detector temperature 80 °C, current was 60 mA. Standard mixture of gases (15,7% H2, 18,7% N2, 20,5% CH4 and 45,1% CO2) was used for calibration performed using the method of surface normalization. The resulting methane yields were normalized to standard conditions and expressed in normalized volume percentage. Ether extraction of SCFAs was performed according to the adapted method.44 SCFAs were determined by GC equipped with a flame ionization detector (FID). Helium was used as a gas carrier. For analysis, we used the following program: injector temperature was 250 °C, column initial temperature 80 °C, column final temperature 160 °C, detector temperature 250 °C, time of maintaining the initial column temperature was 2 minutes and time of final temperature maintenance was 4 minutes. Column was heated at a rate of 15 °C per minute. Quantification was performed by an internal standard method (crotonic acid, 100 mg ml-1). 3. Results and Discussion 3. 1. Biogas and Methane Production from Microalgae 3. 1. 1. Mesophilic Process The highest biogas production under mesophilic conditions resulted from thermal pretreatment of microalgae (TA) with the average production of 452,9 ml per 1 g TVSsubstrate. The lowest production was recorded in case of biological pretreatment of microalgae (P-MZ5), with the average production of 324,5 ml biogas per 1 g TVSsubstrate (Figure 2, A). The highest methane production was recorded for mixtures with thermally pretreated microalgae (TA), with the average production of 273,2 ml of methane per 1 g TVSsubstrate. The lowest production of methane was measured in case of untreated microalgae (A), with average production of 217,2 ml of methane per 1 g TVSsubstrate (Figure 2, B). The average percentage of methane in biogas in mesophilic process on the last day of BMP assay represented 64,1%. The trend showed that each of the pretreatments slightly increased the methane proportion in produced biogas (Table 3). 3. 1. 2. Thermophilic Process The maximal biogas production under thermophilic conditions was measured in case of microalgae biologically pretreated with bacteria P. xylanivorans Mz5T (P-MZ5), with average biogas production of 406,2 ml per 1 g TVSsubstrate. Mixtures with untreated microalgae (A) and different other pretreatments produced from 317 to 386 ml of biogas per 1 g TVSsubstrate. The lowest production was measured in case of thermally pretreated microalgae (TA), with average production of 317,2 ml of biogas per 1 g TVSsubstrate(Figure 2 C). The lowest production of methane was measured in case of untreated microalgae (A), with average production of 176,9 ml of methane per 1 g TVSsubstrate. The highest methane production was recorded for mixtures with biologically pretreated microalgae (P-MZ5), with the average production of 279,9 ml of methane per 1 g TVSsubstrate (Figure 2, D). The average percentage of methane in biogas in thermophilic process on the last day of BMP assay represented 61,1%. The trend also showed that each of the Table 2. BMP assay results showing cumulative methane production (at standard conditions) per 1 g TVSsubstrate (ml) in every experimental mixture for mesophilic and thermophilic anaerobic digestion of untreated, thermally, biologically and thermally-biologi-cally treated microalgae. Legend: A - untreated microalgae, TA -thermally pretreated microalgae, P-MZ5 - biologically pretreated microalgae (Mz5 culture added), P-MZ5a - negative control for biologically pretreated microalgae (autoclaved Mz5 culture added), P-MZ5-T - thermally and biologically pretreated microalgae (Mz5 culture added), P-MZ5a-T - negative control for thermally and biologically pretreated microalgae (autoclaved Mz5 culture added). Cumulative methane production per 1 g Titrate M) Bioreactor Mesophilic process Thermophilic process A 217,2 176,9 TA 273,2 187,1 P-MZ5 230,8 279,9 P-MZ5a 238,6 242,4 P-MZ5-T 254,1 231,7 P-MZ5a-T 240,2 203,8 Table 3. BMP assay results showing increase in biogas and methane production due to different methods of pretreatments in mesophilic and ther-mophilic anaerobic digestion. Effects of pretreatments were reckoned according to the comparison in pairs (e.g. thermally treated microalgae to untreated microalgae, etc.). Differences of cumulative production of biogas and methane per 1 g TVSsubstrate due to pretreatment effects between pairs were later expressed in percentages. Mesophilic process Thermophilic process CH4 Biogas CH4 Biogas Thermal pretreatment 21% 16% 6% 0% Biological pretreatment 0% 0% 13% 5% Thermal and biological pretreatment 6% 6% 12% 11% Vidmar et al.: Influence of Thermal and Bacterial Pretreatment 232 Acta Chim. Slov. 2017, 64, 227-236 A) 450 CO ? 20 30 Time (days) B) 300 250 200 7 150 5 100 £ 50 C) 400 350 in 300 250 E 150 100 200 20 30 Time (days) D) 300 20 40 Time (days) 20 30 Time (days) Figure 2. Cumulative biogas and methane production (at standard conditions) in mesophilic and thermophilic anaerobic digestion of untreated, thermally, biologically and thermally-biologically treated microalgae. A) biogas production per 1 g TVSsubstrate (ml) under mesophilic conditions, B) methane production per 1 g TVSsubstrate (ml) under mesophilic conditions, C) biogas production per 1 g TVSsubstrate (ml) under thermophilic conditions, D) methane production per 1 g TVSsubstrate (ml) under thermophilic conditions. Legend: A - untreated microalgae, TA - thermally pretreated microalgae, P-MZ5 - biologically pretreated microalgae (Mz5 culture added), P-MZ5a - negative control for biologically pretreated microalgae (autoclaved Mz5 culture added), P-MZ5-T - thermally and biologically pretreated microalgae (Mz5 culture added), P-MZ5a-T - negative control for thermally and biologically pretreated microalgae (autoclaved Mz5 culture added). pretreatments slightly increased the methane percentage in produced biogas (Table 3). According to the literature, the thermophilic process shows 25-50% higher anaerobic activity compared to mesophilic.21 The temperature of anaerobic process affects the concentration and presence of individual SCFAs, which indicate that the accumulation of intermediates is in fact different under mesophilic and thermophilic conditions. Research results indicate that this feature depends mainly on the composition of microbial communities.22'23 For optimal process, the concentration of acetic acid should not be higher than 2 g l-1 and concentration of propionic acid higher than 0,9 g l-1. Increased concentration of propionic acid is the most significant indication of process inhibition45 and occurs following the acetic acid accumulation. The highest total concentration of SCFAs in this study under mesophilic conditions was 1,4 g l-1 (up to 1,3 Vidmar et al.: Influence of Thermal andBacterial Pretreatment ... Acta Chim. Slov. 2017, 64, 227-236 233 g l-1 of acetic acid and up to 0,17 g l-1 of propionic acid). In case of thermophilic BMP assay the highest total concentration of SCFAs was 1,3 g l-1 (up to 1 g l-1 of acetic acid and up to 0,22 g l-1 of propionic acid). Acetic acid was the most abundant in all mixtures. SCFAs were within optimal concentration range during both experiments, with the lowest concentration at the end of BMP assays, demonstrating that anaerobic methanogenic degradation ran smoothly and with no inhibitory effects. Optimum pH during anaerobic degradation varies between 6 and 8, with optimum value around pH = 7,5 for thermophilic process46 and pH = 7 for mesophilic process.47 During our experiments the pH value ranged between 7,9 and 8,1 for mesophilic process and 7,8 and 8,2 for thermophilic process. Results were slightly higher than the optimal value, but still appropriate for stable biogas production. Important parameter for determining the process activity is the reduction of the organic substance during anaerobic degradation. The content of TVS in thermophilic process has reduced by 22,3% and only by 9,0% in me-sophilic process. The results indicate that the thermophilic anaerobic digestion is more efficient in decomposition of organic matter, which confirms the known facts about the thermophilic process.20 3. 2. Impact of Microalgae Pretreatments on Biogas and Methane Production 3. 2. 1. Thermal Pretreatment Thermal pretreatment is recognized as possible and effective hydrolysis treatment for microalgae biomass. Higher temperature conditions stimulate cellulose and hemicel-lulose hydrolysis of algal cell wall components (mainly cellulose and hemicellulose), followed by formation and release of range of low molecular weight compounds (sugars, acids, etc.).38,48 Heat also disrupts the hydrogen bonds in crystalline cellulose, causing the biomass to swell.38 It was found, that bonds between and within the molecules forming the microalgae Scenedesmus cell walls were cleaved during the thermal pretreatment at 90 °C, which resulted in increased methane production by 2,2-fold with regard to untreated microalgae.10 It is also known that time period of pretreatment is less important, as the temperature itself.49 The same conclusion had the research of Marsolek et al., where culture of Nannochloropsis oculata was treated at different temperatures.50 Temperatures between 30 and 60 °C did not increase decomposition, yet treatment at 90 °C caused partial decomposition, which allowed up to 36% increase in biogas production. High temperatures disintegrate algae cells already after 30 minutes of pretreatment, proving that thermal treatment improves the cellular contents release into extracellular space.10 The results of our study show that thermal pretreat-ment in mesophilic (37 °C) BMP assay increased the bio- gas production by 16% and methane production by 21% in comparison to untreated microalgae (Table 3). Experimental results show that thermal pretreatment enables more efficient hydrolysis of microalgae cell wall compounds (especially cellulose and hemicellulose) and consequently releases more sugars for further efficient micro-bial transformation to biogas. The thermal pretreatment of microalgae also increased methane percentage in biogas and finally increased methane yield (Table 3). The results of BMP assay under thermophilic conditions (55 °C) did not show similar trends. Thermal pretreatment has not increased biogas production. Nevertheless, the methane production was increased, but only by 6% (Table 3). We can assume hypothetically, why thermal pretreatment has no significant effect on production in thermophilic process. One of the possible reasons may be the relatively low C:N ratio, which can lead to the release and consequent increase in concentration of free ammonium during the thermal pretreatment of microalgae.35 Since thermop-hilic process is performed at higher temperatures than the mesophilic process, the anaerobic degradation of thermally pretreated microalgae can further disintegrate damaged algae cells. That can lead towards ammonium release, too, and thus to partial inhibition of methanogenic activity.19 Koster et al. have demonstrated the impact of free ammonia on anaerobic microorganisms and discovered that it rapidly penetrates through the cell membrane, causes proton imbalance, lack of potassium (K+) and enzyme inhibition.51 From the results we have obtained in our study, we can conclude that thermal pretreatment of microalgae at 90 °C (three hours) for thermophilic process is unnecessary, since the methane yield is not significantly higher than the methane yield from raw and untreated mi-croalgae. 3. 2. 2. Biological Pretreatment Strains of genus Butyrivibrio represent a major proportion (10-30%) of bacteria in the rumen of domestic and wild cattle. Many bacterial species of the genus But-yrivibrio contribute to the decomposition of fiber in the rumen. Most strains synthesize xylanase, amylase and cel-lodextrinase, some also 1,4-0-endoglucanses that can decompose a wide range of polymers.39 P. xylanivorans Mz5T is a close relative of bacterial species of the genus Butyrivibrio. It is a Gram-negative anaerobic bacterium that synthesizes many hydrolytic enzymes and holds excellent enzymatic activities.39,40 The results of BMP assay under mesophilic conditions showed that biological pretreatment of microalgae did not affect the production of biogas or methane (Table 3). It may be that during biological pretreatment a part of presented substrate is used for the growth of the microorganism used for the biological treatment itself, resulting in a loss of monomeric organic compounds left for the following methane production. More tests are needed Vidmar et al.: Influence of Thermal and Bacterial Pretreatment 234 Acta Chim. Slov. 2017, 64, 227-236 to explain this phenomenon. The results of BMP assay under thermophilic conditions were somewhat different. Biological pretreatment increased biogas production by 5% and methane production by 13% (Table 3). The results show that bacterium P. xylanivorans Mz5T managed to break down a certain proportion of hemicellulose, cellulose and xylan molecules in microalgae cell walls. This provided easier nutrient access for methanogenic microbial community during the thermophilic process, what consequently influenced the increase in methane production.20-22 In the case of biological treatment it is more meaningful if we add live hydrolytic bacteria in to the process, which constantly produce extracellular enzymes and allow the hydrolysis of the substrate (bioaugmentation).50 The effect of biological pretreatment of microalgae on biogas production is still poorly understood, mainly due to the complexity of the structure of cell walls and species diversity of microalgae. Substrate that was applied for BMP assays contained mixed culture of microalgae in which certain types predominate, but are also changing seasonally. Therefore it is difficult to accelerate the hydrolysis of cell walls with only one bacterial strain. Mi-croalgae are very diverse, thus we should choose an appropriate microorganism for each type or mixed culture and adjust the process of anaerobic degradation accor- dingly.50 3. 2. 3. Thermal-biological Pretreatment We also tested the influence of the combined pre-treatment (thermal-biological) of microalgae on biogas production by BMP assays. The above-mentioned pre-treatment showed no significant effect on biogas or methane production in mesophilic process (Table 3). Production of biogas and methane was increased by 6%. Results were similar when the microalgae were only biologically pretreated. Comparing all tested pretreatments (thermal, biological, thermal-biological) of microalgae to produce biogas and methane, we found that only thermal pretreat-ment maximizes production in mesophilic process. The results of BMP assay under thermophilic conditions showed that thermal-biological pretreatment increases the biogas production by 11% and methane production by 12% (Table 3). According to the results, combined pre-treatment of microalgae indicates stronger effect on ther-mophilic process than individual pretreatments. This result could be explained by the fact that during biological pretreatment a part of substrate presented after thermal pretreatment was used for the growth of the bacteria used for the biological treatment itself, resulting in loss of substrate in the system left for the following methane production. Although it is generally accepted that thermophilic anaerobic digestion is more efficient than mesophilic anaerobic digestion of the same substrate, our results have not proved that for microalgae. It has been calculated from data in Table 2 that the average cumulative biogas production in mesophilic process is more efficient for 2% and the average cumulative methane production for 9% (Table 2) than in thermophilic process. There were also differences in the methane yield, where the average yield of methane in mesophilic process was higher for 9% in comparison to thermophilic process (Table 3). 4. Conclusions In order to accelerate anaerobic digestion we applied different types of pretreatments of microalgae. Following the obtained results, we can conclude that thermal pretreatment at 90 °C is the most effective method for increasing methane and biogas production under mesophilic conditions. Biogas production was increased by 16% and methane production by 21%. Biological pretreatment with bacterium P. xylanivorans Mz5T is the most effective method for increasing methane and biogas production under thermophilic conditions. Methane production was increased by 13%. Combined (thermal-biological) pretreatment showed the strongest effect in thermophilic process. Biogas production was increased by 11% and methane production by 12%. Further research should be carried out to determine which pretreatments are the most economical for individual biogas plant and which algae species are the best for biofuel production, before we could transfer the research to higher scale. 5. Acknowledgements The authors would like to thank to the two Slovenian biogas plants Koto d.o.o. and Petrol d.d. for their cooperation during this research. 6. References 1. A. Cadenas, S. Cabezudo, Technol. Forecast. Soc. Change. 1998, 58, 83-103. http://dx.doi.org/10.1016/S0040-1625(97)00083-8 2. R. E. H. Sims, W. Mabee, J. N. Saddler, Bioresour. 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Wastes. 1984, 9, 205-216. http://dx.doi.org/10.1016/0141-4607(84)90080-5 ter Pollut. Control Fed. 1978, 50, 73-85. Povzetek Pri iskanju novih alternativnih virov energije ima velik potencial odpadna biomasa. V zadnjem času se pozornost preusmerja tudi na neobičajne vire, na primer mikroalge, ki jih lahko uporabimo kot substrat za proizvodnjo bioplina v anaerobni razgradnji. Mikroalge imajo težko razgradljive celične stene, kar ovira učinkovitost anaerobnega procesa. Za pospešitev hidrolize in povečanje proizvodnje bioplina iz mikroalg smo uporabili dva načina predobdelave - biološko in termično v mezofilnih in termofilnih pogojih. Pri biološkem načinu smo mikroalge pred poskusom inkubirali s hidrolit-skimi bakterijami Pseudobutyrivibrio xylanivorans Mz5T. Pri termični predobdelavi smo mikroalge inkubirali pri 90 °C. Preizkusili smo tudi kombinirano termično-biološko predobdelavo, kjer smo mikroalge po termični obdelavi inkubirali s P. xylanivorans Mz5T. Termična predobdelava je v mezofilnem procesu povečala proizvodnjo metana za 21 %, v termofilnem procesu le za 6%. Biološka predobdelava mikroalg je povečala proizvodnjo metana samo v termofilnih pogojih in sicer za 13% (predobdelava v mezofilnem procesu ni imela večjega vpliva). Termično-biološka predobdelava je v termofilnih pogojih povečala proizvodnjo metana za 12 %, v mezofilnih pogojih pa za 6 %. Vidmar et al.: Influence of Thermal andBacterial Pretreatment ... DPI: I0.l7344/acsi.20l6.3l28_Acta Chirn, Slov. 2017,64, 237-247_©commons 237 Scientific paper Biosorption of 2,4 dichlorophenol Onto Turkish Sweetgum Bark in a Batch System: Equilibrium and Kinetic Study Dilek Yildiz,1'* Feyyaz Keskin1 and Ahmet Demirak2 1 Mugla Sitki Kocman University, Research and Application Centre For Research Laboratories, 48000 Mugla, Turkey 2 Mugla Sitki Kocman University, Department of Chemistry, 48000 Mugla, Turkey * Corresponding author: E-mail: dilekyildiz2003@hotmail.com Tel: +90 0 252 211 1675 Received: 09-12-2016 Abstract In this study, Turkish Sweetgum bark was used as a new biosorbent to investigate the removal of hazardous 2,4 dichlorophenol (2,4-DCP) from aqueous solutions in batch biosoption experiments. The effective usage of Turkish sweetgum bark is a meaningful work for environmental utilization of agricultural residues. The effects of experimental parameters like solution pH, contact time, initial concentration of adsorbate and amount of bisorbent dosage were investigated in a series of batch studies at 25 °C. Taguchi's Orthogonal Array (OA) analysis was used to find the best experimental parameters for the optimum design process in this study. The functional groups and surface properties of biosorbent were characterized by using Fourier transformer infrared (FTIR) and scanning electron microscopy (SEM) techniques. The experimental data were fitted to Langmuir isotherm and Freundlich isotherm models. There is a good agreement between the parameters and this confirms the monolayer adsorption of 2,4-DCP onto sweetgum bark. As a result of kinetic studies, the pseudo-second-order kinetic model was found to be suitable for all the data. Also, the results of the study show that Turkish Sweetgum bark can be potential as a low-cost alternative commercial adsorbents for removal 2,4 dichlorophenol from aqueous solutions. Keywords: 2,4 dichlorophenol; Biosorption; Turkish Sweetgum; Equilibrium; Kinetics; Taguchi's Orthogonal Array 1. Introduction One type of dangerous wastes that are chiefly produced during chemical and many other industrial and agricultural activities is phenols and phenol compounds.1-6 If the low concentrations of pollutants are harmful to organism, these pollutants are considered as priority pollutants. Many of them have potential to harm human health; therefore, they have been classified as hazardous pollutants.7 United State Environmental Protection Agency (USEPA) has registered phenolic compounds as priority pollutants. Most of the phenolic compounds are toxic and hardly biodegradable, and it can be really difficult to get rid of them in the environment. Especially chlorophenols (CPs) are believed to create bad taste and odor in drinking water at concentrations below 0.1 g/L and cause adverse impacts on the environment.8 Some physicochemical and biological methods including adsorption, extraction by chemical solvents, air stripping, freezing and crystallization, chemical oxidation, wet oxidation, advanced oxidation processes, biological degradation biosorption, coagulation, chlorination and liquid membrane permeation have been developed for the removal of phenolic compounds from aqueous solutions.6,7,9,10-13 Among these methods, the ones used for the concentration of the chlorinated phenols on the solid phase are adsorption and ion exchange methods but they are not for complete mineralization. The ones used for complete mineralization and combination of chlorophenols are chemical or biological oxidation methods. While one advantage of chemical oxidation methods is their being fast, they might result in undesirable by-products and they are expensive. Mostly preprocessed and rigid solid bisorbent material was investigated for removal hazardous wastes from aqueous solutions. Pretreatment is certainly advantageous Yildiz et al.: Biosorption of 2,4 dichlorophenol Onto Turkish 238 Acta Chim. Slov. 2017, 64, 237-247 concerning mechanical properties, but it is needed additional resources. Therefore, naturally immobilized biomass in the form of pellets with good biosorption capacities is a type of biosorbent. However, it is a highly porous, soft and mechanically sensitive material, and this might affect the column performance.14 Biosorption of chlorophenols are more specific and relatively cheap than chemical oxidation methods. Biosorption methods of chlorophenols were also investigated by many researchers.710 According to recent studies, some natural minerals, industrial wastes, agricultural wastes, and forest wastes are low-cost adsorbent ma-terials.15-18 Agricultural wastes among them are one of the most promising groups of adsorbent materials. New adsorbents that are locally-easily available, high adsorption capacity and economic materials, or certain waste products from industrial or agricultural operations, may have potential as low-cost sorbents.19-21 Their unique chemical composition makes these wastes economic and eco-friendly alternatives the removal of chlorophenols.6,7,22,23 We are interested in bark of Turkish Sweetgum as biosor-bent. The sweetgum, which is widely known as Turkish sweetgum. is a deciduous tree native to the eastern Mediterranean region Styrax liquidus obtained from sweetgum have been known since very old times and they are known to have been used to mummify pharoses in ancient Egypt . The volatile oil extracted from Styrax liquidus has been utilized for the production of pharmaceutical and cosmetic products and they are made available in Turkey through ex-port.24 The barks of sweetgum are a forest wastes to obtain the export goods from sweetgum plant and Styrax liquidus. Processed Turkish sweetgum barks are left in the forest as waste. These can cause forest fires. So it should be cleaned from the forest. Sweetgum bark consists of tannin compounds. Previous studies have reviewed low-cost adsorbents including bark/tannin, lignin, chitin/chitosan, non-living biomass etc.19 There are main objective of the present study is to explore the ability of sweetgum (Turkish Sweetgum) bark that become forest waste to remove 2,4-DCP from aqueous solutions. For this reason, biosorbent was characterized using Fourier transformer infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). In addition, experimental parameters such as solution pH, contact time, initial concentration of adsorbate and amount of biosorbent dosage were investigated. A statistical optimization was used to determine the optimum biosorption conditions for removal of 2,4-DCP from aqueous solutions in sweetgum bark. Moreover, adsorption isotherm models and kinetics models were studied to understand the biosorption mechanism for theoretical evaluation. 2. Materials and Methods 2. 1. Materials The bark of sweetgum was obtained from the Mugla Manager ship of Governmental Operation of Forestry, Ge- neral Directorate of Forestry, Ministry of Environment and Forestry, Republic of Turkey at November, 2015. The 2,4 dichlorophenol, > 99%, (2,4-DCP) was from Sigma-Aldrich (St. Louis, MO, USA). 4-aminoantipyrine and potassium ferricyanid used in this study were obtained from Merck and were of GR grade. 2. 2. Equipment and Analysis A pH meter (WTW) was used for the measurement of pH. The concentrations of phenol compound were analyzed calorimetrically by using 4-aminoantipyrine and potassium ferriciyanid at pH 7.9 ± 0.1 according to the Standard Methods.25 All the analyses of this study were performed in the laboratory that has a framework of ISO IEC 17025 Laboratory accreditation 2. 3. Biosorbent The sweetgum consists of resin alcohols avaiblable free and combined with cinnamic acid, which makes up 30-45 % of the total weight. Detailed chemical composition of TSB was styrene (1.56); a-pinene (1.02); benzaldehyde (0.47); b-pinene (0.15); benzyl alcohol (1.22); acetophenone (0.19); 1-phenyl-1-ethanol (0.17); hydro-cinnamyl alcohol (41.13); trans-cinnamyl aldehyde (0.24); trans-cinnamyl alcohol (45.07) and bcaryophylle-ne (3.60 %).26 The barks of sweetgum were dried in the oven at 60 °C for 48 h and then passed through a 150 pm size screen to use it in the study. 2. 4. Preparation of Synthetic Sample It was prepared for a stock solution of 2,4-DCP (1000 mg/l) with distilled water. To obtain all the solutions of varying concentrations, the stock solution was used in the current study. The pH of each solution was adjusted to the desired value using 0.1 M HCl and 0.1 M NaOH. 2. 5. Batch Sorption Experiments The batch technique was used to conduct the experiments of sorption in a routine manner. The dry biomass (1,0 g) was shaken with 50 ml of 2,4-DCP solution at a concentration of 150 mg/l in a shaker at room temperature (20 ± 0.5 °C) for about 150 minutes. For the separation of the particles of sweetgum barks by filtration, a 0.45 pm membrane filter was used. The amounts of sweetgum barks adsorbed in each case were measured by calculating the difference between the initial and the final concentrations of 2,4-DCP. By using the difference between the initial concentration and equilibrium (qe) of 2,4-DCP concentration, biosorption capacity at equilibrium time (qe) was calculated as follows: Yildiz et al.: Biosorption of 2,4 dichlorophenol Onto Turkish Acta Chim. Slov. 2017, 64, 237-247 239 = V(Co-Ce)j M (1) Table 2. L25 Experimental and expected results from Taguchi's Orthogonal Array (OA) analysis where V is the sample volume (L), Co is the initial concentration of 2,4-DCP (mg/l), Ce is the equilibrium or final concentration of 2,4-DCP (mg/l), M is the dry weight of (0.5 g for this study), and qe is the biomass biosorption capacity of the biomass at equilibrium time. 2. 