4 n Year 2024, Vol. 71, No. 1 ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChim A cta C him ica Slovenica 71/2024 Pages 1–178 Pages 1–178 n Year 2024, Vol. 71, No. 1 http://acta.chem-soc.si 1 71/2024 1 ISSN 1580-3155 In 2023, 46 study programmes offering environmental sciences with diverse chemistry content were identified in Slovenia, comprising ten in secondary education, ten in short-cycle higher vocational education, nine in bachelor’s programmes, 11 in master’s programmes, and six in doctoral programmes. Environmental Education Programmes EDITOR-IN-CHIEF EDITORIAL BOARD ADVISORY EDITORIAL BOARD ASSOCIATE EDITORS Alen Albreht, National Institute of Chemistry, Slovenia Aleš Berlec, Jožef Stefan Institute, Slovenia Janez Cerkovnik, University of Ljubljana, Slovenia Mirela Dragomir, Jožef Stefan Institute, Slovenia Krištof Kranjc, University of Ljubljana, Slovenia Matjaž Kristl, University of Maribor, Slovenia Maja Leitgeb, University of Maribor, Slovenia Helena Prosen, University of Ljubljana, Slovenia Jernej Stare, National Institute of Chemistry, Slovenia Irena Vovk, National Institute of Chemistry, Slovenia ADMINISTRATIVE ASSISTANT Eva Mihalinec, Slovenian Chemical society, Slovenia 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, University of Ljubljana Janez Košmrlj, University of Ljubljana, Slovenia Mahesh K. Lakshman, The City College and The City University of New York, USA Blaž Likozar, National Institute of Chemistry, Slovenia Janez Mavri, National Institute of Chemistry, Slovenia Jiři Pinkas, Masaryk University Brno, Czech Republic Friedrich Srienc, University of Minnesota, USA Walter Steiner, Graz University of Technology, Austria Jurij Svete, University of Ljubljana, Slovenia David Šarlah, University of Illinois at Urbana-Champaign, USA; Università degli Studi di Pavia, Italy Ivan Švancara, University of Pardubice, Czech Republic Gašper Tavčar, Jožef Stefan Institute, Slovenia Ennio Zangrando, University of Trieste, Italy Polona Žnidaršič Plazl, University of Ljubljana, Slovenia Chairman Branko Stanovnik, Slovenia Members Udo A. Th. Brinkman, The Netherlands Attilio Cesaro, Italy Vida Hudnik, Slovenia Venčeslav Kaučič, Slovenia Željko Knez, Slovenia Radovan Komel, Slovenia Stane Pejovnik, Slovenia Anton Perdih, Slovenia Slavko Pečar, Slovenia Andrej Petrič, Slovenia Boris Pihlar, Slovenia Milan Randić, Des Moines, USA Jože Škerjanc, Slovenia Đurđa Vasić-Rački, Croatia Marjan Veber, Slovenia Gorazd Vesnaver, Slovenia Jure Zupan, Slovenia Majda Žigon, Slovenia FRANC PERDIH University of Ljubjana, Facuty of Chemstry and Chemical Technology, Večna pot 113, SI-1000 Ljubljana, Slovenija E-mail: ACSi@fkkt.uni-lj.si, Telephone: (+386)-1-479-8514 Izdaja – Published by: SLOVENSKO KEMIJSKO DRUŠTVO – SLOVENIAN CHEMICAL SOCIETY Naslov redakcije in uprave – Address of the Editorial Board and Administration Hajdrihova 19, SI-1000 Ljubljana, Slovenija Tel.: (+386)-1-476-0252; Fax: (+386)-1-476-0300; E-mail: chem.soc@ki.si Izdajanje sofinancirajo – Financially supported by: National Institute of Chemistry, Ljubljana, Slovenia Jožef Stefan Institute, Ljubljana, Slovenia Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia Faculty of Chemistry and Chemical Engineering, University of Maribor, Slovenia University of Nova Gorica, Slovenia Acta Chimica Slovenica izhaja štirikrat letno v elektronski obliki na spletni strani http://acta.chem-soc.si. V primeru posvečenih številk izhaja revija tudi v tiskani obliki v omejenem številu izvodov. Acta Chimica Slovenica appears quarterly in electronic form on the web site http://acta.chem-soc.si. In case of dedicated issues, a limited number of printed copies are issued as well. Transakcijski račun: 02053-0013322846 Bank Account No.: SI56020530013322846-Nova Ljubljanska banka d. d., Trg republike 2, SI-1520 Ljubljana, Slovenia, SWIFT Code: LJBA SI 2X Oblikovanje ovitka – Design cover: KULT, oblikovalski studio, Simon KAJTNA, s. p. Grafična priprava za tisk: OSITO, Laura Jankovič, s.p. 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Articles in this journal are published under the   Creative Commons Attribution 4.0 International License – Graphical Contents Graphical Contents ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChimica Year 2024, Vol. 71, No. 1 SCIENTIFIC PAPER 1–7 Inorganic chemistry One-Step Synthesis of Biocompatible Thiosemicarbazone Functionalized Copper Oxide Nanoparticles: Evaluation of Enhanced Antibacterial Activity Seyede Khadije Safavi-Mirmahaleh, Zeinab Moradi-Shoeili, and Mehdi Rassa 9–19 Biochemistry and molecular biology Chemical Composition and In Vitro Biological Activity of Two Thymus L. Varieties Growing in Turkey Turgut Taşkın, Mustafa Öksüz, Bünyamin Bulkurcuoğlu, Sebnem Ercelen, Erkan Rayaman, Mizgin Ermanoğlu, Beyza Nur Yılmaz, Duygu Taşkın, Talip Şahin and Ömer Kılıç 20–25 Inorganic chemistry Synthesis, Crystal Structures and Antibacterial Activity of Nickel(II) and Copper(II) Complexes Derived from (E)-2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methylene)- N-phenylhydrazinecarbothioamide Jing Wang, Haiyang Fei, Min Zhou, Chengcai Zhang, Juan Sun, Yang Zhou and Meng Yang Graphical Contents 47–55 Organic chemistry Nano MgCuAl2O5: Synthesis by Sol-Gel Auto- Combustion Process, Characterization and Reusable Heterogeneous Catalyst for the Hantzsch 1,4-Dihydropyridine Reaction Marzieh Mahmoodi Keshtiban, Abbas Nikoo and Bakhshali Massoumi 39–46 Organic chemistry Synthesis, Characterization, Crystal Structures and Urease Inhibition of Some Thiosemicarbazones Ling-Wei Xue, Qiao-Ru Liu and Yong-Jun Han 26–38 Biomedical applications Brain Targeted Drug Delivery System of Carmustine: Design, Development, Characterization, in vitro, ex vivo Evaluation and in vivo Pharmacokinetic Study Audumbar Mali and Anil Bhanwase 56–65 Chemical education Environmental Education Programmes: A Case Study of Slovenia Janja Vidmar, Jan Hočevar and Ester Heath 66–83 Inorganic chemistry Synthesis of Bone Meal-derived 4-Carboxyphenylboronic Acid Functionalized Sulfur and Nitrogen Co-doped Graphene Quantum Dots Nanoprobe for Sialic Acid Sensing Sopan N. Nangare, Pratik P. Yeole, Zamir G. Khan, Ashwini G. Patil, Bhushankumar S. Sathe Sanjaykumar B. Bari and Pravin O. Patil Graphical Contents 99–109 Chemical, biochemical and environmental engineering Exploring a Substitute for Hydrogen Peroxide in Fenton Process – A Case Study on the COD Removal of Acid Orange 8 Tsungom Mulai, John Elisa Kumar, Wanshanlang Kharmawphlang and Mihir Kumar Sahoo 91–98 Inorganic chemistry Samarium(III) Removal by Weak Acid Exchanger Amberlite IRC-50 in (H+) and (Na+) Forms Afaf Amara Rekkab, and Mohamed Amine Didi 84–90 Chemical education Assessing 15-year-olds’ Understanding of Chemical Concepts in the Context of the Lithosphere and Pedosphere Luka Ribič, Iztok Devetak and Miha Slapničar 110–122 Inorganic chemistry Synthesis, Spectroscopy, X-ray Structures, DNA Binding and Photocatalytic Properties of Two Ni(II) and Co(II) Complexes of a Pyrazolyl Schiff-base Ligand Suman Mandal, David B. Cordes, Alexandra M. Z. Slawin and Nitis Chandra Saha 123–134 Chemical, biochemical and environmental engineering Removal of Methyl Violet 2B and Direct Black 22 from Single and Binary System Using a Magnetic Zeolite/MgO/Starch/Fe3O4 Nanocomposite Serap Fındık Graphical Contents 135–142 Inorganic chemistry Syntheses, Crystal Structures and Antimicrobial Activity of Zinc(II) Complexes Derived from 5-Bromo-2- (((2-piperazin-1-yl)ethyl)imino)methyl)phenol Yin-Bing Chen, Xiao-Yang Qiu*, Meng-Yuan Xu, Fei-Yu Qi, Xin He, Chen Wu and Shu-Juan Liu 143–160 Chemical education Textbook Sets Through the Perspective of the Orientation of the Intended Chemistry Curriculum for Primary and Secondary Schools Špela Hrast and Vesna Ferk Savec 161–169 Biomedical applications Quality by Design Based Development of Electrospun Nanofibrous Solid Dispersion Mats for Oral Delivery of Efavirenz Md. Faseehuddin Ahmed, Kalpana Swain, Satyanarayan Pattnaik and Biplab Kumar Dey 170–178 Biomedical applications QSAR Modeling of Sphingomyelin Synthase 2 Inhibitors for Their Potential as Anti-Atherosclerotic Agents Dejan Petrović, Marina Deljanin Ilić, Dejan Simonović, Zoran Marčetić, Milovan Stojanović, Sanja Stojanović, Nebojša Arsić, Dušan Sokolović and Aleksandar M. Veselinović 1Acta Chim. Slov. 2024, 71, 1–8 Safavi-Mirmahaleh et al.: One-Step Synthesis of Biocompatible Thiosemicarbazone ... DOI: 10.17344/acsi.2023.8067 Scientific paper One-Step Synthesis of Biocompatible Thiosemicarbazone Functionalized Copper Oxide Nanoparticles: Evaluation of Enhanced Antibacterial Activity Seyede Khadije Safavi-Mirmahaleh,1 Zeinab Moradi-Shoeili ,1,* and Mehdi Rassa2 1 Department of Chemistry, Faculty of Sciences, University of Guilan, P.O. Box 41335–1914, Rasht, Iran 2 Department of Biology, Faculty of Sciences, University of Guilan, P.O. Box 41335–1914, Rasht, Iran * Corresponding author: E-mail: zmoradi@guilan.ac.ir Tel.: +98 13 33333262; fax: +98 13 33320066 Received: 02-13-2023 Abstract Organic–inorganic hybrid bioactive nanomaterials were sonochemically synthesized by covalent anchoring of 2-acetylpyridine thiosemicarbazone on the surface of CuO nanoparticles using two different approaches. The prepared nanoparticles were characterized by a combination of physico-chemical and spectroscopic techniques. The synergetic bactericidal activity of CuO and thiosemicarbazone moieties in prepared nanomaterials was tested in vitro using the zone inhibition methods against Gram positive and Gram negative bacterial strains. Additionally, the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were also determined. Results prove that CuO and functionalized CuO nanoparticles synthesized by the sonochemical method in present study show improved antibacteri- al activities and they could be used in the design of more efficient antibacterial materials for pharmaceutical applications. Keywords: Copper oxide; Thiosemicarbazone, Nanoparticles; Antibacterial activity. 1. Introduction In recent years, the need to develop novel drugs with enhanced, targeted bactericidal activity has significantly increased due to concerns regarding drug-resistance in pathogens.1 With recent advances in nanotechnology, na- no-sized materials have received considerable attention as potent antimicrobial agents because of their unusual prop- erties which are distinctly different from those of their mi- crometer-sized counterparts.2–4 As antimicrobial agents, nanomaterials show a diversity of modes of action; such as electrostatic interaction with the bacterial membrane, re- active oxygen species (ROS) production, photoactivation or photocatalism, production of reactive nitrogen species (RNS), production and induction of signal secretion, which may cause membrane damage, hinder protein func- tion, induce DNA destruction and therefore promote ap- optosis (programmed cell death).5 For more than a decade, various types of metals such as silver (Ag)6 and gold (Au)7 and also metal oxide nano- particles such as iron oxide (Fe3O4),8 titanium oxide (TiO2),9 magnesium oxides (MgO),10 aluminum oxide (Al2O3),11 and zinc oxide (ZnO)12 have been the focus of intense research due to their antimicrobial properties. Moreover, copper oxide (CuO) is among a group of metal- lo-drugs which can act as effective antimicrobial and anti- bacterial agents.13 The higher antibacterial activity of CuO nanoparticles compared to metal nanoparticles such as silver can be interpreted by the stronger complexation of amine and carboxyl groups on the bacterial cell walls and CuO nanoparticles.14 The antimicrobial activity of nanomaterials has been observed to vary as a function of environmental factors in- cluding pH, temperature, and solvent as well as size, shape, surface area in contact with the microbe, and composition with other organic or inorganic materials.15–18 In addition, it has been shown that surface chemical modification can im- prove colloidal stability in physiological media, water solu- bility, biocompatibility, and specific targeting ability of nano- partricles.19 After the first report of pyridine-2-carbaldehyde thiosemicarbazone synthesis and its carcinostatic proper- ties,20 the synthesis of different thiosemicarbazone ligands 2 Acta Chim. Slov. 2024, 71, 1–8 Safavi-Mirmahaleh et al.: One-Step Synthesis of Biocompatible Thiosemicarbazone ... and their metal complexes have received considerable atten- tion due to the wide range of applications in pharmacologi- cal fields.21,22 Taking into consideration the advanced appli- cations of nanocomposite materials in the field of antimicrobial chemotherapy, the present work reports a sim- ple and cost effective method for conjugation of bioactive 2-acetylpyridine thiosemicarbazone (TSCPy) on the surface of CuO nanoparticles. CuO nanoparticles containing TSCPy moiety were prepared via two different methods using ultra- sonic irradiations. The first route (method A) involves the co-precipitation method in the presence of glutamic acid as conjugating agents. The TSCPy molecules were then an- chored on the surface of CuO nanoparticles by a condensa- tion reaction between glutamic acid and TSCPy. In the sec- ond route (method B), the TSCPy functionalized CuO nanoparticles were directly synthesized by the co-precipita- tion method in the presence of TSCPy. The prepared faction- alized CuO nanoparticles were characterized by different spectroscopic methods. In addition, the synergetic in vitro antibacterial activity of CuO nanoparticles and TSCPy moi- ety have been screened against a series of Gram positive and Gram negative bacteria, using the zone inhibition method. The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were also investigated. 2. Experimental 2. 1. Materials and Methods All reagents were obtained from commercial sources and used without further purification. Powder X-ray dif- fraction (PXRD) data were collected with a Philips pw 1830 diffractometer (Cu-Kα X-radiation, λ = 1.54 Å). FT‒ IR spectra of samples in the form of KBr pellets were re- corded using an Alpha-Bruker FT-IR spectrophotometer. The scanning electron microscopy (SEM) images were taken on a KYKY‒EM3200 scanning electron microscope. The elemental analysis was recorded with an energy dis- persive X-ray (EDX) analyzer, MIRA3 FEG‒SEM series. 2. 2. Preparation of (E)-2-(1-(pyridin-2-yl) ethylidene)hydrazine-1-carbothioamide (TSCPy) 2-acetylpyridine (0.121 mL, 1 mmol) was added to 20 mL ethanolic solution of the thiosemicarbazide (0.203 g, 1 mmol). The reaction mixture was refluxed for 10 h at 70 °C. A precipitate was formed when the solution was al- lowed to cool at room temperature. The pale yellow precip- itate was filtered, washed with cold ethanol, diethyl ether and dried in air. 2. 3. Preparation of CuO Nanoparticles Aqueous solution of NaOH (1 M) was added drop- wise to 50 mL aqueous solution of CuSO4·5H2O (6.24 g, 25 mmol) while it was positioned in a large-density ultrason- ic probe, operating at 37 kHz with a maximum force out- put of 320 W (pH 11−12). A black precipitate was formed, and the suspension was then sonicated for 2 h at 30 °C to ensure completion of the reaction. The product was sepa- rated by centrifugation, washed with distilled water, etha- nol and diethyl ether and dried in air. 2. 4. Preparation of Functionalized CuO Nanoparticles To prepare functionalized CuO nanoparticles with glutamic acid (CuOGA NPs), glutamic acid (0.92 g, 6 mmol) was dissolved in 15 mL of NaOH solution (0.02 M) and then added to an aqueous solution of CuSO4·5H2O (1.56 g, 6 mmol). The rest of the reaction was carried out according to the procedure described above for CuO nan- oparticles. To conjugate CuOGA NPs with TSCPy, 0.3 g of CuOGA was added to 30 mL ethanol and sonicated for 30 min. Then, 0.3 g of TSCPy was dissolved in DMSO (0.1 mL) and 10 mL ethanol was added to the ethanolic sus- pension of CuOGA. The mixture was sonicated for 60 min and allowed to stir overnight at 40 °C. CuOGA-TSCPy NPs were subsequently washed with ethanol and diethyl ether and dried in oven at 70 °C for 3 h. The synthesis of TSCPy functionalized CuO nano- particles (CuOTSCPy) were performed following the pro- cedure described for CuOGA, except that TSCPy (1.05 g, 4 mmol) was used as the functionalization agent. 2. 5. In-vitro Antibacterial Assay Antibacterial activity of the prepared CuO and func- tionalized CuO NPs were tested in vitro using the zone in- hibition method23 against two Gram positive bacterial strains Micrococcus luteus (ATCC 4698) and Staphylococ- cus aureus (ATCC 29213), and two Gram negative bacteri- al strains Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). The nutrient agar and nutrient broth cultures were prepared according to manufactures’ instructions and were incubated at 37 °C. After incubation for the appropriate time, a suspension of 50 μL of each bac- terial test organism was spread onto the nutrient agar plates. Agar wells were prepared with the help of a steri- lized glass tube. Then 30 μL of the test agents at a concen- tration of 2000 μg/ml in DMSO were added to each well. All the bacterial strains were incubated at 37 °C for 24 h. Clear zones around the wells showed inhibition of bacteri- al growth and turbidity indicated bacterial resistance to the compound at the concentration present in the medi- um. The diameter of inhibition zones was determined in millimeters (mm). The concentration of DMSO in the me- dium did not affect growth of any of the microorganisms tested. All experiments were carried out in triplicate. The results are reported as mean±standard deviation of zone of inhibition in millimeter. Antibacterial activity of each 3Acta Chim. Slov. 2024, 71, 1–8 Safavi-Mirmahaleh et al.: One-Step Synthesis of Biocompatible Thiosemicarbazone ... compound was compared with penicillin G and tetracy- cline as standard drugs. DMSO was used as a negative con- trol. The MIC and MBC were also determined by the dilu- tion method against the tested bacterial species. The MIC is defined as the lowest concentration of compound that inhibited bacterial growth (no turbidity in the tube). Brief- ly, NPs were diluted into concentrations of 20, 10, 5 and 2.5 µg/ml, in nutrient broth tubes inoculated with the test bac- terium. The tubes were incubated at 37 °C for 24 h and thereafter observed for growth or turbidity using unaided eye. The MBC was defined as the lowest concentration of compound lethal to the bacteria. In brief, 50 µL of broth from each test tube showing no visible signs of growth/ turbidity, was inoculated onto a nutrient agar plate and in- cubated further for 24 h at 37 °C. The agar plates were then examined for growth. 3. Result and Discussion 3. 1. Preparation and Characterization of Spinel Ferrite Nanoparticles The thiosemicarbazone ligand (TSCPy) derived from 2-acetopyridine and thiosemicarbazid was synthe- sized following the procedure described previously.24 CuO and also functionalized CuO nanoparticles were synthe- sized using ultrasonic irradiations which significantly re- duced the synthesis time and temperature. CuO nanopar- ticles containing the TSCPy bioactive molecule were prepared by using one of two methods shown in Scheme 1. The first route (method A) involves the synthesis of CuO nanoparticles by the co-precipitation method in the pres- ence of glutamic acid. The covalently grafted TSCPy mole- cules can then be produced via the subsequent condensa- tion reaction between glutamic acid and TSCPy. In the second route (method B), the TSCPy functionalized CuO nanoparticles were directly synthesized by the co-precipi- tation method in the presence of TSCPy. Figure 1 displays the FT–IR spectra of as-prepared samples. Strong bands around 3460 and 1650 cm−1 ap- peared in FT–IR spectra of nano-samples (Figures 1b–1e) corresponding to the vibrational modes of O−H stretching and bending vibrations of surface hydroxyl groups and physisorbed water molecules.25 FT–IR spectrum for un- treated CuO nanoparticles (Figure 1b) displayed strong peaks at 473 cm−1, 526 cm−1 and 618 cm−1, which are asso- ciated with Cu–O vibrational modes. This is in good agree- ment with literature sources.26,27 Glutamic acid and thio- semicarbazone-treated CuO nanoparticles (Figures 1c–1e), in addition to the same two characteristic peaks present in the untreated sample, also showed additional ones. The new bands at about 2850–3050 cm–1 in CuOGA and CuOGA-TSCPy spectra can be attributed to –C–H bond stretching assigned to the alkyl group.28 The peaks at 1113 and 1030 cm–1 in CuOGA spectrum as well as the peaks at 1062 and 1031 cm−1 in CuOGA-TSCPy spectrum are assigned to C−O stretching coordinated to the metal cations.14 A comparison between FT‒IR spectra of thio- semicarbazone ligand (Figure 1a) and synthesized CuO- GA-TSCPy nanoparticles (Figure 1d) indicates the suc- cessful grafting of thiosemicarbazone onto the surface of CuO nanoparticles. The bands at around 3300 cm−1 and 820 cm−1 in the IR spectrum of TSCPy corresponding to Scheme 1. Synthetic pathways for the synthesis of CuO and TSCPy functionalized CuO nanoparticles. 4 Acta Chim. Slov. 2024, 71, 1–8 Safavi-Mirmahaleh et al.: One-Step Synthesis of Biocompatible Thiosemicarbazone ... the ν(NH) and ν(C=S),29–31 respectively (Figure 1a), have also been observed in IR spectrum of CuOGA-TSCPy na- noparticles (Figure 1d). The band observed at 1210 cm−1 and a band at 1556 cm−1 in the spectrum of CuOGA-TSCPy can be assigned to the ν(C‒N),32 indicating the thiosemi- carbazone anchoring on the CuOGA-TSCPy nanoparti- cles via amide linkage between glutamic acid and TSCPy. The FT-IR spectra of CuOGA-TSCPy and CuOTSCPy na- noparticles are very similar. Both exhibit the characteristic CuO peaks as well as peaks at around 817 cm−1 and 3300 cm−1 corresponding to C=S and N-H bonds, respectively. Furthermore, in the TSCPy treated sample (Figure 1e) multiple C-H stretching peaks above and below 3000 cm−1 were observed, indicative of both saturated and unsaturat- ed C-H bond stretching. The aromatic C=C stretching peaks including the pyridine skeleton stretching were ob- served at about 1440 cm−1 for CuOGA-TSCPy and CuOTSCPy nanoparticles.33 Figure 1. FT–IR spectra of (a) TSCPy, (b) CuO, (c) CuOGA, (d) CuOGA-TSCPy and (e) CuOTSCPy. Typical XRD pattern of the synthesized CuO and functionalized CuO nanoparticles are shown in Figure 2. The peaks are indexed as 32.3º (110), 35. 5º (11), 38.7º (111), 49.1º (20), 53.7º (020), 58.2º (202), 61.7º (11), 66.5º (31), 68. 3º (220), 72.6º (311) and 75.5 (004), respectively. These were compared with the Joint Committee on Pow- der Diffraction Standards (JCPDS) card No 48–1548. They suggest a monoclinic structure and the diffraction patterns of the characteristic peaks are in good agreement with data presented previously.34 High intensity and sharpness of CuO XRD characteristic peaks indicate the good quality crystalline structure of nanoparticles. Despite a decrease in intensity observed for the CuOGA-TSCPy sample, it is clear from Figure 2b that the functionalization does not influence the crystal structure. Further, no noticeable peaks such as Cu(OH)2, CuS or other copper compound were observed in CuO and CuOGA-TSCPy XRD patterns, indicating the formation of single-phase CuO with a mon- oclinic structure. The size of the synthesized nanoparticles was calcu- lated using Scherrer’s equation: D = kλ/βcosθ, where, D is the average crystalline size, k the Scherrer constant (0.89), λ the X-ray wavelength used, β the angular line width at half maximum intensity and θ is the Bragg’s angle in de- grees unit.35 The calculated average particle sizes are 13 and 10 nm for CuO and CuOGA-TSCPy, respectively. The XRD pattern obtained for the CuOTSCPy (Figure 2c) is different from those observed for CuO and CuO- GA-TSCPy and is relatively broader. No sharp peaks can be attributed to the amorphous nature of CuOTSCPy powders. In addition, the peaks related to the CuS phase were also identified in Figure 2c.36 These phenomena can be mainly explained by the presence of TSCPy during the formation of CuO phase in the synthesis of CuOTSCPy nanoparticles. As known, thiocarbamates and thiocarba- zides have been used frequently for the synthesis of CuS nanoparticles,37,38 and this can be one of the disadvantages of direct functionalization of CuO nanoparticles using method B. Figure 2. The XRD patterns of the synthesized samples of (a) CuO (b) CuOGA-TSCPy and (c) CuOTSCPy nanoparticles. The morphology and particle size of the synthesized samples were also investigated by scanning electron micros- copy (SEM). SEM images show that the nanoparticles have almost spherical shape (Figure 3). The particles are well sep- arated and uniformly distributed. The average particle sizes 5Acta Chim. Slov. 2024, 71, 1–8 Safavi-Mirmahaleh et al.: One-Step Synthesis of Biocompatible Thiosemicarbazone ... estimated from the SEM images were about 58, 42 and 52 nm for CuO, CuOGA-TSCPy and CuOTSCPy, respectively. The larger size of the functionalized nanoparticles might be due to the capping of nanoparticles by GA and/or TSCPy confirmed by FT−IR analysis. To provide further informa- tion about the elemental composition of the prepared nano- particles, the samples were characterized by energy disper- sive X-Ray (EDX) analysis. As shown in Figure 3, results clearly demonstrated the purity of the synthesized CuO, CuOGA-TSCPy and CuOTSCPy nanoparticles. 3. 3. Antibacterial Activity The antibacterial activities of TSCPy, and also CuO, CuOGA, CuOGA-TSCPy and CuOTSCPy nanoparticles were studied against Gram-positive and Gram-negative bacterial strains including Micrococcus luteus (M. luteus), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), using the zone inhibition method. Penicillin G and tetracycline were used as positive controls. The antibacterial activity of test- ed agents was monitored at a concentration of 2000 μg/mL in DMSO and the experiments were performed in tripli- cate. The trend in antimicrobial activity of four compounds was determined by measuring the inhibition zone around the well and the results are presented in Table 1. The data shows that the synthesized compounds are active against almost all the microorganisms under study. TSCPy free ligand showed the highest antibacterial activity among all of the synthesized compounds. Moreover, antibacterial ac- tivities of glutamic acid functionalized CuO (CuOGA) and CuO nanoparticle were the same against Gram-posi- tive and Gram-negative bacterial strains. The addition of TSCPY to the structure of CuOGA increased the antibac- Figure 3. SEM and EDX images of (a) CuO (b) CuOGA-TSCPy (c) CuOTSCPy nanoparticles. 6 Acta Chim. Slov. 2024, 71, 1–8 Safavi-Mirmahaleh et al.: One-Step Synthesis of Biocompatible Thiosemicarbazone ... terial activity of CuOGA-TSCPy against M. luteus (28 ± 1), S. aureus (21.5 ± 2.5) and E. coli (12 ± 1). Furthermore, CuOTSCPy showed higher antibacterial activity in com- parison with CuO against S. aureus (23 ± 2) and P. aerugi- nosa (19.5 ± 0.5). Results in Table 1 confirm that the func- tionalization of CuO with the bioactive TSCPy moiety can enhance antibacterial activity. Furthermore, the observed MIC and MBC for the respective microorganisms are shown in Table 2. MIC is the lowest concentration of the antimicrobial agent that inhibits microbial growth and MBC was also determined as the lowest bactericidal concentration of the tested com- pound. For CuO, it was found that the MIC and the MBC were 10 μg/mL and 20 μg/mL for M. luteus respectively. The best MIC value for TSCPy (10 μg/mL) was found against S. aureus and E. coli, whereas it was more than 25 µg/ml for CuO. MBC was not reached using CuO against S. aureus, E. coli and P. aeruginosa. The behavior of CuOGA-TSPy and CuOTSPy was similar. The results of our studies showed that the CuOGA-TSPy and CuOTSPy nanoparticles not only showed bacteriostatic effects, but also exhibited bactericidal activity in most cases. Accord- ing to the MBC results, it was found that antimicrobial ac- tivity of nano-sized CuO was enhanced after functionali- zation with the TSCPy moiety (Table 2). The contribution of the size, shape, morphology, and capping agents on bactericidal effect of metal oxide nano- particles and their interaction with microbial membranes have been previously proven.39–42 Previous studies re- vealed that CuO nanoparticles prepared by various proce- dures have shown different physicochemical properties that govern the antibacterial activity (Table 3). The ob- tained values of zone of inhibition demonstrate that CuO, CuOGA-TSCPy and CuOTSCPy nanoparticles prepared by the described method have higher antibacterial activi- ties compared with previously published values.43–47 This can be attributed to their size, shape and high surface to volume ratio, and also the synergetic effect of CuO and thi- osemicarbazone moieties. 4. Conclusions Copper oxide nanoparticles were synthesized by sonochemical method and functionalized with the bioac- tive 2-acetylpyridine thiosemicarbazone molecule through two different methods: A and B routes, yielding the CuO- GA-TSCPy and CuOTSCPy nanoparticles, respectively. In method A, nanoparticles were first functionalized with glutamic acid, followed by a subsequent condensation step between glutamic acid and thiosemicarbazone. In method B, the surface of the CuO nanoparticles is directly modi- fied with 2-acetylpyridine thiosemicarbazone. The bacte- ricidal activity of bare CuO and functionalized CuO nano- materials prepared using A and B routes was tested in vitro using the zone inhibition method against Gram positive Table 1. Antibacterial activity of synthesized compounds and comparison to penicillin and tetracycline. Compounds zone of inhibition (mean ± SD, mm) Gram-positive Gram-negative M. luteus S. aureus E. coli P. aeruginosa TSCPy 53.5 ± 3.5 35.7 ± 4.5 25.5 ± 0.5 27.5 ± 2.5 CuO 21.5 ± 2 13.5 ± 0.5 8.5 ± 0.5 16.5 ± 0.8 CuOGA 21 ± 1.5 8 8 15 ± 1 CuOGA-TSCPy 28 ± 1 21.5 ± 2.5 12 ± 1 12.5 ± 1 CuOTSCPy 16 ± 1.5 23 ± 2 8 19.5 ± 0.5 Penicillin G 50 ± 1 50 ± 1 18 ± 1 – Tetracycline 46 ± 1 41 ± 1 31 ± 1 27 ± 1 Table 2. MIC and MBC values for synthesized compounds against different bacterial strains. Compounds Compound concentration (μg/mL) Gram-positive bacteria Gram-negative bacteria M. luteus S. aureus E. coli P. aeruginosa MIC MBC MIC MBC MIC MBC MIC MBC TSCPy 20 20 10 20 10 20 – – CuO 10 20 – – 20 – – – CuOGA 10 10 – – 10 10 – – CuOGA-TSCPy 2.5 2.5 10 20 20 – – CuOTSCPy 5 2.5 10 10 10 – 10 10 Penicillin G 15.6 15.62 62.5 1000 Tetracycline 31.3 62.5 31.25 15.62 7Acta Chim. Slov. 2024, 71, 1–8 Safavi-Mirmahaleh et al.: One-Step Synthesis of Biocompatible Thiosemicarbazone ... and Gram negative bacterial strains. MIC and MBC values were also determined. The CuOTSCPy nanoparticles pos- sess lower crystallinity and phase purity than CuO- GA-TSCPy nanoparticles. This might be due to the pres- ence of thiosemicarbazone during the formation of CuO phase leading to formation of some impurities such as CuS. Compared with previously published experimental results, CuO, CuOGA-TSCPy and CuOTSCPy nanoparti- cles synthesized by the sonochemical method showed higher antibacterial activities. Moreover, antibacterial ac- tivity of nano-sized CuO was enhanced after functionali- zation with thiosemicarbazone moiety. Acknowledgements The authors are grateful to the University of Guilan for financial support. 5. References 1. A. MacGowan, E. Macnaughton, Medicine 2013, 41, 642–648. DOI:10.1016/j.mpmed.2013.08.002 2. Q. Li, S. Mahendra, D. Y. Lyon, L. Brunet, M. V. Liga, D. 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Chemical Method of synthesis Shape Size Microorganism (growth inhibition Ref. composition (nm) hole, mm) CuO green synthesis spherical Less than 100 E. coli (12.8); S. aureus (12) [43] CuO electrochemical reduction spherical 5–10 nm E. coli (5); S. aureus (12) [44] CuO hydrothermal reaction, nanoflowers, 50–100 S. aureus (nanoflakes: 10 [45] low temperature nanoleaves, nanoflakes, and nanoleaves: 12); S. pneumonia* sonochemical nano rod (nanoflakes: 15 , nanoleaves: 12); S. typhimurium** (nanoflakes: 14, nanoleaves: 16) CuO and biosynthesis spherical Less than 10 S. pneumonia ( CuO:13 [46] Ag/CuO nanocomposite and Ag/CuO: 24 ) polyindole/ reflux condensation plates like, leaf like, Less than 20 E. coli (5); S. aureus (10) [47] Ag–CuO nanocomposite flower buds like CuO sono-chemical spherical 40.4–57.6 S. aureus (13.5); M. luteus This (21.5); P. aeruginosa ( 16.5) study CuOGA- sono-chemical spherical 37.8–52.8 S. aureus (21.5); M. luteus (28); This TSCPy P. aeruginosa ( 12); E. coli (12.5) study CuOTSCPy sono-chemical spherical 49.2–56.3 S. aureus (23); M. luteus (16); This P. aeruginosa (19.5) study * Streptococcus pneumonia ** Salmonella typhimurium 8 Acta Chim. 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Lett. 2017, 196, 78–82. DOI:10.1016/j.matlet.2017.02.111 47. M. Elango, M. Deepa, R. Subramanian, A. M. Musthafa, Polym. Plast. Technol. Eng. 2018, 57, 1440–1451. DOI:10.1080/03602559.2017.1410832 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek S sonokemijsko metodo smo sintetizirali organsko – anorganske hibridne nanomateriale, pri čemer smo uporabili dva različna pristopa za sidranje 2-acetilpiridin tiosemikabazona na površino nanodelcev CuO. Sintetizirane nanodelce smo karakterizirali s kombinacijo fizikalno-kemijskih in spektroskopskih metod. Sinergijsko baktericidno aktivnost CuO in tiosemikabazona smo testirali in vitro proti grampozitivnim in gramnegativnim bakterijam. Določili smo tudi minimal- no inhibitorno koncentracijo in minimalno baktericidno koncentracijo. Rezultati kažejo, da nanodelci CuO in funkcion- alizirani nanodelci CuO, pripravljeni po sonokemijski metodi iz te raziskave, kažejo izboljšano baktericidno aktivnost in bi lahko bili uporabni v pripravi učinkovitejših antibakterijskih materialov za farmacevtsko uporabo. 9Acta Chim. Slov. 2024, 71, 9–19 Taşkın et al.: Chemical Composition and In Vitro Bio ... DOI: 10.17344/acsi.2023.8314 Scientific paper Chemical Composition and In Vitro Biological Activity of Two Thymus L. Varieties Growing in Turkey Turgut Taşkın,1,* Mustafa Öksüz,1,2 Bünyamin Bulkurcuoğlu,2,7 Sebnem Ercelen,2 Erkan Rayaman,3 Mizgin Ermanoğlu,1 Beyza Nur Yılmaz,1 Duygu Taşkın,4 Talip Şahin5 and Ömer Kılıç6 1 Department of Pharmacognosy, Faculty of Pharmacy, Marmara University, Istanbul-Turkey 2 Life Sciences Vice Presidency, TUBITAK MRC, Turkey 3 Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Marmara University, Istanbul-Turkey 4 Department of Analytical Chemistry, Faculty of Pharmacy, University of Health Sciences, Istanbul, Turkey 5 Department of Biology, Institute of Science, Adiyaman University, Adiyaman-Turkey 6 Departmnt of Pharmaceutical Botany, Faculty of Pharmacy, Adıyaman University, Adıyaman-Turkey 7 Institute of Biotechnology, Gebze Technical University, Kocaeli, Turkiye * Corresponding author: E-mail: turguttaskin@marmara.edu.tr Received: 08-05-2023 Abstract Thymus kotschyanus var. kotschyanus (TKK) and T. kotschyanus var. glabrescens (TKG) varieties were used as both spice and medicine by the people in Turkey. It was determined that plants’ methanol extracts had the strongest antioxidant, an- ticholinesterase, antiurease activity and high total phenolic contents. The ethyl acetate and methanol extracts were found to have strong antimicrobial activity. Methanol extracts showed low hemolytic effect against human erythrocytes. It was determined that TKG extract showed higher anti-proliferative effect compared to TKK extract. Both plants extract sig- nificantly decreased reactive oxygen species (ROS) generation in cancer cells. It was determined that amounts of chloro- genic and rosmarinic acid compounds were similar in both plants, but apigenin 7-O-neohesperidoside compound was found in higher amounts in TKK. The findings obtained in this study suggest that methanol and ethyl acetate extracts obtained from these two species can be used as antioxidant, anticholinesterase, antimicrobial and antiurease agents. The findings support the traditional use of these species. Keywords: Thymus species, biological activity, HPLC-DAD 1. Introduction For many years, human beings have used plants not only as a source of food, but also as fuel, clothing raw ma- terial, building material and medicine to prevent and cure diseases. These folk remedies, which have been used by people for centuries, also shed light on the development of modern medicines. In addition, today's research on drug active ingredients with natural origin enriches our current knowledge in this field.1–4 Alzheimer's disease is a neurodegenerative condi- tion that progresses over time and is caused by the death of neurons and synapses in many regions of the central nerv- ous system. It is marked by a decline in cognitive abilities, difficulties with self-care, and a variety of neuropsychiatric and behavioral abnormalities. More than half of all cases of dementia have been linked to Alzheimer's disease. One of the early stages of Alzheimer's disease, oxidative stress plays a pathogenic function in the illness. 5,6 Stone formation in the kidney and urinary system is a significant medical illness that affects 4–20% of the pop- ulation and can result in renal failure if left untreated.7 The nucleation, growth, and aggregation of crystals in saturat- ed urine and epithelial cells of the renal papilla are steps of kidney stone development. Among the other forms (cal- cium oxalate dihydrate, calcium oxalate trihydrate), calci- 10 Acta Chim. Slov. 2024, 71, 9–19 Taşkın et al.: Chemical Composition and In Vitro Bio ... um oxalate monohydrate is the most thermodynamically stable form and has a strong affinity for renal tubular cells. As a result, more research has been done in recent years on the inhibition of calcium oxalate monohydrate crystals.8–12 Antimicrobial resistance against bacteria is increas- ing rapidly around the world, making the treatment of many bacterial diseases, especially infectious diseases, more difficult day. Scientists are working on the discov- ery of new antimicrobial compounds due to antibiotics that have become highly resistant to bacteria. Plants have significant potential for the discovery of these new com- pounds.13 Helicobacter pylori is the first bacterium to be identified as a carcinogen and is a pathogen that colonizes the stomach in more than half of the world's population. One of H. pylori 's most significant virulence factors, the urease enzyme converts urea to ammonia and carbon di- oxide, creating an alkaline environment for the bacteria to thrive in. H. pylori establishes a dwelling space in the tis- sue in this manner without being harmed by stomach acid. Finding substances that specifically affect this bacterium is crucial due to the harmful side effects of the medications used in treatment and the resistance of H. pylori to con- ventional antibiotics.14 It is predicted that more successful results can be obtained in the treatment, especially by us- ing anti-urease effective compounds. Due to the need for new drug active substances in the treatment of infectious diseases, it is emphasized that plants or plant extracts used in natural traditional medicine can be potential antimicro- bial agents that can be used against infections.15 Toxicity of the active ingredients in plant extracts on healthy cells is an important factor in terms of drug effica- cy and their clinical applications. In this respect, hemolyt- ic activity is an important parameter to be investigated. It provides primary information on the interaction between active ingredients and cell membranes at the cellular level. Many plant extracts contain active ingredients that have potential to cause hemolytic activity on human erythro- cytes.16,17 Reactive oxygen species (ROS) production is elevated in tumor cells because of increased metabolic rate, mutations, and relative hypoxia.18 In order to damage proteins, nucleic acids, lipids, membranes, and organelles, several cancer therapeutic strategies attempt to raise cellu- lar ROS levels. This can activate cell death processes such apoptosis at the tumor microenvironment. ROS genera- tion capacity of studied plant extracts on cancer cells was evaluated. ROS formation in HT-29 cells was analyzed with the cell-permeable reagent 2’,7’-dichlorofluorescein (DCFDA). DCFDA is oxidized by ROS and forms a flu- orescent compound. The increased fluorescent emission proves the presence of anticancer activity18. Lamiaceae (Labiatae) family has a very intense distri- bution throughout the world, especially in the Mediterra- nean basin. It is represented by 224 genera and about 5600 species worldwide. The Lamiaceae family is represented by 45 genera and more than 735 taxa in the flora of Turkey. The Thymus genus is a member of Lamiaceae family, with about 300–400 species. Turkey's vegetation includes 37 species and more than 57 taxa, 27 of which are endemic.19 Thymus species were used internally in traditional medi- cine for indigestion and other gastrointestinal disorders; in the treatment of colds, bronchitis, and pertussis; It was used as a mouthwash against laryngitis and tonsillitis. Thy- mus essential oil and thymol were used internally for the treatment of respiratory ailments, and externally they were included in the composition of wound-healing ointments and antiseptic drugs, syrups, and inhalation preparations. Thymus kotschyanus Boiss. Et Hohen. species is a member of the Thymus genus, which is widely distributed in nature. This species was used in folk medicine to treat digestive and respiratory ailments, along with other medicinal uses.20 Therefore, the aim of this study was to evaluate the biological activities of two varieties of Thymus kotshyanus (i), to analyze the phytochemical content of the effective extract qualitatively and quantitatively (iii) and to evaluate the toxicity of the taxa (iii). 2. Materials and Methods 2. 1. Plant Material and Preparation of Extracts Thymus kotschyanus var. glabrescens (TKG) (Her- barium No:27) and Thymus kotschyanus var. kotschyanus (TKK) (Herbarium No:28) species were collected from Adıyaman province and preserved in the Adıyaman Uni- versity Pharmacy Faculty Herbarium. Prof. Dr. Omer Kılıc, a member of the Pharmaceutical Botany Depart- ment of Adıyaman University, made the identification. Plant species were dried in the shade at 25ºC. The crude extracts from the plant's aerial parts were obtained using the sequential maceration process with petroleum ether, ethyl acetate, and methanol. 2. 2. Antioxidant Activity Assays DPPH (2,2-diphenyl-1-picrylhydrazyl) assay: 3.9 mL of DPPH solution (0.1 mM) were added to the 0.1 mL of extracts that had been obtained at various concentration. Before being incubated for 30 minutes at 25 °C, the pro- duced mixtures were stirred for 1 minute. The mixes' ab- sorbance values were measured at 517 nm. Under identical circumstances, the absorbance of the control sample was measured using 0.1 mL of methanol rather than the ex- tract. Using the following formula, the percentage DPPH radical scavenging activity was determined: % DPPH rad- ical scavenging activity= [(A0-A1)/A0]x100 where A0 is the absorbance of the control, and A1 is the absorbance of the extractives/standard. Then % of inhibition was plotted against concentration, and from the graph IC50 was calcu- lated. The information gathered throughout the investiga- tion is provided as IC50 (mg/mL).21 11Acta Chim. Slov. 2024, 71, 9–19 Taşkın et al.: Chemical Composition and In Vitro Bio ... CUPRAC (cupric ion reducing/antioxidant power) test: In this assay, 60 µL of Cu(II).2H2O, 60 µL of neocupro- ine, and 60 µL of 1 M NH4Ac were mixed, then 60 µL of the extracts were added, and finally, 10 µL of ethanol was added to the mixture. The mixes' absorbance was spectro- photometrically evaluated at 450 nm after 60 min against a reference solution that was made by substituting ethanol for the plant extracts. The extracts' CUPRAC values were provided as mg Trolox equivalent (TE)/mg extract. 21 FRAP (iron reducing antioxidant power): Benzie and Strain's (1996) method was used to examine the extracts' ability to reduce ferrous iron. The FRAP reagent was stored at 37 oC for 30 min. It consisted of 25 mL of 300 mM ace- tate buffer (pH 3.6), 2.5 mL of TPTZ solution, and 2.5 mL of 20 mM FeCl3.6H2O. 10 µL of extract were combined with 190 µL of FRAP reagent, and after 4 minutes, the mixture's absorbance at 593 nm was measured. The FRAP values were presented as mM Fe2+/mg extract.22 Plants' phenolic contents: The total amount of phenolic compounds in the extracts was obtained using the Fo- lin-Ciocalteu reagent. 0.1 mL of the diluted plant extracts, 4.5 mL of water, and 0.1 mL of the Folin-Ciocalteu rea- gent were combined, and then 0.3 mL of sodium carbonate solution (2%) was added. One minute of medium-con- tinuous shaking was then performed. After two hours at room temperature, the absorbance at 765 nm was meas- ured using a UV/Vis spectrophotometer. Total phenolic content was calculated as milligrams of Gallic acid equiva- lent (GAE) per gram of plant extract.23 2. 3. Anti-cholinesterase Activity Testing AChE (20 µL) and extracts (20 µL) were added to a phosphate buffer solution (pH 8, 0.1 M, 40 µL). Ten min- utes were spent incubating this mixture at 25°C. Follow- ing incubation, the mixture was combined with substrates DTNB (100 µL) and AcI (20 µL). 5-thio-2-nitrobenzoic acid was measured spectrophotometrically at 412 nm.24 2. 4. Anti-urease Activity Assay Plant extracts (100 µL) and an enzyme solution (500 µL) were combined and maintained in an incubator at 37 °C for 30 minutes. This combination was then given 1.100 µL of urea, and it was allowed to sit in an incubator at 37 °C for 30 minutes. After taking it out of the incu- bator, the mixture was then combined with the reagents R1 (1% phenol, 0.005% sodium nitroprusside) and R2 (0.5% NaOH, 0.1% sodium hypochlorite), and the result- ant mixture was then held at 37 °C in an incubator for 2 hours. The mixture's absorbance was determined (635 nm) in comparison to a reference solution that had been made by substituting a buffer solution for the urease en- zyme solution.21 2. 5. Antimicrobial Activity Antimicrobial activity analysis of plant extracts de- termined primarily by the agar well diffusion method. Minimum inhibitory concentration (MIC) determined for extracts showing antimicrobial activity in agar well diffusion method. Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 11228, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Pro- teus mirabilis ATCC 14153, Klebsiella pneumoniae ATCC 4352, Candida albicans ATCC 10231 were used in the an- timicrobial tests. Agar well diffusion method: Bacteria were inoculated on tryptic soy agar and C. albicans on Sabouraud dextrose agar, incubated at 37 °C for 24 hours. Microorganism suspensions were prepared from colonies in 0.85% NaCl physiological saline solution (PSS). Bacterial suspensions were adjusted to 108 cfu/ml and C. albicans suspensions to a concentration of 106 cfu/mL according to Mc Farland 0.5 standard turbidity. The microorganism suspensions were spread over the sur- face of the Mueller Hinton agar for bacteria and SDA for C. albicans by sterile swabs under aseptic conditions and then 5 mm diameter wells were made surface of the medium with a sterile punch. The wells were filled with 50 µL (50 mg/ mL) of the extracts dissolved in appropriate solvents. In ad- dition, meropenem (10 µg/well) for bacteria, amphotericin B (100 µg/well) as a positive control for the yeast, solvents (DMSO) and PSS were used as negative control. Inoculated petri dishes were incubated at 37°C for 18–24 hours for bac- teria, for 24–48 hours at 35 °C for yeast, and at the end of in- cubation time, inhibition zones were measured in mm. The trials were performed with three replicates and averaged.25 Detection of minimal inhibitory concentration for bac- teria: Detection of MIC for bacteria were performed in ac- cordance with the standards of the Clinical and Laboratory Standards Institute (CLSI). Cation Adjusted Mueller Hinton Broth (CAMHB) was used as medium. Bacteria suspension was prepared from the colonies in the 24-hour bacterial cul- ture according to McFarland 0.5 turbidity and the final in- oculum concentration shall be diluted to 5x105 cfu/ml. The sterile U-based microdilution plates were placed 100 µL of the CAMHB. The extracts were placed 100 µl in the first wells and serial dilutions were made. Then 5 µL of bacterial sus- pension was added to the wells containing the extract and the plates incubated at 37 °C for 24 hours. At the end of the incu- bation, the lowest extracting concentrations with no growth was determined as minimal inhibitory concentration (MIC). Escherichia coli ATCC 25922 was used as a quality control microorganism. CAMHB, DMSO and PSS were used as neg- ative control. Meropenem was used as positive control.26 2. 6. Examination of Phenolic Compounds The approach we have previously described was used to assess the content of the bioactive extracts using 12 Acta Chim. Slov. 2024, 71, 9–19 Taşkın et al.: Chemical Composition and In Vitro Bio ... sulphophenyl)-5 [(phenylamino)carbonyl]-2H-tetrazoli- um hydroxide) were conducted by the manufacturer’s (Ro- che Diagnostics Corporation, Indianapolis, IN) protocol. A volume of 20% of the medium XTT mixture was added to cells and allowed to incubate for 4 hours at 37°C, with 5% CO2. Then absorbance values were measured at 450 nm and 690 nm. Percentage of cell viability was calculated from the absorbance readings and plotted.24 2. 9. DCFDA Assay Cells Oxidative stress that was produced by plant extracts was quantified in HT-29 cells by DCFDA assay. The Mc- Coy's 5A medium, 10% fetal bovine serum (FBS), and 1% antibiotics were used to maintain the HT-29 human colorectal cancer cell line at 37 °C in a 5% CO2 atmosphere with saturated humidity. Cells (1 × 105 cells per well) were seeded into a 96-well plate and grown for 24 hours for the DCFDA test. Plates were then cleaned with PBS before be- ing treated for 24 hours with TKK and TKG extracts at various doses (7.5, 75.0, and 750 g/mL) in medium con- taining 10% FBS. All samples were examined in triplicate and control cells were treated with PBS. Following a PBS wash, the cells were incubated for an additional 45 min in 100 μL of PBS with a final DCFDA concentration of 10 μM. In a fluorescence microplate reader (Cytation 5, Bi- otek, CA, USA), fluorescence emission was measured us- ing an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Results are presented as relative fluorescence intensity.28 2. 10. Statistical Analysis The findings were presented as the mean and stand- ard deviations (SD) of three parallel measurements. Following ANOVA procedures, a one-way analysis of variance was conducted. A Tukey multiple comparison test was used to identify significant differences between means, with a p value of 0.05 being regarded as statistically significant. 3. Results 3. 1. Antioxidant Activity It was determined that TKK (IC50: 0.0630 mg/mL) and TKG (IC50: 0.0436 mg/mL) methanol extracts showed the highest DPPH radical scavenging activity compared to other extracts. These results demonstrated that both plants have very similar potential for radical scavenging activity. Comparing the ascorbic acid (IC50: 0.004 mg/mL) employed as a reference to the radical scavenging potentials of the plant extracts, it was found that all extracts had minimal radical scavenging capacity. The CUPRAC test results showed that TKK methanol (6.658 mM TE/mg extract) and ethyl acetate (4.0827 mM high-performance liquid chromatography with diode-ar- ray detection (HPLC-DAD) (Agilent 1260 Infinity). The chemicals were separated using a C18 reverse phase No- va-Pak analytical column (3.9 mm × 150 mm inner diam- eter, 5 μm). The column temperature was kept at 30 °C. The mobile phases were water (0.05% acetic acid) and (B) acetonitrile. The gradient elution step was used: the mobile phase B was increased from 0% to 20% in 5 minutes, 40% in 10.00 min, 50% in 20.00 min, 60% 30.00 min, 90% B 40.00 min and 45.00 min, 20%. The injection volume was settled as 20 μL.21 2. 7. Hemolytic Activity Preparation of erythrocytes suspension: Blood samples were taken from the arm veins of healthy volun- teer individuals and transferred. Transferred into EDTA containing tubes. In order to separate plasma and erythro- cytes samples were centrifuged at 3000 rpm for 5 minutes (4 °C). Plasma was discarded and pellets, which contain erythrocytes, were washed 2 times with physiological sa- line (pH 7.2 ± 0.2) and centrifuged at 3000 rpm and at 4 °C for 5 min. 27 Hemolytic activity: In vitro hemolytic activity of metha- nol extract of TKK and TKG was performed with spectro- photometer method.27 The erythrocytes were diluted with 4 mL PBS and incubated on a shaker at 37 °C for 1.5 hours with different concentrations of plant extract at 125, 250, 500 and 1000 μg/mL in phosphate buffer saline at equal volume of erythrocytes. PBS and Triton X-100 were used as controls. The sample was centrifuged at 3000 rpm for 5 min at 4 °C. The amount of free hemoglobin in the super- natant was measured with UV spectrophotometer at 540 nm each experiment was performed in triplicates. The lev- el of hemolysis percentage by the extracts was calculated according to the following formula: At is the absorbance of plant extract Ac is the absorbance of Triton X-100 2. 8. Cytotoxic Activity For the cytotoxic activity of TKK and TKG extracts, HT-29 cells (1 × 104) per well were seeded in 96-well plates in fresh complete medium at 37 °C, 5% CO2 for 24 hours before the test. After, cells were rinsed with phos- phate buffered saline (PBS) and incubated for 4 h with a cell culture medium containing increasing concentrations of TKK and TKG extracts (7.5, 75.0, and 750 µg/mL). Control cells were treated with PBS and all samples were studied in triplicate. After incubation of cells with plant extracts, cells were washed with PBS. Cytotoxicity analyzes was performed with XTT (2,3-bis(2-methoxy-4-nitro-5- 13Acta Chim. Slov. 2024, 71, 9–19 Taşkın et al.: Chemical Composition and In Vitro Bio ... TE/mg extract) extracts have a higher Cu(II) to Cu(I) re- duction potential than petroleum ether (0.09443 mM TE/mg extract) extract. The same experiment showed that the CUPRAC values of the TKG methanol (7.097 mM TE/mg extract) and ethyl acetate (5.314 mM TE/mg extract) extracts were higher than petroleum ether ex- tract. These results demonstrated that TKG and TK methanol extracts have higher Cu(II) to Cu(I) reduction potential than the ascorbic acid compound. It was dis- covered that TKG (17.704 mM TE/mg extract) and TKK (16.907 mM TE/mg extract) methanol extracts had more iron reducing antioxidant power than other extracts. Ad- ditionally, it was discovered in this study that TKG meth- anol extract (FRAP: 17.704) had higher FRAP values than BHA compound (FRAP: 16.91), while TKK (FRAP: 16.907) methanol extract displayed FRAP values to BHA compound (Table 1). 3. 2. Determination of Total Phenolic Content (TPC) It is well known that phenolic contents and biologi- cal activity often have a linear relationship.29 Based on this knowledge, the total phenolic contents of several plant ex- tracts were spectrophotometrically measured in this inves- tigation. The results, which corroborated the literature, demonstrated that TKG (105.8 mg GAE/g extract) and TKK (81.6 mg GAE/g extract) methanol extracts had higher phenolic component concentrations than other ex- tracts (Table 2). Table 2. The amount of phenolic compounds contained in the ex- tracts from plants. TPC (mg GAE/g extract) TKK TKG Petroleum ether 25.5±0.2 36.3±0.3 Ethyl acetate 48.1±0.3 60.5±0.2 Methanol 81.6±0.5 105.8±0.7 TKK: Thymus kotschyanus var. kotschyanus; TKG: T. kotschyanus var. glabrescens; TPC, total phenolic contents; GAE, gallic acid equivalent; Values are mean of triplicate determination (n = 3) ± standard deviation Table 1. Antioxidant activity of extracts from plants. DPPH (IC50, mg/mL) CUPRAC (mM TE/mg extract) FRAP (mM TE/mg extract TKK TKG TKK TKG TKK TKG Petroleum ether 0.431±0.033* 1.98±1.40* 0.094±0.108* 2.21±0.20* 7.18±0.29* 6.27±0.55* Ethyl acetate 0.069±0.003* 0.066±0.003* 4.08±0.36* 5.31±0.16* NA 1.49±0.30* Methanol 0.063±0.001* 0.044±0.003 6.66±0.25* 7.10±0.33* 16.91±1.30 17.7±3.5* Ascorbic acid 0.004±0.007 5.92±0.51 BHA 16.91±0.02 TKK: Thymus kotschyanus var. kotschyanus; TKG: T. kotschyanus var. glabrescens, ascorbic acid positive control for DPPH and CUPRAC assays; BHA, butylatedhydroxyanisole, positive control for FRAP assay; DPPH, 2,2-diphenyl-1-picrylhydrazyl; CUPRAC, cupricion reducing/antioxidant power; FRAP, ferric reducing antioxidant power; Values are mean of triplicate determination (n = 3) ± standard deviation; *P < 0.05 compared with the positive control Table 3. Antimicrobial activity of plant extracts on various microorganisms by agar well diffusion method (in mm). Staphylococcus Staphylococcus Escherichia Pseudomonas Proteus Klebsiella Candida aureus epidermidis coli aeruginosa mirabilis pneumoniae albicans TKK Petroleum ether 18.10±0.27 14.30±0.17 0 0 0 0 0 TKK Ethyl acetate 8.82±0.08 13.61±0.13 0 0 0 0 0 TKK Methanol 9.37 ±0.48 17.88±0.04 0 0 0 0 0 TKG Petroleum ether 16.34±0.16 24.71±0.13 0 0 0 0 0 TKG Ethyl acetate 24.10±0.84 41.46±0.23 0 0 0 0 0 TKG Methanol 18.09±0.14 21.10±0.15 0 0 0 0 0 Meropenem 33.90±0.02 47.09±0.21 29.40±0.22 32.81±0.18 31.61±0.12 30.98±0.21 – Amphotericin B – – – – – – 16.42±0.13 TKK: Thymus kotschyanus var. kotschyanus; TKG: T. kotschyanus var. glabrescens, –: not done. 3. 3. Antimicrobial Activity Antimicrobial activities of 6 different extracts ob- tained from TKK and TKG varieties were investigated. All the extracts used in our study showed antimicrobial activity only against S. aureus and S. epidermidis. It has been determined that they were more effective against S. epidermidis. It is particularly interesting for TKG-EA (ethyl acetate) and TKG-M (methanol) against S. aureus and S. epidermidis. When we look at our results, it was concluded that our extracts were effective on gram pos- itive strains and among these positive strains, S. epider- midis was more effective than S. aureus. While the highest 14 Acta Chim. Slov. 2024, 71, 9–19 Taşkın et al.: Chemical Composition and In Vitro Bio ... antimicrobial activity was observed against S. epidermid- is strain of ethyl acetate extract of TKG species, the low- est antimicrobial activity was determined as the activity of ethyl acetate extract of TKK species against S. aureus strain. The diameter of the inhibition zones and the min- imum inhibitory concentration (MIC) values formed by the extracts against the tested strains are shown in Tables 3 and 4, respectively. Table 4. MIC values of plant extracts (mg/mL). Staphylo- Staphylo- Escherichia coccus coccus coli aureus epidermidis TKK Petroleum Ether 6.25 6.25 – TKK Ethyl Acetate 1.56 3.12 – TKK Methanol 3.125 3.12 – TKG Petroleum Ether 1.25 1.25 – TKG Ethyl Acetate 0.78 0.78 – TKG Methanol 0.78 0.78 – Meropenem – – 0.06 µg/mL TKK: Thymus kotschyanus var. kotschyanus; TKG: T. kotschyanus var. glabrescens, –: not done. 3. 4. Enzyme Inhibition Activity of Extracts The findings of comparing the potentials of plant extracts (200 µg/mL) to inhibit the enzymes acetylcho- linesterase and urease are shown in Table 5. The results showed that the acetylcholinesterase enzyme inhibition potentials of various extracts of both plants were ex- tremely like one another. In comparison to the other ex- tracts and the galantamine molecule, the TKG methanol (98.33%) extract was found to have a larger potential for acetylcholinesterase activity. Plant extracts and thiourea compound at a concentration of 12.5 µg/mL were exam- ined for their anti-urease properties. The urease enzyme inhibitory potential of the TKK (34.404%) and TKG (17.726%) methanol extracts was found to be greater than that of other extracts. In this investigation, it was shown that all extracts had lower capacity to inhibit en- zymes than the thiourea (70.05%) molecule employed as a reference (Table 5). 3. 5. Hemolytic Activity There was no report on the hemolytic activity of these Thymus species in the literature. In this study for the first-time hemolytic activity of methanol extracts of Thymus kotschyanus var. kotschyanus and T. kotschyanus Table 5. Enzyme inhibition potential of different extracts from plants. Inhibition of the acetylcholinesterase Inhibition of the urease enzyme (%) enzyme (%) TKK TKG TKK TKG Petroleum ether 95.12 ± 0.60* 90.91 ± 1.87* 12.346 ± 1.623* 6.995 ± 1.138* Ethyl acetate 93.20 ± 0.33 91.41 ± 0.57* 19.846 ± 0.218* 9.834± 0.197* Methanol 95.18± 0.95* 98.33 ±0.66* 34.404 ± 3.869* 17.726± 8.356* Galantamine 97.14 ±0.14 Thiourea 70.05 ±0.01 Figure 1. Hemolytic activity of methanol extracts from plants. A Thymus kotschyanus var. kotschyanus, B T. kotschyanus var. gla- brescens. 15Acta Chim. Slov. 2024, 71, 9–19 Taşkın et al.: Chemical Composition and In Vitro Bio ... var. glabrescens species in the selected dose range against human erythrocytes were examined and compared to Tri- ton X-100 and PBS. The hemolytic activity results of these extracts are shown in Figures 1. The methanol extract of both plant extracts presented low hemolytic activity. How- ever, the hemolytic activity of both extracts increased in a dose-dependent manner as expected. As no significant difference was observed, TKK methanol extract presented rather higher hemolytic activity than TKG at 300–1000 μg/mL. 3. 6. Cytotoxicity Activity The cytotoxic activity of methanol extracts of Thy- mus kotschyanus var. kotschyanus and T. kotschyanus var. glabrescens species were tested on HT-29 colorectal cancer cells. Methanol extract obtained from TKK did not show cytotoxic activity against HT-29 cells. T. kotschyanus var. glabrescens showed relative cytotoxic activity at concentra- tions of 750 µg/ml (Figure 2). Figure 2. Cytotoxicity analysis of methanol extracts of Thymus kotschyanus var. kotschyanus (TK) and T. kotschyanus var. gla- brescens (TKG) in HT-29 cells. The cytotoxicity effect of plant ex- tracts was analysed with in vitro XTT assay. Cells were incubated with 7.5, 75, and 750 µg/mL plant extract for 4 h, and the medium was replaced with fresh complete medium. Cells were then cultured for an additional 24 h before the XTT assay. Control cells were treat- ed with PBS. 3. 7. DCFDA Assay Both plants extract significantly decreased ROS gen- eration in cancer cells. Such dramatic changes in ROS gen- eration in cancer cells are expected to affect cell prolifera- tion profile of cancer cells.30 However, we did not observe a significant difference in terms of cell proliferation with control groups (Figure 3). 3. 8. Phenolic Compounds Analysis The composition of these extracts was examined both qualitatively and quantitatively since methanol ex- tracts from plants show considerable biological activity when compared to other extracts. Compounds of apigenin 7-O-neohesperidoside, chlorogenic acid and rosmarinic acid in both plants were examined and it was shown that apigenin 7-O-neohesperidoside molecule (TKK: 7.250 µg analyte/mg extract; TKG: 6.103 µg analyte/mg extract was dominant in both plant extracts. In addition, chlorogenic acid and rosmarinic acid compounds were found similar amounts in both plant extracts (Figure 4, Table 6). Table 6. Quantitative determination of compounds in methanol ex- tracts of plants. µg analyte/mg extract TKK TKG Apigenin 7-O-neohesperidoside 7.25±0.09 6.10±0.04 Chlorogenic acid 3.60±0.01 3.73±0.01 Rosmarinic acid 2.93±0.37 2.26±0.11 TKK: Thymus kotschyanus var. kotschyanus; TKG: T. kotschyanus var. glabrescens 4. Discussion Egyptians laid the foundation for the widespread usage of Thymus species for medicinal purposes today. Because of its antibacterial and antiviral qualities, they employed it as an antiseptic. Today, it is employed as a mouthwash or a tea to treat inflammation of the upper respiratory tract and cough. Thymus species' flowering stems are used as diuretics, carminatives, and nausea rem- edies. It is applied to eliminate gastrointestinal parasites. It is utilized in several gynecological disorders, including breast cancer, breast swelling, breast inflammation, sick and inflamed uterine treatments, and as a miscarriage Figure 3. ROS generation analysis of methanol extracts of Thymus kotschyanus var. kotschyanus (TK) and T. kotschyanus var. gla- brescens (TKG) in HT-29 cells. The ROS effect of plant extracts was analysed with in vitro DCDFA assay. Cells were incubated with 7.5, 75 and 750 µg/mL plant extract for 45 min and medium was re- placed with fresh complete medium. * p < 0.05 16 Acta Chim. Slov. 2024, 71, 9–19 Taşkın et al.: Chemical Composition and In Vitro Bio ... preventative precaution. It is beneficial in controlling ex- cessive menstrual flow.31 The phenolic components of di- chloromethane, methanol and water extracts obtained by using Thymus argaeus species were determined. The two substances that were most often identified in methanol extract were chlorogenic acid and rosmarinic acid. In wa- ter extract, chlorogenic acid was the most prevalent sub- stance. Sinapic acid and gallic acid were only found in the water extract. Caffeic acid was found to be the most prev- alent substance in dichloromethane extract.32 Thymus mi- gricus species phenolic components were examined using LC-MS/MS. Quinic acid, chlorogenic acid, cinaroside, luteolin, and p-coumaric acid are the major substances included in T. migricus extract.33 LC MS/MS chromatog- raphy was used to evaluate the phenolic components of the water decoction and water infusion extracts made from aerial portions of the Thymus sipyleus species that were collected from the Kaz Mountains within the bound- aries of Balkesir province. The main phenolic compounds in the decoction extract are fumaric acid, rosmarinic acid, and quercitin, whereas the main phenolic compounds in the infusion extract are kaempferol, rosmarinic acid, and fumaric acid. The largest flavonoid derivative substance, kaempferol, the major coumaric acid derivative, ros- marinic acid, fumaric acid, have all been identified in the infusion extract.34 Figure 4. The HPLC-DAD chromatogram of methanol extracts from plants. A: Thymus kotschyanus var. kotschyanus; B: T. kotschyanus var. gla- brescens 17Acta Chim. Slov. 2024, 71, 9–19 Taşkın et al.: Chemical Composition and In Vitro Bio ... The antioxidant activity of extracts from the Thymus argaeus species in dichloromethane, methanol, and water was evaluated using the DPPH and ABTS radical scav- enging assays, as well as the FRAP, phosphomolybdenum and CUPRAC procedures. The water extract of T. argaeus obtained by decoction was found to have the most effec- tive ABTS and DPPH radical scavenging activity. The weakest activity belongs to dichloromethane extract. The methanol extract had the highest CUPRAC, while the wa- ter extract showed the highest activity in the FRAP test. The dichloromethane extract was found to have the low- est activity in both experiments. While the water extract showed the strongest antioxidant activity in the phospho- molybdenum test, the dichloromethane extract showed the least activity.32 The antioxidant activity of 1,2,4 and 8% infusions obtained from aerial parts of Thymus haussknechtii species was tested by DPPH radical scavenging test and H2O2 scavenging tests. In the DPPH radical scavenging test, 8% infusion was the extract with the highest activity with 77.41% inhibition value. In the H2O2 scavenging test, 1% T. haussknechtii extract showed the highest H2O2 scaveng- ing activity.35 Only a few studies have been found in the literature on the two Thymus L. varieties used in this study. In one of them, the DPPH, FRAP activity of the essential oil of the plant was examined and it was determined that it showed significant antioxidant activity. In addition, the es- sential oil of the plant was found to be effective against strains B. cereus, L. monocytogenes, E. coli and S. typhimu- rium.36 In another study, essential oil of Thymus kotschyanus var. kotschyanus and T. kotschyanus var. gla- brescens were analyzed by HS-SPME/GC-MS. In this study thymol, carvacrol, p-cymene and γ-terpinene were detect- ed the main compounds of T. kotschyanus var. kotschyanus; thymol, carvacrol and p-cymene were detected the major constituents of T. kotschyanus var. glabrescens.37 DPPH radical and iron reducing activity of both essential oil and methanol extract of T. kotschyanus species were investigat- ed. It was determined that the DPPH radical scavenging and iron reducing activities of the essential oil were close to the DPPH and FRAP antioxidant activities of the meth- anol extract.38 In a recent study, it was emphasized that the water extract prepared from the leaf of the T. kotschyanus species showed a significant antibacterial effect on gram-negative bacteria.39 For the first time, the biological activities of several extracts from two Thymus L. varieties were thoroughly studied in this work. Analyses of the bioactive extracts' composition and toxicity were conducted. In parallel with the literature, methanol extracts of both plants exhibited significant biological activity, and compounds like phenol- ic compounds previously analyzed from Thymus species were also analyzed in these species. Hemolysis, also known as membrane lipid bilayer lysis, is typically responsible for the destruction of red blood cells and is influenced by the concentration and po- tency of the extract. Furthermore, the chemical contents of the extracts have a direct impact on the hemolytic ac- tivity.40,41 Many of the natural compounds found in me- dicinal plants can alter biological membranes. Several authors have reported that flavonoids and the widely dis- tributed polyphenol subgroup have a positive impact on the stability of the erythrocyte membrane.42 Furthermore, De Freitas et al. report that the flavonoids may be a source of membrane stabilization by intensifying the van der Waals contacts inside the lipid bilayer. 43,44 In this study, using the hemolytic assay, the cytotoxic activity of the ex- tracts was assessed to ascertain the toxicity profile of Thy- mus L. varieties. It seems that the phenolic compounds in the plants' methanol extract affect both the erythrocyte membrane's structural stability and its capacity to stifle free radicals. Nonetheless, the administration of Thymus L. varieties extracts results in heightened erythrocyte re- sistance against hemolysis triggered by free radicals. Our findings demonstrate that these plants in phenolic com- pounds have a cell membrane-stabilizing and protective function. 5. Conclusion In this study, the biological activities of two Thymus kotschyanus varieties and the chemical content of the ac- tive extract were analyzed. It was determined that meth- anol extract of TKG showed more effective antioxidant and anticholinesterase activity than TKK extracts. Ac- cording to the anti-urease activity results, the highest urease enzyme inhibition was detected in TKK. It was determined that TKG methanol and ethyl acetate extracts exhibited strong antimicrobial activity against Staphylo- coccus aureus and Staphylococcus epidermidis strains. It was determined that both plant methanol extracts showed low hemolytic effect against human erythrocytes. 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DOI:10.1016/j.ijbiomac.2006.12.003 41. S. Rafique, M. A. Murtaza, I. Hafiz, K. Ameer, M. N Qayyum, S. Yaqub, I. A. M. Ahmed, Food Sci Nutr 2023, 11, 6303– 6311. DOI:10.1002/fsn3.3569 42. M. Sharma, K. Kishore, S.K. Gupta, S. Joshi, D. S. Arya, Mol Cell Biochem 2001, 225, 75–83. DOI:10.1023/A:1012220908636 43. M.V. de Freitas, R. M. Netto, J.C. da Costa Huss, T.M. de Souza, J. O. Costa, C. B. Firmino, Toxicol In Vitro 2008, 22, 219–224. DOI:10.1016/j.tiv.2007.07.010 19Acta Chim. Slov. 2024, 71, 9–19 Taşkın et al.: Chemical Composition and In Vitro Bio ... 44. M. Ramchoun, K. Sellam, H. Harnafi, C. Alem, M. Benlyas, F. Khallouki, S. Amrani, Asian Pac J Trop Biomed 2015, 5(2), 93–100. DOI:10.1016/S2221-1691(15)30151-9 Povzetek Ljudje v Turčiji so uporabljali sorti Thymus kotschyanus var. kotschyanus (TKK) in T. kotschyanus var. glabrescens (TKG) kot začimbo in zdravilo. Ugotovljeno je bilo, da imajo metanolni izvlečki rastlin najmočnejšo antioksidativno, antiho- linesterazno in antiureazno aktivnost ter visoko vsebnost skupnih fenolov. Ugotovljeno je bilo tudi, da imajo etil acetatni in metanolni izvlečki močno protimikrobno aktivnost. Metanolni izvlečki so izkazovali majhen hemolitični učinek na človeške eritrocite. Ugotovljeno je bilo, da ima ekstrakt TKG večji antiproliferativni učinek v primerjavi z ekstraktom TKK. Oba rastlinska izvlečka sta pomembno zmanjšala nastajanje reaktivnih kisikovih zvrsti (ROS) v rakavih celicah. Ugotovljeno je bilo, da so bile količine klorogenske in rožmarinske kisline v obeh rastlinah podobne, spojina apigenin 7-O-neohesperidosid pa je bila v večjih količinah ugotovljena v TKK. Ugotovitve, pridobljene v tej študiji, kažejo, da se metanolni in etil acetatni izvlečki, pridobljeni iz teh dveh vrst, lahko uporabljajo kot antioksidanti, zaviralci holinester- aze, protimikrobna sredstva in zaviralci ureaze. Ugotovitve podpirajo tradicionalno uporabo teh vrst. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 20 Acta Chim. Slov. 2024, 71, 20–25 Wang et al.: Synthesis, Crystal Structures and Antibacterial Activity of ... DOI: 10.17344/acsi.2023.8397 Scientific paper Synthesis, Crystal Structures and Antibacterial Activity of Nickel(II) and Copper(II) Complexes Derived from (E)-2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methylene)- N-phenylhydrazinecarbothioamide Jing Wang1, Haiyang Fei1, Min Zhou1, Chengcai Zhang1, Juan Sun*,2, Yang Zhou*,3 and Meng Yang*,1 1 School of Pharmaceutical Engineering, Jiangsu Food & Pharmaceutical Science College, Huai’an 223005, China 2 School of Biological & Chemical Engineering, Zhejiang University of Science & Technology, Hangzhou 310023, PR China 3 Cixi Institute of Biomedical Engineering, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo 315300, China * Corresponding author: E-mail: J. Sun (sunjuan18@zust.edu.cn); Y. Zhou (zhouyang876@nimte.ac.cn); M.Yang (yangmenghuaian@163.com) Received: 08-17-2023 Abstract The biosynthesis of fatty acids is essential for the survival of bacteria, and β-ketoacyl-acyl carrier protein synthase III (FabH) is a promising target for antibacterial drug development. Nickel(II) complex [NiL2] (1) and copper(II) complex [CuL2] (2), where L is (E)-2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methylene)-N-phenylhydrazinecarbothioamide, were synthesized and characterized by elemental analysis, IR and 1H NMR spectroscopy and HRMS. Structures of the complexes were further studied by single crystal X-ray determination, which reveals that the nickel and copper atoms in the complexes are in tetrahedral geometry. These compounds were evaluated for their antibacterial and E. coli FabH inhibitory activities. Keywords: 1,4-Benzodioxane, nickel complex, copper complex, antibacterial activity, FabH inhibitory 1. Introduction Coordination complexes have been extensively stud- ied as potential inhibitors of FabH, a key enzyme involved in fatty acid biosynthesis.1 FabH inhibitors have attracted significant attention as potential therapeutic agents for the treatment of bacterial infections.2 Coordination complex- es have been shown to inhibit FabH activity through a va- riety of mechanisms, including binding to the active site of the enzyme and disrupting its catalytic activity.3 Some of the most promising complexes are those containing ruthe- nium, rhodium, cobalt, and platinum. For example, the Copper complexes have been shown to bind to the active site of FabH and inhibit its enzymatic activity.4 Similarly, the cobalt and chromium complexes have also been shown to inhibit FabH activity.5 (E)-2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)meth- ylene)-N-phenylhydrazinecarbothioamide (L) is a poten- tial antibacterial agent that belongs to the class of hydrazi- necarbothioamide derivatives. It contains a dioxin ring system, a phenyl group, and a hydrazinecarbothioamide functional group, which are known to possess antibacteri- al activity.6 The inhibitory activity of L against bacterial growth is believed to be due to its ability to target the FabH enzyme, which is essential for the biosynthesis of fatty ac- ids in bacteria.7 The compound binds to the active site of FabH and inhibits its activity, thereby preventing the pro- duction of fatty acids and disrupting bacterial growth. Overall, L has the potential to serve as a promising anti- bacterial skeleton for the development of new therapeutics targeting FabH. The synthesis of new metal complexes with antibac- terial properties is a prominent subject in the fields of co- ordination chemistry and bioinorganic chemistry.8 Addi- tionally, Ni and Cu metals are frequently employed in 21Acta Chim. Slov. 2024, 71, 20–25 Wang et al.: Synthesis, Crystal Structures and Antibacterial Activity of ... coordination chemistry due to their well-documented bio- logical activities, especially their antimicrobial properties.9 Moreover, previous studies have demonstrated the effec- tive interaction of metals with the active site of FabH, lead- ing to interference with its enzymatic activity. Conse- quently, this study focuses on elucidating the crystal structures of nickel(II) and copper(II) complexes with the ligand L, as well as examining their antibacterial and FabH inhibitory activities. These findings provide valuable in- sight into the potential clinical application of the hydrazi- necarbothioamide complex as an antibacterial agent. 2. Experimental 2. 1. Materials and Methods 1,4-benzodioxane-6-formaldehyde, nickel acetate, copper acetate, hydrazine hydrate and phenyl isothiocy- anate were obtained from Aladdin. All other chemicals were commercial obtained from Anhui Senrise Technolo- gies Co., Ltd. HNMR spectra were measured on a Bruker AV-400 spectrometer at 25 °C and referenced to Me4Si. LC/MS spectra were recorded on a Waters G2-XSQTot Mass spectrometer. Chemical shifts were reported in ppm (δ) using the residual solvent line as internal standard. An- alytical thin-layer chromatography (TLC) was performed on the glassbacked silica gel sheets (silica gel 60 Å GF254). Elemental analyses for C, H and N were performed on a Perkin-Elmer 240C elemental analyzer. FT-IR spectra were obtained on BrukerVertex 70 with samples prepared as KBr pellets. Single crystal X-ray diffraction was carried out on a Bruker SMART 1000 CCD diffractometer. 2. 2 Synthesis of (E)-2-((2,3-dihydrobenzo[b] [1,4]dioxin-6-yl)methylene)-N- phenylhydrazinecarbothioamide (L) 1,4-benzodioxane-6-formaldehyde (0.16 g, 1.0 mmol) and hydrazine hydrate (0.13 g, 4.0 mmol) were mixed in 30 mL ethanol. The mixture was reflux for 4 h and the solvent was evaporated to give solid product b, which was re-crystallized from ethanol, yield 95%. Then the intermediate hydrazine compound b (0.14 g, 0.8 mmol) and phenyl isothiocyanate (0.14 g, 1 mmol) was dissolved in chloroform (30 mL) and refluxed for 4 h. The solvent was evaporated under reduced pressure, and the ligand L was obtained by recrystallized from ethanol. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 10.07 (s, 1H), 8.03 (s, 1H), 7.60–7.52 (m, 3H), 7.36 (m, 2H), 7.28 (m, 1H), 7.20 (m, 1H), 6.89 (d, J = 8.3 Hz, 1H), 4.27 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 176.20, 145.79, 144.16, 143.11, 139.61, 128.46, 127.88, 126.44, 125.72, 122.31, 117.62, 115.96, 64.79, 64.44. Anal. calc. for C16H15N3O2S: C, 61.32; H, 4.82; N, 13.41; found: C, 61.17; H, 4.95; N, 13.56%. HR-MS m/z: 314.0954 (M+H)+, calculated molec- ular weight of C16H16N3O2S+: 314.0885 for (M+H)+. 2. 3. Synthesis of [NiL2] (1) L (0.10 mmol) and nickel acetate (0.10 mmol) mixed in methanol (10 mL) were stirred at room temper- ature for 30 min to give a clear earthy yellow solution. Needle-shaped dark purple crystals suitable for X-ray diffraction were grown from the solution upon slowly evaporation within 2 days. The crystals were isolated by filtration. 1H NMR (400 MHz, DMSO-d6) δ 9.71 (s, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.74 – 7.47 (m, 4H), 7.30 (m, 2H), 7.02 (m, 1H), 6.87 (d, J = 8.5 Hz, 1H), 4.50–4.16 (m, 4H). Anal. calc. for C32H28N6NiO4S2: C, 56.24; H, 4.13; N, 12.30; found: C, 56.12; H, 4.27; N, 12.21%. HR-MS m/z: 683.1014(M+H)+, calculated molecular weight of C32H29N6NiO4S2+: 683.0967 for (M+H)+. 2. 3. Synthesis of [CuL2] (2) L (0.10 mmol) and copper acetate (0.10 mmol) mixed in methanol (10 mL) were stirred at room temperature for 30 min to give a clear green solution. Half of the solvent was slowly evaporated at room temperature for 6 days to give sin- gle crystals. Anal. calc. for C32H28N6CuO4S2: C, 55.84; H, 4.10; N, 12.21; found: C, 565.76; H, 4.25; N, 12.16%. IR data (cm–1): 3474, 3146, 1541, 1509, 1421, 1294, 1063, 963, 811, 679, 626, 553. HR-MS m/z: 688.0915(M+H)+, calculated mo- lecular weight of C32H29N6CuO4S2+: 688.0909 for (M+H)+. 2. 4. X-ray Crstallography X-ray diffraction was carried out at a Bruker APEX II CCD area diffractometer equipped with MoKα radiation (λ = 0.71073 Å). The collected data were reduced with SAINT,10 and multi-scan absorption correction was performed using SADABS. 11 The structures of the complexes were solved by direct method, and refined against F2 by full-matrix least- squares method using SHELXTL.12 For Cu complex, there is a thermal vibration in the crystal as a whole, so during refine- ment, the center Cu is fixed and all organic frameworks com- plexed with Cu are subjected to disordered treatment. The SIMU command allows for the simultaneous optimization of multiple atoms, ensuring the coordination geometry around Cu is accurately represented. The SADI command is em- ployed to constrain the bond length variations between spe- cific atoms, ensuring consistent bond lengths within the com- plex. Lastly, the ISOR command helps model the thermal vibrations of atoms by assigning isotropic displacement pa- rameters to capture their positional uncertainties. The ulti- mate goal of related instructions is to make refinement more perfect and to make the structure converge. Crystallographic data and refinement parameters are given in Table 1, and im- portant interatomic distances and angles are given in Table 2. 2. 5. Antibacterial Assay The in vitro minimal inhibitory concentrations (MICs) of the synthesized derivatives were obtained 22 Acta Chim. Slov. 2024, 71, 20–25 Wang et al.: Synthesis, Crystal Structures and Antibacterial Activity of ... against two clinical Gram-positive bacterial strains: Bacil- lus subtilis (B. subtilis), Staphylococcus aureus (S. aureus) and two clinical Gram-negative bacterial strains: Pseu- domonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) by the agar dilution method recommended by Clini- cal and Laboratory Standards Institute (CLSI) (CLSI 2012). The MIC was the lowest concentration in solid media at which no bacterial growth was observed. 2. 6. Enzyme Assay The experiments were performed according to the previous reports.13 In a final 20 μL reaction, 20 mM Na2H- PO4 containing 0.5 mM DTT, 0.25 mM MgCl2, and 2.5 μM holo-ACP were mixed with 1 nM FabH, and H2O was add- ed to 15 μL. After 1 min incubation, a 2 μL mixture of 25 μM acetyl-CoA, 0.5 mM reduced nicotinamide adenine dinucleotide (NADH), and 0.5 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH) was added for FabH reaction for 25 min. The reaction was stopped by adding 20 μL of ice-cold 50% trichloroacetic acid (TCA), incubating for 5 min on ice, and centrifuging to pellet the protein. The pellet was washed with 10% ice-cold TCA and resuspended with 5 μL of 0.5 M NaOH. The incorporation of the 3H signal in the final product was read by liquid scintillation. When determining the inhibition constant (IC50), inhibitors were added from a concentrated DMSO stock such that the final concentration of DMSO did not exceed 2%. 3. Results and Discussion 3. 1. Chemistry Synthetic route of the ligand (L) was shown in Scheme 1. 1,4-benzodioxane-6-formaldehyde reacts with hydrazine hydrate in ethanol solution to form 1,4-benzo- dioxane-hydrazine, and then the intermediate hydrazine compound is stirred with phenyl isothiocyanate in metha- nol solution to form the final ligand L. Finally, the ligand L and nickel acetate or copper acetate reacted in the ratio of 2:1 separately to get the complex 1 and 2 in ethanol. Single crystals of the complexes were formed by slow evaporation of the solvent at room temperature. shown in Figure 1. The Ni atom is coordinated in square planar geometry, with two S and two imino N atoms from two ligands. The coordination geometry around the Ni1 atom exhibits a planar–square arrangement. Both ligands attached to Ni demonstrate a perfectly symmetric struc- ture. Furthermore, it is evident that the N3–Ni1–N31 angle is 180°, similarly, the S11–Ni1–S1 angle also measures 180°. Additionally, the Ni1 -S1 bond length is determined to be 2.181(2) Å, while the Ni1–N3 bond length measures 1.915(6) Å, consistent with the expected values. Figure 1. A perspective view of the molecular structure of complex 1 with the atom labeling scheme. 3. 3. Structure Description of Complex 2 Selected bond lengths and angles for complex 1 are listed in Table 2. Molecular structure of the complex is shown in Figure 2. The spatial arrangement of compound 2 distinguishes it from compound 1, as evidenced by the crystallographic characterization provided in Figure 2. Notably, the Cu1 atom in compound 2 deviates from pla- nar-square coordination, exhibiting a discernible angular distortion. The confirmation of this deviation stems from the measured S1–Cu1–S2 angle of 174.6 degrees and the N3–Cu1–N4 angle of 167.5 degrees. Additionally, despite the consistent selection of ligands forming metal bonds with Cu2+ in compound 2, the coordinated ligands do not maintain symmetrical structures around the central Cu at- om. This asymmetry is substantiated by the determined bond lengths between the ligands and the metal ion: Cu1– Scheme 1. Synthetic route of Ligand L. 3. 2. Structure Description of Complex 1 Selected bond lengths and angles for complex 1 are listed in Table 2. Molecular structure of the complex is S1 measures 2.185(13) Å, Cu1–S2 measures 2.258(13) Å, Cu1–N3 measures 1.85(2) Å, and Cu1–N4 measures 2.215(19) Å. 23Acta Chim. Slov. 2024, 71, 20–25 Wang et al.: Synthesis, Crystal Structures and Antibacterial Activity of ... Figure 2. A perspective view of the molecular structure of complex 2 with the atom labeling scheme. The origin of this asymmetric coordination of ligands to the copper ion lies in the influence of their orientations and conformations within the molecular framework. While the ligands themselves possess inherent symmetry, their co- ordination to the metal center can occur in variable orienta- tions, thus culminating in an overall loss of symmetry with- in the complex. The disruption of symmetry observed in the compound can be attributed to multiple factors, including the orientation and localized distortion of the ligands. The presence of neighboring molecules or ligands may induce perturbations, leading to local distortions upon coordina- tion with the metal ion. These distortions contribute to spa- tial asymmetry within the system. Additionally, the confor- mational flexibility of the ligands plays a significant role in symmetry disruption. Despite sharing the same chemical formula, the ligands may exhibit diverse conformations, in- fluenced by freely rotating bonds or other conformational degrees of freedom. This inherent flexibility allows the lig- ands to adopt different coordination modes when binding to the metal center, further exacerbating the overall struc- tural asymmetry. Consequently, the investigation and com- prehension of this asymmetry necessitate in-depth explora- tion via comprehensive structural analysis and sophisticated computational methods. Table 2. Selected bond lengths (Å) and angles (o) for complexes 1 Ni1–S1I 2.181(2) Ni1–N3I 1.915(6) Ni1–S1 2.181(2) Ni1–N3 1.915(6) S11–Ni1–S1 180.0 N3–Ni1–S11 94.42(17) N3–Ni1–S1 85.57(17) N31–Ni1–S1 94.42(17) N31–Ni1–S11 85.57(17) N3–Ni1–N31 180.0 2 Cu1–S1 2.185(13) Cu1–N3 1.85(2) Cu1–S2 2.258(13) Cu1–N4 2.215(19) S1–Cu1–S2 174.6(8) S1–Cu1–N4 94.6(8) N4–Cu1–S2 80.2(7) N3–Cu1–S2 88.5(9) N3–Cu1–S1 96.6(11) N3–Cu1–N4 167.5(11) 3. 4. Biological Activity The MIC (Minimum inhibitory concentration, μM) of complex 1 and complex 2 against these bacterial strains Table 1. Crystallographic and refinement data for the complexes Complexes 1 2 Empirical Formula C32H28N6NiO4S2 C32H28CuN6O4S2 Formula Weight 683.43 688.26 Crystal System Triclinic Monoclinic Space group P–1 Cc a (Å) 6.570(4) 19.396(3) b (Å) 7.296(5) 17.857(3) c (Å) 16.768(10) 8.6520(13) α (°) 85.557(18) 90 β (°) 80.676(18) 95.578(4) γ (°) 69.897(18) 90 V (Å3) 744.6(8) 2982.5(8) Z 1 4 Dc (g cm–3) 1.524 1.533 F(000) 354 1420 μ(Mo-Kα) (mm–1) 0.842 0.922 Reflections collected 6565 12152 Data/restraints/parameters 2556/0/205 5060/2208/709 Independent reflections (Rint) 2556 (0.0703) 5060 (0.0657) Goodness-of-fit on F2 1.037 1.027 Final R1, wR2 indexes [I >= 2σ (I)] 0.0780, 0.1930 0.0768, 0.1339 Final R1, wR2 indexes [all data] 0.1504, 0.2661 0.1959, 0.1765 24 Acta Chim. Slov. 2024, 71, 20–25 Wang et al.: Synthesis, Crystal Structures and Antibacterial Activity of ... are tested by MTT method and the activity data was pre- sented in Table 3. Based on the data obtained, we found that the two compounds showed some inhibitory activity. In particular, the inhibitory effect on Gram-negative bac- teria was significantly stronger than that of Gram-positive bacteria, and the inhibitory activity was comparable to the positive control kanamycin. Among them, complex 2 has the highest inhibitory activity against two Gram-negative bacteria (MIC = 3.13 μΜ for P. aeruginosa, MIC = 2.5 μΜ for E. coli). Table 3. Antibacterial activity of synthetic complex 1 and 2. Compound Minimum inhibitory concentration, μM Gram-negative Gram-positive E. coli P. aeruginosa B. subtilis S. aureus 1 6.25 6.25 12.5 12.5 2 2.5 3.13 6.25 12.5 kanamycin 2.5 2.5 2.5 1.25 E. coli FabH inhibition potency of complex 1 and complex 2 was examined and the results are summarized in Table 4. As shown in Table 4, all of the two compounds tested exhibited a certain inhibitory activity against E. coli FabH wherein the compound having the highest inhibito- ry activity remained complex 2 (IC50 = 3.67 μΜ). This re- sult indicates that the 1 E)-2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)methylene)-N-phenylhydrazinecarbothioam- ide complexes can inhibit the function of FabH and the antibacterial effect was produced partly by interaction of FabH protein and the compounds. Table 4. E. coli FabH inhibitory activity of synthetic complex 1 and 2. Compound E. coli FabH (IC50±SD), μM 1 10.15±0.329 2 3.67±0.165 3. Conclusions In this manuscript, we describe a novel hydrazine- carbothioamide metal complex and present our findings on its crystal structures, antibacterial activity, and FabH inhibitory activity. Our results demonstrate that Complex 2 exhibits effective inhibition of FabH activity against E. coli, indicating its potential as a novel FabH inhibitor. Coordination complexes as FabH inhibitors have high surface area-to-volume ratios and heterogeneity, which can enhance their biological availability and effica- cy. However, more studies are needed to evaluate their safety and bioavailability before they can be developed in- to therapeutic agents. Future studies could focus on opti- mizing the pharmacological properties and antimicrobial activity of these complexes, as well as exploring their po- tential as therapeutic agents for the treatment of bacterial infections. Acknowledgement This work was supported by Natural science research plan of Huai’an City (HAB202146); Jiangsu Higher Educa- tion Institutions Basic Science (Natural Science) General Program（22KJD150004）and Qing Lan Project; the S&T Innovation 2025 Major Special Program of Ningbo (2020Z091). Conflict of Interest The authors declare no conflict of interest. Supplementary Data CCDC 2266563 (1) and 2266562 (2) contain the sup- plementary crystallographic data for the compounds. These data can be obtained free of charge via http://www. ccdc.cam.ac.uk/conts/retrieving. html, or from the Cam- bridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk. The spectral data of the compounds can be found in the supporting information file. 5. References 1. (a) A. S. Orabi, K. M. Abou EI-Nour, M. F. Youssef, H. A. Salem, Arab. J. Chem. 2020, 13, 2628-2648. DOI:10.1016/j.arabjc.2018.06.016 (b) A. M. Abbas, S. R. Fisal, A. S. Radwan, M. M. Makhlouf, A. S. Orabi, J. Mol. Liq. 2022, 351, 118333. DOI:10.1016/j.molliq.2021.118333 2. (a) H. J. Zhang, Z. L. Li, H. L. Zhu, Curr. Med. Chem. 2012, 19, 1225-1237. DOI:10.2174/092986712799320484 (b) Y. Zhou, Y. Q. Liang, X. Y. Wang, H. Y. Chang, S. P. Hu, J. Sun, Chem. Pharm. Bull. 2022, 70, 544-549. DOI:10.1248/cpb.c22-00090 (c) H. 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Bruker, SMART and SAINT, Bruker AXS Inc., Madison, 2002. 11. G. M. Sheldrick. SADABS, University of Göttingen, Germany, 1996. 12. G. M. Sheldrick, Acta Crystallogr. 2015, C71, 3–8. 13. J. Sun, C. P. Zhang, C. H. Chen, X. M. Guo, C. S. Liu, Y. Zhou, F. L. Hu, Chem. Biodivers. 2023, 20, e202201060. DOI:10.1002/cbdv.202201060 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Biosinteza maščobnih kislin je bistvenega pomena za preživetje bakterij, β-ketoacil-acyl transportni protein sintaza III (FabH) pa je obetavna tarča za razvoj antibakterijskih učinkovin. Nikljev(II) kompleks [NiL2] (1) in bakrov(II) kompleks [CuL2] (2), kjer je L (E)-2-((2,3-dihidrobenzo[b][1,4]dioksin-6-il)metilen)-N-fenilhidrazinkarbotioamid, sta bila sin- tetizirana in okarakterizirana z elementarno analizo, IR in 1H NMR spektroskopijo ter HRMS. Strukture kompleksov so bile določene z monokristalno rentgensko analizo, ki razkriva, da so nikljevi in bakrovi atomi v kompleksih v tetraedrični geometriji. Določili smo antibakterijsko in FabH inhibitorno delovanje teh dveh spojin na E. coli. 26 Acta Chim. Slov. 2024, 71, 26–38 Mali and Bhanwase: Brain Targeted Drug Delivery System of Carmustine: ... DOI: 10.17344/acsi.2023.8461 Scientific paper Brain Targeted Drug Delivery System of Carmustine: Design, Development, Characterization, in vitro, ex vivo Evaluation and in vivo Pharmacokinetic Study Audumbar Mali *,1 and Anil Bhanwase 2 1 School of Life Sciences, Punyashlok Ahilyadevi Holkar Solapur University, Solapur, 413255, Maharashtra, India. 2 Department of Pharmaceutical Chemistry, SPM’s College of Pharmacy, Akluj-413101, Malshiras, Solapur, Maharashtra, India. * Corresponding author: E-mail: maliaudu442@gmail.com Received: 09-20-2023 Abstract The treatment of gliomas remains difficult task. Carmustine is a drug that is used in the treatment of gliomas. Flexible liposomes embedded in situ thermoreversible nasal gel preparations of Carmustine were studied for in vitro carmustine release, ex vivo carmustine permeation and carmustine release kinetics. The epithelial layers of nasal tissue were found to be intact and undamaged during histological analysis. Intranasal administration of optimized flexible liposomes embed- ded in a nasal gel showed higher Cmax (Approximately two-fold), AUC0→t (Approximately three-fold), AUC0→∞ (Approx- imately six-fold), and lower Tmax (1 h) in the brain, compared to intravenous injection of carmustine. The present study demonstrates that the flexible liposome embedded thermoreversible in situ intranasal gel of carmustine improved the targeted uptake of carmustine in the brain through the nasal delivery system and could be a reliable and effective delivery system for carmustine in the treatment of gliomas. Keywords: Flexible liposomes, carmustine, zeta potential, AUC, Cmax, Tmax. 1. Introduction Glioma is the most critical type of brain tumor among human beings. Patients suffering from glioblasto- ma (GBM) have survival period of 8–14 months. Surgery, chemotherapy, and radiation are the prevailing measures to treat GBM. Endothelial junctions of the blood-brain barrier (BBB) proved major challenge in the treatment of GBM. Many drug molecules are ineffective in clinical tri- als because of their inability to cross BBB. Oral route is not suitable to distribute the therapeutic amount of medica- tion to the brain because of its specific obstacles, viz. BBB, blood-cerebrospinal fluid barrier, and efflux transporters. These obstacles regulate the exchange between the circula- tory system for cerebrospinal fluid and peripheral blood flow. The administration of medicines into the central nervous system (CNS) seems more complicated due to other elements like physicochemical characteristics of the drug.1,2,3 Therefore variety of strategies are being used to target medicines to the brain, including BBB disruption, drug manipulation, as well as alternative routes of drug ad- ministration, viz. olfactory pathways (intranasal route), intrathecal, intra-cerebral, and ventricular. The nasal route is a novel, useful, simple, and efficient way to cross the blood-brain barrier, which has led to its recent rise in pop- ularity. It reduces systemic exposure and, consequently, systemic side effects related to medication use. The drug enters the CNS through the olfactory epithelium region due to the neuronal link between the nasal mucosa and the brain, which serves as a doorway for chemicals entering the central nervous system.4,5,6 Carmustine has been referred for the treatment of gliomas.7–10 However; it has been restricted because of side effects like bone marrow suppression11 and pulmo- nary fibrosis.12 Gliadel wafers13 are impregnated with car- mustine and placed at the tumor site to lower side effects. These gliadel wafers are unsuccessful due to low tumor penetration, insufficiency to stop tumor recurrence, an ab- sence of synergistic activity with other chemotherapeutic medicines including radiotherapeutic agents, and insuffi- cient therapeutic efficacy.14,17 To overcome these glitches, an assortment of drug distribution vehicles has been de- veloped in current days. This contains nanoparticles made of poly (D, L-lactic-co-glycolic) acid, polymeric micelles, liposomes, dendrimers, nanoshells, carbon nanotubes, polyglycolic acid, and polylactic acid.15,16,17 Despite several 27Acta Chim. Slov. 2024, 71, 26–38 Mali and Bhanwase: Brain Targeted Drug Delivery System of Carmustine: ... study designs and research carried out, it is still a challenge to deliver required amount of carmustine to the brain. The present work is designed to formulate Carmus- tine embedded flexible liposomal thermoreversible in situ intranasal gel for better brain targeting and effective thera- peutic outcomes. The transdermal administration of flexible liposomes, viz. ethosomes, has produced some encouraging outcomes.19–23 Researchers have proved the enhanced phar- macokinetic profiles for rizatriptan benzoate, salmon cal- citonin, and galanthamine hydrobromide by transforming the drug into flexible liposomes.24,25,26 Flexible liposomes have more bilayer elasticity because they lack cholesterol and have a higher amount of ethanol (20–40%) than typical liposomes, which are rigid because they contain cholester- ol. Since intercellular pores are smaller than liposomes, the elasticity of liposomes expands penetration through them. By encouraging flux forces of the liposomes at middle-lev- el concentrations, ethanol improves inter-vesicle repulsion and prevents aggregation. These flexible liposomes are, therefore, extra stable compared to regular liposomes. The flexible liposome penetrates the stratum corneum, and re- leases the medication in deepest layers of the skin. Topical application of flexible liposomes reaches therapeutic level in the plasma. Viscosity and mucoadhesive strength for differ- ent thermoreversible gels can be improved by extending the residence time in the intranasal cavity.27,28 Vani et al., 2022 developed nano-sized carmustine liposomes with reasonable entrapment efficiency.42 Hence, there is need to deliver carmustine effectively to the brain through appropriate drug delivery system. 2. Materials and Methods Carmustine was obtained as gift sample from MSN Laboratories Private Limited, Telangana, India. Poloxamer 407 and Carbopol 934 were obtained as gift samples from BASF India Limited, Navi Mumbai, Maharashtra, India and Research Lab Fine Chem Industries, Mumbai, Mahar- ashtra, India respectively. 2. 1. Compatibility Study of Excipients With Carmustine The compatibility between selected excipients along carmustine was evaluated using an FTIR. FTIR spectra of carmustine with a physical blend of carmustine, lipids, polymers, and other excipients were scanned.29 2. 2. Preparation and Characterization of Flexible Liposomes Flexible liposomes were prepared by using ethanol injection method.26,30 Ethanol was mixed using a magnetic stirrer (2 MLH, 220/230 V AC supply, Bio Technics India) to dissolve the carmustine and soya lecithin. Using a sy- ringe, double-distilled water was gradually added to the mixture as a thin stream (500 μl /min), which made up to 30 ml, and the mixture was agitated for 30 minutes at 750 rpm with the help of a magnetic stirrer. To prevent ethanol loss, parafilm was used to cover the dispersion. Through- out the entire process, temperature was maintained at 30oC. The developed flexible liposomes were sonicated using a probe sonicator for three cycles of 5 minutes each, with 5 minutes of rest. The sonication was performed in an icy atmosphere to prevent an excessive rise in the tem- perature during the process. Formulation batches (F1–F9) were prepared by varying soya lecithin and ethanol ratio. 2. 3. Full Factorial Design for Preparation of Flexible Liposomes of Carmustine The 32 full factorial designs were used in the current research work. In this research work, two factors were es- timated, each at three different levels, and experimental trials were accomplished at all nine possible combinations. Particle size (Y1), percent entrapment efficiency (EE) (Y2), and polydispersity index (PDI) (Y3) were used as depend- ent variables. In contrast, % of ethanol (X1) and % of soya lecithin (X2) in the final preparation were used as inde- pendent factors. The three levels for least, adequate, and extreme concentrations were classified as –1, 0, and +1, respectively, and presented in Tables in the supplementary material. Responses of different formulations were meas- ured as per factorial design.26,43 The responses were assessed using an interactive and polynomial statistical model. Y = b0 + b1X1 + b2X2 + b11X12 + b22X22 + b12X1X2 In the above equation, Y is the dependent variable, b0 is the arithmetic mean response for nine runs, and bi (b1, b2, b12, b11, and b22) is the estimated coefficient of cor- responding factors Xi (X1, X2, X1X2, X12, and X22). Critical effect (X1 and X2) signifies average results of changing 1 factor from its lower to higher values simultaneously. The interaction term (X1X2) indicates how the re- sponse changes when two factors are changed concurrent- ly. Polynomial terms X12 and X22 determine the quadratic impact. To analyze how independent variables impact de- pendent variables, the fit summary and analysis of vari- ance (ANOVA) were combined to create the best-fit mod- el. Design-expert software (Stat-Ease® 360) was used to optimize carmustine drug delivery system.26,31,43 3. Characterization of Prepared Flexible Liposomes 3. 1. % Entrapment Efficiency (% EE)26,32 The % EE of liposomal dispersions was determined by separating non-encapsulated carmustine from carmus- 28 Acta Chim. Slov. 2024, 71, 26–38 Mali and Bhanwase: Brain Targeted Drug Delivery System of Carmustine: ... tine liposome dispersion by centrifuging 2 ml of carmus- tine liposomes at 20000 rpm for 15 minutes at 4 °C. The supernatant layer was removed, sediment liposomes were disrupted with 2 ml ethanol to release entrapped Carmus- tine, then diluted using distilled water up to 10.00 ml and estimated for carmustine presence at 231 nm to calculate % EE by using a plotted calibration curve in phosphate buffer saline pH 6.4 (linearity, range = 0.50–2.50 μg/ml, R2 = 0.9996). The amount of carmustine entrapped was calculated: % Entrapment Efficiency = (Wa – Ws) / Wa × 100 Where, Wa is the total quantity of carmustine initially added Ws is the concentration of carmustine in liposomes. 3. 2. Carmustine Liposomes size, PDI, and Zeta Potential1,26 The liposomal size for prepared flexible liposomes was assessed using the dynamic light scattering method and the Malvern Zeta sizer (ZSU3100, Nano ZS, Malvern Instruments Ltd., UK) particle size analyzer. The PDI was computed to examine liposomal size distribution. Malvern zeta sizer was used to calculate the zeta potential of each carmustine batch. 3. 3. Flexible Liposomes Surface Morphology and Shape1,18,26 Surface morphology of liposomes was studied using Atomic Force Microscopy (AFM) and Transmission Elec- tron Microscopy (TEM). For TEM examination, samples were mounted on carbon-coated grids, negatively stained using a phosphotungstic acid solution, and then observed under a microscope at 10,000–60,000 times the original magnification while accelerated at 100 kV. In the non-con- tact approach, silicon nitride cantilevers were used to in- vestigate the Nanosurf Flex AFM model at room temper- ature. 3. 4. Embedded flexible liposomes in thermoreversible in situ gel Stable liposomal dispersions (F1-F9) were converted into thermoreversible in situ gel formulations using a cold technique. Based on preliminary research, poloxamer 407 and carbopol 934 were used to transform the sol into gel under intranasal circumstances. 0.3% carbopol 934 was slowly mixed with distilled water using a stirrer. Then, 20 ml flexible liposome dispersion was mixed using a me- chanical stirrer with a speed of one thousand revolutions per minute for thirty minutes to obtain the last mixture with Carmustine (0.2 mg/ml). Poloxamer 407 (18.00%) was mixed into the mixture. The prepared mixture was left at 4 °C overnight to produce a clear solution. The viscosity, mucoadhesive strength, and other physicochemical prop- erties of the gel were investigated.26,33 4. Evaluation Parameters 4. 1. Physico-chemical Characteristics of Carmustine Nasal Gel The pH of intranasal gel, carmustine concentration, viscosity, and mucoadhesive strength were assessed. The pH of all batches was tested using a pH meter (Equip-Tron- ics EQ- 610). Carmustine content was determined with the help of a UV-visible spectrophotometer (Shimadzu 1800, Japan) at 231nm. At various temperatures (20 °C–40 °C), rheological investigations were carried out using a thermo- statically precise Brookfield viscometer (DV3T Rheome- ter, USA). The mucoadhesive strength was assessed using a Texture Analyzer CT3 (Brookfield, USA) outfitted with a 4.5 kg load cell with Texture Pro CT software.27,34,35,37 4. 2. Spreadability The spreadability of nasal gel preparations was meas- ured by using Whatman filter paper (#0.45 mm). Graduat- ed pipette (1 ml) with a rubber bulb was clamped vertically to stand where its tip was kept 2 cm above the horizontal surface of round filter paper. At the center of the filter pa- per, 0.1 ml of the gel preparation was dropped. At a fixed time interval of 20 seconds, the surface area covered by gel was observed and evaluated.36 4. 3. In vitro Carmustine Release Studies In vitro drug release study was performed by using Franz diffusion cell. Cellophane membrane (molecular weight: – 12,000.00–14,000.00) having a permeation area of 0.8 cm2 was used for permeation study. 15 ml of Phos- phate Buffer Saline (pH 6.4) was retained in the receptor chamber, and carmustine nasal gel containing a carmus- tine equivalent to 1mg was retained in the donor chamber. A 0.5 ml sample was taken from the receptor compartment at predetermined intervals by continuously replacing it with freshly prepared buffer solution for eight hours. Then, samples were diluted and estimated for carmustine content with the help of UV spectrophotometer at 231nm.37,38,39 4. 4. Ex-vivo Carmustine Release Study for Carmustine in situ Nasal Gel The freshly isolated nasal cavity of the sacrificed goat was taken from the local slaughterhouse, and kept in Phosphate buffer saline (pH 6.4). Mucosal membrane was identified, removed, cleaned, and maintained in Phos- phate buffer saline. A Franz diffusion cell with a thermo- 29Acta Chim. Slov. 2024, 71, 26–38 Mali and Bhanwase: Brain Targeted Drug Delivery System of Carmustine: ... stat facility was used to conduct the study. Franz diffusion cells with an actual permeation component of 2.00 × 2.00 cm2 were used to hold tissue sections. Carmustine-loaded thermoreversible nasal gel equivalent to 1 mg of carmus- tine was kept in the donor compartment, and the receptor compartment was filled using 15 ml of Phosphate buffer saline (pH 6.4). The study was carried out at 34 ± 1 °C un- der stirring. Aliquots of 0.5 ml were taken from the recep- tor compartment and substituted for 8 hours using a new buffer. The samples were diluted before being examined with a UV spectrophotometer at 231 nm.37,38,39 4. 5. Release Kinetics of Carmustine All preparations of carmustine were taken to study release kinetics. The release profile was evaluated for best fit model.40 4. 6. Histopathological Study Using Nasal Mucosa Histopathological evaluation of nasal mucosa was carried out after ex-vivo permeation study. The nasal membrane was set aside on the glass slide with the help of a 10% buffered formaldehyde solution. Nasal tissue parts were colored by using hematoxylin with eosin, and then finally seen by using a light microscope to check for signs of tissue damage caused during ex-vivo drug permeation.10 4. 7. In Vivo Pharmacokinetic Study Healthy Wistar rats of 3 to 4 months, weighing 200– 250 grams were included in study. The rats were kept in a neat and hygienic room at 25 ± 1 °C along with humidity of 45–55%, for 12 hrs /12 hrs light and dark conditions. The rats have given free access to food and water. The Ethical clearance (CPCSEA/IAEC/CP-PL/01/2023) was obtained from Institutional Animal Ethics Committee (IAEC) of Sudhakarrao Naik Institute of Pharmacy, Yavatmal, Maha- rashtra, India. The rats were fasted the whole night before the work. Rats were divided in five different groups com- prising three animals in each group.46,47 4. 8. In Vivo Pharmacokinetic Study in Brain All rats were kept at 25 ± 1 °C and fasting overnight. Two groups of rats are as follows: Group 1 – Intranasal administered optimized flexible li- posome embedded in situ thermoreversible nasal gel. Group 2 – The Carmustine-marketed formulation, (Car- mustine for injection USP 100 mg) was administered IV through the tail vein. 20 μl of gel containing carmustine (0.81 mg/kg) was applied to nostrils of each animal in the first group,42 and, marketed carmustine injection was given to each animal in the second group through the tail vein, which containing carmustine corresponding to 0.81 mg/kg. The rats stayed supine for two minutes after taking carmustine prepara- tions. Rats were sacrificed using intraperitoneal urethane (1g/kg). The brain was isolated at different time intervals, viz. 