6. Optimization Study Taguchi is a simple and effective statistical method, which organizes a systematic experimentation to determine the near to optimum settings of design parameters for performance, quality, and cost. In this method, a large number of variables are studied with a small number of experiments using orthogonal arrays.27-32 For this reason this study was carried out using Taguchi statistical method. In the Taguchi approach, an orthogonal arrays and analysis of variance (ANOVA) are used for the analysis of experimentations. By using ANOVA, the effect of factors can be estimated and by orthogonal arrays the minimum number of experiments is needed. In this method variability of parameters is expressed by signal-to-noise (S/N) ratio, which represents the ratio of desirable results (signal) to undesirable results (noise). In this statistical method the S/N ratio is used to measure the quality characteristic derivation from the desired value. The maximum S/N ratio is considered as the optimal condition as the variability is inversely proportional to the S/N ratio.33 The Taguchi experimental design method was used to determine optimum removal conditions. The effect of experimental parameters such as pH, amount of biosor-bent, initial concentration of adsorbate and contact time were investigated using an L25 (55) orthogonal array. One of the main objectives of this research was to apply Taguc-hi statistical approach to optimize the reaction parameters toward higher adsorption efficiency. In this work, the effect of four important factors including pH, amount of biosorbent, initial concentration of adsorbate, contact time and each factor at five levels on the adsorption efficiency of 2,4- DCP were studied using Taguchi's method. The used level setting values of the main factors (A-D) and the L25 (55) matrix employed to Table 1. Factors and levels for experimental parameters used to in sorption capacity test Levels A (pH) B (amount of biosorbent (g) C (initial concentration of adsorbate (mg/L) D (contact time (min) 1 2 0,2 25 30 2 4 0,4 50 60 3 6 0,6 100 90 4 8 0,8 150 120 5 10 1,0 200 150 Experiment no. pH Amount of biosorbent Initial concentration of adsorbate Conta time 1. 1 1 1 1 2. 1 2 2 2 3. 1 3 3 3 4. 1 4 4 4 5. 1 5 5 5 6. 2 1 2 3 7. 2 2 3 4 9. 2 3 4 5 10. 2 4 5 1 11. 2 5 1 2 12. 3 1 3 5 13. 3 2 4 1 14. 3 3 5 2 15. 3 4 1 3 16. 3 5 2 4 17. 4 1 4 2 18. 4 2 5 3 19. 4 3 1 4 20. 4 4 2 5 21. 4 5 3 1 22. 5 1 5 4 23. 5 2 1 5 24. 5 3 2 1 25. 5 4 3 2 26. 5 5 4 3 assign the considered factors are shown in Tables 1 and 2, respectively. The experimental data were analyzed using the statistical software MINITAB 15. The data (y;) and corresponding S/N ratios were calculated on the basis of Taguchi's "larger is better" approach. 2. 7. Scanning Electron Microscopy Scanning Electron Microscopy with Energy Dispersive Spectroscopy (SEM-EDS) was used to characterize the structures of the samples (JEOL SEM 7700F) in the Research Centre Laboratory at Mugla Sitki Koçman University (Turkey). 2. 8. FTIR Analysis FTIR spectrum of the samples were performed in Per-kin Elmer Each spectrum was recorded in a frequency of 400-4000 cm1 using potassium bromide (KBr) disc. The KBr was oven-dired to minimize the interference of water. 2. 9. The Determination of pHpzc Batch equilibrium experiments were used to estimate zero point charge (pHpzc). 50mL of 0.01M NaCl solu- Yildiz et al.: Biosorption of 2,4 dichlorophenol Onto Turkish 240 Acta Chim. Slov. 2017, 64, 237-247 tion was poured into several erlenmeyer flasks. The pH of solution for each flask was adjusted to a value between 2 and 12 by addition of 0.1M HCl or 0.1M NaOH solution. Then, 0.10 g of adsorbent was added to the flasks and the dispersion was stirred for 48 h. After this time the final pH was measured.A plot of the final pHf as a function of the initial pHi provides pHpzc of the adsorbents by the plateau of constant pH to the ordinate.34 3. Results and Discussion 3. 1. Optimization Study As the orthogonal array experimental design method was found to be the most appropriate for the conditions under investigation, it was chosen to determine the experimental plan, L25 (55) (Table 2); four parameters each with five values. The data (yi) and corresponding S/N ratios were calculated on the basis of Taguchi's "larger is better" approach using Eq. 2 S/N Oram = -10.log[X(1/Y2)/n] (2) In order to calculate the effects of parameters, S/N ratio was averaged for each level. The effect of the noise sources on the adsorption process was observed by repeating each experiment twice under the same conditions. The sequence, in which the experiments were carried out, was randomized to avoid any personal or subjective bias. In the proposed method, no interaction between the variables was found in the matrix and the focus was placed on the main effects of the four most important factors. The optimum design for the adsorption of 2,4-DCP by Sweet-gum bark is an important aspect in the production of the adsorption process. It can be concluded that the values of optimum experimental parameters for adsorption capacity of 2,4-DCP are as below: contact time (150 min) , amount biosorbent (1 g), initial concentration of adsorbate (150 mg/L) and pH (2) (figure1). Taguchi method predicted that the adsorption efficiency under the optimum conditions will be 90.2371%. Under these optimum conditions, it was determined that the 2,4- DCP adsorption efficiency was 89.2158%. 3. 2. Influence of pH The previous studies have shown that pH of the solution is a critical parameter affecting biosorption of 2,4-DCP.7,12,35 The pH ranges of 2-10 were used in this study to ensure the presence of the protonated form of 2,4-DCP and the increase of negative charges at the surface of the particles of bark of sweetgum. The initial pH of the solution was increased with the decrease in the adsorption capacity of 2,4-DCP (figure 2). The figure shows that maximum adsorption capacity of 2,4-DCP was observed at a pH of 2.0. Also it was found the same values of initial pH of the solution using Taguchi's Orthogonal Array (OA) analysis (figure 1). Factor levels for predictions Predicted values pH Amount of Initial concentration of Contact time S/N Ratio Mean 1 5 4 5 40,6185 90,2371 Figure 1. Main effects plot for SN ratios, Factor levels for predictions, Predicted values Yildiz et al.: Biosorption of 2,4 dichlorophenol Onto Turkish 241 Acta Chim. Slov. 2017, 64, 237-247 Figure 2. The effect of pH on the equilibrium sorption capacities of sweetgum bark, for 2,4-DCP The Henderson-Hasselbalch equation (pH= pKa + log ^ ^/[AH] 'is usclul f°r estimating the pH of acidic compounds, such as 2.4-DCP. The value of p-Ka for 2,4-DCP which is known to be weak acid is 7.85. The value of pH (2) is lower than p^a (7.85), the dissociation degree of 2,4-DCP to form anions increases. The sweetgum bark consists of hydrolyzable tannin compounds.36 The hydroxyl groups of the carbohydrate in hydrolyzable tannin compounds provide negative charge in surface of the biomass as the pH increases. Consequently, the electrostatic impulse between the identical charged target molecules decreases the adsorption capacity of 2,4-DCP in increasing pH of the 2,4-DCP in aqueous solution. 3. 3. Effect of Contact Time and Initial Concentration The relationship between contact time and 2,4-DCP sorption on sweetgum bark at different initial 2,4- DCP concentrations is presented in Figure 3. The rate of sorption capacities increased slightly at contact time of 150 min. The sorption was not very rapid and the equilibrium time for 2,4-DCP calculated from this study is more than what is reported for phenols onto different biomass.1 The initial concentration of aqueous solution ensures an important locomotive strength to accomplish all mass transfer resistances of adsorbate between the aqueous solid phase and therefore increases the rate of adsorbate molecules passing from the solution to the adsorbent surface.1,37-39 Accordingly, a low initial concentration of 2,4-DCP would decrease the process of adsorption (Figure 3). Also Taguchi's Orthogonal Array (OA) analysis indicates that the optimum of equilibrium time and initial concentration of 2,4-DCP in this study are 150 min and 150 mg/L, respectively (Figure 1). 3. 4. Adsorption Kinetic Models The pseudo - first-order model and the pseudo - second-order model were performed to the experimental pa- Figure 3. The sorption equilibration time of 2,4-DCP by dried sweetgum bark (biomass: 1 g, 2,4-DCP concentration: 25, 50, 100, 150, 200 mg/I: temperature 20 ± 0.5, agitation rate: 125 rpm) Yildiz et al.: Biosorption of 2,4 dichlorophenol Onto Turkish ... 242 Acta Chim. Slov. 2017, 64, 237-247 rameters to evaluate the adsorption kinetics of 2,4-DCP onto sweetgum bark in this study. 3. 4. 1. Pseudo-first-order Model and Pseudo-second-order Model The kinetic of biosorption by any biological material in an aqueous solution has been tested for the pseudofirst-order model equation given by Lagergren. The pseudo-second-order model may provide a better description of the adsorption kinetics.7'22 The pseud-first-order Lagergren equation is: where qe and qt are the amount of 2'4-DCP adsorbed per unit of biomass (mg/g) at equilibrium and at time t, t is the contact time (min) and k is the rate constant of this equation (1/min). The values of K and qecal were calculated from a plot of log (qe-qt) versus t. The pseudo-second-order kinetic equation is22'39 where h represents the initial adsorption rate (mg/g min), and K2 is the rate constant in the pseudo-second-order kinetic model (g / mg.min). The values of qecal, K2 and h can be obtained by a linear plot of t/qt versus t. The linear regression correlation coefficient (R2) values for Lagergren-first order kinetic model ranged from 0.8140 to 0.9922, which was lower than the R2 values for Pseudo-second order kinetic model which ranged from 0.8140 to 0.9999 (Table 3). The reaction involved in present biosorption system may not be of the Lagergren -first-order kinetic model. The whole range of data might not be sufficiently described by the Lagergren-first order kinetics. Moreover, the qecal values for pseudo-second-order kinetic model were closer to the experimental qe values than the calculated qecal values for Lagergren -frist-order kinetic model and, also, calculated qecal values agreed with experimental qe values for pseudo-second-order kinetic (Table 3). These values show that pseudo-second-order kinetic fits for the biosorption of 2,4- DCP on the sweetgum bark. The Pseudo-second-order kinetic model was suitable for all the data. The process of the Pseudofirst-order kinetic model has been used for adsorption of reversible with an equilibrium being established between Table 3. Parameters of Lagergren-first order kinetic model Pseudo-second order kinetic model for 2,4- DCP adsorption onto sweetgum bark (pH: 2; biomass: 1 g, temperature 20 ± 0.5, agitation rate: 125 rpm) (mg/L) Lagergren-first order kinetic model Pseudo-second order kinetic model 2,4 DCP qe(mg/g) K1(min-1) qecal (mg/g) R2 qecal (mg/g) K2 (gmg-1) R2 25 0,269 1,74 0,029 0,8124 0,514 0,0187 0,814 50 0,963 2,43 0,004 0,9922 1,049 0,0257 0,8619 100 2,013 1,74 0,029 0,8124 2,077 0,0984 0,9999 150 5,243 1,28 0,013 0,9609 5,482 0,0182 0,9967 200 4,828 2,16 0,012 0,9483 4,900 0,0584 0,9995 Figure 4. Graphical representation of Pseudo-second order kinetic model Yildiz et al.: Biosorption of 2,4 dichlorophenol Onto Turkish ... Acta Chim. Slov. 2017, 64, 237-243 247 Figure 5. Pseudo-second -order plots at different initial 2.4 DCP concentrations (pH: 2; biomass: 1 g, temperature 20 ± 0.5, agitation rate: 125 rpm) adsorbate and adsorbent systems although the process of the Pseudo-second-order kinetic model demonstrates che-misorptions which control the adsorption such as Vander Waals, hydrogen bonding, ion exchange etc. 40 The process of 2,4- DCP adsorption in sweetgum bark may be chemisorptions. It is possible to see similar adsorbent performance for each of the three plots in initial concentrations 100, 150 and 200 ppm when they are compared with each other's in Pseudo-second-order plots. However, R2 values are different. The maximum R2 value is found at 150 ppm (Table 3, Figure 5). Also, it is possible to say the sorption system reached the final equilibrium plateau after 100 min and it started desorption after 150 minutes for initial concentrations 100, 150 and 200 ppm (Figure 3). This situation may demonstrate that there are surface binding sites on the biomass for the biosortion of 2,4-DCP and a number of biosorption mechanisms that included many factors such as physico-chemical adsorption, com-plexation, ion-exchange and micro-precipitation. 3. 5. Adsorption Isotherm Models Adsorption isotherm models are important in order to describe the sorption process. The data of adsorption isotherm models are also important to predict the adsorption capacity and describe the surface properties and affinity of the adsorbent.22 Two adsorption isotherm models were used to studies in the present study: the Langmuir isotherm model and Freundlich isotherm model. The general Langmuir equation whose linearized form is given as follows: where Ce is the equilibrium concentration of the adsorbate (mg/L), qe is the amount of the adsorbate adsorbed per unit mass of the adsorbent (mg/g), b is the Langmuir adsorption constant (L/mg), and Qm is the maximum adsorption amount (mg/g). Qm and b can be determined from the linear plot of Ce/qe versus Ce.1,22 The dimensionless separation factor or equilibrium constant (RL) describes the essential characteristics of Langmuir isotherm. RL is defined as; where Co is the initial concentration (mg/I), and b is the Langmuir constant. Table 4 indicates dimensionless separation factor. The Freundlich isotherm is an empirical relationship that describes the sorption on a heterogeneous surface. It can be linearized in logarithmic form as follows: where Ce is the equilibrium concentration of the adsorbate (mg/L), qe is the amount of the adsorbate adsorbed per unit mass of the adsorbent (mg/g), Kf and n are the Freundlich constants, whereas Kf and n are indicators of adsorption capacity and adsorption intensity of the sorbents, respectively.18 The regression correlation coefficients (R2) values of Freundlich isotherm model and Langmuir isotherm Table 4. The dimensionless separation factor.39 rl > 1 = 1 097%) from Sigma Aldrich were used for the liquid-liquid extraction. Both Cr(III) and Cr(VI) stock standard solutions containing 1000 mg L1 of Cr were obtained from Fluka. 2. 2. Apparatus To prepare sample solutions ultra-pure water was used, which was made using a Millipore Milli-Q RG apparatus. The pH of the solutions was measured with a pH Kapitany et al.: Separation/preconcentration of Cr(VI) with 250 Acta Chim. Slov. 2017, 64, 248-255 Table 1. Heating program I for the determination of total chromium in aqueous phase with Varian AA-20 GFAAS instrument. Step Temperature [°C] Time [s] Ramp Hold Argon flow rate [cm3 min-1] 1 110 1 15 250 2 130 6 10 250 3 1500 8 10 250 4 2300 0 5 0 5 2450 1 3 250 Table 2. Heating program II for the determination of chro-mium(VI) in chloroform with Varian AA-20 GFAAS instrument. Step Temperature [°C] Time [s] Ramp Hold Argon flow rate [cm3 min-1] 1 45 1 5 250 2 85 6 40 250 3 1000 15 15 250 4 2300 0 5 0 5 2450 1 3 250 meter made by HANNA Instrument. For the chromium analysis a graphite furnace atomic absorption spectrometer (Varian AA-20 + GTA 96) was used. The injected volume of the samples was 20 pL. The temperature program of the furnace was customised for proper determination as seen in Table 1 and Table 2. Chromium measurements were carried out at 357.9 nm wavelength with a spectral bandwidth of 0.5 nm. Argon 99.996% (Linde Hungary) was used as protective gas and integrated absorbance (peak area) was used for the determination. 2. 2. 1. The Modified SDME Cell The aim of the developed extraction cell was to reduce the disadvantages of the SDME. The procedure is as follows: first, the droplet is sitting, not hanging. This configuration increased the stability of the droplet. The new glass cell is hollowed for the organic droplet (Fig 1). It has two main components: an extraction cell and glass stopper. The extraction procedure is the following: the closed cell is filled with distilled water, and after that the cell is opened so that the organic droplet can be placed in it. Then it is closed. The sample solution is introduced into the extraction cell with a syringe pump. After the extraction the organic droplet can be removed with syringe or pipette. The 10 -100 pL micropipette (Biohit) was a better solution. The Hamilton syringe for GC was problematic because it had metal parts and the extraction solvent reacted on it. The results were increased blank values for chromium. The advantages of this cell geometry were the following: first, it stabilised the droplet, increased robustness Figure 1. The new extraction cell of the extraction and ensured a higher flow rate of the sample. Second, the droplet volume was increased to 40 pL to provide a greater contact area between the two phases and a higher extraction efficiency. On the other hand the higher droplet volume was better for GFAAS determination. The droplet could be introduced into the graphite tube or the vials of the autosampler. At this experiment 40 pL of the chloroform droplet was used and the ion-pair agent was dissolved in chloroform to separate and enrich the Cr(VI) content of the sample. 2. 2. 2. The Recirculating Single-drop Microextraction Device This system is an upgraded version of the above mentioned system. It is understood that extraction efficiency can be increased by repeating the procedure. Our aim was to construct an extraction system to multiply the single-drop extraction. The result was the recirculating single drop microextraction system shown in Fig. 2. This system consists of a sample reservoir (25 mL beaker), a peristaltic pump (MTA KUTESZ LS-204), an extraction cell, a Hoffmann clamp and a Tygon tube (i.d.: 0.76 mm). Figure 2. Recirculating single drop micro extraction device with peristaltic pump: 1 sample reservoir, 2 peristaltic pump, 3 extraction chamber, 4 Hoffmann clamp 5 Tygon tube Kapitany et al.: Separation/preconcentration of Cr(VI) with Acta Chim. Slov. 2017, 64, 248-255 251 The Hoffmann clamp was needed to set the back pressure, as the extraction chamber could be fully loaded with the sample solution. The procedure is as follows: first, the beaker is filled with the sample and the tubes are inserted into the sample solution. After that the whole system is filled using the peristaltic pump and finally, the droplet is ready to be inserted into the extraction cell. The additional advantages of this system over the modified SDME are increased extraction efficiency thanks to the recirculating sample and easier sample changing, as the syringe pump is replaced with peristaltic pump and during the clean-out procedure only the inlet tube has to be put into the distilled water. 3. Results and Discussion 3. 1. Method Development The principal steps of the method are: adjusting the pH of the water sample, extraction with the new system and finally GFAAS measurement to determine the chromium concentration in the chloroform. Optimal parameters, such as pH, time, reagent concentration and GFAAS heating programme were explored for each step. The aqueous phase volume was set to 10 mL. 3. 1. 1. Optimization of the Extraction We tested the extraction range of 1.0 - 7.0 pH with 0.5 steps. 1.0 mol/L HCl and 0.1 mol/L NaOH was used to set the pH. The optimum pH range of this extraction was found from 2.0 - 5.0 pH. Thus for all further analyses, we used 4.0 pH, and it was adjusted with acetic acid / sodium acetate buffer (10 mL sample solution + 1 mL buffer). 1 L buffer was prepared from 847 mL 0.1 mol/L acetic acid and 153 mL 0.1 mol/L sodium acetate. Methyltrioctylammonium chloride concentrations in the chloroform were investigated in the range of 0.1 - 5 % (w/w) and the ideal was found at 1 % (w/w). Probably at the high methyltrioctylammonium chloride concentration, there is a negative effect on GFAAS determination, because too much organic material was introduced into the graphite tube and at the ashing step chromium losses occurred. The flow rate of the sample solution was investigated. Previously with the syringe pump the optimal flow rate was 1.0 mL/min with single extraction. The peristaltic pump was used in the range of 2.5 - 14.0 mL/min and the extracted Cr(VI) linearly increased by the flow rate (Fig. 3). At the higher flow rate, the droplet immediately ran out from the extraction cell. The flow rate was reduced to 11.5 mL/min to ensure the stability and repeatability of this method. At this parameter, 10 mL of the sample circulated in the extraction cell 11.5 times in 10 min. This was a remarkable signal increase with GFAAS measurements compared to previous SDME sample preparation. Figure 3. The effect of the flow rate of the sample solution on ab-sorbance. Sample volume was 10 mL, Cr(VI) concentration was 1 |g/L (pH = 4) Extraction time was 10 min (GFAAS, 40 |L droplet volume was diluted to 100 ||L) The extraction time was investigated in the range of 1 - 35 min (Fig. 4). We found that the chromium concentration of the droplet linearly increased in the range of 1 -15 min. 0.480 0.400 0.320 " 0.240 o m si < 0.160 0.080 0.000 10 20 30 Extraction time, minute 40 Figure 4. Extraction time effect on the absorbance at 11.5 mL/min flow rate and 10 mL sample volume, concentration of the Cr(VI) was 1 |g/L (pH = 4) (GFAAS, 40 |L droplet was diluted to volume 100 |L, 0.01 mol/L methyltrioctylammonium chloride in droplet) We limited the extraction time to 10 min to take in account the throughput of this method, and all further measurements were carried out in 10 min. Kapitany et al.: Separation/preconcentration of Cr(VI) with 252 Acta Chim. Slov. 2017, 64, 248-255 2. Two kinds of calibration standards were prepared. One of them was diluted from the Cr(III) stock standard in water. The other was diluted from the Cr(VI) stock standard in water and was extracted by chloroform with methyl-trioctylammonium chloride in 13 mL plastic test tube with screw cap. This liquid-liquid extractions were carried out at an optimum pH and methyltrioctylammonium chloride concentration. The ratio of the phases was 1:1 (3 + 3 mL). Heating programme I was used for the water phase and programme II was used for organic solutions. The results are shown in Fig 6. The sensitivity of the chromium determination was decreased in organic media. Therefore, the extraction calibration had to be used for Cr(VI) determination. This calibration method was used for all further measurements at Cr(VI) determination. Figure 5. Effect of droplet volume on the relative absorbance (0.25 Hg/L pH = 4), flow rate was 11 mL/min (GFAAS, droplet volume was diluted to 100 ^L) The volume of the droplet was investigated between from 10 - 70 pL and the ideal volume was found at 40 pL (Fig 5). At a 70 pL droplet volume, the efficiency of the extraction was decreased. 3. 1. 2. Optimisation of the Heating Programme The graphite heating was optimized for organic media with a high-concentration of methyltrioctylammonium chloride. The right drying and ashing steps had to be used to maximise the chromium(VI) signal at GFAAS determination. The modified programme is shown in Table 3. 2. Method Validation The 1000 mg/L Cr(VI) stock standard was used and the calibration standards were established by dilution with distilled water. These prepared solutions (10 mL sample + 1 mL buffer) were extracted by this method and the chloroform phase was analysed by the GFAAS. The recovery analysis was carried out with three different known quantities of Cr(VI). These spiked samples were processed as normal samples. The method was validated for linearity with 0.14 - 5.00 pg/L Cr(VI). The equation of the calibration curve was y = 0.2958x + 0.0395 (R2 = 0.99), where 'y' is the peak-area and 'x' is the concentration of Cr(VI). The detection limit of the method was 42 ng/L. The recovery of Cr(VI) in spiked tap water samples ranged from 97% to 101.1% and the precision of the measurements was from 2.38% to 2.81% (Table 3). Regarding the result for the repeatability of this method, 2.53% was observed. Table 3. Cr(VI) recovery of the developed method (3 replicates) Sample Cr(VI) in aqueous phase |g L-1 Added Determined Recovery, % RSD % 1 1.00 0.97 97.0 2.53 2 3.00 2.96 98.6 2.81 3 8.00 8.09 101.1 2.38 Figure 6. GFAAS calibration curve for Cr(III) in water phase and Cr(VI) in organic phase with 1 % (w/w) methyltrioctylammonium chloride in chloroform. 3. 3. An optimised Method for Cr(VI) Analysis The optimised procedure was: first, the sample pH was set to 4 with acetic acid and sodium acetate buffer. A 10 mL sample and 1 mL buffer were introduced to the beaker, the flow rate was set to 11.5 mL/min and the extraction time was 10 min. In this procedure, the methyl-trioctylammonium chloride concentration in the chloro- Kapitany et al.: Separation/preconcentration of Cr(VI) with Acta Chim. Slov. 2017, 64, 248-255 253 form droplet was 1 % (w/w). After the extraction, 40 pL of chloroform was diluted to 100 pL to ensure enough sample volume for the autosampler. Finally 40 pL of the sample was introduced into the graphite tube and the chromium content was determined with the optimized heating programme. At this method the enrichment factor (EF) was 100. 3. 4. Analysis of the Real Samples The developed method was tested with sea water samples. The water samples were collected from same location at different time. The results were summarized in Table 4. Table 4. Bulgarian Black Sea water samples 2016 (n = 3, RSD < 3%) Date Cr(VI) (pg/L) Total Cr (pg/L) I.16. 0.28 0.72 III. 26. 0.24 0.97 V. 28. 0.17 0.28 VI. 29. 0.17 0.66 VII. 20. 0.17 0.45 VIII. 21. 0.21 0.41 The Cr(VI) and total chromium concentrations were determined in sea water samples and the results were in good agreement with other research.42 3. 5. Comparison to Other Methods Our developed method has very good limit of detection compared to other cited methods in Table 5. The advantage of the developed method is that the sample volume is freely variable. Large amount of water sample can be used to increase the enrichment. The higher volume of the droplet and the recirculation of the sample solution around the droplet leads to higher efficiency of extraction than possible with the normal SDME. This off-line method can be easily adapted to any GFAAS instrument and there is no need to modify the expensive instrument. This SDME technique can be applied to other analytical task, where a high enrichment of the analyte is important. There are a number of potential drawbacks to the SDME method. Low sample throughput as it takes 10 min, but other SDME methods require the same extraction time. Highly skilled lab worker is needed to set the droplet and this device. The chloroform is volatile, therefore the temperature has to be controlled and before the GFAAS the sample vials have to be sealed to avoid evaporation of the organic solvent. This effect can cause unexpected increase of the chromium concentration. Currently, extraction cell is not commercially available, because it has to be made manually. Table 5. Comparison of Cr(VI) determination methods for water samples Extraction method Analytical method LOD (re/L) Automation approach Reference Continuous This SDME GFAAS 0.042 research SPE ICP-AES 0.200 FIA 43 CME ICP-MS 0.018 FIA 20 SPE FAAS 0.034 FIA 44 SPE FAAS 0.8 FIA 45 SPE FAAS 0.3 SIA 23 SPE GFAAS 0.02 SIA 24 SPE FAAS 45 46 SPE GFAAS 0.027 47 CPE FAAS 0.18 48 CPE GFAAS 0.01 49 - UV/VIS 23 SIA 21 - UV/VIS 5.6 |SIA-LOV 26 LLE UV/VIS 7.5 FIA 50 LLME UV/VIS 0.26 SIA 22 DLLME FAAS 0.08 51 DLLME TXRF 0.8 52 Thermal GFAAS 0.7 38 CME: capillary microextraction, SDME: single drop microextraction, CPE: cloud point extraction, SPE: solid phase extraction, LLE: liquid-liquid extraction, LLME: liquid-liquid microextraction, DLLME: dispersive liquid-liquid microextraction, FAAS: flame atomic absorption spectrometry, GFAAS: graphite furnace atomic absorption spectrometry, TXRF: total reflection X-ray fluorescence spectrometry, ICP-AES, Inductively coupled plasma atomic emission spectroscopy, FIA: flow injection analysis, SIA: sequential injection analysis, LOV: Lab-On-Valve 3. 6. Automatization Currently, the peristaltic pump had a timer to switch off after 10 min. We are planning to increase the automati- Figure 7. The proposed semi-automatic apparatus for chromium speciation method SY: syringe + steeper motor, HC: holding coil, DV: distribution valve (Hamilton), W: waste, S: organic solvent, PP: peristaltic pump, EC: extraction cell, AS: auto-sampler, SC: sample collector, |C: microcontroller (Arduino) Kapitany et al.: Separation/preconcentration of Cr(VI) with 254 Acta Chim. Slov. 2017, 64, 248-255 zation degree of this process. The notion is based on semiautomatic chromium speciation approach with pSIA technique. First, the Arduino microcontroller coordinates the syringe, distribution valve, peristaltic pump and tip of the extraction cell. The next step is to couple the developed device with an autosampler and sample collector (Fig. 7). This system is containing new and salvaged parts to reduce the cost. The Arduino microcontroller is easily programmable, cheap and easy to connect the display, relay control and motor driver boards. Further plan is to replace the sample collector (SC) with the autosampler of the GFAAS to achieve the full automatization. The planned fully automated sample preparation system will be useful to determine the Cr(VI) by GFAAS. 4. Conclusions In this study a novel single drop microextraction (SDME) technique is presented for chromium speciation. The advantages of this method are, in addition to minimal organic solvent consumption and the need for only one droplet per sample to extract, the higher stability of the drop and the possibility of high enrichment of the analysed elements. The higher volume of the droplet in a modified cell and the recirculation of the sample solution around the droplet leads to higher efficiency of the extraction than is possible with the normal SDME. 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Acta, 2009, 166, 69-75. https://doi.org/10.1007/s00604-009-0167-x 52. Z. Bahadir, V. N. Bulut, M. Hidalgo, M. Soylak, E. Margui, Spectrochim. Acta B, 2016, 115, 46-51. https://doi.org/10.1016/j.sab.2015.11.001 Povzetek Razvili smo metodo za speciacijo in predkoncentracijo kroma, ki uporablja tehniko atomske absorpcijske spektrometrije z grafitno kiveto (GFAAS). Metoda je osnovana na tehniki mikroekstrakcije v kapljico (SDME). Dandanašnji so mi-kroekstrakcije postale popularne, saj je za separacijo potrebna majhna količina organskega topila. Vzorec je v ekstrakcij-ski celici v stiku z eno samo kapljico kloroforma. Za separacijo in obogatitev kromovih zvrsti smo uporabili ionsko-par-no spojino. Po ekstrakciji smo vsebnost kroma v kapljici določili z GFAAS. Analizna občutljivost se je izboljšala glede na standardno SDME tehniko zaradi večjega volumna organske faze in zaradi kroženja vzorca. Zaradi večje stične površine in razvite ekstrakcijske naprave je bila tudi stabilnost kaplice znatno večja. Kot primer uporabe smo določili vsebnost Cr(VI) v morski vodi s tehniko GFAAS in razvito separacijsko/ekstrakcijsko metodo. Pri optimiziranih ekstrakcij-skih pogojih je bilo za Cr(VI) linearno območje 0,14-5,00 |g/L, meja zaznave (S/N = 3) 0,042 |g/L in natančnost (RSD, n = 3) < 3,0 %. Prednosti metode so naslednje: cenovna učinkovitost, visoka obogatitev kromovih zvrsti in enostavna uporaba v povezavi z GFAAS tehniko. Koncentracijo kromovih zvrsti tako lahko določimo na ng/L nivoju. Kapitany et al.: Separation/preconcentration of Cr(VI) with 256 DPI: 10.17344/acsi.20l6.2947_Acta Chirn. Slov. 2017, 64, 256-260_©commons Short communication About the Randi} Connectivity, Modify Randi} Connectivity and Sum-connectivity Indices of Titania Nanotubes TiO2(m,n) Wei Gao,1 Mohammad Reza Farahani2 and Muhammad Imran3'4* 1 School of Information Science and Technology, Yunnan Normal University, Kunming 650500, China 2 Department of Applied Mathematics, Iran University of Science and Technology (IUST), Narmak, Tehran 16844, Iran 3 Department of Mathematical Sciences, United Arab Emirates University, P. O. Box 1551, Al Ain, United Arab Emirates 4 School of Natural Sciences, National University of Sciences and Technology, Sector H-12, P.O. 44000, Islamabad, Pakistan * Corresponding author: E-mail: imrandhab@gmail.com Phone: +97 137136389, Fax: +971 3 7671291 Received: 28-09-2016 Abstract The Randic Connectivity Index R(G) is one of the oldest connectivity index, introduced by Randic in 1975. Another connectivity indices is the Sum-Connectivity Index X(G) introduced in 2008 by Zhou and Trinajstic. Recently in 2011, a modification of the Randic Connectivity Index of a graph G was introduced by Dvorak et al. In this paper, we compute these connectivity topological indices for a family of molecular graphs known as titania nanotubes TiO2(m,n). Keywords: Molecular graph, Nanotubes, Titania nanotubes TiO2(m,n), Topological indices, Randic index, Sum-connectivity index, Modify Randic index, Zagreb index, Multiple Zagreb index. 1. Introduction A graph is a collection of points and lines connecting a subset of them. The points and lines in a graph are respectively called vertices and edges of the graph. An edge in E(G) with end vertices u and v is denoted by uv. Two vertices u and v are said to be adjacent if there is an edge between them. In chemical graph theory, the vertices of molecular graph G correspond to the atoms and its edges correspond to the chemical bonds. We denoted the order and size and degree of a vertex/atom v of a molecular graph G by \V(G)\, \E(G)\ and dv, respectively. The set of all vertices adjacent to a vertex v in V(G) is said to be the neighborhood of v, denoted as N(v). The number of vertices in N(v) is said to be the degree of v. The minimum and maximum vertex degrees in a graph G denoted by 8(G) and A(G), respectively and are defined as min{dv \ veV(G)} and max{dv \ ve V(G)}, respectively. Our notation is standard and mainly taken from standard books of chemical graph theory.1-3 We have many connectivity topological indices, for an arbitrary graph with connected structure in chemical graph theory. The oldest of them is Randic Connectivity Index which has shown to reflect molecular branching, introduced by Milan Randic in 1975,4 and defined as (1) where, du and dv are the degrees of the vertices u and v, respectively. Another connectivity indices is the Sum-Connectivity Index that was introduced by Zhou and Trinajstic in 2008.5'6 The sum-connectivity index X(G) is defined as the sum over all edges of the graph of the terms du + dv)-2/2 and is equal to (2) Recently in 2011, Dvorak et al. introduced a modification of the Randic Connectivity Index of G and is defined as Gao et al.: About the Randic Connectivity, Modify Randic Acta Chim. Slov. 2017, 64, 256-260 257 ff'(G) ^ I uvEE(G) 1 max{du dp}' (3) that is more tractable from computational point of view. It is much easier to compute Modify Randic index R'(G) than Randic index R'(G) (see7 for more details). Some basic properties of these indices can be found in the recent letters. For more study, see reference.8-13 In this paper, we investigate the topological Connectivity indices, and compute some formulas for the Randic, Sum-Connectivity and Modify Randic indices of a family of molecular graphs that called titania na-notubes TiO2(m,n) for positive integers n, m (see Figure 1). 2. Main results and Discussion In this section, we compute the Randic, Sum-connectivity and Modify Randic Indices for the titania nano-tubes TiO2(m,n) (V m,neN). Titania nanotubes were systematically synthesized during the last 10-15 years using different methods and carefully studied as prospective technological materials. Since the growth mechanism for TiO2 Nanotubes is still not well defined, their comprehensive theoretical studies attract enhanced attention. The TiO2 sheets with a thickness of a few atomic layers were found to be remarkably stable.14-17 Molecular graphs titania TiO2(m,n) is a family of nanotubes, such that the structure of this family of nanotubes consist of the cycles with length four C4 and eight C8. Several topological indices of titania nanotubes (TiO2) have been studied in the literature.18-20 Let us denote the number of Octagons or cycles C8 in the first row and column of the 2- Dimensional lattice of TiO2 nanotubes (Figure 1) by m and n, respectively. Theorem 1. Let TiO2(m,n) be the titania nanotubes for positive integers m,n. Then the following indices are calculated by formulas: Figure 1. A 2-Dimensional Lattice of the titania nanotubes TiO2(m,n) (V m, neN).17 - The Randic Connectivity index R(Ti02(m,n))=[^^m + L 5 / 4E1/2 + 3VÏQ+ S1/3+2V1S\ 1 — )]n (4) + is - The Sum-Connectivity index X(Ti02(m,n)) = 3V2 4V7\ -+- m + 2 7 J (5) - The Modify Randic index R'(TiO2(m,n)) = 2n(m +1). (6) Before we prove the main results, let us introduce some definitions. Definition 1. Consider the graph G = (V, E), then we divide the vertex set V(G) and edge set E(G) of G into several partitions based on the degrees of vertices/atoms in G as follows.9 = (uv e E(G)|du X dv = j}. (7) Where du(1 < dv < n - 1) be the degrees of ve V(G) and S and A are the minimum and maximum, respectively. In particular, let G = (V, E) be a connected molecular graph or nanotubes, then we can divide the vertex set and edge set of G in following partitions: (8) Since the degree of an atom (or vertex) of the molecular graph is equal to 1, 2,..., 5 and the hydrogen atoms (with degree 1) in G are often omitted. In particular, let TiO2(m,n) be the titania nanotubes (Vm,«eN) with 6n(m+1) vertices and 10mn+8n edges, then from its structure, the vertex and edge partitions of Gao et al.: About the Randic Connectivity, Modify Randic 258 Acta Chim. Slov. 2017, 64, 256-260 the vertex set V(TiO2(m,n)) and edge set E(TiO2(m,n)) and their order and size are as follow.17 and Es = {uv £ E(Ti02(m, n))|du +dv = ó}, EB* = {uv E E(Ti02(m,n))|du X dv = 8}, |Eb*| = 6n (9) (10) By above mentioned formulas, one can see that |v(Ti02(m, n))| = (2mn + 4n) + 2mn + |E(Ti02(m,n))| = 1/2 [2 X (2mn + 4n) + -I- 3 X 2mn + 4 X 2n + 5 X 2mn] = lOmn + 8n. Now, we have the following computations of the Randic, Sum-connectivity and Modify Randic Indices for the titania nanotubes TiO2(m,n) V m,n e N. R(Ti02(m,n)) = ^ —= uvEE(Ti02 [m.,?l]) yj du ^p y i | y i | y 1 | y 1 ¿->uvEEs*jdudv ¿->uviE10> ^Jdudv ¿->uvEE12" jdudv ^«rE£ls*1/dBd„ =(6n) x (vfe)+2n(2m +1} x bk)+ C2n) * (A)+C6mn"2n) x hk) (12) r n(2m + 1)VTÖ V3 V15 = 3nv2 H---1--n H--(6mn — 2n) 5 3 15 2 , r— ,—, / r VlÖ V3 2VÍ5A - (Vl5 + VIO)mn + 3\2 + —— H--H--— n. 5 y 5 3 15 y Thus the Randic connectivity index of TiO2(m,n) nanotubes is equal to (13) Gao et al.: About the Randic Connectivity, Modify Randic Acta Chim. Slov. 2017, 64, 256-260 259 Also, X(Ti02(m,n)) = ^ vEEtJiO- liueEfi V ^ MUÉE, V^" + uueEn V+ ^ 6ti 4mn -I- 2n 2n 6mn — 2n (14) V 2+4 V2 + 5 V3 +4 a/3 4- 5 ,— 4îi(jn+l)V7 i/2 = \ 6n H--Ji H--Ji(3m — 1). 7 2 Hence the Sum-Connectivity index of TiO2(m,n) nanotubes is (15) Now, by using Definition 1, we see that there are two modify edges partitions E4+and E5+ for the titania nanotubes TiO2(m,n) (Vm,neN) as: Es+ = [uv e E(Ti02(m,n))jMax{2,5} Therefore the Modify Randic index of TiO2(m,n) is equal to: l R'(Ti02(mjn)) = uv£E(Ti02[m,n]) max{du, dv} I : + max{du, dv} max{du, dv} UVGE4 UVÊEs I edges. For enough large integer number m and n, the Randic, Sum-connectivity and Modify Randic Indices of TiO2(m,n) are equal to: (1) The Randic Connectivity index R(Ti02A[m,n]) fa (2.814m + 5.9688)n. (2) The Sum-Connectivity index X(Ti02"[m,n]) Rf (3.6332m + 3.2542)n. (3) The Modify Randic index Corollary 2. 2. Consider TiO2(m,n) nanotubes, Corollary 1 implies that for enough large integer number m,ne N, XfTiOjfnijn)) > fl(Ti02(m,n)) > R'(Ti02(m,n)). 3. Discussion Now we study the change of the values of Randic, Sum-connectivity and Modify Randic Indices of TiO2(m,n) nanotubes when the parameters m and n are slightly changed. The graphs of these nanotubes corresponding to some small values of m and n are shown in Figure 2. Similarly, the values of the studied topological indices corresponding to small change in the values of m and n is summarized in Table 1. (17) 811 lOmn - = 2mn + 2n = 2n(m + 1). Figure 2. The graph of titania nanotubes TiO2(m,n) for m = 2,4 and n = 2,4. Here, we complete the proof of main theorem of this article and all main results are computed. Corollary 2.1. Consider the titania nanotubes TiO2(m,n) Vm,neN (Figure 1), with 6n(m+1) vertices and 10mn+8n 4. Conclusion In this paper, we considered an infinite class of the titania nanotubes TiO2(m,n), that were systematically Gao et al.: About the Randic Connectivity, Modify Randic 260 Acta Chim. Slov. 2017, 64, 256-260 Table 1. Some values of Randic, Sum-connectivity and Modify Randic Indices of TiO2(m,n) nanotubes corresponding to the change in m and n. (m, n) n = 1 n = 2 n = 3 n = 4 R(G) m = 3 14.4112 28.8223 43.2335 57.6446 m = 4 17.225 34.4505 51.6758 68.9010 X(G) m= 3 14.1538 28.3076 42.4613 56.6151 m= 4 17.7869 35.5739 53.3609 71.1478 R'(G) m= 3 8 16 24 32 m= 4 10 20 30 40 synthesized during the last 10-15 years using different methods and carefully studied as prospective technological materials. We computed its connectivity topological indices including Randic index R(G) = '"" ' , Sum-Connectivity index X(G) = ' »sW' u 1 and Modify Randic index R '(G) = " "" K I, that du and dv are the degrees of the vertices u and v, respectively. 4. References 1. H. Wiener, J. Am. Chem. Soc. 1947, 69, 17-20. https://doi.org/10.1021/ja01193a005 2. R. Todeschini, V. Consonni, Handbook of Molecular Descriptors, Wiley-VCH, Weinheim, 2000. https://doi.org/10.1002/9783527613106 3. N. Trinajstic, Chemical Graph Theory, CRC Press, Boca Raton, FL, 1992. 4. M. Randic, J. Am. Chem. Soc. 1975, 97, 6609-6615. https://doi.org/10.1021/ja00856a001 5. B. Zhou, N. Trinajstic, J. Math. Chem. 2009, 46, 1252-1270. https://doi.org/10.1007/s10910-008-9515-z 6. B. Zhou, N. Trinajstic, J. Math. Chem. 2010, 47, 210-218. https://doi.org/10.1007/s10910-009-9542-4 7. Z. Dvorak, B. Lidicky, R. Skrekovski, Eur. J. Comb. 2011, 52, 434-442. https://doi.org/10.1016Zj.ejc.2010.12.002 8. F. Ma, H. Deng, Math. Comput. Model 2011, 24, 497-507. https://doi.org/10.1016/j.mcm.2011.02.040 9. M. R. Farahani, Acta Chim. Slov. 2012, 59, 779-783. 10. M. R. Farahani, Adv. Mater. Corrosion 2012, 1, 57-60. 11. M. R. Farahani, M. P. Vlad, Studia UBB Chemia 2015, 60, 251-258. 12. M. R. Farahani, K. Kato, M. P. Vlad, Studia UBB Chemia 2013, 58, 127-132. 13. G. Sridhara, M. R. Rajesh Kanna, R. S. Indumathi, J. Nano-mater 2015,16, 292. 14. R. A. Evarestov, Y. F. Zhukovskii, A. V. Bandura, S. Pisku-nov, Cent. Eur. J. Phys. 2011, 9, 492-501. 15. M. Ramazani, M. Farahmandjou, T. P. Firoozabadi, Int. J. Nanosci. Nanotechnol. 2015, 11, 115-122. 16. A. Subramaniyan, R. Ilangovan, Int. J. Nanosci. Nanotech-nol. 2015, 11, 59-62. 17. M. A. Malik, M. Imran, Acta Chim. Slov. 2015, 62, 973-976. https://doi.org/10.17344/acsi.2015.1746 18. M. R. Farahani, R. P. Kumar, M. R. R. Kanna, S. Wang, Int. J. Pharma. Sci. Res. 2016, 21, 3734-3741. 19. J. B. Liu, W. Gao, M. K. Siddiqui, M. R. Farahani, AKCE Int. J. Graphs Comb. 2016, 13, 255-260. 20. W. Gao, M. R. Farahani, M. K. Jamil, M. K. Siddiqui, Open Biotechnol. J. 2016, 10, 272-277. https://doi.org/10.2174/1874070701610010272 Povzetek Randicev indeks povezanosti R(G) je eden izmed najstarejših indeksov povezanosti, ki ga je uvedel Randic leta 1975. Drug indeks povezanosti je indeks vsote povezanosti X(G), ki sta ga leta 2008 uvedla Zhou in Trinajsti}. Nedavno, leta 2011 so Dvorak in sod. uvedli modificiran Randicev indeks povezanosti grafa G. V tem prispevku smo izračunali navedene topološke indekse povezanosti za družino molekulskih grafov znanih kot nanocevke TiO2(m,n). Gao et al.: About the Randic Connectivity, Modify Randic DOI: 10.17344/acsi.2016.3036 Acta Chirn, Slov. 2017,64, 261-265_©commons 261 Short communication A Rarely Seen Phenolato and Azido-Bridged Polymeric Cadmium(II) Complex Derived from 2-Bromo-6-[(2-isopropylaminoethylimino)methyl]phenol Guo-Ping Cheng,1 Ling-Wei Xue1* and Cai-Xia Zhang2 1 College of Chemistry and Chemical Engineering, Pingdingshan University, Pingdingshan Henan 467000, P. R. China 2 Coal Chemical Industry Branch of Shenhua Ningxia Coal Group, Yinchuan Ningxia 750411, P. R. China * Corresponding author: E-mail: pdsuchemistry@ 163.com Received: 03-11-2016 Abstract A rarely seen phenolato and azido-bridged polymeric cadmium(II) complex derived from the Schiff base ligand 2-bro-mo-6-[(2-isopropylaminoethylimino)methyl]phenol (HL) has been prepared and characterized by elemental analysis, IR spectroscopy, and single crystal X-ray diffraction. The Schiff base ligand coordinates to the Cd atom through the NNO donor set. The Cd atom is hexa-coordinated in an octahedral geometry. Adjacent two Cd atoms are bridged by two phenolato groups generating a dimer with Cd-Cd distance of 3.475(1) A. The dimers are further linked via azido bridges forming 2D sheets parallel to the bc plane. Keywords: Self-assembly; Crystal structure; Schiff base; Cadmium complex; Thermal analysis. 1. Introduction The self-assembly and construction of metal-organic frameworks is currently a hot research field due to their fascinating structures and potential applications.1 Schiff bases have long been received much attention for their preparational accessibilities, structural varieties and biological properties.2 Tri-dentate salen-type Schiff bases are capable of forming complexes with certain metal atoms which can exhibit unusual coordination, high ther-modynamic stability and kinetic inertness.3 Preparation of one-, two- or three-dimensional polymeric network by suitable metal and ligand coordination is the special area of current research because of their interesting properties, such as electrical conductivity, magnetism, host-guest chemistry, molecular separation, gas storage, sensors and catalysis.4 Among the various transition and non-transition metal atoms cadmium is an extremely toxic element that is naturally present in the environment and also as a result of human activities. The development of chelating agents is essential for the treatment of cadmium intoxication. Schiff bases have been proved to be a kind of interesting chelating agents for cadmium. A number of cadmium complexes with Schiff bases have been reported.5 Cadmium(II) due to its d10 electronic configuration, is particularly suitable for the construction of coordination polymers and networks. The spherical d10 configuration is associated with a flexible coordination environment so that geometries of these complexes can vary from tetrahe-dral to octahedral and severe distortions in the ideal polyhedron occur easily.6 The terminal or blocking co-ligands, which are usually used along with the bridging ligand to complete the metal coordination sphere, can alter the su-pramolecular assembly and consequently the type of structure formed taking the advantage of the flexibility of the coordination sphere.5a A detailed study of such complexes indicates that thiocyanate ligand is readily coordinate to the Cd atom, either through terminal mode or through bridging modes.7 As a comparison, azide, a simi- H Br Scheme 1. The Schiff base HL. Cheng et al.: A Rarely Seen Phenolato and Azido-Bridged 262 Acta Chim. Slov. 2017, 64, 261-265 lar pseudohalide group to thiocyanate, is rarely seen in the Schiff base cadmium complexes.8 As a continuation of our work on Schiff base complexes9 we report herein a rarely seen phenolato and azido-bridged polymeric cadmium(II) complex derived from the Schiff base ligand 2-bromo-6-[(2-isopropylaminoethylimino)methyl]phenol (HL; Scheme 1). 2. Experimental 2. 1. Material and Methods 3-Bromosalicylaldehyde and V-ethylethane-1,2-dia-mine were purchased from Fluka. Cadmium nitrate and other reagents were analytical grade and used without further purification. The Schiff base HL was prepared by the condensation of equimolar quantities of 3-bromosa-licylaldehyde with N-ethylethane-1,2-diamine in methanol. Elemental (C, H and N) analyses were made on a Per-kin-Elmer Model 240B automatic analyser. Infrared spectrum was recorded on an IR-408 Shimadzu 568 spectrophotometer. X-ray diffraction was carried out on a Bruker SMART 1000 CCD diffractometer. Thermal analysis was performed on a Perkin-Elmer Pyris Diamond TG-DTA thermal analyses system. Caution! Azido compounds of metal ions are potentially explosive especially in presence of organic ligands. Only a small amount of material should be prepared and it must be handled with care. 2. 2. Preparation of the Complex Schiff base HL (0.271 g, 1.0 mmol) was diluted by methanol (20 mL), to which was added with stirring a methanol solution (10 mL) of cadmium nitrate tetrahydrate (0.309 g, 1.0 mmol) and an aqueous solution (5 mL) of ammonium thiocyanate (0.076 g, 1.0 mmol). The mixture was stirred for 1 h at ambient temperature to give a colorless solution. Colorless block-shaped single crystals suitable for X-ray diffraction were formed by slow evaporation of the solution in air for a week. The crystals were filtered off and washed with cold methanol. Yield 51% (based on HL). Analysis calculated for C12H16BrCdN5O: C, 32.86; H, 3.68; N, 15.97%; found: C, 32.72; H, 3.77; N, 15.83%. Selected IR data (cm1): 3266 (N-H), 2066 (N3), 1643 (C=N). 2. 3. X-ray Diffraction Diffraction intensities for the crystal were collected at 298(2) K using a Bruker Apex II diffractometer with MoKa radiation (k = 0.71073 Â). The collected data for the complex was processed with SAINT10 and corrected for absorption using SADABS.11 The absorption correction was applied with y-scans.12 Structure of the complex was solved by direct method using the program SHELXS- 97, and was refined by full-matrix least-squares techniques on F2 using anisotropic displacement parameters.13 All hydrogen atoms were placed at the calculated positions. Idealized H atoms were refined with isotropic displacement parameters set to 1.2 (1.5 for methyl groups) times the equivalent isotropic U values of the parent carbon or nitrogen atoms. The C-H distances for CH2 and CH3 are constrained to 0.97 and 0.96 Â, respectively. The remaining C-H distances are constrained to 0.93 Â. The crystallographic data for the complex are listed Table 1. Table 1. Crystal and structure refinement data for the complex Empirical formula C12H16BrCdN5O Colour; habit Block, colorless Formula weight 438.6 Temperature (K) 298(2) Crystal system Monoclinic Space group P21/c Unit cell dimensions a (Ä) 12.142(1) b (Ä) 12.492(1) c (Ä) 10.385(1) ß(°) 106.649(3) V (Ä3) 1509.2(3) Z 4 Density (mg cm-3) 1.930 Absorption coefficient (mm-1) 4.097 Reflections collected 13740 Independent reflections 2816 Observed reflections [I > 2a(I)] 2366 Parameters/restraints 183/0 Rv wR2 [I > 2o(I)]a 0.0394, 0.0916 R1, wR2 (all data)a 0.0523, 0.1017 Goodness-of-fit 1.078 a Ri = S | | Fo|-| Fc| | /X | Fo| , WR2 = [Sw(Fo2-Fc2)2/Sw(Fo2)2]1' 3. Results and Discussion 3. 1. Description of the Crystal Structure of the Complex An ORTEP representation of the asymmetric unit of the complex is shown in Figure 1, with selected bond distances and bond angles listed in Table 2. The Schiff base acts as a tridentate ligand and chelates the Cd atoms through phenolate oxygen, imino nitrogen, and amino nitrogen, forming a five-membered chelate ring N1-Cd1-N2 and a six-membered chelate ring O1-Cd1-N1. The coordination mode of the Schiff base ligand is similar to the tri-dentate Schiff bases we reported recently.913,14 The phenola-te group of the Schiff base ligand binds two Cd atoms, generating a dinuclear subunit with Cd-Cd distance of 3.475(1) Â. The dinuclear subunits are further linked through end-to-end azido bridges forming two-dimensional sheets parallel to the bc plane (Figure 2). The azido- Cheng et al.: A Rarely Seen Phenolato and Azido-Bridged Acta Chim. Slov. 2017, 64, 261-265 263 bridged Cd—Cd distance is 6.559(2) A. Adjacent four di-nuclear subunits are linked via end-to-end azido bridges forming a 20-membered ring with dimensions of 10.36 A x 6.66 A (Figure 3). The Cd atoms are all six coordinated with distorted octahedral geometry having N4O2 donor set. The equatorial plane of the octahedral geometry is formed by pheno-lato oxygen (O1), imino nitrogen (N1) and amino nitrogen (N2) of the Schiff base ligand, and terminal nitrogen (N5A) of bridging azido ligand. The two axial positions are occupied by the phenolato oxygen (O1B) and terminal nitrogen (N3) with a trans angle, N3-Cd1-O1B, of 164.42(16)°. The distortion of the geometry from regular octahedron is evidenced from the respective cis- and trans-angles about the metal center. The N-N-N bond an- Table 2. Coordinate bond distances (A) and angles (°) for the complex Bond lengths Cd1-O1 2.269(3) Cd1-N1 2.304(4) Cd1-N2 2.365(4) Cd1-N3 2.432(5) Cd1-N5A 2.210(5) Cd1-O1B 2.455(3) Bond angles N5-Cd1-O1A 102.24(17) N5-Cd1- -N1A 174.07(18) O1-Cd1-N1 77.75(15) N5-Cd1- -N2A 105.72(18) O1-Cd1-N2 150.96(14) N1-Cd1- -N2 75.25(16) N5-Cd1-N3A 92.05(19) O1-Cd1- -N3 91.63(17) N1-Cd1-N3 82.04(18) N2-Cd1 -N3 95.04(18) N5A-Cd1-O1B 103.53(16) O1-Cd1- -O1B 85.37(12) N1-Cd1-O1B 82.39(14) N2-Cd1 -O1B 80.68(13) N3-Cd1-O1B 164.42(16) Symmetry codes: A) x, V - y, -V + z; B) 2 - x, 1 - y, - z. Figure 1. ORTEP view of the complex with atom labels. Displacement ellipsoids are shown at 30% probability level. The carbon hydrogen atoms are omitted for clarity. Atoms labeled with the suffix A are at the symmetry position: x, V - y, -V + z. Figure 2. Crystal packing of the complex viewed along the a axis. Figure 3. The azido-bridged 20-membered chelate ring. gle in the azido ligand is 178.1(6)°, slightly deviated from the linearity. The Cd-O and Cd-N distances are within normal ranges as compared to other Schiff base cadmium complexes.7 the Cd-N As expected, the Cd-Nimino is shorter than 3. 2. IR Spectrum of the Complex The solid state infrared spectrum (Figure 4) of the complex is consistent with its crystal structure result. The Cheng et al.: A Rarely Seen Phenolato and Azido-Bridged 264 Acta Chim. Slov. 2017, 64, 261-265 W avenu m be r (cm ) Figure 4. IR spectrum of the complex. weak and sharp band at 3266 cm-1 is assigned to the N-H vibration. The single and intense absorption band at 2066 cm-1 is assigned to the stretching vibrations of the azide groups. The strong absorption band centered at 1643 cm-1 is assigned to the azomethine group, v(C=N). The v(Cd-O) mode is present as a medium band at 1296 cm1. The vibrations of the Cd-O and Cd-N bonds are located at the low wave numbers of 400-700 cm-1.7 4. Conclusion A rarely seen phenolato and azido-bridged polymeric cadmium(II) complex was obtained by reaction of 2-bro-mo-6-[(2-isopropylaminoethylimino)methyl]phenol with cadmium nitrate and sodium azide in methanol. The Schiff base ligand coordinates to the Cd atom through the NNO donor set, and the azido ligands bridge Cd atoms, to form a polymeric structure. The complex is stable up to 190 °C. 5. Supplementary Material CCDC-945893 contains the supplementary crystal-lographic data for this paper. These data can be obtained free of charge at http://www.ccd.ccam.ac.uk/const/retrie-ving.html or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033 or e-mail: deposit-@ ccdc.cam.ac.uk. 6. 