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 8 hours. Brain samples were homogenized in methanol and mixed with acetonitrile. Homogenate was filtered and examined using HPLC.48 4. 9. Statistical Examination PK Solver software was used for statistical analysis. 5. Results 5. 1. Compatibility Study of Excipients with Carmustine The drug-excipient compatibility was assessed us- ing an FTIR spectrophotometer. The infrared spectrum of carmustine and physical mixture of carmustine with excipients were compared (Figure 1). No variations were observed in the spectrum of carmustine. This indicates that carmustine is compatible with a mixture of excipi- ents. The distinctive peaks of the carmustine FTIR spec- trum may correspond to secondary amine groups at 3331 cm–1 and to the C=O stretch at 1708 cm–1. In addition, peaks at 1380 cm–1, and 2973 cm–1 for N=O and aliphatic C-H stretch were observed respectively. The C-O stretch was observed at 1087 cm–1 and 1045 cm–1, C-X (chloride) was observed at 803.78 cm–1 and 654 cm–1 respectively C-N stretch (amines) was observed at 1274 cm–1 and 1329 cm–1 respectively. Typical characteristic peaks of the car- mustine were also seen in the FTIR spectrum of the phys- ical mixture with no noticeable change from the spectra of the separate carmustine and excipients. This demon- strated that carmustine and excipients did not interact chemically.41 5. 2. Evaluation for Carmustine Flexible- Liposomes Carmustine flexible liposomes were prepared by using an ethanol injection sonication method. Probe sonication causes a cavitation effect, where sonic vibra- tions are translated into dispersion, which forms several tiny bubbles. The internal pressure of system is raised due to these tiny bubbles leading collision of particles and re- duction of their size to nanoscale. Carmustine flexible liposomes were stored at cold temperature (2–8 °C) for further study. Table 1 depicts the liposomal particle size, % of EE, PDI, and zeta potential. 30 Acta Chim. Slov. 2024, 71, 26–38 Mali and Bhanwase: Brain Targeted Drug Delivery System of Carmustine: ... 5. 3. 32 Full Factorial Designs for the Formulation of Flexible Liposomes A 32 full factorial design was applied to study the ef- fect of factors systematically. With the help of Design Ex- pert® software (Stat-Ease® 360), the impact of independent variables such as % Ethanol (X1) and % Soya Lecithin (X2) was examined by contour plots and response surface plots by application of ANOVA (Table 1). The following equations were formed via regression along with a graphical examination of results obtained in experimental values, where F ratios were statistically signifi- cant (p < 0.05), and Adjusted-R2 values reached from 0.9880 to 0.9327. The results were well-fit by these model equations. Table 1. Characterization of flexible liposomes Batches Coded values Particle Size Entrapment Efficiency PDI Y3 Zeta Potential code Ethanol Soya Lecithin (nm) Y1 (%)Y2 (mV) (%) X1 (%) X2 F1 –1 –1 146.8 ± 10.2 96.9± 1.2 0.1 –28.3 ± 4.2 F2 –1 0 149.8 ± 16.0 94.9± 1.1 0.2 –50.2 ± 4.8 F3 –1 +1 180.3 ± 10.2 94.4± 1.7 0.2 –20.3 ± 2.9 F4 0 –1 181.1 ± 14.3 98.6± 1.1 0.3 –47.8 ± 3.5 F5 0 0 180.5 ± 21.4 96.9± 1.2 0.2 –26.2 ± 4.9 F6 0 +1 197.7 ± 14.1 96.9± 1.2 0.4 –24.0 ± 5.2 F7 +1 –1 187.8 ± 20.2 91.9± 1.1 0.4 –67.5 ± 6.2 F8 +1 0 180.5 ± 18.7 92.2± 1.6 0.4 –48.7 ± 4.7 F9 +1 +1 192.3 ± 12.9 91.0± 1.4 0.4 –39.4 ± 5.9 Note: (n = 3, mean± Standard Deviation (SD)) Where: Independent Variables = X1 – % of ethanol, X2 – % of soya lecithin, Dependent Variables = Y1 – Particle size (nm), Y2 – Percentage EE, Y3 – PDI. Figure 1. (A) FTIR spectrum of Carmustine API in Ethanol (Carmustine solution), (B) FTIR spectrum of Carmustine solution + Soya Lecithin, (C) FTIR spectrum of Carmustine solution + Soya Lecithin + water, (D) FTIR spectrum of Carmustine solution + Poloxamer 407, (E) FTIR spectrum of Carmustine solution + Carbopol 934, (F) FTIR spectrum of Carmustine + All excipients 31Acta Chim. Slov. 2024, 71, 26–38 Mali and Bhanwase: Brain Targeted Drug Delivery System of Carmustine: ... The impact on particle size (Y1), % EE (Y2), and PDI (Y3) were observed to be significant by ANOVA, and the quadratic equation as below: Y1 = 1167.61X1 + 496.86X2 + 365.40X12 + 230.41X22 + 210.25X1X2 (1) Y2 = 20.42X1 + 4.49X2 + 31.23X12 + 0.1780X22 + 0.6806X1X2 (2) Y3 = 0.0865X1 + 0.0050X2 + 0.0002X12 + 0.0021X22 + 0.0004X1X2 (3) Flexible liposomes are seen in TEM photomicro- graphs to be unilamellar and almost spherical. The flexible liposomes deviate from the typical spherical shape of con- ventional liposomes due to lack of cholesterol. Cholesterol makes the liposomal bilayer dispersion rigid, its absence and a higher alcohol concentration make it flexible and cause it to deviate from the typical spherical shape. These observations are consistent with the earlier findings by Touitou et al.30 and Kempwade et al.26 Figure 3 depicts a TEM image of camustine flexible liposomes. Figure 2. Contour plots (A, C, E) and Surface response plots (B, D, F) for particle size, % EE, and PDI respectively. Figure 3. TEM results of carmustine flexible liposomes 32 Acta Chim. Slov. 2024, 71, 26–38 Mali and Bhanwase: Brain Targeted Drug Delivery System of Carmustine: ... The physicochemical properties of the produced flexible liposomes implanted in in situ nasal gels were eval- uated. It was seen that the gelation time was less than 15 seconds. The gel developed right away as the temperature reached 32 to 34 °C. It was observed that the mucoad- hesive strength was 3726.52 to 4667.96 dynes/ cm2. The formulation’s viscosities ranged from 6579 ± 49.90 cps to 7032 ± 80.62 at 30 °C±1 °C. The pH of optimized formu- lations was observed from 5.50±0.38 to 6.02 ± 0.58. The % carmustine content of optimized in situ nasal gel prepara- tions was 97.00 ± 2.18 to 99.34±1.97. The spreadability of optimized formulations was observed from 16.28 ± 2.05 to 18.75 ± 1.89. 5. 4. In vitro Carmustine Release Study Flexible liposomes embedded in situ nasal gel is re- quired to release drug slowly for longer time. Therefore, these formulations of carmustine were studied for release kinetics by performing an in vitro drug release study for eight hours. Samples were withdrawn at intervals of 15 minutes, 30 minutes, one hour, two hours, four hours, six hours, and eight hours. In nine different formulations, the TG7 formulation showed the lowest cumulative % drug release, observed to be 83.7%, whereas TG4 showed the highest cumulative % drug release, observed to be 96.2% (Figure 5 A). The comparative in vitro release profile of carmustine API solution, flexible liposomes of carmustine, in situ nasal gel of carmustine, and flexible liposomes em- bedded in situ nasal gel of carmustine followed zero order kinetics. Carmustine API solution showed lowest cumula- tive % drug release which was observed to be 56.2%. How- ever; flexible liposomes embedded in situ nasal gel showed the highest cumulative % drug release which was observed to be 96.1% (Figure 5 B). 5. 5. Ex-vivo Carmustine Permeation Study Ex-vivo carmustine permeation study was per- formed using a nasal membrane. Drug permeation was Figure 4 depicts AFM pictures of flexible liposomes. Flexible liposomes of carmustine underwent an AFM examination to evaluate their surface topography and size. Uniformly distributed, roughly spherical-shaped liposomes can be seen in the AFM pictures. 33Acta Chim. Slov. 2024, 71, 26–38 Mali and Bhanwase: Brain Targeted Drug Delivery System of Carmustine: ... Figure 5. (A) % Cumulative drug release of flexible liposomes embedded in situ in thermoreversible nasal gel (TG1-TG9), (B) % Cumulative drug release of Carmustine API solution, flexible liposomes, in situ nasal gel of carmustine and TG4 Figure 6. (A) Cumulative % drug permeation of flexible liposomes embedded in situ in thermoreversible nasal gel (TG1-TG9), (B) Cumulative % drug permeation of Carmustine API solution, flexible liposomes, in situ nasal gel of carmustine and TG4 34 Acta Chim. Slov. 2024, 71, 26–38 Mali and Bhanwase: Brain Targeted Drug Delivery System of Carmustine: ... assessed for eight hours at specified time intervals. Maxi- mum drug permeation was observed in case of TG4; how- ever, TG7 showed minimum drug permeation across the goat nasal membrane. In nine different formulations, the TG7 formulation showed the lowest drug permeation, ob- served to be 87.5%, whereas TG4 showed the highest drug permeation, observed to be 97.4% (Figure 6 A). Comparative ex-vivo permeation of the carmustine across goat nasal membrane for TG4 with other formula- tions viz. carmustine API solution, flexible liposomes, and in situ nasal gel of carmustine followed zero order kinet- ics. TG4 showed the highest drug permeation (97.4%) and carmustine API solution showed the lowest drug perme- ation (58.1%) through the nasal membrane. (Figure 6 B). 5. 6. Determination of Carmustine Release Kinetics Dissolution profile of different carmustine formula- tions were compared by using model dependent (Curve fitting) methods followed by statistical analysis. Higuchi’s equation was the best-fit model as r2 = 0.9848 for the in vitro carmustine release profile; zero-order and higuchi matrix kinetics were the best-fit models as r2 = 0.9912 for ex-vivo carmustine release profile (Table 2). The flux values of different flexible liposomes em- bedded in situ nasal gel formulations were obtained from 1.6119 (μg/cm) 2/min to 1.8491 (μg/cm) 2/min, and en- hancement ratios for various formulations were obtained from 2.1483 to 2.4644. TG7 showed lowest flux value, whereas TG8 showed highest flux value. TG7 showed low- est enhancement ratio, whereas TG8 showed highest en- hancement ratio. 5. 7. Histopathological Study of Nasal Mucosa Histopathological analysis was performed to veri- fy cellular damage to goat nasal mucosa after an ex-vivo study. Nasal goat mucosa retained in phosphate buffer sa- line (SPBS) having pH 6.4 was a standard control. Pseu- dostratified columnar ciliated epithelium and lamina pro- pria with mucus acini were normal. The epithelium layer of normal goat nasal tissue and tissue used for the perme- ation study of carmustine was observed to be intact and without cellular damage Figures 7(A) and 7(B). Figure 7. Histopathological study of goat mucosal membrane: (A) Nasal mucosal membrane kept in SPBS having pH 6.4, and (B) Na- sal mucosal membrane used for permeation of TG4 5. 8. In vivo Pharmacokinetic Study The drug concentration – time profile of carmustine flexible liposomes embedded in situ thermoreversible na- sal gel (TG4) and marketed formulation is illustrated in Figure 8, Table 3. It was observed that, absorption via nasal route of optimized flexible liposomes embedded in situ thermore- versible intranasal gel appears to be fast, along with more concentration of carmustine accomplishment in the brain within 0.25 h (55.1 % release), as compared to marketed intravenous drug delivery system (11.73 % release). The fact that the Tmax following intranasal formulations was shorter (1 h) than that following IV administration (2 h) (Table 3) suggests that carmustine is rapidly transported to the brain through the nose. Nasal administration for TG4 observed approx. 2-fold higher Cmax value in the na- sal route than the IV route of the marketed formulation of carmustine injection (Table 3). Table 2. Drug release kinetic models for optimized flexible liposomes embedded in situ nasal gel of carmustine For In vitro drug release Formulations Zero Order First Order Higuchi Matrix Korsmeyer Peppas Hixson-Crowell Best fit model Kinetics Kinetics Model. TG4 0.9848 0.8525 0.9848 0.9564 0.9271 Higuchi Matrix Kinetics For Ex-vivo drug release TG4 0.9912 0.7767 0.9912 0.9789 0.8916 Zero Order & Higuchi Matrix Kinetics 35Acta Chim. Slov. 2024, 71, 26–38 Mali and Bhanwase: Brain Targeted Drug Delivery System of Carmustine: ... The bioavailability of TG4 optimized formulation through nasal delivery was observed to be higher ap- prox.6.0 folds more than the marketed formulation of carmustine injection through the IV route (Table 3). This might be because of poor transport of the carmustine via the BBB.48,49 A relative relationship of bioavailability of TG4 quantified through AUC0→t, indicated 3-fold more in the brain than the marketed formulation of carmustine injection (Table 3). Hence, it shows the potential ability and practical suitability of TG4 for effective delivery of carmustine to brain. In general, the pharmacokinetic pa- rameters for intranasal administration of the TG4 nasal gel proved significant enhancement in the brain bioavailabil- ity of carmustine as compared to commercial IV injection of carmustine through the IV route. 6. Discussion FTIR spectra of carmustine did not show any dis- tinctive alteration when mixed with different ingredients like soya lecithin, and polymers. This indicates retention of structural and chemical integrity of the Carmustine after mixing all excipients. Ethanol and soya lecithin, at varying amounts; give a positive association concerning the particle size of car- mustine-embedded liposomes. The results revealed that ethanol is responsible for carmustine liposome size, PDI, and zeta potential with % EE. The liposomal size was in- creased with the increase in amount of ethanol. This may be because of the high ethanol amount, which affects bi- layer solubilization and serves to change the morpholog- ical characteristics of carmustine embedded liposomes. Another cause might be the rise in inter-vesicle repul- sion with rise in intermediate ethanol amount by helping fluctuation force. More amount of ethanol affects liposo- mal fusion (due to too robust fluctuations or bilayer par- tial or local solubilization). Similar outcomes for several researchers have previously been observed.26, 44, 45 The amount of ethanol was similarly correlated with a slight but substantial decrease in percent entrapment efficiency (p < 0.05). The adjusted determination coefficient (R2 = 0.9880, 0.9558, and 0.9327 for Y1, Y2, and Y3) and predicted deter- mination coefficient (R2 = 0.9516, 0.8179, and 0.9077 for Y1, Y2, and Y3) results were comparative and give higher significance of the model. By rejecting the null hypothesis, “P” values of 0.05 showed a significant interaction between selected inde- pendent variables. For Y1, the model F-value of 132.81 shows that the model is significant. In this study, ethanol, soya lecithin, interaction terms, and polynomial terms significantly im- pact the particle size of liposomes. The Y2 model’s F-value of 35.62 shows that the mod- el is significant. In this study, ethanol, soya lecithin, and polynomial terms significantly impact % entrapment effi- ciency. The Y3 model’s F-value of 56.46 shows that the mod- el is significant. Only ethanol significantly impacts PDI in this study. The “P” values for particle size, percentage entrap- ment efficiency, and PDI were 0.0010, 0.0071, and 0.0109, respectively. For 32 factorial design models, the sum of “P” and the adjusted R2 values shows a substantial synergistic association between both independent variables at P < 0.05. TG7 showed the lowest cumulative percentage of drug release (83.676%). TG4 showed the highest cumula- tive percentage of drug release; however, the carmustine API solution showed the lowest cumulative percentage drug release. In the ex-vivo carmustine permeation study, maximum drug permeation was observed in the case of TG4; however, TG7 showed minimum drug permeation across the goat nasal mucosa. TG4 showed the highest drug permeation, and carmustine API solution showed the lowest carmustine permeation across goat nasal mu- cosa From the in vitro dissolution and ex-vivo Carmustine permeation study, it was observed that the final optimized Table 3. Pharmacokinetic parameters obtained from drug concentration in brain-time profile curve in Wistar rats. Sr. No. Formulations Route of Administration Cmax (µg/ml) Tmax (h) AUC0→t (µg/h/ml) AUC0→∞ (µg/h/ml) 01 TG4 Nasal 7.60±0.49 1 1.38±0.20 14.65±0.25 02 Marketed Formulation Intravenous 3.93±0.36 2 0.45±0.21 2.46±0.24 (Carmustine for injection USP 100 MG) Figure 8. Drug concentration in brain time profile of carmustine formulation (TG4) and marketed formulation in rats 36 Acta Chim. Slov. 2024, 71, 26–38 Mali and Bhanwase: Brain Targeted Drug Delivery System of Carmustine: ... TG4 preparation showed maximum cumulative % Car- mustine release and maximum Carmustine permeation. Histopathological study revealed that the intranasal administration of flexible liposomes embedded thermore- versible gel is safe.42 The crucial target of the current work was to increase brain bioavailability of carmustine optimizing flexible li- posomes embedded in situ nasal gel formulation. The BBB may diverge drug concentration-time profile in the brain significantly. Medicines in the brain are distributed and eliminated through various mechanisms, including dif- fusion, bulk flow of cerebrospinal fluid, extracellular-in- tracellular exchange, brain extracellular fluid, and metab- olism in brain tissue. To predict the desired therapeutic effect, it is crucial to establish a link between the medi- cine-distribution processes throughout the brain, and the amount of the medicine in the brain. The outcome of a medicine that targets the brain could be reliably predicted by mathematical models depicting medicine transport via the brain capillary system, medicine transport over BBB, intra-extracellular interchange, medicine binding inside the brain, and medicine metabolism in the brain.48,50,51 TG4 showed higher permeation of carmustine with higher Cmax. Pharmacokinetic studies showed the importance of the nasal route administration of carmustine and signifi- cance of the TG4 to deliver carmustine effectively to brain. 7. Conclusion Intranasal route of drug administration is considered as an effective methodology for transporting the therapeu- tic agents to the brain in managing brain tumors. Carmus- tine-embedded flexible liposomes-based in situ nasal gel formulations were developed and optimized. TG4 nasal gel of carmustine can improve carmustine delivery to the brain by increasing gel retention in the nasal membrane, and therefore increasing carmustine transport. Hence, in the current study, the TG4 was formulated, and assessed for its brain-targeting potential. The in vivo pharmacoki- netics of TG4 showed more amount of carmustine is de- livered to brain via nasal route. TG4 was proven to be safe for nasal mucosal tissue, and would be a safe, reliable, and convenient method of treating GBM. List of abbreviations TEM: – Transmission electron microscopy AFM: – Atomic Force Microscopy GBM: – Glioblastoma BBB: – Blood-Brain Barrier CNS: – Central Nervous System FTIR: – Fourier Transform Infrared Spectrophotometer PDI: – Polydispersity Index SD: – Standard Deviation SPBS: – Saline Phosphate Buffer Solution IV: – Intravenous Author contribution statement Each mentioned contributor contributed substan- tially to this manuscript’s idea and writing. Declarations Conflict of Interest The authors declare that they have no conflict of in- terest. 8. 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Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Zdravljenje gliomov ostaja zahtevna naloga. Karmustin je zdravilo, ki se uporablja pri zdravljenju gliomov. Fleksibilne liposome, vgrajene v in situ termoreverzibilen nazalni gel, so proučevali z vidika sproščanje karmustina in vitro, per- meacije karmustina ex vivo in kinetike sproščanja karmustina. Med histološko analizo so ugotovili, da so epitelne plasti nosnega tkiva intaktne in nepoškodovane. Intranazalna aplikacija optimiziranih fleksibilnih liposomov, vgrajenih v naz- alni gel, je v primerjavi z intravensko aplikacijo karmustina pokazala višje Cmax (približno dvakrat), AUC0→t (približno trikrat), AUC0→∞ (približno šestkrat) in nižji Tmax (1 h) v možganih. Pričujoča študija dokazuje, da je termoreverzibilni intranazalni gel karmustina s fleksibilnimi liposomi izboljšal ciljno absorpcijo karmustina v možganih prek nazalnega dostavnega sistema in bi lahko predstavljal zanesljiv in učinkovit dostavni sistem za karmustin pri zdravljenju gliomov. 39Acta Chim. Slov. 2024, 71, 39–46 Xue et al.: Synthesis, Characterization, Crystal Structures ... DOI: 10.17344/acsi.2023.8027 Scientific paper Synthesis, Characterization, Crystal Structures and Urease Inhibition of Some Thiosemicarbazones Ling-Wei Xue*, Qiao-Ru Liu and Yong-Jun Han School of Chemical and Environmental Engineering, Pingdingshan University, Pingdingshan 467000, P.R. China * Corresponding author: E-mail: pdsuchemistry@163.com Received: 01-25-2023 Abstract Six new thiosemicarbazones were prepared and structurally characterized by elemental analysis, NMR and IR spec- tra and single-crystal X-ray diffraction. The compounds were evaluated for their Jack bean urease inhibitory activities. Among the compounds, those with hydroxyl and chloro substituent groups have effective activity with IC50 values of 1.8-12.7 μmol L–1. Docking simulations were performed to insert the molecules of the compounds into the active urease site determined by the crystal structure to determine their probable binding modes. Keywords: Thiosemicarbazone, Crystal structure, Urease inhibition, Molecular docking study 1. Introduction Urease is an enzyme that catalyzes the hydrolysis of urea to NH3 and CO2.1 This process has negative effects in the fields of medicine and agriculture.2 Therefore, it is nec- essary to control the activity of urease. In recent years, a variety of urease inhibitors have been reported, including acetohydroxamic acid, 1,4-benzoquinone, humic acid, and some natural products.3 Schiff base compounds have in- teresting biological activities.4 Aroylhydrazones are a kind of special Schiff base compounds with –C(O)–NH– N=CH– groups, which can be easily prepared by conden- sation of carbonyl-containing compounds with acroylhy- drazines. The compounds have attracted considerable attention due to their diverse biological activities, such as antibacterial,5 antifungal,5c,6 antitumor,7 anti-inflammato- ry,8 and cytotoxic.9 Thiosemicarbazones have broad bio- logical activity.10 Recent reports indicated that hydrazone compounds have urease inhibitory activity.11 As a contin- uation of the work to explore new urease inhibitors, six new thiosemicarbazones were prepared and evaluated for their urease inhibitory activities in this work. 2. Experimental 2. 1. Materials and Measurements 3-Chlorobenzaldehyde, 2-chloro-4-fluorobenzalde- hyde, 2,3-difluorobenzaldehyde, 3-methoxybenzaldehyde, 4-pyridinecarboxaldehyde, 5-fluoro-2-hydroxybenzalde- hyde and AR grade thiosemicarbazide were from Sig- ma-Aldrich, and were used as received. Elemental analyz- es were performed using a Perkin-Elmer 240C elemental analyzer. IR spectra were recorded using a Jasco FT/IR- 4000 spectrometer with KBr pellets. 1H and 13C NMR spectra were recorded with a Bruker 300 MHz instrument. X-ray diffraction was performed using a Bruker APEX II CCD area diffractometer with MoKα radiation. 2. 2. General Method for Synthesizing the Compounds Equimolar amounts (1.0 mmol each) of aldehyde and thiosemicarbazide were dissolved in methanol (30 mL). The mixtures were stirred at room temperature for 30 minutes to obtain a clear solution. Slow evaporation of the solution in air over several days resulted in the formation of single crystals of X-ray quality. 3-Chlorobenzaldehyde thiosemicarbazone (1) Colorless crystals. Yield: 0.19 g, 90%. Anal. calcd. for C8H8ClN3S: C, 44.97; H, 3.77; N, 19.66; found C, 45.21; H, 3.86; N, 19.53%. IR (ν, cm–1): 3438 (m), 3337 (w), 3066 (w), 2927 (w), 2850 (w), 1640 (s), 1552 (s), 1450 (m), 1408 (w), 1370 (w), 1306 (m), 1226 (w), 1154 (w), 1087 (w), 938 (w), 781 (w), 748 (w), 705 (w), 646 (w), 528 (w). 1H NMR (300 MHz, DMSO-d6): δ 11.52 (s, 1H, NH), 8.27 (s, 1H, 40 Acta Chim. Slov. 2024, 71, 39–46 Xue et al.: Synthesis, Characterization, Crystal Structures ... ArH), 8.21 (s, 1H, CH=N), 8.08 (s, 1H, NH2), 8.03 (s, 1H, NH2), 7.67 (d, 1H, ArH), 7.45-7.43 (m, 2H, ArH). 13C NMR (126 MHz, DMSO-d6): δ 181.22, 164.34, 135.27, 133.17, 130.89, 130.32, 126.78, 125.62. 2-Chloro-4-fluorobenzaldehyde thiosemicarbazone (2) Colorless crystals. Yield: 0.22 g, 96%. Anal. calcd. for C8H7ClFN3S: C, 41.47; H, 3.05; N, 18.14; found C, 41.38; H, 3.12; N, 17.98%. IR (ν, cm–1): 3417 (m), 3252 (m), 3150 (m), 3032 (w), 2985 (w), 2808 (w), 1602 (s), 1539 (s), 1480 (m), 1366 (w), 1293 (m), 1239 (m), 1103 (w), 1035 (w), 913 (w), 857 (m), 815 (w), 705 (w), 617 (w), 515 (w). 1H NMR (300 MHz, DMSO-d6): δ 11.62 (s, 1H, NH), 8.43 (s, 1H, NH2), 8.38 (s, 1H, NH2), 8.31 (s, 1H, ArH), 8.17 (s, 1H, CH=N), 7.52 (d, 1H, ArH), 7.30 (d, 1H, ArH). 13C NMR (126 MHz, DMSO-d6): δ 178.22, 162.89, 160.35, 145.46, 127.12, 126.03, 118.85, 117.91. 2,3-Difluorobenzaldehyde thiosemicarbazone (3) Colorless crystals. Yield: 0.19 g, 88%. Anal. calcd. for C8H7F2N3S: C, 44.64; H, 3.28; N, 19.52; found C, 44.55; H, 3.21; N, 18.63%. IR (ν, cm–1): 3434 (m), 3252 (m), 3150 (m), 3015 (w), 2977 (w), 2863 (w), 1594 (s), 1518 (s), 1471 (s), 1378 (w), 1289 (m), 1209 (w), 1107 (w), 1056 (m), 938 (w), 820 (w), 773 (w), 710 (w), 625 (w), 523 (m). 1H NMR (300 MHz, DMSO-d6): δ 11.65 (s, 1H, NH), 8.37 (s, 1H, NH2), 8.28 (s, 1H, NH2), 8.15 (s, 1H, CH=N), 8.08 (q, 1H, ArH), 7.45 (d, 1H, ArH), 7.23 (d, 1H, ArH). 13C NMR (126 MHz, DMSO-d6): δ 179.13, 163.46, 161.46, 137.16, 133.76, 129.25, 116.87, 115.14. 3-Methoxybenzaldehyde thiosemicarbazone (4) Colorless crystals. Yield: 0.18 g, 86%. Anal. calcd. for C9H11N3OS: C, 51.65; H, 5.30; N, 20.08; found C, 51.50; H, 5.33; N, 20.16%. IR (ν, cm–1): 3396 (m), 3277 (m), 3155 (m), 3003 (w), 2969 (w), 2837 (w), 1594 (s), 1534 (s), 1467 (m), 1361 (w), 1277 (s), 1162 (w), 1095 (m), 1044 (m), 930 (w), 836 (w), 777 (w), 689 (w), 617 (w), 557 (m). 1H NMR (300 MHz, DMSO-d6): δ 11.44 (s, 1H, NH), 8.24 (s, 1H, NH2), 8.07 (s, 1H, NH2), 8.03 (s, 1H, CH=N), 7.46 (s, 1H, ArH), 7.33-7.27 (m, 1H, ArH), 6.98 (d, 1H, ArH), 3.82 (s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6): δ 177.67, 147.86, 145.94, 139.47, 120.73, 118.91, 118.13, 112.81, 55.85. Isonicotinaldehyde thiosemicarbazone (5) Colorless crystals. Yield: 0.15 g, 83%. Anal. calcd. for C7H8N4S: C, 46.65; H, 4.47; N, 31.09; found C, 46.53; H, 4.55; N, 30.92%. IR (ν, cm–1): 3417 (m), 3265 (m), 3155 (w), 2947 (w), 2796 (w), 1598 (s), 1539 (s), 1450 (m), 1412 (w), 1361 (w), 1293 (s), 1243 (w), 1171 (w), 1108 (m), 1065 (m), 989 (w), 921 (w), 879 (m), 828 (w), 748 (w), 634 (w), 516 (m). 1H NMR (300 MHz, DMSO-d6): δ 11.70 (s, 1H, NH), 8.61 (q, 2H, PyH), 8.41 (s, 1H, NH2), 8.23 (s, 1H, NH2), 8.02 (s, 1H, CH=N), 7.80 (q, 2H, PyH). 13C NMR (126 MHz, DMSO-d6): δ 178.55, 149.71, 141.67, 139.27, 121.16. 5-Fluoro-2-hydroxybenzaldehyde thiosemicarbazone (6) Colorless crystals. Yield: 0.20 g, 94%. Anal. calcd. for C8H8FN3OS: C, 45.06; H, 3.78; N, 19.71; found C, 45.15; H, 3.83; N, 19.58%. IR (ν, cm–1): 3425 (m), 3315 (m), 3129 (w), 2985 (w), 2880 (w), 1615 (s), 1534 (m), 1488 (m), 1445 (m), 1357 (w), 1264 (w), 1163 (m), 1078 (m), 951 (w), 857 (w), 701 (w), 638 (w), 540 (w). 1H NMR (300 MHz, DM- SO-d6): δ 11.43 (s, 1H, NH), 9.92 (s, 1H, OH), 8.31 (s, 1H, NH2), 8.15 (s, 1H, NH2), 8.11 (s, 1H, CH=N), 7.89 (s, 1H, ArH), 7.05 (d, 1H, ArH), 6.85 (d, 1H, ArH). 13C NMR (126 MHz, DMSO-d6): δ 177.87, 156.65, 154.79, 152.63, 137.69, 121.74, 117.51, 111.57. 2. 3. Urease Inhibitory Activity Assay Urease inhibitory activity was measured according to the method described in the literature.12 The test mix- ture containing 75 μL of Jack bean urease and 75 μL of test- ed compounds at various concentrations (dissolved in DMSO) was pre-incubated on a 96-well assay plate for 15 minutes. Acetohydroxamic acid was used as a reference. Then 75 μL of phosphate buffer with a pH of 6.8 contain- ing phenol red (0.18 mmol L–1) and urea (400 mmol L–1) were added and incubated at room temperature. The reac- tion time required for sufficient ammonium carbonate to form to raise the pH of the phosphate buffer from 6.8 to 7.7 was measured using a microplate reader (560 nm), with the endpoint determined by the color change of the phenolred indicator. 2. 4. Crystal Structure Determination The diffraction intensities for the compounds were collected at 298(2) K using a Bruker Apex II diffractometer with MoKα radiation (λ = 0.71073 Å). The collected data were reduced using SAINT,13 and multi-scan absorption correction was performed using SADABS.14 The struc- tures of the compounds were solved using direct methods and refined against F2 using the full-matrix least-squares method with SHELXTL.15 All non-hydrogen atoms were refined anisotropically. The amino and water H atoms in the compounds were localized using Fourier difference maps and isotropically refined, limiting the N–H and H···H distances to 0.90(1) and 1.43(2) Å, respectively. The remaining hydrogen atoms were placed at calculated posi- tions and restricted to their parent atoms. The crystallo- graphic data for the compounds are summarized in Tables 1 and 2. CCDC-2049283 (1), 2049284 (2), 2049285 (3), 2049286 (4), 2049288 (5), 2049289 (6) contain the supple- mentary crystallographic data for this work. These data can be obtained free of charge from http://www.ccdc.cam. ac.uk/const/retrieving.html or from the Cambridge Crys- tallographic Data Centre (CCDC), 12 Union Road, Cam- bridge CB2 1EZ, UK; fax: +44(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk. 41Acta Chim. Slov. 2024, 71, 39–46 Xue et al.: Synthesis, Characterization, Crystal Structures ... Table 1. Crystallographic and experimental data for the compounds 1–3. Compound 1 2 3 Formula C8H8ClN3S C8H7ClFN3S C8H7F2N3S Mr 213.7 231.7 215.2 Crystal shape/color block/colorless block/colorless block/colorless Crystal size (mm3) 0.18 × 0.17 × 0.17 0.30 × 0.27 × 0.27 0.32 × 0.28 × 0.27 Crystal system Monoclinic Triclinic Monoclinic Space group P21/c P-1 P21/c a (Å) 12.944(3) 6.1342(8) 11.4737(7) b (Å) 8.149(2) 7.352(1) 11.6382(8) c (Å) 10.454(2) 12.348(2) 6.9680(5) α (°) 90 113.277(2) 90 β (°) 105.579(2) 93.331(2) 102.820(2) γ (°) 90 93.039(2) 90 V (Å3) 1013.0(4) 518.9(1) 929.2(1) Z 4 2 4 Dc (g cm–3) 1.401 1.483 1.539 µ (Mo-Kα) (mm-1) 0.539 0.546 0.340 F(000) 440 236 440 Reflections collected 7283 4565 8793 Unique reflections 1795 1924 1731 Observed reflections (I ≥ 2σ(I)) 924 1298 1461 Parameters 127 136 136 Restraints 4 4 4 Goodness-of-fit on F2 1.033 1.067 1.085 R1, wR2 [I ≥ 2σ(I)]a 0.0685, 0.1302 0.0533, 0.1089 0.0321, 0.0790 R1, wR2 (all data)a 0.1533, 0.1660 0.0910, 0.1244 0.0413, 0.0841 aR1 = Fo – Fc/Fo, wR2 = [∑ w(Fo2 – Fc2)/∑ w(Fo2)2]1/2 Table 2. Crystallographic and experimental data for the compounds 4–6. Compound 4 5 6 Formula C9H11N3OS C7H8N4S C8H8FN3OS Mr 209.3 180.2 213.2 Crystal shape/color block/colorless block/colorless block/colorless Crystal size (mm3) 0.31 × 0.28 × 0.27 0.22 × 0.20 × 0.20 0.19 × 0.18 × 0.18 Crystal system Monoclinic Monoclinic Monoclinic Space group P21/c P21/n C2/c a (Å) 11.8192(9) 7.2403(4) 28.017(2) b (Å) 5.6785(5) 13.9456(7) 6.914(2) c (Å) 15.240(1) 8.4144(5) 19.586(2) α (°) 90 90 90 β (°) 90.248(2) 90.863(2) 92.162(2) γ (°) 90 90 90 V (Å3) 1022.8(1) 849.51(8) 3791.0(12) Z 4 4 16 Dc (g cm–3) 1.359 1.409 1.494 µ (Mo-Kα) (mm–1) 0.287 0.328 0.326 F(000) 440 376 1760 Reflections collected 8581 7890 10005 Unique reflections 1803 1576 3513 Observed reflections (I ≥ 2σ(I)) 1701 1390 2788 Parameters 137 118 273 Restraints 4 4 8 Goodness-of-fit on F2 1.086 1.055 1.156 R1, wR2 [I ≥ 2σ(I)]a 0.0748, 0.1839 0.0313, 0.0837 0.0532, 0.1319 R1, wR2 (all data)a 0.0773, 0.1854 0.0364, 0.0876 0.0714, 0.1418 aR1 = Fo – Fc/Fo, wR2 = [∑ w(Fo2 – Fc2)/∑ w(Fo2)2]1/2 42 Acta Chim. Slov. 2024, 71, 39–46 Xue et al.: Synthesis, Characterization, Crystal Structures ... 3. Results and Discussion 3. 1. Chemistry Compounds 1–6 were synthesized by reaction of equimolar amounts of thiosemicarbazide with various al- dehydes in methanol at room temperature (Scheme 1) in high yields (over 90%). All compounds crystallized as well-formed single crystals, which were soluble in metha- nol, ethanol, acetonitrile, and chloroform. The C, H, and N analyzes are in agreement with the chemical formulae ob- tained from X-ray analysis of the single crystals. Scheme 1. Synthesis of the compounds. 3. 2. IR and 1H NMR Spectra The characteristic intense bands in the range 1594– 1640 cm–1 are assigned to the ν(C=N) vibrations.16 In the spectrum of compound 6, the absorption at 3425 cm–1 can be assigned to the hydrogen-bonded phenol group. The sharp band at 3315 cm–1 can be assigned to the ν(N–H) vibration. In the 1H NMR, the peaks for NH protons are in the range of δ = 11.43–11.70 ppm. The imine CH protons in the range of δ = 8.02–8.21 ppm confirm the formation of the compounds. The signals of aromatic protons are found with different frequencies in their respective re- gions, confirming their respective substitution patterns. 3. 3. Crystal Structure Description The molecular structures of compounds 1–6 are shown in Figure 1. Selected bond lengths are listed in Table 3. The asymmetric unit of compound 6 consists of two in- dependent molecules. All molecules of the compounds adopt the E-configuration with respect to the methylidene units. The distances of the methylidene bonds, which are between 1.26 and 1.29 Å, confirm that they are typical double bonds. The shorter distances of the C–N bonds and Table 3. Selected bond lengths (Å) for the compounds 1–6. 1 C7–N1 1.267(5) N1–N2 1.367(5) N2–C8 1.332(5) C8–S1 1.688(4) C8–N3 1.302(5) 2 C7–N1 1.271(4) N1–N2 1.375(3) N2–C8 1.342(3) C8–S1 1.690(3) C8–N3 1.313(3) 3 C7–N1 1.273(2) N1–N2 1.373(2) N2–C8 1.343(2) C8–S1 1.690(2) C8–N3 1.315(2) 4 C7–N1 1.283(5) N1–N2 1.370(5) N2–C8 1.347(5) C8–S1 1.691(4) C8–N3 1.298(6) 5 C7–N1 1.274(2) N1–N2 1.366(2) N2–C8 1.356(2) C8–S1 1.676(2) C8–N3 1.320(2) 6 C7–N1 1.284(4) N1–N2 1.382(3) N2–C8 1.340(4) C8–S1 1.692(3) C8–N3 1.315(4) C15–N4 1.284(4) N4–N5 1.378(4) N5–C16 1.343(4) C16–S2 1.690(3) C16–N6 1.315(4) the longer distances of the C=O bonds for the –C(O)– NH– units than usual, suggest the presence of conjugation effects in the molecules. The bond lengths in the com- pounds are comparable to each other, and are within nor- mal values.17 The crystal structures of the compounds are stabilized by intermolecular hydrogen bonds (Table 4). 3. 4. Pharmacology and Molecular Docking Study The inhibitory effect of Jack bean urease was meas- ured three times in parallel. The percentage of inhibition at a concentration of 100 μmol L–1 for the compounds against urease are shown in Table 5. Compound 6 has significant inhibition of urease with IC50 value of 1.8 μmol L–1. Com- pound 1 has moderate activity with IC50 value of 12.7 μmol L–1. However, the remaining compounds exhibit weak activity. Acetohydroxamic acid (AHA) was used as a reference with an IC50 value of 36.3 μmol L–1. The results show that the compound with the o-OH group has the strongest urease inhibitory activity. The compounds with a chloro group also have urease inhibitory activity. Com- pound 1 has an m-Cl group, while compound 2 has an o-Cl group. The fluorine group does not appear to have positive effect on urease inhibition. 43Acta Chim. Slov. 2024, 71, 39–46 Xue et al.: Synthesis, Characterization, Crystal Structures ... Figure 1. Perspective views of the molecular structures of the compounds with the atomic labeling scheme. The thermal ellipsoids are drawn with a probability of 30 %. Table 4. Hydrogen bond distances (Å) and bond angles (°) for the compounds 1–6. D–H∙∙∙A d(D–H) d(H∙∙∙A) d(D∙∙∙A) Angle (D–H∙∙∙A) 1 N3–H3A∙∙∙S1#1 0.90(1) 2.43(1) 3.318(4) 173(4) N2–H2A∙∙∙S1#2 0.90(1) 2.56(2) 3.404(4) 156(4) 2 N2–H2∙∙∙S1#3 0.90(1) 2.46(1) 3.346(2) 171(3) N3–H3A∙∙∙S1#4 0.90(1) 2.48(1) 3.366(3) 177(3) 3 N3–H3A∙∙∙S1#5 0.90(1) 2.53(1) 3.412(2) 178(2) N2–H2∙∙∙S1#6 0.90(1) 2.51(1) 3.356(2) 158(2) 4 N2–H2∙∙∙S1#7 0.90(1) 2.50(2) 3.359(4) 159(5) N3–H3B∙∙∙S1#8 0.90(1) 2.55(2) 3.430(4) 169(6) N3–H3A∙∙∙S1#9 0.90(1) 2.72(4) 3.393(4) 133(5) 5 N3–H3A∙∙∙S1#10 0.90(1) 2.63(1) 3.500(1) 173(2) N2–H2∙∙∙N4#11 0.90(1) 2.09(1) 2.958(2) 162(2) 6 N6–H6B∙∙∙O1 0.90(1) 2.03(1) 2.908(4) 168(4) N3–H3B∙∙∙O2#12 0.90(1) 2.13(2) 2.994(4) 163(4) N3–H3A∙∙∙S2 0.90(1) 2.65(3) 3.311(3) 132(3) N6–H6A∙∙∙S1#9 0.90(1) 2.61(3) 3.289(3) 134(3) N5–H5∙∙∙S2#13 0.90(1) 2.5191) 3.393(3) 168(4) N2–H2∙∙∙S1#14 0.90(1) 2.49(2) 3.356(3) 162(4) O2–H2A∙∙∙N4 0.82 1.93 2.642(3) 145(4) O1–H1∙∙∙N1 0.82 1.95 2.666(3) 145(4) Symmetry codes: #1: 1 – x, 1/2 + y, 3/2 – z; #2: 1 – x, –1/2 + y, 3/2 – z; #3: 1 – x, 1 – y, – z; #4: 2 – x, 2 – y, – z; #5: 1 – x, 1 – y, 2 – z; #6: x, 3/2 – y, –1/2 + z; #7: – x, 3 – y, 1 – z; #8: – x, –1/2 + y, 3/2 – z; #9: x, –1 + y, z; #10: 2 – x, – y, 2 – z; #11: 3/2 – x, –1/2 + y, 1/2 – z; #12: x, 1 + y, z; #13: 1/2 – x, –1/2 – y, 1 – z; #14: – x, y, 1/2 – z. Table 5. Inhibition of urease by the materials tested. Tested materials Percentage Inhibition# IC50 (μmol L–1) 1 86.3 ± 2.2 12.7 ± 1.3 2 57.2 ± 2.5 83.2 ± 2.7 3 45.5 ± 1.8 – 4 49.7 ± 1.6 – 5 55.3 ± 2.3 – 6 97.0 ± 2.4 1.8 ± 1.3 Acetohydroxamic acid 85.8 ± 3.2 36.3 ± 3.5# The concentration of the tested material is 100 μmol L–1. The molecular docking study was performed to in- vestigate the binding effects between compounds 1 and 6 with the active sites of urease. Figures 2 and 3 show the binding models of the compounds to the active site of the urease enzyme. The docking scores are –5.43 for 1 and –5.87 for 6. For comparison, the docking score for AHA is –5.01. The values of the docking scores are approximately consistent with the inhibitory activities observed in the ex- periment. The docking score values are roughly consistent with the inhibitory activities observed in the experiment. The molecules of the compounds bind to the urease via hydrogen bonds. 4. Conclusion In the present study, the syntheses, structures and urease inhibitory activity of six thiosemicarbazones are described. The structures of the compounds were investi- gated by single crystal X-ray diffraction. All compounds 44 Acta Chim. Slov. 2024, 71, 39–46 Xue et al.: Synthesis, Characterization, Crystal Structures ... Figure 2. Binding mode of 1 with Jack bean urease. Figure 3. Binding mode of 6 with Jack bean urease. were analyzed for their urease inhibitory activities. Two of the compounds have effective activities. The urease inhibi- tory activities and the molecular docking studies of the compounds against Jack bean urease suggest that hydroxyl and chloro groups in the aromatic rings of the compounds may be necessary for the exploration of new urease inhib- itors. 5. References 1. (a) L. V. Modolo, A. X. de Souza, L. P. Horta, D. P. Araujo, A. de Fatima, J. Adv. Res. 2015, 6, 35–44; DOI:10.1016/j.jare.2014.09.001 (b) H. Cantarella, R. Otto, J. R. Soares, A. G. D. 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Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek V prispevku je predstavljena sinteza šestih novih tiosemikarbazonov, ki so tudi strukturno okarakterizirani z elementno analizo, NMR in IR spektroskopijo, ter rentgensko difrakcijo analizo na monokristalih. Raziskana je tudi njihova inhib- itorna aktivnosti na Jack bean ureazo. Med pripravljenimi spojinami imajo največjo aktivnost tiste z vezanimi hidroksil- nimi in kloro skupinami z IC50 vrednostmi 1,8−12,7 μmol L–1. Avtorji so izvedli tudi simulacije prileganja teh molekul na aktivno mesto urease, določenega na osnovi kristalne strukture, z namenom da bi določili njihov najbolj verjeten način vezave. 47Acta Chim. Slov. 2024, 71, 47–55 Keshtiban et al.: Nano MgCuAl2O5: Synthesis by Sol-Gel Auto-Combustion ... DOI: 10.17344/acsi.2023.8133 Scientific paper Nano MgCuAl2O5: Synthesis by Sol-Gel Auto-Combustion Process, Characterization and Reusable Heterogeneous Catalyst for the Hantzsch 1,4-Dihydropyridine Reaction Marzieh Mahmoodi Keshtiban,1 Abbas Nikoo2,* and Bakhshali Massoumi1,* 1 Department of Chemistry, Payame Noor University, Tehran, Iran 2 Department of Organic Chemistry, Faculty of chemistry, Urmia University, Urmia, Iran * Corresponding author: E-mail: b_massoumi@pnu.ac.ir a.nikoo@urmia.ac.ir Received: 03-11-2023 Abstract For the first time, MgCuAl2O5 heterogeneous nanocatalysts were prepared by sol-gel auto-combustion method, which displayed great yield and suitable activity for the preparation of 1,4-DHP derivatives with high efficiency in green solvent ethanol/water (1:1) at 80 °C under one pot condition. The synthesis materials were separated in short reaction times with excellent yield (80–95) %. Moreover, the synthesized nanocatalyst was easily retrieved and continuously reused for six re- actions without a noticeable significant loss of proficiency. The solid catalyst was confirmed by XRD, BET, FTIR, FESEM and EDX, and the substituted 1,4-dihydropyridines were characterized by melting point, 1H and 13C NMR. Keywords: Heterogeneous catalyst, Sol-Gel auto-combustion, Recyclable, One-pot synthesis, 1,4-Dihydropyridine. 1. Introduction In modern synthetic organic chemistry, one-pot Multi-component reactions (MCR) are a powerful syn- thetic tool that produces complex heterocyclic molecules and pharmaceutical compounds they have many advan- tages over classical multistep reactions, including easy handling and efficiency, no need for excess separation steps and economy of materials, energy and time.1–3 In the path of achieving green chemistry, the synthe- sis process of organic compounds has undergone changes in recent decades. It is progressing towards being environ- mentally friendly, although laboratory research for the in- novation of new methods is expanding consequently.4 The design and development of new synthetic methods that do not have a negative impact on humans and the environ- ment, as well as the recycling of materials, are among the goals of green chemistry.5,6 The usage of heterogeneous catalysts in one-pot conditions is one of the important fac- tors for achieving green chemistry.7 Heterogeneous nanocatalysts have attracted atten- tion in the past years in various chemical industries and research centers due to their properties such as selectivity, activity and stability. The most obvious feature is easy recy- cling, which is confirmed by the exponential increase in the number of patents and technologies.8–10 Arthur Rudolf Hantzsch opened a new horizon in the synthesis of drugs by synthesizing 1,4-dihydropyridine compounds, which are useful and effective antiviral,11 an- tidepressant,12 anti-inflammatory,13 anti-mutagenic,14 an- ti-diabetic,15 anti-hypertensive,16 sedative,17 vasodilator,18 and antibacterial19 drugs, research to improve and modify Hantzsch's method by organic chemists is expanding. Several methods with various catalysts and in the different reaction conditions for the synthesis of 1,4-dihy- dropyridine have been reported in scientific research arti- cles such as ZnFe2O4,20 La1–xSrxMn1–yZnyO3,21 Fe3O4@ Co(BDC)NH2,22 Fe2O3/ZrO2,23 Fe3O4@SiO2@ ADMPT/ H6P2W18O62,24 Sulphated Tin Oxide,25 Fe3O4 supported glutathione,26 sulphated poly borate,27 DBU,28 SBA–DAB- CO,29 SiO2,30 trimethylamine,31 Fe-CuZSM-5,32 CoFe2O4@ SiO2-NH2-CoII33 and graphene oxide/Cu NPs.34 However, many reported methods suffer from long reaction times, low efficiencies, moisture sensitivity, difficult synthesis processes, as well as expensive materials used and non-re- cyclable catalysts.35 In this research, MgCuAl2O5 heterogeneous nano- catalyst was synthesized for the first time by sol–gel au- 48 Acta Chim. Slov. 2024, 71, 47–55 Keshtiban et al.: Nano MgCuAl2O5: Synthesis by Sol-Gel Auto-Combustion ... to-combustion method with a simple, cheap and non-tox- ic preparation method. This solid nanocatalyst does not lose its effect in the long term and is stable and does not deteriorate under the influence of heat, air and moisture. Next, by one-pot reaction, 1,4-DHP derivatives were pre- pared in green solvent ethanol/water (1:1) with high effi- ciency and short duration. It is worth noting that the cata- lytic property was maintained in six consecutive reactions, and the MgCuAl2O5 nanocatalyst can be separated after the end of the reaction and reused. 2. Experimental Section 2. 1. Materials Ethyl acetoacetate, Urea, Ammonium acetate, Mg(NO3)2·4H2O, Cu(NO3)2·3H2O, Al(NO3)3·9H2O, Alde- hyde derivatives, and the solvents were used during the reaction were purchased from reputable companies such as Merck and Sigma-Aldrich, which were used without the need for purification. D500 diffractometer (Siemens) was employed for the pattern X-ray diffraction (XRD) of the MgCuAl2O5 heterogeneous nanocatalysts at 25 °C using Cu Kα radiation (λ = 0.154 nm) in a scanning rate of 2° per min, at 40 kV. Data have been collected with a step size of 0.05° degrees and a temperature range of 10–80°, nominal time per step of 1 s, and slit width of 5 nm to confirm the type of structure and check the purity of synthesized nan- oparticles. Information about the morphology and size of the MgCuAl2O5 heterogeneous nanocatalysts was ob- tained with the device field emission scanning electron microscopy (FESEM, TESCAN MIRA III) with 20 kV ac- celerating voltage. X-ray electron dispersive spectroscopy (EDX) using a SAMX detector has been employed to ana- lyze and specify the relative abundance of elements. Spec- troscopy Fourier transforms infrared (FTIR) was used on a Thermo AVATAR spectrometer in the range of 4000–400 cm−1 with a KBr disk. Specific BET surface area (SBET) amounts were computed with 0.05 < P/P0 < 0.30. The total pore volume (Vt) was estimated from the adsorption data at a P/P0 value of 0.99. To determine the porosity of the catalyst surface, spectroscopy BET in a Micrometrics Gemini surface area analyzer was used under liquid nitro- gen. Catalyst capacity was specified by atomic absorption method with BEL SORP MINI II absorption appliance. Low-temperature N2 adsorption–desorption isotherms of the MgCuAl2O5 heterogeneous nanocatalysts were ob- tained on a BEL PREP VAC II analyzer. The MgCuAl2O5 heterogeneous nanocatalysts were outgassed at 450 °C overnight Prior to N2 adsorption. The multipoint Brunau- er–Emmett–Teller (BET) method was operated for the to- tal specific surface area. Continuously, the progress pro- cess of the reaction was prosecuted by TLC plates (silica-gel PolyGram SILG/UV254) and relevant tests. Finally, the reaction completion time was determined. By using differ- ent equipment and spectroscopic techniques, the obtained products were identified. Melting points were recorded by open capillary on Electrothermal 9200, and contrasted with reference models. 1H and 13C spectra were registered with a Bruker Avance-300 MHz spectrometer. Chemical shifts were described in ppm using TMS as an internal standard, and CDCl3 was used as solvent at room temper- ature. All yields mention separate products. 2. 2. Synthesis of MgCuAl2O5 NPs A sol–gel auto-combustion method with a ratio of Mg(NO3)2·6H2O (25 mmol), Al(NO3)3·9H2O (50 mmol), and Cu(NO3)2·3H2O (25 mmol) at the presence of Urea as a fuel for the synthesis of MgCuAl2O5 NPs was applied. In 1000 ml distilled water, 6.40 g Mg(NO3)2·6H2O, 18.75 g Al(NO3)3·9H2O and 6.05 g Cu(NO3)2·3H2O were dis- solved. 25 g of Urea was dissolved in 250 ml of distilled water then added to the mixture, and stirred for 3 h. The formation of a gel happened by evaporation of the solu- tion. The obtained gel was heated at 80 °C overnight by an oven and calcined at 750 °C for 4 h in air. 2. 3. Preparation of 1,4-Dihydropyridines Considering the time required for each reaction, a mixture of aryl aldehyde (2.5 mmol), NH4OAc (3.75 mmol), ethyl acetoacetate (5 mmol) and MgCuAl2O5 na- nocatalyst (37.5 mg) in ethanol/water (1:1) was heated un- der 80 °C. After the completion of the reaction, as moni- tored by TLC (ethyl acetate/petroleum ether 1:3), the reaction temperature was down to 25 °C, and 25 mL of water was added to the reaction mixture. The catalyst was detached for use in the next reaction by ordinary centrifu- gation. The 1,4-DHP derivatives were extracted with ethyl acetate (3 × 25 mL), and the organic layer was dried with anhydrous Na2SO4 (50 g). Ethyl acetate was separated from the product under reduced pressure, and 1,4-DHP derivatives with 80–95% yields were afforded by recrystal- lization of residue from ethanol. 3. Results and Discussion 3. 1. Characterization The structural property of MgCuAl2O5 NPs was ana- lyzed by powder XRD (Figure 1). X-Ray diffraction pat- tern of MgCuAl2O5 NPs proved that the presence of only phase with 2θ = 31.5, 35.6, 37.0, 38.8, 45.0, 49.0, 59.5 and 65.5 after calcination at 750 °C. The data was analyzed in the 2θ degree range from 10° to 80° with the scanning step of 0.5 per sec. The XRD pattern is the replica of the JCPDS pattern with reference code 96-901-6435. This indicates the formation of single-phase pure cubic MgCuAl2O5 NPs crystallites. The crystal size of MgCuAl2O5 NPs was com- puted with the Debye Scherrer equation (Particle Size = 0.9 × λ/dcosθ), where λ = 1.54060 Å (in the case of Cu Kα), 49Acta Chim. Slov. 2024, 71, 47–55 Keshtiban et al.: Nano MgCuAl2O5: Synthesis by Sol-Gel Auto-Combustion ... d = the full width at half maximum intensity of the peak in radian and θ is the Braggs' angle in degree. The particle size of MgCuAl2O5 NPs was calculated to be about 43 nm. Figure 1. XRD pattern of MgCuAl2O5 NPs By scanning electron microscopy, the size distribu- tion, surface morphology of this particle and particle shape were investigated (FESEM; Figure 2), which showed the nanostructure of MgCuAl2O5 NPs. The FESEM picture shows that the average size of MgCuAl2O5 is about 43 nm, which is thoroughly consistent with the XRD pattern and the number obtained from the Debye-Scherr formula. The state of aggregation of MgCuAl2O5 NPs shows that the materials are crystalline and homogeneous, which indi- cates the dominance of active sites and the effectiveness of catalyst becoming more. The stoichiometry and chemical purity of the MgC- uAl2O5 NPs were checked by energy dispersive X-ray spectroscopy (EDX) studies. The elemental composition of nanoparticles is O, Mg, Cu and Al, which was determined by the EDX spectrum as the just main ingredients of Mg- CuAl2O5 (Figure 3). Figure 4. shows the Fourier transform infrared (FT- IR) spectrum of MgCuAl2O5 NPs. In this spectrum, the Figure 2. FESEM images of MgCuAl2O5 NPs Figure 3. EDX spectrum of MgCuAl2O5 NPs 50 Acta Chim. Slov. 2024, 71, 47–55 Keshtiban et al.: Nano MgCuAl2O5: Synthesis by Sol-Gel Auto-Combustion ... wide peak that emerged in the confine of 3434 cm–1 is re- lated to the stretching disposition of the absorbed water. The stretching vibration of the Al–O band is in 1572 cm–1, while the bending vibration is at 782 cm–1. 1416 and 697 cm–1 are respectively related to the stretching and bending vibrations of the Mg–O band. The stretching vibration of the Cu–O is 1114 cm–1, and the bending vibration of it is 512 cm–1. Figure 5. shown the N2 adsorption-desorption iso- therm of the MgCuAl2O5 NPs. N2 adsorption-desorption isotherms were used to measure the size distribution, spe- cific surface area and pore volume of MgCuAl2O5 NPs at 77 K (ASAP 2010 Micromeritics). The pore size distribu- tion and pore volume were computed by using the BJH method, and the surface area was computed by using the BET equation. In order to evacuate the physisorbed mois- ture, before the BET test, the MgCuAl2O5 nanocatalyst was degassed at 120 °C for 4 h under vacuum. The type IV absorption isotherm (range 2 P/Po 0.990) affirmed the na- ture of the sample, which was mesoporous. The surface area of the catalyst was 6.3636 m2 g–1, and the pore size of 252.31 Å with a pore volume of 0.040139 cm3 g–1. 3. 2. Catalytic Activity In the present work, 20 mol% of MgCuAl2O5 NPs was used throughout the experiments. 2.5 mmol of differ- ent aromatic aldehydes and 3.75 mmol ammonium acetate with 5 mmol of ethyl acetoacetate were stirred at 80 °C in the presence of MgCuAl2O5 nanocatalyst to produce the 1,4-DHP derivatives in excellent yield (Scheme 1). The re- action progress was regularly checked by TLC plates and observed by UV (254nm) light. After completion of the reaction, the 1,4-DHP derivatives were extracted by ethyl acetate. In continuation of this research, to select the appro- priate solvent, water and ethanol/water (1:1) were used in the model reaction in the presence of MgCuAl2O5 nano- catalyst at 80 °C. As said in Table 1, we found that ethanol/ water (1:1) is the most efficient solvent for synthesizing 1,4-DHP derivatives, due to the hydrophobicity of the cat- alyst and organic reactant materials, giving the product in 90% yield (Table 1, entry 3). In the early stages without a catalyst, no product was synthesized in the reaction mix- ture even after stirring for 60 minutes. In order to achieve the desired product with the best yield, the reaction was optimized by using different amounts of catalyst (18.75, 37.5 and 56.25 mg). For each of these quantities, the yields were 75, 90 and 89%, respectively. The best efficiency was obtained with 37.5 mg of catalyst at 80 °C in ethanol/water solvent with a ratio of 1:1 (Table 1). After stirring the reac- tion mixture for 60 minutes, the yield increased to 90%, which is a great improvement. In fact, by increasing the quantity of catalyst from 18.75 to 37.5 mg, the yield raised Figure 4. FT-IR spectrum of MgCuAl2O5 NPs Scheme 1. Synthesis of 1,4-DHP derivatives 51Acta Chim. Slov. 2024, 71, 47–55 Keshtiban et al.: Nano MgCuAl2O5: Synthesis by Sol-Gel Auto-Combustion ... from 75 to 90%. The further use of more than 37.5 mg of catalyst facilitated neither the yield nor the reaction time. By increasing the amount of catalyst, there was no change in efficiency. Based on the experiments, it was determined that adding 37.5 mg of MgCuAl2O5 nanocatalyst leads to the best results. The good results obtained from benzaldehyde en- couraged us to continue proceedings on other aldehydes, including aldehydes with electron donating and elec- tron-withdrawing groups, which were reacted mildly with ammonium acetate and ethyl acetoacetate to completed the synthesis of 1,4-DHP derivatives with good to excel- lent yields. All the reactions were accomplished in the Figure 5. N2 adsorption-desorption isotherms of MgCuAl2O5 NPs 52 Acta Chim. Slov. 2024, 71, 47–55 Keshtiban et al.: Nano MgCuAl2O5: Synthesis by Sol-Gel Auto-Combustion ... presence of MgCuAl2O5 using in catalytic quantity (20 mol%) in ethanol/water (1:1) at 80 °C. The results of the experiments, which are also mentioned in the table, show that the yield and the reaction rate changes with the varia- tion of the functional groups on benzaldehyde, so that the electron-withdrawing functional groups lead to an in- crease in efficiency and the reaction rate. However, the electron-donating functional groups cause a decrease in it. Generally, 45 to 140 mins are needed to complete all the reactions. The synthesis of 1,4-DHP derivatives was ob- tained in 80–95% yields (Table 2). Based on what is proposed in various scientific arti- cles for synthesizing 1,4-DHP derivatives, we also present a proposed mechanism (Scheme 2). In the first step, the MgCuAl2O5 nanocatalyst led to the production of ester enamine from β-ketoester. Then, β-ketoester and aryl al- dehyde produced chalcone intermediate during aldol condensation in the presence of MgCuAl2O5 nanocata- lyst. In the end, ester enamine and chalcone produce 1,4- DHP derivatives with the Michael addition by removing the water. Table 1. Optimization of MgCuAl2O5 nanocatalyst a Entry Solvent Catalyst Time Yields (mol%) (min) (%) 1 ethanol/ water (1:1) – 60 Trace 2 ethanol/ water (1:1) 10 60 75 3 ethanol/ water (1:1) 20 60 90 4 ethanol/ water (1:1) 30 60 89 5 water – 60 Trace 6 water 10 60 18 7 water 20 60 42 8 water 30 60 57 a 2.5 mmol benzaldehyde, 5 mmol ethyl acetoacetate, 3.75 mmol ammonium acetate under 80 °C Scheme 2. The Suggested mechanism for synthesis of 1,4-DHP derivatives 53Acta Chim. Slov. 2024, 71, 47–55 Keshtiban et al.: Nano MgCuAl2O5: Synthesis by Sol-Gel Auto-Combustion ... 