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Mathews, Acta Crystallogr. 1968, A24, 351-355. https://doi.org/10.1107/S0567739468000707 13. G.M. Sheldrick, SHELXL-97, Program for the Refinement of Crystal Structures, Gottingen (Germany): University of Gottingen, 1997. 14. (a) X.-M. Hu, L.-W. Xue, C.-X. Zhang, W.-C. Yang, Synth. React. Inorg. Met.-Org. Nano-Met. Chem. 2015, 45, 17131716; https://doi.org/10.1080/15533174.2013.867881 (b) L.-W. Xue, X. Wang, G.-Q. Zhao, Synth. React. Inorg. Met.-Org. Nano-Met. Chem. 2012, 42, 1334-13 Povzetek Predstavljamo redek primer polimernega kadmijevega(II) kompleksa z mostovnima fenolato in azido skupinama z uporabo Schiffove baze 2-bromo-6-[(2-izopropilaminoetilimino)metil]fenola (HL) kot liganda. Kompleks smo okarakteri-zirali z elementno analizo, IR spektroskopijo in monokristalno rentgensko difrakcijo. Schiffova baza je koordinirana na Cd atom preko NNO donorskega seta. Cd atom je heksakoordiniran z oktaedrično geometrijo. Sosednja dva Cd atoma sta mostovno povezana preko dveh fenolatnih skupin, pri čemer tvorita dimer s Cd-Cd razdaljo 3,475(1) A. Dimeri so nadalje povezani preko azido mostov in tvorijo 2D plasti vzporedne z bc ravnino. Cheng et al.: A Rarely Seen Phenolato and Azido-Bridged Acta Chim. Slov. 2017, 64, (1), Supplement S55 DRU[TVENE VESTI IN DRUGE AKTIVNOSTI SOCIETY NEWS, ANNOUNCEMENTS, ACTIVITIES Vsebina Doktorska in magistrska dela, diplome v letu 2016 ........................... S2 Koledar važnejših znanstvenih srečanj s področja kemije, kemijske tehnologije in kemijskega inženirstva............................. S49 Navodila za avtorje ................................................... S56 Contents Doctoral theses, master degree theses, and diplomas in 2016 ................... S2 Scientific meetings - chemistry, chemical technology and chemical engineering . . S49 Instructions for authors ............................................... S56 Društvene vesti in druge aktualnosti S2 Acta Chim. Slov. 2017, 64, (1), Supplement S55 UNIVERZA V LJUBLJANI FAKULTETA ZA KEMIJO IN KEMIJSKO TEHNOLOGIJO 1. januar - 31. december 2016 DOKTORATI DOKTORJI ZNANSOTI KEMIJA Petra ZALAR TERMODINAMSKE IN TRANSPORTNE LASTNOSTI POLIANETOLSULFONATOV Z RAZLIČNIMI PROTIIONI Mentor: prof. dr. Ciril Pohar Datum zagovora: 30. 9. 2016 Slavko KLOBČAR IZOLACIJA STRUKTURNO SORODNIH NEČISTOČ ATORVASTATINA S SUPERKRITIČNO TEKOČINSKO KROMATOGRAFIJO Mentorica: prof. dr. Helena Prosen Datum zagovora: 7. 7. 2016 Nina FRANČIČ SOL-GEL NANOS Z ENCIMOM His6-OPH ZA DETEKCIJO ORGANOFOSFATOV 6 Mentorica: prof. dr. Aleksandra Lobnik Somentorica: prof. dr. Brigita Lenarčič Datum zagovora: 6. 4. 2016 DOKTORSKI ŠTUDIJSKI PROGRAM KEMIJSKE ZNANOSTI KEMIJA Tomaž FAKIN NAPREDNI GRANULIRANI ZEOLITI S HIERARHIČNO STRUKTURO POR NA POL-INDUSTRIJSKEM NIVOJU Mentor: prof. dr. Venčeslav Kaučič Somentor: prof. dr. 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Anton Meden Datum zagovora: 20. 9. 2016 Nika OSTERMAN RAZVOJ NIKLJEVEGA KATALIZATORJA NA ZEOLITNEM NOSILCU ZA DEOKSIGENACIJO ODPADNIH OLJ Mentor: prof. dr. Anton Meden Datum zagovora: 12. 9. 2016 Silvija BAJUK FUNKCIONALNI PREMAZI ZA ZAŠČITO HISTORIČNIH MATERIALOV Mentorica: doc. dr. Romana Cerc Korošec Somentorica: doc. dr. Andrijana Sever Škapin Datum zagovora: 28. 10. 2016 Matjaž GRČMAN DOLOČEVANJE IZBRANIH OGLJIKOVIH HIDRATOV Z UPORABO ANIONSKO IZMENJEVALNE KROMATOGRAFIJE S PULZNO AMPEROMETRIČNO DETEKCIJO Mentor: izr. prof. dr. Matevž Pompe Datum zagovora: 25. 11. 2016 Veronika ROVANŠEK POLIMERNI NOSILCI ZA DOSTAVO RUTENIJEVIH KOMPLEKSOV Mentor: prof. dr. Iztok Turel Datum zagovora: 13. 5. 2016 Patricija HRIBERŠEK SINTEZA IN PRETVORBE ALKIL 5-SUBSTITUIRANIH-4-OKSO-4,5-DIHIDRO-1H-PIROL-3-KARBOKSILATOV Mentor: doc. dr. Uroš Grošelj Datum zagovora: 9. 9. 2016 Ana KOVAČIČ SINTEZA IN DOLOČANJE ANTIOKSIDATIVNE AKTIVNOSTI DERIVATOV RESVERATROLA Mentor: izr. prof. dr. Franci Kovač Datum zagovora: 30. 8. 2016 Igor ZELENOVIC FUNKCIONALIZACIJA EPOKSIDOV Z RAZLIČNIMI NUKLEOFILI, VSEBUJOČIMI AZIDNO ALI ALKINSKO SKUPINO Mentor: prof. dr. Darko Dolenc Datum zagovora: 17. 10. 2016 Aleš POLOVIČ SINTEZA POTENCIALNIH NEČISTOT PRI ZDRAVILNI UČINKOVINI Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 19. 7. 2016 Jasna DRUŠKOVIČ ODSTRANITEV ORGANSKIH ONESNAŽEVAL IZ ODPADNE VODE Z UPORABO BIOLOŠKIH IN NAPREDNIH OKSIDACIJSKIH POSTOPKOV ČIŠČENJA Mentorica: prof. dr. Helena Prosen Datum zagovora: 27. 6. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Esmira NIKOČEVIC PREUČEVANJE VPLIVOV POSPEŠENEGA STARANJA NA HIDROKSIPROPIL CELULOZO IN ŠKROB Mentor: doc. dr. Iztok Prislan Datum zagovora: 22. 12. 2016 Tjaša LUŠINA OKSIDACIJA a-HIDROKSIKARBOKSILNIH KISLIN Z MOLIBDENOVIMI(V) IN (VI) SPOJINAMI Mentor: prof. dr. Darko Dolenc Datum zagovora: 20. 4. 2016 Damjana HRIBERŠEK SINTEZA IN KARAKTERIZACIJA CINKOVIH KARBOKSILATNIH KOMPLEKSOV Mentor: prof. dr. Alojz Demšar Datum zagovora: 16. 9. 2016 Urška LEBAR KOORDINACIJSKE SPOJINE 3D ELEMENTOV Z 1-HIDROKSIBENZOTRIAZOLOM Mentor: doc. dr. Bojan Kozlevčar Datum zagovora: 14. 10. 2016 Janja LAKNER SINTEZA TRIMETILSILILNIH ESTROV KARBOKSILNIH KISLIN POD REAKCIJSKIMI POGOJI BREZ TOPIL Mentor: izr. prof. dr. Marjan Jereb Datum zagovora: 23. 9. 2016 Maruša ŠKALER DOLOČANJE SORODNIH SUBSTANC NATRIJEVE SOLI NAPROKSENA Z UPORABO SUPERKRITIČNE KROMATOGRAFIJE Mentorica: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 15. 9. 2016 Bruno Aleksander MARTEK PRIKONDENZIRANI PIRAZINI KOT SUBSTRATI V REAKCIJI AKTIVACIJE C-H VEZI Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 9. 9. 2016 Anja KRISTL ŠTUDIJ ZADRŽEVANJA ZVRSTI NA KROMATOGRAFSKI KOLONI Z VEČ SEPARACIJSKIMI MEHANIZMI Mentor: izr. prof. dr. Matevž Pompe Datum zagovora: 14. 6. 2016 Zala GOMBAČ KOMPLEKSI KOBALTOVEGA, NIKLJEVEGA IN CINKOVEGA KLORIDA S HIDROKSI DERIVATI PIRIDINA Mentorica: doc. dr. Saša Petriček Datum zagovora: 16. 9. 2016 Sandi BRUDAR UPORABA TEKOČINSKE KALORIMETRIJE IN KROMATOGRAFIJE ZA ANALIZO PROTEOMA ČLOVEŠKE KRVNE plazme Mentor: doc. dr. Iztok Prislan Datum zagovora: 9. 9. 2016 Taja VEROVŠEK DOLOČANJE HLAPNIH SPOJIN V PROPOLISU Mentorica: prof. dr. Helena Prosen Datum zagovora: 15. 9. 2016 Tilen ZORE KOORDINACIJSKE SPOJINE CINKA IN VANADIJA Z DERIVATI PIRIDIN-2,6-DIKARBOKSILNE KISLINE S POTENCIALNIM ANTIDIABETIČNIM DELOVANJEM Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 5. 9. 2016 Eva PETKOVŠEK DOLOČEVANJE AMINOKISLIN IN MAŠČOBNIH KISLIN V PREHRANSKIH IZDELKIH Mentor: izr. prof. dr. Matevž Pompe Datum zagovora: 30. 9. 2016 Sarah MERLINI FUNKCIONALIZACIJA EPOKSI-SUBSTITUIRANIH POLIMERNIH NOSILCEV S KARBOKSILNIMI SKUPINAMI Mentor: prof. dr. Darko Dolenc Datum zagovora: 9. 11. 2016 Hermina HUDELJA NAČRTOVANJE PROCESA KRISTALIZACIJE AKTIVNE FARMACEVTSKE UČINKOVINE Z UPORABO IN-LINE TEHNIK Mentor: prof. dr. Marijan Kočevar Somentor: doc. dr. Blaž Likozar Datum zagovora: 12. 9. 2016 Uroš PRAH VPLIV DOPIRANJA NA LASTNOSTI PRAHOV IN TANKIH PLASTI TITANOVEGA DIOKSIDA Mentorica: doc. dr. Irena Kozjek Škofic Datum zagovora: 2. 9. 2016 MAGISTRSKI ŠTUDIJSKI PROGRAM 2. stopnja - KEMIJSKO INŽENIRSTVO Nina PETERLIN Urban VERBIČ MOŽNOSTI RAZGRADNJE BIOPLASTIKE Z REAKCIJA VODNEGA PLINA NA Cu-ZnGaOx LIGNINOLITIČNIMI ENCIMI GLIVE DICHOMITUS KATALIZATORJU SQUALENS Mentor: prof. dr. Janez Levec Mentor: prof. dr. Aleksander Pavko Datum zaključka: 17. 6. 2016 Datum zaključka: 8. 6. 2016 Društvene vesti in druge aktualnosti S8 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Rajko VNUK VPLIV MAKROOKSIGENACIJE PRI ZORENJU MLADEGA VINA Mentor: prof. dr. Marin Berovič Somentorca: prof. dr. Tatjana Košmerl Datum zaključka: 7. 10. 2016 Katja JURAČ MATEMATIČNO MODELIRANJE INTERAKCIJE MED BAKTERIJO IN BAKTERIOFAGI Mentor: doc. dr. Aleš Podgornik Datum zaključka: 23. 12. 2016 Jasmina SEDMAK IZBIRA KLJUČNIH VSTOPNIH MATERIALOV V RAZVOJU IN PROIZVODNJI FARMACEVTSKIH UČINKOVIN Mentor: prof. dr. Aleksander Pavko Datum zaključka: 19. 5. 2016 Eva UDOVIČ SUBMERZNA KULTIVACIJA GLIVE HERICIUM ERINACEUS V LABORATORIJSKEM BIOREAKTORJU Mentor: prof. dr. Marin Berovič (N) Datum zaključka: 29. 9. 2016 Urban BORŠTNIK OPTIMIZACIJA PROCESA KONTINUIRNE TER POLŠARŽNE SUSPENZIJSKE POLIMERIZACIJE MIKROSFERNIH AKRILATNIH LEPIL Mentor: doc. dr. Jernej Kajtna Datum zaključka: 6. 12. 2016 Kaja JAVORŠEK GOJENJE GLIVE HERICIUM ERINACEUS NA TRDNEM SUBSTRATU IN DOLOČEVANJE VSEBNOSTI FENOLOV, FENOLSNIH KISLIN IN FLAVONOIDOV Mentor: prof. dr. Marin Berovič Datum zaključka: 28. 10. 2016 Katja LOVRIN RAZVOJ MATEMATIČNEGA MODELA ZA NAPOVED DELOVANJA KOMUNALNIH ČISTILNIH NAPRAV V POREČJU DRAVE - ZGORNJA DRAVA Mentor: prof. dr. Igor Plazl Datum zaključka: 21. 10. 2016 Blaž KOMAR IMOBILIZACIJA O-TRANSAMINAZ S POZITIVNO NABITIMI OZNAČEVALCI V MIKROREAKTORJIH Mentorica: prof. dr. Polona Žnidaršič Plazl Datum zaključka: 8. 7. 2016 Aljaž PETANČIČ ZAMENJAVA KOROZIJSKEGA INHIBITORJA V ZAPRTEM HLADILNEM SISTEMU Mentor: prof. dr. Igor Plazl Datum zaključka: 10. 6. 2016 Klemen BOGOVIČ KOROZIJA V UPARJALNIKIH V NUKLEARNI ELEKTRARNI KRŠKO Mentor: prof. dr. Igor Plazl Datum zaključka: 14. 7. 2016 Luka NOČ REOLOŠKA IN APLIKATIVNA KARAKTERIZACIJA DISPERZIJSKIH OMETOV Mentorica: prof. dr. Urška Šebenik Datum zaključka: 20. 9. 2016 Nika ŽGAJNAR EMULZIJSKA POLIMERIZACIJA NA PRITISK OBČUTLJIVIH LEPIL Z DODATKOM NANOGLINE Mentor: doc. dr. Jernej Kajtna Datum zaključka: 23. 11. 2016 David BAJEC KARAKTERIZACIJA SAMOCELJENJA NA OSNOVI DIELS-ALDER REAKCIJE V EPOKSIDNIH SMOLAH Mentorica: prof. dr. Urška Šebenik Datum zaključka: 6. 9. 2016 Anže PRAŠNIKAR SIMULACIJA REAKCIJE IN PRENOSA SNOVI Z MREŽNO BOLTZMANNOVO METODO Mentor: prof. dr. Igor Plazl Somentor: izr. prof. dr. Tomaž Urbič Datum zaključka: 16. 9. 2016 Barbara JOZINOVIC DEGRADACIJA KATALIZATORJEV RuO2 in IrO2 Z UPORABO METODE IDENTIČNE LOKA2CIJE IL2-SEM Mentor: prof. dr. Miran Gaberšček Datum zaključka: 15. 11. 2016 Filip STRNIŠA UPORABA MREŽNE BOLTZMANNOVE METODE ZA MODELIRANJE TRANSPORTNIH POJAVOV V MIKROFLUIDNIH NAPRAVAH Mentor: prof. dr. Igor Plazl Somentor: izr. prof. dr. Tomaž Urbič Datum zaključka: 13. 9. 2016 Nina SLAPŠAK FUNKCIONALIZACIJA NANODELCEV ZA UPORABO V KOZMETIKI Mentor: doc. dr. Boštjan Genorio Datum zaključka: 22. 9. 2016 Anja PAJNTAR KARAKTERIZACIJA MIKROREAKTORJEV S STRNJENIM SLOJEM ZA IZVEDBO ENCIMSKO KATALIZIRANE SINTEZE KRATKOVERIŽNIH ESTROV Mentorica: prof. dr. Polona Žnidaršič Plazl Datum zaključka: 2. 12. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 MAGISTRSKI ŠTUDIJSKI PROGRAM 2. stopnja - TEHNIŠKA VARNOST Tine PANJTAR UGOTAVLJANJE KAKOVOSTI ZRAKA IN ŠKODLJIVOSTI NA DELOVNEM MESTU LAKIRCA Mentorica: prof. dr. Marija Bešter Rogač Datum zagovora: 11. 5. 2016 Anja LEŠNJAK POJAVNOST PSIHOSOCIALNIH TVEGANJ V EVROPSKIH DELOVNIH OKOLJIH Mentorica: doc. dr. Marija Molan Datum zagovora: 14. 7. 2016 Nina ČESNIK ŠIRJENJE PLINOV V MODELU GARAŽE Mentor: doc. dr. Jože Šrekl Datum zagovora: 12. 7. 2016 Tomaž VOŠNER SOCIALNO ZAVAROVANJE ZA PRIMER POŠKODB PRI DELU IN POKLICNIH BOLEZNI ŠTUDENTOV Mentor: Luka Tičar Datum zagovora: 8. 1. 2016 Jernej FORSTNER VPLIV KOMUNIKACIJE V DELOVNIH PROCESIH NA PODROČJE VARNOSTI Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 14. 7. 2016 Admir BABIC LASERSKA VARNOST NA DELOVNEM MESTU Mentor: prof. dr. Marjan Bilban Somentor: pred. dr. Grega Bizjak Datum zagovora: 13. 1. 2016 Nina JALEN KARAKTERIZACIJA VZORCEV IZ DIMNIKOV IN PRIMERJAVA NJIHOVE POTENCIALNE NEVARNOSTI ZA POŽAR Mentor: doc. dr. Saša Petriček Datum zagovora: 19. 10. 2016 Peter KASTRIN MERITVE TRDNIH DELCEV IN ČRNEGA OGLJIKA V AVTOMOBILSKIH IZPUHIH IN DOLOČITEV EMISIJSKIH FAKTORJEV Mentorica: znan. svet. dr. Irena Grgic Somentorica: prof. dr. Marija Bešter Rogač Datum zagovora: 18. 11. 2016 Društvene vesti in druge aktualnosti S10 Acta Chim. Slov. 2017, 64, (1), Supplement S55 DIPLOME - UNIVERZITETNI ŠTUDIJ KEMIJA Mirjam PROSENC KONCENTRACIJE ELEMENTOV NA SUSPENDIRANIH DELCIH PRI POJAVU POVIŠANIH VOD REKE SAVE Mentor: prof. dr. Marjan Veber Datum zagovora: 30. 9. 2016 Nataša MEŽNAR RAZVOJ KROMATOGRAFSKE METODE ZA DOLOČANJE KONCENTRACIJE VIRUSNIH DELCEV INFLUENCE A V LIZATU VERO CELIC Mentor: izr. prof. dr. Matevž Pompe Datum zagovora: 19. 9. 2016 Helena LAHOVEC STRUKTURNO PODPRTO MODELIRANJE INHIBITORJEV KINAZE B-RAF Mentor: doc. dr. Črtomir Podlipnik Datum zagovora: 22. 9. 2016 Mateja NOGRAŠEK RAZVOJ METODE ZA DOLOČANJE KLORIRANIH FENOLOV V VODAH Mentorica: prof. dr. Helena Prosen Datum zagovora: 22. 9. 2016 Dren ROLLKA KVANTITATIVNA IN KVALITATIVNA FAZNA ANALIZA KOMPOZITOV IZ ZEMLJINE STARE CINKARNE CELJE IN PEPELA VIPAP Mentor: prof. dr. Anton Meden Datum zagovora: 26. 9. 2016 Nastja KOTNIK FLUORIMETRIČNO DOLOČEVANJE TIAMINA PO OKSI-DACIJI S HEKSACIANOFERATOM (III) Mentor: izr. prof. dr. Matevž Pompe Datum zagovora: 30. 9. 2016 Tomaž KORITNIK MERJENJE KONTAKTNEGA KOTA TEKOČIN (NA HIDROFOBNIH POVRŠINAH) Z RAČUNALNIŠKO ANALIZO OBLIKE SEDEČE KAPLJICE Mentor: prof. dr. Ciril Pohar Datum zagovora: 30. 9. 2016 Tomaž ZORNIK RAZVOJ POSTOPKA ZA INDUSTRIJSKO SINTEZO CINKOVEGA FOSFATA Mentor: prof. dr. Iztok Turel Datum zagovora: 30. 9. 2016 Polona ŠMRGUT DOLOČEVANJE SINTEZNIH PRODUKTOV MONOBUTI-RINA NA PLINSKEM KROMATOGRAFU Mentor: izr. prof. dr. Matevž Pompe Datum zagovora: 30. 9. 2016 Tomaž JANŽEKOVIČ INTERAKCIJE NATRIJEVEGA POLISTIREN SULFONATA Z DEVTERIRANIMI IN/ALI FLUORIRANIMI SURFAK-TANTI Mentorica: prof. dr. Ksenija Kogej Datum zagovora: 30. 9. 2016 Uroš LIPOVŠEK PRIPRAVA IN KARAKTERIZACIJA TANKIH PLASTI TiO. FOTOKATALITSKO AKTIVNIH PRI OBSEVANJU Z VIDNO SVETLOBO Mentorica: doc. dr. Romana Cerc Korošec Datum zagovora: 28. 9. 2016 KEMIJA - 1. stopnja Polona RUDOLF ŽIGON REAKCIJE SEMIKARBAZIDOV Z NEKATERIMI KOVINSKIMI IONI Mentor: doc. dr. Andrej Pevec Datum zagovora: 24. 8. 2016 Mateja HOZJAN UPORABA POTENCIOMETRIČNIH METOD PRI DOLOČEVANJU STABILNOST KOORDINACIJSKIH SPOJIN Mentor: izr. prof. dr. Mitja Kolar Datum zagovora: 25. 11. 2016 Klara KRAPEŽ VREDNOTENJE V CELOTI TRDNIH ELEKTROD S PVC-MEMBRANO Mentorica: izr. prof. dr. Nataša Gros Datum zagovora: 31. 8. 2016 UrhJEVŠOVAR OPTIMIZACIJA MIKROVALONE EKSTRAKCIJE LOVILCEV SEKUNDARNIH ORGANSKIH AEROSOLOV Mentor: izr. prof. dr. Matevž Pompe Datum zagovora: 8. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Uroš KLOPČIČ MIKROEKSTRAKCIJA ZA DOLOČANJE KLORIRANIH ONESNAŽEVAL V VODI Mentorica: prof. dr. Helena Prosen Datum zagovora: 5. 2. 2016 Jakob MAKOVAC MONTE CARLO SIMULACIJA SISTEMA ELEKTROLIT-MEMBRANA Mentor: doc. dr. Miha Lukšič Datum zagovora: 15. 9. 2016 Katja VOVČKO RAZISKAVA VEZAVE LIGANDA TMPyP4 NA OLIGONUKLEOTID ČLOVEŠKEGA ZAPOREDJA Tel22 Z UPORABO SPEKTROFLUORIMETRA Mentor: doc. dr. Matjaž Bončina Datum zagovora: 19. 9. 2016 Miha NOSAN TITRACIJSKO OBNAŠANJE POLIKARBOKSILNIH KISLIN Mentorica: prof. dr. Ksenija Kogej Datum zagovora: 12. 9. 2016 Mitja OVEN FUGATIVNOSTNI KOEFICIENT RAZLIČNIH PLINOV Mentor: prof. dr. Andrej Jamnik Datum zagovora: 13. 9. 2016 Anja DEBELJAK FAZNA ANALIZA RAZLIČNIH VZORCEV TAL Z RENTGENSKO PRAŠKOVO ANALIZO Mentorica: izr. prof. dr. Amalija Golobič Datum zagovora: 4. 2. 2016 Urška ZAPLOTNIK SINTEZA IN KARAKTERIZACIJA FOTOKATALITSKO AKTIVNIH TANKIH PLASTI TITANOVEGA DIOKSIDA Mentorica: doc. dr. Romana Cerc Korošec Datum zagovora: 4. 2. 2016 Robert KRBAVČIČ ORGANOKATALIZIRANE ADICIJE 1-(TERC-BUTIL) 3-METIL 5-FENIL-4,5-DIHIDRO-4-OKSO-1H-PIROL-1,3-DIKARBOKSILATA NA NITROALKENE Mentor: doc. dr. Uroš Grošelj Datum zagovora: 12. 9. 2016 Tjaša GOGNJAVEC PRETVORBE ORGANSKIH SPOJIN POD KLASIČNIMI IN ZELENIMI POGOJI Mentor: izr. prof. dr. Marjan Jereb Datum zagovora: 13. 9. 2016 Anže PAVLIN SINTEZA IZBRANIH HIDRAZONOFORMAMIDOV Mentor: prof. dr. Janez Košmrlj Datum zagovora: 16. 9. 2016 Eva ŽOS REAKCIJE ETOKSIMETILEN HIDRAZONOV Z DUŠIKOVIMI NUKLEOFILI Mentor: prof. dr. Janez Košmrlj Datum zagovora: 16. 9. 2016 Nik RUS SINTEZE SUBSTITUIRANIH 2H-PIRAN- 2-ONOV S POSEBNIM POUDARKOM NA POIZKUSU SINTEZE DERIVATOV, KI VSEBUJEJO 2-FLUORO- 4-METOKSIFENILNO SKUPINO Mentor: doc. dr. Krištof Kranjc Datum zagovora: 13. 9. 2016 Aleš ŠAVRIČ DEHIDROGENACIJA 5,6-DIFENIL-2,3-DIHIDROPIRAZINA Z UPORABO AKTIVNEGA OGLJA KOT KATALIZATORJA Mentor: doc. dr. Krištof Kranjc Datum zagovora: 13. 9. 2016 Bine LEDINEK KATALITSKO ARILIRANJE C-H VEZI 2-FENILPIRIMIDINA V VODI Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 15. 12. 2016 David SMODIŠ KINAZOLIN KOT USMERJAJOČA SKUPINA V KATALITSKI AKTIVACIJI C-H VEZI Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 13. 9. 2016 Blaž HODNIK UPORABA Cu(0)-GRAFITNEGA KATALIZATORJA V ORGANSKI KEMIJI Mentor: prof. dr. Jurij Svete Datum zagovora: 12. 9. 2016 Anže ZUPANC UPORABA BAKRA NA ŽELEZU KOT KATALIZATORJA V ORGANSKIH REAKCIJAH Mentor: prof. dr. Jurij Svete Datum zagovora: 9. 9. 2016 Jana ČIMŽAR NEKATERE SELEKTIVNE PRETVORBE 8-HIDROKSIKINOLINA Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 15. 9. 2016 Jure ZEKIČ Katarina DOLES PRETVORBE NEKATERIH ORGANSKIH SPOJIN POD PRIPRAVA FENIL SUBSTITUIRANIH KLASIČNIMI IN ALTERNATIVNIMI POGOJI IZOKSAZOLIDINSKIH SISTEMOV Mentor: izr. prof. dr. Marjan Jereb Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 12. 9. 2016 Datum zagovora: 6. 9. 2016 Društvene vesti in druge aktualnosti S12 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Hana HACE SUZUKI-MIYAURA REAKCIJA 3-BROMOKINOLINA S HETEROARIL BOROVIMI KISLINAMI Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 14. 9. 2016 Marija KISILAK AKTIVACIJA NEREAKTIVNIH C-H VEZI Z ŽELEZOVIMI(II) IN ŽELEZOVIMI(III) KOMPLEKSI Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 12. 9. 2016 Žan TESTEN SUZUKI-MIYAURA REAKCIJA 2-BROMOKINOLINA S HETEROARIL BOROVIMI KISLINAMI Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 14. 9. 2016 Matej REBERC SINTEZA IN KARAKTERIZACIJA KOVINSKO- ORGANSKIH MATERIALOV Z VGRAJENIMI HIDRANTI LAHKIH KOVIN Mentor: prof. dr. Anton Meden Datum zagovora: 15. 9. 2016 Katja VRABEC SPOJINE NIKLJA S 3-HIDROKSIPIRIDIN-2-ONOM Mentorica: doc. dr. Saša Petricek Datum zagovora: 15. 9. 2016 Aja Ana PAVLIČ VPLIV VSEBNOSTI ŽVEPLA NA FOTOKATALITSKO AKTIVNOST TiO2 Mentorica: doc. dr. Romana Cerc Korošec Datum zagovora: 1. 7. 2016 Nina PODJED SINTEZA IN KAPSULACIJA ORGANORUTENIJEVIH SPOJIN Mentor: prof. dr. Iztok Turel Datum zagovora: 15. 9. 2016 Simona GRIČAR SINTEZA DIKETONOV Z AMINSKIMI SUBSTITUENTI IN NJIHOVIH RUTENIJEVIH KOMPLEKSOV Mentor: prof. dr. Iztok Turel Datum zagovora: 15. 9. 2016 Maja TIHOMIROVIC KOORDINACIJSKE SPOJINE BAKRA, CINKA IN KOBALTA Z 1,2,4-TRIAZOLOM Mentor: doc. dr. Bojan Kozlevcar Datum zagovora: 15. 9. 2016 Anja SEDMINEK SINTEZA IN KARAKTERIZACIJA NEKATERIH TIOSEMIKARBAZONOV KOT POTENCIALNIH KELATNIH LIGANDOV Mentor: doc. dr. Andrej Pevec Datum zagovora: 16. 9. 2016 Monika HORVAT KOORDINACIJSKE SPOJINE VANADIJA IN CINKA S 6-SUBSTITUIRANIMI PIRIDIN-2-KARBOKSILATI S POTENCIALNIM ANTIDIABETIČNIM DELOVANJEM Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 14. 9. 2016 Nejc BUTALA SINTEZA VEČVEZNIH LIGANDOV S PIRIDIN-2 -KARBOKSILATNO SKUPINO ZA KOORDINACIJO LANTANOIDNIH IONOV Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 15. 9. 2016 Polona ŠKRINJAR PROUČEVANJE ADSORPCIJSKIH LASTNOSTI BIOOGLJA Mentorica: doc. dr. Marija Zupancic Datum zagovora: 16. 9. 2016 Natalija POGORELC PRODUKTI REAKCIJ SOLI KOVIN ČETRTE PERIODE Z GLICINOM Mentorica: doc. dr. Nives Kitanovski Datum zagovora: 14. 9. 2016 Kristina MAGDALENIC VPLIV DEBELINE PLASTI TiO2 NA RAZGRADNJO BARVILA PLASMOCORINTH B2 Mentorica: doc. dr. Irena Kozjek Škofic Datum zagovora: 10. 11. 2016 Aleksandar DJURDJEVIC VPLIV SILICIJA NA PRETVORBO ANATASA V RUTIL Mentorica: doc. dr. Irena Kozjek Škofic Datum zagovora: 16. 9. 2016 Anja KRAMER UPORABA KONJUGIRANIH POLIMEROV V FOTONAPETOSTNIH CELICAH Mentor: doc. dr. Janez Cerar Datum zagovora: 15. 9. 2016 Ema SLEJKO AKTIVNOSTNI KOEFICIENT PROPANOJSKE KISLINE V ADSORBENTU Mentorica: prof. dr. Barbara Hribar Lee Datum zagovora: 14. 9. 2016 Martin KOŠIČEK DISOCIACIJSKA RAVNOTEŽJA V VODNIH RAZTOPINAH ENOSTAVNIH IN POLIMERNIH KARBOKSILNIH KISLIN Mentorica: prof. dr. Ksenija Kogej Datum zagovora: 12. 9. 2016 Anja KOS STRUKTURNE IN TERMODINAMSKE ZNAČILNOSTI PREPOZNAVANJA IN VEZANJA NETROPSINA NA DNA Mentor: prof. dr. Jurij Lah Datum zagovora: 13. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Danijela HODNIK STRUKTURNE IN TERMODINAMSKE ZNAČILNOSTI PREPOZNAVANJA DNA Z LIGANDOM 360A-Br Mentor: prof. dr. Jurij Lah Datum zagovora: 13. 9. 2016 Blaž ZDOVC KVANTNO - KEMIJSKI PRISTOPI K NAPOVEDI ABSORPCIJSKIH SPEKTROV KONJUGIRANIH BARVIL Mentor: doc. dr. Miha Lukšič Datum zagovora: 15. 9. 2016 Tomislav KOSTEVC ISKANJE INHIBITORJEV VIRUSA ZIKA Z UPORABO METOD MOLEKULSKEGA MODELIRANJA Mentor: doc. dr. Črtomir Podlipnik Datum zagovora: 16. 9. 2016 Jaka ŠTIRN VPLIV PODROBNOSTI MODELOV TETRAMETILAMONIJEVE SOLI POLIANETOLSULFONSKE KISLINE NA RAZREDČILNE ENTALPIJE Mentor: izr. prof. dr. Jurij Reščič Datum zagovora: 13. 9. 2016 Klavdija MIRTIČ INTERAKCIJE MED SURFAKTANTI IN BIOLOŠKIMI MEMBRANAMI Mentor: Bojan Šarac Datum zagovora: 16. 9. 2016 Robert KOMAN SUPERHIDROFOBNE POVRŠINE IN PREMAZI NA TRDNIH MATERIALIH: LASTNOSTI, IZDELAVA IN APLIKACIJE Mentor: Bojan Šarac Datum zagovora: 14. 9. 2016 Matjaž SIMONČIČ KEMIJSKE REAKCIJE V MEDZVEZDNEM PROSTORU Mentor: izr. prof. dr. Tomaž Urbič Datum zagovora: 15. 9. 2016 Matjaž DLOUHY STRUKTURA IN INTERAKCIJA METIL RADIKALA Mentor: izr. prof. dr. Tomaž Urbič Datum zagovora: 15. 9. 2016 Petra PAPEŽ MONTE CARLO SIMULACIJA DVODIMENZIONALNIH MODELOV ALKOHOLOV Mentor: izr. prof. dr. Tomaž Urbič Datum zagovora: 13. 9. 2016 Domen KASTELIC OPTIMIZACIJA INSTRUMENTA ZA MASNO SPEKTROMETRIJO Z INDUKTIVNO SKLOPLJENO PLAZMO Mentor: prof. dr. Marjan Veber Datum zagovora: 15. 9. 2016 Ema GRIČAR POTENCIOMETRIČNE TITRACIJE PRI ŠTUDIJU INTERAKCIJ FITATOV Z IZBRANIMI KOVINSKIMI IONI Mentor: izr. prof. dr. Mitja Kolar Datum zagovora: 15. 9. 2016 Jan GAČNIK DOLOČANJE RADIJEVIH IZOTOPOV V VODI Z METODO TEKOČINSKE SCINTILACIJE Mentorica: prof. dr. Helena Prosen Datum zagovora: 12. 9. 2016 Tina GRUBAR DOLOČANJE TRIAZINSKIH PESTICIDOV V VODI Z DLLME-HPLC Mentorica: prof. dr. Helena Prosen Datum zagovora: 12. 9. 2016 Urša KOŠAK SEKVENČNA INJEKCIJSKA ANALIZA Mentorica: izr. prof. dr. Nataša Gros Datum zagovora: 31. 8. 2016 Maja ŠUŠTERŠIČ DERIVATIVNA SPEKTROMETRIJA Mentorica: izr. prof. dr. Nataša Gros Datum zagovora: 9. 9. 2016 Anja PIRC ŠTUDIJ DEGRADACIJE VITAMINA D2 Mentor: izr. prof. dr. Matevž Pompe Datum zagovora: 15. 9. 2016 Nika SIMONIČ UPORABA VISKOZIMETRIJE ZA DOLOČANJE STOPNJE POLIMERIZACIJE CELULOZE V PAPIRJIH Z VISOKO VSEBNOSTJO LIGNINA Mentorica: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 12. 9. 2016 Društvene vesti in druge aktualnosti S14 Acta Chim. Slov. 2017, 64, (1), Supplement S55 KEMIJSKO INZENIRSTVO Sara SEVER PRETVORBA POLŠARŽNEGA PROCESA Z IZMENJAVO MEDIJA V PERFUZIJSKI PROCES NA PRIMERU BIOPROCESA S SESALČJO CELIČNO KULTURO Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 24. 3. 2016 Jan DEBEVEC ANOKSIČNI PRETOČNI BIOREAKTOR S STRNJENIM SLOJEM BIOMASE: KINETIČNI MODEL OKSIDACIJE MRAVLJINČNE KISLINE Mentor: prof. dr. Igor Plazl Datum zagovora: 16. 9. 2016 Marija OBLAK PONOVNA UPORABA TEHNOLOŠKEGA ODPADKA ELASTOMEROV Mentorica: prof. dr. Urška Šebenik Datum zagovora: 8. 9. 2016 Matej BIRK PRIMERJAVA GOJENJA CELIČNE LINIJE CHO V RAZLIČNIH BIOREAKTORJIH Mentor: prof. dr. Aleksander Pavko Datum zagovora: 13. 9. 2016 Anže DOLINŠEK OPREDELITEV PRIMERNIH LABORATORIJSKIH METOD ZA DOLOČITEV OPTIMALNIH VOZNIH LASTNOSTI RADIALNE MOTORSKE PNEVMATIKE Mentorica: prof. dr. Urška Šebenik Datum zagovora: 7. 9. 2016 Uroš JANICUEVIC PORABA ENERGIJE V KOMUNALNI ČISTILNI NAPRAVI Mentorica: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 23. 6. 2016 Boris PEKLAR VPLIV OBRATOVALNIH POGOJEV NA KRISTALIZACIJO DERIVATA PIPERAZINA Mentor: doc. dr. Aleš Podgornik Somentor: doc. dr. Blaž Likozar Datum zagovora: 13. 9. 2016 Ana Tea KOS RAZVOJ PREVODNIH GRAFENSKIH KOMPONENT ZA FUNKCIONALNE BARVE Mentor: doc. dr. Boštjan Genorio Datum zagovora: 23. 9. 2016 Marko VIDIC OKSIDACIJA LEVULINSKE KISLINE Z UPORABO HETEROGENEGA RUTENIJEVEGA KATALIZATORJA Mentor: prof. dr. Aleksander Pavko Somentor: doc. dr. Blaž Likozar Datum zagovora: 13. 9. 2016 Noel AVBELJ VREDNOTENJE MORFOLOŠKIH KARAKTERISTIK PRI INDUSTRIJSKI PRIPRAVI VODNE SUSPENZIJE CaCO3 Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 29. 9. 2016 Ana Roza MEDVED MOKRI POSTOPEK KARBONIZACIJE ELEKTROFILTRSKEGA PEPELA IN KARAKTERIZACIJA PRODUKTOV Mentorica: doc. dr. Barbara Novosel Datum zagovora: 6. 5. 2016 Rebeka GREGORČIČ PRIMERJAVA FIZIKALNO-KEMIJSKIH POSTOPKOV ČIŠČENJA IZCEDNIH VOD KOMUNALNE DEPONIJE Mentorica: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 19. 5. 2016 Irena IHAN KINETIKA NASTAJANJA POROZNEGA POLIMETAKRILATA Mentor: doc. dr. Aleš Podgornik Datum zagovora: 16. 6. 2016 Rok PUCER KRIOMACERACIJA GROZDJA Mentor: prof. dr. Marin Berovic Somentor: prof. dr. Mojmir Wondra Datum zagovora: 7. 9. 2016 Tanja BRESKVAR POLIOLNA SINTEZA AZO SPOJIN Z MIKROVALOVI Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 8. 9. 2016 Rok KLOBČAR KINETIKA SINTRANJA KERAMIKE NA OSNOVILANTAN-STRONCIJ-KROM-MANGANMEŠANEGA OKSIDA Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 8. 9. 2016 Bernarda ANZELAK IZLOČANJE PROTEINOV IZ BIOLOŠKIH MEDIJEV Z UPORABO MAGNETNIH NANODELCEV Mentor: prof. dr. Marin Berovic Somentor: Darko Makovec Datum zagovora: 13. 9. 2016 Brina ZUPANČIČ BIOSINTEZA KORDICEPINA GLIVE Cordyceps militaris S KULTIVACIJO NA TRDNEM SUBSTRATU Mentor: prof. dr. Marin Berovic Somentor: prof. dr. Samo Kreft Datum zagovora: 13. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Marko ŠKRILEC UPORABA OZONA ZA DEZINFEKCIJO ZAPRTIH PROSTOROV Mentorica: izr. prof. dr. Andreja Zgajnar Gotvajn Datum zagovora: 16. 9. 2016 Vinko ŠUCUR VPLIV GLINE NA SINTEZE NANOKOMPOZITNIH UV ZAMREZLJIVIH AKRILATNIH LEPIL Mentor: doc. dr. Jernej Kajtna Datum zagovora: 22. 9. 2016 Jasmina VALENČAK GRAFTIRANJE POROZNIH METAKRILATNIH NOSILCEV Mentor: doc. dr. Aleš Podgornik Datum zagovora: 22. 9. 2016 Borut MLAKAR IZBOLJŠANJE MEHANSKIH LASTNOSTI ALUMINIJEVE ZLITINE ZA VISOKO TLAČNO LITJE Mentorica: doc. dr. Klementina Zupan Datum zagovora: 22. 9. 2016 Irena PRIMC DOLINŠEK ANALIZA STROŠKOV PROCESA TRANSAMINACIJE Z RAZLIČNIMI OBLIKAMI BIOKATALIZATORJA Mentorica: prof. dr. Polona Znidaršic Plazl Datum zagovora: 23. 9. 2016 Jan ČERNELČ ENCIMSKO KATALIZIRANA SINTEZA IZOAMIL ACETATA V PRETOČNEM SISTEMU Z INTENZIVNIM KONTAKTIRANJEM DVEH KAPLJEVIN Mentorica: prof. dr. Polona Znidaršic Plazl Datum zagovora: 23. 9. 2016 KEMIJSKO INŽENIRSTVO - 1. stopnja Anže URANKAR UTRJEVANJE EPOKSIDNE SMOLE Z RAZLIČNIMI KATALIZATORJI Mentorica: prof. dr. Urška Šebenik Datum zagovora: 1. 2. 2016 Miha ŠVAGELJ VEČANJE PROSOJNOSTI NARAVNO PRESEVNIH MATERIALOV Z UPORABO POLIMEROV KOT POLNILCEV RAZPOK Mentor: doc. dr. Aleš Podgornik Datum zagovora: 25. 7. 2016 Rene OBLAK PRIPRAVA METAKRILATNIH POLYHIPE MONOLITOV Mentor: doc. dr. Aleš Podgornik Datum zagovora: 14. 9. 2016 Elizabeta STEKLASA SPOSOBNOST SAMOCELJENJA POLIMERA Mentorica: prof. dr. Urška Šebenik Datum zagovora: 16. 9. 2016 Dejan MIŠIC POLIMERNI MATERIALI S SPOMINSKIM UČINKOM Mentorica: prof. dr. Urška Šebenik Datum zagovora: 16. 9. 2016 Klemen ZLATNAR POLŠARZNA suspenzijska polimerizacija MIKROSFERNIH AKRILATNIH LEPIL Mentor: doc. dr. Jernej Kajtna Datum zagovora: 20. 9. 2016 Matjaž VELKOVRH POLŠARZNA SUSPENZIJSKA POLIMERIZACIJA MIKROSFERNEGA AKRILATNEGA LEPILA Mentor: doc. dr. Jernej Kajtna Datum zagovora: 21. 9. 2016 Rok MRAVLJAK PRODUKCIJA BIODIZLA Mentor: prof. dr. Marin Berovic Datum zagovora: 10. 6. 2016 Milena BEVK IMOBILIZACIJA ENCIMOV NA OSNOVI UPORABE MAGNETNIH NANODELCEV Mentorica: prof. dr. Polona Znidaršic Plazl Datum zagovora: 12. 9. 2016 Kevin JERIČ UPORABA HOMOGENE IN HETEROGENE FENTONOVE OKSIDACIJE ZA ČIŠČENJE ODPADNIH VOD Mentorica: izr. prof. dr. Andreja Zgajnar Gotvajn Datum zagovora: 16. 9. 2016 Monika KRIVEC ZAKAJ IN KAKO OBLOŽIMO AKTIVNO UČINKOVINO V ZDRAVILU Mentor: prof. dr. Radovan Stanislav Pejovnik Datum zagovora: 27. 