3. 3. Reusability of the Catalyst To prove the reusability of the MgCuAl2O5 nanocat- alyst in the synthesis of 1,4-DHP derivatives, it was tested during optimal reactions. MgCuAl2O5 nanocatalyst can catalyze several reactions without significantly lossing of its catalytic activity. As will explained in the experimental section, after the end of each reaction, the catalyst, by sim- ple filtration, was recycled for the subsequent reaction. The separated MgCuAl2O5 nanocatalyst was washed with wa- ter, ethanol and ethyl acetate, dried at 80 °C under vacuum for 2 h, and reused further in the next reaction without further modification. This process was performed over six runs with only a slight reduction in the catalytic activity, showing that all of the reactions were performed at favora- ble yields. In the specific time, the yield obtained for the reusability of the catalyst can be compared (Table 3), which shows that the recycling and reusability were accomplished without considerable loss of catalytic activity. According to the outcomes, the MgCuAl2O5 nanocatalyst showed great stability and excellent activity during several successive re- actions, so that after six consecutive reactions for the preparation of diethyl 2,6-dimethyl-4-phenyl-1,4-dihy- dropyridine-3,5-dicarboxylate, the yield of the catalyst de- creased by only 6%. These meager amounts reflect that the MgCuAl2O5 nanocatalyst preserves its activity and dura- bility during recycles. Figure 6. Reusability of MgCuAl2O5 nanocatalyst 4. Conclusion In conclusion, in this article, an efficient and conven- ient process has been used to prepare various symmetrical 1,4-DHPs from the one-pot reaction of ethyl acetoacetate, different aryl aldehydes, and ammonium acetate using 20 mol% MgCuAl2O5 nanocatalyst as a new heterogeneous catalyst at 80 °C in ethanol/water (1:1), which has many privileges such as ease of preparation, insolubility in most organic solvents, being eco-friendly and green process cat- alysts for green synthesis of 1,4-DHP derivatives in excel- lent yields. Also, the MgCuAl2O5 nanocatalyst could be successfully recovered and recycled at least for six runs without significant loss in activity. The method offers sev- eral advantages, including more efficient and economical than previous ones and offers advantages such as fewer re- action times, more yields, an environmentally friendly procedure, easy isolation of catalyst, and simple work-up procedure. The MgCuAl2O5 nanocatalyst was identified by XRD, SEM, EDX and FTIR techniques. The structure of 1,4-DHP derivatives has been specified by 1H and 13C NMR. Acknowledgments The authors are grateful to the Payame Noor University of Tabriz Research Council for providing a fellowship for the pres- ent work. We are also thankful to the Department of Chemistry, Urmia University, for the support of this work. Table 2. Synthesis of 1,4-DHP derivatives a Entry R Time Yield MP (oC) (min) (%) Found Reported36–46 1 3-Me-Ph 110 85 121–123 122–124 2 4-Me-Ph 130 83 135–137 137–139 3 4-MeO-Ph 120 82 161–163 158–160 4 2-HO-Ph 140 80 159–161 162–163 5 3-HO-Ph 100 88 181–183 180–182 6 4-HO-Ph 120 81 228–230 226–228 7 2-O2N-Ph 100 89 119–121 118–120 8 3-O2N-Ph 55 91 166–168 163–165 9 4-O2N-Ph 45 93 126–128 129–131 10 2-Cl-Ph 80 90 143–145 144–145 11 4-Cl-Ph 50 95 155–157 158–161 12 2,4-diCl-Ph 70 89 239–241 241–242 a 2.5 mmol aldehyde, 5 mmol ethyl acetoacetate, 3.75 mmol ammo- nium acetate and 37.5 mg of MgCuAl2O5 nanocatalyst in ethanol/ water (1:1) at 80 °C Table 3. Reusability of MgCuAl2O5 nanocatalyst a Entry Time (min) Yield (%) 1 60 90 2 60 88 3 60 86 4 60 86 5 60 85 6 60 84 a 2.5 mmol benzaldehyde, 5 mmol ethyl acetoacetate, 3.75 mmol ammonium acetate and 37.5 mg of MgCuAl2O5 nanocatalyst in eth- anol/water (1:1) at 80 °C 54 Acta Chim. 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DOI:10.1016/j.tetlet.2017.02.038 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Avtorji v prispevku poročajo o pripravi MgCuAl2O5 heterogenega nanokatalizatorja s sol-gel metodo samozgorevanja. Nanokatalizator je pokazal odlično aktivnost in visoko učinkovitost pri enostopenjski sintezi derivatov 1,4-dihidropirid- ina (1,4-DHP) v zmesi etanol/voda (1:1) kot zelenem topilu pri 80 °C. Reakcije potekajo hitro in z odličnimi izkoristki (80–95%). Poleg tega je nanokatalizator mogoče hitro obnoviti in ponovno uporabiti v najmanj šestih zaporednih ciklih brez znatne izgube aktivnosti. Nanokatalizator je bil karakteriziran z XRD, BET, FTIR, FESEM in EDX, ter temperaturo tališča. Substituirane 1,4-dihidropiridine pa so okarakterizirali z 1H in 13C NMR spektroskopijo. 56 Acta Chim. Slov. 2024, 71, 56–65 Vidmar et al.: Environmental Education Programmes: ... DOI: 10.17344/acsi.2023.8585 Scientific paper Environmental Education Programmes: A Case Study of Slovenia Janja Vidmar, 1,2,4* Jan Hočevar3,4 and Ester Heath 1,2,4,5 1 Jožef Stefan Institute, Department of Environmental Sciences, Jamova cesta 39, Ljubljana, Slovenia 2 Jožef Stefan International Postgraduate School, Jamova cesta 39, Ljubljana, Slovenia 3 University of Ljubljana, Faculty of Chemistry and Chemical Technology, Večna pot 113, Ljubljana, Slovenia 4 Section for the Environment, Slovenian Chemical Society, Hajdrihova 19, Ljubljana, Slovenia 5 Division of Chemistry and the Environment, European Chemical Society, Rue du Trône 62, Brussels, Belgium * Corresponding author: E-mail: janja.vidmar@ijs.si Telephone: +386 1 477 3542 Received: 12-13-2023 Abstract Environmental chemistry plays a vital role in the assessment of chemical pollution of the environment and thus con- tributes to the protection of ecosystems and human health. For this reason, it is important to provide future generations with the necessary knowledge and skills in environmental chemistry. The overall aim of this study was to assess the state of environmental chemistry education in Slovenia in 2023 by providing an overview of Slovenian study programmes in environmental science and identifying the significance of chemistry for secondary, short-cycle higher vocational, and higher education (including bachelor’s, master’s, and PhD studies). A total of 46 study programmes offering environ- mental science were identified, with wide variability in their chemistry content at different levels of education. This study provides valuable information on environmental chemistry education programmes in Slovenia to students and scientists interested or engaged in environmental science. Keywords: environmental programmes, environmental chemistry programmes, Slovenia, secondary education, short-cycle high- er vocational education, higher education 1. Introduction Given the increasing global awareness of environ- mental issues, environmental sciences are crucial in under- standing and addressing pressing and complex environ- mental issues. Protecting the environment requires an explicitly multidisciplinary approach, encompassing envi- ronmental research, natural sciences, ecology, technology, conservation, management and policy development. Therefore, adequate and trained experts with multidiscipli- nary knowledge in environmental sciences are needed to solve environmental problems at both national and Euro- pean levels. Among the disciplines of environmental sci- ence, environmental chemistry is of particular importance. According to the definition of S. Manahan,1 “environmental chemistry is the discipline that describes the origin, transport, reactions, effects and fates of chemical species in the hydro- sphere, atmosphere, geosphere, biosphere, and anthropo- sphere”. Environmental chemistry, therefore, plays an im- portant role in protecting ecosystems, the climate, human health, and the assessment of chemical pollution. The emergence of environmental chemistry as a dis- cipline in European education was revealed by a 2014 sur- vey on higher education in environmental sciences with an emphasis on chemistry.2 It demonstrated that nearly all Eu- ropean countries (28 in total) offer programmes in envi- ronmental chemistry, comprising 152 bachelor's and 181 master's programmes and two diploma and six advanced study programmes. Since some programmes recognized in the 2014 survey may have become outdated, and new ones have likely emerged in the past decade, there is a need for a new evaluation of environmental science education. To es- timate the state of environmental education in Europe in 57Acta Chim. Slov. 2024, 71, 56–65 Vidmar et al.: Environmental Education Programmes: ... 2023, two online databases, Study.eu (https://www.study. eu/)3 and Educations.com (https://www.educations.com/),4 which provide information on universities and their bach- elor’s, master’s and doctoral programmes in Europe and worldwide, were used. According to these databases, the number of environmentally related programmes has dou- bled over the last ten years. Namely, depending on the spe- cific online database used, a total of 183 or 354 bachelor's degree programmes and 555 or 704 master's degree pro- grammes were identified in 2023 (Table S1). It should be emphasized that the data retrieved from the online data- bases cannot be directly compared to that of the 2014 sur- vey owing to the varying environmental disciplines includ- ed, which is a result of the different methodologies employed for data collection (questionnaires in the 2014 survey versus programmes searched by disciplines accessi- ble through the online databases). Nevertheless, the find- ings undoubtedly demonstrate a significant increase in the number of bachelor’s and master’s programmes linked to environmental sciences across Europe in the past decade. Slovenia, a small European country with a popula- tion of approximately 2.1 million and situated between the Alps, the Adriatic Sea and the Pannonia Plain, presents a unique context combining rich biodiversity, historical landscapes, and contemporary environmental concerns. In 2004, Slovenia joined the European Union (EU), which triggered, among others, an increased need for the imple- mentation of sustainable development principles into Slo- venia’s research and education strategy. As a result, envi- ronmental science topics and subjects have been incorporated into its education system since then. A 2014 study identified two bachelor's and two master's pro- grammes in environmental chemistry,2 while one bache- lor's, two master's and one doctoral programme in envi- ronmental studies were retrieved from the two online databases in 2023 (Table S1). Nevertheless, the data on environmental education programmes in Slovenia provid- ed in these overviews has either been underestimated, in- complete, or outdated. Given the significance of this infor- mation for advancing strategic planning and progress in sustainable development, there is a need for a comprehen- sive and systematic evaluation of the present state of edu- cation programmes in environmental chemistry within the Slovenian context. In this work, we aimed to provide an overview of the study programmes in the field of environmental sciences in Slovenia in 2023 and to determine the importance of chemistry in these study programmes. Unlike 2014 study and 2023 database that included higher education pro- grammes (bachelor's, master's, and doctoral studies), this overview included also secondary education and short-cy- cle higher vocational education, which we believe are equally important. The study was conducted by members of the Section for the Environment, founded in 2022 with- in the Slovenian Chemical Society5 with the vision of be- coming one of the leading associations of experts in Slove- nia dealing with environmental chemistry topics. The mission of this Section is to bring together members of the Slovenian Chemical Society who are interested in or in- volved with environmental chemistry topics. The goal is to promote cooperation, networking and knowledge sharing to improve the understanding and perception of environ- mental chemistry among various stakeholders. The Sec- tion also aims to encourage the proper use of chemistry in evaluating and resolving environmental issues and ad- dresses those aspects of environmental chemistry requir- ing regulation. In addition, the Section’s work programme also includes promoting the integration of new environ- mental chemistry content into Slovenian education and cooperation with international environmental organiza- tions, particularly with the Division of Chemistry and the Environment (DCE) of the European Chemical Society. In line with the latter two objectives, the Section aimed to identify the current situation and potential gaps in envi- ronmental chemistry study programmes in Slovenia by following the example of the 2014 survey on higher educa- tion programmes in environmental chemistry in Europe conducted by the DCE.2 2. Methodology 2. 1. Slovenian education System The Slovenian education system has three levels: pri- mary, secondary, and tertiary (Figure 1). Briefly, secondary education is provided by (upper) secondary schools offering general or technical upper sec- ondary education (4-year programme), short upper sec- ondary vocational education (2-year programme), and upper secondary vocational (3-year programme) or tech- nical education (2-year programme). Tertiary education consists of short-cycle higher vocational education (2-year programmes) and higher education, which is part of the Bologna Process and includes undergraduate programmes (3–4 years of bachelor’s – first cycle), postgraduate pro- grammes (2 years of master’s – second cycle) and doctoral programmes (3–4 years of PhD – third cycle). The educa- tion system in Slovenia is organized mainly as a public ser- vice rendered by public and private institutions providing officially recognized or accredited programmes. Primary education is mandatory and funded by the government in accordance with the Constitution of the Republic of Slove- nia, which guarantees the right to free education. Howev- er, both public and private institutions offer further levels of education such as upper secondary schools and higher education studies.6 2.2. Data Collection An overview of the study programmes in environ- mental science available in Slovenia was obtained for up- per secondary technical education, short-cycle higher vo- 58 Acta Chim. Slov. 2024, 71, 56–65 Vidmar et al.: Environmental Education Programmes: ... Figure 1: Schematic representation of the Slovenian education system, including primary, secondary, and tertiary education.6 59Acta Chim. Slov. 2024, 71, 56–65 Vidmar et al.: Environmental Education Programmes: ... cational education, and higher education that includes bachelor's, master's, and doctoral studies. First, existing educational institutions offering environmental studies were overviewed with the help of online databases. Rele- vant study programmes in upper secondary technical and short-cycle higher vocational education were obtained from the Institute of the Republic of Slovenia for Vocation- al Education and Training (CPI) website.7 The CPI is a central national research and development institution in vocational education and training, which hosts several na- tional coordination points and education and counselling centers in this domain. The list of higher education study programmes was compiled with the help of the GOV.SI Portal of the Slovenian government administration.8 The GOV.SI Portal is a central website which provides compre- hensive information on the organization and operation of the state administration, including education. Next, pro- grammes with significant coverage of environmentally re- lated subjects were deemed relevant. Identification of “en- vironment/environmental”, “ecotechnology”, “water management”, and “nature conservation/preservation” in the title of the study programme was considered necessary for the programme to be reported as relevant. In some cas- es, programmes focused on related fields, such as, for ex- ample, ecology, biology, geotechnology, and agriculture, were also included in the overview. We acknowledge that the data collected herein may be incomplete, as our selec- tion criterion may result in overlooking programmes that cover environmental chemistry in their curricula but do not explicitly feature environmentally related topics in their titles. However, this methodology had to be selected to narrow down the search within the extensive database of the Slovenian education system, which would otherwise require reviewing the curricula of approximately 560 study programmes across different levels of education. 2. 3. Data Analysis From the collected data, a list of study programmes was constructed containing various information, includ- ing the name of the educational institution, the title of the programme, a summary of the programme's content, the resulting professional or scientific title obtained, the loca- tion of the programme, the language of instruction, the institution type (public or private), the tuition fees (if ap- plicable), the year the programme was established, and the study programme website (Table S3–S5). The overview al- so includes the professional or scientific titles obtained for each study programme, which were translated into English using the Slovenian Qualifications Framework (SQF)9 for the benefit of non-Slovenian readers. However, it should be emphasized that these translations have no legal status under Slovenian legislation, which prohibits translating professional and academic titles into a foreign language. The study programmes’ start year was determined with the help of a Register of educational institutions and educa- tional programmes10 and based on their first accreditation, provided by the Slovenian Quality Assurance Agency for Higher Education.11 The Agency is responsible for quality assurance in Slovenian higher education. General infor- mation applicable to all study programmes at each level of education is summarized in Table S2, which comprises de- tails on the type of education, programme duration, ECTS credits obtained and entry-level requirements. Detailed information on the curriculum offered by each study programme, including course descriptions, was gathered from the websites of the respective educational institutions. To assess the significance of chemistry and the environment in selected programmes, we first identified all courses, including both compulsory and elective ones, that focus on either chemistry or the environment. The share of the identified courses in relation to the total num- ber of courses offered by a given study programmes, here- after referred to as the percentage of courses with chemis- try or environment content, was determined by considering the number of hours (for secondary education) or credit points (for tertiary education) assigned to each respective course. The courses assigned to chemistry and environ- ment are shown in Table S6. It is important to stress that this overview presents the status of the existing programmes as of 2023, which are likely to change in the future, especially in light of the forthcoming reform of primary schools, general upper secondary education and higher vocational study pro- grammes initiated by the Ministry of Education Science and Sport in 2021. The modernization of the programmes by updating the curriculum prioritizing the inclusion of competencies and qualifications for the digital and green transition also became an integral part of the National Re- covery and Resilience plan, as confirmed by both the Gov- ernment and the EU Council in 2021.12 3. Results and Discussion 3.1. Distribution and Diversity of Programmes Forty-six relevant environmental science pro- grammes were identified at all levels of education: ten in secondary education, ten in short-cycle higher vocational education, nine in bachelor’s programmes, 11 in master’s programmes, and six in doctoral programmes (Table 1). A detailed list of identified programmes is presented in the Supplementary Material (Table S2–S5). The identi- fied study programmes are offered by 26 different educa- tional institutions (ten offer upper secondary technical education, nine short-cycle higher vocational education, and ten higher education). This finding means that envi- ronmental study programmes are offered in about 19% of the existing institutions of short-cycle higher vocational education in Slovenia (considering 29 public and 18 pri- 60 Acta Chim. Slov. 2024, 71, 56–65 Vidmar et al.: Environmental Education Programmes: ... vate higher vocational schools in Slovenia)13 and in about 22% of the existing higher education institutions in Slove- nia (considering three public and three private universi- ties, one independent public higher education institution and 39 private higher education institutions in Slovenia).13 Five out of six universities in Slovenia (University of Lju- bljana, University of Maribor, University of Nova Gorica, University of Primorska and University of Novo mesto) offer at least one higher education programme related to environmental sciences. The institutions hosting these programmes are correspondingly diverse and include dis- ciplines from the natural sciences (chemistry, biology), environmental sciences (earth sciences, biotechnology, ag- riculture, geology, sustainable development), civil or me- Table 1: Number of relevant environmental science programmes per Slovenian statistical region. Slovenian statistical region Secondary Short-cycle Higher higher vocational Bachelor Master PhD Total 1. Pomurska 2. Podravska 2 2 3 3 2 12 3. Koroška 1 1 4. Savinjska 2 3 1 1 7 5. Zasavska 6. Posavska 7. Jugovzhodna Slovenija 1 2 1 1 5 8. Osrednjeslovenska 2 1 2 4 3 12 9. Gorenjska 1 2 1 4 10. Primorsko-Notranjska 11. Goriška 1 1 1 1 4 12. Obalno-Kraška 1 1 Total 10 10 9 11 6 46 Figure 2: A map showing the geographical distribution of relevant environmental science programmes offered by educational institutions in differ- ent Slovenian statistical regions. The numbers correspond to statistical regions that are in alignment with Table 1. 61Acta Chim. Slov. 2024, 71, 56–65 Vidmar et al.: Environmental Education Programmes: ... chanical engineering and even business sciences (Tables S3–S5). Twenty of the identified educational institutions are public institutions, and six private institutions, including two private institutions of short-cycle higher vocational education (Institute for Education Erudio and DOBA Vo- cational School) and four private higher education institu- tions (Jožef Stefan International Postgraduate School, B&B College of Sustainable Development, Alma Mater Euro- paea and Postgraduate School ZRC SAZU). Private higher vocational colleges and higher education institutions charge tuition fees for all enrolled students or they can be publicly funded, provided that they hold a concession for their full-time study programmes. Meanwhile, public edu- cational institutions in Slovenia charge tuition fees for part-time enrolled students, all international students (ex- cept for those coming from EU member states, Bosnia and Herzegovina, Montenegro, Kosovo, Serbia and North Macedonia), doctoral students, full-time students who al- ready hold an equivalent degree or international students applying for certain English-taught degrees at the Univer- sity of Maribor. The tuition fees for the environmental sci- ence programmes in those cases range between 1,045 and 2,000 €/year for short-cycle higher vocational education, 2,000 and 11,000 €/year for bachelor’s degrees, 2,600 and 11,000 €/year for master’s degrees and 3,000 and 4,500 €/ year for doctoral degrees (Table S3–S5). As expected, the highest number of environmental science programmes was identified in Slovenia’s two larg- est cities, Ljubljana and Maribor (Figure 2). These two cit- ies are Slovenia’s largest university cities, reflected in the highest number of study programmes allocated to higher education, i.e., nine in Ljubljana and seven in Maribor. A relatively high number of environmental programmes in secondary and short-cycle higher education were also identified in Novo mesto (three in total) and Velenje (three in total). The number of bachelor’s (9) and master’s (11) envi- ronmental science programmes per number of inhabitants is in this study significantly higher (i.e., 9.5 programmes per million inhabitants) than the highest number of rele- vant higher education programmes found in the 2014 sur- vey (i.e., 1.2–1.3 programmes per million inhabitants identified for Finland, Norway, Sweden and Serbia).2 This can be partly explained by the outdated information from 2014 since it can be expected that new programmes have been created in the last ten years, both in Slovenia and in other European countries. Nevertheless, the outstanding number of environmental study programmes per number of inhabitants found in Slovenia in 2023 indicates a robust educational base in environmental sciences in a country as small as Slovenia. Most of the programmes in secondary education and short-cycle higher vocational education were established between 2008 and 2014, with the most recent programme in Nature Conservation being established in 2018/2019 at the School of Machinery, Geotechnics and Environment, School Centre Velenje (secondary education) (Table S3 and S4). Higher education programmes in environmental sciences have similarly long tradition, with most pro- grammes established between 2005 and 2012. Some of the most recently established programmes include Environ- mental Management at the University of Novo mesto, Fac- ulty of Business and Management Sciences, with bache- lor's and master's degrees initiated in 2020 and 2021, respectively. The Postgraduate school ZRC SAZU also in- troduced a master's programme in Earth and Environ- mental Sciences and a doctoral programme in Environ- mental and Regional Studies, both launched in 2021. Over the past decade, several programmes have undergone re- newal due to the launch or renaming of new programmes or the transformation of a higher education institution (Table S5). Next, subjects that were identified from the titles of environmental science programmes were listed and grouped into various categories (Table 2). In this context, subjects represent the disciplines, sub-disciplines, and fields of study that can be identified within the environ- mental sciences. Fifty-seven subjects were recognized in 46 programmes, as specific programme titles were classi- fied under two distinct subjects. The identified subjects are very diverse. “Environmental Protection” is the subject most frequently identified (15 times, corresponding to 33% of all programmes), followed by “Nature Conserva- tion/Preservation” (11 times, corresponding to 24% of all programmes), “Environmental Engineering/Ecotechnolo- gy” (nine times, corresponding to 20% of all programmes) and “Environment/Environmental Studies/Environmental Science” (eight times, corresponding to 17% of all the pro- grammes). None of the study programmes are explicitly entitled “Environmental Chemistry”. As can be further seen from Table 2, only two distinct programme subjects, Environmental Protection and Nature Conservation/Pres- ervation, were identified for secondary and short-cycle higher vocational education. In contrast, higher education study programmes include a much more comprehensive range of subjects, such as Environmental Engineering/ Ecotechnology, Environment (including similar terms in programme titles) and Ecology/Biology (and similar terms), which reflects the interdisciplinary nature of envi- ronmental study programmes in the higher education sys- tem in Slovenia. The subjects identified in the programmes titles evi- dently do not cover all aspects of environmental sciences, notably lacking subjects such as air quality, meteorology, and soil science. Since the programme titles may not fully reflect the diversity of their content, examining the courses offered within these programs can provide a more com- prehensive overview of the programme's content. For in- stance, courses such as “Air protection", "Air pollution and meteorology", "The Atmosphere: Gases, Aerosols and Cli- mate Change", “Soil and Environment”, “Soil Conserva- 62 Acta Chim. Slov. 2024, 71, 56–65 Vidmar et al.: Environmental Education Programmes: ... tion”, “Soil Ecology”, “Soil pollution”, etc. were frequently identified in the course titles (Table S6), demonstrating their inclusion in the curricula of the programmes exam- ined in this study. 3. 2. Significance of Chemistry in Programmes The significance of chemistry in environmental sci- ence programmes presented as the percentage of courses assigned to chemistry topics was calculated as described in section 2.3. (Data analysis). From a list of compulsory and elective chemistry courses presented for all levels of educa- tion (Table S6), it can be seen that the most common sub- jects identified in the course titles at all levels of education were “environment” or “environmental” (74 times), fol- lowed by “chemistry” or “chemical” (30 times), “technolo- gies” (23 times), “water” (21 times), “waste” (17 times), “management” (14 times), “materials” (12 times), and pro- tection and pollution (each 11 times). The most common chemistry-related courses covered different areas of chem- istry (including analytical, inorganic, organic, colloidal, bio-, geo- and radiochemistry) or were related to the envi- ronmental chemistry (Environmental Chemistry and Technology, (Principles of) Environmental Chemistry, Chemistry and Environmental Technology, Chemistry in Environmental Protection, Chemistry of Environmental Systems, Chemistry of Pollutants, Chemistry of the Agri- cultural Environment, Colloid Chemistry in the Environ- ment, Environmental Analytical Chemistry, Organic Chemistry for Sustainable Development, and Green Chemistry (Table S6). Only three courses within the bach- elor’s programmes are explicitly entitled “Environmental Chemistry”. A similarly low percentage of environmental chemistry was found in 2014, in which only six universi- ties included in the survey offered environmental chemis- try as a specific programme subject.2 Other chemistry-re- lated courses that were associated with the application of chemistry to solve environmental problems were entitled Ecoremediation, Environmental Science/Technologies/ Management/Monitoring/Engineering, Air Protection, Air/Water/Soil/Environmental Pollution (including simi- lar terms), Environmental/Research/Experimental/Instru- mental Methods(ology) (including similar terms), Table 2: Identification of subjects (disciplines, sub-disciplines and fields of study) in programme titles that offer environmental science and the corresponding number of study programmes. Short-cycle Higher Subject Secondary higher vocational Bachelor Master PhD Env Engineering, Ecotechnology 3 4 2 Env Management 1 1 Env Protection 5 6 2 1 1 Water Science/Water Management 1 1 Environment, Env Studies, Env Science 3 3 2 Geotechnology 1 1 Agriculture 1 Ecology, Eco Sciences, Biology, Evolution, Biodiversity 1 3 2 Nature Conservation, Preservation 5 3 1 2 Earth 1 Figure 3: Histogram showing the number distribution of A) secondary programmes (n = 10) and short-cycle higher vocational programmes (n = 10) and B) higher education programmes (bachelor’s (n = 9), master’s (n = 11) and doctoral (n = 6)) that include a certain percentage of compulsory and elective courses given for chemistry. 63Acta Chim. Slov. 2024, 71, 56–65 Vidmar et al.: Environmental Education Programmes: ... Waste(water) Management/Treatment, Basic/Applied/ Fundamentals of Ecology or Marine/Freshwater Ecology, and Ecotoxicology. It is evident that the diversity of cours- es in higher education programmes was greater than in secondary and short-cycle higher vocational education programmes (Table S6). The distribution (number of courses) with a certain percentage of chemistry for all education levels studied is shown in Figure 3 and allocated to the subjects identified in the programme titles in Table 3. Most programmes have a relatively low percentage of chemistry in their curricula: 31% in secondary education, 26% in short-cycle higher vocational education, 21% in bachelor’s programmes, 30% in master’s programmes and 23% in doctoral programmes (Figure 3). These values are comparable to the percentage of chemistry content identified in bachelor’s (15±11%) and master’s (28±21%) programmes in Europe in 2014.2 The distribution of chemistry content is more diverse in sec- ondary and short-cycle higher vocational programmes than in higher education programmes. When considering the programme subjects, it is evident that, regardless of the level of education, the highest proportion of courses with chemistry content is to be found in the fields of Environ- mental Engineering/Ecotechnology, Environmental Pro- tection, Environment (including similar terms) and Na- ture Conservation/Preservation, with an average of around 30% of courses allocated to chemistry. 3. 3. Significance of Environment in Programmes Similar to chemistry, the significance of the environ- ment in the identified environmental science programmes was evaluated. The course titles (Table S6) of the pro- grammes across all educational levels most frequently con- tained the word “environment” or “environmental” (202 times), followed by other commonly found terms, such as “ecology” or “ecological” (74 times), “biology” or “biologi- cal” (48 times), “nature” or “natural” (42 times), “manage- ment” and “conservation” (each 39 times), “protection” (36 times), “technologies” (32 times), “chemistry” or “chemi- cal” (30 times), and “sustainable” (24 times). Of the envi- Table 3: Percentage of courses (compulsory and elective) with at least 5% allocated to chemistry (%, mean) for the study programmes grouped into different subjects. The number of programmes is given in brackets. Short-cycle Higher Subject Secondary higher vocational Bachelor Master PhD Env Engineering, Ecotechnology <30% (3) <30% (4) <40% (2) Env Management <10% (1) <10% (1) Env Protection <40% (5) <30% (6) <30% (2) <30% (1) <10% (1) Water Science/Water Management <20% (1) <10% (1) Environment, Env Studies, Env Science <20% (3) <40% (3) <20% (2) Geotechnology <10% (1) <10% (1) Agriculture <10% (1) Ecology, Eco Sciences, Biology, Evolution, <20% (1) <20% (3) <10% (2) Biodiversity Nature conservation, Preservation <20% (5) <50% (3) <20% (1) <30% (2) Earth <20% (1) Figure 4: Histogram showing the number distribution of A) secondary programmes (n = 10) and short-cycle higher vocational programmes (n = 10) and B) higher education programmes (bachelor’s (n = 9), master’s (n = 11) and doctoral (n = 6)) that include a certain percentage of compulsory and elective courses given for environment. 64 Acta Chim. Slov. 2024, 71, 56–65 Vidmar et al.: Environmental Education Programmes: ... ronmental matrices, water (i.e., waste, drinking, process, ground, surface and freshwater) was the matrix most fre- quently identified in the course titles (31 times), followed by soil (16 times) and air (10 times). Again, the variety of courses, specifically elective courses, was much greater in tertiary education programmes than in secondary and short-cycle higher vocational education programmes. The average percentage of courses allocated to the environment was 43% in secondary education, 59% in short-cycle higher vocational education, 62% in bachelor’s programmes, 85% in master’s programmes and 50% in doctoral programmes (Figure 4 and Table 4). It is evident that the environmental content of programmes is much higher than their chemistry content and varies considera- bly among different levels of education (Figure 4) and sub- jects (Table 4). A closer look at the programmes grouped by subject area shows that the significance of the environ- ment is highest in Environmental Management, Environ- ment (including similar terms), and Ecology (and similar terms), with an average of around 70% of courses allocated to the environment. These numbers adequately reflect the interdisciplinary nature of environmental science pro- grammes. 3. 4. Teaching Language None of the secondary or short-cycle higher voca- tional programmes offer courses in English. In higher ed- ucation programmes, all courses in two bachelor's, five master's, and three doctoral programmes are offered in Slovene and English. The language is switched to English when non-native students are enrolled. This finding indi- cates that a relatively large proportion of the identified en- vironmental programmes are taught in English, more so at the master's and doctoral levels (45–50%) than at the bachelor's level (22%). The percentage of courses taught entirely in English is higher in Slovenia than at the Euro- pean level in 2014, when 15% of bachelor's and 24% of master's programmes were taught in English.2 4. Conclusions We showed that many environmental science study programmes are offered in Slovenian educational institu- tions at all levels of education, including secondary educa- tion, short-cycle higher vocational education and higher education. The high number of environmental pro- grammes per capita (9.5 programmes per million inhabit- ants) indicates a robust educational base in environmental sciences in a country as small as Slovenia. The identified programmes offer a broad and diverse range of subjects, particularly in higher education, reflecting the interdisci- plinary nature of environmental science programmes. The environmental content of the identified pro- grammes is much higher than their chemistry content and varies considerably among different levels of education and subjects. Only three courses within the bachelor’s pro- grammes are explicitly entitled “Environmental Chemis- try”. This fact suggests that despite a strong educational foundation in environmental sciences in Slovenia, envi- ronmental chemistry as a programme subject is less repre- sented. The programmes identified in this study are estab- lished after 2005. A significant share of courses within higher education programmes (an average of around 40%) are taught in English or offer the possibility to be taught in English. This number reflects the mobility of students within the EU, which has already been implemented in higher education in line with the Bologna objectives. As programmes have a life cycle between accredita- tions, it is reasonable to assume that some information (e.g., programme titles and tuition fees) may have become outdated or changed by the time of publication of this work. Nevertheless, the overview of environmental science programmes provided in this study gives valuable infor- mation for students, academics and researchers interested in environmental chemistry and environmental sciences. In addition, it is believed that the results of this study will help raise the visibility and importance of this discipline at national and international levels. Table 4: Percentage of courses (compulsory and elective) with at least 20% allocated to environment (%, mean) for the study programmes grouped into different subjects. The number of programmes is given in brackets. Short-cycle Higher Subject Secondary higher vocational Bachelor Master PhD Env Engineering, Ecotechnology <50% (3) <60% (4) <40% (2) Env Management <80% (1) <70% (1) Env Protection <50% (5) <60% (6) <70% (2) <80% (1) <80% (1) Water Science/Water Management <40% (1) <60% (1) Environment, Env Studies, Env Science <70% (3) <80% (3) <40% (2) Geotechnology <20% (1) <40% (1) Agriculture <100% (1) Ecology, Eco Sciences, Biology, Evolution, <100% (1) <80% (3) <50% (2) Biodiversity Nature Conservation, Preservation <40% (5) <80% (3) <100% (1) <80% (2) Earth <70% (1) 65Acta Chim. Slov. 2024, 71, 56–65 Vidmar et al.: Environmental Education Programmes: ... Acknowledgements We acknowledge the Division of Chemistry and the Environment at European Chemical Society for inspiring us to conduct this study. Additionally, we would like to thank Dr David Kocman from the Department of Envi- ronmental Sciences, Jožef Stefan Institute, for creating the visuals for the map of Slovenia. Special thanks go to Dr David Heath from the Department of Environmental Sciences, Jožef Stefan Institute, for English correction of the manuscript. 5. References 1. S. Manahan, Environmental Chemistry, Tenth Edition, CRC Press, 2017. DOI:10.1201/9781315160474 2. G. Lammel, E. J. Comas, I. Ivancev-Tumbas, Environ. Sci. Pol- lut. Res., 2014, 21, 7211–7218. DOI:10.1007/s11356-014-2737-7 3. Study.eu, Study in Europe. Bachelors, Masters, PhDs, https:// www.study.eu/, (accessed 8 December 2023). 4. EMG – Educations Media Group AB, Education Abroad: University & College Study Abroad Programs, https://www. educations.com/, (accessed 8 December 2023). 5. Slovenian Chemical Society, https://www.chem-soc.si/, (ac- cessed 8 December 2023). 6. Ministry of Education Science and Sport of the Republic of Slovenia, The education system in the Republic of Slovenia 2018/2019, 2019. 7. CPI – Institute of the RS for Vocational Education and Train- ing, https://cpi.si/en/, (accessed 8 December 2023). 8. Government Communication Office, Education, science and sport | GOV.SI, https://www.gov.si/en/policies/educa- tion-science-and-sport/, (accessed 8 December 2023). 9. Center RS za poklicno izobraževanje, Slovensko ogrodje kvalifikacij | Enotni sistem kvalifikacij v Republiki Sloveniji, https://www.nok.si/en, (accessed 8 December 2023). 10. MIZŠ RS, Evidenca vzgojno-izobraževalnih zavodov in vzgo- jno-izobraževalnih programov, https://paka3.mss.edus.si/ registriweb/ZavodiPodrobno.aspx, (accessed 13 December 2023). 11. Nacionalna agencija Republike Slovenije za kakovost v vi- sokem šolstvu, NAKVIS, https://www.nakvis.si/?lang=en, (accessed 8 December 2023). 12. Republic of Slovenia Gov.si, Prenova sistema vzgoje in izo- braževanja v Sloveniji, https://www.gov.si/zbirke/projek- ti-in-programi/prenova-sistema-vzgoje-in-izobrazevan- ja-v-sloveniji/, (accessed 8 December 2023). 13. Eurydice, Tertiary Education in the Republic of Slovenia, 2022. Povzetek Okoljska kemija ima pomembno vlogo pri ocenjevanju kemijske onesnaženosti okolja, s tem pa prispeva k varovanju ekosistemov in zdravja ljudi. Zato je bistvenega pomena, da prihodnjim generacijam zagotovimo potrebno znanje in veščine s področja okoljske kemije. Splošni cilj te raziskave je bil oceniti stanje izobraževanja na področju okoljske kemije v Sloveniji v letu 2023, s pregledom slovenskih študijskih programov s področja okoljskih ved in opredelitvijo pomena kemije v srednješolskem, višješolskem strokovnem in visokošolskem izobraževanju (vključno z dodiplomskim, magis- trskim in doktorskim študijem). Identificirali smo skupno 46 študijskih programov, ki ponujajo okoljske vede, z veliko raznolikostjo v vsebini kemije na različnih stopnjah izobraževanja. Ta raziskava nudi študentom in raziskovalcem, ki jih zanima ali se ukvarjajo z okoljsko kemijo, dragocene informacije o izobraževalnih programih s področja okoljske kemije v Sloveniji. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 66 Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... DOI: 10.17344/acsi.2023.8436 Scientific paper Synthesis of Bone Meal-derived 4-Carboxyphenylboronic Acid Functionalized Sulfur and Nitrogen Co-doped Graphene Quantum Dots Nanoprobe for Sialic Acid Sensing Sopan N. Nangare,1# Pratik P. Yeole,1# Zamir G. Khan,1 Ashwini G. Patil,1 Bhushankumar S. Sathe,2 Sanjaykumar B. Bari1 and Pravin O. Patil1,* 1 Department of Pharmaceutical Chemistry, H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur-425405, Dist: Dhule (MS); India 2 VYWS Institute of Diploma in Pharmacy, Borgaon, Wardha (MS)- 442001; India * Corresponding author: E-mail: rxpatilpravin@yahoo.co.in Received: 08-09-2023 # These authors contributed equally as the first authors. Abstract Detection of sialic acid using advanced sensors in milk-based products is essential in the food industry. Therefore, the present work reports the sulfur and nitrogen-doped graphene quantum dots from bone meal functionalized with bo- ronic acid (Boro-S/N-dGQDs) nanoprobe for sialic acid sensing applications. Briefly, S/N-dGQDs were functionalized with 4-carboxyphenylboronic acid to improve performance of fluorescent sensors toward the detection of sialic acid. Here, boronic acid surface decoration on S/N-dGQD was confirmed by several spectral characterizations. The addition of different quantities of sialic acid results in a directly proportionate correlation to fluorescence quenching. It gives a broad linear range of 50 ng/mL to 1000 ng/mL and a limit of detection of 6.04 ng/mL. Also, it displayed remarkable se- lectivity, likely due to interaction of sialic acid-containing 1,2-diol with hydroxyl group of Boro-S/N-dGQDs nanoprobe. Designed sensor demonstrated good stability and reproducibility. Real-time analysis of sialic acid in different milk-based products confirmed practicability of Boro-S/N-dGQDs. Keywords: Bone meal; Boro-S/N-dGQDs; boronic acid; sialic acid; milk products; fluorescent sensor 1. Introduction Sialic acid is a negatively charged monosaccharide that is present at the ends of glycolipids and glycoproteins on the cell surface.1 In addition, sialic acid is present in milk, meat, and other foods.2 According to the literature, sialic acid is present in mammalian glycoconjugates. Here, the hydroxylated form of sialic acid is absent in humans. On the contrary, sialic acid is present in mammals. There- fore, the consumption of milk-based products and meat with high concentrations of a hydroxylated form of sialic acid (non-human sialic acid) can pose health risks.3 Here, the chronic consumption of the hydroxylated form of sial- ic acid can result in chronic inflammation, leading to dif- ferent types of diet-associated cancers, as well as other types of health issues.4,5 Therefore, there is a need to mon- itor the level of the hydroxylated form of sialic acid in milk products. To date, several techniques for detecting sialic acid have been recorded. In brief, electrochemical methods such as potentiometric, non-enzymatic electrochemical,6 organic electrochemical transistors,7 electrochemical im- pedance spectroscopy,8 etc. have been documented for the detection of sialic acid. Other methods for the detection of sialic acid include chromatography, colorimetry, and spec- trofluorimetry.1,9 Despite their several advantages, there are some disadvantages, such as the usage of harmful and expensive chemicals in the sensor's construction, limited selectivity, poor sensitivity, time-consuming, complex processing, and so on. Therefore, there is a pressing urgen- cy to produce a highly sensitive, green-made, simple, high- ly selective, cost-effective, quick sensor for sialic acid iden- tification in milk-based products. The adoption of a fluorescence-based sensor over- comes various demerits of the previously utilized approach 67Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... for the identification of interest compounds.10 In the case of fluorescence-based sensors, the scientific community has established a strong preference for carbon-mediated zero-dimensional nanosized fluorescent particles known as graphene quantum dots (GQDs) for a variety of applica- tions. The nanosize design of GQDs is a crystalline gra- phitic material founded on sp2-hybridized carbon.11 Fur- thermore, owing to their edge effects and strong quantum confinement, GQDs have excellent stability, minute toxic- ity, and good fluorescence.12 As well, it has strong biocom- patibility, great solubility, an adjustable band gap, and oth- ers.13 During the last few decades, GQDs have been modified for sensing applications employing several types of heteroatoms.14 According to the literature, heteroa- tom-doped GQDs may efficiently modify their chemical and physical properties.13 In this shade, GQD doping al- lows for changes in energy density, band gap, and other properties.12 As explained, heteroatom doping in GQDs raised the aggregate capability of fluorescence-based sensors for ex- tremely sensitive and selective analyte recognition.15,16 For the design of heteroatom-doped GQDs, the preference for green precursor has been reported17 to avoid exposure to chemicals followed by toxicity.10 Moreover, the use of bio- mass such as honey, orange juice, green tea extract, rice husk, etc. for the design of GQDs18 provides several advan- tages including abundant surface functionality, high car- bon composition, cost-effectiveness, eco-friendly, etc.10 Finally, the green-made doped GQDs showcased upgrad- ed sensor performance.10,17 As a result, we prefer to devel- op green-made doped GQDs for sialic acid sensing appli- cations. Of particular importance, nitrogen (N) and sulfur (S) are the most widely employed doping agents for the production of doped GQDs.17,19 Because of their five va- lence electrons and equivalent atomic size, 'N' doping is capable of bonding with the carbon backbone of GQDs. According to the literature, the design of ‘N’ doping in a GQD lattice can drastically affect the electrical and chem- ical characteristics, as well as impart a large number of sites for additional adjustments.13 Likewise, the preference for ‘S’ doping in GQDs has been reported.20 In this case, the doping of S’ can aid in improving the electron transfer process.21 On this basis, the design of ‘S’ and ‘N’ doped GQDs (S/N-dGQDs) have been majorly documented for plentiful sensing22 and other applications.21 Despite the considerable degree of customization in GQDs utilizing various dopants, high sensitivity, and selectivity are critical tasks in sensing applications.23 In recent years, the func- tionalization of fluorescent nanoparticles such as GQDs has been described as offering an improved presentation for the detection of interest analytes.24 In short, green- made GQDs with appropriate functionalizing agents have been described for sensing applications25,26 that furnish advanced sensing performance. In this light, the selectivity for the analyte of interest in the involvement of various in- terfering agents has been explained as acceptable for func- tionalizing agents.23,25,26 More importantly, the design of surface functionalized S/N-dGQDs for sialic acid sensing in milk-based products has yet to be released. As a result, the synergistic benefits of surface functionalized S/N- dGQDs may grant increased sensitivity and selectivity than the previously published techniques. Therefore, the current study presents a simplistic, highly sensitive, highly selective, green-made, and stable Boro-S/N-dGQDs-based fluorescence sensor for sialic acid detection in milk products. In summary, the hydro- thermal approach was adopted to achieve a green synthe- sis of S/N-dGQDs as a fluorescent agent, with bone meal serving as both a green precursor and a dopant source. Subsequently, the generated S/N-dGQDs were functional- ized with boronic acid, and fluorescence analysis con- firmed enhanced fluorescence compared to the S/N- dGQDs. FT-IR, PXRD, HR-TEM, XPS, Raman, zeta potential, fluorescence research, and other techniques were employed to verify the successful synthesis of S/N- dGQDs and Boro-S/N-dGQDs. The sialic acid sensing us- ing Boro-S/N-dGQDs exhibited a proportional connec- tion to fluorescence quenching. It demonstrated a wide linear concentration range and a detection limit of 50 ng/ mL for sialic acid. Moreover, it displayed significant an- ti-interference potential, attributed to the interaction of sialic acid-containing 1,2-diol with a hydroxyl group of Boro-S/N-dGQDs. Consequently, the study confirmed the synergistic effect of dopants and functionalizing agents used in GQDs to enhance fluorescence sensor perfor- mance for sialic acid detection, surpassing previously re- ported approaches. At last, the different local milk prod- ucts were analyzed confirming the real-time applications of the proposed sensor for the detection of sialic acid. Overall, Boro-S/N-dGQDs offer several advantages, in- cluding enhanced sensitivity, excellent selectivity, superior stability, eco-friendliness, cost-effectiveness, simplicity, and more, which will pave the way for future monitoring safety of milk-based products. 2. Materials and Methods 2. 1. Materials The bone meal was collected from the local market in Shirpur, Maharashtra, India. Sialic acid (C11H19NO9, N-acetylneuraminic acid, Mol. Wt: 309.27 g/mol] was pur- chased from Tokyo Chemical Industry Co. Ltd. Chennai, Tamil Nadu, India. 4-carboxyphenylboronic acid (C7H7BO4; 4-CPBA; Mol. Wt: 165.94 g/mol] was purchased from To- kyo Chemical Industry Co. Ltd. Chennai, Tamil Nadu, In- dia. The citric acid (C6H8O7; 2-hydroxypropane-1,2,3-tri- carboxylic acid; Mol. Wt: 192.12 g/mol], sodium chloride (NaCl), uric acid, ferric chloride (FeCl3), magnesium sulfate (MgSO4), and potassium chloride (KCl), etc. were pur- chased from Loba Chemie Pvt. Ltd. Mumbai, India. Double 68 Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... distilled water (DDW) was purchased from Ranchem Pvt. Ltd. India. For this research work, all chemicals and reagents were utilized exactly as they were obtained. 2. 2. Methods 2. 2. 1. Synthesis of Bare GQDs In this work, the synthesis of bare GQDs was per- formed utilizing a previously published method. In brief, 2 g citric acid was added to 30 mL of DDW. After that, the resulting solution underwent a hydrothermal procedure in a stainless steel autoclave coated with Teflon. In this case, the temperature of the operation was sustained at 160 °C for 8 h in a programmed hot air oven (Bio-Technics In- dia)16. Following the completion of the hydrothermal pro- cedure, the yellow-colored GQDs solution was filtered us- ing 0.22 μm membrane filter paper. Herein, cold centrifugation was used to concentrate the filtrate at 14000 rpm for 45 min at 20 °C. After this, a freeze-drying proce- dure was used to finish the drying. Finally, bare GQDs were employed to validate fluorescence amplification fol- lowing doping and functionalization of GQDs. 2. 2. 2. Green Synthesis of S/N-dGQDs In this step, the green synthesis of S/N-dGQDs was completed from bone meal. In brief, bone meal (1 g) was blended in a glass beaker carrying 50 mL of DDW. Follow- ing this, the produced dispersion was exposed to a sin- gle-step hydrothermal procedure in a stainless steel auto- clave lined with Teflon. The hydrothermal procedure was carried out for 9 h at 160 °C in a laboratory vacuum oven.16 In this part of the process, the initially produced disper- sion of bone meal was altered to a dark brown color. Next, filtration was performed on the treated dispersion using 0.22 μm membrane filter paper. Then, the filtrate was con- centrated using cold centrifugation at 14000 rpm for 45 min at 20 °C. Last, the freeze-drying method was em- ployed to dry up the produced S/N-dGQDs.27 2. 2. 