1. 2016 Katarina SHOAIB UPORABA TiO2 v NANOT TEHNOLOGIJI Mentor: prof. dr. Radovan Stanislav Pejovnik Datum zagovora: 16. 3. 2016 Maša KLENOVŠEK STRUKTURE KARAKTERISTIČNIH OKSIDNIH ANODNIH MATERIALOV V KERAMIČNIH GORIVNIH CELICAH Mentorica: doc. dr. Klementina Zupan Datum zagovora: 5. 9. 2016 Društvene vesti in druge aktualnosti S16 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Kevin STOJANOVSKI ANALIZA MIKROSTRUKTURE KOMPOZITNEGA ANODNEGA MATERIALA ZA SREDNJE-TEMPERATURNE KERAMIČNE GORIVNE CELICE Mentorica: doc. dr. Klementina Zupan Datum zagovora: 16. 9. 2016 Dominika ZORMAN KARAKTERIZACIJA PEROVSKITNEGA ANODNEGA MATERIALA ZA VISOKOTEMPERATURNE GORIVNE CELICE Mentorica: doc. dr. Klementina Zupan Datum zagovora: 5. 9. 2016 Marko FIRM MAGNEZIJEVE ELEKTRODE ZA UPORABO V SEKUNDARNIH BATERIJAH Mentor: prof. dr. Miran Gaberšček Datum zagovora: 15. 9. 2016 Katja BALANTIČ ENCIMSKE REAKCIJE Mentor: prof. dr. Aleksander Pavko Datum zagovora: 11. 7. 2016 Matevž PODOBNIK PRIMERJAVA ŠARŽNIH, POLŠARŽNIH IN KONTINUIRNIH PROCESOV PRI PROIZVODNJI MONOKLONSKIH PROTITELES Mentor: prof. dr. Aleksander Pavko Datum zagovora: 11. 7. 2016 Nina KUZMIC MEMBRANSKE SEPARACIJSKE METODE V BIOTEHNOLOGIJI IN FARMACIJI Mentor: prof. dr. Aleksander Pavko Datum zagovora: 12. 9. 2016 Tomaž PIRMAN NIZKOTEMPERATURNI PARNI REFORMING METANOLA ZA PROIZVODNJO VODIKA Z UPORABO Cu-Zn KATALIZATORJEV Mentor: prof. dr. Igor Plazl Datum zagovora: 14. 7. 2016 Timotej GALUN NANOEMULZIJE ZA PROIZVODNJO IZOLACIJSKIH MATERIALOV Mentor: prof. dr. Igor Plazl Datum zagovora: 13. 9. 2016 Živa BREČKO UPORABA DVOFAZNIH VODNIH SISTEMOV Z MICELI ZA ČIŠČENJE PROTEINOV Z MIKROFLUIDNIMI NAPRAVAMI Mentorica: prof. dr. Polona Žnidaršič Plazl Datum zagovora: 12. 9. 2016 Ana OBERLINTNER BIOSENZORJI V MIKROFUIDIKI Mentorica: prof. dr. Polona Žnidaršič Plazl Datum zagovora: 16. 9. 2016 Damjan KODER VPLIV FARMACEVTSKE ODPADNE VODE NA BIOLOŠKO ČISTILNO NAPRAVO Mentorica: izr. prof. dr. Andreja Zgajnar Gotvajn Datum zagovora: 7. 9. 2016 Gaja TOMSIČ KATALITSKA DEPOLIMERIZACIJA NAJLONA 6 S PIROLIZNO METODO Mentorica: izr. prof. dr. Andreja Zgajnar Gotvajn Datum zagovora: 7. 9. 2016 Matej ŠADL KARAKTERIZACIJA DEBELIH PLASTI BIZMUTOVEGA FERITA, PRIPRAVLJENIH Z METODO SITOTISKA, NA PODLAGI IZ KERAMIKE Z NIZKO TEMPERATURO ZGANJA Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 22. 9. 2016 Erik STARC PRIDOBIVANJE IN UPORABA MOLEKULARNO VTISNJENIH POLIMEROV Mentor: doc. dr. Aleš Podgornik Datum zagovora: 13. 9. 2016 Tina PALJK POROZNI PIEZOELEKTRIČNI MATERIALI Mentor: doc. dr. Aleš Podgornik Datum zagovora: 14. 9. 2016 Tilen KOPAČ VPLIV RAZMERJA MONOMEROV TER MOLEKULSKE MASE NA LEPILNE LASTNOSTI PRI POLŠARZNI SUSPENZIJSKI POLIMERIZACIJI MIKROSFERNIH AKRILATNIH LEPIL Mentor: doc. dr. Jernej Kajtna Datum zagovora: 6. 9. 2016 Lorena KUNC INŽENIRSKI VIDIKI PROIZVODNJE BIOPLINA Mentor: doc. dr. Aleš Podgornik Datum zagovora: 12. 9. 2016 Matjaž ŠKEDELJ TEORETIČNA OBRAVNAVA IN PRISTOP K MODELIRANJU LASTNOSTI POLIMERNIH MATERIALOV S SPOSOBNOSTJO SAMOCELJENJA Mentor: doc. dr. Aleš Rucigaj Datum zagovora: 16. 9. 2016 Matej BENEDIK PERFUZIJSKI BIOREAKTOR ZA GOJENJE SESALSKIH CELIC Mentor: doc. dr. Aleš Podgornik Datum zagovora: 16. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 BIOKEMIJA_ Tjaša KORBAR VPLIV INVERZIJE POLARNOSTI V DNA ZAPOREDJU NA VEZAVO IN PREMIKANJE KATIONOV Mentor: prof. dr. Janez Plavec Datum zagovora: 21. 9. 2016 Irena GRŽINA STRUKTURNA IN BIOFIZIKALNA ANALIZA CELICO PENETRIRAJOČEGA PEPTIDA TP 10 IN NJEGOVEGA DIMERA Mentor: prof. dr. Roman Jerala Somentor: prof. dr. Gregor Anderluh Datum zagovora: 6. 5. 2016 Jaka CEVC RAZVOJ LC-MS METODE ZA DOLOČANJE VSEBNOSTI ELAIOFILINA V SALINOMICINU Mentorica: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 8. 6. 2016 Maja DEBELJAK OCENA DIFERENCIACIJE MODELA ČREVESNEGA EPITELIJA NA OSNOVI CELIC CACO-2 S SLEDENJEM IZRAŽANJA GENOV Mentor: prof. dr. Gregor Anderluh Datum zagovora: 21. 9. 2016 Uroš JAVORNIK ANALIZA SUPRAMOLEKULARNIH STRUKTUR DVEH DIASTEREOIZOMEROV MODIFICIRANEGA GVANINSKEGA DINUKLEOTIDA Z NMR-SPEKTROSKOPIJO V RAZTOPINI Mentor: prof. dr. Janez Plavec Datum zagovora: 29. 2. 2016 Samo MARINČ TVORBA G-KVADRUPLEKSOV IZ ZAPOREDJA PROMOTORJA GENA KRAS Mentor: prof. dr. Janez Plavec Datum zagovora: 30. 9. 2016 Anja JEŠE PRIMERJAVA KLASIČNEGA IN MODIFICIRANEGA DROZGANJA V PIVOVARSKI TEHNOLOGIJI Mentorica: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 21. 9. 2016 Kristina URBAS SINTEZA IN PRETVORBE (R)-2-(2,2-DIMETIL-3-METILENCIKLOPENTIL)-ALKILAMINA Mentor: doc. dr. Uroš Grošelj Datum zagovora: 17. 2. 2016 Branislav LUKIC SINTEZA 5-AMINOETIL SUBSTITUIRANIH KARBOKSAMIDO PIRAZOLO [ 1,5-A] PIRIMIDONOV Mentor: prof. dr. Jurij Svete Datum zagovora: 9. 5. 2016 Mateja KRŽIŠNIK PRIPRAVA CISTEINSKIH ANALOGOV TNF-a ZA MESTNOSPECIFIČNO PEGILACIJO Mentor: prof. dr. Roman Jerala Datum zagovora: 8. 9. 2016 Nina RAZGORŠEK ANAEROBNA STRUPENOST IN BIORAZGRADLJIVOST IZCEDNIH VOD KOMUNALNE DEPONIJE Mentorica: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 8. 9. 2016 Janja ZALETEL SINTEZA, ALKILIRANJE IN AMIDIRANJE METIL 7-OKDO-4H-PIRAZOLO[1,5-A]PIRIMIDIN-3- KARBOKSILATA Mentor: prof. dr. Jurij Svete Datum zagovora: 14. 9. 2016 Staša MATJAŽ STABILNOST KLOROFILINA V IZBRANIH RAZTOPINAH Mentorica: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 14. 9. 2016 Janja REBEC DOLOČEVANJE SUPEROKSID DISMUTAZNE AKTIVNOSTI KOVINSKIH KSANTURENATOV Z UPORABO METODE NBT Mentorica: doc. dr. Elizabeta Tratar Pirc Datum zagovora: 15. 9. 2016 Ajda ŽAGER VPLIV SULINDAK SULFIDA NA DIMERIZACIJO TUMORSKEGA OZNAČEVALCA EPCAM Mentor: doc. dr. Miha Pavšič Datum zagovora: 21. 9. 2016 Gregor MAZOVEC ŠTUDIJ HIDROFOBNIH INTERAKCIJ OB UKRIVLJENI POVRŠINI Z MERCEDES-BENZ MODELOM VODE Mentorica: prof. dr. Barbara Hribar Lee Datum zagovora: 27. 9. 2016 Sonja CIMERMAN BIOLOŠKA IN KATALITSKA AKTIVNOST TANKIH PLASTI PLATINE Mentorica: doc. dr. Irena Kozjek Škofic Datum zagovora: 27. 9. 2016 Aleš KERMELJ PRIPRAVA TUMORSKIH OZNAČEVALNIH PROTEINOV EpCAM IN Trop2 V KVASOVKI PICHIA PASTORIS Mentor: doc. dr. Miha Pavšič Datum zagovora: 27. 9. 2016 Blaž KRŽAN ŠTUDIJ TERMODINAMSKE STABILNOSTI PRI RAZVOJU UČINKOVINE IFN-a1A Mentor: prof. dr. Jurij Lah Datum zagovora: 28. 9. 2016 Društvene vesti in druge aktualnosti S18 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Gregor MURN CITOTOKSIČNOST KOMPOZITNIH FIBROINSKIH NOSILCEV IN NJIHOVEGA VPLIVA NA OSTEOGENO DIFERENCIACIJO PRI ZDRAVLJENJU OSTEOHONDRALNIH POŠKODB Mentor: doc. dr. Miha Pavšič Datum zagovora: 29. 9. 2016 BIOKEMIJA - 1. stopnja Rok FERENC ANALIZA VLOGE KINAZE MKK6 V OBRAMBI KROMPIRJA PROTI VIRUSU PVY Z VIRUSNIM UTIŠANJEM Mentorica: prof. dr. Kristina Gruden Datum zagovora: 13. 9. 2016 Primož TIČ ŠTUDIJE RUTENIJEVIH SPOJIN KOT MOŽNIH INHIBITORJEV ENCIMOV AKR1C Mentor: prof. dr. Iztok Turel Datum zagovora: 16. 9. 2016 Sabina ŠTUKELJ GENOMSKA IN TRANSKRIPTOMSKA ANALIZA ANTIMIKROBNIH PEPTIDOV (AMP) PRI NAJSTAREJŠIH SKUPINAH VRETENČARJEV - NOV POGLED NA NASTANEK IN EVOLUCIJO AMP DRUŽIN PRI VRETENČARJIH Mentor: izr. prof. dr. Dušan Kordiš Datum zagovora: 14. 9. 2016 Marija SRNKO VPLIV FOSFORILACIJE C-KONČNEGA TIROZINSKEGA OSTANKA V PROTEINU FUS NA NJEGOVO CELIČNO RAZPOREDITEV Mentor: izr. prof. dr. Boris Rogelj Datum zagovora: 14. 9. 2016 Tomaž ŽAGAR PRIPRAVA MONOMERNIH MUTANT PROTEINA EpCAM Mentorica: prof. dr. Brigita Lenarčič Datum zagovora: 12. 9. 2016 Urška ČERNE PRIPRAVA FUZIJSKEGA PROTEINA NANOTELESA M33 Z MCHERRY ZA FLUORESCENČNO DETEKCIJO MLKL Mentor: doc. dr. Gregor Gunčar Datum zagovora: 15. 9. 2016 Monika PEPELNJAK IZRAŽANJE ORTOKASPAZ CIANOBAKTERIJE MICROCYSTIS AERUGINOSA PCC 7806 V BAKTERIJI ESCHERICHIA COLI Mentor: izr. prof. dr. Marko Dolinar Datum zagovora: 25. 8. 2016 Anja TANŠEK VPLIV OKOLJSKIH VOD IN KOVINSKIH IONOV NA RAST CIANOBAKTERIJ SYNECHOCYSTIS SP. PCC 6803 TER PREVERJANJE UČINKOVITOSTI SINTEZNOBIOLOŠKIH UBIJALSKIH STIKAL Mentor: izr. prof. dr. Marko Dolinar Datum zagovora: 25. 8. 2016 Aneja TAHIROVIC OPTIMIZACIJA IN UPORABA TESTA MTT ZA DOLOČANJE PREŽIVETJA SINTEZNOBIOLOŠKO SPREMENJENIH CIANOBAKTERIJ SYNECHOCYSTIS SP. PCC 6803 PO INDUKCIJI GENOV ZA SPROŽITEV CELIČNE SMRT Mentor: izr. prof. dr. Marko Dolinar Datum zagovora: 25. 8. 2016 Tadej ULČNIK FUNKCIJSKA ANALIZA NEKATERIH MUTANT KATEPSINA K Mentor: doc. dr. Marko Novinec Datum zagovora: 15. 9. 2016 Jernej VIDMAR NAČRTOVANJE IN PRIPRAVA KONSTITUTIVNO MONOMERNE OBLIKE PROTEINA TROP2 Mentor: doc. dr. Miha Pavšič Datum zagovora: 15. 9. 2016 Katjuša TRIPLAT PRIMERJAVA SERUMSKIH KONCENTRACIJ TUMORSKEGA OZNAČEVALCA OSTEOPONTINA IN CELOKUPNIH PROTEINOV PRI BOLNICAH Z RAKOM JAJČNIKA OB POSTAVITVI DIAGNOZE IN PO ZDRAVLJENJU Mentorica: Katarina Černe Datum zagovora: 12. 9. 2016 Amadeja LAPORNIK VPELJAVA IN OPTIMIZACIJA VERIŽNE REAKCIJE S POLIMERAZO ZA DOKAZ ALFAVIRUSOV Mentorica: prof. dr. Tatjana Avšič Zupanc Datum zagovora: 15. 9. 2016 Jerneja KOCUTAR UGOTAVLJANJE MIOTOKSIČNOSTI NOVIH OBLIK ANESTETIKOV Mentor: prof. dr. Tomaž Marš Datum zagovora: 15. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Inge SOTLAR TRANSKRIPTOMSKI VIDIK URAVNAVANJA METABOLIZMA GLICEROLA PRI KVASOVKAH RODU AUREOBASIDIUM Mentorica: Martina Turk Datum zagovora: 12. 9. 2016 Bine TRŠAVEC IZRAŽANJE REKOMBINANTNEGA ČLOVEŠKEGA MCSF IN ŠTUDIJA NJEGOVEGA PROTEOLITSKEGA PROCESIRANJA Mentor: prof. ddr. Boris Turk Datum zagovora: 15. 9. 2016 Dominik DEKLEVA VPLIV SREBROVIH NANODELCEV NA SESALSKE CELICE V KULTURI Mentor: prof. dr. Peter Verami Datum zagovora: 12. 9. 2016 Matic KOVAČIČ STABILIZACIJA G-KVADRUPLEKSA TROMBIN-VEZAVNEGA APTAMERA S PIRENSKIMI SKUPINAMI Mentor: prof. dr. Janez Plavec Datum zagovora: 12. 9. 2016 Katja MALOVRH VPLIV GLUKOZNEGA METABOLIZMA NA NASTANEK LIPIDNIH KAPLJIC V PODGANJIH KORTIKALNIH ASTROCITIH V KULTURI Mentorica: doc. dr. Nina Vardjan Datum zagovora: 12. 9. 2016 Enja KOKALJ VPLIV ADRENERGIČNIH RECEPTORJEV IN RECEPTORJA GPR40 NA TVORBO LIPIDNIH KAPLJIC V PODGANJIH ASTROCITIH V KULTURI Mentorica: doc. dr. Nina Vardjan Datum zagovora: 12. 9. 2016 KEMIJSKO IZOBRAŽEVANJE_ Lea ZAJEC OPTIMIZACIJA ANALIZNEGA POSTOPKA ZA DOLOČANJE IZOTOPSKEGA RAZMERJA 87 SR/86 SR V OKOLJSKIH VZORCIH S KVADRUPULNIM ICP-MS Mentor: prof. dr. Marjan Veber Datum zagovora: 6. 7. 2016 Doroteja ŠPEC CIKLOADICIJE DIALKIL AZODIKARBOKSILATOV NA 2H-PIRAN-2-ONE Mentor: prof. dr. Marijan Kocevar Datum zagovora: 30. 9. 2016 Marko MERMAL OKSIDACIJA VINILNIH ETROV Z DIMETILDIOKSIRANOM IN NEKATERE PRETVORBE NASTALIH EPOKSIDOV Mentor: izr. prof. dr. Franci Kovac Datum zagovora: 30. 9. 2016 Elma LJUTIC ŠTUDIJ VODNIH RAZTOPIN ATAKTIČNE POLIMETAKRILNE KISLINE Z METODAMI SIPANJA SVETLOBE Mentorica: prof. dr. Ksenija Kogej Datum zagovora: 30. 9. 2016 Doris POTOČNIK ENANTIOSELEKTIVNA REDUKCIJA 2-BENZILIDENCIKLOALKANONOV Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 30. 9. 2016 TEHNIŠKA VARNOST - 1. stopnja_ Nejc JUHART OKSIDATIVNI STRES V DELOVNEM OKOLJU Mentor: prof. dr. Marjan Bilban Datum zagovora: 13. 1. 2016 Mateja KOČEVAR PROMOCIJA ZDRAVJA V PODJETJU ISKRA IP D. O. O. Mentor: prof. dr. Marjan Bilban Datum zagovora: 12. 9. 2016 Klementina RADANOVIČ STRES IN IZGORELOST V DEJAVNOSTI GOSTINSTVA IN TURIZMA Mentor: prof. dr. Marjan Bilban Datum zagovora: 29. 9. 2016 Maruša SVETINA ERGONOMSKE MERITVE DELOVNEGA MESTA NATAKAR Mentorica: doc. dr. Klementina Zupan Datum zagovora: 18. 11. 2016 Tomaž ČRNIGOJ ERGONOMIJA V ULTRALAHKIH LETALIH Mentorica: prof. dr. Simona Jevšnik Datum zagovora: 6. 9. 2016 Nina MONETA PRIMERJAVA IN MERITVE HRUPA V OKOLICI IZOBRAŽEVALNIH USTANOV Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 23. 11. 2016 Društvene vesti in druge aktualnosti S20 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Janja TORI SPRINKLERSKI SISTEMI V LESENIH OBJEKTIH Mentor: doc. dr. Domen Kušar Datum zagovora: 29. 9. 2016 Nastja SMOLNIKAR VPLIV DODATKOV NA MINIMALNO VŽIGNO ENERGIJO FARMACEVTSKIH AKTIVNIH UČINKOVIN Mentorica: doc. dr. Barbara Novosel Datum zagovora: 15. 9. 2016 Polona STARAŠINIČ UČINKOVITOST DIPHOTERINA ZA NEVTRALIZACIJO JEDKOVIN Mentorica: doc. dr. Barbara Novosel Datum zagovora: 15. 9. 2016 Laura BOROŠ EVAKUACIJA IZ 3C LAMELE NOVE FAKULTETE ZA KEMIJO IN KEMIJSKO TEHNOLOGIJO Mentorica: doc. dr. Saša Petriček Datum zagovora: 18. 7. 2016 Luka MAKAROVIČ ERGONOMSKE MERITVE POLOŽAJEV PACIENTA IN ZDRAVSTVENEGA OSEBJA Mentorica: doc. dr. Klementina Zupan Datum zagovora: 4. 2. 2016 Tina ROBNIK ERGONOMIJA DELA IN ERGONOMSKE MERITVE POLOŽAJEV PRI MOLŽI KRAV Mentorica: doc. dr. Klementina Zupan Datum zagovora: 21. 9. 2016 Anže ŠPEHAR MERITVE UČINKOVITOSTI ZGLOBNIH ODSESOVALNIH ROK Mentorica: prof. dr. Marija Bešter Rogač Datum zagovora: 4. 7. 2016 Neja JEKOVEC PRAVNA UREDITEV VARSTVA PRED HRUPOM Mentor: Grega Strban Datum zagovora: 13. 6. 2016 Midhat AHMETOVIC PRAŠNE EKSPLOZIJE V PREHRAMBNI INDUSTRIJI Mentor: doc. dr. Jože Šrekl Datum zagovora: 15. 9. 2016 Primož VRBINC IZBOLJŠANE POŽARNE ODPORNOSTI Z MATERIALI, KI PRI FAZNI PRETVORBI PORABLJAJO TOPLOTO Mentorica: doc. dr. Klementina Zupan Datum zagovora: 29. 6. 2016 Ana ŽABJEK SPECIALNI BETONI ZA DVIG PROTIPOŽARNE VARNOST Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 15. 9. 2016 Gregor HORVAT MATERIALI ZA TOPLOTNO ZAŠČITO V OSEBNI VAROVALNI OPREMI Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 15. 9. 2016 Eva FURLAN NEVTRALIZACIJSKA SPOSOBNOST DIPHOTERINA, RAZTOPINE KLOROVODIKOVE KISLINE IN NATRIJEVEGA HIDROKSIDA Mentor: doc. dr. Bojan Kozlevčar Datum zagovora: 16. 9. 2016 Rok REPINC HRUP V PAPIRNI INDUSTRIJI Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 21. 10. 2016 Kaja BERKOPEC ERGONOMIJA V ZDRAVSTVENI NEGI Mentor: prof. dr. Marjan Bilban Datum zagovora: 12. 9. 2016 Urška KOŽELJ NOČNO DELO IN VPLIV NA ZDRAVJE Mentor: prof. dr. Marjan Bilban Datum zagovora: 12. 9. 2016 Maja PORENTA VPLIV UPORABE MOBILNIH TELEFONOV NA VARNOST V CESTNEM PROMETU Mentor: prof. dr. Marjan Bilban Datum zagovora: 12. 9. 2016 Sabina TURK ZAGOTAVLJANJE VARNOSTI ELEKTRIČNIH DVIGAL Mentor: doc. dr. Boris Jerman Datum zagovora: 16. 9. 2016 Valerija PRIMOŽIČ VPLIV INDUSTRIJE V MESTNIH JEDRIH NA VARNOST LJUDI IN OKOLJA Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 5. 9. 2016 Martina ČEČ RAVEN VARNOSTNE KULTURE ŠTUDENTOV RAZLIČNIH FAKULTET Mentorica: doc. dr. Marija Molan Datum zagovora: 14. 9. 2016 Karmen KORENIČ VARNOSTNA KULTURA V RAZLIČNIH EKIPNIH ŠPORTNIH PANOGAH Mentorica: doc. dr. Marija Molan Datum zagovora: 14. 9. 2016 Renata MEGLEN POJAVLJANJE NADURNEGA DELA V KOVINSKI INDUSTRIJI Mentorica: doc. dr. Marija Molan Datum zagovora: 14. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Jerneja PAVLIČ POSREDOVANJE GASILCEV OB NEZGODAH Z NEVARNIMI SNOVMI V PODJETJU ZA PREDELAVO JEKLA Mentorica: doc. dr. Barbara Novosel Datum zagovora: 26. 9. 2016 Urška POJE NEVARNOSTI IN UPORABA PIROTEHNIČNIH IZDELKOV Mentorica: doc. dr. Barbara Novosel Datum zagovora: 28. 9. 2016 Karin LAZAR DOLOČITEV MINIMALNE VŽIGNE ENERGIJE LESNIH PRAHOV SMREKE IN HRASTA Mentorica: doc. dr. Barbara Novosel Datum zagovora: 15. 9. 2016 Tajda AHČIN ZAGOTAVLJANJE POŽARNE VARNOSTI V INDUSTRIJSKEM OBJEKTU STOLARNE Mentor: prof. dr. Simon Schnabl Datum zagovora: 5. 9. 2016 Tanja ČERNOŠA ANALIZA EVAKUACIJSKEGA ČASA V STAVBI OPERE S PROGRAMOM PATHFINDER Mentor: prof. dr. Simon Schnabl Datum zagovora: 5. 9. 2016 Rok GREGORIN ZVOK IN GAŠENJE, ZATIRANJE PLAMENA IN DINAMIKA POŽAROV Mentor: prof. dr. Simon Schnabl Datum zagovora: 16. 9. 2016 Tadej LESJAK OGNJEMETI IN NJIHOV VPLIV NA PRISOTNOST TRDNIH DELCEV V ZRAKU Mentorica: prof. dr. Marija Bešter Rogač Datum zagovora: 16. 9. 2016 Miržel COCIC ODPADNE VODE NA FAKULTETI ZA KEMIJO IN KEMIJSKO TEHNOLOGIJO Mentor: prof. dr. Marjan Veber Datum zagovora: 20. 9. 2016 Društvene vesti in druge aktualnosti S22 Acta Chim. Slov. 2017, 64, (1), Supplement S55 DIPLOME - VISOKOŠOLSKI STROKOVNI ŠTUDIJ KEMIJSKA TEHNOLOGIJA Vlasta ROZMAN PREVERJANJE STABILNOSTI PUFRNIH RAZTOPIN IN INDIKATORJEV, KI VSTOPAJO V ANALIZNI PROCES Mentorica: izr. prof. dr. Nataša Gros Datum zagovora: 18. 5. 2016 Jernej VARGA DOLOČANJE VSEBNOSTI VODE V GRANULATIH Mentorica: izr. prof. dr. Nataša Gros Datum zagovora: 27. 9. 2016 Jasna DRAGAN PRIMERJAVA DVEH VIROV KONCENTRATOV ZA PRIPRAVO MEDIJEV ZA RAZTAPLANJE TRDNIH FARMACEVTSKIH OBLIK Mentor: izr. prof. dr. Matevž Pompe Datum zagovora: 30. 9. 2016 Anica JOVANDARIC PREGLED OBRATOVANJA KOMUNALNIH ČISTILNIH NAPRAV S PRIMERJAVO DEJANSKIH IN PROJEKTIRANIH VREDNOSTI PARAMETROV NA IZTOKU Mentorica: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 23. 6. 2016 Ksenja AHČIN VPLIV INTERFERENC NA POTENCIOMETRIČNE MERITVE Mentorica: izr. prof. dr. Nataša Gros Datum zagovora: 30. 9. 2016 Janez ILERŠIČ REGENERACIJA IZOPROPILACETATA IN METILENKLORIDA Mentor: prof. dr. Aleksander Pavko Datum zagovora: 5. 1. 2016 Irena BRODARIČ OPTIMIZACIJA DELEŽA POSPEŠEVALCA V AKRIALTIVNEM LEPILU Mentorica: prof. dr. Urška Šebenik Datum zagovora: 22. 9. 2016 Mitja NOVAK ŠTUDIJA IZLUŽEVALNIH KARAKTERISTIK FOSFATOV IZ RAZLIČNIH VRST BIOOGLJA Mentorica: doc. dr. Marija Zupančič Datum zagovora: 27. 9. 2016 Darja PALATINUS VPLIV UPORABE NANOCELULOZNIH MATERIALOV NA LASTNOSTI PREMAZANEGA PAPIRJA Mentor: prof. dr. Igor Plazl Datum zagovora: 23. 9. 2016 Ema KEMPERLE LASNOSTI ASFALTNIH ZMESI Z DODANIM HIDRIRANIM APNOM Mentorica: doc. dr. Klementina Zupan Datum zagovora: 16. 9. 2016 Denis PUNGERČAR KVALIFIKACIJA LC/MS SISTEMA ZA POTREBE SPREMLJANJA REAKCIJ Mentor: prof. dr. Janez Košmrlj Datum zagovora: 26. 9. 2016 Alenka PAPEŽ ZNAČILNOSTI SUSPENZIJE ZA PRIPRAVO VEČPLASTNEGA VARISTORJA Mentorica: doc. dr. Klementina Zupan Datum zagovora: 28. 9. 2016 Jure ZAJC POTENCIOMETRIČNE MERITVE Z ELEKTRODAMI V CELOTI V TRDNEM STANJU Mentorica: izr. prof. dr. Nataša Gros Datum zagovora: 11. 5. 2016 Elvis DEŽMAN OPTIMIZACIJA REAKCIJE HIDROGENIRANJA LIPSTATIN OLJA Mentor: prof. dr. Matjaž Krajnc Datum zagovora: 16. 5. 2016 Andreja MAVEC DOLOČANJE VSEBNOSTI MIDAZOLAMIJEVEGA KLORIDA Mentor: izr. prof. dr. Matevž Pompe Datum zagovora: 26. 9. 2016 Anja ŠTEBE PRIPRAVA NEIDEALNIH DVOKOMPONENTNIH RAZTOPIN: VOLUMSKI EFEKTI V MEŠANICAH VODA-ETANOL Mentor: izr. prof. dr. Jurij Reščič Datum zagovora: 28. 9. 2016 Ben KRISTAN NADOMESTEK NONILFENOLNIH ETOKSILATOV Mentorica: prof. dr. Ksenija Kogej Datum zagovora: 21. 6. 2016 Simona MATKO PRIPRAVA IZBRANIH 1,3-DIARILTRIAZENOV Mentor: prof. dr. Janez Košmrlj Datum zagovora: 4. 7. 2016 Maja MITROVIC DOLOČITEV UČINKOVITOSTI PRIMARNE SEPARACIJE V BIOPROCESIH S SESALČJO CELIČNO KULTURO Mentor: prof. dr. Aleksander Pavko Datum zagovora: 8. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Jernej URH RAZBARVANJE RAZTOPINE VANKOMICINA Z ALUMINIJEVIM OKSIDOM IN AKTIVNIM OGLJEM Mentor: prof. dr. Aleksander Pavko Datum zagovora: 8. 9. 2016 Mojca ŠUTAR DOLOČEVANJE RIBOFLAVINA Z METODO TEKOČINSKE KROMATOGRAFIJE VISOKE LOČLJIVOSTI V PREHRANSKIH DOPOLNILIH IN BIOLOŠKIH VZORCIH Mentorica: dr. Tatjana Zupančič Datum zagovora: 26. 9. 2016 Alisa CEHIC VALIDACIJA HPLC METODE ZA DOLOČANJE OSTANKOV HORMONSKEGA PREPARATA NA BRISIH PO ČIŠČENJU PROIZVODNE OPREME Mentorica: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 26. 9. 2016 Nataša KOROŠEC STABILIZACIJA ČRNEGA PIGMENTA V VODNEM PREMAZU Z UPORABO FIZIKALNO-KEMIJSKIH METOD Mentor: dr. Branko Alič Datum zagovora: 27. 9. 2016 Mateja FIDERŠEK OVREDNOTENJE RAZLIČNIH NAČINOV SPEKTROMETRIČNEGA MERJENJA Mentorica: izr. prof. dr. Nataša Gros Datum zagovora: 27. 9. 2016 Maja OBERČ SPREMLJANJE INTERAKCIJ PRI KEMIJSKI STABILIZACIJI ZDRAVILNE UČINKOVINE V FORMULACIJI Mentor: prof. dr. Janez Plavec Datum zagovora: 27. 9. 2016 Darja MOHORČIČ SINTEZA IN KARATKTERIZACIJA PRIRDINSKIH DERIVATOV Z 1H-BENZIMIDAZOL-2-TIOLOM Mentor: prof. dr. Marijan Kočevar Datum zagovora: 27. 9. 2016 Anja PLAHUTA RAZVOJ MIKROSTRUKTURE OKSIDNE ANODE ZA KERAMIČNE GORIVNE CELICE Mentorica: doc. dr. Klementina Zupan Datum zagovora: 27. 9. 2016 Mojca MATOH SINTEZA IN KARAKTERIZACIJA KOMPLEKSOV KOBALTOVEGA IN MANGANOVEGA BROMIDA Z ACETONITRILNIM LIGANDOM Mentor: prof. dr. Alojz Demšar Datum zagovora: 28. 9. 2016 Lidija BOŽIČ MRKONJIČ SPREMLJANJE SEKUNDARNIH FAZ V ANODNIH MATERIALIH NA OSNOVI LANTAN STRONCIJ KROM MANGAN OKSIDA ZA KERAMIČNE GORIVNE CELICE Mentorica: doc. dr. Klementina Zupan Datum zagovora: 29. 9. 2016 Vladimira OBRANOVIČ PRIPRAVA KOVINSKIH PRAHOV S TERMIČNIM RAZKROJEM MEŠANIH HIDRAZIN-KARBOKSILATOV Mentorica: doc. dr. Barbara Novosel Datum zagovora: 29. 9. 2016 Janez VOLMAJER PRIMERJAVA DOLOČANJA VLAŽNOSTI PAPIRJA PO STANDARDNI METODI S POMOČJO BLIŽNJE INFRARDEČE SPEKTROMETRIJE Mentor: izr. prof. dr. Matevž Pompe Somentorica: Jana Kolar Datum zagovora: 30. 9. 2016 Matjaž MALAVAŠIČ IZOLACIJA INHIBITORJEV AMINOPEPTIDAZE N IZ FILTRATA KULTURE STREPTOMYCES RIMOSUS Mentorica: Metka Renko Datum zagovora: 30. 9. 2016 Mojca PERPAR VPLIV STOPENJSKEGA ZNIŽEVANJA pH VREDNOSTI NA PROCES INKAPSULACIJE BUTIL STEARATA Z MELAMINSKO-FORMALDEHIDNO SMOLO Mentor: dr. Branko Alič Datum zagovora: 30. 9. 2016 KEMIJSKA TEHNOLOGIJA - 1. stopnja Blaž ŠPRAJCER ČIŠČENJE IZCEDNE VODE ZAPRTEGA ODLAGALIŠČA ODPADKOV S FENTONOVO OKSIDACIJO Mentorica: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 25. 2. 2016 Miha SOLDAT PRIPRAVA SILIL SUBSTITUIRANIH BENZOJSKIH KISLIN Mentor: izr. prof. dr. Janez Cerkovnik Datum zagovora: 16. 9. 2016 Rok PIKON TVORBA C-C VEZI NA DERIVATU NIKOTINA Mentor: prof. dr. Janez Košmrlj Datum zagovora: 14. 9. 2016 Urška TRBOVC SINTEZA IN NADALJNJA PRETVORBA FENIL-SUBSTITUIRANIH PIRAZINOV IN KINOKSALINOV Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 5. 7. 2016 Društvene vesti in druge aktualnosti S24 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Maja BRINOVEC SINTEZA IN KARAKTERIZACIJA (3R*,4R*)-4- BENZILOKSIKARBONILAMINO-3-IZOPROPIL-5- OKSOPIRAZOLIDINONA Mentor: prof. dr. Jurij Svete Datum zagovora: 6. 7. 2016 Aleš GABER UV-VIDNI SPEKTRI KROMOVIH(III) KOORDINACIJSKIH SPOJIN Mentorica: doc. dr. Barbara Modec Datum zagovora: 4. 3. 2016 Andrej GNIDOVEC RAZVOJ IN VALIDACIJA METODE ZA DOLOČEVANJE OSTANKOV ANTIBIOTIKA V BRISIH PO ČIŠČENJU PROIZVODNE OPREME Mentorica: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 23. 12. 2016 Barbara MENCIN DOLOČITEV POGOJEV ZA PREVERJANJE KVALITETE VELIKIH KROMATOGRAFSKIH MONOLITOV Mentor: prof. dr. Marjan Veber Datum zagovora: 20. 9. 2016 Simon LONČARIČ VPLIV MLETJA IN RAZKLOPA RAZLIČNIH ILMENITNIH RUD NA PROCES PRIDOBIVANJA TITANOVEGA DIOKSIDA PO SULFATNEM POSTOPKU Mentorica: doc. dr. Barbara Novosel Datum zagovora: 21. 1. 2016 Dominika ZORMAN ANALIZA UV-SUŠEČIH LEPIL ZA UPORABO V ZAŠČITNIH TISKOVINAH Mentor: prof. dr. Miran Gaberšček Datum zagovora: 1. 2. 2016 Sanja POPOVIC TEMPERATURNA ODVISNOST GOSTOT ALKOHOLOV IN MEŠANIC ALKOHOLOV TER VODE Mentor: izr. prof. dr. Tomaž Urbič Datum zagovora: 15. 9. 2016 Tinkara ČUČNIK BUDIŠA CINKOVE KOORDINACIJSKE SPOJINE Z INDOL-3-OCETNO IN INDOL-3-PROPANOJSKO KISLINO S POTENCIALNIM ANTIDIABETIČNIM DELOVANJEM Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 7. 4. 2016 Anja MODIC RAZVOJ IN UPORABA ICP-OES METOD ZA DOLOČANJE SREBRA V REALNIH VZORCIH Mentor: izr. prof. dr. Mitja Kolar Datum zagovora: 30. 9. 2016 Mirjana BARBORIČ RAZVOJ ANALIZNE METODE ZA DOLOČEVANJE SORODNIH SUBSTANC KLOPIDOGRELIJEVEGA HIDROGENSULFATA S TANKOPLASTNO KROMATOGRAFIJO Mentorica: prof. dr. Helena Prosen Datum zagovora: 21. 10. 2016 Tina KOZJEK DOLOČEVANJE ORGANSKIH KISLIN V AEROSOLIH S TEKOČINSKO KROMATOGRAFIJO Mentor: izr. prof. dr. Matevž Pompe Datum zagovora: 5. 9. 2016 Aleksandra HUDAK PREVERJANJE ALTERNATIVNIH HPLC KOLON ZA DOLOČANJE DERIVATA BENZIZOKSAZOLA Mentorica: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 3. 3. 2016 Eva RIFELJ IZDELAVA POSTOPKA VREDNOTENJA REFERENČNIH SUBSTANC V FARMACEVTSKI INDUSTRIJI Mentorica: prof. dr. Helena Prosen Datum zagovora: 7. 7. 2016 Maja ANTONIC SINTEZA NEČISTOČ DIMETIL AMLODIPINA IN DIETIL AMLODIPINA Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 28. 9. 2016 Karmen SIMONČIČ ADSORPCIJA METANOJSKE, ETANOJSKE, PROPANOJSKE IN BUTANOJSKE KISLINE NA AKTIVNO OGLJE Mentor: doc. dr. Miha Lukšič Datum zagovora: 15. 9. 2016 Sabina JENSTERLE SPREMLJANJE OKOLJSKIH PODATKOV Z RAZVOJNO PLOŠČICO ARDUINO Mentor: doc. dr. Črtomir Podlipnik Datum zagovora: 15. 9. 2016 Tanja BIZJAK VISKOZNOST IN GOSTOTA VODNIH RAZTOPIN 1,2-DIMETILIMIDAZOLIJEVEGA KLORIDA Mentorica: prof. dr. Marija Bešter Rogač Datum zagovora: 12. 9. 2016 Katja KERT TEMPERATURNA ODVISNOST NAVIDEZNIH MOLSKIH VOLUMNOV KOMPLEKSA MED DODECILTRIMETIL AMONIJEVIM KATIONOM IN POLIAKRILATNIM ANIONOM V ETANOLNIH RAZTOPINAH Mentorica: prof. dr. Ksenija Kogej Datum zagovora: 23. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Neža BREZOVAR TEMPERATURNA ODVISNOST NAVIDEZNIH MOLSKIH VOLUMNOV KOMPLEKSA MED KATIONSKIM SURFAKTANTOM IN POLISTIRENSULFONATNIM ANIONOM V ETANOLNIH RAZTOPINAH Mentorica: prof. dr. Ksenija Kogej Datum zagovora: 8. 9. 2016 Alen HONSIC POSKUSI PRIPRAVE MODIFICIRANIH DERIVATOV FULERENA C60 Mentor: izr. prof. dr. Janez Cerkovnik Datum zagovora: 16. 9. 2016 Denis HONSIC POSKUSI PRIPRAVE MODIFICIRANIH DERIVATOV FULERENA Mentor: izr. prof. dr. Janez Cerkovnik Datum zagovora: 16. 9. 2016 Domen ŽIGANTE SINTEZA N-PROPARGILMALEIMIDA Mentor: prof. dr. Darko Dolenc Datum zagovora: 30. 6. 2016 Benjamin ODORČIC AMIDIRANJA (S)-(2,2-DIMETIL-3-METILENCIKLOPENTIL) METANAMINA Mentor: doc. dr. Uroš Grošelj Datum zagovora: 3. 6. 2016 Katarina ŽAGAR SINTEZA IN PRETVORBE B-KETO ESTRA PRIPRAVLJENEGA IZ Boc-Aib-OH Mentor: doc. dr. Uroš Grošelj Datum zagovora: 5. 9. 2016 Žiga BREGAR VZDRŽEVANJE IN RAVNANJE Z NMR INŠTRUMENTOM Mentor: prof. dr. Andrej Petric Datum zagovora: 6. 6. 2016 Anže JAKLIČ SINTEZA IMINOV IZ ALDEHIDOV IN KETONOV Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 25. 1. 2016 Gregor PAVEC SINTEZA IN KARAKTERIZACIJA KINOKSALINSKIH DERIVATOV Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 25. 1. 2016 Tomaž ZUPANČIČ SINTEZA IN KARAKTERIZACIJA KOORDINACIJSKIH SPOJIN NIKLJA(II) S TIOCIANATNIM LIGANDOM IN 4-PIRIDINOLOM Mentor: izr. prof. dr. Boris Čeh Datum zagovora: 27. 9. 2016 Jaka ZEMLJAK REAKCIJA MANGANOVEGA KLORIDA DIHIDRANTA IN PIPERAZINA Mentorica: doc. dr. Saša Petricek Datum zagovora: 29. 1. 2016 Tim KNIFIC SINTEZA BAKROVIH(II) KOORDINACIJSKIH SPOJIN S KINALDINSKO KISLINO IN PIRIDINSKIMI LIGANDI Mentorica: doc. dr. Barbara Modec Datum zagovora: 1. 9. 2016 Pandi BUKLESKI SINTEZA IN KARATKTERIZACIJA NEKATERIH AMINOMETILPIRIDINIJEVIH HEKSAFLUORIDOTITANATOV Mentor: doc. dr. Andrej Pevec Datum zagovora: 2. 9. 2016 Tina ŠIMUNOVIC SPOJINE KOVIN ČETRTE PERIODE Z O-AMINOBENZATOM Mentorica: doc. dr. Nives Kitanovski Datum zagovora: 2. 9. 2016 Domen OTOREPEC DOLOČITEV MINIMALNE VŽIGNE ENERGIJE LESNIH PRAHOV Mentorica: doc. dr. Barbara Novosel Datum zagovora: 9. 9. 2016 Martina PETELINC NEVARNOST PRAŠNIH EKSPLOZIJ V FARMACEVTSKI INDUSTRIJI Mentorica: doc. dr. Barbara Novosel Datum zagovora: 9. 9. 2016 Sabrina ČERMELJ TEMPERATURNA ODVISNOST KRITIČNEMICELNE KONCENTRACIJE SURFAKTANTA N-DODECILPIRIDINIJEVEGA KLORIDA, DOLOČENA S SPEKTROFOTOMETRIČNO METODO Mentorica: prof. dr. Barbara Hribar Lee Datum zagovora: 29. 9. 2016 Marko GAŠPERIČ NOVI KATODNI MATERIALI ZA VISOKOTEMPERATURNE GORIVNE CELICE Mentorica: doc. dr. Klementina Zupan Datum zagovora: 29. 6. 2016 Sandi JAKLIČ OPTIMIZACIJA SINTEZE ORGANSKIH LIGANDOV ZA PRIPRAVO CINKOVIH KOORDINACIJSKIH SPOJIN Z ANTIDIABETIČNIM DELOVANJEM Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 15. 11. 2016 Žiga KASTELIC TRANSFORMACIJE EVGENOLA Z RAZLIČNIMI REAGENTI Mentor: izr. prof. dr. Franci Kovac Datum zagovora: 23. 9. 2016 Društvene vesti in druge aktualnosti S26 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Draženko LONČAR PRIPRAVA TH-SIMETRIČNIH HEKSASUBSTITUIRANIH DERIVATOV FULERENA C60 Mentor: izr. prof. dr. Janez Cerkovnik Datum zagovora: 27. 9. 2016 Gregor BORDON UPORABA AZOMETIN IMINOV ZA SINTEZO BICIKLIČNIH HETEROCIKLOV Mentor: prof. dr. Jurij Svete Datum zagovora: 6. 9. 2016 Rok REMŠAK REAKCIJE MED MODERNIMI MATERIALI ZA VISOKOTEMPERATURNE GORIVNE CELICE PRI POVIŠANIH TEMPERATURAH Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 18. 