3. Synthesis of Boro-S/N-dGQDs In this step, the synthesis of fluorescent Boro-S/N -dGQDs was accomplished (Scheme 1) for the detection of sialic acid. In short, 4 mL of S/N-dGQDs (100 ng/mL) and phosphate-buffered solution (PBS, pH 7.4) were appropri- Scheme 1: Synthesis of Boro-S/N-dGQDs from bone meal-derived S/N-dGQDs from bone meal powder 69Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... ately mixed (1:1). After that, the solution was treated with 24 mg of 4-CPBA to form Boro-S/N -dGQDs. Here, the resulting mixture was incubated at room temperature for 3 h with constant stirring at 100 rpm. Followed by, the ob- tained Boro-S/N-dGQDs were filtered using a membrane filter whereas the filtrate was dialyzed for 48 h against PBS (pH 7.4) in a dialysis bag (Mol Wt. cut-off: 3500 Da) to provide pure Boro-S/N -dGQDs.23 At last, the freeze-dry- ing method was employed to dry the produced Boro-S/N- dGQDs. 2. 2. 4. Characterizations of Bare GQDs, S/N-dGQDs, Boro-S/N-dGQDs The ultraviolet-visible (UV-Vis) spectrophotometer (UV 1800 Shimadzu, Japan) was chosen to ensure the syn- thesis of bare GQDs, S/N-dGQDs, and Boro-S/N-dGQDs using a quartz cuvette (width: 1 cm) and a scanning wave- length range of 200 nm to 800 nm. The fluorescence study of bare GQDs, S/N-dGQDs, and Boro-S/N-dGQDs was performed using a UV cabinet (Southern scientific lab in- strument, India) and spectrofluorometer spectrophotome- ter (Jasco, FP-8200, Japan). In addition, the excitation and emission of Boro-S/N-dGQDs were reported using a spec- trofluorometer. The excitation wavelength-dependent emission of Boro-S/N-dGQDs was measured by altering the excitation wavelength (range 310 nm − 370 nm) using a spectrofluorometer. The Fourier transform infrared (FT- IR, Bruker, ALPHA II Compact FT-IR Spectrometer) spectrophotometer was used to confirm the functionality present in bare GQDs, S/N-dGQDs, and Boro-S/N-dGQDs. In FT-IR analysis, the samples were scanned from a range of 600 nm to 4000 nm with 22 scans. The particle size, zeta potential, and polydispersity index (PDI) of obtained na- nomaterials bare GQDs, S/N-dGQDs, and Boro-S/N -dGQDs were assessed using a particle size analyzer (Nan- oPlus3, Micromeritics, USA). The crystalline nature of prepared modified nanomaterials was evaluated using a powder X-ray diffractometer (Bruker Kappa Apex II). The elemental composition of bone meal was confirmed using Energy-dispersive X-ray analysis (EDAX, Jeol/OXFORD XMX N). (EDAX)The Raman analysis of S/N-dGQDs and Boro-S/N-dGQDs was completed using via Raman spec- troscopy (Renishaw). The X-ray photoelectron spectrosco- py (XPS, Physical Electronics, PHI 5000 Versa Probe III) was used to verify the surface functionality of S/N-dGQDs and Boro-S/N-dGQDs. The particle size and morphology of S/N-dGQDs and Boro-S/N-dGQDs were validated us- ing high-resolution-transmission electron microscopy [HR-TEM, Joel/JEM 2100] in which LaB6 light was pre- ferred as an electron gun. 2. 2. 6. pH-dependent Fluorescence Stability In this step, the impact of pH on the fluorescence property of Boro-S/N-dGQDs was confirmed. At first, the Boro-S/N-dGQDs (100 ng/mL) were freshly prepared us- ing DDW as a stock. After this, 5 mL of Boro-S/N-dGQDs was added into a separate test tube for further analysis. Herein, different pH (range from pH 3, 5, 7, 9, and 11) of Boro-S/N-dGQDs was adjusted using 1 M NaOH and 1 M HCl. Afterwards, the solutions were kept for 30 min at be- low 25 °C temperature. Finally, the impact of pH on the fluorescent property of Boro-S/N-dGQDs was assessed using a spectrofluorometer at λmax of 360 nm (excitation wavelength). After this, the UV cabinet was preferred to inspect the change in fluorescence of the Boro-S/N-dGQDs sensor under different lights such as visible light, short wavelength (λmax = 254 nm), and long wavelength (λmax = 365 nm). As well, pH-based fluorescence correlation was verified via zeta potential analysis. 2. 2. 7. Determination of Percent Quantum Yield (% QY) In this study, the % of QY of bare GQDs, S/N- dGQDs, and Boro-S/N-dGQDs was calculated using the formerly reported method.26 The ‘% QY’ of bare GQDs, S/N-dGQDs, and Boro-S/N-dGQDs was determined us- ing the following Equation 1, (1) In this equation 1, ‘φ’ denotes the QY. As well, the subscripts 'x' and 'st' stand for a test and standard, accord- ingly. The ‘η’ denotes the refractive index (RI) of solvents. 2. 2. 8. Sensing of Sialic Acid At first, Boro-S/N-dGQDs (100 µg/mL) were pre- pared freshly for sensitivity application. In concisely, 5 mL of prepared Boro-S/N-dGQDs was added into each test tube as a fluorescent sensing probe for the recognition of sialic acid. Then, the selected range of sialic acid concen- tration from 50 ng/mL to 1000 ng/mL was prepared using pH 7.4 phosphate buffer in a separate sample tube. The first concentration of sialic acid was added into the Boro- S/N-dGQDs and then it was kept aside for 10 min to com- plete the reaction. After this, the probe was subjected to a fluorescence study to verify the suppression of fluores- cence of Boro-S/N-dGQDs. Similarly, different concentra- tions of sialic acid were added to the probe solution to ob- tain the linearity. Finally, the relation of concentration of sialic acid vs fluorescence quenching of Boro-S/N-dGQDs was investigated. As well, the limit of detection (LOD) and limit of quantification (LOQ) was assessed using slope and standard deviation. Equation 2 and Equation 3 were used for LOD and LOQ measurements.28 (2) (3) 70 Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... 2. 2. 9. Selectivity, Stability, and Reproducibility Analysis In this study, different interfering agents were pre- ferred to confirm the selectivity of designed Boro-S/N -dGQDs for sialic acid in the complex sample containing agents. In brief, diverse types of interfering agents such as NaCl, KCl, uric acid, MgSO4, and ferric chloride were add- ed into the separate test tube containing pH 7.4 phosphate buffer (100 ng/mL). After this preparation step, 100 µL of each interfering agent was added to the 5 mL of Boro-S/N- dGQDs in a separate test tube. After 10 min, each sample was examined for change in fluorescence using a spectro- fluorometer (n = 3). Similarly, 100 ng/mL of sialic acid (100 μL) was added to the 5 mL of Boro-S/N-dGQDs to compare the selectivity in the presence of other interfering substances. In addition, the stability of fluorescence of Boro-S/N-dGQDs was studied at different time points at 25 °C. Similarly, the stability analysis of Boro-S/N-dGQDs as a sensor in the presence of sialic acid was confirmed. In this study, 100 ng/mL of sialic acid was added to the 5 mL of Boro-S/N-dGQDs sensor solution (n = 3). After com- pletion of the redox reaction, the sensor was subjected to confirm the fluorescence quenching at different time in- tervals at controlled room temperature (25 °C). After this analysis, the reproducibility of the anticipated Boro-S/N- dGQDs-based fluorescent sensor was confirmed. In brief, 100 ng/mL of sialic acid was added to the 5 mL of Boro- S/N-dGQDs (n = 6) in triplicate. This sensor was exam- ined for fluorescence quenching in the occurrence of the same concentrations of sialic acid. Here, the percent rela- tive standard deviation (% RSD) was calculated to confirm the stability and reproducibility of the Boro-S/N-dGQDs sensor for the recognition of sialic acid. 2. 2. 10. Real-time Analysis of Sialic Acid The real-time analysis of sialic acid in local milk- based products, such as cheese, flavored milk, yogurt, and butter was accomplished using a designed fluores- cence-based Boro-S/N-dGQDs sensor. In brief, the milk products were collected from the North Maharashtra state. After that, 10 mg of each milk-based product (cheese) was mixed with dilute hydrochloric acid (45 mM) in separate 50 mL of water in a glass beaker for 75 min at 80 °C. Here, hydrolyzation using dilute hydrochloric acid gives the free form of sialic acid from their conjugates.29 Next, 0.1 mL of the sample was added to the test containing 5 mL of Boro- S/N-dGQDs (n = 3), separately. After 5 min, the fluores- cence of the sensor was monitored to ensure the quench- ing of fluorescence due to the presence of sialic acid in the milk product. At last, the sialic acid concentration was calculated using a calibration curve containing the slope and intercept. A similar method was used for other report- ed milk products such as yogurt, butter, and flavor milk29. 3. Results and Discussion The bone meal underwent PXRD and EDAX analy- ses (Figure S1). In Figure S1A, the diffractogram revealed Figure 1: (A) UV Vis spectra of bare GQDs, S/N-dGQDs, and Boro-S/N-dGQDs. (B) UV cabinet pictures of bare GQDs, S/N-dGQDs, and Boro- S/N-dGQDs at visible light (i), short wavelength (λmax = 254 nm-ii), and long wavelength (λmax = 365 nm-iii). 71Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... sharp intense peaks at 2θ values of 19.94°, 20.64°, 23.56°, 26.38°, 32.32°, 40.19°, 47.28°, etc., indicating its crystalline nature. The EDAX spectrum in Figure S1A displayed the elemental composition of the bone meal, showing the presence of carbon (C), nitrogen (N), oxygen (O), phos- phorus (P), sulfur (S), and calcium, constituting 40.77 wt%, 12.84 wt%, 9.49 wt%, 1.08 wt%, 3.45 wt%, respective- ly. In summary, EDAX analysis confirmed the diverse ele- mental composition present in the bone meal. 3. 1. UV Vis Spectroscopy Figure 1A represents the UV Vis spectra of bare GQDs, S/N-dGQDs, and Boro-S/N-dGQDs. In brief, the green-prepared GQDs showed two absorption peaks. Herein, the peak at 215 nm confirmed the π- π* transition of C=C while the peak at 335 nm indicates the n-π* of -C=O and -OH. Overall, it verifies the presence of carbox- ylic functional groups in bare GQDs. In the second spec- tra, the UV Vis spectrum of S/N-dGQDs showed the peaks at 222 nm while it also demonstrates the decreased ab- sorption peaks around 292 nm (π-π* transition of C=C bond) and 330 nm (n-π* transition of C=N, -C=O and -OH). Therefore, it confirmed the presence of amine and carboxylic functionality on the surface of doped GQDs. As well, the no absorption peak for ‘S’ at near about 550 nm to 595 nm was found, which may be because of the identical electronegativity of ‘C’ and ‘S’. In conclusion, it confirmed the synthesis of S/N-dGQDs using a bone meal via the hy- drothermal method.30,31 In the case of third spectra, the UV Vis absorption spectra of Boro-S/N-dGQDs displayed two peaks around wavelengths 226 nm and 340 nm, which are ascribed to the π-π* and n-π* transitions of carboxylic and amine functionality, respectively. In these spectra, the reduction in peak intensity at 292 nm was obtained which may be because of structural changes. Furthermore, the shift in UV absorption peaks from 222 nm and 330 nm to 226 nm and 340 nm was obtained, which may be because of functionalization using 4-CPBA. Overall, the UV Vis analysis confirmed the synthesis of Boro-S/N-dGQDs. 3. 2. Fluorescence study of GQDs, S/N-dGQDs, and Boro-S/N-dGQDs Figure 1B depicts the fluorescence of bare GQDs us- ing a UV cabinet. In brief, the freshly prepared solutions of bare GQDs, S/N-dGQDs, and Boro-S/N-dGQDs were analyzed for changes in fluorescence in different lights. In brief, bare GQDs exhibited no color (transparent), faintly blue, and blue fluorescence at visible light, short wave- length (λmax = 254 nm), and long wavelength (λmax = 365 nm), respectively.26 Figure 1B displays the pictures of the S/N-dGQDs in different lights. In concise, it shows a slight orange color, green luminescence, and prominent blue flu- orescence in the visible range, short wavelength (λmax = 254 nm), and long wavelength (λmax = 365 nm), respec- tively. At this instant, it confirmed the significant incre- ment in the fluorescence behavior of S/N-dGQDs. Figure 1B demonstrates the fluorescence behavior of Boro-S/N- dGQDs in different lights. In brief, it shows the orange color, greenish-orange fluorescence, and bright bluish flu- orescence under visible light, short wavelength (λmax = 254 nm), and long wavelength (λmax = 365 nm), respectively. Here, the functionalization of S/N-dGQDs using 4-CPBA offered a boost in the fluorescence property of Boro-S/N- dGQDs more than the S/N-dGQDs and bare GQDs. In a nutshell, this study confirmed the functionalization of S/N-dGQDs. After confirmation of fluorescence changes of prepared bare GQDs, S/N-dGQDs, and Boro-S/N -dGQDs, the spectrofluorometer was preferred to ensure the fluorescence characteristic of bare GQDs, S/N-dGQDs, and Boro-S/N-dGQDs. In brief, the diverse types of mod- ification strategies such as doping and functionalization of bare GQDs resulted in the boost in fluorescence properties of GQDs. The % QY was found to be 8.9 %, 25.36 %, and 68.69 % for bare GQDs, S/N-dGQDs, and Boro-S/N -dGQDs, accordingly. Herein, the augment in % QY of Boro-S/N-dGQDs confirmed the improvement in optical properties such as the fluorescence of the sensor. Figure 2A depicts the excitation wavelength-dependent emission spectra of Boro-S/N-dGQDs. In brief, the change in ex- citation wavelength from 310 nm to 350 nm resulted in a shift in the emission wavelength of Boro-S/N-dGQDs to- wards a longer wavelength. At an excitation wavelength of 360 nm, it demonstrated an intense emission peak at 425 nm. After an increment in excitation wavelength from 360 nm to 370 nm, the reduction in emission peak with a slight shift towards a longer wavelength was obtained. Overall, the excitation and emission of Boro-S/N-dGQDs were ob- tained at 360 nm and 425 nm, respectively (Figure 2B). Figure 2C displays the fluorescence behavior of GQDs, S/N-dGQDs, and Boro-S/N-dGQDs. Here, there was a significant difference in the fluorescence behavior of Boro- S/N-dGQDs obtained as compared to the S/N-dGQDs and bare GQDs. Interestingly, it may be because of the combined benefits of ’S’ and ‘N’ atom doping in graphitic structure as well as the functionalization of S/N-dGQDs using 4-CPBA. Overall, Boro-S/N-dGQDs presented a highly fluorescent sensing system for the revealing of in- terest analytes that can assist in improving sensitivity pa- rameters. 3. 3. pH study of Boro-S/N-dGQDs The UV cabinet study of Boro-S/N-dGQDs at differ- ent pH at longer wavelength light (λmax = 365 nm) reveals the changes in fluorescence behavior. Figure 3A illustrates the UV cabinet pictures of Boro-S/N-dGQDs at different pH ranges. In brief, the change in pH from pH 3 to pH 5 displayed an increase in the fluorescence intensity of Boro- S/N-dGQDs. On the contrary, the adjustment in pH from pH 9 to pH 11 demonstrated the reduction in fluorescence 72 Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... intensity of Boro-S/N-dGQDs. As well, at pH 7, Boro-S/N- dGQDs displayed a high fluorescent intensity. The impact of different pH ranges on the fluorescence property of de- signed Boro-S/N-dGQDs may be because of protonation and deprotonation. In conclusion, it confirmed the impact of pH on the fluorescence characteristics of Boro-S/N -dGQDs. After qualitative confirmation, the quantitative confirmation of pH impact on fluorescence was confirmed using a spectrofluorometer. Later, the impact of pH of flu- orescence of Boro-S/N-dGQDs was examined using a spectrofluorometer (Figure 3B). In concisely, the fluores- cence spectra revealed a rise in fluorescence after adjusting the pH from the acidic range (pH 3 to pH 5). At pH 7, the fabricated Boro-S/N-dGQDs sensor disclosed a higher flu- orescence intensity. After that, with the change in pH from pH 9 to pH 11 (basic), there was a reduction in fluores- cence intensity of the Boro-S/N-dGQDs sensor. Possibly, the protonation or de-protonation and aggregation of the Boro-S/N-dGQDs may be responsible for the pH-based changes in the fluorescence properties of Boro-S/N -dGQDs.32 Figure 3C disclosed the impact of pH on the zeta potential of the Boro-S/N-dGQDs sensor solution. In concisely, the zeta potential of the Boro-S/N-dGQDs sen- sor was found to be + 30.58 mV, and + 15.28 mV, for pH 3, and pH 5, accordingly. After changes in pH from the neu- tral to the basic, the zeta potential of Boro-S/N-dGQDs was obtained to be –34.8 mV, –26.4 mV, and –39.88 mV at pH 7, 9, and 11, respectively. In this, the change in the zeta potential of Boro-S/N-dGQDs with respective adjusted pH of solution confirmed the protonation and deprotona- tion of carboxylic functionality present in the exterior of Boro-S/N-dGQDs. As well, it ensured the good stability of designed Boro-S/N-dGQDs in an aqueous environment. 3. 4. Zeta Potential and Particle Size Analysis The zeta potential of nanomaterials is measured to ensure their stability in a particular solvent solution. Figure 2: (A) Excitation wavelength dependant emission of Boro-S/N-dGQDs. (B) Excitation and emission spectra of designed Boro-S/N-dGQDs. (C) Fluorescence comparison of bare GQDs, S/N-dGQDs, and Boro-S/N-dGQDs 73Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... Figure 4 depicts the zeta potential of bare GQDs, Boro- S/N-dGQDs, and Boro-S/N-dGQDs.33 In concisely the zeta potential of citric acid-produced bare GQDs was determined to be –31.79 mV. As a result, it proved that the negative zeta potential was due to the presence of carboxylic functionality on the surface of GQDs, such as carboxyl, hydroxyl, epoxy, etc. It also ensured the stability of GQDs in the aqueous system. The zeta po- tential of S/N-dGQDs was also discovered to be –20.96 mV. Here, the decrease in zeta potential is caused by the incorporation of 'N' and 'S' into the graphitic struc- ture of GQDs. Furthermore, it demonstrated the stabil- ity of S/N-dGQDs in aquatic environments. Boro-S/N- dGQDs had a zeta potential of –19.62 mV after the functionalization of the S/N-dGQDs. As a result, it implies that Boro-S/N-dGQDs are stable in specific solvent systems. 3. 5. FT-IR Spectroscopy Analysis Figure 5A displays the FT-IR spectrum of bare GQDs, S/N-dGQDs, and Boro-S/N-dGQDs. In brief, the peak intensity at 3236 cm–1, 1637 cm–1, 1524 cm–1, 1445 cm–1, 1395 cm–1, and 1233 cm–1 indicates the OH stretch- ing, C=O stretching, aromatic C=C stretching, OH bend- ing, C-O stretching of COOH, and C-O-C stretching vi- brations, respectively. Hence, it confirmed the presence of carboxylic functionality in citric acid-made bare GQDs. The peak intensity at 3186 cm–1, 3045 cm–1, 1680 cm–1, 1355 cm–1, and 1170 cm–1 confirmed the OH/NH₂ stretch- ing, –CH stretching, C=O stretching, C-N stretching, and C-S stretching vibrations, respectively. On the whole, the FT-IR analysis confirmed the presence of carbon, oxygen, nitrogen, and sulfur-based functionality in S/N-dGQDs. The peak intensity at 3275 cm–1, 2975 cm–1, 1700 cm–1, 1555 cm–1, 1338 cm–1, 1254 cm–1, and 1170 cm–1 desig- Figure 3: Impact of pH on fluorescence behavior of Boro-S/N-dGQDs. (B) Fluorescence spectra of Boro-S/N-dGQDs in different pH solutions. (C) Zeta potential of Boro-S/N-dGQDs at different pH 74 Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... nates the -OH/NH stretching, C-H stretching, C=O stretching, C-N stretching, B-O-H bending, C-O-C stretching, and C-S stretching vibrations, respectively. Overall, FT-IR analysis confirmed the synthesis of Boro- S/N-dGQDs. 3. 6. Powder X-ray Diffraction (PXRD) In this work, PXRD analysis ensured the fabrication of the graphitic architecture of S/N-dGQDs and Boro-S/ N-dGQDs. In brief, Figure 5B reveals the diffractogram of Figure 5: (A) Overlay of FTIR spectra of overlay of bare GQDs, S/N-dGQDs, and Boro-S/N-dGQDs. (B) Diffractogram of S/N-dGQDs and Boro- S/N-dGQDs Figure 4: Zeta Potential analysis of (A) bare GQDs, (B) S/N-dGQDs, and (C) Boro-S/N-dGQDs 75Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... Figure 6: (A) XPS survey scan spectra of S/N-dGQDs. The deconvoluted high-resolution XPS spectra of (B) C1s, (C) S2p, (D) N1s, and (E) O1s S/N-dGQDs in which a wide diffraction peak was ob- tained at 2θ = 19.25° and 23.69°.30 Here, it confirmed the occurrence of S/N-dGQDs in the amorphous form of a graphene-like structure. Possibly, the doping of the ‘N’ and ‘S’ components in graphitic structure enhanced the lattice voids and structural defects. The diffractogram of Boro-S/ 76 Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... N-dGQDs is presented in Figure 5B. It displays the peaks at 2θ = 17.70°, 24.19°, 28.70°, 29.99°, 31.02°, 32°, 34.31°, 35.52°, 36.72°, 38.74°, 40.87°, 46.04°, 46.74°, 47.29°, 50.54°, 55.36°, 59.05°, 60.36° and 66.75°. Here, the crystallinity of S/N-dGQDs was increased after functionalization by 4-CPBA (‘d’ spacing: 0.36). Possibly, the functionality of 4-CPBA provides the separation of graphitic flakes that may part in a boost in the crystalline nature of the sensor. Overall, it confirmed the synthesis of Boro-S/N-dGQDs from S/N-dGQDs. Figure 7: (A) XPS survey scan of Boro-S/N-dGQDs. The deconvoluted high-resolution XPS spectra of (B) B1s, (C) C1s, (D) N1s, (E) O1s, and (F) S2p 77Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... 3. 7. X-Ray Photoelectron Spectroscopy Figure 6 represents the XPS spectra of S/N-dGQDs. The survey scan spectrum showed the peaks at binding en- ergy 159.5 eV, 285 eV, 400.5 eV, and 531.5 eV for ‘S2p, C1s, N1s, and O1s,’ respectively. Hence, it confirmed the pres- ence of ‘S, C, N, and ‘O’-based functionality in S/N- dGQDs.34 In brief, ‘Cls’ deconvoluted high-resolution peaks showed the binding energies at 284.48 eV, 285.33 eV, and 288.43 eV for C=C, C-O/C-N, and O-C=O respective- ly. As a result, it assured the presence of carbon-based functionality in S/N-dGQDs. The ‘O1s’ deconvoluted high-resolution XPS spectra demonstrated the intensity peaks at binding energies of 531.71 eV and 532.67 eV for C-OH and C-O-C, respectively. Hence, it confirmed the existence of oxygen-based functionality in S/N-dGQDs. The ‘S2p’ deconvoluted high-resolution XPS spectra showed peaks at binding energies of 163.48 eV and 163.53 eV for C-S (S2p½) and C-S (S2p3/2), respectively. Hence, it verified the doping of the ‘S’ element into the graphitic structure of GQDs. The ‘N1s’ high-resolution peaks illus- trated the binding energies at 399.92 eV for N-H that en- sured the presence of ‘N’ in graphitic frameworks of GQDs. In conclusion, the XPS analysis of S/N-dGQDs val- idated the synthesis of heteroatom-doped GQDs from Figure 8: HR-TEM images of (A, B) S/N-dGQDs and (C, D) Boro-S/N-dGQDs 78 Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... bone meal. Figure 7 represents the XPS spectra of Boro-S/ N-dGQDs. The survey scan spectrum of Boro-S/N-dGQDs displayed the major peaks at near about 154 eV, 192.5 eV, 284 eV, 399 eV, and 531.5 eV indicating the S2p, B1s, C1s, N1s, and O1s, respectively. Hence, it confirmed the occur- rence of ‘B1s, S2p, C1s, N1s, and O1s’ after the functional- ization of S/N-dGQDs.34 In brief, the ‘S2p’ deconvoluted high-resolution XPS spectra demonstrated the peaks at the binding energies of 164.05 eV and 168.49 eV for -SO3H and -SH, respectively. The ‘B1s’ deconvoluted high-resolu- tion XPS spectra disclosed the peaks at binding energies of 190.99 eV, 193.07 eV, and 192.15 eV for B-N, C-B-N, and B-O, respectively. Therefore, it confirmed the functionali- zation of S/N-dGQDs using 4-CPBA. In short, the ‘Cls’ deconvoluted high-resolution XPS spectra revealed the binding energies at 284.88 eV, 287.42 eV, and 295.36 eV for C-C, C-N, and C-B-N, respectively. The ‘O1s’ deconvolut- ed high-resolution XPS spectra showed the peaks at 532.11 eV for O-B and C-O confirming the presence of oxy- gen-based functionality. In addition, the ‘N1s’ deconvolut- ed high-resolution XPS spectra endow with the peaks at binding energies of 399.95 eV and 401.36 eV for N-B and N-C, respectively. Consequently, it confirmed the func- tionalization of S/N-dGQDs using 4-CPBA. 3. 8. HR-TEM Analysis The particle size and shape of S/N-dGQDs and Boro- S/N-dGQDs were confirmed using HR-TEM analysis. In brief, Figures 8A and B displayed the HR-TEM images of S/N-dGQDs. In this, the average particle size of S/N- dGQDs was found to be less than 10 nm. As well, the shape of S/N-dGQDs was found to be spherical with proper dis- tribution in the aqueous system. Overall, it confirmed the synthesis of nanosized and non-aggregated S/N-dGQD.35 Figures 8C and D illustrate the HR-TEM image of Boro- S/N-dGQDs. Here, the average particle size of Boro-S/N- dGQDs was found to be upto 20 nm. As well, it depicts the spherical shape along with homogenous dispersion in an aqueous environment. Here, the increase in average parti- cle size of Boro-S/N-dGQDs was obtained from the S/N- dGQDs which may be because of the decoration of 4-CPBA on the surface of S/N-dGQDs. Taken as a whole, the HR-TEM analysis proved the synthesis of nano dimen- sion and uniform distribution of Boro-S/N-dGQDs from bone meal.35 3. 9. Raman Spectroscopy Figure 9 depicts the Raman spectra of as-synthe- sized S/N-dGQDs and Boro-S/N-dGQDs. In brief, the Raman spectra of S/N-dGQDs revealed the two bands namely the ‘D’ band and the ‘G’ band at near about 1327.77 cm–1 and 1583.39 cm–1 respectively. The ratio of the intensity of the ‘D’ band and the ‘G’ band (ID/IG) was obtained to be 1.64. Here, the disordered structure of the ‘D’ band is related to hetero-atom doping faults and size reduction. On the contrary, the crystal-like character of carbon material is accompanied by the ‘G’ band.36,37 In the case of Raman spectra of Boro-S/N-dGQDs, the ‘D’ band, and the ‘G’ band were obtained at 1327.70 cm–1 and 1587.44 cm–1, respectively. The ratio of ID/IG (ID/ IG:1.14) was found to be reduced than the S/N-dGQDs, which may be because of the functionalization of S/N- dGQDs, surface using 4-CPBA.36,37 Overall, the Raman analysis confirmed the synthesis of S/N-dGQDs and Boro-S/N-dGQDs. 3. 10. Sensing of Sialic Acid using Fluorescence-Based Boro-S/N-dGQDs In this step, sialic acid was detected using a fabricat- ed highly fluorescent Boro-S/N-dGQDs sensor. To begin, different concentrations of sialic acid were incubated in a separate test tube containing the Boro-S/N-dGQDs sen- sor. Here, the reaction between sialic acid and 4-APBA of functionalized doped GQDs was achieved. As an output, the screening of fluorescence of the Boro-S/N-dGQDs sensor in the occurrence of sialic acid revealed fluores- cence quenching. Similarly, an increase in sialic acid con- centration has a directly proportional relationship with fluorescence suppression of the Boro-S/N-dGQDs sensor (Figure 10A). The linearity range (y = 6.71x + 0.1089, R2 = 0.99) of sialic acid appears here from 50 ng/mL to 1000 ng/ mL (Figure 10B). The LOD and LOQ for sialic acid were then determined to be 6.04 ng/mL and 14.81 ng/mL, re- spectively. It ensured that the doping of heteroatoms like ‘N’ and ‘S’, as well as the boronic acid functionalization of GQDs, provides increased sensitivity to sialic acid. In terms of sensing, the Boro-S/N-dGQDs sensor provides boronic acid functional groups on the surface. Important- ly, it generates a reversible covalent contact with sialic acid. Figure 9: Raman spectra of S/N-dGQDs and Boro-S/N-dGQDs. 79Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... Figure 10: (A) Fluorescence spectra of sialic acid concentrations based on quenching of Boro-S/N-dGQDs fluorescence. (B) Graph of linear corre- lation between concentrations of sialic acid and fluorescence of Boro-S/N-dGQDs. (C) Selectivity study of Boro-S/N-dGQDs (probe) for sialic acid (SA) in the presence of interfering agents As a result, it aids in modulating the fluorescence intensity of the Boro-S/N-dGQDs sensor.38,39 Table 1 summarizes the comparison of formerly reported methods for sensing sialic acid. 3. 11. Selectivity Study and Other Analytical Parameters To verify the anti-interference potential of the Boro-S/N-dGQDs sensor, the selectivity study was per- Table 1: Summary of SA detection using fluorescence-mediated sensing methods Sr. No. Material used Method used LOD Linearity range Ref. 1. Boronic acid Spectrofluorimetric 54 µM 80 µM to 4000 µM 40 functionalized carbon dots 2. Gold nanoparticles Colorimetric 68 µM 80 µM to 2000 µM 41 3. – Liquid chromatography 0.003 mg/ mL 0.1 μg/mL to 10 µg/mL 42 fluorescence detection 4. – Spectrophotometric 0.239 mg/mL 1 mg/mL to 10 mg/mL 43 5. Zirconium metal-organic Spectrofluorimetric 0.15 μM 1 μM to 100 μM 44 framework 6. Boro-S/N-dGQDs Spectrofluorimetric 6.04 ng/mL 50 ng/mL to 1000 ng/mL Present work 80 Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... formed using different interfering agents. Figure 10C il- lustrates the selectivity potential of the Boro-S/N-dGQDs sensor for sialic acid in the occurrence of interfering sub- stances. In short, it verifies the fluorescence quenching of the Boro-S/N-dGQDs sensor after the addition of sialic acid. On the contrary, there was no response was found for the addition of the mentioned interfering molecules. Moreover, the addition of sialic acid with a mixture of interfering agents did not demonstrate a significant change in fluorescent intensity than the Boro-S/N-dGQDs Figure 11: (A) Schematic representation of sensing of sialic acid using designed Boro-S/N-dGQDs based fluorescence turn ‘On-Off ’ sensor. (B) Fluorescence stability of Boro-S/N-dGQDs. (C) Sensor stability of Boro-S/N-dGQDs after the addition of sialic acid at different intervals. (D) Repro- ducibility study of Boro-S/N-dGQDs for detection of sialic acid (n = 6). 81Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... sensor with a sialic acid response. Importantly, the fabri- cated Boro-S/N-dGQDs sensor demonstrated the high selectivity for sialic acid only due to the esterification re- action between boronic acid-containing functional groups and sialic acid. After this, stability and reproduci- bility analysis was performed as an important analytical parameter of the sensor. The scheme of the detection mechanism for sialic acid using a highly fluorescent Boro-S/N-dGQDs sensor is revealed in Figure 11A. In brief, the boronic functional group of prepared Boro-S/ N-dGQDs formed the cyclic ester with a sialic acid-con- taining dial group that may be the possible reason for the quenching of fluorescence behavior of Boro-S/N-dGQDs. At first, the fluorescence stability (Figure 11B) was per- formed in the absence of sialic acid wherein it proved good stability for up to 6 days (n = 3). After this, a decline in fluorescence intensity was obtained, which may be be- cause of the protonation or deprotonation of Boro-S/N- dGQDs in the solution. As well, the Boro-S/N-dGQDs sensor stability was performed with sialic acid (Figure 11C). Herein, the addition of sialic acid into the fluores- cent Boro-S/N-dGQDs sensor shows the quenching of fluorescence. The stability study ensured that the slight recovery of suppressed fluorescence of Boro-S/N -dGQDs-SA was found after 48 h. Hence, it confirmed the good stability (% RSD: 0.0068%) of Boro-S/N -dGQDs-SA samples. The reproducibility analysis of the Boro-S/N-dGQDs sensor provided the 0.0067% of % RSD (less than 5) confirmed good reproducibility (Figure 11D). Overall, the designed Boro-S/N-dGQDs-based flu- orescent sensor exhibited high sensitivity, high selectivi- ty, good stability, and reproducibility for the recognition of sialic acid. In the future, there is a need to conduct spiked sample analysis and preclinical studies to ensure the practical applicability of the proposed Boro-S/N -dGQDs fluorescent-mediated sensor for sensing sialic acid. 3. 12. Real-time Analysis of Sialic Acid The real-time analysis of sialic acid in local milk- based products was accomplished using a fluorescence Boro-S/N-dGQDs sensor. In summary, the total amount of sialic acid in cheese and butter was found to be 11 ± 1.10 ng/mL and 7.5 ± 1.85 ng/mL, respectively. Similarly, the analysis of flavored milk and yogurt confirmed the pres- ence of 6.4 ± 2.41 ng/mL and 9.74 ± 2.14 ng/mL of sialic acid, respectively.29 In conclusion, the Boro-S/N-dGQDs sensor confirmed the presence of sialic acid in milk prod- ucts of north Maharashtra, India. In the future, there is a need to explore the reported Boro-S/N-dGQDs on a larger scale for the detection of sialic acid in different milk prod- ucts. Furthermore, the validation of the proposed sensor using another method is needed for the detection of sialic acid. 4. Conclusion This work aimed to construct an extremely luminous Boro-S/N-dGQDs sensor for highly sensitive and selective detection of sialic acid in milk-based products. In concise, the bone meal was effectively used as both a dopant and precursor in the single-step hydrothermal synthesis of S/N-dGQDs, resulting in improved fluorescence com- pared to bare GQDs. Subsequently, 4-CPBA was used to functionalize the surface of S/N-dGQDs, leading to even higher fluorescence levels than S/N-dGQDs alone. Next, the spectral study confirmed the formation of stable, na- nosized, spherical Boro-S/N-dGQDs with appropriate functional groups required for sialic acid detection. Upon the addition of sialic acid to Boro-S/N-dGQDs, the fluo- rescence exhibited a 'turn ON-OFF' behavior, providing a wide linear range from 50 ng/mL to 1000 ng/mL and a re- duced detection limit of 6.04 ng/mL in phosphate buffer at pH 7.4. The sensor also demonstrated strong selectivity for sialic acid due to the interaction of sialic acid-containing 1,2-diol with a hydroxyl group of Boro-S/N-dGQDs. Fur- thermore, the Boro-S/N-dGQDs design exhibited high stability and repeatability. The real-time analysis con- firmed the presence of sialic acid in cheese, butter, flavored milk, and yogurt, thus affirming the practicality of the de- signed Boro-S/N-dGQDs sensor for detecting and moni- toring sialic acid in milk-based products. In conclusion, Boro-S/N-dGQDs offer a highly selective, sensitive, sim- plistic, eco-friendly, and cost-effective platform for sens- ing sialic acid in milk-based products. 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Sun, Talanta 2019, 197, 548–552. DOI:10.1016/j.talanta.2019.01.074 41. S. Sankoh, C. Thammakhet, A. Numnuam, W. Limbut, P. Kanatharana, P. Thavarungkul, Biosens. Bioelectron. 2016, 85, 743–750. DOI:10.1016/j.bios.2016.05.083 42. F. Zhao, B. Chen, K. Li, X. Wang, Sh Kexue/Food Sci. 2021, 42, 313–318. DOI: 10.7506/spkx1002-6630-20191008-031 43. J. B. Costa, N. T. de Paula, P. A. da Silva, G. C. de Souza, A. P. S. Paim, A. F. Lavorante, Microchem J. 2019, 147, 782–788. DOI:10.1016/j.microc.2019.03.086 44. Q. Cao, Y. Peng, Q. Yu, Z. Shi, Q. Jia, Dyes Pigm. 2022, 197, 109839. DOI:10.1016/j.dyepig.2021.109839 83Acta Chim. Slov. 2024, 71, 66–83 Nangare et al.: Nanoprobe for Sialic Acid Sensing ... Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Detekcija sialne kisline v mlečnih proizvodih z naprednimi senzorji je ključna v živilski industriji. Namen predstavljene- ga dela je uporaba grafenskih kvantnih pik, pridobljenih iz kostne moke, dopiranih z žveplom in dušikom ter funkcional- iziranih z boronsko kislino (Boro-S/N-dGQDs) kot nanosenzorji za določanje sialne kisline. Nanodelce S/N-dGQDs smo funkcionalizirali s 4-karboksifenilboronsko kislino z namenom izboljšanja flurescenčnih lastnosti senzorja za določanje sialne kisline. Vezavo boronske kisline na površino S/N-dGQD smo potrdili z različnimi spektralnimi metodami karak- terizacije. Dodatek različnih množin sialne kisline je povzročil proporcionalno korelacijo s fluorescenčnimi lastnostmi. Meritve kažejo široko linearno območje od 50 ng/mL do 1000 ng/mL in mejo določljivosti 6.04 ng/mL. Metoda izkazu- je tudi dobro selektivnost, najverjetneje zaradi interakcije med 1,2-diolom sialne kisline in hidroksilno skupino Boro- S/N-dGQDs nanosenzorja. Pripravljeni senzor kaže dobro stabilnost in ponovljivost. Analize sialne kisline v različnih mlečnih proizvodih v realnem času potrjujejo uporabnost senzorjev na osnovi Boro-S/N-dGQDs. 84 Acta Chim. Slov. 2024, 71, 84–90 Ribič et al.: Assessing 15-year-olds’ Understanding of Chemical ... DOI: 10.17344/acsi.2024.8610 Scientific paper Assessing 15-year-olds’ Understanding of Chemical Concepts in the Context of the Lithosphere and Pedosphere Luka Ribič,1* Iztok Devetak1 and Miha Slapničar 1,2* 1 University of Ljubljana, Faculty of Education, Kardeljeva ploščad 16, 1000 Ljubljana, Slovenia 2 BEC Ljubljana, Cesta v Mestni log 47, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: luka.ribic@pef.uni-lj.si; miha.slapnicar@pef.uni-lj.si Received: 01-08-2024 Abstract The aim of this study was to determine the understanding of environmental chemistry content related to lithosphere and pedosphere, such as soil and soil types, soil horizons, rock and rock types, weathering, minerals, coal, and soil erosion, and to investigate misconceptions among 9th grade lower secondary school students. 503 students (254 male and 249 female) from 14 different primary schools and 8 different regions of Slovenia participated in this study. A three-tier achievement test (with 10 three-tier tasks to identify misconceptions) was used to collect the data. The results show that the Slovenian students’ knowledge of the lithosphere and pedosphere is adequate. On average, students achieved 55.6% of all possible points. The lowest level of knowledge was found for the topic of soil formation. The number of misconcep- tions on this topic is low and does not exceed 30% for any task. The highest number of misconceptions was found for the topic of soil formation and pollution. Keywords: Three-tier diagnostic test, environmental chemistry, environmental education, lithosphere, pedosphere, mis- conceptions. 1. Introduction The Slovenian environmental curriculum is interdis- ciplinary in its structure, i.e., it contains a list of objectives and recommendations. The reason for such a curriculum lies in the complexity of environmental problems, whose explanation and solution lies at the intersection of several sciences.1 One of the multidisciplinary sciences that is part of environmental education and combines physics, chem- istry, biology, etc. is environmental chemistry.2 Environ- mental chemistry also includes the topics of soil litera- cy,3,4,5 such as the topic of lithosphere3,6 and pedosphere.6 These two topics also lend themselves to the integration of physics, chemistry, and mathematics.7 In Slovenian school system topic of lithosphere and pedosphere is taught in natural science in 6th grade.8 It is teachers’ responsibility to connect this content from science and geography, so that the students acquire broader picture of these content.9 People need adequate and quality knowledge about environmental factors to protect the environment, explain environmental problems, and create a healthy environ- ment for future generations.10 To this end, environmental education must provide students with soil literacy. Soil lit- eracy is a combination of attitudes, behaviours and skills that ultimately contribute to the well-being of the natural environment.6 Experts believe, that we need a methodo- logical approach if we want to measure the effectiveness of environmental education.11 A well-known barrier to science learning are mis- conceptions.12 Misconceptions are cognitive structures that are persistent and can become an obstacle when students want to learn more complex concepts. It is very important that we review possible misconceptions be- fore we begin teaching new content.13,14 Misconceptions formed in school are the result of misleading explanations of concepts where we find oversimplifications and gener- alization.15 The study of misconceptions is of interest to researchers. Misconceptions can be uncovered with writ- ten tests of knowledge, such as: achievement tests with multiple-choice questions, multiple-tier tests of knowl- edge, concept maps, interviews, etc.16 The limiting factor 85Acta Chim. Slov. 2024, 71, 84–90 Ribič et al.: Assessing 15-year-olds’ Understanding of Chemical ... in diagnostic multiple-choice tests is the high probability of guessing. Therefore, diagnostic tests began to gain base- line knowledge by requiring an explanation for the answer choice in addition to the answer. This form of testing al- lows for exploration of the reasons for the occurrence of misconceptions.17 Cetin – Dindar and Geban18 developed a diagnostic knowledge test with three-tier tasks to deter- mine students’ knowledge of acids and bases. This test was used to test how much more accurate it is compared to the two-tier and one-tier diagnostic knowledge test. Re- liability was measured using the Cronbach’s alpha coeffi- cient. This showed that the reliability of the first part of the knowledge tests (alpha coefficient value) was 0.58, the reliability of the second part was 0.59, and the reliability of the third part was 0.72. According to Milenković et al.17 0 to 30% of students indicate a low number of misconcep- tions, 31 to 60% of students indicate a medium number of misconceptions and more than 61% of students indicate a high number of misconceptions on a given topic. Research by Borghini et al.14 and Dove19 has shown that students have misconceptions about earth science. Misconceptions exists for several topics related to earth science, such as rocks, earthquakes, volcanoes, the struc- ture of the earth, landforms, weathering and erosion, and soil. Borghini et al.14 cited the short time devoted to earth science, absence of geological background of teachers, dif- ficulty in understanding complex topics, ineffective teach- ing and learning methods, etc., as reasons for the high number of misconceptions in this area. Francek20 found a high number of misconceptions in the topic of tecton- ic plates followed by the topic of weathering/erosion. As Monteiro et al.21 found, students also have problems with the definitions of minerals. The study found that more than 92% of students have misconceptions about miner- als. Given the wide variety of minerals and rocks that can appear, this is to be expected.19 Study by Putri et al.22 also showed that type of task can be problematic. Students usu- ally have problems interpreting social problems or mathe- matical data in graphs. Misconceptions about the rock cycle often stem from students inability to understand the rock cycle.20 The problem of understanding the rock cycle among students can address many misconceptions about rocks, but stu- dents have trouble connecting the three major rock cat- egories.23 Rather than seeing a connection between rock classes and the rock cycle, students view the rock cycle as the cause of rock formation.24 Weathering and erosion are also part of the rock cycle and allow rocks to change from one form to another.23 Unable to connect different rock types24 students view erosion and weathering as two unrelated processes and do not connect them to the for- mation of soil.20 Rock classification and formation is also problematic because students use observable character- istics such as colour, shape, and size to identify specific rock types. However, these features are not used in rock identification. Therefore, students’ perceptions of rocks they know from previous experience are not met and they remain unidentified.19 There was confusion among students about what soil is made of and how long it takes to form.20 The same problem was found among teachers in a study by Hayhoe et al.25 where teachers had difficulty defining soil as a com- position of solid particles with spaces for air and water. Students often believe that soil extends for miles below the surface.20 This may be due to the difficulty in visualizing cross-sections of soil as soil profiles that are not easily ob- servable.19 Russel et al.26 conducted a study that found that upper-level students do not understand the nature of soil and cannot relate to soil composition. 2. Research Problem and Research Questions The environmental program was introduced in the Slovenian school system in 2008.1 Part of environmen- tal education is also environmental chemistry,2 which covers the topics of lithosphere and pedosphere.3,4,5 To our knowledge, no research has been conducted on the performance of environmental education and students’ misconceptions about environmental chemistry topics such as lithosphere and pedosphere. In subject of natural sciences students should be introduced to the key con- cepts earth science and also reflect on the main causes of soil pollution.8 However, we do not have enough data to evaluate students’ basic understanding of environmental issues.28 The aim of the present research is to identify the level of knowledge that 9th grade primary school students pos- sess about the lithosphere and pedosphere. Two research questions were formulated for this purpose: (1) What is the level of knowledge of 15-year-old students about the lithosphere and pedosphere? (2) Do students have misconceptions about the lith- osphere and pedosphere? 3. Method A quantitative and cross-sectional research approach was used in this study, non-experimental and descriptive methods were used to determine students’ knowledge of the lithosphere and pedosphere. 3. 1. Participants A total of 503 students (254 males and 249 females, M = 15 years, SD = 6.0 months) attending 14 different ele- mentary schools in 8 different statistical regions of Slove- nia participated in the study. This sample represent 2.53% of the entire population of 9th grade students in that year29. Participation in the study was voluntary and anonymous. 86 Acta Chim. Slov. 2024, 71, 84–90 Ribič et al.: Assessing 15-year-olds’ Understanding of Chemical ... Prior to implementation, a letter was sent to the school and parents or caregivers of ninth graders informing them of the study. School principals, teachers, students, and their parents or caregivers agreed to participate in the study and informal consents were signed by students’ parents or car- egivers. 3. 2. Instruments The data was collected using instrument comprised of two parts: (1) information about the participants (IP), that include general information about the participants (e.g., gender, school, region and grades in biology, chem- istry, and physics; (2) diagnostic instrument entitled How Well do I Know Soil and Rocks (HWiKSR), which measured students’ knowledge about lithosphere and pedosphere and consist of 10 three-tier multiple-choice tasks, of specific environmental phenomena such as: soil, rocks, soil pollution, rock formation, erosion, soil struc- ture and soil formation. The content validity of the instruments was con- firmed by six independent experts in chemistry and envi- ronmental education. The full texts of the instrument can be obtained by request from the corresponding author. HWiKSR tasks differ in level of complexity and specificity according to Krathwohl27. According to Bloom taxonomy each task has been defined in which level it be- longs according to this taxonomy. Each tasks topic and Bloom’s cognitive level is shown in Table 1. Each task as shown in Figure 1 includes three-tiers: a multiple-choice answer tier (tier 1), a reasoning tier (tier 2) describing an expected reason for the students’ answer selected in tier 1 and a six-point confidence scale (tier 3) – the answers obtained in the six-point confidence scale correspond to “1-just guessing”, “2-very unconfident”, “3-unconfident”, “4-confident”, “5-very confident” and “6-absolutely confi- dent” and expresses the students’ confidence in giving the answer and the reason for it (tiers 1 and 2). To simplify the discussion, the following answers from the confidence scale were merged as follows: ˝Not Sure˝, when students choose “1”, “2” or “3” and ˝Sure˝ when students pick “4”, “5” or “6” on the confidence scale. The overall response possibilities in the HWiKSR (first, second, and third tiers together) resulted in the following categories according to Milenković et al.17: (i) a combination of correct (tier 1) and correct (tier 2) and sure (tier 3) answers was treat- ed as knowledge (ii) a combination of correct (tier 1) and correct (tier 2) and not sure (tier 3) answers was treated as luck (iii) a combination of incorrect (tier 1) and cor- rect (tier 2) and not sure (tier 3) answers was treated as guessing (iv) a combination of correct (tier 1) and incor- rect (tier 2) and not sure (tier 3) answers was treated as guessing (v) a combination of incorrect (tier 1) and incor- rect (tier 2) and not sure (tier 3) answers was treated as lack of knowledge (vi) a combination of correct (tier 1) and incorrect (tier 2) and sure (tier 3) answers was treated as misconception (vii) a combination of incorrect (tier 1) and correct (tier 2) and sure (tier 3) answers was treated as misconception (vii) and a combination of incorrect (tier 1) and incorrect (tier 2) and sure (tier 3) answers was treat- ed as misconception. The answer to an item was correct if both first and second tiers were correctly answered. The HWiKSR diagnostic instrument not only identifies mis- conceptions of 15-year-old students, but also differenti- ates them from their lack of knowledge about the litho- sphere and pedosphere. Students could achieve maximum 20 points solving the tasks on HWiKSR (10 for answer tier, 10 for reason tier). Table 1 Specification table of HWiKSR diagnostic instrument tasks. Number Topic Question Bloom’s cognitive of task level 1. Soil properties Soils contain different proportions of water and air. Which soil can be the Understanding most breathable and contain the most water? 2. Soil properties Does soil type increase the biotic diversity of plants? Understanding 3. Rocks What is rock? Remembering 4. Soil properties The figure shows the root system of an oak tree. An adaptation to which Understanding environmental factor do roots represent? 5. Pollution The graph shows the amount of mined lignite in the Velenje coal mine from Analyse 1950 to 2018. Assume that all lignite burned, which pollutes the environment. During which period did lignite mining have the greatest environmental impact? 6. Pollution How does a fuel oil spill affect soil fertility? Understanding 7. Rocks What do we call rocks that form from cooled magma below the surface Remembering of the earth? 8. Formation of soil Erosion is defined as the process of furrowing action of external forces on the Apply surface and removal of material. In what way can we most effectively reduce erosion in nature? 9. Soil properties The picture shows the soil profile. What layers or horizons characterize the Analyse soil layer? 10. Formation of soil Which process of soil formation is shown in the picture? Analyse 87Acta Chim. Slov. 2024, 71, 84–90 Ribič et al.: Assessing 15-year-olds’ Understanding of Chemical ... 3. 3. Research design Data collection took place between April 5 and April 23, 2021, in elementary schools throughout Slovenia, fol- lowing the ethical principles of educational research. The IP and HWiKSR were applied anonymously in groups, and all the participants had similar classroom conditions while completing both instruments. They spent an average of 30 minutes completing the two instruments. Participants were informed that the data would be used for research purposes only and the main objective of the study was ex- plained. The research was conducted in accordance with ethical standards for educational research. Data was ana- lysed using descriptive statistics (mean M, standard devi- ations SD) to determine the level of students’ understand- ing of the lithosphere and pedosphere and confidence in solving the specific tasks in the HWiKSR; the data were analysed using Excel. 4. Results and Discussion 4. 1. Students’ Knowledge About Lithosphere and Pedosphere The HWiKSR answers and reason responses (i.e., tier 1 and tier 2 responses) indicated low level of student understanding of the lithosphere and pedosphere. Accord- ing to Milenković et al.17, Slovenian students’ knowledge of the lithosphere and pedosphere is somehow adequate. 31.0% of students did not reach the arbitrary limit of pos- itive evaluation according to rules of evaluation in Slove- nian school system.8 Students scored an average of 55.6% of all possible points on HWiKSR, which is equivalent to 11.2 points. These results are encouraging when compared to the results of the study by Borghini et al.,14 in which stu- dents scored an average of 44.0% of all possible points on the lithosphere and pedosphere achievement test. Tasks 1., 2., 4. and 9. in the HWiKSR, referred to knowledge of soil properties. The results show that 46.5% of students have knowledge of soil properties. However, 30.1% of the students showed knowledge deficits in these tasks. These results support the idea by Russel et al.26 who found that students do not understand the composition of soil, these problems may originate from findings by Hay- hoe et al.25 who found that teachers also had difficulties defining soil as composition of solid particles with spaces for air and water. In task 9., only 21.5% of students chose the correct answer in tier 1 and tier 2. A possible explana- tion for the low level of knowledge could be that, accord- ing to Krathwohl27 this task is at a higher cognitive level of Bloom. In tasks 8. and 10. that referred to soil formation, the results show that 37.8% of students have knowledge. On the other hand, 28.7% of students showed a lack of knowledge of soil formation processes, both tasks being at a higher Bloom’s cognitive level according to Krathwohl.27 In addition, students have difficulty linking the stages of the rock cycle23, therefore they do not see weathering and erosion as processes of soil formation and have problems linking these two processes. In tasks 3. and 7., that referred to rocks, the students’ level of knowledge is very different: 15.5% of the students expressed knowledge in task 3 and 38.6% in task 7. Both tasks were at lower cognitive level according to Bloom’s taxonomy27. One explanation for the students’ low level of knowledge in task 3 could be that the task asked what type of rock is formed from cold lava. Students learn this topic in 6th grade in natural sciences8 and the participants in the study were 9th graders, so it is possible that they forgot what they learned. A possible ex- planation could also be that rock classification was defined as problematic due to the type of characteristics we use for classification19. Tasks 5. and 6. referred to soil pollution, Figure 1. An example of the task no. 3 in HWiKSR; 1st tier (3), 2nd tier (3.1); 3rd tier (3.2.); the correct answer and the correct reason are presented in bold. 88 Acta Chim. Slov. 2024, 71, 84–90 Ribič et al.: Assessing 15-year-olds’ Understanding of Chemical ... and the results show that the students’ knowledge level is the lowest for this topic. 21.9% of students answered 1st and 2nd tier of the task correctly. For task 5. alone, only 15.5% of students expressed knowledge. This could be due to the problems that students have in interpreting mathe- matical and social problems using graphs as stated by Putri et al.21 The average performance of students to each task is shown in Table 2. 4. 2. Students’ Misconceptions About Lithosphere and Pedosphere The analysis of three-tier tasks on the HWiKSR di- agnostic instrument showed that Slovenian students that participated in the study have misconceptions. These re- sults are in line with the findings of Borghini et al.17 and Dove19 who also found misconceptions about soil, rock, weathering, and erosion among students in earth sciences. Francek20 also found that students have misconceptions about weathering and erosion. However, the number of misconceptions in the HWiKSR was below 30.0% for each task, which according to Milenković et al.17 represents a low number of misconceptions. As shown in Table 2, the highest number of misconceptions was found in task 7 (25.6%), where students had to name the rocks that are formed from cold lava. As mentioned above, one expla- nation for the high number of misconceptions in this task could be students learn this topic in 6th grade, but this study was conducted with 9th grade students. Monteiro et al.21 also found that students have misconceptions about minerals and rocks due to the wide variety of minerals and rocks that can occur. Students’ inability to understand the rock cycle20 and to connect different types of rocks24 could also be an explanation for the higher number of miscon- ceptions in this task. The problem of understanding the rock cycle may address many misconceptions about rocks, as Francek20 noted. Students’ inability to connect different types of rocks and the rock cycle may also explain the high number of misconceptions in task 9 (23.5%), as students often believe that soil extends for miles below the surface25 and do not understand the composition of soil and its depth.26 However, according to Dove19 students also have problems visualizing cross-sections, which could also be an explanation for the high number of misconceptions in task 9. For task 5 (24.5%), the explanation for the higher number of misconceptions could be that students solve so- cial and mathematical problems by reading graphs as Putri et al.22 found. These types of problems are also more dif- ficult to solve as they are higher on Bloom’s cognitive the- ory level.28 The number of misconceptions is also higher than 20.0% in task 10. As students are not able to connect different types of rocks to each other, they see weathering and erosion as two unrelated processes and to not connect them to soil formation.20 Students also have problems see- ing erosion and weathering as processes that allow rocks to change from one form to another.23 In other tasks, the number of misconceptions was below 20.0%. In task 3 19.1% of students showed misconceptions, probably due to the wide variety of rocks and minerals and problems with the definition of minerals.21 As mentioned above, the overall number of miscon- ceptions was low according to the literature.17 However, the main cause of misconceptions arising in the topic of lithosphere and pedosphere is the short time devoted to earth science, as this topic is only covered in 6th grade. Borghini et al.14 found that the short time devoted to a par- ticular topic is one of the main reasons for the formation of misconceptions. The same applies to the lack of geological knowledge among teachers. The topic of lithosphere and pedosphere is covered in 6th science, and the teachers who teach these topics are not geology or geography teachers. 5. Conclusions The purpose of this study was to determine wheth- er Slovenian 15-year-old students have sufficient knowl- edge about the lithosphere and pedosphere, and if they possess any misconceptions about this topic. The three- tier HWiKSR diagnostic instrument was used to obtain Table 2 The proportion of knowledge, lack of knowledge, guessing, luck and misconceptions according to students’ responses on HWiKSR test. Knowledge Lack of knowledge Guessing Luck Misconceptions Number f f% f f% f f% f f% f f% of task 1. 86 17.1 184 36.6 26 5.20 154 30.6 53 10.5 2. 154 30.6 119 23.7 36 7.20 172 34.2 21 4.2 3. 78 15.5 146 29.0 91 18.1 92 18.3 96 19.1 4. 126 25.0 108 21.5 45 8.9 147 29.2 77 15.3 5. 78 15.5 177 35.2 68 13.5 57 11.3 123 24.5 6. 142 28.2 91 18.1 54 10.7 195 38.8 21 4.2 7. 194 38.6 53 10.5 60 11.9 67 13.3 129 25.6 8. 134 26.6 153 30.4 57 11.3 116 23.1 43 8.5 9. 59 11.7 196 39.0 84 16.7 45 8.9 118 23.5 10. 81 16.1 136 27.0 134 26.6 49 9.7 103 20.5 89Acta Chim. Slov. 2024, 71, 84–90 Ribič et al.: Assessing 15-year-olds’ Understanding of Chemical ... information about their understanding of the soil, rocks, weathering and erosion and soil pollution. An addition- al instrument to gather students’ background informa- tion was also used. It can be concluded that the students’ knowledge of the lithosphere and pedosphere is adequate. However, according to the rules of evaluation in Sloveni- an school system, the average performance of students is just above the positive evaluation standards of 50.0% of all possible points. 50.0% of all tasks in the HWiKSR were solved correctly by less than 50.0% of the participants. The lowest level of knowledge was found in the tasks on soil formation, where students had to connect weathering and erosion as processes of soil formation and understand the structure of soil. Moreover, 21.1% of students showed knowledge of the properties of soil. The highest level of knowledge was found for the topic of rocks. The highest number of misconceptions appeared in the topic of rocks, soil formation and pollution. The results show that in no task did the number of misconceptions exceed 30.0%, which is a low number of misconceptions. The present study highlights important issues in the current basic school curricula and points to directions in further research into the content of lithosphere and pedosphere. We must be aware that this topic is part of environmental chemistry and people need this knowledge to explain environmental problems, to protect the envi- ronment and to create healthy environment for the future. Therefore, it is essential to include environmental topics about lithosphere and pedosphere in curriculum in the upper grades, which, however, would require a change at the national level. The introduction of such changes may be chaotic at the beginning and thus demand high level of cooperation among all the stakeholders involved. There are some limitations of this research. The first one can be found in the analysis of the students’ responses on all three tiers identifying the proportion of specific mis- conceptions about lithosphere and pedosphere at the end of the contemporary education in Slovenia. The second limitation lies in the fact that the HWiKSR was applied only at one level of education, and it can be also imple- mented at the end of secondary education as well as at the beginning or/and at the end of university teacher educa- tion. Also, students from all regions should be included in further studies, with a larger sample, in order to be able to generalize the data to the entire population. This data can provide more a detailed picture of students’ and teachers’ understanding of specific environmental phenomena and help preparing curriculum changes for all levels of educa- tion in Slovenia. In this way, a significant impact can be made on improving students’ knowledge of the content covered in this article, while at the same time reducing the number of misconceptions about these topics. Consider- ing the limitation of this research some further research on this topic can be conducted. For instance, research should be also conducted at the end of grade 7 when students finish the subject natural science, where these topics are covered. Therefore, we can assume that less knowledge is lost due to forgetting. It is also important to analyse the correlations between answer, reason and confidence tier. The level of teachers’ environmental literacy, how they apply environmental issues in their teaching even when the specific curriculum aim is suggested can be studied. More detailed textbooks analysis regarding environmental issues is necessary to interpret the data in more detail. 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Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Članek predstavlja rezultate raziskave, ki je med slovenskimi devetošolci ugotavljala razumevanje področja litosfere in pedosfere. Raziskava vključuje razvoj tristopenjskega diagnostičnega inštrumenta sestavljenega iz desetih vprašanj z naslovom Kako dobro poznam prst in kamnine (HWiKSR). V raziskavi so sodelovali skupno 503 učenci iz osmih različnih regij v Sloveniji, ki so v šolskem letu 2021 obiskovali 9. razred osnovne šole. Podatki pridobljeni s HWiKSR so omogočili proučevanje razumevanja in prepričanosti  učencev o prsti in kamninah. Rezultati kažejo, da imajo učenci 9. razredov osnovne šole v Sloveniji ustrezno znanje s področja o litosferi in pedosferi. V povprečju so učenci dosegli 55,6 % vseh točk na HWiKSR. Najnižja raven znanja je bila ugotovljena pri temi nastanka tal. Število napačnih predstav učencev o litosferi in pedosferi je nizko in ne presega 30 % pri nobeni nalogi. Največ napačnih predstav je bilo ugotovljenih pri temi nastanka tal in onesnaževanja. 91Acta Chim. Slov. 2024, 71, 91–98 Rekkab and Didi: Samarium(III) Removal by Weak Acid Exchanger ... DOI: 10.17344/acsi.2023.8099 Scientific paper Samarium(III) Removal by Weak Acid Exchanger Amberlite IRC-50 in (H+) and (Na+) Forms Afaf Amara Rekkab, 1,2,* and Mohamed Amine Didi2 1 Institute of Science and Technology, Department of Hydraulics, University center of Maghnia Zouia maghnia street – 13000, Algeria 2 Laboratory of Separation and Purification Technologies, Department of Chemistry – Faculty of Sciences, Box 119, University of Tlemcen – 13000, Algeria * Corresponding author: E-mail: amarafaf@yahoo.fr; afaf.amara@cumaghnia.dz Received: 02-22-2023 Abstract Adsorption of samarium(III) on a weakly macroporous cation exchanger Amberlite IRC-50 (H+) and (Na+) forms is studied as a function of the initial pH of the aqueous solution, time and temperature, initial samarium(III) ion concen- tration, and the amount of resin at a fixed temperature (20 ± 1 °C). The concentration range was between 0.1–5 mmol/L, the pH range was between 1.8 and 10.5; the stirring time was between 2 and 60 min; and the amount of resin was between 0.025 and 0.15 g. Both the film and particle diffusion equations are applied to explain the kinetic data. The rate constant values for samarium(III) adsorption were calculated for both film and particle diffusion processes. It is observed to follow the order (Na+) > (H+). Temperature is found to have an insignificant effect on both diffusional processes. Various thermodynamic parameters (ΔH°, ΔS°, and ΔG°) from samarium(III) exchange on the resin were calculated. The optimum conditions were found to be a concentration of 1 mmol/L, pH of 9.3, stirring time of 20 and 5 min for Amberlite IRC-50 (H+) and (Na+) forms, respectively, and 0.15 g of resin. The equilibrium extraction of samarium was 22.2 mg/g for Amberlite IRC-50 (H+) and 21.9 mg/g for Amberlite IRC-50 (Na+) at an initial concentration of 1 mmol/L. The results obtained show that the Amberlite IRC-50 weak cati- on-exchange resin performed well for the removal and recovery of samarium(III). The optimization procedure provides access to industrial-scale Sm(III) removal processing. Keywords: Samarium, adsorption, ion exchange, Amberlite IRC-50, metal removal, kinetics study 1. Introduction In recent years, rare earth elements (REE) have been regarded as important components from an industrial point of view. The major causes for this stem from the high application interest of the REE in many fields, as these ele- ments and their compounds find various commercial ap- plications, knowing that a large deposit of REEs, which is located in Ihouhouan in Algeria, is not yet exploited. The recovery of REE from large quantities of pro- cessing solutions and industrial wastewater is of particular importance to protect the environment and meet the de- mand for green and sustainable products in energy pro- duction. REE can be introduced in small quantities into the human body with water or at the workplace when the work is connected to the relative production dealing with REEs. Being heavy metals, rare-earth elements can accu- mulate in biological systems, replacing calcium. Samarium is primarily used in the production of sa- marium–cobalt permanent magnets, which are used in lightweight electronic equipment where size or space is a limiting factor and where functioning at high tempera- tures is of great concern. Because of its weak spectral ab- sorption band, samarium is used in the filter glass on Nd:YAG solid-state lasers to surround the laser rod to im- prove efficiency by absorbing stray emissions. Stable sa- marium– titanate compounds with useful dielectric prop- erties are suitable for coatings and capacitors at microwave frequencies. The specific applications of samarium in dif- ferent fields of technology have turned it into an industrial material of outstanding significance.1,2 However, samari- um(III) is also toxic to health and, at the same time, pre- cious and expensive; therefore, it must be recovered through recycling processes to protect the environment and reduce costs. In addition, it is essential to separate and 92 Acta Chim. Slov. 2024, 71, 91–98 Rekkab and Didi: Samarium(III) Removal by Weak Acid Exchanger ... recover Sm(III) ions from refuse because samarium is one of the most important rare earth elements.3 Many removal techniques have been proposed for the removal of samarium(III), including solvent extrac- tion, molecular imprinting, ion exchange, co-precipita- tion, membrane processes, oxidation, and adsorption. Among these methods, ion exchange is highly popular and has been widely practiced for metal ion removal.4–7 Furthermore, organic ion exchange resins are more suit- able for the removal of toxic elements because of their faster kinetics, ease of regeneration, and high exchange capacity.8 While several studies have been reported for the exchange removal of monovalent and divalent metal cati- ons from aqueous solutions,9 very little is reported about the exchange of trivalent metal cations such as Sm3+ and La3+.10–13 Other research indicates that solutions contain- ing Sm3+ ions were treated with different resins, and the results obtained showed that the resin has a strong affinity for these ions.14,15 Synthetic resins are readily available, biodegrada- ble, and extendable. These resins, Amberlite IRC-50, are small, synthetic, porous, crystalline solids. Negative charge compensation cationic systems give the adsorbent cationic polymer resin exceptional properties that lead to many ap- plications, especially in the areas of catalysis, absorption, and as a cationic exchange-acid.16,22 A review of the pertinent literature indicates the ab- sence of studies regarding the activation of this resin at its basic level. Hence, this aspect has been thoroughly investi- gated and elaborated upon in this paper. The main goal of this study was to examine the fac- tors that affect ion exchange, such as the initial solution pH, agitation time, concentration of samarium(III) ions, temperature, and amount of resin. In addition, equilibri- um, kinetics, and thermodynamic parameters were deter- mined on the basis of measurements of ion exchange. 2. Materials and Methods Amberlite IRC-50 (H+) (C23H37Cl2N3O, M = 442.5 g/mol; Scheme 1) (supplied by Fluka) is a macroporous weak acid cation-exchange resin with a methacrylic ac- id-DVB structure and is available in the form of spherical beads. The maximum temperature it can tolerate is 120 °C. It works in the pH range of 5–14. The particle size varies Scheme 1: Chemical structure of Amberlite IRC-50. from 0.297 to 1.190 mm. The exchange capacity of the res- in is 9.5 mg/g. The moisture content is 10% by weight.10 2. 1. Conversion of Amberlite IRC-50 (H+) into (Na+) A 100 g sample of the hydrogen ion form of Amber- lite IRC-50 was treated with a solution of sodium hydrox- ide at 85 g/L in Erlenmayer flasks. After stirring intermit- tently for 2 h, the resin was filtered off, re-treated with a fresh sodium hydroxide solution, filtered again, thorough- ly washed with water, and desiccated.23 2. 2. Ion Exchange Studies The removal of Sm(III) with Amberlite IRC-50 as a function of contact time was investigated. An exactly weighed amount (0.1 g) of Amberlite IRC-50 in (H+) and (Na+) forms was mixed with 5 mL of Sm2(CO3)3 solution dissolved in 4 mL nitric acid and diluted with distilled water to obtain a concentration of 1 mmol/L, which had attained the desired temperature (293–313 K). The stirring rate was 1000 rpm. The concentration of Sm(III) in the aqueous phase was analyzed with a SPECORD 210 plus spectrophotometer using the method described in the lit- erature.24 The percent Sm(III) extraction (%) was determined as follows: (1) The adsorption amount was calculated as follows: (2) where qt is the adsorption amount (mg/g), w is the weight of the Amberlite IRC-50 (g), M is the molar mass (g/mol), V is the volume of solution (L), and C0 and C are the con- centrations (mol/L) of samarium ions before and after ad- sorption, respectively. The effect of solution pH on the equilibrium uptake of samarium(III) from aqueous solution by Amberlite IRC-50 resin in (H+) and (Na+) forms was investigated be- tween pH 1.8 and 10.5 for 15 min. The experiments were performed by adding a known weight of the resin (0.1 g) into six 10 mL Erlenmayer flasks containing 5 mL of sa- marium(III) solution. Dilute nitric acid or sodium hydrox- ide was used to adjust the pH of the samarium solutions using a pH meter (model WTW, PH 3310 SET 2, Germa- ny). The flasks were shaken for 15 min at 1000 rpm and 20±1 °C. Kinetic experiments were carried out by agitating 5 mL of samarium(III) solution of concentration ranging from 0.01 to 5 mmol/L with 0.1 g of Amberlite IRC-50 resin (H+) and (Na+) forms in a 10 mL Erlenmayer flask at 20±1 °C at pH 9.3 and at constant agitation speed of 1000 rpm. 93Acta Chim. Slov. 2024, 71, 91–98 Rekkab and Didi: Samarium(III) Removal by Weak Acid Exchanger ... The effect of the adsorbent amount was studied with a 5 mL solution of 1 mmol/L samarium(III) solution and varying amounts of adsorbent from 0.025 to 0.15 mg at equilibrium time. 3. Results and Discussion 3. 1. Effect of pH In the adsorption operation, the solution pH plays an important role in controlling the high adsorption capac- ity and selectivity of the target lanthanide ions.25–27 This is partly because hydrogen ions themselves are strongly competitive with adsorbents.28 Figure 1: Effect of initial pH on efficient extraction of samarium(I- II). Amount of resin 0.1 g, volume of ion-exchange medium 5 mL, T 20 ± 1 °C, stirring time 1000 rpm, initial concentration of Sm(III) 1 mmol/L, and contact time 15 min. To determine the optimum pH for the adsorption of Sm(III) ions onto Amberlite IRC-50, the percentage removal of Sm(III) ions as a function of hydrogen ion concentration was examined at an initial concentration of 1 mmol/L. In Fig. 1, both adsorbents show a decrease in the removal rate of Sm(III) ions at lower pH conditions. At lower pH, hydrogen ions occupy most of the adsorption sites on the surface of the adsorbent, resulting in very low adsorption of Sm(III) ions due to electrostatic repulsion. However, increasing the pH of the solutions results in a decrease in the competition of hydrogen ions with Sm(III) ions for adsorption sites, thus facilitating a higher rate of removal of Sm(III) ions. The opti- mum pH for both beads was found to be 9.3, with maximum percentage removal of 63% and 88% onto Amberlite IRC-50 in (H+) and (Na+) forms, respectively. Moreover, increasing the pH to above 9.3 resulted in the precipitation of insoluble samarium hydroxide, causing a decrease in the removal of Sm(III) ions.27 Thus, this pH was selected for our subsequent investigations in the following experiments.26 3. 2. Kinetic Curves Figure 2 shows the results of the study on how quick- ly samarium adsorbs to different types of resin Amberlite IRC-50 at 293 K. The maximum percent Sm(III) extrac- tions were 93% and 90% obtained at 20 and 5 min for the (H+) and (Na+) forms, respectively, which are suitable con- tact times for samarium(III) adsorption. Thereafter, it be- comes slower near equilibrium. Amberlite IRC-50, being a good exchanger, has the fastest kinetics for Sm(III) adsorp- tion in the (Na+) form, followed by the (H+) form. Between these final and initial stages of adsorption, the rate is virtu- ally consistent. This is obvious from the fact that numerous vacant surface sites are available for adsorption during the initial stage, and after a period of time, the residual vacant sites are difficult to occupy due to repulsive forces between the solute molecules in the solid and bulk phases. No sig- nificant change in samarium removal was observed after approximately 20 and 5 min by the two types of Amberlite IRC-50 in (H+) and (Na+) forms, respectively. The results of the kinetic study are presented in Fig- ure 3. The equilibrium is attained within 20, 60, and 30 min at 293, 313, and 333 K, respectively. The extraction of samarium sorbed after equilibrium is 97% at 333 K using Amberlite IRC-50 in the (H+). Figure 3: Effect of contact time on the ion exchange of Sm(III) us- ing Amberlite IRC-50 in the (H+) form at different temperatures. The initial concentration of Sm(III) 1 mmol/L, the amount of resin 0.1 g, the volume of ion-exchange medium 5 mL, T = 20 ± 1 °C, stirring time was 1000 rpm, and the initial pH was 9.3. Figure 2: Effect of contact time on the ion exchange of Sm(III) us- ing Amberlite IRC-50 in the (H+) and (Na+) forms. The initial con- centration of Sm(III) 1 mmol/L, the amount of resin 0.1 g, the vol- ume of ion-exchange medium 5 mL, T 20 ± 1 °C, stirring time was 1000 rpm, and the initial pH was 9.3. 94 Acta Chim. Slov. 2024, 71, 91–98 Rekkab and Didi: Samarium(III) Removal by Weak Acid Exchanger ... The kinetics of samarium adsorption on Amberlite IRC-50 (H+) can be described using two types of equa- tions: film diffusion and particle diffusion equations. The expression for the film diffusion equation is given as fol- lows: 29 (3) where F is the ratio of the amount adsorbed after time t to the amount adsorbed at equilibrium, and Ku is the rate constant. According to Eq. (3), when the kinetic data ob- tained for a series of F values are plotted against t, a straight line is obtained with a slope equal to the rate constant, as shown in Fig. 4. This indicates that on Amberlite IRC-50 (H+) resin, the mechanism of samarium adsorption is the diffusion of samarium through a thin covering liquid film. Similarly, for the particle diffusion equation, the Bt values can be calculated using the following equations: (4) (5) where Bt is equal to Dπ2/r2, D is the particle diffusion coef- ficient and r its radius. Figure 4: Film diffusion plots for Sm(III) adsorption on Amberlite IRC-50 (H+) at 293 K. The initial concentration of Sm(III) 1 mmol/L, the amount of resin 0.1 g, the volume of ion-exchange me- dium 5 mL, T 20 ± 1 °C, stirring time was 1000 rpm, and the initial pH was 9.3. Eq. (4) is used for values of F from 0 to 0.85 and Eq. (5) is used for values of F from 0.86 to 1 according to the simplification given by Reichenberg.30 The Bt values calcu- lated from Eqs. (4) and (5) are plotted against t, and again, a straight line is obtained. The values of the rate constant Bt are calculated from the slope in Fig. 5. The plot of Bt versus t was linear, and a correlation coefficient of 0.953 indicated that the adsorption processes were controlled by film dif- fusion for the adsorption of samarium(III), as indicated by R2 values (R2 = 0.968). Figure 5: Particle diffusion plots for Sm(III) adsorption on Amber- lite IRC-50 (H+) at 293 K. The initial concentration of Sm(III) 1 mmol/L, amount of resin 0.1 g, volume of ion-exchange medium 5 mL, T 20 ± 1 °C, stirring time was 1000 rpm, and initial pH 9.3. Figure 6 shows how the contact time affects the batch adsorption of samarium on the resin Amberlite IRC- 50 (Na+) at 293 K. It is obvious that with an increase in contact time, the percentage removal of Sm(III) was en- hanced significantly. Initial rapid adsorption gives a very slow approach to equilibrium. The nature of the adsorbent and its available adsorption sites affected the time required to reach equilibrium. The desorption of samarium at 333 K for a time interval of 5 to 15 min may be due to resin shrinkage at high temperatures and for a long time of con- tact, which limits Sm3+ adsorption. The equilibrium times for the adsorption of Sm(III) were 5 min at 293, 313, and 333 K. Figure 6: Effect of contact time on the ion exchange of Sm(III) us- ing Amberlite IRC-50 in the (Na+) form at 293 K. The initial con- centration of Sm(III) 1 mmol/L, the amount of resin 0.1 g, the vol- ume of ion-exchange medium 5 mL, T 20 ± 1 °C, stirring time was 1000 rpm, and the initial pH was 9.3. Film and particle diffusion kinetic models were ap- plied against the kinetic data, and it was observed that both film and particle diffusion models were the best choices for explaining the kinetic parameters. The ion exchange adsorption of metal cations has been reported in the lit- 95Acta Chim. Slov. 2024, 71, 91–98 Rekkab and Didi: Samarium(III) Removal by Weak Acid Exchanger ... erature31,32 to be controlled either by the film, particle dif- fusion, or both. According to equation (3), when ln(1−F) is plotted against t, the intercepts of the plots do not equal zero, as shown in Fig. 7. Similarly, for the particle diffusion equation, the Bt values are calculated using equations (4) and (5). Figure 7: Film diffusion plots for Sm(III) adsorption on Amberlite IRC-50 (Na+) at 293 K. The initial concentration of Sm(III) 1 mmol/L, the amount of resin 0.1 g, the volume of ion-exchange me- dium 5 mL, T 20 ± 1 °C, stirring time was 1000 rpm, and the initial pH was 9.3. Unfortunately, these simplifications are commonly used as ‘‘different’’ methods to determine Ku or incorrectly used to determine the rate-limiting step without consider- ing the surface coverage range for which the approxima- tions were originally derived. Eq. (3) is commonly used as a litmus test to determine the rate-limiting mechanism. If plotting -ln (1-F) vs. t, Eq. (3), yields a linear relation through the origin; this is seen as evidence for mass trans- fer control. It can be judged from Figs. 7 and 8 that the film dif- fusion equation (R2 = 0.968) is well-fitted to the data with relatively high R2 values and low intercepts compared to the particle diffusion equation (R2 = 0.933). This indicates that the film diffusion process is the rate-limiting step dur- ing Sm(III) adsorption. The values of Ku and Bt obtained from both diffusional equations at 293 K are presented in Table 1. 3. 3. Effect of Samarium Concentration Figure 9 shows Sm3+ removal efficiency and ad- sorption capacity for Amberlite IRC-50 in (H+) and (Na+) forms. It is clear that the (%) removal efficiency of Sm3+ increases with increasing initial concentration of sa- marium(III). This may be due to the presence of more ac- tive adsorption sites for Sm3+. The extraction of samarium sorbed after equilibrium is 22.2 and 21.9 mg/g for Amber- lite IRC-50 (H+) and (Na+) forms, respectively, at an initial concentration of 1 mmol/L. Figure 9: Effect of the initial concentration of Sm(III) adsorption on Amberlite IRC-50 in (H+) and (Na+) forms. Amount of resin 0.1 g, volume of ion-exchange medium 5 mL, T 20 ± 1 °C, stirring time 1000 rpm, initial pH 9.3, contact time 20 min for (H+) and 5 min for (Na+). Figure 9 also demonstrates that Sm3+ adsorption ca- pacity decreases as the initial concentration increases. This effect can be explained as follows: at low metal/sorbent ratios, there are several adsorption sites in the Amberlite Table 1. Values for film and particle diffusion processes on Amberlite IRC-50 in the (H+) and (Na+) forms Temperature (K) (H+) form (Na+) form Rate constants (min−1) Film diffusion (Ku) Particle diffusion (Bt) Film diffusion (Ku) Particle diffusion (Bt) 293 0.01 1 0.9 0.01 Figure 8: Particle diffusion plots for Sm(III) adsorption on Amber- lite IRC-50 (H+) at 293 K. The initial concentration of Sm(III) 1 mmol/L, the amount of resin 0.1 g, the volume of ion-exchange me- dium 5 mL, T 20 ± 1 °C, stirring time was 1000 rpm, and the initial pH was 9.3. 96 Acta Chim. Slov. 2024, 71, 91–98 Rekkab and Didi: Samarium(III) Removal by Weak Acid Exchanger ... IRC-50 structure. As the metal/sorbent ratio increases, ad- sorption sites become saturated, resulting in a decrease in adsorption efficiency.33 3. 4. Effect of the Resin Dosage The resin amount is an important parameter for determining the quantitative uptake of metal ions. The retention of the metals was examined in relation to the amount of resin. Fig. 10 shows the removal of Sm(III) as a function of resin dosage using Amberlite IRC-50 in the (H+) and (Na+) forms. The resin amount varied from 0.025 to 0.15 g and was equilibrated for 20 and 5 min at an initial metal ion concentration of 1 mmol/L solution. The equilibrium concentration in the liquid phase and the contact time required to reach equilibrium decrease with increasing resin doses for a given initial metal concentra- tion. These results were anticipated because increasing the adsorbent dose could provide a large surface area or ion-exchange sites for a fixed initial solute concentration. Figure 10: Effect of resin amount on ion exchange Sm(III) adsorp- tion on Amberlite IRC-50 in (H+) and (Na+) forms. Amount of res- in 0.1 g, volume of ion-exchange medium 5 mL, T 20 ± 1 °C, stirring time 1000 rpm, initial pH 9.3, contact time 20 min for (H+) and 5 min for (Na+). It may also be concluded that the removal efficien- cy increases and the ion-exchange density decreases with increasing adsorbent dose. The decrease in ion-exchange density can be attributed to the fact that some of the ion exchangers remain unsaturated during the adsorption process, whereas the number of available ion-exchange sites increases with resin dosage, resulting in an increase in removal efficiency.34 It is clear from Fig. 10 that for the quantitative removal of 1 mmol/L samarium in a 5 mL solution, a minimum resin dosage of 0.15 g in the (H+) and (Na+) forms is required. For this amount of resin, the adsorption values were 99%. 3. 6. Thermodynamic Studies Thermodynamic parameters, such as the Gibbs en- ergy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°), are deter- mined using the following equations: 10,35 (6) (7) (8) where R (8.3145 J/mol K) is the ideal gas constant, T (K) is the absolute temperature, and Kd is the thermodynamic equilibrium constant. The values of changes in enthalpy (ΔH°) and entropy (ΔS°) are calculated from the slopes and intercepts of the plot of lnKd vs. 1/T using Eq. (8). The calculated values of the thermodynamic param- eters are given in Table 2. The negative value for the Gibbs energy change for the two resins shows that the adsorption process is feasible and thermodynamically spontaneous. Furthermore, the decrease in ΔG° values with increasing temperature indicates that adsorption is not favorable at higher temperatures. Table 2. Gibbs free energy, enthalpy, and entropy changes for Sm(III) adsorption on Amberlite IRC-50 Resin ΔH°.104 ΔS° ΔG° .105 (kJ/mol) (kJ/mol) (J/K mol) 293 K 303 K 333 K Amberlite +21 +95 –8 –10 –12 IRC-50 (H+) Amberlite –19 +32 –9 –9 –8 IRC-50 (Na+) The enthalpy of the adsorption, ΔH°, is a measure of the energy barrier that must be overcome by reacting molecules.25 The values of ΔH° for the adsorption of Sm3+ by Amberlite IRC-50 in (H+) are positive, indicating that the extraction procedure of samarium is endothermic in nature, unlike the values of ΔH° for the adsorption of Sm3+ by Amberlite IRC-50 in (Na+), which indicate the exothermic nature of the adsorption process of Sm(III) at 20–60 °C. The value of ΔS° can be used to identify whether the adsorption reaction is attributed to an associative or dissociative mechanism. Generally, entropy change ΔS° > −10 J/mol K implies a dissociative mechanism.20 Before adsorption occurs, the heavy metal ions near the surface of the adsorbent will be more ordered than in the subse- quent adsorbed state, and the ratio of free heavy metal ions to ions interacting with the adsorbent will be higher than that in the adsorbed state. As a result, the distribution of rotational and translational energy among a few molecules will increase with increasing adsorption by producing a positive value of ΔS° and randomness will increase at the solid-solution interface during the process of adsorption. The entropy changes in this work are all positive for the 97Acta Chim. Slov. 2024, 71, 91–98 Rekkab and Didi: Samarium(III) Removal by Weak Acid Exchanger ... two resins, implying that the dissociative mechanism is in- volved in the adsorption processes. The negative values of ΔG° also indicate that the pro- cess of extraction by the two resins is spontaneous. 3. 7. Probabilities of the Mechanism Samarium ions may exist in the aqueous phase in different ionic forms. Any of these forms will predomi- nate over other forms of samarium depending on the total amount of samarium and the pH of the aqueous phase. Sm(III) cation prevails in an acidic or slightly basic solu- tion, whereas different samarium cations dominate in a basic solution. Therefore, in this study, the samarium ion will be in the form of Sm(OH)2+, as shown in Fig. 11. Figure 11: Distribution diagrams of samarium using the Medusa and Hydra programs36 To explain the observed behavior of Sm(III) removal with varying pH, it is necessary to examine various mech- anisms, such as electrostatic attraction/repulsion, chemi- cal interaction, and ion exchange, that are responsible for adsorption on sorbent surfaces. Therefore, the following mechanisms can be pro- posed for the adsorption of samarium(III) by Amberlite IRC-50 (Na+): Sm3+ + 2 H2O → Sm(OH)2+ + 2H+ (9) R–H + Na+ → R−Na + H+ (10) R–Na + Sm(OH)2+ → R−Sm(OH)2 + Na+ (11) Similar competition was observed by Mohan et al.37 and Chanda and Rempel38 while studying Cr(III) adsorp- tion on weak acid exchangers. 4. Conclusions The present study deals with the adsorption of Sm(I- II) on Amberlite IRC-50 in (H+) and (Na+) forms from aqueous solutions. The effects of pH, contact time, kinetics, and thermodynamics are examined in batch experiments. Amberlite IRC-50 is a weak cationic resin with good ca- pability and efficiency. The ideal conditions for achieving the highest adsorption capacity of samarium(III) were de- termined. At a temperature of 293 K, the kinetic analysis indicates that the rate of adsorption is primarily limited by film diffusion. Acknowledgements Memorial to the beloved Professor Mohamed Amine DIDI, who passed away on January 17, 2023. You will nev- er be forgotten, dear Professor. Competing interests The authors declare that no conflict of interest would prejudice the impartiality of this scientific work. 5. References 1. R.Torkaman, M. A.Moosavian, M.Torab-Mostaedi, J. Safdari, Hydrometallurgy 2013, 137, 101–107. DOI:10.1016/j.hydromet.2013.04.005 2. M. E. Mahmoud, G. M. Nabil, S. M.T. Elweshahy, Powder Tech- nol. 2021, 378, 246–254. DOI:10.1016/j.powtec.2020.09.058 3. A. I. Rasee, E. Awual, A. I. Rehan, M. S. Hossain, R.M. Wa- liullah, K. T. Kubra, Md. C. Sheikh, Md. S. Salman, Md. N. Hasan, Md. M. Hasan, H. M. Marwani, A. Islam, Md. A.Kha- leque, Md. R. Awual, Surf. Interfaces. 2023, 103276. DOI:10.1016/j.surfin.2023.103276 4. S. Kocaoba, G. Akcin, Desalination. 2005, 180, 151–156. DOI:10.1016/j.desal.2004.12.034 5. A. Amara-Rekkab, M. A. Didi, D. Villemin, Eur. Chem. Bull. 2015, 4, 190–195. 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Mater. 2013, 252, 313–320. DOI:10.1016/j.jhazmat.2013.03.020 28. E. Igberase, P. Osifo, A. Ofomaja, J. Environ. Chem. Eng. 2014, 2, 362–369. DOI:10.1016/j.jece.2014.01.008 29. L. Dandan, C. Xijun, H. Zheng, W. Qihui, L. Ruijun, C. Xiaoli, Talanta 2011, 83, 1742–1747. DOI:10.1016/j.talanta.2010.12.012 30. F. J. Alguacil, M. Alonso, L. J. Lozano, Chemosphere 2004, 57, 789–793. DOI:10.1016/j.chemosphere.2004.08.085 31. A. Chatterjee, Chem. Eng. J. 2014, 244, 105–116. DOI:10.1016/j.cej.2013.12.017 32. M. Amara, H. Kerdjoudj, Desalination 2004, 168, 195–200. DOI:10.1016/j.desal.2004.06.187 33. S. A. Cavaco, S. Fernandes, M. Margarida, Q. M. F. Licinio, J. Hazard. Mater. 2007, 144, 634. DOI:10.1016/j.jhazmat.2007.01.087 34. Y. Zhihui, Q. Tao, Q. Jingkui, W. Lina, C. Jinglong, J. Hazard. Mater. 2009, 167, 406–412. DOI:10.1016/j.jhazmat.2008.12.140 35. S. Mustafa, K. H. Shah, A. Naeem, T. Ahmad, M. Waseem, Desalination 2010, 264, 108–114. DOI:10.1016/j.desal.2010.07.012 36. I. Puigdomenech, “HYDRA (Hydrochemical Equilibrium Constant Database) and MEDUSA (Make Equilibrium Di- agrams Using Sophisticated Algorithms) Programs,” Royal Institute of Technology, Sweden. http://www.kemi.kth.se/ medusa. 37. D. Mohan, K. P. Singh, V. K. Singh, J. Hazard. Mater. 2006, 135, 280–295. DOI:10.1016/j.jhazmat.2005.11.075. 38. M. Chanda, G. L. Rempel, Ind. Eng. Chem. Res. 1997, 36, 2184–2189. DOI:10.1021/ie960525t. Povzetek Preučevali smo adsorpcijo samarija(III) na šibki makroporozni kationski izmenjevalec Amberlite IRC-50 v (H+) in (Na+) oblikah kot funkcijo začetnega pH vodne raztopine, časa in temperature, začetne koncentracije samarijevih(III) ionov in količine smole pri stalni temperaturi (20 ± 1 °C). Koncentracijsko območje je bilo med 0,1 in 5 mmol/L, pH območje med 1,8 in 10,5; čas mešanja med 2 in 60 min; količi- na smole med 0,025 in 0,15 g. Za razlago kinetičnih podatkov smo uprabili tako filmsko-plastno enačbo kot enačbo za di- fuzijo delcev. Vrednosti hitrostne konstante za adsorpcijo samarija(III) smo izračunali tako za filmsko-plastni proces kot za difuzijo delcev. Sledi vrstnemu redu (Na+) > (H+). Temperatura ima insignifikanten učinek na oba difuzijska procesa. Izračunali smo različne termodinamske parametre (ΔH°, ΔS° in ΔG°) za izmenjavo samarija(III) na smoli. Optimalni pogoji so bili koncentracija 1 mmol/L, pH 9,3, čas mešanja 20 min za Amberlite IRC-50 (H+) in 5 min za (Na+) obliko, ter 0,15 g smole. Ravnotežna ekstrakcija samarija je bila 22,2 mg/g za Amberlite IRC-50 (H+) in 21,9 mg/g za Amberlite IRC-50 (Na+) pri začetni koncentraciji 1 mmol/L. Pridobljeni rezultati so pokazali, da se šibki kationski izmenjevalec Amberlite IRC-50 dobro obnese za odstranjevanje in ekstrakcijo samarija(III). Z optimizacijo je možno pridobiti pogoje za odstranjevanje Sm(III) v industrijskem merilu. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 99Acta Chim. Slov. 2024, 71, 99–109 Mulai et al.: Exploring a Substitute for Hydrogen Peroxide in Fenton ... DOI: 10.17344/acsi.2023.8183 Scientific paper Exploring a Substitute for Hydrogen Peroxide in Fenton Process – A Case Study on the COD Removal of Acid Orange 8 Tsungom Mulai,1 John Elisa Kumar,2 Wanshanlang Kharmawphlang1 and Mihir Kumar Sahoo1,* 1 Department of Chemistry, North-Eastern Hill University, Shillong– 793 022, India 2 Department of Chemical Engineering, Institute of Chemical Technology, Mumbai – 400 019, India * Corresponding author: E-mail: mksahoo@nehu.ac.in el.: +91-364-2722632; Cell: +91-9436706767; Fax: +91-364-2551634 Received: 12-04-2023 Abstract Hydrogen peroxide (HP) is widely used in advanced oxidation processes (AOPs). This study evaluates the chemical oxy- gen demand (COD) removal efficiency of Acid Orange 8 (AO 8) at a higher concentration by modified Fenton processes using substituted HP, such as tert-butyl hydroperoxide (TBHP), sodium perborate (SPB), and sodium persulphate (SPS) as oxidising agents. Under optimal conditions, COD removal was found to be 72.8 and 58.9% at pH 3.0 and 6.5 in the Fenton process in 300 min. The COD removal efficiency of different systems is in the order: Fe2+/SPB > Fe2+/HP > Fe2+/ TBHP > Fe2+/SPS indicating the possibility of using SPB as a substitute for HP and SPS. The order of efficiency is attribut- ed, among other factors, to their ability to produce HO• radicals. Various anions are shown to exhibit an inhibitory effect in the order: I– > Br– > F– > Cl– > SO42– > NO3–. The inhibitory effect of Cl– is observed at higher concentrations than F– and Br–, even though I– displays inhibition at all concentrations. Finally, COD removal kinetics and the degradation mechanism, based on the identified intermediate products, were determined in this study. Keywords: Fenton and modified Fenton processes; Substitute of hydrogen peroxide; tert-Butyl hydroperoxide; Sodium Perborate; COD removal kinetics; Identification of products and degradation mechanism 1. Introduction Synthetic dyes are widely used in different industries, such as textile, cosmetic, printing, drug, food processing, tannery, leather etc.1 Such dyes are stable and resistant to biodegradation causing discoloration of wastewater.2 The colour tends to persist even after different convention- al treatment process such as coagulation,3 adsorption,4 membranes processes,5 biological processes,6 etc. Among the various AOPs, Fenton (Fe2+/HP) has been proved to be convenient, efficient and cost effective.7–12 Fenton process utilises highly reactive hydroxyl radicals (HO•), generated by the decomposition of HP by Fe2+ (Eq. (1)). Hydroxyl radicals are non-selective and have high oxidation poten- tial of 2.8V vs. normal hydrogen electrode (NHE) with rate constant in the order of 107 to 1010 M–1s–1.13 There- fore, they have excellent ability to mineralize organic com- pounds including azo dyes to carbon dioxide, water and inorganic salts. However, the effectiveness of this process has been limited by its narrow working pH range.7 Thus, there is a need for search of alternate oxidant to be used in the Fenton process. Persulphate (PS), e.g. sodium persul- phate (SPS) has long been used successfully and efficiently in place of HP in a process, called modified Fenton process (Fe2+/PS).14–16 This process utilises reactive sulphate radi- cals (SO4•–), which are generated by the activation of PS by Fe2+ (Eq. (2)). The advantage of this process is its ability to work in a broad pH range.17 (1) (2) It has been reported that tert-Butyl hydroperoxide (TBHP), an organic peroxide and Sodium perborate (SPB) are potential oxidants to replace HP in the Fenton pro- 100 Acta Chim. Slov. 2024, 71, 99–109 Mulai et al.: Exploring a Substitute for Hydrogen Peroxide in Fenton ... modified Fenton process have not been paid due attention. It is expected that different groups adjacent to the peroxo group as in TBHP, SPB and HP would greatly influence the cleavage of peroxo bond and subsequent generation of HO• through Fe2+ catalysed activation. To augment these two lacunae found in the literature, the present work has been devoted the establish the effect of various oxidants such as SPS, TBHP and SPB on the colour and COD re- moval of a higher concentration (0.3 mM) of Acid Or- ange 8 (AO 8) solution, a representative organic pollutant in the modified Fenton processes. This is proposed to be achieved through optimising operational parameters such as concentration of catalyst and oxidant, and pH for the effective decolorization and COD removal of AO 8 in Fenton (Fe2+/HP) and modified Fenton processes (Fe2+/ TBHP, Fe2+/SPB and Fe2+/SPS). Kinetics of COD remov- al and HP consumption has been studied in detail. Effort has been made to identify intermediate ions and products formed during the reaction. Based on the identified ions and intermediate products, a mechanism of degradation of AO 8 has been proposed. 2. Materials and Methods 2. 1. Reagents The anionic water-soluble diazo dye, Acid Orange 8 (AO 8) was procured from sigma aldrich (Germany). The molecular structure, chemical and physical properties of AO 8 are described in Table S1. The other chemicals used in this work, viz. iron (II) sulphate (FeSO4.7H2O, GR), HP (H2O2, 30% w/w purified, GR), methanol (CH3OH, GR), sodium hydroxide (NaOH, GR), sulphuric acid (H2SO4, GR), sodium nitrate (NaNO3, GR), sodium chloride (NaCl, GR), anhydrous sodium sulphate (NaSO4, GR), tert-Butanol ((CH3)3COH, GR), sodium peroxodisulphate (Na2S2O8, AR) were procured from Merck. Sodium perborate tetrahy- drate (NaBO3.4H2O, GR), sodium fluoride (NaF, AR), sodi- um iodide (NaI, AR) were obtained from Himedia (India). Sodium bromide (NaBr, AR) and tert-Butyl hydroperoxide ((CH3)3COOH, 70% aqueous solution, AR) were obtained from Nice and Avra (India), respectively. Solutions of mer- curic sulphate and sulphuric acid (Solution 1) and silver sulphate, chromic acid, sulphuric acid and demineralized water (Solution 2) used for the measurement of low range COD was obtained from HACH (USA). Methane sulphonic acid (CH3SO3H) and sodium hydroxide (NaOH, 50–52% in water) used in ion chromatographic analyses of intermedi- ate products were procured from Himedia (India) and Sig- ma (Germany), respectively. All the chemicals were used as received without further purification. 2. 2. Procedure The procedure for this work is similar to our ear- lier work published recently.27 However, for a clear un- cess.18–20 The difference among these three oxidants is the presence of different groups adjacent to the peroxo-bond (–O–O–). While it is hydrogen atoms on both sides of the peroxo-bond in HP, tert-butyl on one side in TBHP and dihydroxyboranyl on both sides in SPB (Fig. 1). Figure 1. Structure of oxidants: (a) HP; (b) TBHP; (c) SPB (dimer) TBHP is widely used as a radical initiator in polym- erization reaction and as a cross linking agent in unsatu- rated polyester.21,22 It is also used as a source of tert-butyl derivatives.21 When used as an oxidant in the modified Fenton process (Fe2+/TBHP), TBHP generates tert-butox- yl radical ((CH3)3CO•, t-BuO•) as the major species along with tert-butylperoxyl radical ((CH3)3COO•, t-BuOO•) (Eqs. (3) and (4)).18,19,23 However, generation of HO• was not reported in those studies. The generation of t-BuO• radicals is analogous to the classical Fenton reaction (Eq. (1)). (3) (4) The use of SPB has been widely seen in ophthalmolo- gy, dental industry, pulp bleaching, and detergent industry as a bleaching agent.24 The reagent possesses low toxicity and a longer self-life and has been used as a substitute for HP in organic synthesis.20 SPB when dissolved in water re- leases sodium metaborate and H2O2 (Eq. (5)).20,24 Thus, the presence of catalysts like Fe2+ in aqueous acidic SPB solution would establish Fenton process, which leads to the generation of HO•.25 The degradation of cytarabine an- tineoplastic was studied by the photoactivation of TBHP and SPB26 and the efficiency of oxidants was established to be: HP>SPB>TBHP. Further, SPS was shown to be most effective among all the oxidants. (5) A thorough literature survey reveals that the concen- tration of pollutants used in the degradation process varies in the range 10 to 30 ppm.10 Further, the degradation and/ or COD/TOC removal of organic pollutants using sub- stituted hydrogen peroxides such as TBHP and SPB in a 101Acta Chim. Slov. 2024, 71, 99–109 Mulai et al.: Exploring a Substitute for Hydrogen Peroxide in Fenton ... Where, COD0 and A0 are initial COD and absorb- ance at time ‘t’ = 0 and CODt and At are COD and ab- sorbance at time ‘t’, respectively. The slope of the straight line obtained by plotting –ln (CODt/COD0) vs. time is considered as the first order rate constant (kCOD) for COD removal. Concentration of residual hydrogen peroxide, or- ganic acids, cations, anions, were analysed with the help of ion chromatography system (ICS) manufactured by Ther- mo Scientific, USA (Dionex, ICS-1100). The details of ion chromatographic techniques was described in our recent publication10 and therefore, are not described here. 3. Results and Discussion 3. 1. Spectral Analysis and Decolorization Study The UV-Vis absorption spectra of aqueous AO 8 shows four absorption bands – three appearing at 241, 263, 312 nm in the UV region and one at 417 nm in the visible region (Fig. 2). The presence of absorption peaks at 241, 263 and 312 nm representing π → π* transitions indi- cate the presence of aromatic rings in the AO 8. The 311 nm band represents benzene and/or naphthalene rings at- tached to the azo bond. The peak at 471 nm, representing n → π* transitions of C=O, C=N and N=N chromophore groups is responsible for the color of the AO 8 solution. Figure 2. UV-Vis absorption spectrum of pure AO 8: [AO 8] = 0.3 mM The decolorization was measured by monitoring the decrease in absorbance at 471 nm. The cleavage of −N=N− bonds with a probability of 60% is considered as the initial step in the degradation of azo dyes.28 The gradual cleavage of −N=N− bond decreases the intensity of color in the dye solution. It was observed that absorption maxima of the peak at 471 nm decreased to 89.6% in 5 min and 97.3% in 10 min in Fe2+/HP system (Fig. 3a). Needless to mention derstanding of the readers, we are presenting a brief de- scription here. Aqueous solution of desired concentration of AO 8 was prepared in Millipore water (Elix3 Century, Millipore India, Bengaluru). The reactions were carried out in the presence of air and at room temperature (21 ± 2 °C) by placing required volume of the dye solution of required concentration in amber borosilicate bottles. Each bottle was designated to be used for analysis after a pre- determined reaction period. Each bottle was sealed with aluminium foil and three holes were pierced through it to allow free passage of air. The desired pH of the solu- tion was maintained by adding H2SO4 or followed by the sequential addition of 0.5 ml of desired Fe2+ solution of concentration 60 mM and 0.5 ml of the HP and TBHP was taken from 1400 mM stock solution which corresponds to 7.0 mM in the resulting 100 ml dye solution in Fe2+/HP and Fe2+/TBHP systems. The only change in procedure for Fe2+/SPB system is the concentration of the oxidants, i.e. 5.0 ml of the desired concentration of SPB was added de- pending upon their required concentration. The pH was measured using a digital pH meter (Eutech instruments, Singapore). In all the reactions the volume was made upto 100 ml by adding water. All the solutions used in this work were freshly prepared except the dye solution, which was stored at 4 °C and used within three days. No adjustment of pH was done during the course of the reaction and any change in pH during the reactions was noted regularly. 2. 3. Analytical Techniques Decolorization studies were carried out by measur- ing the absorbance at 471 nm with the help of a UV-Vis spectrophotometer (HACH, USA; DR 6000). The COD was measured by following the procedure prescribed by HACH, USA and reported by Kumar et al.10 In short, a mixture of 2.0 ml of a given sample, 0.25 ml of ‘Solution 1’ and 2.8 ml of ‘Solution 2’ (low range) was digested in a COD digester (HACH, USA; DRB 200) at 150 °C for 2 hrs. The digested samples were cooled to room tempera- ture and analysed at 420 nm for COD measurement with the help of a UV-Vis spectrophotometer (HACH, USA; DR 6000). The data presented in the text and figures were analysed by standard deviation using ‘Origin 7’ (Microcal Inc.) and has been rounded up to significant values. Decolorization and COD removal efficiency (CO- Deff) was calculated using (Eqs. (6) and (7)), respectively. The rate constant for COD removal was obtained accord- ing to the pseudo-first-order rate law (Eq. (8)). (6) (7) (8) 102 Acta Chim. Slov. 2024, 71, 99–109 Mulai et al.: Exploring a Substitute for Hydrogen Peroxide in Fenton ... here that although 97.3% decolorization was achieved in only 10 min, it takes further 20 min for the rest 2.7% de- colorization. In other words, complete decolorization was achieved in 30 min. Thus, there is a rapid cleavage of the azo bond during the first 10 min of the reaction followed by slow cleavage during the next 20 min. On the other hand the peak at 311 nm decreases very slowly as com- pared to the peak at 471 nm. This may be due to the forma- tion and accumulation of intermediate aromatic products in the system.27 However, in the case of Fe2+/SPB system, the decolorization at 5 min was a mere 20.0% which in- creased to 64.9% at 10 min and 97.3% in 90 min (Fig. 3b). No further change in decolorization was observed till 300 min of the reaction. Thus, the decolorization is faster in the initial stages in Fe2+/HP system than in Fe2+/SPB system, although at the end of the reaction not much dif- ference was observed in both the processes. The decolori- zation in Fe2+/TBHP system was found to be slower than in other two systems yielding only 5.5% in 5 min, which increased to 7.6 and 70.4% in 10 min and 300 min, respec- tively (Fig. 3c). Fe2+/SPS system has been earlier proved to be the most efficient process due to the higher oxidation potential of persulphate (2.5 to 3.1 V Vs. normal hydrogen electrode (NHE)) and its ability to operate at all pH val- ues.14,29,30 Under optimum conditions, the decolorization in Fe2+/SPS system at pH 3.0 reached up to 75.3 and 93.5%, respectively, in 90 and 300 min. Thus, the degree of decol- orization in all the systems studied is in the order: Fe2+/HP ≈ Fe2+/SPB > Fe2+/SPS > Fe2+/TBHP (Fig. 3d). 3. 2. Optimization of Operational Parameters and COD Removal Study 3. 2. 1. Fe2+/HP System The optimization of operational parameters such as Fe2+ dosage, concentration of HP and pH for a 0.3 mM solution of AO 8 was done by determining the CODeff at 90 min of treatment. The COD was measured by varying one parameter while maintaining the other two constant.27 Figure 3. (a–c): UV-Vis absorption spectra of AO 8 in different systems (d) decolorization in different systems. [AO 8] = 0.3 mM; [Fe2+] = 0.3, 0.2, 0.3 and 0.3 mM for Fe2+/HP, Fe2+/TBHP, Fe2+/SPB and Fe2+/SPS systems respectively; [HP] = 7.0 mM; [TBHP] = 7.0 mM; [SPB] = 2.0 mM; [SPS] = 7.0 mM; pH = 3.0. 103Acta Chim. Slov. 2024, 71, 99–109 Mulai et al.: Exploring a Substitute for Hydrogen Peroxide in Fenton ... The [Fe2+] was varied from 0.05 to 0.7 mM, [HP] from 1.0 to 10.0 mM and pH from 3.0 to 11.0. Using this strategy, the optimum parameters were established as [Fe2+] = 0.3 mM; [HP] = 7.0 mM; pH = 3.0. It is observed that both decolorization and CODeff show a decreasing trend once the operational parameters exceed the optimized values (Fig. 4a–4c). These inhibiting effects at higher [Fe2+] on the re- moval efficiency is due to scavenging of HO• by Fe2+. Further the higher concentration of Fe2+ leads to a higher generation of Fe3+ which scavenges HO• to form monohy- droxy complex (Eqs. (9) and (10)).31,32 (9) (10) At higher [HP], HO• radicals are scavenged to form hydroperoxyl radicals (HO2•) in accordance to Eqs. (11) and (12), which are less reactive and do not contribute to the degradation of organic molecules and recombination of HO• takes place (Eq. (13)).7 Figure 4. (a–c) Optimization of operational parameters in Fe2+/HP system (d) Variation of CODeff and decolorization with treatment period in Fe2+/ HP system: [AO 8] = 0.3 mM (11) (12) (13) The maximum efficiency at pH 3.0 is attributed to the high oxidation potential (2.8 V vs. NHE) of HO• rad- icals13,33,34 and the lower efficiency at pH > 3.0 is due to the formation of Fe(OH)3, which leads to a decrease in the generation of HO• due to the unavailability of Fe2+ ions.35 Additionally at higher pH, the formation of HO• also re- tards due to the decomposition of HP to O2 gas.36 Under the optimized parameters, a maximum CODeff of 66.2% was achieved in 90 min. This is attributed to the degradation of dye solutions by HO• radicals and the forma- tion of intermediates. To further increase the magnitude of CODeff, the treatment period was increased up to 300 min. But a mere 6.6% additional increase in CODeff was observed at the end of 300 min. Nevertheless, the CODeff remained constant from 120 min onwards (Fig. 4d). The CODeff con- 104 Acta Chim. Slov. 2024, 71, 99–109 Mulai et al.: Exploring a Substitute for Hydrogen Peroxide in Fenton ... sists of 2 stages: the initial fast stage (0 to 60 min) with a CODeff of 60.3% is followed by a slow step (120 to 300 min) with a CODeff of 72.8% (Fig. 5). The fast stage is due to the rapid consumption of HP (97.3% in 5 min and 100% in 10 min). The minor increase in CODeff (12.5%) in the 2nd stage probably is due to the presence of negligible amount of reactive species after the first stage (Fig. 5). As established earlier, the presence of residual HP leads to over estimation of COD of a given solution.37,38 This effect of excess HP on COD has also been verified by us recently.10,27 Therefore, the excess use of HP is not recommended for the degradation process. Since 97.3% HP is consumed in 5 min and 100% in 10 min, it may be concluded that presence of HP has no visible effect in the COD value. In an attempt to provide a cost effective method for COD removal, the experiment was carried out at the natu- ral pH (where no external reagent is required to adjust the pH to desired value) of the dye, i.e. at pH 6.5 under optimal parameters (Fig. 4d). The CODeff progressively increased from 6.0% at 10 min to 58.9% at 300 min. A comparison of CODeff at both pH reveals that COD removal is more effective at pH 3.0 than at 6.5. Colour removal studies un- der similar parameters indicate that almost complete de- colorization was achieved from 30 min onwards at pH 3.0. On the contrary only 85.0% decolorization was achieved in 30 min and almost complete decolorization in 90 min of treatment at pH 6.5 (Fig. 4d). 