5. 2016 Taja VODOPIVEC PRIPRAVA IN ANALIZA TEHNOLOŠKE VODE Mentorica: doc. dr. Barbara Novosel Datum zagovora: 21. 10. 2016 Klemen VRBANČIČ SINTEZA SREBROVIH KOORDINACIJSKIH SPOJIN Z BENZOATNIM IN 2,6-BIS(TRIFLUOROMETIL) BENZOATNIM LIGANDOM Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 6. 10. 2016 Blaž FON SINTEZA KURKUMINA MODIFICIRANEGA S PROPANOJSKO KISLINO Mentor: prof. dr. Darko Dolenc Datum zagovora: 1. 9. 2016 Lenart DEBELAK ORGANOKATALIZIRANE REAKCIJE 1,3-DIKARBONILNIH SPOJIN Z ORTO-SUBSTITUIRANIMI DERIVATI TRANS-A-NITROSTIRENA Mentor: doc. dr. Uroš Grošelj Datum zagovora: 13. 9. 2016 Janez JAVORNIK PRIPRAVA NEKATERIH DERIVATOV BENZOTIAZOLA IN IZBRANE PRETVORBE Mentor: izr. prof. dr. Franci Kovač Datum zagovora: 9. 9. 2016 Eva ŠTRAKL SUBSTITUIRANI 3-ACILAMINO-2H-PIRAN-2-ONI KOT DIENI V DIELS-ALDERJEVIH REAKCIJAH Mentor: doc. dr. Krištof Kranjc Datum zagovora: 16. 9. 2016 Peter SEBANC ČIŠČENJE IN SUŠENJE NEKATERIH ORGANSKIH TOPIL Mentor: prof. dr. Andrej Petrič Datum zagovora: 5. 9. 2016 Karmen ŽBOGAR SINTEZA VSEH IZOMEROV DINITROBENZENA Mentor: prof. dr. Andrej Petrič Datum zagovora: 19. 9. 2016 Marjana VRHOVEC SINTEZA CIKLIČNIH AZOMETIN IMINSKIH SUBSTRATOV Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 16. 9. 2016 Maruša BREGAČ REAKCIJE CINKOVEGA ALI KROMOVEGA KLORIDA Z MORFOLINOM Mentorica: doc. dr. Saša Petriček Datum zagovora: 26. 10. 2016 Daniela MIKIC PRIPRAVA KOORDINACIJSKIH SPOJIN CINKA(II) S KINALDINATOM Mentorica: doc. dr. Barbara Modec Datum zagovora: 24. 10. 2016 Jaka ŠTURM REAKCIJE MOLIDBENA(V) Z N,O-DONORSKIMI LIGANDI Mentorica: doc. dr. Barbara Modec Datum zagovora: 30. 11. 2016 Tilen SIMŠIČ ELEKTROKROMNE LASTNOSTI NIKELJ-OKSIDNIH TANKIH PLASTI Mentorica: doc. dr. Romana Cerc Korošec Datum zagovora: 2. 9. 2016 Klavdija KOČNAR VPLIV PARAMETROV V RAZTOPINI PLASMOCORINTHA B NA UČINKOVITOST NJEGOVE FOTOKATALITSKE RAZGRADNJE S TiO2 Mentorica: doc. dr. Romana Cerc Korošec Datum zagovora: 27. 9. 2016 Tina BREC FAZNA ANALIZA RAZLIČNIH VZORCEV PUDRA Z RENTGENSKO PRAŠKOVNO DIFRAKCIJO Mentorica: izr. prof. dr. Amalija Golobič Datum zagovora: 2. 9. 2016 Tina Melisa ŠIMIČ MANGANOVE SPOJINE Z 1-HIDROKSIBENZOTRIAZOLOM Mentor: doc. dr. Bojan Kozlevčar Datum zagovora: 31. 8. 2016 Patricia TANDARA PIRIDINSKI DERIVATI TIOSECNINE KOT KATIONI V HEKSAFLUORIDOTITANATNIH SOLEH Mentor: doc. dr. Andrej Pevec Datum zagovora: 7. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Renata BEVEC SINTEZE LIGANDOV IZ DIPIKOLINSKE KISLINE ZA VEZAVO NA CINKOVE IN VANADIJEVE IONE Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 2. 9. 2016 Neža GARTNAR RAZISKAVA PIROLIZNIH LASTNOSTI MESNO-KOSTNE MOKE Mentorica: doc. dr. Marija Zupančič Datum zagovora: 2. 9. 2016 Natalija PUCIHAR PRODUKTI SOLVOTERMALNE SINTEZE Z 2-AMINOBENZOJSKO KISLINO Mentorica: doc. dr. Nives Kitanovski Datum zagovora: 16. 9. 2016 Nina ZALETEL VPLIV pH RAZTOPINE BARVILA PLASMOCORINTH B NA NJEGOVO RAZGRADNJO Mentorica: doc. dr. Irena Kozjek Škofic Datum zagovora: 2. 9. 2016 Neja LOMBERGAR ZMRZIŠČE, ZAŠČITA PRED ZMRZOVANJEM IN STRDIŠČE HLADILNIH TEKOČIN Mentor: dr. Andrej Godec Datum zagovora: 1. 9. 2016 Kaja PURKAT LASTNOSTI RAZTOPIN KALCIJEVEGA HIDROKSIDA V RAZLIČNIH TOPILIH Mentorica: prof. dr. Barbara Hribar Lee Datum zagovora: 28. 9. 2016 Mojca ZALOKAR TEMPERATURNA ODVISNOST TOPLOTNE KAPACITETE PLINOV Mentor: prof. dr. Andrej Jamnik Datum zagovora: 13. 9. 2016 Simona PUST PROUČEVANJE INTERAKCIJ LEKTINA FIMH IN RASTLINSKIH POLIFENOLOV Z METODAMI MOLEKULSKEGA MODELIRANJA Mentor: doc. dr. Črtomir Podlipnik Datum zagovora: 21. 12. 2016 Urša SEDMAK DOLOČEVANJE KLORIRANIH SPOJIN Z MIKROEKSTRAKCIJO NA TRDNO FAZO Mentorica: prof. dr. Helena Prosen Datum zagovora: 9. 9. 2016 Miha ŠEST KVANTITATIVNO DOLOČANJE LIMONENA Z IR SPEKTROSKOPIJO Mentor: izr. prof. dr. Mitja Kolar Datum zagovora: 14. 9. 2016 Friderik ŠTENDLER MEŠANJE IN SNOVNI PRENOS KISIKA V BIOREAKTORJIH ZA SUBMERZNO GOJENJE Mentor: prof. dr. Aleksander Pavko Datum zagovora: 9. 12. 2016 Evgen ZORC REGULACIJA NAKLONA LAMEL BRISOLEJA NA NOVI STAVBI FKKT Mentor: doc. dr. Janez Cerar Datum zagovora: 25. 11. 2016 VARSTVO PRI DELU IN POŽARNO VARSTVO Natalija HRASTOVEC VZROKI NASTANKA VELIKIH KOLIČIN MEŠANIH KOMUNALNIH ODPADKOV V ZDRAVSTVENI USTANOVI Mentor: doc. dr. Jože Šrekl Datum zagovora: 28. 9. 2016 Bojan BORIŠEK OCENA TVEGANJA PRI STRUŽNICI PRVOMAJSKA TN-TNP 250 Mentor: doc. dr. Boris Jerman Somentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 23. 9. 2016 Luka ŠKARJA GAŠENJE POŽAROV V VISOKIH STAVBAH Mentor: izr. prof. dr. Matija Tomšič Datum zagovora: 23. 9. 2016 Aleksander ŽAGAR IZBOR SISTEMA ZA ODKRIVANJE POŽARA GLEDE NA PRIČAKOVANO VRSTO POŽARA IN VRSTO OBJEKTA Mentor: doc. dr. Tomaž Hozjan Datum zagovora: 26. 9. 2016 Darko STOLNIK IZBOLJŠANJE VARNOSTI STROJA ZA PROIZVODNJO VALOVITEGA KARTONA Mentor: doc. dr. Boris Jerman Datum zagovora: 30. 9. 2016 Tilen BRECELJ ERGONOMSKO OBLIKOVANO DELOVNO MESTO OPERATERJA DELOVNIH STROJEV IN KONTROLORJA KAKOVOSTI Mentorica: prof. dr. Simona Jevšnik Datum zagovora: 22. 9. 2016 Ivo LOZEJ MERJENJE IN ZMANJŠEVANJE HRUPA V KOVINSKI PROIZVODNJI Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 4. 3. 2016 Igor JUSTIN ANALIZA IZPOSTALJENOSTI HRUPU IN SANACIJSKI UKREPI V PROIZVODNJI PIJAČ Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 4. 3. 2016 Društvene vesti in druge aktualnosti S28 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Maja KOŽUH SAMOSTOJNI PODJETNIK IN OCENA TVEGANJA Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 26. 9. 2016 Tine MAZALOVIC OCENA PROIZVODNJE IN KORISTNA UPORABA DEPONIJSKEGA PLINA NA DEPONIJI Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 28. 9. 2016 Primož JAGRIČ POZARNA VARNOST V GUMARSKI INDUSTRIJI Mentor: prof. dr. Simon Schnabl Datum zagovora: 13. 6. 2016 Aljaž KRIŽMAN OSEBNA VAROVALNA OPREMA PRI FORENZIČNIH PREISKAVAH POZAROV Mentor: izr. prof. dr. Matija Tomšic Datum zagovora: 26. 9. 2016 Saša BAŽDAR PROMOCIJA ZDRAVJA PRI DELU V DRUZBI ELES D. O. O. Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 21. 7. 2016 Vojko CIGAN PREVOZ NEVARNEGA BLAGA V CESTNEM PROMETU Mentorica: doc. dr. Barbara Novosel Datum zagovora: 13. 6. 2016 Mateja KOCMAN VARNOSTNI PREGLED DVEH SREDNJEŠOLSKIH LABORATORIJEV Mentorica: doc. dr. Barbara Novosel Datum zagovora: 5. 7. 2016 Zdenka PETERLE NADZOR NAD OGLJIKOVIM MONOKSIDOM V OBJEKTIH - NORMATIVNE ZAHTEVE IN PRAKTIČNE IZVEDBE Mentor: pred. dr. Aleš Jug Datum zagovora: 5. 7. 2016 Milena HVALIČ ARHAR OKVARA HRBTENICE IN GIBALNEGA SISTEMA PRI NEGOVALNEM OSEBJU Mentor: prof. dr. Marjan Bilban Datum zagovora: 5. 7. 2016 Lidija KUNŠIČ MOZNOST IZRABE BIOPLINA V OBČINI GORJE Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 9. 9. 2016 Maja MLADENOVIC VPLIV STRESA IN IZGORELOST NA MOTIVACIJO ZAPOSLENIH Mentor: prof. dr. Marjan Bilban Datum zagovora: 13. 7. 2016 Stanko MOČNIK UPORABA ELEKTRIČNIH AGREGATOV V GASILSTVU Mentor: pred. dr. Aleš Jug Datum zagovora: 15. 7. 2016 Mojca BAHUN PRESOJA ZDRAVEGA IN VARNEGA DELA V LABORATORIJU Mentorica: doc. dr. Barbara Novosel Datum zagovora: 2. 9. 2016 Blaž RAČNIK RAVNANJE Z ODPADKI S STRATEGIJO BREZ ODPADKOV V SLOVENIJI Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 2. 9. 2016 Mladen BJEGOJEVIC NAVODILA ZA VARNO DELO, PREIZKUS ZNANJA, TER UPORABA OVO NA ZNAŠALNO ŠIVALNEM STROJU Müller Martini PRIMA Mentor: prof. dr. Jože Horvat Datum zagovora: 2. 9. 2016 Helena FRANK VARNO DELO V MARKETU Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 2. 9. 2016 Alan RATNIK ANALIZA ZDRAVJA IN POČUTJA TAKSI VOZNIKOV ZARADI DELOVNEGA MESTA Mentor: prof. dr. Marjan Bilban Datum zagovora: 2. 9. 2016 Janko SEKNE PRAKTIČNA UPORABA POŽARNIH NAČRTOV PRI GASILCIH V REPUBLIKI SLOVENIJI Mentor: doc. dr. Jože Šrekl Datum zagovora: 5. 9. 2016 Igor KRAJNC OPERATER PREDVAJANJA PROGRAMA Mentor: doc. dr. Jože Šrekl Datum zagovora: 5. 9. 2016 Marko RUŽIČ OBVLADOVANJE TVEGANJA PRI DELU Z NEVARNIMI SNOVMI V TISKARNI Mentorica: doc. dr. Barbara Novosel Datum zagovora: 5. 9. 2016 Miha KUKOVICA SAMOVŽIG LESNE BIOMASE Mentor: doc. dr. Jože Šrekl Datum zagovora: 9. 9. 2016 Mateja MALAVAŠIČ ERGONOMSKO OBLIKOVANJE DELOVNEGA MESTA NATAKARJA Mentorica: doc. dr. Klementina Zupan Datum zagovora: 9. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Marinko MAKSIMOVIČ UPORABA NEVARNIH KEMIKALIJ V GRADBENIŠTVU Mentorica: doc. dr. Barbara Novosel Datum zagovora: 9. 9. 2016 Mitja PLANINŠEK VARNOSTNI UKREPI PRI SKLADIŠČENJU IN DISTRIBUCIJI GORIV Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 9. 9. 2016 Ivica COSIC ZBIRANJE, ODDAJANJE IN PREDELAVA ELEKTRONSKIH ODPADKOV Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 15. 9. 2016 Tinka KERN ZAGOTAVLJANJE VARNOSTI DELOVNE OPREME Mentor: doc. dr. Boris Jerman Datum zagovora: 15. 9. 2016 Vesna MARKOVIC VARNOST VODOVODNIH SISTEMOV Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 15. 9. 2016 Nejc PLEČKO MIKROPLASTIKA V OKOLJU Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 16. 9. 2016 Ervin MUJKIC GRADBENO DOVOLJENJE ZA DEPONIJE Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 16. 9. 2016 Luka BALAS ANALIZA VŽIGA KOMPRESORJA Mentorica: doc. dr. Barbara Novosel Datum zagovora: 21. 9. 2016 Dušan ŠUKLJE SREDSTVA ZA PASIVIZACIJO V SPECIALNI ENOTI SLOVENSKE POLICIJE Mentorica: doc. dr. Barbara Novosel Datum zagovora: 21. 9. 2016 Alenka KOS IZPOSTAVLJENOST DELAVCA OGLJIKOVEMU OKSIDU MED VARJENJEM Mentorica: doc. dr. Barbara Novosel Datum zagovora: 22. 9. 2016 Valentina KOLMAN ANALIZA INTERVENCIJ PGD ŠKOFJA VAS Mentor: prof. dr. Simon Schnabl Datum zagovora: 22. 9. 2016 Tanja VIDMAR STALIŠČA DO RABE ALKOHOLA NA DELOVNEM MESTU V MIKRO IN MALIH PODJETJIH NA DOLENJSKEM Mentorica: doc. dr. Marija Molan Datum zagovora: 22. 9. 2016 Sandi LEPOŠA OBREMENJENOST SLOVENSKIH POKLICNIH GASILCEV Mentorica: doc. dr. Marija Molan Datum zagovora: 22. 9. 2016 Maja ROJC VPLIV PSIHOSOCIALNIH TVEGANJ NA ZAPOSLENE V PODJETJU KOLEKTOR ETRA Mentorica: doc. dr. Marija Molan Datum zagovora: 22. 9. 2016 Zoran MAČKOVIC PSIHOSOCIALNA TVEGANJA IN OBVLADOVANJE LE TEH V KOVINSKI IN ELEKTRO INDUSTRIJI TER V INDUSTRIJI KOVINSKIH MATERIALOV IN LIVARN Mentorica: doc. dr. Marija Molan Datum zagovora: 22. 9. 2016 Bojan POLAJŽER USKLAJENOST OTROŠKIH IGRAL Z VARNOSTNIMI PREDPISI Mentor: doc. dr. Jože Šrekl Datum zagovora: 23. 9. 2016 Nuša DROBNAK OSEBNA VAROVALNA OPREMA V KROVSTVU Mentor: izr. prof. dr. Matija Tomšič Datum zagovora: 23. 9. 2016 Miha LEVEC POŽARNA VARNOST V DOMOVIH STAREJŠIH OBČANOV Mentor: prof. dr. Simon Schnabl Datum zagovora: 23. 9. 2016 Rudolf VOLČINI POŽARNA VARNOST V INDUSTRIJI PREMAZOV Mentor: prof. dr. Simon Schnabl Datum zagovora: 23. 9. 2016 Zdenko ZUPAN POŽARNA VARNOST V PLANINSKIH KOČAH Mentor: prof. dr. Simon Schnabl Datum zagovora: 23. 9. 2016 Matej TURK RAČUNALNIŠKI MODELI ZA IZRAČUN TOKSIČNOSTI KEMIJSKIH SPOJIN Mentor: doc. dr. Črtomir Podlipnik Datum zagovora: 26. 9. 2016 Društvene vesti in druge aktualnosti S30 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Rok SEVER ZMANŠEVANJE TVEGANJA ZA NASTANEK PRAŠNE EKSPLOZIJE PRI PRAŠNEM LAKIRNJU Mentorica: doc. dr. Barbara Novosel Datum zagovora: 26. 9. 2016 Matija GOMILAR SANACIJA SISTEMA ČIŠČENJA ODPADNIH VOD IZ PROIZVODNJE TEHNIČNE KERAMIKE Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 26. 9. 2016 Matej PRELEC OBRATOVANJE KOMUNALNE ČISTILNE NAPRAVE Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 26. 9. 2016 Robert MIHELČIČ STATISTIKA NEZGOD PRI DELU V PODJETJU ZA PROIZVODNJO SLAŠČIC IN PEKOVSKEGA PECIVA Mentor: doc. dr. Jože Šrekl Datum zagovora: 26. 9. 2016 Gordana DOBRIHA VARNOST ELEKTRIČNE PEČI ZA TALJENJE STEKLA Mentor: doc. dr. Boris Jerman Datum zagovora: 26. 9. 2016 Andraž SLAK TRDNI DELCI IN NJIHOVI VPLIVI NA OKOLJE IN NA ZDRAVJE LJUDI Mentorica: prof. dr. Marija Bešter Rogač Datum zagovora: 27. 9. 2016 Daniel RADONIC ANALIZA IN OPREDELITEV ONESNAŽEVANJA ZRAKA NA PRIMERU LONDONA Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 28. 9. 2016 Jože DERNAČ HRUP V PROIZVODNJI PAPIRNATIH VREČK Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 28. 9. 2016 Igor ŠTEBLAJ ANALIZA VARNOSTI VERTIKALNIH IN HORIZONTALNIH OPAŽEV VISOKIH OBJEKTOV Mentor: prof. dr. Simon Schnabl Datum zagovora: 28. 9. 2016 Miha PEČENIK NESREČE NA NAFTNIH PLOŠČADIH IN NJIHOV VPLIV NA OKOLJE NA JADRANU Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 28. 9. 2016 Mateja ŠTEFANČIČ PREGLED METOD IZOBRAŽEVANJA IN USPOSABLJANJA MLADIH V GASILSTVU Mentor: prof. dr. Simon Schnabl Datum zagovora: 28. 9. 2016 Klemen ŠKRJANEC ANALIZA POŽARNE VARNOSTI OBJEKTA NA MESTNEM TRGU V ŠKOFJI LOKI Mentor: prof. dr. Simon Schnabl Datum zagovora: 28. 9. 2016 Jerneja KOZOROG VARNO IN ZELENO DELOVNO OKOLJE V PISARNI Mentorica: doc. dr. Klementina Zupan Datum zagovora: 28. 9. 2016 Aleksander VRABIČ PROBLEMATIKA HRUPA V LESNI INDUSTRIJI Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 28. 9. 2016 Igor SAMOTORČAN VARNOSTNI UKREPI PRI POLNJENJU, TESTIRANJU IN ROKVANJU S TLAČILNIMI POSODAMI Mentorica: doc. dr. Barbara Novosel Datum zagovora: 28. 9. 2016 Erika POTRČ HRIBAR PREGLED PREVENTIVNIH UKREPOV OMEJEVANJA POŽAROV V NARAVNEM OKOLJU Mentor: prof. dr. Simon Schnabl Datum zagovora: 29. 9. 2016 Dijana PETKOVIC ANALIZA POŽARNE VARNOSTI V DOMU STAREJŠIH OBČANOV Mentor: prof. dr. Simon Schnabl Datum zagovora: 29. 9. 2016 Anita ČUK POZNAVANJE ZAPOSLENIH GLEDE VARSTVA IN ZDRAVJA PRI DELU V MESTNI OBČINI LJUBLJANA Mentor: doc. dr. Jože Šrekl Datum zagovora: 29. 9. 2016 Martina KERMAVNER KOLMAN PREPREČEVANJE ZDRAVSTVENIH TEŽAV SKOZI CELOTNO POKLICNO ŽIVLJENJE Mentor: doc. dr. Jože Šrekl Datum zagovora: 29. 9. 2016 Nina Mirjam MATKOVIČ OBREMENITVE VZGOJITELJIC NA DELOVNEM MESTU Mentor: doc. dr. Jože Šrekl Datum zagovora: 29. 9. 2016 Igor KOSI VARNOST IN ZDRAVJE PRI DELU PRI MONTAŽI BETONSKIH KONSTRUKCIJSKIH ELEMENTOV Mentor: doc. dr. Jože Šrekl Datum zagovora: 29. 9. 2016 Mario MAJKIC VPLIV DELOVNIH POGOJEV NA ZDRAVJE GRADBENIH DELAVCEV Mentor: doc. dr. Domen Kušar Datum zagovora: 29. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Nina KOTNIK VPETOST JEDRSKE ELEKTRARNE V LOKALNO SKUPNOST Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 29. 9. 2016 Robert TIVADAR DVIG VARNOSTNE KULTURE PRI ROKOVANJU Z OROŽJEM Mentor: doc. dr. Jože Šrekl Datum zagovora: 29. 9. 2016 Mojca BARIČIČ NIZKOTEMPERATURNI SISTEMI Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 29. 9. 2016 Brigita BARIČIČ RAVNANJE Z ODPADKI V OBČINI ILIRSKA BISTRICA Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 29. 9. 2016 Aleš VOVK VARNO DELO S KEMIČNIMI SNOVMI V AVTOLIČARSKI DELAVNICI Mentorica: doc. dr. Barbara Novosel Datum zagovora: 29. 9. 2016 Barbara PUC CELOVITO RAVNANJE Z ODPADKI Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 29. 9. 2016 Barbara DERNAČ RAVNANJE Z ODPADKI V MANJŠI OBČINI Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 29. 9. 2016 Blaž FINK POTENCIALNE NEVARNOSTI PRI MANIPULACIJI IN HRAMBI NAFTNIH DERIVATOV Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 29. 9. 2016 Ajša TOLA OBNOVLJIVI VIRI ENERGIJE V SLOVENIJI IN NJIHOVA UPORABA Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 29. 9. 2016 Anton TEGELJ ANALIZA SISTEMA RAVNANJA Z OKOLJEM EMAS IN NJEGOVA UPORABA V AVTOKLEPARSKEM PODJETJU Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 29. 9. 2016 Mateja SAVŠEK INTERVENCIJSKA TOLERAČNA VREDNOST Mentorica: doc. dr. Barbara Novosel Datum zagovora: 30. 9. 2016 Klemen AVBELJ NEZGODA DEEPWATER HORIZON V MEHIŠKEM ZALIVU Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 30. 9. 2016 Janez GERČAR IZVAJANJE VARNOSTNIH MERITEV NA ELEKTRIČNIH STROJIH Mentor: pred. dr. Grega Bizjak Datum zagovora: 30. 9. 2016 Herman PEČEVNIK OBREMENITVE DELOVNEGA OKOLJA NA DELAVCA V KOVINO OBDELOVALNEM OBRATU Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 30. 9. 2016 Roman KURALT PROBLEMATIKA PREUREJANJA MANJŠIH NESTANOVANJSKIH KMETIJSKIH STAVB V INDUSTRIJSKE STAVBE Z VIDIKA POŽARNE VARNOSTI Mentor: doc. dr. Domen Kušar Datum zagovora: 30. 9. 2016 Iva PERČIC RAVNANJE Z ODPADKI V ZDRAVSTVU Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 30. 9. 2016 Robert TEŽAK VOŽNJA VOZIL S PREDNOSTJO Mentor: pred. dr. Aleš Jug Datum zagovora: 30. 9. 2016 Branislav ORLIC RAZISKAVE POŽAROV Mentor: doc. dr. Tomaž Hozjan Datum zagovora: 30. 9. 2016 Društvene vesti in druge aktualnosti S32 Acta Chim. Slov. 2017, 64, (1), Supplement S55 UNIVERZA V MARIBORU FAKULTETA ZA KEMIJO IN KEMIJSKO TEHNOLOGIJO 1. januar - 31. december 2016 DOKTORATI ENOVIT DOKTORSKI ŠTUDIJ Gregor FERK SINTEZA IN KARAKTERIZACIJA MAGNETNIH NANODELCEV ZA UPORABO V SAMOREGULATIVNI MAGNETNI HIPERTERMIJI Mentor: red. prof. dr. Miha Drofenik Datum zagovora: 5. 7. 2016 Albin MATAVŽ VPLIV STARANJA NA MEHANSKE LASTNOSTI SMOLNO VEZANIH BRUSOV S KORUNDNIMI IN SiC ZRNI Mentor: izr. prof. dr. Darko Goričanec Somentor: red. prof. dr. Jurij Krope Datum zagovora: 25. 8. 2016 Nataša SOVIČ OCENA KAKOVOSTI PODATKOV PRIDOBLJENIH V PROGRAMIH SPREMLJANJA PODZEMNIHVOD IN UPORABA KEMOMETRIJSKIH METOD ZA DOLOČITEV MERILNIH MEST Mentorica: red. prof. dr. Darinka Brodnjak-Vončina Somentor: dr. Mitja Kolar Datum zagovora: 15. 6. 2016 Janez ŽLAK OKOLJSKO SPREJEMLJIVA ENERGIJSKA IZRABA MULJA KOMUNALNIH ČISTILNIH NAPRAV Mentor: red. prof. dr. Jurij Krope Somentor: izr. prof. dr. Darko Goričanec Datum zagovora: 12. 7. 2016 DOKTORSKI ŠTUDIJ Marko AGREŽ SIMULACIJA UPLINJANJA ENERGENTOV ZA PROIZVODNJO ENERGIJE IN SINTETIČNIH GORIV Mentor: izr. prof. dr. Darko Goričanec Somentor: red. prof. dr. Jurij Krope Datum zagovora: 2. 9. 2016 Janja MAJER SINTEZA IN FUNKCIONALIZACIJA MAKROPOROZNIH POLIAKRILATOV Mentor: red. prof. dr. Peter Krajnc Datum zagovora: 27. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 DOKTORSKI ŠTUDIJ - 3. stopnja Darija COR EKSTRAKCIJE BIOLOŠKIH MATERIALOV S SUBKRITIČNIMI IN SUPERKRITIČNIMI FLUIDI Mentor: red. prof. dr. Željko Knez Somentorica: red. prof. dr. Mojca Škerget Datum zagovora: 20. 4. 2016 Urban FEGUŠ RAZVOJ PILOTNE NAPRAVE ZA ENKAPSULACIJO AROMATIČNIH SUBSTANC V TALINO OGLJIKOVIH HIDRATOV Z UPORABO VISOKOTLAČNEGA HOMOGENIZATORJA Mentor: red. prof. dr. Željko Knez Datum zagovora: 15. 7. 2016 Jernej HOSNAR REKONSTRUKCIJSKI PRINCIPI IN STRATEŠKE ODLOČITVE V OBSTOJEČIH INDUSTRIJSKIH PROCESIH Mentorica: doc. dr. Anita Kovač Kralj Somentor: red. prof. dr. Zdravko Kravanja Datum zagovora: 28. 6. 2016 Maja LEŠNIK MEHANIZEM DOPIRANJA ULTRAFINEGA RUTILNEGA TiO2 ZA SPREMINJANJE FOTOKATALITSKE AKT2IVNOSTI Mentor: red. prof. dr. Miha Drofenik Datum zagovora: 20. 9. 2016 Matej RAVBER SUBKRITIČNA VODA KOT ZELENI MEDIJ ZA EKSTRAKCIJO IN PROCESIRANJE NARAVNIH MATERIALOV Mentor: red. prof. dr. Mojca Škerget Somentor: red. prof. dr. Željko Knez Datum zagovora: 6. 6. 2016 Jana SIMONOVSKA OLEORESINI IZ RDEČE PEKOČE PAPRIKE -EKSTRAKCIJA IN UPORABA Mentor: red. prof. dr. Željko Knez Somentorica: red. prof. dr. Mojca Škerget Datum zagovora: 21. 10. 2016 Nina TRUPEJ TERMODINAMSKE IN TRANSPORTNE LASTNOSTI SISTEMOV POLIMEROV IN BIOLOŠKO AKTIVNIH SPOJIN S SUPERKRITIČNIMI FLUIDI Mentor: red. prof. dr. Željko Knez Somentorica: red. prof. dr. Mojca Škerget Datum zagovora: 20. 5. 2016 Društvene vesti in druge aktualnosti S34 Acta Chim. Slov. 2017, 64, (1), Supplement S55 MAGISTRSKI ŠTUDIJ MAGISTRSKI ŠTUDIJ Andrej CAF IZKORIŠČANJE NIZKOTEMPERATURNIH VIROV ENERGIJE PLINSKIH KOGENERACIJSKIH MOTORJEV Mentor: izr. prof. dr. Darko Goricanec Somentor: red. prof. dr. Jurij Krope Datum zagovora: 27. 9. 2016 Vanja FORJAN RAZVOJ, VALIDACIJA IN PRIMERJAVA BIOANALIZNIH METOD HPLC IN LC-MS/MS ZA DOLOČANJE KANDESARTANA V HUMANI PLAZMI Mentorica: red. prof. dr. Darinka Brodnjak-Voncina Somentorica: red. prof. dr. Helena Prosen Datum zagovora: 8. 7. 2016 Dušica IFKO PRIPRAVA MAGNETNIH ZAMREŽENIH ENCIMSKIH SKUPKOV IZ ENCIMA CELULAZA IN OPTIMIRANJE PARAMETROV Mentorica: red. prof. dr. Maja Leitgeb Datum zagovora: 26. 9. 2016 Bojana KRAJNC GALUNDER KEMIJSKA ANALIZA IN KEMOMETRIJSKA KARAKTERIZACIJA KVALITETE VODE REKE MURE Mentorica: red. prof. dr. Darinka Brodnjak-Voncina Somentor: dr. Mitja Kolar Datum zagovora: 27. 9. 2016 Gorazd PECKO ŠKOF SINTEZA SEPARACIJSKIH PROCESOV ZA ČIŠČENJE ODPADNIH OLJNIH EMULZIJ Mentor: red. prof. dr. Zdravko Kravanja Somentorica: red. prof. dr. Zorka Novak Pintaric Datum zagovora: 30. 9. 2016 Metka PEŠL ODZIVNE FUNKCIJE IN TOPLOTNI VPLIV RAZLIČNIH KONFIGURACIJ VERTIKALNIH TOPLOTNIH PRENOSNIKOV V VRTINI V SISTEMIH ZEMELJSKIH TOPLOTNIH ČRPALK Mentor: izr. prof. dr. Darko Goricanec Somentor: red. prof. dr. Jurij Krope Datum zagovora: 12. 7. 2016 Sašo POBERŽNIK VSEŽIVLJENJSKO VREDNOTENJE STROŠKOV ENERGIJE PRI KLASIČNO IN TRAJNOSTNO NARAVNANI GRADNJI STANOVANJSKEGA OBJEKTA Mentor: red. prof. dr. Jurij Krope Somentor: izr. prof. dr. Darko Goricanec Datum zagovora: 12. 7. 2016 David ŠIROVNIK SINTEZA SISTEMOV Z MAKSIMIRANJEM TRAJNOSTNE NETO SEDANJE VREDNOSTI Mentor: red. prof. dr. Zdravko Kravanja Somentorica: doc. dr. Lidija Čucek Datum zagovora: 30. 9. 2016 Lidija ŠKODIČ ČIŠČENJE ODPADNIH TEKSTILNIH VOD Z UV/H2O2 POSTOPKOM 2 2 Mentorica: red. prof. dr. Darinka Brodnjak-Voncina Somentorica: red. prof. dr. Alenka Majcen Le Marechal Datum zagovora: 27. 9. 2016 Andreja ZEMLJIČ UČINKOVITA RABA ENERGIJE V KLIMATIZACIJSKIH SISTEMIH Mentor: izr. prof. dr. Darko Goricanec Somentor: red. prof. dr. Jurij Krope Datum zagovora: 27. 9. 2016 MAGISTRSKI ŠTUDIJ - 2. stopnja_ Filip AMBROŽ RAZVOJ ELEKTROKEMIJSKEGA ČIPA ZA IN-SITU PROIZVODNJO AKTIVNEGA KLORA Mentor: doc. dr. Matjaž Finšgar Somentorica: doc. dr. Irena Ban Datum zagovora: 1. 9. 2016 Klara BIGEC FORMULACIJA BIOLOŠKO AKTIVNIH UČINKOVIN S SUPERKRITIČNIMI FLUIDI Mentor: red. prof. dr. Željko Knez Somentorica: doc. dr. Maša Knez Hrnčič Datum zagovora: 21. 9. 2016 Selena BOŠNJAK OKSALATI IMOBILIZIRANI NA VINILBENZIL KLORIDNI NOSILEC Mentor: izr. prof. dr. Jernej Iskra Somentor: red. prof. dr. Peter Krajnc Datum zagovora: 7. 9. 2016 Mitja BUKOVEC ANALIZA EMAJLIRANEGA NERJAVNEGA JEKLA Mentor: doc. dr. Matjaž Finšgar Somentorica: red. prof. dr. Andreja Goršek Datum zagovora: 21. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Alja GABOR IZRAŽANJE GENOV TNFAIP6, S100A8, IL-11, G0S2 IN S100A9 V KRVNIH LIMFOCITIH IN ČREVESNI SLUZNICI BOLNIKOV S CROHNOVO BOLEZNIJO KOT NAPOVEDNI BIOOZNAČEVALEC ODZIVA NA ZDRAVLJENJE Z ADALIMUMABOM Mentor: red. prof. dr. Uroš Potočnik Somentor: mag. Peter Skok Datum zagovora: 1. 9. 2016 Natalija GRAH ANALIZA AMINOV NA JEKLU Mentor: doc. dr. Matjaž Finšgar Somentorica: izr. prof. dr. Regina Fuchs Godec Datum zagovora: 7. 9. 2016 Kaja GROBELNIK AKTIVNOST ENCIMOV IZ WALLEMIE ICHTHYOPHAGE PO IZPOSTAVITVI V SC CO2 Mentorica: doc. dr. Mateja Primožič Somentorica: red. prof. dr. Maja Leitgeb Datum zagovora: 17. 2. 2016 Jasna GROMAN RAZVOJ METODE ZA VODENO SPROŠČANJE OGLJIKOVEGA DIOKSIDA MED FERMENTACIJO PROBIOTIČNEGA NAPIKA Mentorica: red. prof. dr. Andreja Goršek Somentorica: doc. dr. Darja Pečar Datum zagovora: 17. 2. 2016 Mateja GRUŠOVNIK MAKROPOROZNI POLIMERI IZ KROGLIČNIH ŠABLON Mentor: red. prof. dr. Peter Krajnc Somentorica: asist. dr. Muzafera Paljevac Datum zagovora: 23. 2. 2016 Doroteja GSELMAN GENOTIPIZACIJA SLOVENSKIH BOLNIKOV Z REVMATOIDNIM ARTRITISOM ZA DNA POLIMORFIZME PREDHODNO POVEZANE Z BOLEZNIJO V ASOCIACIJSKIH ŠTUDIJAH V CELOTNEM GENOMU Mentor: red. prof. dr. Uroš Potočnik Somentor: izr. prof. dr. Artur Pahor Datum zagovora: 1. 9. 2016 Ivana HOHNJEC DOLOČANJE OSTANKOV PESTICIDOV V RIBAH IN ŠKOLJKAH S PLINSKO KROMATOGRAFIJO IN MASNO SPEKTROMETRIJO Mentorica: red. prof. dr. Darinka Brodnjak-Vončina Somentor: dr. Mitja Kolar Datum zagovora: 23. 3. 2016 Neja HROVAT UPORABA ZEOLITOV IN RAZVOJ ANALIZNIH METOD ZA SPREMLJANJE UČINKOVITOSTI ČIŠČENJA KOMUNALNIH ODPADNIH VOD Mentor: doc. dr. Matjaž Finšgar Somentorica: Mojca Poberžnik Datum zagovora: 21. 9. 2016 Maša IRŠIČ VPLIV RAZLIČNIH NAČINOV PREDOBDELAVE SUROVE CELULOZE NA UČINKOVITOST ENCIMSKE HIDROLIZE Mentorica: doc. dr. Darja Pecar Somentorica: red. prof. dr. Andreja Goršek Datum zagovora: 13. 7. 2016 Mirjana JEREMIC ODSTRANJEVANJE ATRAZINA IZ PITNE VODE Z VLAKNI IZ AKTIVNEGA OGLJA Mentorica: izr. prof. dr. Marjana Simonic Somentorica: red. prof. dr. Andreja Goršek Datum zagovora: 21. 12. 2016 Gregor JEZERNIK VPLIV POLIMORFIZMOV V CELOTNEM GENOMU NA PROFILE MAŠČOBNIH KISLIN PRI BOLNIKIH S KRONIČNO VNETNO ČREVESNO BOLEZNIJO Mentor: red. prof. dr. Uroš Potocnik Somentorica: doc. dr. Katja Repnik Datum zagovora: 1. 9. 2016 Kaja KAJZER VPLIV SC CO2 NA ODPIRANJE CELIC ČRNE KVASOVKE PHAEOTHECA2 TRIANGULARIS Mentorica: doc. dr. Mateja Primožic Somentorica: red. prof. dr. Maja Leitgeb Datum zagovora: 23. 3. 2016 Monika KOROŠA FERMENTACIJA SIROTKE Z NARAVNO STARTER KULTURO Mentorica: red. prof. dr. Andreja Goršek Somentorica: doc. dr. Darja Pecar Datum zagovora: 31. 9. 2016 Alja KOŠTOMAJ MERJENJE PORAZDELITVE IN VELIKOSTI DELCEV TiO2 PIGMENTA Z APARATURO MASTERSIZER 3000 Mentorica: doc. dr. Irena Ban Somentor: doc. dr. Matjaž Kristl Datum zagovora: 21. 9. 2016 Lucija KRIŽNIK RAZLIČNE TEHNIKE IMOBILIZACIJE ENCIMA a- GALAKTOZIDAZE Mentorica: red. prof. dr. Maja Leitgeb Somentorica: doc. dr. Mateja Primožic Datum zagovora: 23. 3. 2016 Žiga KVAR VPLIV NUKLEATORJEV NA LASTNOSTI POLIPROPILENA Mentorica: red. prof. dr. Andreja Goršek Somentor: IZTOK ŠVAB Datum zagovora: 21. 9. 2016 Aleš LORBER PRIPRAVA ODPADNE VODE ZA PONOVNO UPORABO V TEHNOLOŠKEM PROCESU Mentorica: izr. prof. dr. Marjana Simonic Somentorica: red. prof. dr. Zorka Novak Pintaric Datum zagovora: 21. 12. 2016 Društvene vesti in druge aktualnosti S36 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Evelina MOHORKO RAZVOJ IN VALIDACIJA PLINSKIH SENZORJEV ZA MEDICINSKE APLIKACIJE Mentor: dr. Mitja Kolar Somentor: Andrej Holobar Datum zagovora: 20. 1. 2016 Petra NOVINA IZOLACIJA ANTIOKSIDANTOV IZ JABOLK Mentor: red. prof. dr. Zeljko Knez Somentorica: red. prof. dr. Mojca Škerget Datum zagovora: 23. 3. 2016 Barbara PETOVAR ELEKTROKEMIJSKA IN POVRŠINSKA ANALIZA AZOLOV NA JEKLU Mentor: doc. dr. Matjaž Finšgar Somentor: izr. prof. dr. Urban Bren Datum zagovora: 17. 2. 2016 Tjaša PETROVIČ SIMULACIJE IN OPTIMIZACIJE ODSTRANJEVANJA HLAPNIH ORGANSKIH SNOVI IZ ODPADNIH TOKOV Mentorica: red. prof. dr. Zorka Novak Pintarič Somentorica: red. prof. dr. Mojca Škerget Datum zagovora: 17. 2. 2016 Darja PREDIKAKA SINTEZA IN KARAKTERIZACIJA BIOOGLJA PRIDOBLJENEGA IZ RAZLIČNIH VRST ODPADNE BIOMASE S SUBKRITIČNO VODO Mentorica: red. prof. dr. Mojca Škerget Somentor: red. prof. dr. Zeljko Knez Datum zagovora: 22. 6. 2016 Saša PUŠAVER DOLOČANJE IZBRANIH MONOSAHARIDOV V EKSOPOLISAHARIDIH S PLINSKO KROMATOGRAFIJO IN MASNO SPEKTROMETRIJO Mentor: doc. dr. Matjaž Finšgar Somentorica: doc. dr. Maša Islamčevic Razboršek Datum zagovora: 23. 3. 2016 Barbara SITAR ODSTRANJEVANJE CINKA IN BAKRA IZ VODE Z MODIFICIRANIMI VLAKNI IZ AKTIVNEGA OGLJA Mentorica: izr. prof. dr. Marjana Simonič Somentorica: red. prof. dr. Lidija Fras Zemljič Datum zagovora: 23. 3. 2016 Violeta TRAJKOVSKA MODELIRANJE ZA HITRO OCENJEVANJE POSLEDIC KEMIJSKIH NESREČ Mentorica: red. prof. dr. Zorka Novak Pintarič Somentor: doc. dr. Matjaž Finšgar Datum zagovora: 23. 11. 2016 Jožica ULČNIK HIDROLIZA GLIKOZIDNO VEZANIH ANTIOKSIDANTOV V ČEBULNEM EKSTRAKTU S SUBKRITIČNO VODO Mentorica: red. prof. dr. Mojca Škerget Somentor: red. prof. dr. Zeljko Knez Datum zagovora: 13. 7. 2016 Sabina VERBUČ SINTEZE KOORDINACIJSKIH SPOJIN Co, Cu, Ni IN NEKATERIH LANTANOIDOV Z MEŠANIMI N-DONORSKIMI LIGANDI: AMINOPIRIDINI IN PIKOLINSKO KISLINO Mentor: doc. dr. Matjaž Kristl Somentorica: doc. dr. Irena Ban Datum zagovora: 17. 2. 2016 Nika VERDELJ RAZVOJ IN VALIDACIJA SPEKTROFOTOMETRIČNE METODE ZA DOLOČANJE BORA V REALNIH VZORCIH TAL IN RASTLINSKIH TKIV Mentor: doc. dr. Jože Košir Somentor: dr. Mitja Kolar Datum zagovora: 21. 9. 2016 Tadeja VOLAUŠEK OCENA MOŽNOSTI ONESNAŽENJA TAL IN PODZEMNE VODE NA OBMOČJU NAPRAVE S KEMIČNO NEKOVINSKO PROIZVODNJO Mentorica: red. prof. dr. Zorka Novak Pintarič Somentor: doc. dr. Matjaž Finšgar Datum zagovora: 1. 9. 2016 Marko ŽIŽEK ANALIZA SISTEMOV ZA DOSTAVO ZDRAVILNIH UČINKOVIN IZ MEDICINSKIH IMPLANTATOV Mentor: doc. dr. Matjaž Finšgar Somentor: doc. dr. Uroš Maver Datum zagovora: 21. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 DIPLOME - UNIVERZITETNI ŠTUDIJ UNIVERZITETNI ŠTUDIJ Tjaša AHEJ UPORABA TERMOGRAVIMETRIČNE METODE PRI KARTAKTERIZACIJI KOORDINACIJSKIH SPOJIN Mentor: doc. dr. Matjaž Kristl Somentorica: doc. dr. Irena Ban Datum zagovora: 7. 9. 2016 Simon CEGLAR IZBIRA NAJPRIMERNEJŠEGA HLADIVA ENOSTOPENJSKE ALI DVOSTOPENJSKE VISOKOTEMPERATURNE TOPLOTNE ČRPALKE Mentor: izr. prof. dr. Darko Goričanec Somentor: asist. dr. Peter Trop Datum zagovora: 20. 4. 2016 Šolasta ČUČEK REŠEVANJE MEŠANO CELOŠTEVILSKIH NELINEARNIH PROBLEMOV Z DEKOMPOZICIJSKIMI IN RELAKSACIJSKIMI METODAMI Mentorica: red. prof. dr. Zorka Novak Pintarič Somentor: red. prof. dr. Zdravko Kravanja Datum zagovora: 26. 9. 2016 Irena FERLAN ANALIZA OBDELAVE POLIZDELKOV V GALVANSKEM OBRATU Mentor: doc. dr. Anita Kovač Kralj Somentor: doc. dr. Matjaž Kristl Datum zagovora: 30. 9. 2016 Maja FERLEŽ BIOINFORMATSKA ANALIZA MOLEKULARNO BIOLOŠKIH POTI RAKA MATERNIČNEGA VRATU Mentor: red. prof. dr. Uroš Potočnik Somentorica: doc. dr. Katja Repnik Datum zagovora: 21. 9. 2016 Mateja FLIS OPTIMIZACIJA PROCESOV RAZKROJA IN REDUKCIJE V ZAPRTEM SISTEMU ZA DOLOČITEV MASNE KONCENTRACIJE TITANOVEGA DIOKSIDA V REALNIH VZORCIH Mentor: dr. Mitja Kolar Somentor: doc. dr. Matjaž Kristl Datum zagovora: 20. 1. 2016 Kristjan GROBIN OBDELAVA ODPADNE VODE IZ PROIZVODNJE NITROOKSINA Mentorica: izr. prof. dr. Marjana Simonič Somentor: mag. Tomaž Mesar Datum zagovora: 21. 9. 2016 Estera HABJANIČ SOČASNO DOLOČANJE IZBRANIH FLAVONOIDOV V RASTLINSKIH EKSTRAKTIH S HPLC Mentor: doc. dr. Matjaž Finšgar Somentorica: doc. dr. Maša Islamčevic Razboršek Datum zagovora: 7. 9. 2016 Marjan HORVAT RAZVOJ METODE ZA DOLOČEVANJE VISKOZNOSTI SUBSTANC V SISTEMIH S SUPERKRITIČNIMIFLUIDI Mentor: red. prof. dr. Željko Knez Somentor: doc. dr. Maša Knez Hrnčič Datum zagovora: 26. 9. 2016 Helena HRIBERNIK ČIŠČENJE ODPADNE VODE IZ PODJETJA NA KOROŠKEM Mentorica: izr. prof. dr. Marjana Simonič Somentorica: asist. dr. Irena Petrinic Datum zagovora: 21. 9. 2016 Sanja KELBIČ ČIŠČENJE KOMUNALNE ODPADNE VODE Z MEMBRANSKIM BIOREAKTORJEM Mentorica: izr. prof. dr. Marjana Simonič Somentorica: Cimermančič Bernardka, univ. dipl. biol. Datum zagovora: 21. 9. 2016 Timi KOKOL ŠTUDIJE MOŽNOSTI TEHNOLOGIJ ZA ZAJEMANJE CO2 Mentorica: red. prof. dr. Zorka Novak Pintarič Somentor: red. prof. dr. Zdravko Kravanja Datum zagovora: 21. 9. 2016 Nataša KORAŽIJA RAZVOJ IN OPTIMIZACIJA ANALIZNIH METOD PRI SPROŠČANJU KOVIN IZ MATERIALOV NAMENJENIH STIKU Z ŽIVILI Mentor: dr. Mitja Kolar Somentorica: red. prof. dr. Karin Stana Kleinschek Datum zagovora: 20. 1. 2016 Sara KUGLER PRIMERJAVA NATANČNOSTI DVEH METOD TESTIRANJA ZA PRISOTNOST VIRUSA HEPATITISA C PRI KRVODAJALCIH Mentorica: izr. prof. dr. Marjana Glaser Kraševac Somentorica: doc. dr. Špela Stangler Herodež Datum zagovora: 20. 4. 2016 Tjaša LEMUT KREIRANJE IN UPORABA INTERAKTIVNEGA MULTIMEDIJSKEGA UČNEGA GRADIVA PRI USVAJANJU NUMERIČNIH METOD Mentorica: doc. dr. Majda Krajnc Somentorica: doc. dr. Anita Kovač Kralj Datum zagovora: 13. 7. 2016 Društvene vesti in druge aktualnosti S38 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Igor LENKIČ ANALIZA UPORABE EMAJLA KOT ZAŠČITE ZA BETONSKO JEKLO Mentorica: red. prof. dr. Andreja Goršek Somentor: doc. dr. Matjaž Finšgar Datum zagovora: 21. 9. 2016 Mirjana LUKIC ZNIZEVANJE VSEBNOSTI KOVIN IZ KOMPOSTNE IZCEDNE VODE Z ZEOLITI Mentorica: izr. prof. dr. Marjana Simonic Somentorica: red. prof. dr. Lidija Fras Žemljic Datum zagovora: 18. 5. 2016 Julija MACUH PRIDOBIVANJE KLOROFILA IN DERIVATOV KLOROFILA Mentor: red. prof. dr. Zeljko Knez Somentorica: red. prof. dr. Mojca Škerget Datum zagovora: 26. 9. 2016 Laura MAKSIMOVIC eQTL ANALIZA KROMOSOMSKIH REGIJ 17q21 IN 2q12 TER NJUN VPLIV NA RAZVOJ IN POTEK ASTME PRI SLOVENSKIH OTROCIH Mentor: red. prof. dr. Uroš Potocnik Somentorica: doc. dr. Katja Repnik Datum zagovora: 7. 9. 2016 Matjaž MARKUŠ ANALIZA VPLIVA ČASA NA VISKOZNOST KOZMETIČNIH POLIZDELKOV Mentorica: doc. dr. Anita Kovac Kralj Somentor: doc. dr. Matjaž Kristl Datum zagovora: 21. 9. 2016 Luka MLINARIČ POVEZAVA MED POLIMORFIZMI IN IZRAZANJEM GENOV TRIM35 IN EPHX2 Z OTROŠKO ASTMO Mentor: red. prof. dr. Uroš Potocnik Somentor: doc. dr. Vojko Berce Datum zagovora: 1. 9. 2016 Peter PALLER SUŠENJE ODPADNEGA KOMUNALNEGA MULJA Z MIKROKOGENERACIJO NA DEPONIJSKI PLIN IN ODPADNA OLJA Mentor: izr. prof. dr. Darko Goricanec Somentor: asist. dr. Peter Trop Datum zagovora: 7. 9. 2016 Lidija PODJAVERŠEK OBLIKOVANJE POSLOVNEGA MODELA OBSTOJEČEGA IZDELKA ZA ŠIRITEV TRGA Mentor: doc. dr. Dušan Klinar Somentor: Datum zagovora: 21. 9. 2016 Barbara POLANIČ POVRŠINSKA OBDELAVA SILIKONSKEGA MATERIALA Mentor: doc. dr. Matjaž Finšgar Somentorica: red. prof. dr. Lidija Fras Žemljic Datum zagovora: 21. 9. 2016 Vesna REBIC TERMOGRAVIMETRIČNA ANALIZA SADRE IZ TERMOELEKTRARNE ŠOŠTANJ Mentorica: red. prof. dr. Andreja Goršek Somentorica: doc. dr. Darja Pecar Datum zagovora: 7. 9. 2016 Anita ROGAČ DOLOČANJE ORTOFOSFATA, AMONIJAKALNEGA DUŠIKA, MBAS INDEKSA IN FENOLNEGA INDEKSA V VODAH, S PRETOČNIM ANALIZATORJEM Mentor: doc. dr. Matjaž Finšgar Somentorica: izr. prof. dr. Marjana Simonic Datum zagovora: 13. 7. 2016 Borut ROŽMAN IZDELAVA VEZIVA ZA ASFALT - BITUMNA IZ OBNOVLJIVIH VIROV - S POMOČJO PIROLIZE NA LABORATORIJSKI NAPRAVI Mentor: doc. dr. Dušan Klinar Somentor: dr. Marjan Tušar Datum zagovora: 21. 9. 2016 Polona ROŽMAN INHIBICIJSKE LASTNOSTI NEIONSKEGA SURFAKTANTA POLIOKSIETILEN (40) IZOBUTIL ETER PRI POVIŠANI TEMPERATURI Mentorica: izr. prof. dr. Regina Fuchs Godec Somentor: doc. dr. Matjaž Finšgar Datum zagovora: 21. 9. 2016 Anja SEVER SONOKEMIJSKA SINTEZA IN KARAKTERIZACIJA INDIJEVIH IN GALIJEVIH SULFIDOV Mentor: doc. dr. Matjaž Kristl Somentorica: doc. dr. Irena Ban Datum zagovora: 14. 9. 2016 Ines ŠPINDLER SEPARACIJA MONOTERPENOV IZ SEMEN KUMINE (CARUM CARVIL.) SEMEN NAVADNEGA KOPRA (ANETHUM GRAVEOLENS L.) IN LISTOV ZELENE METE (MENTHA CORDIFOLIA L.) Mentorica: red. prof. dr. Mojca Škerget Somentor: red. prof. dr. Zeljko Knez Datum zagovora: 21. 9. 2016 Žiga ŠUT PROIZVODNJA BIOPLINA S SOSUBSTRATOM KORUZNO SLAMO Mentorica: doc. dr. Lidija Čucek Somentor: red. prof. dr. Zdravko Kravanja Datum zagovora: 21. 9. 2016 Aleksandra TURK ENERGETSKA IN EKONOMSKA OCENA SANACIJE STANOVANJSKE STAVBE Mentor: izr. prof. dr. Darko Goricanec Somentorica: asist. dr. Danijela Urbancl Datum zagovora: 23. 3. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Tanja TURK DOLOČEVANJE BIOMASE ALG IN KLOROFILA V RASTNEM MEDIJU Mentorica: izr. prof. dr. Marjana Simonič Somentorica: red. prof. dr. Andreja Goršek Datum zagovora: 12. 7. 2016 Matej ŽULJAN UČINEK ČIŠČENJA ODPADNE VODE V SEKVENČNEM BIOLOŠKEM REAKTRORJU PRI RAZLIČNIH TEMPERATURNIH POGOJIH Mentorica: izr. prof. dr. Marjana Simonič Somentor: Aljaž Klasinc, univ. dipl. inž. str. Datum zagovora: 21. 9. 2016 UNIVERZITETNI ŠTUDIJ - 1. stopnja — Nuša CMAGER SINTEZA ZAMREŽENEGA POLI(4-VINILPIRIDINA) Mentor: izr. prof. dr. Jernej Iskra Somentor: red. prof. dr. Peter Krajnc Datum zagovora: 7. 9. 2016 Suzana CVIJETINOVIC ANALIZA ENERGTSKIH POTREB RASTLINJAKOV Mentor: izr. prof. dr. Darko Goričanec Somentorica: asist. dr. Danijela Urbancl Datum zagovora: 13. 7. 2016 Matej FUREK SIMULACIJA OBRATOVALNIH KARAKTERISTIK ABSORPCIJSKE TOPLOTNE ČRPALKE Mentor: izr. prof. dr. Darko Goričanec Somentor: asist. dr. Peter Trop Datum zagovora: 7. 9. 2016 Tamara GOVEJŠEK DOLOČANJE VSEBNOSTI P-GLUKANOV V SLADICI IN PIVU Mentor: doc. dr. Jože Košir Somentor: doc. dr. Matjaž Finšgar Datum zagovora: 1. 9. 2016 Barbara GRABROVEC DOLOČANJE LOKALNE GENSKE EKSPRESIJE V POSTOPKU CELJENJA RAN Mentor: red. prof. dr. Uroš Potočnik Somentor: doc. dr. Uroš Maver Datum zagovora: 7. 9. 2016 Andreja HORVAT EKSTRAKCIJA BIOLOŠKO AKTIVNIH SPOJIN IZ RAZLIČNIH VRST GOB DRUŽINE POLYPORACEAE (LUKNJARKE) Mentor: red. prof. dr. Željko Knez Somentor: Gregori Andrej Datum zagovora: 1. 9. 2016 Maša HREN UPORABA OPLAŠČENEGA ZEOLITA ZA ODSTRANJEVANJE ORGANSKE SNOVI IZ KOMPOSTNE VODE Mentorica: izr. prof. dr. Marjana Simonič Somentor: Fakin Tomaž Datum zagovora: 7. 9. 2016 Žan HRIBAR VLOGA RECEPTORJEV ENDOKANABINOIDNEGA SISTEMA CB1 IN CB2 PRI INHIBICIJI CITOKINA TNF-a V MONONUKLEARNIH LIMFOIDNIH CELICAH BOLNIKOV S CROHNOVO BOLEZNIJO Mentor: red. prof. dr. Uroš Potočnik Somentor: asist. ddr. Matjaž Deželak Datum zagovora: 7. 9. 2016 Maja IVANOVSKI KONTROLA ELASTIČNIH LASTNOSTI BETONA Z DODATKOM GUMENIH SEKANCEV Mentorica: red. prof. dr. Andreja Goršek Somentor: doc. dr. Samo Lubej Datum zagovora: 7. 9. 2016 Kaja JEROMEL PRIPRAVA MAGNETNIH ZAMREŽENIH ENCIMSKIH SKUPKOV (mCLEA) IZ P-GALAKTOZIDAZE Mentorica: red. prof. dr. Maja Leitgeb Somentorica: asist. Katja Vasic Datum zagovora: 7. 9. 2016 Sabina JURAK VPLIV Br- IN I- IONOV NA INHIBICIJSKO UČINKOVITOST NEIONSKEGA TIPA PAS V KISLEM MEDIJU Mentorica: izr. prof. dr. Regina Fuchs Godec Somentor: doc. dr. Matjaž Finšgar Datum zagovora: 7. 9. 2016 Tina KEGL MERJENJE FIZIKALNO-KEMIJSKIH IN TRANSPORTNIH LASTNOSTI SISTEMA POLIMER/SCF Mentor: red. prof. dr. Željko Knez Somentorica: doc. dr. Maša Knez Hrnčič Datum zagovora: 1. 9. 2016 Alain KERHE AKTIVNOST IN STABILNOST PROTEINOV V GELIH ZA KOZMETIČNE IN MEDICINSKE APLIKACIJE Mentorica: red. prof. dr. Maja Leitgeb Somentorica: doc. dr. Mateja Primožič Datum zagovora: 7. 9. 2016 Društvene vesti in druge aktualnosti S40 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Nika KODBA VPLIV VELIKOSTI ZRNATOSTI PŠENIČNIH OTROBOV NA RAST GLIVE PLEUROTUS OSTREATUS Mentorica: red. prof. dr. Maja Leitgeb Somentorica: doc. dr. Mateja Primožic Datum zagovora: 22. 6. 2016 Katarina KORES RAČUNALNIŠKE SIMULACIJE VPLIVA METILACIJE CITOZINA NA VEZAVO AFLATOKSINA B1 V DVOVERIŽNO DNK Mentor: izr. prof. dr. Urban Bren Datum zagovora: 9/1/2016 Julij LOZINŠEK OBSTOJNOST PROTIKOROZIJSKIH HIDROFOBNIH PREVLEK PRI POVIŠANI TEMPERATURI Mentorica: izr. prof. dr. Regina Fuchs Godec Somentor: doc. dr. Matjaž Finšgar Datum zagovora: 13. 7. 2016 Aljaž MARIN PROJEKTIRANJE IN ZAGON TESTNE PROGE ZA IZVAJANJE MERITVE PRETOKA ODPADNIH VOD Mentorica: doc. dr. Mateja Primožic Somentorica: red. prof. dr. Maja Leitgeb Datum zagovora: 7. 9. 2016 Azra OSMIC OPTIMIZACIJA REAKCIJSKIH POGOJEV ZA KONTROLO POROZNOSTI poli(HEMA-ko-EA) MATERIALOV Mentor: red. prof. dr. Peter Krajnc Somentorica: asist. dr. Muzafera Paljevac Datum zagovora: 7. 9. 2016 Rok PETRIJAN UČINEK AGONISTOV IN ANTAGONISTOV ENDOKANABINOIDNIH RECEPTORJEV CB1 IN CB2 NA IZRAŽANJE NEKATERIH CITOKINOV V CELIČNIH KULTURAH GOJENIH LIMFOIDNIH CELIC BOLNIKOV Z ASTMO Mentor: red. prof. dr. Uroš Potocnik Somentor: asist. ddr. Matjaž Deželak Datum zagovora: 7. 9. 2016 Tanja POPOVIC REPLIKACIJA DNA POLIMORFIZMOV POVEZANIH Z MULTIPLO SKLEROZO V ASOCIACIJSKIH ŠTUDIJAH V CELOTNEM GENOMU PRI SLOVENSKIH BOLNIKIH Mentor: red. prof. dr. Uroš Potocnik Somentorica: izr. prof. dr. Tanja Hojs - Fabjan Datum zagovora: 21. 9. 2016 Maja PRESKAR PIROLIZA LESNE BIOMASE Mentor: izr. prof. dr. Darko Goricanec Somentorica: asist. dr. Danijela Urbancl Datum zagovora: 20. 1. 2016 Tina RAJH SINTEZA IN KARAKTERIZACIJA KOORDINACIJSKIH SPOJIN PREHODNIH KOVIN (Co,Ni,Cu) Z MELAMINOM Mentor: doc. dr. Matjaž Kristl Somentorica: doc. dr. Irena Ban Datum zagovora: 1. 9. 2016 Nina RIBIČ UPORABA ALGINATNIH NOSILCEV ZA ODSTRANJEVANJE ONESNAŽIL IZ VODE Mentorica: izr. prof. dr. Marjana Simonic Somentorica: doc. dr. Irena Ban Datum zagovora: 21. 9. 2016 Luka ROMANIC EKSTRAKT ROŽMARINA, KOT INHIBITOR KOROZIJSKIH PROCESOV Mentorica: izr. prof. dr. Regina Fuchs Godec Somentor: izr. prof. dr. Urban Bren Datum zagovora: 7. 9. 2016 Barbara SKOK POLIMERIZACIJA OLIGOMERNIH AKRILATOV V EMULZIJAH Mentor: red. prof. dr. Peter Krajnc Somentorica: asist. dr. Muzafera Paljevac Datum zagovora: 7. 9. 2016 Anita SOVIČ UPORABA SUROVEGA GLICEROLA ZA PROIZVODNJO BIOPLINA Mentorica: doc. dr. Lidija Čucek Somentor: red. prof. dr. Zdravko Kravanja Datum zagovora: 7. 9. 2016 Rok ŠPINDLER KRIOGENA AKUMULACIJA ENERGIJE Mentor: izr. prof. dr. Darko Goricanec Somentor: asist. dr. Peter Trop Datum zagovora: 7. 9. 2016 Jadranka ŠVIGELJ ODPIRANJE CELIC HALOFILNE GLIVE HORTAEA WERNECKII S HOMOGENIZATORJEM IN ZASLEDOVANJE AKTIVNOSTI PRISOTNIH ENCIMOV Mentorica: red. prof. dr. Maja Leitgeb Somentorica: asist. Maja Čolnik Datum zagovora: 7. 9. 2016 Nina URBIČ TOPNOST ORGANSKIH TOPIL V PLINIH PRI NIZKIH TLAKIH Mentorica: red. prof. dr. Mojca Škerget Somentorica: doc. dr. Maša Knez Hrncic Datum zagovora: 7. 9. 2016 Sara VOZLIČ FUNKCIONALIZACIJA POLIAKRILNE KISLINE DO KISLINSKEGA KLORIDA IN ŠTUDIJA STABILNOSTI Mentor: red. prof. dr. Peter Krajnc Somentorica: asist. dr. Muzafera Paljevac Datum zagovora: 7. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Tina ZOREC STABILNOST KURKUMINOIDOV V SUBKRITIČNI VODI: DOLOČANJE MEHANIZMOV IN HITROST REAKCIJ RAZGRADNJE Mentorica: red. prof. dr. Mojca Skerget Somentor: red. prof. dr. Zeljko Knez Datum zagovora: 7. 9. 2016 Taja ŽITEK ANTIOKSIDATIVNE LASTNOSTI EKSTRAKTOV NEKATERIH RASTLINSKIH MATERIALOV Mentor: red. prof. dr. Zeljko Knez Somentorica: red. prof. dr. Mojca Skerget Datum zagovora: 22. 6. 2016 VISOKOŠOLSKI STROKOVNI ŠTUDIJ — Marjan BALOH VPLIV PROCESNIH PARAMETROV NA EKSTRAKCIJO MAKROLID LAKTAMOV IZ FERMENTACIJSKE BROZGE S TOLUENOM Mentorica: red. prof. dr. Mojca Skerget Somentor: red. prof. dr. Zeljko Knez Datum zagovora: 23. 3. 2016 Otmar BEVK OPTIMIRANJE POSTOPKOV KEMIJSKE PRIPRAVE VODE IN KONDICIONIRANJE TEHNOLOŠKE VODE V TERMOELEKTRARNI TRBOVLJE Mentorica: red. prof. dr. Andreja Goršek Somentorica: izr. prof. dr. Marjana Simonič Datum zagovora: 12. 7. 2016 Dušanka BOHINC OVREDNOTENJE ENERGIJE RAZTEGOVANJA PmB BITUMNA NA RAZLIČNIH DOLZINAH RAZTEGA Mentor: izr. prof. dr. Urban Bren Somentor: dr. Marjan Tušar Datum zagovora: 1. 9. 2016 Robert BREMŠAK IZOLACIJA - REKOMBINANTNEGA FLAGELINA Mentor: red. prof. dr. Uroš Potočnik Somentorica: dr. Karolina Ivičak Kocjan Datum zagovora: 21. 9. 2016 Simona BREŽNIK ANALIZE LASTNOSTI NANOSA TISKARSKIH BARV Mentorica: doc. dr. Anita Kovač Kralj Somentor: doc. dr. Matjaž Kristl Datum zagovora: 21. 9. 2016 Marjeta BRODAR PRIPRAVA STANDARDOV PIMEKROLIMUSOVIH NEČISTOČ Mentor: doc. dr. Matjaž Finšgar Somentor: dr. Gregor Kopitar Datum zagovora: 21. 9. 2016 Zlatka CAFUTA PREVOLŠEK ADSORPCIJA IN DESORPCIJA NEKATERIH NARAVNIH SPOJIN NA RAZLIČNE ADSORBENTE Mentorica: red. prof. dr. Mojca Skerget Somentorica: asist. dr. Amra Perva-Uzunalic Datum zagovora: 21. 9. 2016 Petra DREVENŠEK VEČKRITERIJSKO OPTIMIRANJE BIOPROCESOV Z UPORABO TRAJNOSTNEGA KAZALCA NA EKONOMSKI RAVNI Mentorica: doc. dr. Lidija Čuček Somentor: red. prof. dr. Zdravko Kravanja Datum zagovora: 21. 9. 2016 Robert FERLINC UPORABA ANALIZNIH METOD ZA DOLOČANJE UČINKOVITOSTI MALIH KOMUNALNIH ČISTILNIH NAPRAV Mentor: dr. Mitja Kolar Somentorica: izr. prof. dr. Marjana Simonič Datum zagovora: 20. 1. 2016 Jana FERME MERILNI PROTOKOL TESTIRANJA ULTRAFILTRACIJSKE NAPRAVE Mentorica: red. prof. dr. Andreja Goršek Somentor: Boštjan Zigon, univ. dipl. inž. kem. str. Datum zagovora: 7. 9. 2016 Ksenija FLEISINGER ENERGETSKA PRENOVA VEČSTANOVANJSKE STAVBE IN NJEN VPLIV NA KVALITETO BIVALNEGA PROSTORA Mentor: izr. prof. dr. Darko Goričanec Somentorica: asist. dr. Danijela Urbancl Datum zagovora: 21. 9. 2016 Klara FRANGEŽ DOLOČANJE VSEBNOSTI LIGNINA V HMELJU Mentor: dr. Iztok Jože Košir Somentor: doc. dr. Matjaž Finšgar Datum zagovora: 1. 9. 2016 Breda GAŠPAR VZPOSTAVITEV NOTRANJEGA NADZORA PITNE VODE PO SISTEMU HACCP Mentorica: red. prof. dr. Andreja Goršek Somentorica: izr. prof. dr. Marjana Simonič Datum zagovora: 12. 7. 2016 Društvene vesti in druge aktualnosti S42 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Marija GOLOB PRIPRAVA IN TESTIRANJE ODSTRANJEVALCA PREMAZOV NA OSNOVI NADOMESTNIH TOPIL ZA METILENKLORID IN N-METIL-2-PIROLIDON (NMP) Mentorica: red. prof. dr. Mojca Škerget Somentorica: mag. Tina Razboršek Datum zagovora: 1. 9. 2016 Teo IVANČIČ KOROZIJSKA OBSTOJNOST NANOSA AEROSOLNEGA RAZPRŠILNIKA »PLASTI-DIP« V AGRESIVNEM MEDIJU Mentorica: izr. prof. dr. Regina Fuchs Godec Somentor: izr. prof. dr. Urban Bren Datum zagovora: 27. 9. 2016 Roman JANKOVIČ PRIMERJAVA UČINKOVITOSTI RAZLIČNIH MAKROZAMREŽENIH POLIMERNIH XAD ADSORBENTOV PRI ADSORPCIJI VANKOMICINA Mentor: izr. prof. dr. Urban Bren Somentor: David Senica, univ. dipl. inž. kem. inž. Datum zagovora: 27. 9. 2016 Darko KERŽAN RAZVOJ IN VALIDACIJA GC/FID METODE ZA DOLOČEVANJE ALKOHOLOV V VINU Mentor: doc. dr. Matjaž Finšgar Somentorica: doc. dr. Maša Islamčevic Razboršek Datum zagovora: 18. 5. 2016 Peter KLADNIK EPIMERIZACIJA ERGOT ALKALOIDOV IN BROM -ERGOT ALKALOIDOV Mentor: red. prof. dr. Peter Krajnc Somentorica: asist. dr. Muzafera Paljevac Datum zagovora: 7. 9. 2016 Veronika KOLAR VPLIV OBRATOVALNIH POGOJEV NA HITROST PRENOSA VODE PRI PROCESU OSMOZE Mentorica: asist. dr. Irena Petrinic Somentorica: izr. prof. dr. Marjana Simonič Datum zagovora: 21. 9. 2016 Helena KOTAR ANALIZE ČIŠČENJA INDUSTRIJSKIH ODPADNIH VOD NA INDUSTRIJSKI ČISTILNI NAPRAVI Mentorica: doc. dr. Anita Kovač Kralj Somentorica: izr. prof. dr. Marjana Simonič Datum zagovora: 21. 9. 2016 Gašper KOZLOVIČ ANALIZA ŽIVLJENJSKEGA CIKLA PROCESOV PROIZVODNJE BIOETANOLA S PROGRAMSKIM ORODJEM OpenLCA Mentorica: doc. dr. Lidija Čuček Somentor: red. prof. dr. Zdravko Kravanja Datum zagovora: 21. 9. 2016 Martina KŠELA PODGORNIK UPORABA SIMULATORJA SKL2 ZA KALIBRACIJO MERILNIKOV pH, PREVODNOSTI IN KONCENTRACIJE KISIKA Mentor: doc. dr. Matjaž Finšgar Somentorica: doc. dr. Maša Islamčevic Razboršek Datum zagovora: 21. 9. 2016 Tomaž KUMER VPLIV TEMPERATURE NA PROCES UTRJEVANJA DVOKOMPONENTNIH AKRILNIH PREMAZOV Mentor: red. prof. dr. Peter Krajnc Somentorica: asist. dr. Muzafera Paljevac Datum zagovora: 7. 9. 2016 Marko LAVRIH POSLOVNI MODEL NOVEGA PROGRAMA IN IZDELKA V OBSTOJEČEM PODJETJU Mentor: doc. dr. Dušan Klinar Datum zagovora: 21. 9. 2016 Dejan MENONI KRISTALIZACIJA NATRIJEVEGA ACETATA TRIHIDRATA Mentorica: red. prof. dr. Mojca Skerget Somentor: red. prof. dr. Zeljko Knez Datum zagovora: 13. 7. 2016 Robert PERETIN PRIPRAVA SAMOGASNE MEŠANICENA OSNOVI BLOK KOPOLIMEROV Z NE-HALOGENSKIMI ZAVIRALCI GORLJIVOSTI Mentorica: red. prof. dr. Andreja Goršek Somentor: red. prof. dr. Peter Krajnc Datum zagovora: 7. 9. 2016 David PILINGER VALIDACIJA HPLC METODE ZA DOLOČEVANJE OSTANKOV AKTIVNE FARMACEVTSKE UČINKOVINE LIDOKAINIJEV HIDROKLORID NA PROIZVODNI OPREMI Mentor: doc. dr. Matjaž Finšgar Somentorica: doc. dr. Maša Islamčevic Razboršek Datum zagovora: 1. 9. 2016 Ines POLAK PRIMERJAVA MERITEV VISKOZNOSTI Z APARATOMA HAAKE VT550 IN HAAKE VISCOTESTER IQ Mentor: izr. prof. dr. Urban Bren Somentorica: Tatjana Jambrovič, univ. dipl. inž. kem. tehnol. Datum zagovora: 27. 9. 2016 Lidija PUNGERŠEK REŠEVANJE KEMIJSKO - TEHNIŠKIH PROBLEMOV S PROGRAMOM MS EXCEL Mentorica: doc. dr. Majda Krajnc Somentorica: izr. prof. dr. Petra Zigert Pleteršek Datum zagovora: 21. 9. 2016 Tomaž ROZONIČNIK VPLIV PIROGENEGA SiO2 IN Al2O3 NA REOLOŠKE LASTNOSTI PRAŠKASTEGA LAKA Mentorica: red. prof. dr. Andreja Goršek Somentorica: doc. dr. Darja Pečar Datum zagovora: 7. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 Dejan SAKULAC OPTIMIZACIJA REGENERACIJE CIKLOHEKSANA NA REKTIFIKACIJSKI KOLONI Mentorica: red. prof. dr. Mojca Škerget Somentor: red. prof. dr. Željko Knez Datum zagovora: 26. 9. 2016 Nataša STAROVERŠKI PREUČEVANJE VSEBNOSTI SNOVI V FLOKULIRANI KOMPOSTNI VODI Mentorica: izr. prof. dr. Marjana Simonič Somentor: dr. Karli Udovičič Datum zagovora: 21. 9. 2016 Sanja STRAH PRIMERJAVA RAZPRŠEVANJA ZRAKA V AERACIJSKIH BAZENIH KOMUNALNIH ČISTILNIH NAPRAV Mentor: izr. prof. dr. Darko Goričanec Somentorica: asist. dr. Danijela Urbancl Datum zagovora: 21. 9. 2016 Ernest ŠIMON VARNO DELO S KEMIKALIJAMI IN PRIPRAVA NAVODIL ZA VARNO DELO V KEMIJSKEM LABORATORIJU Mentorica: red. prof. dr. Zorka Novak Pintarič Somentorica: doc. dr. Julija Volmajer Valh Datum zagovora: 21. 9. 2016 Nuša ŠKERLAK MATEMATIČNI MODEL REZULTATOV ANALIZ V PREHRAMBENI INDUSTRIJI Mentorica: doc. dr. Anita Kovač Kralj Somentorica: doc. dr. Irena Ban Datum zagovora: 21. 9. 2016 Aleksandra TROKŠAR ANALIZA NEČISTOČ KOVINSKIH ULITKOV Mentorica: doc. dr. Anita Kovač Kralj Somentor: doc. dr. Matjaž Kristl Datum zagovora: 21. 9. 2016 Lucija TURNŠEK PRIMER MULTIMEDIJSKEGA UČNEGA GRADIVA IN ELEKTRONSKO PREVERJANJE ZNANJA PRI PREDMETU GRADIVA Mentorica: doc. dr. Majda Krajnc Somentorica: red. prof. dr. Andreja Goršek Datum zagovora: 21. 9. 2016 Oliver TUTIC MIKROFILTRACIJA FERMENTACIJSKE BROZGE Mentorica: red. prof. dr. Mojca Škerget Somentor: Aljaž Kajtna, univ. dipl. inž. kem. tehnol. Datum zagovora: 21. 9. 2016 Marjeta UMEK RAZVOJ IN VALIDACIJA ANALIZNE METODE B PO STANDARDU SIST EN ISO 7887:2012 ZA DOLOČANJE BARVE VODE Mentor: doc. dr. Matjaž Finšgar Somentorica: asist. dr. Amra Perva-Uzunalic Datum zagovora: 13. 7. 2016 Dragica VALEK DOLOČEVANJE SPOSOBNOSTI DISPERGIRANJA PIGMENTNEGA TITANOVEGA DIOKSIDA V ALKIDNI SMOLI Mentorica: red. prof. dr. Andreja Goršek Somentorica: mag. Mojca Pustoslemšek Datum zagovora: 7. 9. 2016 Stjepan ZAGORŠČAK SINTEZA IN LASTNOSTI TRIBAZIČNEGA BAKROVEGA SULFATA Mentorica: red. prof. dr. Andreja Goršek Somentor: doc. dr. Matjaž Kristl Datum zagovora: 7. 9. 2016 Mateja ZVER OD VZORCA DO ANTIBIOGRAMA ENTEROBAKTERIJ V ENEM DNEVU - EVALUACIJA METODE Z VZPOREDNIM KULTIVIRANJEM SEČA V BUJONU V PRIMERJAVI S STANDARDNIM POSTOPKOM Mentorica: red. prof. dr. Maja Leitgeb Somentor: mag. Iztok Štrumbelj Datum zagovora: 21. 9. 2016 VISOKOŠOLSKI STROKOVNI ŠTUDIJ - 1. stopnja Alenka BAKLAN Amanda FERLEŽ ELEKTROKEMIJSKA ANALIZA ALUMINIJEVE ZLITINE VERIŽNI UČINEK ZA POŽAR IN EKSPLOZIJO PRI 6082 V KLORIDNEM MEDIJU SKLADIŠČENJU NAFTNIH DERIVATOV Mentor: doc. dr. Matjaž Finšgar Mentorica: red. prof. dr. Zorka Novak Pintarič Mentorica: izr. prof. dr. Regina Fuchs Godec Mentorica: Jasmina Karba Datum zagovora: 21. 9. 2016 Datum zagovora: 7. 9. 2016 Društvene vesti in druge aktualnosti S44 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Marijana HITER VPLIV SKUPNEGA IONA NA VISKOZNOST ELEKTROLITSKIH MEŠANIC Mentorica: doc. dr. Mojca Slemnik Mentorica: izr. prof. dr. Marjana Simonič Datum zagovora: 6. 1. 2016 Špela KOPRIVC PROGRAMIRANJE V EXCELU VBA IN UPORABA V KEMIJSKI TEHNIKI Mentorica: red. prof. dr. Zorka Novak Pintarič Mentor: doc. dr. Miloš Bogataj Datum zagovora: 21. 12. 2016 Mojca SLANC ODSTRANJEVANJE ŽELEZA IZ PITNE VODE Z UPORABO IMOBILIZIRANIH ALG Mentorica: izr. prof. dr. Marjana Simonič Mentorica: red. prof. dr. Andreja Goršek Datum zagovora: 21. 9. 2016 Peter STRMŠEK KOROZIJA BIOKOMPATIBILNIH KOVIN IN ZLITIN V UMETNI SLINI Mentorica: doc. dr. Mojca Slemnik Mentorica: izr. prof. dr. Regina Fuchs Godec Datum zagovora: 21. 9. 2016 Katja LEČNIK MEHANOKEMIJSKE SINTEZE SULFIDOV PREHODNIH KOVIN 4. PERIODE (MxSy; M = Zn, Ni, Co) Mentor: doc. dr. Matjaž Kristl Mentorica: doc. dr. Irena Ban Datum zagovora: 7. 9. 2016 Maja MAZEJ INHIBICIJSKE LASTNOSTI MEŠANICE POLIOKSIETILEN (40) IZOBUTILFENIL ETRA Z DODATKOM HALOGENIDNIH IONOV V KLOROVODIKOVI KISLINI Mentorica: izr. prof. dr. Regina Fuchs Godec Mentor: izr. prof. dr. Urban Bren Datum zagovora: 7. 9. 2016 Maja MEŽNAR IDENTIFIKACIJA NEKATERIH TEHNIČNO POMEMBNIH POLIMEROV NA OSNOVI NJIHOVIH FIZIKALNO- KEMIJSKIH LASTNOSTI Mentorica: red. prof. dr. Andreja Goršek Mentorica: doc. dr. Darja Pečar Datum zagovora: 7. 9. 2016 Tina OPREŠNIK VSEBNOST FENOLNIH SPOJIN V SADNIH IN ZELIŠČNIH PIJAČAH Mentorica: red. prof. dr. Mojca Škerget Mentorica: asist. Tina Perko Datum zagovora: 7. 9. 2016 Katja RIBIČ PRIMERJAVA REZULTATOV NATEZNEGA PREIZKUSA ZA PREIZKUŠANCE IZ ZLITINSKIH MATERIALOV Mentorica: doc. dr. Darja Pečar Mentorica: red. prof. dr. Andreja Goršek Datum zagovora: 7. 9. 2016 Anže ŠIMIC FOTOLUMINISCENČNE IN ELEKTRIČNE LASTNOSTI PLASTOVITIH STANATOV Srn + 1SnnO3n + 1 (n = , 1 in 2) DOPIRANIH Z LANTANOIDI Mentor: doc. dr. Matjaž Kristl Mentor: Aivaras Kareiva Datum zagovora: 23. 11. 2016 Jure ŠKORJA EKONOMSKE IN OKOLJSKE ANALIZE POSTOPKOV ZA IZRABO ODPADNEGA GLICEROLA Mentorica: red. prof. dr. Zorka Novak Pintarič Mentor: red. prof. dr. Peter Krajnc Datum zagovora: 1. 9. 2016 Tamara ŠUSTER ŠTUDIJA PRISOTNOSTI MIKROBNIH POPULACIJ V NARAVNIH BAZENIH Mentorica: red. prof. dr. Maja Leitgeb Mentorica: izr. prof. dr. Marjana Simonič Datum zagovora: 7. 9. 2016 Katja VODOPIVEC SINTEZA IN KARAKTERIZACIJA Bi2WO6 NANODELCEV Mentor: doc. dr. Matjaž Kristl Mentorica: doc. dr. Irena Ban Datum zagovora: 7. 9. 2016 Alenka ZADRAVEC UPORABA METODE DREVO ODPOVEDI V KEMIJSKIH PROCESIH Mentorica: red. prof. dr. Zorka Novak Pintarič Mentorica: red. prof. dr. Andreja Goršek Datum zagovora: 21. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 UNIVERZA V NOVI GORICI FAKULTETA ZA PODIPLOMSKI ŠTUDIJ 1. januar - 31. december 2016 DOKTORATI PODIPLOMSKI ŠTUDIJSKI PROGRAM ZNANOSTI O OKOLJU Lucija RASPOR DALL'OLIO SYMBIOSIS ECOLOGY OF SELECTED SCYPHOZOA Mentorica: doc. dr. Andreja Ramšak Somentorica: prof. dr. Alenka Malej Datum zagovora: 30. 9. 2016 PODIPLOMSKI ŠTUDIJSKI PROGRAM Karmen BIZJAK BAT CHARACTERIZATION OF SLOVENIAN APPLE JUICE WITH RESPECT TO ITS GEOGRAPHICAL ORIGIN AND AGRICULTURAL PRODUCTION PRACTICE Mentorica: prof. dr. Branka Mozetič Vodopivec Somentorica: prof. dr. Nives Ogrinc Datum zagovora: 2. 6. 2016 iOSTI O OKOLJU - 3. stopnja Martina JAKLIČ ECOLOGICAL NICHE RELATIONS OF INDIGENOUS AND INVASIVE CRAYFISH (ASTACOIDEA) IN SLOVENIA Mentor: prof. dr. Anton Brancelj Datum zagovora: 30. 8. 2016 Društvene vesti in druge aktualnosti S46 Acta Chim. Slov. 2017, 64, (1), Supplement S55 MAGISTERIJI PODIPLOMSKI ŠTUDIJSKI PROGRAM ZNANOSTI O OKOLJU Peter BOHINEC THE EFFECTS OF MIXED COMMUNAL WASTE RECYCLING MANAGEMENT IN SLOVENIA: A CASE STUDY Mentor: dr. Marko Vudrag Datum zagovora: 19. 7. 2016 Renata Janja SLOVŠA ANALYSIS OF ALTERNATIVE CHANCES FOR SLUDGE TREATMENT OF NEW CENTRAL WASTE WATER TREATMENT PLANT Mentor: prof. dr. Viktor Grilc Datum zagovora: 19. 7. 2016 Janez ŠKARJA THE STUDY OF OPTIMAL TECHNOLOGICAL PROCEDURES OF INTERNAL PLUMBING SYSTEM DISINFECTION FACILITIES IN USE BY THE SENSITIVE HUMAN POPULATIONS Mentor: doc. dr. Darko Drev Datum zagovora: 31. 8. 2016 Patrik BAKSA EVALUATION OF MARINE SEDIMENTS FROM THE PORT OF LUKA KOPER FROM THE ENVIRONMENTAL PERSPECTIVE AND IN TERMS OF THEIR USABILITY IN THE BRICK INDUSTRY Mentorica: doc. dr. Rebeka Kovačič Lukman Somentorica: dr. Vilma Ducman Datum zagovora: 2. 9. 2016 Janez PAGON FLOODPLAIN FORESTS OF SOČA RIVER BETWEEN KOBARID AND CONFLUENCE WITH RIVER TOLMINKA: CURRENT SITUATION AND DEVELOPMENT Mentor: prof. dr. Marko Debeljak Datum zagovora: 15. 9. 2016 Boštjan KEPIC TIME RESTRICTIONS IN FOREST OPERATIONS PLANNING Mentor: prof. dr. Janez Krč Datum zagovora: 15. 9. 2016 Nataša SMREKAR ASSESSMENT OF EFFECTIVE DOSES BASED ON VARIOUS RADON MEASURING TECHNIQUES Mentorica: prof. dr. Janja Vaupotič Datum zagovora: 23. 9. 2016 Sebastijan REP THE ROLE OF SPECT/CT SCINTIGRAPFY IN LOCALIZATION OF PARATHYROID ADENOMAS Mentorica: prof. dr. Janja Vaupotic Somentor: prof. Marko Hočevar Datum zagovora: 23. 9. 2016 Mojca NOVAK PREVENTION AND MANAGEMENT OF LEGIONELLA SPP. SPREAD IN HOSPITAL WATER SYSTEM (ESTABLISHING AN EFFECTIVE SYSTEM WITHOUT USING CHEMICALS IN UNIVERSITY CLINIC OF RESPIRATORY AND ALLERGIC DISEASES GOLNIK) Mentorica: doc. dr. Viktorija Tomič Datum zagovora: 29. 9. 2016 Slavica ILC ASSESSMENT OF THE DEVELOPMENT POTENTIAL OF FOREST - WOOD PROCESSING CHAIN Mentor: doc. dr. Henrik Gjerkeš Datum zagovora: 29. 9. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 UNIVERZA V NOVI GORICI FAKULTETA ZA ZNANOSTI O OKOLJU 1. januar - 31. december 2016 MAGISTERIJI STUDIJSKI PROGRAM OKOLJE - 2. stopnja Breda POGLAJEN OVREDNOTENJE VPLIVA EKSPERIMENTALNIH DEJAVNIKOV NA IZMERJENE VREDNOSTI RESPIRACIJSKE AKTIVNOSTI AT4 Mentor: doc. dr. Andrej Kržan Datum zagovora: 21. 6. 2016 Jacopo SEGATO SYNTHESIS OF NOVEL GROUP 3 AND LANTHANIDE COMPLEXES CONTAINING THE FERROCENYL MOIETY Mentor: prof. dr. Marco Bertoluzzi Datum zagovora: 27. 10. 