3. 2. 2. Fe2+/SPS System For a better comparison of the efficiency of oxidants, we have employed similar parameters ([Fe2+] = 0.3 mM; [SPS] = 7.0 mM; pH = 3.0; [AO 8] = 0.3 mM) for COD re- moval study in Fe2+/SPS system. As described earlier (Sec 3.1), near complete decolorization was achieved in this system. Further, to our disbelief COD removal was com- pletely inhibited at pH ≥ 3.0. Thus, our finding is in good agreement with the earlier report from our laboratory.39 Thus, it may be concluded that persulphate system inhibit COD removal of the target molecule at higher concentra- tion. It is for this reason that further study on this system was abandoned. 3. 2. 3. Fe2+/TBHP System It was our curiosity to understand the effect of re- placing one hydrogen of HP with tert-butyl group (as in TBHP) on the COD removal process by a modified Fen- ton process (Fe2+/TBHP). Since TBHP contains one OH- group, it is expected that hydroxyl radicals are generated when activated by Fe2+. The evidence for the formation of hydroxyl radical by TBHP has been discussed briefly in Sec. 3.3. In this system, the optimization of the catalyst, [Fe2+] and the oxidant, TBHP was done pH = 3.0 for 0.3 mM of AO 8 (Fig. S1). Thus the optimized parameters for this system was established as: [Fe2+] = 0.2 mM; [TBHP] = 7.0 mM; pH = 3.0. Under the optimized parameters, a maximum CODeff of 56.3% was achieved in 90 min at pH 3.0 and decolorization of 69.5 and 70.4% in 90 and 300 min, respectively. As in Fe2+/HP system, the CODeff in Fe2+/TBHP system follows a two stage process. The first stage lasts up to 60 min yielding a CODeff of 45.6%. The CODeff value was 69.5% at the end of 300 min in the sec- ond stage (Fig. 5). As already discussed, t-BuO• is a major radical spe- cies in this system26 and it contributes to the degradation of cytarabine antineoplastic. Thus, the decrease in CO- Deff at higher [Fe2+] may be due to the mutual scaveng- ing of [Fe2+] and t-BuO• (Eq. (14)).19 At higher [TBHP], excess generation of t-BuO• would lead to its fragmenta- tion and form acetone and ethane (Eqs. (15) and (16)).18 In addition, presence of excess of t-BuO• may lead to the formation of t-BuOO• (Eq. (4)), which ultimately leads to unreactive non-radical species (Eq. (17)). However, there is a concurrent opposing factor to this retarding effect, whereby Fe3+ is reduced to regenerate Fe2+ and accelerate the degradation process (Eq. (4)). Thus, it is expected that CODeff would be higher in HP than in TBHP. This has been verified in Sec. 3.3. Figure 5. CODeff in different systems at pH 3.0: [AO 8] = 0.3 mM; [Fe2+] = 0.3 mM (for Fe2+/HP and Fe2+/SPB systems) and 0.2 mM (for Fe2+/TBHP system); [HP] = 7.0 mM; [SPB] = 2.0 mM; [TBHP] = 7.0 mM (14) (15) (16) (17) In acidic condition the rapid fragmentation of t-BuO• (Eq. (15)) prevents the subsequent oxidation of Fe2+ as in (Eq. (14)). Regardless of that t-BuOO• will be generated as 105Acta Chim. Slov. 2024, 71, 99–109 Mulai et al.: Exploring a Substitute for Hydrogen Peroxide in Fenton ... corresponding decolorization was 98.3%. We assume it as 100% decolorization as it falls within ±5% error. A closure look at Fig. 5 reveals that the COD removal efficiency of different systems is in the order: Fe2+/SPB > Fe2+/HP > Fe2+/TBHP > Fe2+/SPS. The higher efficiency of Fe2+/HP system than Fe2+/TBHP has already been dis- cussed in Sec. 3.2.3. The other important factor lies in their ability to generate hydroxyl radicals. While HP system generates two equivalents of HO• radicals, TBHP only one. SPB system, on the other hand, generates four equivalents of HP, which generates four equivalents of HO• radicals in the in situ established Fenton process. 3. 3. COD Removal Kinetics As discuss in the above section, the COD remov- al in all the processes consists of two steps except in Fe2+/ SPB where a single step removal process (5 to 240 min) was observed. The rate constant in Fe2+/HP system was found to be 11.67 and 00.39 (10–3min–1) for first and second step, respectively (Fig. S3). The initial rate in Fe2+/HP system is higher than that in Fe2+/TBHP system and overall rate in Fe2+/SPB system. However, the rate is reversed in the sec- ond step. In Fe2+/TBHP system, 45.6% COD removal was obtained in the first step and 23.9% in the second step with a rate constant of 8.24 and 2.03 (10–3min–1), respectively (Fig. S4 and Table S2). The reaction in Fe2+/SPB system is a one step process, proceeds linearly and rapidly rapid by follow- ing pseudo-first order kinetics with a rate constant of 08.24 (10–3min–1) (Fig. S5). The COD removal increases from 2.6 to 92.7% when the treatment period is increased from 5 to 300 min. The linear progress in the COD removal may be due to the constant production of HO• in the system. A comparison of rate constant (Table S2) and the COD removal data (Fig. 5) reveals that compared to Fe2+/ HP system, the reacting species in Fe2+/TBHP system re- acts slowly in the first step. As evident from the rate con- stant, HO• in Fe2+/HP system is generated abundantly in the first 60 min of the reaction (first stage with higher rate constant). As for Fe2+/TBHP system, apart from the active radicals i.e., HO•, t-BuO• radicals act as a subsidiary radi- cal which maybe produced in the second phase of the reac- tion due to which the rate is higher than Fe2+/HP system. 3. 4. Effect of Presence of Anions on the COD Removal Processes Dye wastewater released from dye and textile indus- tries mostly contains inorganic anions such as Cl–, NO3–, and SO42–.26 Hence, there is a possibility that these ions might affect the colour and COD removal processes. We have, therefore, undertaken the study of effect of such ions on the colour and COD removal processes. Apart from inorganic anions, halide ions are also predominant in the effluent.26 They scavenge the HO• radicals and ad- versely affect the treatment process by forming radical and given in (Eq. (15)).19 As already discussed in Sec. 1, some authors have not reported the generation of HO• in Fe2+/ TBHP system. However, Pérez et al.26 in their study have of course reported the role played by HO• in the degra- dation of cytarabine antineoplastic. In order to verify the role played by HO• in the decolorization of AO 8, we have carried out the reaction with t-BuOH which is an efficient scavenger of HO•. The decolorization decreases from 69.4 to 51.2 and 14.4% in the presence of 0.1 and 0.7 molL–1 of t-BuOH, respectively. The generation of HO• is well estab- lished and hence need not be verified again. It is expected that HP and TBHP will generate two and one equivalents of HO•, respectively. Hence the degradation efficiency of HP is expected to be higher than TBHP. This is supported by the COD removal studies involving these two systems as discussed in Sec. 3.3. 3. 2 .4. Fe2+/SPB System The optimization process was similar to those de- scribed in Fe2+/HP system (Sec. 3.2.1). The optimized pa- rameters were found to be [Fe2+] = 0.3 mM; [SPB] = 2.0 mM; pH = 3.0 for 0.3 mM of AO 8. The effect of pH was established in the pH range from 3.0 to 11.0 at the treat- ment period of 90 min. Very interestingly it was found that it works only in pH 3.0 with 97.3 and 58.3% decoloriza- tion and CODeff respectively. At higher pH, decoloriza- tion was found to be in the range of 6.5 to 11.5% due to which no CODeff was observed. The observed CODeff at pH 3.0 is due to the in situ establishment of Fenton reac- tion as already described in Sec. 1. It was also reported by Kurin-Csörgei et al.25 that in acidic medium the perborate species (H3BO3) is in equilibrium with H2O2 in the ratio 1:1 (Eqs. (18) and (19)). At higher pH range, the species (HO)3B(OOH)– and (HO)2B(OOH)2– exist in relatively higher ratio (Eqs. (19) and (20)).25 In other words, HP is scavenged at higher pH leading to a retardation of CODeff. (18) (19) (20) The CODeff was found to be 0.7 and 58.3% at 0.05 and 0.3 mM of Fe2+, respectively, and shows a decreasing trend on further increasing the dosage to 0.7 mM (Fig. S2 (a)). As for CODeff it was found to increase from 20.5 to 58.3% when the [SPB] was increased from 0.5 to 2.0 mM, respectively (Fig. S2 (b)). The retarding effect of higher concentration of Fe2+ and SPB is due to the fact that the reaction is governed by the in situ established Fenton pro- cess in the system and the effect of different parameters are also applicable here (Sec. 3.2.1). It is pertinent to note that 92.7% COD removal was observed in 300 min (Fig. 5). The 106 Acta Chim. Slov. 2024, 71, 99–109 Mulai et al.: Exploring a Substitute for Hydrogen Peroxide in Fenton ... Figure 6. The inhibitory effect of various anions on CODeff in the Fenton process: [AO 8] = 0.3 mM; [Fe2+] = 0.3 mM; [HP] = 7.0 mM; pH = 3.0; Treatment period = 120 min non-radical Reactive Halogen Species (RHS). The concen- tration of various inorganic ions such as Cl–, NO3–, and SO42– was varied from 1.0 to 9.0 g L–1 and halide ions such as F– and Br– from 0.01 to 0.07 g L–1 in all systems at 120 min of treatment period. 3. 4. 1. Effect of Anions in Fe2+/Oxidant System In the presence of Cl–, the CODeff decreased gradually as the concentration was increased from 1.0 to 7.0 g L–1, be- yond which a complete inhibitory effect was observed (Fig. 6a). The decrease in CODeff in the presence of Cl– is due to the scavenging of HO• leading to the formation of less reac- tive ClOH•– and Cl2•– radicals (Eqs. (21) – (23)).40,41 (21) (22) (23) The inhibitory effect of Cl– on the COD removal effi- ciency may also be due to the conversion of non-selective HO• to selective RHS such as ClOH•–, Cl•, Cl– and Cl2•– (Eqs. (24) and (26)). These species attack the electron-rich compounds in the effluent, rather than electron-deficient compounds.42 Further, these species having low oxidation potential which does not contribute in the COD remov- al.43 The formation of RHS may be shown as below: (24) (25) (26) Decolorization was not affected in the presence of SO42– and NO3–. There is a marginal decrease of 10 and 5% in CODeff in the presence of 1.0 g L–1 of SO42– and NO3–, respectively. No further change in CODeff was observed on increasing the concentration of these anions (Fig. 6a). Based on the observations, we may conclude that the in- hibiting effect of anions on CODeff is in the order: Cl– > SO42– > NO3–. The decrease in the CODeff in the presence of SO42– is due to the reaction between SO42– and HO• (Eq. (27)) leading to the formation of SO4•– radicals, which di- merises to form less reactive peroxydisulphate ions (Eq. (28)).44 The inhibitory effect of NO3– is due to the scav- enging of HO• (Eq. (29)).45 (27) (28) (29) In order to know the effect of other halogens on CODeff, reactions were carried out in the presence of F–, Br– and I–. In general, a decreasing trend in decolorization was observed with the increase in the concentration of the halogens. The decolorization was completely inhibited in the presence of I– with a concentration of 0.3 g L–1. How- ever, an inhibition of 50% was recorded in the presence of 9.0 g L–1 of Cl– and Br– and 70% in the presence of 9.0 g L–1 of F–. Thus, the inhibitory effect of halogens on decol- orization is in the order: I– > F– > Cl– ≈ Br–. As far as I– is concerned, it inhibits CODeff at all concentrations. While Cl– shows inhibitory effect at higher concentrations, F– and Br– show at lower concentrations (Fig. 6b). As seen in the figure, the rate of inhibition is higher with Br– than F–. Near or complete inhibition is shown at 9.0 g L–1 of Cl–, 0.05 g L–1 of Br– and 0.07 g L–1 of F–. Thus, the inhibitory effect of the halogens on CODeff is in the order: I– > Br– > F– > Cl–. Although similar trends are observed with TBHP and SPB systems, the inhibitory effect is more pronounced at higher than in lower concentrations of anions. 107Acta Chim. Slov. 2024, 71, 99–109 Mulai et al.: Exploring a Substitute for Hydrogen Peroxide in Fenton ... 3. 5. Ion Chromatographic Analysis of Intermediate Products and Ions Ion chromatography technique was used to identify various ions and intermediate compounds generated in the degradative process. As the products identified in all the systems were same, the product identified in Fe2+/HP system only are listed in Table S3. The ions, Na+ and SO42– are identified in all the systems. Na+ has been identified as the dissociation product of AO 8. The attack of SO3– group by HO• leads to the formation of SO42–. As shown in Fig. 2 the dye has one source of nitrogen i.e., −N=N− bond and successive addition of HO• radicals to the − N=N− bond results in the formation of aryl products, nitroso and nitro aromatic compounds.46 Nitro aromatic compounds further undergoes oxidation by HO• to give substituted phenols, which ultimately form aliphatic ac- ids through ring opening.47,48 Aliphatic acids are further degraded into CO2 and H2O. The probable mechanism of degradation of AO 8 based on the intermediate products and ions and literature review has been proposed and is presented in (Fig. S6). 4. Conclusion Fenton oxidation, by far, is considered as the most studied and cost effective treatment process for the remov- al of various pollutants. However, there is a lack of elabo- rate study to find a suitable substitute for HP as an oxidant in the Fenton process. The substitutes were explored by in- serting different substituents on the peroxo bond (–O–O–) in HP. The substituted peroxides used in the present study on colour and COD removal in AO 8 were SPS, TBHP, and SPB. Further, a literature survey indicates that the concen- tration of organic pollutants used in degradation studies varies in the range 10 to 30 ppm. For industrial applica- tions, a higher concentration of target pollutant is essential and therefore, a higher concentration of AO 8 (0.3 mM) was used in this study. The optimal parameters for 0.3 mM of AO 8 in different systems were established as: Fe2+/HP system – [Fe2+] = 0.3 mM; [HP] = 7.0 mM; pH = 3.0; Fe2+/ TBHP system – [Fe2+] = 0.2 mM; [TBHP] = 7.0 mM; pH = 3.0; Fe2+/SPB system – [Fe2+] = 0.3 mM; [SPB] = 2.0 mM; pH = 3.0. The decolorization and COD removal efficien- cy of the systems under optimal parameters follow the order: Fe2+/HP ≈ Fe2+/SPB > Fe2+/SPS > Fe2+/TBHP and Fe2+/SPB > Fe2+/HP > Fe2+/TBHP > Fe2+/SPS, respective- ly. However, SPS completely inhibit COD removal at any pH. This is a significant finding considering the fact that SPS was described as the most powerful oxidant at all pH in the degradation process. Thus, SPB may be considered as a substitute for HP and SPS in Fenton and Fenton-type processes respectively. In the HP system, complete decol- orization was achieved in 30 min at pH 3.0, while at nat- ural pH of the dye, i.e. pH 6.5 it is 85% in 30 min and ≈ 100% in 90 min of treatment. Under optimized parame- ters, COD removal was found to be 66.2 and 58.8% at pH 3.0 and 6.5, respectively, in Fenton process. The reactivity of different systems towards COD removal efficiency may be ascribed to their ability to generate HO• radicals – two equivalents in HP system, one in TBHP and four in SPB system. Another factor responsible for the lower reactivity of TBHP is the generation of unreactive non-radical spe- cies through the formation of t-BuOO•. It is an established fact that presence of residual HP leads to over estimation of COD. To determine the effect of residual HP on COD in our study, the concentration of HP at different stages of the treatment was estimated using ion chromatography. It was found that 97.3% HP is consumed in 5 min and 100% in 10 min. This leads to the conclusion that the presence of HP has no visible effect in the COD values. The effect of various anions including halogens, gen- erally present in the effluents of textile and dye stuff in- dustries, are also established in this study. All the target anions show inhibitory effect on colour and COD removal and their inhibition effect follow the order: Cl– > SO42– > NO3–. Among halogens, Br– and F– show inhibition at low- er concentration and Cl– at higher concentration. Near or complete inhibition is shown at 9.0 g L–1 of Cl–, 0.05 g L–1 of Br– and 0.07 g L–1 of F–. It is important to note here that I– display inhibitory effect on colour and COD removal at all concentrations. Thus, the inhibitory effect of halo- gens follow the order: I– > Br– > F– > Cl–. Although similar trends are observed with TBHP and SPB systems, the in- hibitory effect is more pronounced at higher than in lower concentrations of anions. The kinetics of COD removal was determined in dif- ferent systems. COD removal in all the systems proceeds through two steps except in Fe2+/SPB where a single step removal process (5 to 240 min) was observed. The rate constant in Fe2+/HP system was found to be 11.67 and 00.39 (10–3min–1) for first and second step, respectively. The initial rate in Fe2+/HP system is higher than that in Fe2+/TBHP and Fe2+/SPB systems. The various intermediate ions such as Na+, NH4+, SO42–, NO2–, NO3– and different aliphatic acids such as formic acid, malonic acid, maleic acid, and fumaric acid were identified using ion chromatography. Based on these data, a degradation mechanism of AO 8 has been pro- posed. Acknowledgement The authors gratefully acknowledge use of facilities acquired through the DAE-BRNS grant (2013/36/50- BRNS/2485, dated 05.12.2013) to MKS; DST-FIST grant (SR/FST/CSI-194-2008) of the Department of Science and Technology, Govt. of India, and UGC-SAP CAS-I grant (F.540/21/CAS/2013(SAP I)) of UGC to the Department of Chemistry, North-Eastern Hill University (NEHU), Shillong. Discussion on the relative reactivity of oxidants 108 Acta Chim. Slov. 2024, 71, 99–109 Mulai et al.: Exploring a Substitute for Hydrogen Peroxide in Fenton ... with Prof. G. Bez from the Dept. of Chemistry, NEHU is greatly acknowledged. Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest. 5. References 1. H. B. Slama, A. C. Bouket, Z. Pourhassan, F. N. Alenezi, A. Silini, H. Cherif-Silini, T. Oszako, L. Luptakova, P. Golińska, L. Belbahri, Appl. Sci. 2021, 11, 6255. DOI:10.3390/app11146255 2. Q. Zeng, J. Fu, Y. Zhou, Y. Shi, H. 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Slov. 2024, 71, 99–109 Mulai et al.: Exploring a Substitute for Hydrogen Peroxide in Fenton ... Shahzad, A. Shakeel, S. Lyu, J. Environ. Chem. Eng. 2022, 10, 107196. DOI:10.1016/j.jece.2022.107196 44. C. L. Clifton, R. E. Huie, Int. J. Chem. Kinet. 1989, 21, 677– 687. DOI:10.1002/kin.550210807 45. R. G. Zepp, J. Hoigne, H. Bader, Environ. Sci. Technol. 1987, 21, 443–450. DOI:10.1021/es00159a004 46. J. M. Joseph, H. Destaillats, H. –M. Hung, M. R. Hoffmann, J. Phys. Chem. A. 2000, 104, 301–307. DOI:10.1021/jp992354m 47. J. H. Fendler, G. L. Gasowski, J. Org. Chem. 1968, 33, 1865– 1868. DOI:10.1021/jo01269a035 48. M. K. Sahoo, Res. J. Chem. Environ. 2011, 15, 96–112. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Vodikov peroksid (HP) se pogosto uporablja v naprednih oksidacijskih procesih (AOP). Ta študija ocenjuje kemijsko po- trebo po kisiku (KPK) učinkovitosti odstranjevanja barvila Acid Orange 8 (AO 8) pri višji koncentraciji z modificiranim Fentonovem postopku ter uporabo substituiranega HP, kot sta tert-butil hidroperoksid (TBHP) in natrijev perborat (SPB) ter natrijev persulfat (SPS) kot oksidant. Pri optimalnih pogojih je bilo ugotovljeno, da je bila KPK odstranitve 72,8 in 58,9 % pri pH 3,0 in 6,5 v Fentonovem procesu v 300 minutah. KPK učinkovitosti odstranjevanja različnih sistemov je v vrstnem redu: Fe2+/SPB > Fe2+/HP > Fe2+/TBHP > Fe2+/SPS, kar kaže na možnost uporabe SPB kot nadomestka za HP in SPS. Vrstni red učinkovitosti je med drugim pripisan njihovi sposobnosti proizvajanja HO• radikalov. Pokazalo se je, da različni anioni izkazujejo zaviralni učinek v vrs– tnem redu: I– > Br– > F– > Cl–> SO42– > NO3–. Zaviralni učinek Cl– opazimo pri višjih koncentracijah kot F– in Br–, vendar I– zavira pri vseh koncentracijah. Na koncu sta bila v tej študiji določena kinetika KPK odstranjevanja in mehanizem razgradnje na podlagi identificiranih vmesnih produktov. 110 Acta Chim. Slov. 2024, 71, 110–122 Mandal et al.: Synthesis, Spectroscopy, X-ray Structures, DNA Binding ... DOI: 10.17344/acsi.2023.8392 Scientific paper Synthesis, Spectroscopy, X-ray Structures, DNA Binding and Photocatalytic Properties of Two Ni(II) and Co(II) Complexes of a Pyrazolyl Schiff-base Ligand Suman Mandal,1 David B. Cordes,2 Alexandra M. Z. Slawin2 and Nitis Chandra Saha 1,* 1 Department of Chemistry, University of Kalyani, Nadia, West Bengal-741235, India 2 School of Chemistry, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK * Corresponding author: E-mail: nitissaha@klyuniv.ac.in Phone: +91-33-2582-8750 (Extn: 309) Received: 08-13-2023 Abstract Two new nickel(II) and cobalt(II) complexes, [Ni(MPAFA)3](BF4)2 (I) and [Co(MPAFA)3](BF4)2 (II) were synthesized from a pyrazole containing ‘NN’ bidentate Schiff-base ligand, N-(furan-2-ylmethyl)-1-(5-methyl-1H-pyrazol-3-yl)meth- animine, (MPAFA) (L). The complexes I and II were characterized by various physico-chemical and spectral parameters. Both I and II were 1:3 (M:L) coordination complexes and behaving as 1:2 electrolytes. Single crystal X-ray diffraction studies revealed that both of them were distorted octahedral in nature with a N6 donor set. The binding interactions of the complexes with CT-DNA were studied by UV-Vis and fluorescence spectroscopic methods. I was found to bind with CT-DNA in a partial intercalative mode, whereas II bound via the groove-like manner in solutions. The ligand and the complexes were shown to have potential photocatalytic activity in degrading methylene-blue (MB) under UV-Vis light irradiation. Keywords: Pyrazole, Schiff-base ligand, Ni(II) and Co(II) complexes, X-ray structures, CT-DNA, Photocatalytic activity. 1. Introduction The amazing biological capabilities of Schiff bases and their transition metal complexes, as well as their ex- tensive applicability in other domains such as pigments and dyes, catalyst carriers, corrosion inhibitors, polymer stabilizers and thermos-stable substances etc., attracted a lot of attention to their chemistry.1–3 Normally Schiff base ligands and their metal ion complexes exhibit a wide range of significant biological activities and also possess many important therapeutic applications in a variety of fields, including antibacterial, anticancer, antifungal, anti-malar- ial, antiproliferative, antiviral, antipyretic and anti-inflam- matory activities.4–7 Transition metal complexes of Schiff base ligands belong to an important class that plays a key role in biology because of their unique photochemical or electrochemical properties, well-defined coordination pat- terns, and tendency to interact with DNA.8,9 Due to their ease in forming stable complexes with the majority of tran- sition metals, Schiff bases play a significant role in inor- ganic chemistry also.10 Recent years have witnessed a tre- mendous development in interactions between metal ions and nucleic acids, which has created a challenging research area in inorganic and structural chemistry.11 In coordina- tion chemistry, considering the synthetic work, catalytic activity, bioactivity and physico-chemical study, N and O donor Schiff base ligands with their metal complexes have long been crucial.12,13 The wide-ranging pharmacophoric characteristics of such Schiff bases are in the creation of a variety of top biologically active compounds.14 The metal complexes of these ligands have the potential to bind DNA molecules precisely and be developed as pharmaceuticals. Several scientists have shown their keen interest in met- al-containing medications and their modes of interaction with proteins and DNA.15–17 The fundamental mechanism for the cytotoxic activities of some metal-based medica- tions is assumed to be ROS generation and subsequent de- struction of the DNA helix and/ or mitochondrial mem- brane potential, leading to the induction of apoptosis. In 111Acta Chim. Slov. 2024, 71, 110–122 Mandal et al.: Synthesis, Spectroscopy, X-ray Structures, DNA Binding ... order to produce more potent medications that would tar- get DNA, the association of bioinorganic compounds with DNA has drawn increased interest.18,19 It is known that human physiology depends largely on a number of trace elements like Ni, Co, Zn etc.20,21 The bio- logical significance of nickel is gradually becoming under- stood.22,23 Nickel complexes of the Schiff base ligands have displayed impressive antioxidant,24 antifungal,25 anticancer,26 antibacterial27 and antiproliferative28 activities in the hunt for new metal-based medications. Cobalt plays a decisive role in numerous biologically important processes in human body, and majority of it is present in the form of vitamin B12 (co- balamin).29 Because of their powerful antibacterial, antiviral, antifungal, antiprotozoal and anticancer properties, Schiff base complexes with cobalt have drawn much interest for their interactions with biomolecules such as proteins and nucleic acids, and a large number of therapeutically relevant cobalt complexes have been prepared.30–34 In the present days organic dyes, one of the main pollutants in wastewater, have received a great deal of at- tention due to their reduction in water quality and harmful effects on aquatic creatures as well as on human health.35,36 Degradation/ destruction of various organic dyes repre- sents a big challenge to human civilization, and coordina- tion compounds have recently gained a lot of attention for the investigation of photocatalytic degradation of such or- ganic dyes in water.37–43 Coordination polymers (Cps) ob- tained from reactions between Schiff bases and transition metals, are now being used as catalysts in photocatalytic dye degradation, which is decisive for industrial waste wa- ter treatment. There have been considerable efforts in the treatment of industrial wastewater based on adsorption and separation,44,45 chemical treatment46 and photocata- lytic methods.47 Among them, photocatalysis is a practi- cal, economical and reliable method that has been used to remove toxins like organic dyes safely and effectively from the environment.48–51 Schiff base coordinated Ni(II) and Co(II) complexes show promising photocatalytic activities for the degradation of organic dyes.52–54 In this submission, we have described the synthesis, characterization, spectroscopy and structural elucidation of two new Ni(II) and Co(II) complexes, [Ni(MPAFA)3] (BF4)2 (I) and [Co(MPAFA)3](BF4)2 (II) of a ‘NN’ biden- tate Schiff base ligand, N-(furan-2-ylmethyl)-1-(5-methyl- 1H-pyrazol-3-yl)methanimine, (MPAFA). DNA binding interaction of the complexes with CT-DNA and photo- catalytic degradation of methylene blue (MB) dye by the ligand and the complexes have also been reported here. 2. Experimental All reagents were of AR/GR grade and obtained from commercial sources and used without further purification. The metal salts and other organic chemicals and solvents were purchased from SIGMA ALDRICH CHEMICALS PVT. LTD. For conductance and spectral measurements, Spectro-grade methanol purchased from SPECTRO- CHEM was used. 2. 1. Synthesis of the Ni(II) and Co(II) Complexes: [Ni(MPAFA)3](BF4)2 (I) and [Co(MPAFA)3](BF4)2 (II) The Ni(II) and Co(II) complexes were synthesized by refluxing a 3:1 molar mixture of the ligand55 (0.3968 gm, 0.0021 mol) and Ni(BF4)2.6H2O (for I) and Co(B- F4)2.6H2O (for II) salts (0.0007 mol each) in ethanol for about an hour on a boiling water bath. On slow evapora- tion of the resulting greenish yellow / reddish solutions, the desired Ni(II) and Co(II) complexes crystallized, they were filtered off, washed with ethanol, dried over anhy- drous CaCl2 (yield ~ 76–80%). X-ray quality single crys- tals of [Ni(MPAFA)3](BF4)2 (I) and [Co(MPAFA)3](BF4)2 (II) were obtained from chloroform-n hexane mixture by solvent diffusion technique. Anal. Calcd. (%) for C30H33B- 2F8N9NiO3 (I): C, 45.2; H, 4.3; N, 15.8; Ni, 7.5. Found (%): C, 45.0; H, 4.1; N, 15.7; Ni, 7.3. Λm (MeOH): 196 Ω–1 cm2 mol–1 at 30 °C. µeff 3.01 BM at 300 K. IR (KBr) ν (cm−1): 1634 (νCH=N, azomethine), 1577 (νC=N, pyrazole), 1049 (νN–N, pyra- zole) and 476 (νNi–N, azomethine). UV-Vis. (MeOH, λmax, nm): 217 (π→π*), 239 (n→π*), 552 (d→d). Anal. Calcd. (%) for C30H33B2F8N9CoO3 (II): C, 45.2; H, 4.2; N, 15.9; Co, 7.6. Found (%): C, 45.1; H, 4.0; N, 15.6; Co, 7.4. Λm (MeOH): 101 Ω–1 cm2 mol–1 at 30 °C. µeff 1.98 BM at 300 K. IR (KBr) ν (cm−1): 1634 (νCH=N, azomethine), 1576 (νC=N, pyrazole), 1081 (νN–N, pyrazole) and 459 (νCo–N, azomethine). UV-Vis. (MeOH, λmax, nm): 218 (π→π*), 240 (n→π*), 585 (d→d). 2. 2. Physical Measurements The molar conductance values of the complexes in methanol were measured using a Systronics 308 dig- ital conductivity metre. A Perkin-Elmer 2400 CHNS/O analyser was employed to carry out the elemental analyses (C, H, and N). The nickel and cobalt contents of the com- plexes were determined gravimetrically as dimethylglyox- imatonickel(II) and anhydrous CoSO4, respectively. Using KBr pellets, IR spectra (4000−450 cm−1) of the complexes were measured on a Perkin Elmer Model Spectrum Two FT-IR spectrophotometer. Magnetic susceptibilities were measured in the polycrystalline state on a PAR 155 sample vibrating magnetometer. The UV-Vis spectral study was performed on a Shimadzu UV-1900i spectrophotometer in MeOH. The fluorescence spectra of the complexes in meth- anol were recorded using a Hitachi F-7100 Fluorescence Spectrometer. A Shimadzu UV-1900i spectrophotometer was used to study the photocatalytic degradation of Meth- ylene Blue (MB) and a UV-400 type photochemical reactor equipped with 400 W mercury lamp was used as the UV and Visible light source during the irradiation process. 112 Acta Chim. Slov. 2024, 71, 110–122 Mandal et al.: Synthesis, Spectroscopy, X-ray Structures, DNA Binding ... 2. 3. Crystallographic Measurements Diffraction data for complexes I and II were col- lected at 173 K using a Rigaku FR-X Ultrahigh Brilliance Microfocus RA generator/confocal optics with XtaLAB P200 diffractometer [Mo Kα radiation (λ = 0.71073 Å)]. All intensity data were collected at 173 K, using either both ω and φ steps or just ω steps, accumulating area de- tector images spanning at least a hemisphere of recipro- cal space. Data were collected using CrystalClear56 and processed (including correction for Lorentz, polarization and absorption) using CrysAlisPro.57 Structures were solved by dual-space (SHELXT)58 or direct (SIR2004)59 methods and refined by full-matrix least-squares against F2 (SHELXL-2018/3).60 Non-hydrogen atoms were refined anisotropically, and carbon-bound hydrogen atoms were refined using a riding model. Hydrogen atoms bound to heteroatoms were located from the difference Fourier map and refined isotropically subject to a distance restraint. The structures of both complexes showed disorder in their an- ions, these were modelled over two sites with occupancies of 0.91:0.09 and 0.79:0.21 for I and 0.88:0.12 and 0.78:0.22 for II. Fluorine atoms in the minor component of the dis- order were refined isotropically, and restraints to bond distances and thermal motion were used. All calculations were performed using the Olex261 interface. 2. 4. DNA binding Studies 2. 4. 1. Absorption Spectral Studies UV-Vis titration of a tris-HCl buffer (30 mM) at pH 7.5 at room temperature was used to assess the DNA binding characteristics of complexes I and II. The titration experiment was carried out in a quartz cuvette holding a constant concentration of each complex (1.25 × 10−4 M) and a changing concentration of CT-DNA (0–5.769 × 10−5 M). The concentration of the CT-DNA solution was deter- mined by absorption spectroscopy using 13,600 M–1 cm−1 molar extinction coefficient at 260 nm.62 To eliminate the DNA's particular absorbance, equal quantities of CT-DNA solution were added to the complex and standard solu- tions. Each complex received a DNA addition in Tris-HCl buffer, and the resultant solution was allowed to reach equilibrium at 25 °C for 10 minutes. The absorbances for I and II were calculated while being scanned at 240 and 244 nm, respectively. 2. 4. 2. Emission Spectral Studies The fluorescence displacement assays with ethidi- um bromide (EB) were performed at 25 oC in a 30 mM Tris-HCl buffer (pH 7.5). First, the CT-DNA was incu- bated in a darkened atmosphere for around 30 minutes at 35 °C with ethidium bromide ([EB]/[DNA] = 0.1).63 The resultant complex was then adjusted from 0–6.725 × 10−3 M in an EB-bound CT-DNA solution. The effects of flu- orescence quenching were determined by observing how the spectrum of fluorescence emission changed at varying concentrations of complexes. After excitation of the sam- ple solutions at 510 nm, the fluorescence intensities for I and II were measured at 591 nm and 590 nm, respectively. 2. 4. 3. Viscosity Measurement A thermostatic water bath was used to evaluate the viscosity in a buffer containing 30 mM Tris-HCl at a con- stant temperature of 25 °C (pH 7.5). The plots of binding ratio ([complex]/[DNA]) vs. relative specific viscosity {(η/ η0)1/3} were obtained for I and II, where [complex]/[DNA] = 0, 0.2, 0.6, 1.0, 1.4, 2.0; η and η0 were the specific viscosi- ties of CT-DNA in the presence and in absence of the com- plexes, respectively. The equation η = (t – t0)/t0 was used to calculate the relative viscosity, where t was the flow time of the CT-DNA solution in the absence or presence of the complex and t0 represented the flow time of the Tris-HCl buffer solution. CT-DNA was present at a concentration of 25 × 10−5 M. The flow time of each sample was measured three times with a digital stopwatch, and the average flow time was calculated. 2. 5. Photocatalytic Experiment Methylene Blue (MB) was used as the target dye in the investigation of photocatalytic activity of the ligand (L) and its complexes (I and II). Each compound could be well dispersed in the dye solution prior to the photocatalytic degradation experiment. 0.015 mmol of solid compound was added into 100 mL of MB aqueous solution (10 mg/L). The compounds were magnetically stirred for 30 minutes in the dark until an adsorption-desorption equilibrium was reached before applying UV radiation. The mixture was then exposed to UV and visible light for140 minutes. Samples were taken every 20 minutes interval throughout this time, and their absorbances were continually meas- ured. To determine the photosensitivity of MB, the same experimental setup was used for the blank experiment (without the addition of compounds). The following equa- tion was used to compute the degradation efficiencies of photocatalysts: D% = At /A0 x100% D% = the degradation efficiencies of photocatalysts. A0 = the initial absorbance values of the MB aqueous solution. At = the absorbance values of the MB aqueous solu- tion at time t. 3. Results and Discussion 3. 1. Synthesis and Characterization The two new mononuclear complexes, [MII(MPA- FA)3](BF4)2 (where, M = Ni and Co for I and II, respec- 113Acta Chim. Slov. 2024, 71, 110–122 Mandal et al.: Synthesis, Spectroscopy, X-ray Structures, DNA Binding ... tively) were prepared by refluxing ethanol solution of three equivalent of the ligand, MPAFA and one equivalent of re- spective metal tetrafluoroborate salt in each case (Scheme 1). Elemental analyses of the complexes were in good agreement with the molecular structures determined by the single crystal X-ray studies. 3. 2. IR spectra Upon comparison of the infrared bands (4000−450 cm−1) of the complexes with those of the free ligand, valua- ble information on the bonding sites of the primary ligand molecule was obtained. A negative shift in ν(CH=N, azomethine) (1650 cm−1) band in the spectrum of the free ligand to low- er values1633−1634 cm−1 in the complexes was consistent with the coordination of the azomethine nitrogen to the central metal ion. The pyrazolyl tertiary ring nitrogen atom (2N) as a potential binding site was indicated by the shifting of the ν(C=N, pyrazole ring) bands of the complexes to a higher frequency range 1576−1581 cm−1 than the free ligand it- self at 1540 cm−1. A relatively strong IR band at 1010 cm−1 in the free ligand, due to ν(N–N, pyrazole) vibration, was also found to shift to the higher wave numbers 1049−1081 cm−1 in the metal complexes. This offered additional evidence that the tertiary nitrogen (2N) atom of pyrazole ring par- ticipated in bonding.64 The appearance of new IR bands at 459−481 cm−1 in the spectra of the complexes were then assigned to ν(M–N) vibrations (Figures S1 and S2). 3. 3. UV-Vis Spectra The electronic absorption spectra of the free Schiff base ligand and its Ni(II) and Co(II) complexes were measured in methanol. The electronic spectrum of the free ligand exhibited a band at 368 nm, assigned to the (n→π*) transition of the azomethine group. A noticeable band ob- served at 235 nm, which might be a (π→π*) transition.65 The ligand to metal charge transfer (LMCT) bands for the Ni(II) and Co(II) complexes were visible at 217–218 and 239–240 nm for (π→π*) and (n→π*) transitions, respective- ly, as well as a low intensity major band for the d–d tran- sition of a metal ion at 552–585 nm was also observed66 (Figures S3 and S4). 3. 4. Fluorescence Property Scheme 1. Synthetic procedure of the complexes I and II. Figure 1. Fluorescence spectra of the complexes in I and II in MeOH 114 Acta Chim. Slov. 2024, 71, 110–122 Mandal et al.: Synthesis, Spectroscopy, X-ray Structures, DNA Binding ... The fluorescence spectra of the complexes were re- corded in methanol at a concentration of 2 × 10−5 M. The complex species displayed distinctive fluorescence traits. When the complexes were stimulated at wavelengths be- tween 247 and 239 nm, the emission bands were found to be discernible between 453 and 456 nm (Table S1, Figure 1). Among the complexes, II was more fluorescent than I. It demonstrated a strong emission band at the highest emission wavelength of 456 nm at the excitation wave length of 239 nm. The probable causes for the emission phenomenon displayed by the complexes might be due to the ligand to metal charge transfer (LMCT). The data sug- gested that both I and II in particular, might be the suita- ble candidate for a photoactive molecule. 3. 5. Structural Description ORTEP-367 plots of the complexes, [Ni(MPAFA)3] (BF4)2 (I) and [Co(MPAFA)3](BF4)2 (II) together with the atom numbering schemes are shown in Figures 2 and 3, respectively. The crystallographic data and refinement pa- rameters are summarized in Table 1. The asymmetric unit of each structure consists of a [M(MPAFA)3]2+ cation, and two BF4– counter ions, and complexes I and II are iso- structural to the related complex [Ni(MPAFA)3](ClO4)2.55 The two complex cations reported here and the isostruc- tural complex55 are geometrically very similar; selected bond distances and bond angles in the structures of I, II and [Ni(MPAFA)3](ClO4)2 are compiled in Table 2. Like the isostructural complex,55 the metal centres, in both the complexes I and II, display a distorted octahedral geometry and three neutral MPAFA molecules upon coordination to the respective metal centres, generate a N6 donor set. Each bidentate ligand molecule bonded to the metal ion via the azomethine and the pyrazolyl (tertiary) nitrogen atoms; two of the pyrazolyl nitrogen atoms (N16 and N30 for I; N2 and N30 for II), two of the azomethine nitrogen atoms (N7 and N35 for I; N7 and N21 for II) and the remaining pyrazolyl nitrogen and the azomethine nitrogen atoms (N2 and N21 for I; N16 and N35 for II) coordinate to Ni1/ Co1 in a trans, cis and cis manner, respectively. Figure 3. Ortep-3 diagram (30% probability ellipsoids) of complex II with atom numbering scheme (hydrogen atoms are omitted for clarity) Strong N-H···F hydrogen bonding interactions are observed in the crystal lattices of both I and II. The tetrafluoroborate anions and the pyrazolyl N-H groups play significant role in the formation of H-bonding interactions as shown in Figures 4 and 5. The details of the hydrogen bonding interactions observed in I and II are summarized in Table 3. π···π stacking interactions have also been identi- fied in both the complex species. In I, intramolecular offset π···π stacking interaction is observed between the pyrazole ring (N29-N30-C31-C32-C33) of one ligand and the furan ring (C9-O10-C11-C12-C13) of another ligand; while in II, the same is observed between the rings (N1-N2-C3- C4-C5) and (C23-O24-C25-C26-C27). The distances be- tween the centroids of pyrazole and furan rings involved in the stacking interactions are 3.658 and 3.650 Å, and angles between the mean planes of the rings are 7.81 and 7.02º, and the offset between the centroids (in the plane of one ring) are 0.98 and 0.88 Å for I and II, respectively (Figures 4 and 5). The stacking interactions, in both the complex species, are quite strong,68,69 given the offset as well as the relatively short distance between centroids, making the complex molecules more stable. Crystal packing of I and II are shown in Figures S5 and S6, respectively. 3. 6. DNA binding Performance 3. 6. 1. Stability of the Complexes In research on biological activity, dimethyl sulphox- ide (DMSO) is frequently used as a co-solvent. Time-de- Figure 2. Ortep-3 diagram (30% probability ellipsoids) of complex I with atom numbering scheme (hydrogen atoms are omitted for clarity). 115Acta Chim. Slov. 2024, 71, 110–122 Mandal et al.: Synthesis, Spectroscopy, X-ray Structures, DNA Binding ... pendent UV-Vis spectroscopy was used to determine the stability of the Ni(II) and Co(II) complexes at room temperature in DMSO and DMSO/Tris-HCl buffer (1:1 V/V). The complexes were dissolved in DMSO or DMSO/ Tris-HCl buffer (1:1 V/V) at a concentration of 10–5 M, over a period of 48 hours, and the stability of I and II was Table 1. Crystal data and structure refinement parameters for complexes I and II Crystal data [Ni(MPAFA)3](BF4)2 (I) [Co(MPAFA)3](BF4)2 (II) Empirical formula C30H33B2F8N9NiO3 C30H33B2F8N9CoO3 Formula weight 799.96 800.20 Temperature/K 173 173 Crystal system Monoclinic Monoclinic Space group P21/c P21/c a/Å 13.0905(5) 13.1847(4) b/Å 15.1604(5) 15.1337(4) c/Å 18.2845(7) 18.4095(6) α/° 90.0000 90.0000 β/° 105.003(4) 105.613(4) γ/° 90.0000 90.0000 Volume/Å3 3505.0(2) 3537.77(19) Z 4 4 ρcalcg/cm3 1.516 1.502 μ/mm1 0.643 0.574 F(000) 1640.0 1636.0 Reflections collected 44820 45274 Independent reflections (Rint) 8150 (0.0658) 8179 (0.0495) Data/restraints/parameters 8150/206/523 8179/181/523 Goodness-of-fit on F2 1.034 1.028 R1, wR2 [I ≥ = 2σ (I)] 0.0502, 0.1027 0.0473, 0.1033 R1, wR2 [all data] 0.0919, 0.1146 0.0877, 0.1159 Largest diff. peak/hole / e Å–3 0.52/–0.39 0.47/–0.30 Table 2. Selected bond lengths (Å) and bond angles (°) of I, II and [Ni(MPAFA)3](ClO4)2 I II [Ni(MPAFA)3](ClO4)255 Bond length (Å) Bond length (Å) Bond length (Å) Ni1−N2 2.077(2) Co1−N2 2.0919(19) Ni1−N2 2.0825(18) Ni1−N7 2.120(2) Co1−N7 2.161(2) Ni1−N7 2.1262(17) Ni1−N21 2.105(2) Co1−N21 2.1631(18) Ni1−N21 2.1120(18) Ni1−N16 2.062(2) Co1−N16 2.117(2) Ni1−N16 2.0552(18) Ni1−N30 2.053(2) Co1−N30 2.0999(19) Ni1−N30 2.0638(18) Ni1−N35 2.112(2) Co1−N35 2.1534(19) Ni1−N35 2.1127(17) Bond angle (°) Bond angle (°) Bond angle (°) N2−Ni1−N7 77.95(8) N2−Co1−N7 76.46(7) N2−Ni1−N7 77.83(7) N7−Ni1−N21 167.04(8) N7−Co1−N21 98.32(7) N7−Ni1−N21 97.10(7) N21−Ni1−N16 77.85(8) N21−Co1−N16 76.89(7) N21−Ni1−N16 77.84(7) N16−Ni1−N35 97.32(8) N16−Co1−N35 95.13(7) N16−Ni1−N35 99.46(7) N35−Ni1−N2 171.50(8) N35−Co1−N2 101.37(7) N35−Ni1−N2 93.53(7) N30−Ni1−N7 91.63(8) N30−Co1−N7 98.25(8) N30−Ni1−N7 91.80(7) N30−Ni1−N35 78.00(8) N30−Co1−N35 76.70(7) N30−Ni1−N35 77.90(7) N30−Ni1−N21 99.32(8) N30−Co1−N21 91.47(7) N30−Ni1−N21 96.95(7) N2−Ni1−N21 94.02(8) N2−Co1−N21 91.10(7) N2−Ni1−N21 171.87(7) N16−Ni1−N30 174.50(8) N16−Co1−N30 89.35(8) N16−Ni1−N30 174.13(7) N35−Ni1−N7 96.71(8) N35−Co1−N7 91.00(7) N35−Ni1−N7 166.70(7) N2−Ni1−N16 89.52(8) N2−Co1−N16 96.07(8) N2−Ni1−N16 95.80(7) N16−Ni1−N7 91.78(8) N16−Co1−N7 171.17(7) N16−Ni1−N7 91.54(7) N35−Ni1−N21 92.36(8) N35−Co1−N21 165.93(7) N35−Ni1−N21 92.54(7) N30−Ni1−N2 95.41(8) N30−Co1−N2 174.41(8) N30−Ni1−N2 89.62(7) 116 Acta Chim. Slov. 2024, 71, 110–122 Mandal et al.: Synthesis, Spectroscopy, X-ray Structures, DNA Binding ... confirmed by their UV-Vis spectral patterns (which were almost same, no significant changes were noticed, Figures S7 and S8). 3. 6. 2. Absorption Spectral Studies The UV-Vis spectroscopic technique was used to evaluate the binding characteristics of I and II with CT- DNA. Observing the changes in the absorption spectra of the complexes upon addition of increasing amounts of DNA is one of the most extensively utilised approaches for analysing their binding abilities. Figures 6 and 7 depicted the absorption spectra of I and II at fixed concentrations and in the presence of increasing concentrations of CT- DNA, respectively. As increasing amounts of DNA were added, the UV-Vis spectra of I were found to exhibit a hypochromic effect, while the same for II demonstrated a hyperchromic effect of the charge transfer region. Hence, a 2 nm red-shift of I absorption maximum (λmax I = 240 nm) when CT-DNA bound I (λmax = 242 nm; Figure 6) and a 4 nm blue-shift of II absorption maximum (λmax II = 244 nm) when CT-DNA bound II (λmax = 240 nm; Figure 7) were observed. These changes highlighted the unique- ness of the complexes that interacted with CT-DNA via non-covalent and / or covalent interactions.70 The hyper- chromism or hypochromism, as well as significant red or blue shifts for I and II, indicated that DNA was interact- ing with the complexes in solution. As complexes I and II exhibited hypochromism and hyperchromism effects, re- spectively, it might be concluded that I and II were bound with CT-DNA via the partial intercalative mode71 and the groove binding mode,72 respectively. By the help of eqn. (1), one can calculate the intrinsic binding constant (Kb) values from the plots of [DNA] versus [DNA] / (εa–εf) for I and II in order to understand the strength of the binding between DNA and the complexes. 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DOI:10.1016/j.poly.2020.114431 Povzetek Z ligandom N-(furan-2-ilmetil)-1-(5-metil-1H-pirazol-3-il)metanimin, (MPAFA) (L), ki deluje kot Schiffova baza s pi- razolnim obročem, smo sintetizirali dva nova kompleksa niklja(II) in kobalta(II), [Ni(MPAFA)3]2BF4 (I) in [Co(M- PAFA)3]2BF4 (II). Obe spojini smo karakterizirali z različnimi fizikalno-kemijskimi in spektralnimi metodami. Obe spojini, I in II, imata razmerje M:L = 1:3 in se obnašata kot 1:2 elektrolita. Strukturna analiza na monokristalu kaže za oba kompleksa popačeno oktaedrično zgradbo z N6 donorskimi atomi. Interakcije pri vezavi kompleksa s CT-DNA smo preučevali z UV-Vis in fluorescenčno spektroskopijo. Ligand in kompleksi imajo potencialno fotokatalitsko aktivnost pri razgradnji metilenskega modrila (MB) pod obsevanjem z UV-Vis svetlobo. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 123Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... DOI: 10.17344/acsi.2023.8440 Scientific paper Removal of Methyl Violet 2B and Direct Black 22 from Single and Binary System Using a Magnetic Zeolite/MgO/Starch/Fe3O4 Nanocomposite Serap Fındık Hitit University, Engineering Faculty, Chemical Engineering Department, Kuzey Yerleskesi, Çevre Yolu Bulvarı, 19030, Çorum, Türkiye * Corresponding author: E-mail: serapfindik@hitit.edu.tr Received: 09-13-2023 Abstract This study focuses on the preparation and characterization of magnetic zeolite (FSM-Zeo) using starch, magnesium oxide, and Fe3O4. Various analyses, including BET, FTIR, SEM, EDS, XRD, Zeta potential, and VSM, were conducted to assess the properties of FSM-Zeo. The adsorption capacity of FSM-Zeo was investigated for methyl violet (MV-2B) and direct black 22 (DB-22) in both single and binary dye solutions. Key parameters such as adsorbent amount, initial dye concentration, contact time, temperature, initial pH, and ionic strength were examined in the single system. Kinetic and isotherm studies revealed that DB-22 and MV-2B adsorption followed the pseudo-second-order model. Moreover, Freundlich and Langmuir models were confirmed for MV-2B and DB-22 adsorption on FSM-Zeo, respectively. In the binary system, the presence of MV-2B enhanced the adsorption of DB-22, resulting in higher removal compared to the single dye solution. A synergistic effect was observed due to the interaction between DB-22 and MV-2B, promoting the adsorption of DB-22 on FSM-Zeo. Keywords: Adsorption; direct black 22; methyl violet 2B; magnetic zeolite; magnetite 1. Introduction The dyes used in industries such as textiles, print- ing, plastics and paper have a complex aromatic structure. Among these industries, the textile industry is one of the largest consumers of water containing various dyes. If dye-containing wastewater is discharged into the receiv- ing environment without treatment, it causes a decrease in photosynthesis and sunlight penetration. In addition, the dye in water produces toxic and harmful compounds through oxidation, hydrolysis or chemical reactions. These dyes are dangerous for humans, aquatic organisms and other living organisms due to their cancerogenic, muta- genic and toxic effects.1,2 In order to eliminate the negative effects of the dye on the environment and living organ- isms, wastewater containing dyes must be treated before being discharged into the environment. There are several treatment methods for the elim- ination of dyes from the wastewater such as chemical treatment (ozonation, photolysis, photocatalysis etc.), physicochemical treatment (adsorption, ion exchange, membran filtration etc.) and biological treatment (aerobic and anaerobic degradation). However, these methods have some limitations, including high operating costs, produc- tion of toxic by-products, generation of sludge, and dis- posal issues.3 Amoung these methods, adsorption is the most suitable method due to its flexibility, simplicity, low cost, and absence of toxic sludge.4 Adsorption is a mass transfer process in which a solid substance called adsor- bent collects dissolved substances in aqueous solution on its surface. The efficiency of adsorption depends on vari- ous factors related to the adsorbent, including its molecu- lar structure, molecular weight, surface area, particle size, cost, availability, and ease of use.3 There are different types of adsorbents that can be used to remove dyes, such as ag- ricultural wastes, industrial and urban wastes, clays, and natural polymers.1 Bio-adsorbents such as starch, lignin, chitosan, cellu- lose, and pectin are widely used due to their low cost, biodeg- radability and non-toxicity. Starch, which is one of the most abundant biopolymers, contains hydroxyl groups in its struc- ture. However, starch has low mechanical strength, which can be improved through modification.5,6 MgO is common- 124 Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... ly used in polymer metal matrix composites because of its excellent heat resistance, good thermal properties, and high tensile strength.7 Zeolite is a naturally abundant adsorbent with a porous structure consisting of crystalline alumina sil- icate. There are more than 40 types of naturally occurring zeolites, and clinoptilolite is one of the most abundant types with low cost and a high surface area. Zeolite can be used in its natural form or modified to enhance its adsorption capac- ity through thermal or chemical methods.8 The use of magnetic nanoparticles as adsorbents has been increasing due to their functional groups, active sites, adsorption capacity, surface area and easy of separation.4 Among magnetic particles, iron oxides, such as magnetite (Fe3O4), hematite (α–Fe2O3), and maghemite (γ–Fe2O3), have garnered attention due to their ease of synthesis, magnetic susceptibility, biocompatibility, and low cost. Among these, Fe3O4 is the most extensively studied iron oxide. It can be easily modified, and new nanoadsorbents can be developed using low-cost conventional materials.9 Numerous studies in the literature have explored adsorbents incorporating clay, starch, and iron oxide for various applications. Noteworty examples include the re- moval of methyl violet using clay/starch/Fe3O4,10 the re- moval of sunset yellow and Nile blue using clay/starch/ MnFe2O4,5 the removal of methylene blue, methyl violet and crystal violet using clinoptilolite/starch/CoFe2O4,11 the removal of methylene blue and methyl violet using Montmorillonite clay/starch/CoFe2O4,1 and the removal of anionic Biebrich Scarlet using magnetic Fe3O4 zeolite 13X.12 However, a significant gap exists in the literature regarding composites comprising starch, Fe3O4, MgO, and zeolite. This study introduces an environmentally friendly and novel magnetic adsorbent composed of zeolite, starch, MgO, and Fe3O4. The innovative composite shows prom- ise for efficiently removing dyes from aqueous solutions, filling a critical research void, and demonstrating signifi- cant potential in advancing dye removal technologies. In this study, a magnetic zeolite (FSM-Zeo) was prepared using starch, MgO and Fe3O4. The adsorption ability of FSM-Zeo was investigated for the removal of the cationic dye methyl violet (MV-2B) and the anionic dye direct black 22 (DB-22) in single and binary dye solutions. FSM-Zeo was characterized using various analyses such as SEM, EDS, XRD, BET, Zeta potential, VSM, and FTIR. The effects of adsorption parameters, including contact time, initial dye concentration, FSM-Zeo amount, ionic strength, temperature, and initial pH of the solution on the adsorption of DB-22 and MV-2B were studied. Addi- tionaly, equilibrium and kinetic studies were performed. 2. Materials and Methods 2. 1. Materials In the study, zeolite (a commercial product ob- tained from a company in Türkiye), FeSO4.7H2O (Merck), FeCl3.6H2O (Sigma Aldrich), MgSO4.7H2O (Merck, 99 %), ethyl alcohol (Merck, 96 %) and starch (Carlo Erba, code 417587) were used to prepare magnetic composite. Adsorption experiments were performed using MV-2B (C.I. 42535, Isolab) and DB-22 (commercial name Di- rect Black 22 VSF 1600, supplied from a company named “HNY” in Turkey). All the chemicals were used without purification. 2. 