2016 DIPLOME UNIVERZITETNI STUDIJSKI PROGRAM OKOLJE Bojan ŠUC IDENTIFIKACIJA, PORAZDELITEV IN VEZAVNE OBLIKE ŽELEZA V RIŽU (ORYZA SATIVA L. ) Z RENTGENSKO ABSORPCIJSKO IN EMISIJSKO MIKRO-SPEKTROSKOPIJO Mentorica: prof. dr. Katarina Vogel Mikuš Somentor: prof. dr. Iztok Arčon Datum zagovora: 5. 9. 2016 Vanja KRISTANČIČ VPLIV KOPALCEV NA BENTOŠKE NEVRETENČARJE V OBALNEM PASU BOHINJSKEGA JEZERA Mentor: prof. dr. Anton Brancelj Datum zagovora: 27. 9. 2016 Mateja PETAVS KRISTANČIČ UGOTAVLJANJE STRUPENOSTI ACETAMIPRIDA NA KOPENSKE ENAKONOŽNE RAKE VRSTE PORCELLIO SCABER (ISOPODA, CRUSTACEA) Mentorica: prof. dr. Polonca Trebše Datum zagovora: 29. 9. 2016 Društvene vesti in druge aktualnosti S48 Acta Chim. Slov. 2017, 64, (1), Supplement S55 ŠTUDIJSKI PROGRAM OKOLJE - 1. stopnja Lucija VODIR VPLIV HIDROLOŠKIH RAZMER NA KAKOVOST KRAŠKIH VODNIH VIROV - PRIMER IZVIRA RIŽANE Mentorica: prof. dr. Metka Petric Datum zagovora: 19. 1. 2016 Tine BIZJAK OBČUTLJIVOST MODELA ZA DOLOČANJE VIROV AEROSOLIZIRANEGA ČRNEGA OGLJIKA NA IZBRANE VHODNE PARAMETRE Mentor: doc. dr. Griša Mocnik Datum zagovora: 19. 1. 2016 Tamara GAJŠT ANALIZA OSTANKOV PLASTIKE V KOMERCIALNEM KOMPOSTU Mentor: doc. dr. Andrej Kržan Datum zagovora: 19. 1. 2016 Sara PRIBOVŠEK VPLIV ONESNAŽIL IZ OKOLJSKIH AEROSOLOV NA TARČNE CELICE V PLJUČIH Mentorica: doc. dr. Martina Bergant Marušic Datum zagovora: 8. 3. 2016 Polona PETERNELJ PREGLED STANJA IN PREDLOG SPREMEMB SISTEMA RAVNANJA Z ODPADNO EMBALAŽO V RS Mentor: doc. dr. Andrej Kržan Datum zagovora: 21. 4. 2016 Andrej JERKIČ DOLOČEVANJE KONCENTRACIJ IN TESTIRANJE BAKTERICIDNEGA DELOVANJA KOLOIDNEGA SREBRA V VODI Mentorica: doc. dr. Dorota Korte Datum zagovora: 31. 5. 2016 Jasna GELATI STABILNOST IN DETEKCIJA ŽELEZOVIH IONOV V VODI IZ OBLAKOV Mentorica: doc. dr. Dorota Korte Datum zagovora: 5. 9. 2016 Mojca GRMEK ITALIJANSKI VRABEC (PASSERITALIAE) V VIPAVSKI DOLINI Mentor: prof. dr. Davorin Tome Datum zagovora: 6. 9. 2016 Sandra DUKIC UČINKI HERBICIDA GLIFOSATA V ČISTI OBLIKI IN V PRIPRAVKU NA DEŽEVNIKE (EISENIA ANDREI) Mentorica: doc. dr. Suzana Žižek Datum zagovora: 14. 9. 2016 Monika FERFOLJA FIZIKALNA, KEMIJSKA IN BIOLOŠKA ANALIZA REKE IDRIJCE OD IZVIRA DO IZLIVA Mentorica: izr. prof. dr. Tanja Pipan Datum zagovora: 28. 9. 2016 Urban ČESNIK RAZMNOŽEVANJE TIGRASTEGA KOMARJA (AEDES ALBOPICTUS) V NOVI GORICI Mentorica: dr. Jana Laganis Datum zagovora: 28. 9. 2016 Tjaša STEINMAN OPTIMIRANJE ZBIRANJA KOMUNALNIH ODPADKOV Mentor: doc. dr. Andrej Kržan Datum zagovora: 29. 9. 2016 Aleš GRAHOVAC DOLOČANJE SREBROVIH ZVRSTI V TEKOČIH VZORCIH Mentorica: doc. dr. Dorota Korte Datum zagovora: 29. 9. 2016 Grega SARKA KAKOVOST TAL V MESTNIH VRTOVIH NA OBMOČJU NOVE GORICE Mentorica: doc. dr. Suzana Žižek Datum zagovora: 10. 11. 2016 Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 KOLEDAR VAŽNEJŠIH ZNANSTVENIH SREČANJ S PODROČJA KEMIJE IN KEMIJSKE TEHNOLOGIJE SCIENTIFIC MEETINGS -CHEMISTRY AND CHEMICAL ENGINEERING 2017 April 2017 3 - 5 SOLUTIONS FOR DRUG-RESISTANT INFECTIONS (SDRI 2017) Brisbane, Australia Information: http://www.sdri2017.org/ 5 - 6 2nd INTERNATIONAL CONFERENCE ON nanomaterials, nanodevices, FABRICATION AND CHARACTERIZATION (ICNNFC'17) Barcelona, Spain Information: http://icnnfc.com/ 5 - 6 2nd INTERNATIONAL CONFERENCE ON nanobiotechnology (ICNB'17) Barcelona, Spain Information: http://nbconference.com/ 5 - 6 2nd INTERNATIONAL CONFERENCE ON nanotechnology MODELING AND SIMULATION (ICNMS'17) Barcelona, Spain Information: http://icnms.net/ 10 - 13 14th UNESCO/IUPAC WORKSHOP AND CONFERENCE ON MACROMOLECULES & MATERIALS Stellenbosch, South Africa Information: http://academic.sun.ac.za/unesco/ 10 - 13 ELECTROSTATICS 2017 Frankfurt am Main, Germany Information: http://www.dechema.de/en/electrostatics2017.html 19 - 22 25th CROATIAN MEETING OF CHEMISTS AND CHEMICAL ENGINEERS Porec, Croatia Information: http://25hskiki.org/en/homepage/ May 2017 7 - 11 SETAC EUROPE 27th ANNUAL MEETING Brussels, Belgium Information: http://www.setac.org/events/EventDetails.aspx?id=683532&group= 14 - 17 2nd GREEN AND SUSTAINABLE CHEMISTRY CONFERENCE Berlin, Germany Information: http://www.greensuschemconf.com/ 16 - 19 ISGC, THE INTERNATIONAL SYMPOSIUM ON GREEN CHEMISTRY La Rochelle, France Information: https://www.isgc-symposium.com/welcome/ 17 - 18 STAT TEST IN CLINICAL LABORATORY Barcelona, Spain Information: http://www.acclc.cat/ Društvene vesti in druge aktualnosti S50 Acta Chim. Slov. 2017, 64, (1), Supplement S55 17 - 19 Information: 18 - 21 Information: 21 - 25 Information: 23 - 25 Information: 25 - 27 Information: 25 - 27 Information: 28 - 31 Information: FIFTH INTERNATIONAL SYMPOSIUM FRONTIERS IN POLYMER SCIENCE Seville, Spain http://www.frontiersinpolymerscience.com/ 29th EUROPEAN SYMPOSIUM ON APPLIED THERMODYNAMICS Bucharest, Romania http://jetc2017.hu/ 12th ADVANCED POLYMERS VIA MACROMOLECULAR ENGINEERING (APME 2017) Ghent, Belgium http://www.ldorganisation.com/apme2017 14th JOINT EUROPEAN THERMODYNAMICS CONFERENCE 2017 Budapest, Hungary http://jetc2017.hu/ MaCKiE-2017 - INTERNATIONAL CONFERENCE ON MATHEMATICS IN CHEMICAL KINETICS AND ENGINEERING (MaCKiE) Budapest, Hungary http://www.mackie-workshops.com/ 7th SLOVENIAN-SERBIAN-CROATIAN SYMPOSIUM ON ZEOLITES Ljubljana, Slovenia http://zeo2017.ki.si/ BIOHETEROCYCLES 2017 - XVII INTERNATIONAL CONFERENCE ON HETEROCYCLES IN BIOORGANIC CHEMISTRY Galway, Ireland http://www.conference.ie/Conferences/index.asp?Conference=442 June 2017 6 - 10 Information: 11 - 15 Information: 11 - 16 Information: 12 - 14 Information: 13 - 15 Information: 18 - 22 Information: 19 - 21 Information: 19 - 23 Information: 8th INTERNATIONAL SYMPOSIUM ON MACRO- AND SUPRAMOLECULAR ARCHITECTURES AND MATERIALS Sochi, Russian Federation www.mam-17.org EUROMEDLAB ATHENS 2017 Athens, Greece www.athens2017.org COLLOQUIUM SPECTROSCOPICUM INTERNATIONALE XL (CSI-XL) Pisa, Italy www.csi-conference.org INORGANIC CHEMISTRY DAYS Nynashamn, Sweden http://www.oorgan.se/ V INTERNATIONAL SYMPOSIUM ON RELIABLE FLOW OF PARTICULATE SOLIDS Skien, Norway http://www.relpowflo.no/ 16th INTERNATIONAL CONFERENCE ON CHEMISTRY AND THE ENVIRONMENT (ICCE 2017) Oslo, Norway http://icce2017.org/welcome/ 6th EUROPEAN DRYING CONFERENCE Liège, Belgium http://efce.info/EuroDrying+2017.html 9th INTERNATIONAL SYMPOSIUM ON MOLECULAR MOBILITY AND ORDER IN POLYMER SYSTEMS Saint-Petersburg, Russian Federation https://iupac.org/event/mmops2017/ Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 25 - 29 Information: 28 Information: 28 - 30 Information: INTERNATIONAL SYMPOSIA ON ORGANOMETALLIC CHEMISTRY DIRECTED TOWARDS ORGANIC SYNTHESIS (OMCOS 19) Jeju Island, Republic Of Korea https://iupac.org/event/omcos-19/ 7th EUROVARIETY - 7th EUROPEAN VARIETY IN UNIVERSITY CHEMISTRY EDUCATION Belgrade, Serbia http://www.chem.bg.ac.rs/eurovariety/ 4th INTERNATIONAL WORKSHOP ON PERICYCLIC REACTIONS AND SYNTHESIS OF HETERO- AND CARBOCYCLIC SYSTEMS Milan, Italy http://sites.unimi.it/cirp_workshop/ July 2017 _ 2 - 5 Information: 2 - 6 Information: 2 - 7 Information: 2 - 8 Information: 3 - 7 Information: 7 - 10 Information: 9 - 13 Information: 9 - 13 Information: 9 - 14 Information: 23 - 29 Information: 4th EUCHEMS INORGANIC CHEMISTRY CONFERENCE - EICC-4 Copenhagen, Denmark http://www.euchems.eu/events/4th-euchems-inorganic-chemistry-conference-eicc-4/ INTERNATIONAL SYMPOSIUM ON MACROCYCLIC AND SUPRAMOLECULAR CHEMISTRY IN CONJUNCTION WITH ISACS: CHALLENGES IN ORGANIC MATERIALS & SUPRAMOLECULAR CHEMISTRY Cambridge, United Kingdom http://www.rsc.org/events/detail/17933/international-symposium-on-macrocyclic-and- supramolecular-chemistry-in-conjunction-with-isacs-challenges-in-organic-materials-and- supramolecular-chemistry 16th EUROPEAN POLYMER CONGRESS Lyon, France http://www.europolyfed.org/home 3rd INTERNATIONAL MASS SPECTROMETRY SCHOOL (IMSS) Dubrovnik, Croatia http://www.imss.nl/ ISSNP 2017 - INTERNATIONAL SUMMER SCHOOL ON NATURAL PRODUCTS Naples, Italy http://www.issnp.org/ 10th INTERNATIONAL SYMPOSIUM ON CATALYSIS IN MULTIPHASE REACTORS (CAMURE-10) & 9th INTERNATIONAL SYMPOSIUM ON MULTIFUNCTIONAL REACTORS (ISMR-9) Tsingtao (Qingdao), PR China http://camure2017.csp.escience.cn/dct/page/! 16th INTERNATIONAL MEETING ON BORON CHEMISTRY (IMEBORON XVI) Hong Kong, China www.imeboron16.org EuCOMC 2017 - 22nd EUROPEAN CONFERENCE ON ORGANOMETALLIC CHEMISTRY Amsterdam, The Netherlands http://www.eucomc2017.amsterdam/ 46th IUPAC WORLD CHEMISTRY CONGRESS (IUPAC-2017) Sao Paulo, Brazil www.IUPAC2017.org RACI CENTENARY CONGRESS Melbourne, Australia http://www.racicongress.com Društvene vesti in druge aktualnosti S52 Acta Chim. Slov. 2017, 64, (1), Supplement S55 24 - 26 Information: 5th INTERNATIONAL CONFERENCE ON GREEN CHEMISTRY AND TECHNOLOGY Rome, Italy http://greenchemistry.alliedacademies.com/ August 2017 13 - 17 Information: 16 - 18 Information: 20 - 23 Information: 27 - 30 Information: 28 - 31 Information: 28 - Sept. 1 Information: 28 - Sept. 2 Information: SE2017 - 200 YEARS OF SELENIUM RESEARCH Stockholm, Sweden http://se2017.se/ CHEMICAL IDENTIFIER Bethesda, MD United States http://www.inchi-trust.org GLS-13 - 13th INTERNATIONAL CONFERENCE ON GAS-LIQUID AND GAS-LIQUID-SOLID REACTOR ENGINEERING (GLS-13) Brussels, Belgium http://www.gls13.com/ EUROPACAT 2017 Florence, Italy http://www.europacat2017.eu/index.html 17th IUPAC INTERNATIONAL SYMPOSIUM ON MACROMOLECULAR COMPLEXES (MMC-17) Tokyo, Japan http://www.waseda.jp/assoc-mmc17/ EuroAnalysis 2017 Stockholm, Sweden http://euroanalysis2017.se/ 11ICHC - 11th INTERNATIONAL CONFERENCE ON THE HISTORY OF CHEMISTRY Trondheim, Norway http://www.ntnu.edu/11ichc September 2017 3 - 6 Information: 3 - 8 Information: 5 - 8 Information: 10 - 13 Information: 17 - 20 Information: 17 - 22 Information: 3rd EuGSC - 3rd EuCheMS CONGRESS ON GREEN AND SUSTAINABLE CHEMISTRY York, UK http://www.euchems.eu/events/3rd-eugsc-3rd-euchems-congress-on-green-adn-sustainable -chemistry/ 21st EUROPEAN CONFERENCE ON THERMOPHYSICAL PROPERTIES Graz, Austria http://ectp2017.tugraz.at/ THERMODYNAMICS 2017 Edinburgh, UK http://www.thermodynamics2017.efconference.co.uk/ GDCh SCIENTIFIC FORUM CHEMISTRY 2017 - ANNIVERSARY CONGRESS »GDCh -150 YEARS Berlin, Germany https://veranstaltungen.gdch.de/tms/frontend/index.cfm?l=7210&modus= BloodSurf2017 Clemson, SC United States http://www.ireviakine.net/Bloodsurf/ INTERNATIONAL SYMPOSIUM ON IONIC POLYMERIZATION - IP 2017 Durham, United Kingdom https://www.dur.ac.uk/soft.matter/ip2017/ Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 19 Information: 20 - 22 Information: 27 - 29 Information: CUTTING EDGE 2017 Ljubljana, Slovenia http://www.cutting-edge.si/ SLOVENIAN CHEMICAL DAYS 2017 Portoroz, Slovenia http://chem-soc.si/slovenski-kemijski-dnevi 11th INTERNATIONAL SYMPOSIUM ON BIOORGANIC CHEMISTRY (ISBOC-11) Konstanz, Germany https://www.uni-konstanz.de/isboc-11/about-isboc-11/ October 2017 1 - 5 Information: 1 - 5 Information: 1 - 5 Information: 2 - 5 Information: 4 - 6 Information: 9 - 12 Information: 9 - 13 Information: 11 - 13 Information: 12 - 14 Information: EPIC 2017 - 6th EUROPEAN PROCESS INTENSIFICATION CONFERENCE 2017 Barcelona, Spain http://www.wcce10.org/index.php/en/ WCCE10 - 10th WORLD CONGRESS OF CHEMICAL ENGINEERING INCORPORATING THE 11th EUROPEAN CONGRESS OF CHEMICAL ENGINEERING (ECCE11) Barcelona, Spain http://www.wcce10.org/index.php/en/ 4th EUROPEAN CONGRESS OF APPLIED BIOTECHNOLOGY - ECAB3 Barcelona, Spain http://www.wcce10.org/index.php/en/ 7th IUPAC INTERNATIONAL CONFERENCE ON GREEN CHEMISTRY Moscow, Russian Federation http://greeniupac2017.muctr.ru XIXth EUROFOODCHEM CONFERENCE Budapest, Hungary http://www.eurofoodchem2017.mke.org.hu/index.php 9th WORKSHOP ON PROFICIENCY TESTING IN ANALYTICAL CHEMISTRY, MICROBIOLOGY AND LABORATORY MEDICINE Portorož, Slovenia http://eurachempt2017.eu/ POLYCHAR 25 - 25th ANNUAL WORLD FORUM ON ADVANCED MATERIALS Kuala Lumpur, Malaysia http://www.25POLYCHAR.org.my IUPAC-FAPS 2017 POLYMER CONGRESS ON SMART MATERIALS FOR EMERGING TECHNOLOGY Jeju Island, Republic of Korea http://www.faps2017.org EWCC 2017 - EAST-WEST CHEMISTRY CONFERENCE 2017 Skopje, Macedonia http://ewcc2017.org/ November 2017 5 - 9 HPLC 2017 - THE 46th INTERNATIONAL SYMPOSIUM ON HIGH PERFORMANCE LIQUID PHASE SEPARATIONS AND RELATED TECHNIQUES Jeju Island, Republic Of Korea Information: http://www.hplc2017-jeju.org Društvene vesti in druge aktualnosti S54 Acta Chim. Slov. 2017, 64, (1), Supplement S55 2018 February 2018 21 - 23 Information: ChemCYS 2018 - 14th CHEMISTRY CONFERENCE FOR YOUNG SCIENTISTS Blankenberge, Belgium http://chemcys.be/ June 2018 4 - 6 Information: IIS PRAGUE 2018 - 13th INTERNATIONAL SYMPOSIUM ON THE SYNTHESIS AND APPLICATIONS OF ISOTOPES AND ISOTOPICALLY LABELLED COMPOUNDS Prague, Czech Republic http://www.iis-prague2018.cz/ September 2018 16 - 19 Information: DISTILLATION & ABSORPTION CONFERENCE 2018 Firenze, Italy http://www.aidic.it/da2018/ October 2018 14 - 18 Information: 14th iupac international congress of pesticide chemistry Rio de Janeiro, Brazil https://iupac.org/event/14th-iupac-international-congress-of-pesticide-chemistry/ Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement_ S55 Društvene vesti in druge aktualnosti S56 Acta Chim. Slov. 2017, 64, (1), Supplement S55 Acta Chimica Slovenica Author Guidelines Submissions Submission to ACSi is made with the implicit understanding that neither the manuscript nor the essence of its content has been published in whole or in part and that it is not being considered for publication elsewhere. All the listed authors should have agreed on the content and the corresponding (submitting) author is responsible for having ensured that this agreement has been reached. The acceptance of an article is based entirely on its scientific merit, as judged by peer review. There are no page charges for publishing articles in ACSi. Submission material Typical submission consists of: • full manuscript (Word file, with title, authors, abstract, keywords, figures and tables embedded, and references); • supplementary files: - Statement of novelty (Word file), - List of suggested reviewers (Word file), - ZIP file containing graphics (figures, illustrations, images, photographs), - Graphical abstract (single graphics file), - Proposed cover picture (optional, single graphics file), - Appendices (optional, Word files, graphics files). Submission process Submission process consists of 5 steps. Before submission, authors should go through the checklist at the bottom of these guidelines page and prepare for submission: Step 1: Starting the submission • Choose one of the journal sections. • Confirm all the requirements of the checklist. • Additional plain text comments for the editor can be provided in the relevant text field. Step 2: Upload submission • Upload full manuscript in the form of a Word file (with title, authors, abstract, keywords, figures and tables embedded, and references). Step 3: Enter metadata • First name, last name, contact email and affiliation for all authors, in relevant order, must be provided. Corresponding author has to be selected. Full postal address and phone number of the corresponding author has to be provided. • Title and abstract must be provided in plain text. • Keywords must be provided (max. 6, separated by semicolons). • Data about contributors and supporting agencies may be entered. • References in plain text must be provided in the relevant text filed. Step 4: Upload supplementary files • Statement of novelty in a Word file must be uploaded • List of suggested reviewers with at least three reviewers must be uploaded as a Word file. • All graphics have to be uploaded in a single ZIP file. Graphics should be named Figure 1.jpg, Figure 2.eps, etc. • Graphical abstract image must be uploaded separately. • Proposed cover picture (optional) should be uploaded separately. • Any additional appendices (optional) to the paper may be uploaded. Appendices may be published as a supplementary material to the paper, if accepted. • For each uploaded file the author is asked for additional metadata which may be provided. Depending of the type of the file please provide the relevant title (Statement of novelty, List of suggested reviewers, Figures, Graphical abstract, Proposed cover picture, Appendix). Step 5: Confirmation • Final confirmation is required. Article Types Review articles are welcome in any area of chemistry and may cover a wider or a more specialized area, if a high impact is expected. Manuscripts normally should not exceed 40 pages of one column format (letter size 12, 33 lines per page). Authors should consult the ACSi editor prior to preparation of a review article. Scientific articles should have the following structure: 1. Title (max. 150 characters), 2. Authors and affiliations, 3. Abstract (max. 1000 characters), 4. Keywords (max. 6), 5. Introduction, 6. Experimental (Results and Discussion), 7. Results and Discussion (Experimental), 8. Conclusions, 9. Acknowledgements (if any), 10. References. The sections should be arranged in the sequence generally accepted for publications in the respective fields. Scientific articles should report significant Društvene vesti in druge aktualnosti Acta Chim. Slov. 2017, 64, (1), Supplement S55 and innovative achievements and exhibit a high level of originality. Short communications generally follow the same order of sections, but should be short (max. 2500 words) and report a significant aspect of research work meriting separate publication. Technical articles report applications of an already described innovation. Typically, technical articles are not based on new experiments. Preparation of Submissions Text of the submitted articles must be prepared with Word for Windows. Normal style set to single column, 1.5 line spacing, and 12 pt Times New Roman font is recommended. Line numbering (continuous, for the whole document) must be enabled to simplify the reviewing process. For any other format, please consult the editor. Articles should be written preferably in English. Correct spelling and grammar are the sole responsibility of the aut-hor(s). Papers should be written in a concise and succinct manner. The authors shall respect the ISO 80000 standard, and IUPAC Green Book rules on the names and symbols of quantities and units.The Système International d'Unités (SI) must be used for all dimensional quantities. Graphics (figures, graphs, illustrations, digital images, photographs) should be inserted in the text where appropriate. The captions should be self-explanatory. Lettering should be readable (suggested 8 point Arial font) with equal size in all figures. Use common programs such as Word Excel to prepare figures (graphs) and ChemDraw to prepare structures in their final size (8 cm for single column width or 17 cm for double column width) so that neither reduction nor enlargement is required. In graphs, only the graph area determined by both axes should be in the frame, while a frame around the whole graph should be omitted. The graph area should be white. The legend should be inside the graph area. The style of all graphs should be the same. Figures and illustrations should be of sufficient quality for the printed version, i.e. 300 dpi minimum. Digital images and photographs should be of high quality (minimum 250 dpi resolution). On submission, figures should be of good enough resolution to be assessed by the referees, ideally as JPEGs. High-resolution figures (in JPEG, TIFF, or EPS format) might be required if the paper is accepted for publication. Tables should be prepared in the Word file of the paper as usual Word tables. The captions should above the table and self-explanatory. References should be numbered and ordered sequentially as they appear in the text, likewiise methods, tables, figure captions. When cited in the text, reference numbers should be superscripted, following punctuation marks. It is the sole respon- sibility of authors to cite articles that have been submitted to a journal or were in print at the time of submission to ACSi. Formatting of references to published work should follow the journal style; please also consult a recent issue: 1. J. W. Smith, A. G. White, Acta Chim. Slov. 2008, 55, 1055-1059. 2. M. F. Kemmere, T. F. Keurentjes, in: S. P. Nunes, K. V. Peinemann (Ed.): Membrane Technology in the Chemical Industry, Wiley-VCH, Weinheim, Germany, 2008, pp. 229-255. 3. J. Levec, Arrangement and process for oxidizing an aqueous medium, US Patent Number 5,928,521, date of patent July 27, 1999. 4. L. A. Bursill, J. M. Thomas, in: R. Sersale, C. Col-lela, R. Aiello (Eds.), Recent Progress Report and Discussions: 5th International Zeolite Conference, Naples, Italy, 1980, Gianini, Naples, 1981, pp. 25-30. 5. J. Szegezdi, F. Csizmadia, Prediction of dissociation constant using microconstants, http://www. chemaxon.com/conf/Prediction_of_dissociation _constant_using_microco nstants.pdf, (assessed: March 31, 2008) Titles of journals shoud be abbreviated according to Chemical Abstracts Service Source Index (CASSI). Special Notes • Complete characterization, including crystal structure, should be given when the synthesis of new compounds in crystal form is reported. • Numerical data should be reported with the number of significant digits corresponding to the magnitude of experimental uncertainty. • The SI system of units and IUPAC recommendations for nomenclature, symbols and abbreviations should be followed closely. Additionally, the authors should follow the general guidelines when citing spectral and analytical data, and depositing crystallographic data. • Characters should be correctly represented throughout the manuscript: for example, 1 (one) and l (ell), 0 (zero) and O (oh), x (ex), D7 (times sign), B0 (degree sign). Use Symbol font for all Greek letters and mathematical symbols. • The rules and recommendations of the IUBMB and the International Union of Pure and Applied Chemistry (IUPAC) should be used for abbreviation of chemical names, nomenclature of chemical compounds, enzyme nomenclature, isotopic compounds, optically active isomers, and spectroscopic data. • A conflict of interest occurs when an individual (author, reviewer, editor) or its organization is involved in multiple interests, one of which could possibly corrupt the motivation for an act in the other. Financial relationships are the most easily identifiable conflicts of interest, while conflicts can occur also as personal relationships, academic competition, etc. The Edi- Društvene vesti in druge aktualnosti S58 Acta Chim. Slov. 2017, 64, (1), Supplement S55 tors will make effort to ensure that conflicts of interest will not compromise the evaluation process; potential editors and reviewers will be asked to exempt themselves from review process when such conflict of interest exists. When the manuscript is submitted for publication, the authors are expected to disclose any relationships that might pose potential conflict of interest with respect to results reported in that manuscript. In the Acknowledgement section the source of funding support should be mentioned. The statement of disclosure must be provided as Comments to Editor during the submission process. • Published statement of Informed Consent. 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If doubt exists whether the research was conducted in accordance with the Helsinki Declaration, the authors must explain the rationale for their approach and demonstrate that the institutional review body explicitly approved the doubtful aspects of the study. When reporting experiments on animals, authors should indicate whether the institutional and national guide for the care and use of laboratory animals was followed. • Contributions authored by Slovenian scientists are evaluated by non-Slovenian referees. • Papers describing microwave-assisted reactions performed in domestic microwave ovens are not considered for publication in Acta Chimi-ca Slovenica. • Manuscripts that are not prepared and submitted in accord with the instructions for authors are not considered for publication. Appendices Authors are encouraged to make use of supporting information for publication, which is supplementary material (appendices) that is submitted at the same time as the manuscript. It is made available on the Journal's web site and is linked to the article in the Journal's Web edition. The use of supporting information is particularly appropriate for presenting additional graphs, spectra, tables and discussion and is more likely to be of interest to specialists than to general readers. When preparing supporting information, authors should keep in mind that the supporting information files will not be edited by the editorial staff. In addition, the files should be not too large (upper limit 10 MB) and should be provided in common widely known file formats so as to be accessible to readers without difficulty. All files of supplementary materials are loaded sepa-ratly during the submission process as supplementary files. Proposed Cover Picture and Graphical Abstract Image Authors are encouraged to submit illustrations as candidates for the journal Cover Picture as well as graphical abstracts. Graphical abstract contains an image that appears as a part of the entry in the table of contents in both online and printed edition. The pictures may be the same. The illustrations must be related to the subject matter of the paper. Usually both proposed cover picture and picture for graphical abstract are the same, but authors may provide different pictures as well. Graphical content: an ideally full-colour illustration of resolution 300 dpi from the manuscript must be proposed with the submission. Graphical abstract pictures are printed in size 6.5 x 4 cm (hence minimal resolution of 770 x 470 pixels). Cover picture is printed in size 11 x 9.5 cm (hence minimal resolution of 1300 x 1130 pixels). Statement of novelty Statement of novelty is provided in a Word file and submitted as a supplementary file in step 4 of submission process. Authors should in no more then 100 words emphasize the scientific novelty of the presented research. 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Slov. 2017, 64, (1), Supplement S55 Koristni naslovi Slovensko kemijsko društvo www.chem-soc.si e-mail: chem.soc@ki.si Wessex Institute of Technology www.wessex.ac.uk SETAC www.setac.org European Water Association http://www.ewa-online.eu/ :URDPËRM )CIEÍ\JCE European Science Foundation "OUWDRTION www.esf.org European Federation of Chemical Engineering https://efce.info/ International Union of Pure and Applied Chemistry https://iupac.org/ Novice europske zveze kemijskih društev (EuCheMS) najdete na: EuCheMS: Brussels News Updates http://www.euchems.eu/newsletters/ Društvene vesti in druge aktualnosti TINYCLAVE Kovinska posoda 10 ml Do 100 bar -20 do +300 °C € 2.310+ddv MINICLAVE Kovinska posoda 100 ml Do 100 bar -20 do +300 °C € 2.708 + DDV Izmenljiva reaktorska posoda, dobavljiva v stekleni ali kovinski izvedbi (nerjaveče jeklo s PTFE insertom oz. Hastelloy). Vse posode so izmenljive in kompatibilne z osnovnim pokrovom. 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Redna uporaba Bilobila: razširi krvne žile in izboljša pretok krvi v možganih, krepi delovanje možganskih celic, saj izboljša izrabo kisika in glukoze, varuje možganske celice pred škodljivimi vplivi radikalov. Bilobil vsebuje izvleček iz listov Ginkga biJobe. Bilobil' trde kapsule Ginltgo biloboe folii Butraclum siccum 60 trdih IojhuI M peiorotno- uporabo www.bilobil.si Bilobil Noša inovativnost in znanje za učinkovite in varne izdelke vrhunske kakovosti. Pred uporabo natančno preberite navodilo! 0 tveganju in neželenih učinkih se posvetujte z zdravnikom ali s farmacevtom. ActaChimica Slovenica Acta ChimicaSlovenica The figure showed that MnO2 submircoparticles arrayed densely on the eggshell membrane along with the fiber-like protein (scale bar: 20 ^m). The size of the spherical particles was about 710 nm, which was a good consistency with the microstructured biotemplate. 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