2. Preparation of the Adsorbent and Characterization The adsorbent used in the study was prepared using the chemical coprecipitation method.5,10,13 Iron II sulphate heptahydrate (FeSO4.7H2O) and iron III chloride hex- ahydrate (FeCl3.6H2O) were dissolved in 100 mL of ethyl alcohol with a molar ratio of 1:1. The mixture was stirred for 10 minutes using a magnetic stirrer. MgSO4 . 7H2O was added to the iron solution and stirred for additional 10 min- utes. Next, starch was added to the solution and stirred for 5 minutes. Zeolite was subsequently added to the solution and stirred for 30 minutes at a temperature of 70–75 °C. The weight ratio of starch to zeolite was 1:1. The pH of the solu- tion was adjusted to 11 using 3 M NaOH solution. After ad- justing the pH, stirring continued for one hour at a tempera- ture of 75–80 °C. The prepared adsorbent was left overnight, washed several times with distilled water, and filtered using filter paper (Whatmann-40). Finally, it dried at 90 °C for 70 hours. The resulting adsorbent was coded as FSM-Zeo. FSM-Zeo was characterized by BET (Quantachrome Nova Touch LX4), XRD (Bruker D8 Advence), FTIR (Bruker, Alpha), VSM (Lake Shore 7407), Zeta poten- tial (Malvern ZetaSizer Nano ZSP), SEM and EDS (FEI, Quanta FEG250) analyses. 2. 3. Adsorption Experiments In this study, process variables such as the contact time (0–90 min), initial pH (3.5–9), initial concentration of the dye solution (10–40 mg/L), NaCl amount (0.1–0.7 g/100 mL), Na2SO4 amount (0.1–0.7 g/100 mL), amount of adsorbent (0.1–0.5 g/100 mL) and temperature (22–50 °C) were investigated. A known amount of FSM-Zeo was added to 100 mL dye solution with a known concentration and stirred at 300 rpm. Samples were taken from the dye solution and centri- fuged at 3500 rpm for 10 minutes to separate the adsorbent. The concentrations of MV-2B and DB-22 were determined by measuring the absorbance of the sample using UV spec- trophotometer (Hach, DR-2400). The absorbance of MV-2B was recorded at a wavelength of 584 nm, while the absorb- ance of DB-22 was measured at a wavelength of 481 nm. All the experiments were repeated at least three times. The dye removal (R), was calculated using Eq. 1 R, % = [(C0 – Ct) / C0]*100 (1) 125Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... The adsorption capacity of the FSM-Zeo at equilibri- um (qe, mg/g) was calculated using Eq. 2 (2) where V is the volume of the dye solution (L), W is the weight of the FSM-Zeo (g), C0, Ct and Ce are the concen- tration of dye (mg/L) at initial, at any time and at equilib- rium respectively. 3. Results and Discussion 3. 1. Characterization of the FSM-Zeo The structure of FSM-Zeo and elemental distribu- tion were examined using SEM-EDS analyses. Figure 1a and 1b show the SEM and EDS analyses of the zeolite (Z) and FSM-Zeo. In Figure 1a, the zeolite exhibits a porous, layered structure with a non-uniform surface. After the synthesis of FSM-Zeo, particles of various sizes can be ob- served on its surface, which can be attributed to the pres- ence of Fe3O4, MgO, and starch. The weight percentages of the elements in Z and FSM-Zeo are presented in Table 1. The results indicate that FSM-Zeo has been successfully loaded with iron and magnesium. The BET specific surface area of Z and FSM-Zeo was determined as 16.79 m2/g and 34.26 m2/g respectively. These results indicate that the sur- face area of FSM-Zeo is enhanced compared to zeolite. Similar findings have been reported in the literature. For instance, Foroutan et al.11 determined the surface area of clinoptilolite as 18.82 m2/g in their study. Another study by Ahmedi et al.1 reported the BET surface area of mont- morillonite clay and the montmorillonite clay/starch/ CoFe2O4 composite as 3.167 m2/g and 27.27 m2/g, respec- tively. Figure 2 shows the XRD patterns of Z and FSM- Zeo. In the XRD diffraction pattern of Z, the main peaks observed at 2θ = 9.85°, 22.4°, 22.73°, 26.6°, 28.15°, 30.2° and 31.7° correspond to clinoptilolite (PDF#39-1383). In the XRD pattern of FSM-Zeo, the main peaks associated with clinoptilolite remained unchanged, suggesting the crystal structure of clinoptilolite remained stable during the synthesis process. Additionally, new diffraction peaks appeared in the FSM-Zeo XRD pattern at 30.19°, 35.8°, 43.3°, 57.5°, and 63.05°, which are characteristic peaks of magnetite (Fe3O4). These magnetite diffraction peaks are Fig. 1. The SEM and EDS spectra of (a) Z and (b) FSM-Zeo 126 Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... in accordance with the PDF card 75–0449 and have been reported in previous studies.14–16 Furthermore, a diffrac- tion peak at 75.15° can be attributed to MgO, and it match- es with the PDF card 45-0946. Figure 3a shows the VSM (Vibrating Sample Mag- netometer) analysis of FSM-Zeo. The magnetic satura- tion value of FSM-Zeo was determined as 8.41 emu/g at room temperature under a magnetic field of ± 20000 Qe. The magnetization curve of FSM-Zeo exhibits a S-shaped curve, indicating superparamagnetic behavior. This be- havior is characterized by zero coercivity and remanence at room temperature. The superparamagnetic properties of FSM-Zeo enable it to be easily separated from the dye solution using a magnet after the adsorption process. This magnetic separation capability has been reported in previ- ous studies.17,18 Figure 3b presents the FTIR spectra of Z, FSM-Zeo, FSM-Zeo after the adsorption of MV-2B, DB-22, and MV- 2B/DB-22. In the spectrum of Z, the peaks observed at 3395 cm–1 and 1632 cm–1 can be attributed to the hydrox- yl (OH) groups present on the surface of clinoptilolites. These peaks indicate the presence of adsorbed water mole- cules.8,11,19 The absorption peak at 1025 cm–1 corresponds to the stretching vibration of silicate groups in the struc- ture of Z.11,19 The absorption peaks at 450 cm–1 and 790 cm–1 indicate the vibrations of Si-O or Al-O bonds in the clinoptilolite structure.11,19 Similar peaks to Z were observed after the synthesis of FSM-Zeo, indicating a favorable interaction between starch and Fe3O4 in the FSM-Zeo structure. The peak at 605 cm–1 in FSM-Zeo corresponds to the Fe-O bond.19 Fig- Table 1. Results of the EDS analysis Z FSM-Zeo Element Weight (%) Weight (%) O 50.3 30.75 Si 37.0 9.4 Al 6.7 2.33 Ca 2.4 0.84 K 1.7 0.72 Mg 1 3.08 Fe – 27.92 Cl – 7.62 Na – 13.40 S – 3.93 Fig. 3. (a) VSM analysis of FSM-Zeo at room temperature (b) FTIR analysis Fig. 2. (a) XRD analysis of Z and (b) FSM-Zeo 127Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... ure 3b demonstrates that there are no significant changes in the peaks of FSM-Zeo before and after the adsorption of dyes. However there are changes in the peak intensities. The variations in the intensity of the bands after the ad- sorption of dyes onto FSM-Zeo may indicate the interac- tion of the dyes with the FSM-Zeo structure.11 3. 2. Effect of FSM-Zeo Amount In the study, the effect of FSM-Zeo amount on the removal of MV-2B and DB-22 was investigated at 20 mg/L initial dye concentration, 22 °C temperature, original pH and 60 min contact time. Figure 4a shows the results. The removal of MV-2B and DB-22 increased from 45.5% and 20.8% to 62.2% and 61% with increasing FSM-Zeo amount from 0.1 g/100 mL to 0.4 g/100 mL respectively. After 0.4 g/100 mL, the removal rates of MV-2B and DB-22 slight- ly decreased to 61.2% and 60.2% respectively. Therefore, the optimum adsorbent amount was determined to be 0.4 g/100 mL under the studied conditions. The increase in removal rate with the increasing amount of adsorbent can be attributed to the availability of more active sites and a sufficient specific surface area for the adsorption of dye molecules.10,20 However, beyond the optimum adsorbent amount, there is no significant change in the removal of dyes. This could be due to the accumula- tion of the adsorbent particles, which may hinder the ac- cessibility of dye molecules to active sites, resulting in a de- crease in the active surface area available for adsorption.21 3. 3. Effect of Initial pH and Temperature The effect of initial dye solution pH was examined in the range of 3.5–9. The original pH of the MV-2B and Fig. 4. (a) Effect of FSM-Zeo amount on removal rate (initial dye concentration: 20 mg/L, temperature: 22 °C, initial pH: original, contact time: 60 min), (b) effect of pH (initial dye concentration: 20 mg/L, temperature: 22 °C, FSM-Zeo amount: 0.4 g/100 mL, contact time: 60 min), (c) zeta potential of FSM-Zeo (d) effect of tempera- ture (initial dye concentration: 20 mg/L, pH: original, FSM-Zeo amount: 0.4 g/100 mL, contact time: 60 min), (e) effect of ionic strenght (initial dye concentration: 20 mg/L, pH: original, FSM-Zeo amount: 0.4 g/100 mL, contact time: 60 min, temperature: 22 °C) 128 Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... DB-22 solutions were 5.8 and 7.5 respectively. The pH of the dye solution was adjusted before starting the ex- periment. It was not controlled during the experiment. Figure 4b shows the effect of pH on dye removal. MV-2B removal was found to be 60.7%, 62.2%, 60.3%, and 65.3 at solution pH 3.5, 5.8, 7.5 and 9 respectively. There was no significant change on the removal rate of MV-2B between pH 3.5–7.5. At pH 9 removal rate of MV-2B increased to 65.3%. On the other hand, removal of DB-22 increased from 39.2% and 55.8% to 61% and 61.5 % with increasing pH from 3.5 to 9. The point of zero charge (pHpzc) refers to the pH at which the surface’s electrical charge density becomes zero. In the adsorption process, the pHpzc value provides in- formation about the electrostatic interaction between the surface and solute.22 When medium pH > pHpzc, surface of the adsorbent has negative charges while when medi- um pH < pHpzc, the adsorbent surface has positive surface charges.22,23 Figure 4c displays the Zeta potential results of FSM-Zeo, indicating that the pHpzc value for FSM-Zeo was determined to be 3.5. Adsorption of MV-2B and DB-22 on the FSM-Zeo surface can be explained by various mechanism such as pore saturation, electrostatic interaction between the ad- sorbent surface and dye molecules, hydrogen bonding, and π–π interactions between functional grups of the adsorbent and dye molecules.1,24 One of the effective mechanisms in removing MV-2B and DB-22 is the presence of pores and porosity in the adsorbent. SEM analysis confirms the presence of pores, providing sites for the adsorbing MV- 2B and DB-22 molecules, the removal of these dyes might occur through the pore saturation mechanism.1 The mech- anism of MV-2B and DB-22 dye adsorption at different pH values may be explained as follows. MV-2B molecules dissociate in water, forming positively charged molecules. At pH > pHpzc, an electrostatic attraction occurs between the positively charged MV-2B molecules and the negative- ly charged surface of FSM-Zeo. Between pH 3.5 and 7.5, the adsorption rate of MV-2B shows no significant change. As the pH increases to 9, the concentration of OH– ions increases, leading to more dominant role for electrostatic interaction.1 Below pH 3.5, the high concentration of H+ ions increases the competition between dye molecules and H+ ions for adsorption onto the surface of the adsorbent.11 π–π interaction, hydrogen bonding and pore saturation may play a significant role in the adsorption of MV-2B at pH values below 3.5.1 On the other hand, DB-22 is an an- ionic dye consisting of negatively charged molecules. At pH > pHpzc, the negative charges on the surface of the ad- sorbent result in no electrostatic interaction between DB- 22 and the surface of FSM-Zeo. However, above the pHpzc, π–π interaction and the hydrogen bonding with FSM-Zeo may play dominant roles in the adsorption of DB-22.24 The effect of temperature on the adsorption of MV- 2B and DB-22 was examined at temperatures of 22, 30, 40 and 50 °C at 20 mg/L initial dye concentration, original pH, 0.4 g/100 mL adsorbent amount and 60 min contact time. Figure 4d shows the results. For DB-22, there was no significant change in its removal with increasing solution temperature from 22 °C to 40 °C. The removal rates of DB- 22 were found to be 64.7%, 63.7%, and 65% at 22 °C, 30 °C, and 40 °C respectively. However, at 50 °C, the removal of DB-22 increased to 80.2%. A similar observation was reported by Fındık25 in a study on the adsorption of DB-22 using synthesized magnetic kaolin supported zinc ferrite. The decrease in solution viscosity with increasing tem- perature leads to enhanced mobility of the dye molecules, resulting in more interactions between the dye molecules and the free active sites on the FSM-Zeo surface. On the other hand, the removal of MV-2B remained nearly constant with increasing temperature. The removal rates of MV-2B were found to be 62.2% , 66.5%, 61.7% and 65.8% at 22 °C, 30 °C, 40 °C and 50 °C respectively. The stable performance efficiency observed between 22–50 °C. In literature, Chung et al.26 obtained similar results for the adsorption of methylene blue. They reported that the near- ly constant removal rate indicated the adsorptive stability of the adsorbent under various temperature changes. Sim- ilarly, Kanwall et al.27 investigated the effect of temperature on the adsorption of crystal violet using native clay and Cl pretreated clay, and they observed no significant change in crystal violet adsorption with increasing temperature. The removal rate remained unchanged at higher temperature due to stability of the adsorbent. Overall, the effect of tem- perature on the adsorption of MV-2B and DB-22 varied. While DB-22 exhibited increased removal at higher tem- peratures, MV-2B showed a stable removal rate across the temperature range studied. 3. 4. Effect of Co-Existing Ions In the study, the effect of ionic strength on the re- moval of dye was investigated using NaCl and Na2SO4 at different amounts. Figure 4e shows the results. For MV-2B, the removal rate decreased with increasing NaCl amount. However, the addition of 0.1 g/100 mL Na2SO4 did not sig- nificantly affect the removal rate of MV-2B. At 0.4 g/100 mL and 0.7 g/100 mL Na2SO4 amounts, the removal rate of MV-2B was lower than without Na2SO4. This observation suggests that there is competition between ions and dye molecules to be adsorbed onto the FSM-Zeo surface. This phenomenon can affect the availability of active sites for dye adsorption.11 On the other hand, the removal of DB-22 decreased with the addition of 0.1 g/100 mL NaCl or Na2SO4, but the removal rate increased with increasing salt concentration. DB-22 removal rates were found to be 83.5% with addition of 0.4 g/100 mL NaCl and 61% without salt. In literature, Olesegun and Mohallem28 observed that the adsorption of Congo red increased with increasing NaCl concentration. They suggested that the increase in removal rate may be attributed to enhanced hydrophobic interactions, which 129Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... lead to the shielding of intermolecular repulsion between the dye and the nanocomposite. The increase in removal rate with increasing salt concentration offers an advantage in treating textile wastewater that contains high levels of salt. Overall, it can be concluded that FSM-Zeo effectively removes DB-22 in the presence of NaCl and Na2SO4. 3. 5. Effect of Contact Time and Initial MV-2B Concentration Figure 5 shows effect of the contact time and initial dye concentration on the removal of MV-2B and DB-22 using 0.4 g/100 mL FSM-Zeo amount at 22 ° C and origi- nal pH. The initial dye concentration was varied between 10–40 mg/L. The removal rates of MV-2B and DB-22 de- creased from 73 % and 72 % to 51 % and 40.7% respec- tively, as the initial concentration increased from 10 mg/L to 40 mg/L. This decrease in removal efficiency can be at- tributed to the rapid saturation of active sites on FSM-Zeo and a reduction in the number of available active sites. The limited number of active sites on FSM-Zeo leads to a de- crease in dye removal efficiency.1,11 The effect of contact time on dye removal was stud- ied over a range of 5–90 minutes. The results show that initially, the removal of dyes was rapid, indicating the pres- ence of suitable and unsaturated active sites for adsorption. However, after a contact time of 45 minutes, the removal rate reached a plateau, and further contact time did not significantly affect the removal efficiency. This observation is consistent with the findings reported by Foroutan et al.11 in their study on the adsorption of methyl violet, crys- tal violet, and methylene blue onto clinoptilolite/starch/ CoFe2O4. 3. 6. Adsorption Isotherms The adsorption isotherm determines the interaction between the adsorbent and the adsorbate. That is, it is used to examine the relationship between the adsorbent and the dye it adsorbs.11,29 The common isotherm models such as Langmuir, Freundlich and Temkin were applied to analyze adsorption of MV-2B and DB-22 onto FSM-Zeo in the Fig. 5. Effect of initial concentration on the removal of (a) MV-2B (b) DB-22 (pH: original, FSM-Zeo amount: 0.4 g/100 mL, temperature: 22 °C) range of 10–40 mg/L initial concentrations while the other factors were kept constant (temperature: 22 °C, initial pH: original, FSM-Zeo amount: 0.4 g/100 mL). The linear form of the Langmuir, Freundlich and Temkin models, and as well as the separation factor (RL) for Langmuir isotherm are given in Table 2.18,29,30 When fitting different models to experimental data, it may not be sufficient to use only the R2 value to compare these models. Evaluation can be made by using the sum of the squared errors (SSE) and the R2 value together. SSE value can be calculated using Eq. 3.29 (3) where qcal and qexp are the calculated and experimental values of q, respectively. The calculated parameters of the isotherm models for the adsorption of MV-2B and DB-22 are presented in Table 2. The Freundlich model provided the best fit for the adsorption of MV-2B with the highest correlation coef- ficient (R2 = 0.9921) and lowest SSE value. On the other hand, the Langmuir isotherm model exhibited good fitting for the adsorption of DB-22 with a R2 value of 0.9695 and SSE value of 0.27. Figure 6a and 6b show the linear forms of the Freundlich and Langmuir isotherms for the adsorp- tion of MV-2B and DB-22, respectively. These results indicate that the adsorption of MV- 2B occurs on a heterogeneous surface while the adsorp- tion of DB-22 takes place on a homogeneous surface. The RL values for the adsorption process of MV-2B and DB-22 dyes were in the range of 0–1, which indicates that the adsorption process can be desirable.4 The KL values for the MV-2B and DB-22 adsorption onto FSM- Zeo were determined as 0.117 L/mg and 0.276 L/mg, respectively. The higher KL value for DB-22 suggests a stronger affinity between DB-22 molecules and the FSM-Zeo surface compared to MV-2B.4 The Freundlich coefficient (n) for MV-2B and DB-22 adsorption were found to be 2.03 and 3.42, respectively. The value of n was greater than 1, it indicated that adsorption was physical and desirable.4,5 130 Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... 3. 7. Kinetic Analysis The adsorption kinetic analysis of MV-2B and DB- 22 onto FSM-Zeo was performed using common kinetic models such as pseudo first order (Ps.FO), pseudo second order (Ps.SO) and intraparticle diffusion models. The lin- ear forms of these models21 and the corresponding kinetic parameters obtained from the experimental data are pre- sented in Table 3. The regression coefficient (R2) value in- dicates the agreement between the calculated qe values and the experimental qe values obtained from the experiments. A higher R2 value suggests a better fit of the kinetic model to the adsorption process. The results of the Ps.SO model and intraparticle diffusion model for MV-2B and DB-22 adsorption are shown in Figure 7. According to Table 3, the Ps.SO model exhibited higher correlation coefficients (R2 : 0.993–0.999 for MV- 2B and R2 : 0.988–0.998 for DB-22) compared to the Ps.FO model (R2 : 0.715–0.939 for MV-2B and R2 : 0.720–0.997 for DB-22). Additionally, there was a significant deviation between the calculated and experimental q values in the Ps.FO model, while the qe,exp and qe,cal values were closer in the Ps.SO model. These results indicate that the Ps.SO model provided the best fit for describing the adsorption kinetics of MV-2B and DB-22 on FSM-Zeo. In the Ps.SO model, the rate controlling step was determined to be a chemical reaction.18 As shown in Figure 7c and 7d, the relation of qt against t0.5 was linear. These results showed that the adsorption pro- cess was controlled by intraparticle diffusion mechanism. However, a deviation from linearity was observed within the 30–40 mg/L range, suggesting that adsorption on the layers surrounding the adsorbent influenced the overall ad- sorption process. Consequently, the intraparticle diffusion mechanism alone cannot fully account for the adsorption process at high initial dye concentrations.21 Table 2. Isotherm parameters for the adsorption of MV-2B and DB-22 (pH: original, FSM-Zeo amount: 0.4 g/100 mL, temperature: 22 °C) Isotherm Linear form of Constants V-2B DB-22 isotherm model Langmuir qmax (mg/g) 7.16 4.43 KL (L/mg) 0.117 0.276 RL 0.18–0.43 0.083–0.24 R2 0.9777 0.9695 SSE 0.12 0.27 Freundlich n 2.03 3.42 Kf (mg/g) 1.178 1.574 R2 0.9921 0.8513 SSE 0.056 0.33 Temkin qe = β1lnKT + β1lnCe β1 1.625 0.845 KT (L/mg) 1.069 4.279 R2 0.9733 0.8607 SSE 0.139 0.28 Fig. 6. Linear form of the (a)Freundlich isotherm for MV-2B and (b) Langmuir isotherm for DB-22 onto FSM-Zeo (pH: original, temperature: 22 °C, FSM-Zeo amount: 0.4 g/100 mL) 131Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... 3. 8. Adsorption Studies on the Binary System To investigate the adsorption of dyes in binary systems, a dye solution was prepared using DB-22/MV-2B with var- ying initial concentrations ratios such as 10/10, 10/20 and 20/10. The adsorption experiments for the binary system were performed using 0.4 g/100 mL of FSM-Zeo amount, at 22 °C and the original solution pH. The experimental studies followed the same procedure as for the single dye solution. In binary systems the concentration of each dye was calculated using equations 4 and 5.17,31 (4) (5) Table 3. Kinetic models constants for adsorption of MV-2B and DB-22 using the adsorbent FSM-Zeo (FSM-Zeo amount: 0.4 g/100 mL, pH: original, temperature: 22 °C) Dye Initial (qe)Exp Pseudo-first order Pseudo-second order Intraparticle diffusion Conc. ln (qe–qt) = ln qe – k1 t (t/qt) = (1/k2qe2) + (t/qe) qt = kpt1/2 + I (mg/L) k1 qe R2 k2 qe R2 kp I R2 MV-2B 10 2.06 0.0882 0.945 0.896 0.220 2.111 0.993 0.0606 1.557 0.880 20 3.11 0.0647 1.832 0.939 0.069 3.264 0.999 0.1604 1.766 0.926 30 4.30 0.1135 8.950 0.715 0.022 4.769 0.994 0.3342 1.438 0.957 40 5.05 0.0975 6.758 0.783 0.026 5.459 0.997 0.3368 2.198 0.948 DB-22 10 2.04 0.0915 3.465 0.727 0.0349 2.316 0.988 0.1745 0.524 0.963 20 3.22 0.0318 2.103 0.997 0.0246 3.532 0.994 0.2570 0.901 0.981 30 3.29 0.0368 2.625 0.975 0.0155 3.858 0.998 0.3229 0.466 0.936 40 4.05 0.0343 2.676 0.987 0.0203 4.450 0.995 0.3253 1.155 0.963 Fig. 7. Ps.SO model for the adsorption of (a) MV-2B, (b) DB-22 onto FSM-Zeo, intraparticle diffusion model for the adsorption of (c) MV-2B, (d) DB-22 onto FSM-Zeo 132 Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... where A1 and A2 represent the total absorbance at wave- lengths λ1max and λ2max and kA1, kB1, kA2, kB2 are the calibra- tion constants for components A and B at λ1max and λ2max. The effect of both DB-22 and MV-2B dyes in binary system on removal performance of FSM-Zeo were deter- mined using the ratio of adsorption capacities (Rq) as fol- lows:32,33 Rq = qb,i/qm,i (6) where qb,i is the adsorption capacity for dye i in the binary system (mg/g) and qm,i is the adsorption capacity for dye i with the same initial concentration in a mono-com- ponent system. If Rq > 1, the adsorption of component i was enhanced by the other pollutant; if Rq = 1, the adsorp- tion of component i was not affected by other pollutant; if Rq < 1, the adsorption of component i was suppressed by the other pollutant. The effect of initial concentration on the removal of single dye solution and binary dye solution are presented in Table 4. The removal of DB-22 in binary dye solution was higher than the removal of DB-22 in single dye solu- tion. The removal of DB-22 in binary dye solution found to be 85.6%, 97.9% and 80.5% at 10/10, 10/20 and 20/10 ini- tial concentration of DB-22/MV-2B solution respectively. The ratio of adsorption capacities for DB-22 were higher than 1 at the studied conditions There was a synergistic effect due to the interaction between DB-22 and MV-2B, which promotes the adsorption of DB-22 on FSM-Zeo in binary systems. It means adsorption of DB-22 enhanced in the presence of MV-2B. On the other hand, removal of MV-2B in binary dye solution found to be 59.9%, 61.4% and 67.3% at 10/10, 10/20 and 20/10 initial concentration of DB-22/MV-2B solution respectively. At 10/20 initial concentration of DB- 22/MV-2B, the ratio of adsorption capacity for MV-2B was 0.99. It was very close to one and the presence of DB-22 in the solution did not effect the adsorption of MV-2B. Rq was 0.73 and 0.82 at 10/10 and 20/10 initial concentration of DB-22/MV-2B respectively. The Rq value showed that the presence of DB-22 suppressed the MV-2B adsorption at 10/10 and 20/10 initial concentration of DB-22/MV-2B. In conclusion, considering the removal rates and Rq values in binary systems, it can be concluded that FSM- Zeo exhibited suitability for binary solutions under the investigated conditions. Furthermore, the results suggest that the adsorption of the anionic dye DB-22 from binary solutions is notably improved compared to the single-com- ponent system. In their study, Nicola et al.17 investigated the removal of dyes from solutions containing both anion- ic and cationic dyes using magnetic mesoporous silica. In their work conducted at pH 4.5 and 6.3, they observed a decrease in the removal of the anionic dye Congo red (CR) and the cationic dye methylene blue (MB) compared to the single-component system. When comparing these results with the findings of this study, the obtained outcomes are advantageous for systems containing two dyes and for tex- tile wastewater containing a high number of dyes. 3. 9. Comparison of FSM-Zeo with Other Adsorbents and Cost Analysis The maximum adsorption capacity of the adsorbent depends on various parameters such as active surface sites, type of pollutants, modification methods, and precursor material of adsorbent.5 Numerous studies have recent- ly focused on developing low-cost adsorbents with high adsorption capacities. In this study, zeolite and starch bi- opolymer were utilized as cost-effective materials, while Fe3O4 and MgO were incorporated to gain magnetic prop- erties and enhance the adsorption capacity of zeolite. The adsorption capacity of FSM-Zeo was compared to other adsorbents in existing literature (Table 5). FSM-Zeo ex- hibited a maximum adsorption capacity of 7.16 mg/g for MV-2B adsorption and 4.43 mg/g for DB-22 adsorption. Although magnetic property has gained with addition of Fe3O4 to zeolite, the maximum adsorption capacity re- mained relatively low. Moreover, it was observed that the higher amounts of adsorbent led to a lower adsorption ca- pacities. Consequently, this study was limited to laborato- ry-scale experiments, and no investigations were carried out using real wastewater samples. Due to the low adsorp- tion capacity of FSM-Zeo, it has no potential practical us- age. However, the removal of DB-22 in binary dye solu- Table 4. Removal of dyes and ratio of adsorption capacities in binary system Initial dye Removal of dye Rq concentration (%) (mg/L) DB-22 MV-2B DB-22 MV-2B DB-22 MV-2B 10 0 72.0 – – – 20 0 65.0 – – – 0 10 – 73.0 – – 0 20 – 61.7 – – 10 10 85.6 59.9 97.9 1.05 0.73 10 20 61.4 1.2 0.99 20 10 80.5 67.3 1.25 0.82 133Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... tions demonstrated higher efficiency compared to single dye solutions, suggesting its usefulness in treating waste- water containing multiple dyes. Furthermore, the removal efficiency of DB-22 in single dye solution increases with an increase in the salt concentration, particularly with the addition of NaCl. Considering that NaCl is widely used in the textile industry, this is seen as an advantage. Further research can be conducted to improve the adsorption ca- pacity of FSM-Zeo and reduce the required amount. The data obtained from this research may prove valuable to other researchers in the field. NaCl amount, Na2SO4 amount, temperature, and initial pH of the solution for the adsorption of DB-22 and MV- 2B. The results indicated that the adsorption of DB-22 and MV-2B followed the pseudo second order model. Iso- therm studies showed that the adsorption of MV-2B and DB-22 onto FSM-Zeo followed Freundlich and Langmuir models, respectively. Notably, the study observed a trend of low adsorption capacity with a high adsorbent amount. In the binary-dye system, the presence of MV-2B en- hanced the adsorption of DB-22, leading to higher removal efficiency compared to the single-dye solution. Consider- Table 5. Comparison of the FSM-Zeo with other adsorbents Adsorbent Dye Adsorbent amount (g/L) Contact time (min) qmax (mg/g) References Clay/starch/Fe3O4 Methyl violet 1.5 150 29.67 10 CLN/starch/CoFe2O4 Methylene blue 1.2 60 29.62 11 Methyl violet 1.2 60 27.72 11 Crystal violet 1.2 60 30.92 11 ZIF-8 Methylene blue 0.5 120 7.88 18 Erichrome blackT 0.5 120 8.1 18 ACL/Fe3O4 Crystal violet 1.25 60 35.21 23 FSM-Zeo MV-2B 4 90 7.16 This study DB-22 4 90 4.43 This study The cost of FSM-Zeo includes raw materials, chem- icals, and energy costs. This study aimed to minimize ma- terial costs by utilizing zeolite, a low-cost material. The magnetic adsorbent was synthesized using the co-precip- itation method. When it comes to large-scale production of adsorbents, it is crucial to employ an easy preparation method and cost-effective materials in order to keep the adsorbent cost low. A recent study conducted by Augusto et al.34 focused on the design, development, and economic analysis of large-scale plants for nanomagnetic materials used in environmental applications. Based on their liter- ature review, they noted that the cost of magnetic nano- particles can vary depending on their specific application, characteristics, and presentation form. For commercially available nanoparticles without any functionalization, the cost can range from $380 / kg (for iron oxides, including magnetite and maghemite) to $2255/kg (for nZVI). 4. Conclusion In this study, a magnetic adsorbent called FSM-Zeo was synthesized and used for the adsorption of Direct black 22 (DB-22) and Methyl violet 2B (MV-2B) from both single and binary solutions. The structure of FSM- Zeo was characterized through analyzes such as BET, FT- IR, SEM, EDS, XRD, Zeta potential and VSM. The study investigated various adsorption parameters, including contact time, initial dye concentration, FSM-Zeo amount, ing the dye removal rate and the ratio of adsorption capac- ities (Rq), it can be concluded that FSM-Zeo was effective in removing the DB-22/MV-2B mixture. Moreover, the re- moval efficiency of DB-22 in the single system increased with an increase in salt concentration, particularly with the addition of NaCl, presenting an advantageous aspect given the widespread use of NaCl in the textile industry. Further research is recommended to enhance the adsorption ca- pacity of FSM-Zeo and reduce the required amount. The findings obtained from this research will contribute valu- able insights to researchers in the field of dye adsorption. Acknowledgement The author thanks to Hitit University for their finan- cial support of this project under contract of MUH19001.21.003. 5. References 1. A. Ahmadi, R. Foroutan, H. Esmaeili, S. J. Peighambardoust, S. Hemmati, B. Ramavandi, Mat. Chem. Phys. 2022, 284, 126088. DOI:10.1016/j.matchemphys.2022.126088 2. K. K. Kefeni, B. B. Mamba, T. A. M. Msagati, Sep. Pur. Techn. 2017, 188, 399–422. DOI:10.1016/j.seppur.2017.07.015 3. A. Kausar, M. Iqbal, A. Javeda, K. Aftab, Z. Nazli, H. N. Bhatti, S. Nouren, J. Molec. Liq. 2018, 256, 395–407. DOI:10.1016/j.molliq.2018.02.034 134 Acta Chim. Slov. 2024, 71, 123–134 Fındık: Removal of Methyl Violet 2B and Direct Black 22 ... 4. R. Foroutan, S. J. Peighambardoust, Z. Esvandi, H. Khatooni, B. Ramavandi, J. Env. Chem. Eng. 2021, 9, 104752. DOI:10.1016/j.jece.2020.104752 5 . Z. Esvandi, R. Foroutan, S. J. Peighambardoust, A. Akbari, B. Ramavandi, Surf. Interfa. 2020, 21, 100754. DOI:10.1016/j.surfin.2020.100754 6. S. S. Hosseini, A. Hamadi, R. Foroutan, S. J. Peighambardoust, B. Ramavandi, J. Water Proc. Eng. 2022, 48, 102911. DOI:10.1016/j.jwpe.2022.102911 7. Q. Yuan, H. Huang, W. Wang, G. Zhou, L. Luo, X. Zeng, Y. Liu, J. Allo. Comp. 2020, 854, 153889. 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Todea, R. Ianos, C. Pscurariu, J. Chem. 2018, 6249821. DOI:10.1155/2018/6249821 32. J-H. Deng, X-R. Zhang, G-M. Zeng, J-L. Gong, Q-Y. Niu, J. Liang, Chem. Eng. J. 2013, 226, 189–200. DOI:10.1016/j.cej.2013.04.045 33. R. Tovar-Gómez, D. A. Rivera-Ramírez, V. Hernández-Mon- toya, A. Bonilla-Petriciolet, C. J. Durán-Valle, M. A. Mon- tes-Morán, J. Haz. Mat. 2012, 199–200, 290–300. DOI:10.1016/j.jhazmat.2011.11.015 34. P. A. Augusto, T. Castelo-Grande, D. Vargas, A. Pascual, L. Hernández, A. M. Estevez, D. Barbosa, Mater. 2020, 13, 2477. DOI:10.3390/ma13112477 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Študija je osredotočena na pripravo in karakterizacijo magnetnega zeolita (FSM-Zeo) z uporabo škroba, magnezijevega oksida in Fe3O4. Za oceno lastnosti FSM-Zeo so bile izvedene različne analize, vključno z BET, FTIR, SEM, EDS, XRD, Zeta potencialom in VSM. Adsorpcijska kapaciteta FSM-Zeo je bila raziskana za metil vijolično (MV-2B) in neposredno črno 22 (DB-22) v singularnih in binarnih raztopinah barvil. V singularnem sistemu so bili preučeni ključni parametri, kot so količina adsorbenta, začetna koncentracija barvila, kontaktni čas, temperatura, začetni pH in ionska moč. Ki- netične in izotermne študije so pokazale, da je adsorpcija DB-22 in MV-2B sledila modelu psevdodrugega reda. Poleg te- ga sta bila Freundlichov in Langmuirjev model potrjena za adsorpcijo MV-2B oziroma DB-22 na FSM-Zeo. V binarnem sistemu je prisotnost MV-2B povečala adsorpcijo DB-22, kar je povzročilo večjo odstranitev v primerjavi z raztopino z enim barvilom. Opažen je bil sinergistični učinek zaradi interakcije med DB-22 in MV-2B, ki spodbuja adsorpcijo DB-22 na FSM-Zeo. 135Acta Chim. Slov. 2024, 71, 135–142 Chen et al.: Syntheses, Crystal Structures and Antimicrobial ... DOI: 10.17344/acsi.2023.8588 Scientific paper Syntheses, Crystal Structures and Antimicrobial Activity of Zinc(II) Complexes Derived from 5-Bromo-2- (((2-piperazin-1-yl)ethyl)imino)methyl)phenol Yin-Bing Chen, Xiao-Yang Qiu*, Meng-Yuan Xu, Fei-Yu Qi, Xin He, Chen Wu and Shu-Juan Liu Ningbo Key Laboratory of Agricultural Germplasm Resources Mining and Environmental Regulation, College of Science & Technology, Ningbo University, Ningbo 315315, P. R. China * Corresponding author: E-mail: xiaoyang_qiu@126.com Received: 12-17-2023 Abstract Three new zinc(II) complexes, [ZnCl2L]·CH3OH (1), [ZnClL(NCS)]·2CH3OH·0.5H2O (2), and [ZnL(NCS)2]·CH3OH·H2O (3), where L is the zwitterionic form of 5-bromo-2-(((2-piperazin-1-yl)ethyl)imino)methyl)phenol, NCS is thiocyanate anion, were facile prepared by reaction of different molar ratio of L, zinc chloride and ammonium thiocyanate in meth- anol. The complexes were characterized by IR and UV-Vis spectroscopy. Detailed structures of the three complexes were confirmed by single crystal X-ray determination. The Zn atoms in the complexes are in tetrahedral coordination. The Schiff base ligand coordinates to Zn atom through phenolate oxygen atom and amino and imino nitrogen atoms. The remaining two sites are occupied by two Cl for 1, one Cl and one NCS for 2, and two NCS for 3. The compounds show significant antimicrobial activities. Keywords: Schiff base, zinc complex, crystal structure, antimicrobial activity 1. Introduction The preparation of metal complexes with new structures and biological activities is a hot topic in bio- inorganic and coordination chemistry. Among various ligands, Schiff bases due to their facile synthesis and in- teresting biological activities, have received particular attention in the construction of metal complexes.1 Zinc complexes with Schiff base ligands are reported to have various biological activities, and are used as excellent alternatives for classic organic type antifungal, antibac- terial and antitumor agents.2 Despite the large number of metal complexes with antibacterial activities, it is necessary to prepare new zinc complexes with high ac- tivity. It has been reported that compounds with elec- tron-withdrawing groups can improve their antimicro- bial ability.3 The compounds with chloro, fluoro, iodo and bromo groups have shown remarkable antimicrobi- al activities.4 Thiocyanate anion is readily coordinate to metal atoms to form complexes with interesting struc- tures.5 Recently, we have reported some Schiff base complexes with interesting biological activities.6 More- over, the complexes with ligands containing piperazine group have effective antibacterial activities.7 In pursuit of new Schiff base complexes with potential antimicrobi- al activity, three new zinc complexes [ZnCl2L]·CH3OH (1), [ZnClL(NCS)] · 2CH3OH · 0.5H2O (2), and [Zn- L(NCS)2] · CH3OH · H2O (3), where L is the zwitterionic form of 5-bromo-2-(((2-piperazin-1-yl)ethyl)imino)me- thyl)phenol (Scheme 1), NCS is thiocyanate anion, and their antimicrobial activities are present. The compounds show significant antimicrobial activities. Scheme 1. The Schiff base L. 136 Acta Chim. Slov. 2024, 71, 135–142 Chen et al.: Syntheses, Crystal Structures and Antimicrobial ... 2. Experimental 2. 1. Materials and Methods 4-Bromosalicylaldehyde, N-(2-aminoethyl)pipe- ra zine, zinc chloride and ammonium thiocyanate were obtained from Sigma-Aldrich. All other chemicals were commercial obtained from Xiya Chemical Co. Ltd. The Schiff base L was prepared according to the literature method.8 Elemental analyses of C, H and N were carried out in a Perkin-Elmer automated model 2400 Series II CHNS/O analyzer. FT-IR spectra were obtained on a Perkin-Elmer 377 FT-IR spectrometer with samples prepared as KBr pellets. UV-Vis spectra were obtained on a Lambda 35 spectrometer. Molar conductivities of the complexes in DMSO solutions (10–3 M) at room temperature were measured using a Systronic model 303 direct reading conductivity meter. 2. 2. Synthesis of [ZnCl2L]·CH3OH (1) HL (0.10 mmol, 31 mg) and zinc chloride (0.10 mmol, 14 mg) were mixed in methanol (20 mL). The mixture was stirred at 25 °C for 20 min to give a colorless solution. Block single crystals were formed upon slow evaporation. The crystals were obtained by filtration. Yield: 32 mg (67%). Anal. calc. for C14H22BrCl2N3O2Zn: C, 34.99; H, 4.61; N, 8.74; found: C, 35.12; H, 4.55; N, 8.66%. Characteristic IR data (cm–1): 1633 (νC=N), 1577, 1529, 1452, 1406, 1351, 1289, 1268, 1185, 1141, 1070, 1019, 910, 855, 805, 763, 733, 610, 596, 534, 479, 449. UV-Vis data (MeOH, λmax (nm), ε (L mol–1 cm–1)): 227, 2.32 × 103; 245, 1.91 × 103; 267, 1.23 × 103; 366, 6.35 × 102. Molar conductance (10–3 mol L–1 in DMSO): 32 Ω–1 cm2 mol–1. 2. 3. Synthesis of [ZnClL(NCS)]·2CH3OH·0.5H2O (2) HL (0.10 mmol, 31 mg), zinc chloride (0.10 mmol, 14 mg) and ammonium thiocyanate (0.10 mmol, 7.6 mg) were mixed in methanol (20 mL). The mixture was stirred at 25 °C for 20 min to give a colorless solution. Block single crystals were formed upon slow evaporation. The crystals were obtained by filtration. Yield: 35 mg (64%). Anal. calc. for C16H27BrClN4O3.5SZn: C, 35.31; H, 5.00; N, 10.29; found: C, 35.22; H, 5.11; N, 10.20%. Characteristic IR data (cm–1): 2088 (νNCS), 1632 (νC=N), 1581, 1523, 1471, 1452, 1411, 1352, 1293, 1247, 1187, 1139, 1065, 1049, 1021, 915, 843, 788, 760, 730, 677, 620, 606, 585, 532, 465. UV-Vis data (MeOH, λmax (nm), ε (L mol–1 cm–1)): 227, 2.41 × 103; 245, 2.09 × 103; 273, 1.03 × 103; 367, 6.96 × 102. Molar conductance (10–3 mol L–1 in DMSO): 28 Ω–1 cm2 mol–1. 2. 4. Synthesis of [ZnL(NCS)2]·CH3OH·H2O (3) HL (0.10 mmol, 31 mg), zinc chloride (0.10 mmol, 14 mg) and ammonium thiocyanate (0.20 mmol, 15 mg) were mixed in methanol (20 mL). The mixture was Table 1. Crystallographic and refinement data for the complexes Complex 1 2 3 Formula C14H22BrCl2N3O2Zn C16H27BrClN4O3.5SZn C16H24BrN5O3S2Zn Formula weight 480.53 544.21 543.80 Crystal system Monoclinic Monoclinic Monoclinic Space group P21/n P21/c P21/c a (Å) 7.7284(12) 9.3833(13) 9.5085(12) b (Å) 21.3267(16) 20.2828(18) 20.7468(15) c (Å) 11.4218(13) 11.7620(15) 11.8697(13) α (º) 90 90 90 β (º) 90.00(2) 95.904(2) 97.085(1) γ (º) 90 90 90 V (Å3) 1882.6(4) 2226.7(5) 2323.7(4) Z 4 4 4 Dcalc (g cm–3) 1.695 1.623 1.554 µ(Mo Kα) (mm–1) 3.721 3.137 2.981 F(000) 968 1108 1104 Measured reflections 6661 11590 12096 Unique reflections 2533 4155 4325 Observed reflections (I ≥ 2σ(I)) 1512 2292 2329 Parameters 210 260 258 Restraints 0 15 12 GOOF 0.0878 1.048 1.032 R1, wR2 [I ≥ 2σ(I)]a 0.0470, 0.1021 0.0746, 0.1676 0.0536, 0.1290 R1, wR2 (all data)a 0.0840, 0.1109 0.1461, 0.1996 0.1224, 0.1558 a R1 = ∑||Fo| – |Fc||/∑|Fo|, wR2 = {∑[w(Fo2 – Fc2)2]/∑[w(Fo2)2]}1/2 137Acta Chim. Slov. 2024, 71, 135–142 Chen et al.: Syntheses, Crystal Structures and Antimicrobial ... methanol (20 mL). The mixture was stirred at 25 °C for 20 min to give a colorless solution. Block single crystals were formed upon slow evaporation. The crystals were obtained by filtration. Yield: 27 mg (50%). Anal. calc. for C16H24BrN5O3S2Zn: C, 35.34; H, 4.45; N, 12.88; found: C, 35.43; H, 4.37; N, 12.75%. Characteristic IR data (cm–1): 2073 (νNCS), 1634 (νC=N), 1579, 1527, 1469, 1402, 1349, 1289, 1257, 1196, 1185, 1139, 1072, 1017, 910, 839, 795, 760, 730, 610, 543, 485, 447. UV-Vis data (MeOH, λmax (nm), ε (L mol–1 cm–1)): 228, 2.45 × 103; 245, 2.41 × 103; 277, 1.10 × 103; 367, 7.13 × 102. Molar conductance (10–3 mol L–1 in DMSO): 25 Ω–1 cm2 mol–1. 2. 5. X-ray Crystallography X-ray diffraction was done with a Bruker APEX II CCD area diffractometer equipped with Mo-Kα radiation (λ = 0.71073 Å). The collected data were reduced with SAINT.9 Multi-scan absorption correction was performed with SADABS.10 Structures of the three zinc complexes were solved by direct method, and refined against F2 by full-matrix least-squares method with SHELXTL.11 All non-hydrogen atoms were refined anisotropically. All hydrogen atoms were placed in calculated positions and constrained to ride on their parent atoms. The crystallographic data and refinement parameters for the complexes are listed in Table 1. 2. 6. Antimicrobial Aassay The three zinc complexes were assayed against bacteria strains Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas fluorescence using MH (Mueller-Hinton) medium. The compounds were also assayed against fungi Candida albicans and Aspergillus niger using RPMI-1640 medium. The MIC values were determined by a colorimetric method using MTT.12 A stock solution of the compound at concentration of 150 μg mL–1 in DMSO was prepared and graded quantities (75, 37.5, 18.8, 9.4, 4.7, 2.3, 1.2, and 0.59 μg mL–1), which were incorporated in specified quantity of the corresponding sterilized liquid medium. A specified quantity of the medium containing the compound was poured into micro-titration plates. Suspension of the microorganism was prepared to contain 1.0 × 105 cfu mL–1 and applied to micro-titration plates with serially diluted compounds in DMSO to be tested and incubated at 37 °C for 24 h and 48 h for bacteria and fungi, respectively. Then the MIC values were visually determined on each of the microtitration plates, 50 μL of PBS (phosphate buffered saline 0.01 mol L–1, pH = 7.4) containing 2 mg of MTT mL–1 was added to each well. Incubation was continued at room temperature for 4–5 h. The content of each well was removed and 100 μL solution of 95% isopropanol and 1 mol L–1 5% HCl was added to extract the dye. After 12 h of incubation at room temperature, the optical density was measured with a microplate reader at 550 nm. 3. Results and Discussion 3. 1. Synthesis and Characterization The three zinc complexes were facile prepared by reaction of the Schiff base ligand, zinc chloride and ammonium thiocyanate in molar ratio of 1:1:0, 1:1:1 and Scheme 2. The synthetic procedure for the complexes. 138 Acta Chim. Slov. 2024, 71, 135–142 Chen et al.: Syntheses, Crystal Structures and Antimicrobial ... 1:1:2, respectively in methanol (Scheme 1). Interestingly, complex 2 can be prepared by reaction of equimolar quantities of complex 1 with ammonium thiocyanate. Complex 3 can be prepared by reaction of equimolar quantities of complex 2 with ammonium thiocyanate, or 1:2 molar ratio of complex 1 with ammonium thiocyanate. Single crystals of the three complexes were obtained from their methanolic solution. Elemental analyses of the complexes are in accordance with their molecular structures determined by single crystal X-ray analysis. 3. 2. Spectroscopic Studies The intense absorptions at 1632–1634 cm–1 for the complexes are generated by the vibrations of the C=N bonds of the Schiff base ligands which are formed from the condensation reaction of 4-bromosalicylaldehyde and N-(2-aminoethyl)piperazine.13 The strong bands at 2088 cm–1 for complex 2 and 2073 cm–1 for complex 3 can be assigned to thiocyanate ligands.14 In the electronic spectra of the three complexes, the bands at 360‒370 nm are attributed to azomethine chromophore π→π* transition.15 The bands at higher energies (220‒230 and 240‒280 nm) are associated with benzene π→π* transition.15 3. 3. Structure Description of the Complexes The bond lengths and angles related to the Zn atoms for the three compounds are listed in Table 2. Molecular structures of the compounds are shown in Figures 1, 2 and 3, respectively. Compound 1 contains a [ZnCl2L] complex molecule and a methanol molecule of crystallization. Compound 2 contains a [ZnClL(NCS)] complex molecule, two methanol molecules and half water molecule of crystallization. Compound 3 contains a [ZnL(NCS)2] complex molecule, a methanol molecule and a water molecule of crystallization. The Zn atom in each complex is in trigonal bipyramidal coordination, with the equatorial plane defined by the imino nitrogen (N1) of the Schiff base ligand and two Cl or N atoms of the thiocyanate ligands, viz. Cl1 and Cl2 for 1, Cl1 and N4 for 2, N4 and N5 for 3. The two axial positions are occupied by the phenolate oxygen (O1) and amino nitrogen (N2) of the Schiff base ligands. The definition of the trigonal bipyramidal coordination is based on index facor τ (0.55 for 1 and 2, 0.66 for 3).16 The Schiff base acts as a tridentate ligand, chelating the Zn atom by generating one five and one six-membered rings with bite angles of 77.24(16)° and 90.00(16)° (1), 75.4(3)° and 90.2(3)° (2), and 75.9(2)° and 91.19(17)° (3). The bond angles in the equatorial planes are 110.60(14)-134.01(14)° (1), 106.8(3)-132.4(2)° (2) and 109.5(3)-127.7(2)° (3), and those between the apical donor atoms are 167.09(14)° (1), 165.5(3)° (2) and 167.0(2)° (3), indicating slight distortion of the coordination from ideal square pyramidal geometry. The coordinate bond lengths and angles in the three complexes are similar to each other, and are comparable to those in reported Schiff base zinc(II) complexes.17 In the crystal structures of the three complexes, the methanol and water molecules are linked to complex molecules through intermolecular hydrogen bonds (Table 3), The molecules are linked through hydrogen bonds (Table 3) to form three dimensional networks (Figures 4, 5 and 6). Figure 1. A perspective view of complex 1 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level. Figure 2. A perspective view of complex 2 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level. Figure 3. A perspective view of complex 3 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level. 139Acta Chim. Slov. 2024, 71, 135–142 Chen et al.: Syntheses, Crystal Structures and Antimicrobial ... Figure 4. The crystal structure of complex 1, viewed along the a axis. Hydrogen bonds are shown as dashed lines. Figure 5. The crystal structure of complex 2, viewed along the a axis. Hydrogen bonds are shown as dashed lines. Figure 6. The crystal structure of complex 3, viewed along the a axis. Hydrogen bonds are shown as dashed lines. Table 2. Selected bond distances (Å) and angles (°) for the complex- es 1 Zn1–N1 2.032(4) Zn1–O1 2.063(4) Zn1–N2 2.580(5) Zn1–Cl1 2.2726(16) Zn1–Cl2 2.2643(16) N1–Zn1–O1 90.00(16) N1–Zn1–Cl2 134.01(14) O1–Zn1–Cl2 93.82(12) N1–Zn1–Cl1 110.60(14) O1–Zn1–Cl1 95.66(14) Cl2–Zn1–Cl1 114.56(6) N1–Zn1–N2 77.24(16) O1–Zn1–N2 167.09(14) Cl2–Zn1–N2 93.69(11) Cl1–Zn1–N2 90.74(10) 2 Zn1–N1 1.999(7) Zn1–O1 2.001(6) Zn1–N2 2.685(6) Zn1–Cl1 2.209(3) Zn1–N4 2.009(9) N1–Zn1–O1 90.2(3) N1–Zn1–N4 118.6(4) O1–Zn1–N4 98.3(4) N1–Zn1–Cl1 132.4(2) O1–Zn1–Cl1 97.0(2) N4–Zn1–Cl1 106.8(3) N2–Zn1–N1 75.4(3) N2–Zn1–O1 165.5(3) N2–Zn1–N4 87.8(3) N2–Zn1–Cl1 93.7(3) 3 Zn1–N1 2.008(4) Zn1–O1 2.008(4) Zn1–N4 1.961(6) Zn1–N5 1.984(7) Zn1–N2 2.674(6) N4–Zn1–N5 109.5(3) N4–Zn1–N1 127.7(2) N5–Zn1–N1 120.0(2) N4–Zn1–O1 97.3(2) N5–Zn1–O1 98.9(2) N1–Zn1–O1 91.19(17) N2–Zn1–O1 167.0(2) N2–Zn1–N1 75.9(2) N2–Zn1–N4 91.5(2) N2–Zn1–N5 87.0(2) Table 3. Hydrogen bond distances (Å) and angles (°) for the com- plexes D–H∙∙∙A d(D–H) d(H∙∙∙A) d(D∙∙∙A) Angle (D–H∙∙∙A) 1 N3–H3A∙∙∙O2#1 0.90 2.12 2.994(10) 163(6) N3–H3B∙∙∙O1#2 0.90 1.85 2.736(6) 168(6) O2–H2∙∙∙Cl2#3 0.82 2.62 3.349(8) 150(6) 2 O2–H2A∙∙∙O3#4 0.82 2.15 2.702(10) 125(5) O3–H3C∙∙∙O1#5 0.82 1.79 2.589(9) 165(5) O4–H4A∙∙∙Br1#6 0.85 2.79 3.563(13) 151(6) O4–H4B∙∙∙Cl1 0.85 1.65 2.435(14) 151(6) N3–H3A∙∙∙O2#1 0.90 1.88 2.752(10) 162(5) N3–H3B∙∙∙O3 0.90 2.06 2.827(10) 143(6) N3–H3B∙∙∙Cl1#5 0.90 2.97 3.529(8) 122(7) 3 N3–H3A∙∙∙O2#7 0.90 2.02 2.827(7) 148(6) N3–H3B∙∙∙O3#8 0.90 1.93 2.799(7) 163(6) O3–H3C∙∙∙O2 0.82 1.95 2.751(7) 164(7) O2–H2B∙∙∙O1 0.85 1.86 2.661(6) 158(7) Symmetry codes: #1: x, y, 1 + z; #2: 3/2 + x, ½ – y, ½ + z; #3: 3/2 + x, ½ – y, –½ + z; #4: x, ½ – y, –½ + z; #5: x, ½ – y, ½ + z; #6: 2 – x, –½ + y, ½ – z; #7: x, 3/2 – y, –½ + z; #8: x, y, –1 + z. 140 Acta Chim. Slov. 2024, 71, 135–142 Chen et al.: Syntheses, Crystal Structures and Antimicrobial ... 3. 4. Antimicrobial Activity The compounds and related starting materials were assayed for antibacterial activities against Gram positive bacterial strains Bacillus subtilis and Staphylococcus aureus, and Gram negative bacterial strains Escherichia coli and Pseudomonas fluorescence by MTT method. The MIC (minimum inhibitory concentration, μg mL–1) values against the bacteria are summarized in Table 4. Penicillin G was used as a reference. The three zinc complexes have better activities against all the bacteria strains than the free Schiff base and zinc chloride. Complexes 1 and 2 show strong activity against B. subtilis, S. aureus and E. coli, while medium activity against P. fluorescence. Complex 3 shows similar activities against S. aureus and E. coli as complex 2, but lower activity against B. subtilis and P. fluorescence. Complexes 1 and 2 have stronger or similar activity against all the bacteria than Penicillin G. Complex 3 has stronger activity against E. coli and P. fluorescence, while weaker or similar activity against B. subtilis and S. aureus than Penicillin G. However, the three complexes have no activity on the fungal strains Candida albicans and Aspergillus niger. The complexes have similar antimicrobial activities with the zinc complexes derived from 5-bromo-2-((cychlopentylimino)methyl)phenol,6d and higher activities than the nickel complexes with Schiff base ligands.18 Table 4. Antibacterial activities of the assayed compounds (MIC, μg mL–1) Tested B. subtilis S. aureus E. coli P. fluorescence material 1 1.2 2.3 9.4 18.8 2 2.3 4.7 9.4 18.8 3 4.7 4.7 9.4 37.5 L 9.4 18.8 37.5 > 150 ZnCl2 18.8 18.8 75 > 150 Penicillin G 2.3 4.7 >150 > 150 4. Conclusion In this paper, three new zinc complexes were synthesized from Schiff base 5-bromo-2-(((2-piperazin- 1-yl)ethyl)imino)methyl)phenol with zinc chloride in the absence or presence of ammonium thiocyanate. The complexes were characterized by physico-chemical methods. X-ray single crystal structure determination indicates that the zinc atoms in the complexes are in square pyramidal coordination. The chloride ligand can be replaced by thiocyanate ligand. The complexes have strong activities against bacteria B. subtilis, S. aureus and E. coli, which deserve further study. Acknowlegments This work was financially supported by Ningbo Public Welfare Funds (Project Nos. 202002N3056 and 2021S142). Supplementary Data CCDC 2314879 (1), 2314880 (2) and 2314881 (3) contain the supplementary crystallographic data for the compounds. These data can be obtained free of charge via http://www.ccdc.cam.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 e-mail: deposit@ccdc.cam.ac.uk. 5. References 1. (a) E. Aguilar-Llanos, S. E. Carrera-Pacheco, R. Gonza- lez-Pastor, J. Zuniga-Miranda, C. Rodriguez-Polit, A. 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Povzetek Sintetizirali smo tri nove cinkove(II) komplekse [ZnCl2L]·CH3OH (1), [ZnClL(NCS)]·2CH3OH·0.5H2O (2) in [ZnL(NCS)2]·CH3OH·H2O (3), kjer je L zwitterionska oblika 5-bromo-2-(((2-piperazin-1-il)etil)imino)metil)fenola, NCS pa je tiocianatni anion, v različnih molskih razmerij L, cinkovega klorida in amonijevega tiocianata v metanolu. Kompleksi so bili okarakterizirani z IR in UV-Vis spektroskopijo. Strukture treh kompleksov so bile določene z monono- kristalno rentgensko analizo. V kompleksih so atomi Zn v tetraedrični koordinaciji. Ligand Schiffove baze se koordinira na atom Zn prek fenolatnega kisikovega atoma ter amino in imino dušikovega atoma. Preostali dve mesti zasedata dva Cl za 1, en Cl in en NCS za 2 ter dva NCS za 3. Spojine imajo protimikrobno aktivnost. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 143Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the DOI: 10.17344/acsi.2024.8628 Scientific paper Textbook Sets Through the Perspective of the Orientation of the Intended Chemistry Curriculum for Primary and Secondary Schools Špela Hrast* and Vesna Ferk Savec University of Ljubljana, Faculty of Education, Kardeljeva ploščad 16, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: pela.hrast@pef.uni-lj.si Received: 01-18-2024 Abstract Textbooks have a central role in chemistry education and represent the intended chemistry curriculum at the national level. This paper focuses on analysing the intended chemistry curriculum as represented by the visual representations and the activities for students in the textbook sets in relation to the topics of the Slovenian National Chemistry Curric- ulum both at the primary and secondary school levels. The analysis involved all textbook sets approved by the national representatives for the 2021/2022 school year. The results revealed that in most of the curriculum topics in the analysed Slovenian chemistry textbook sets, the curriculum orientation structure of the discipline prevails and the everyday life orientation is present for both primary and secondary schools. To improve the relevance of the textbook sets for students, the currently rare presence of history of chemistry, environmental orientation, and technology and industry orientation and the lack of the use of socio-scientific orientation should be overcome. It would be valuable if further studies in textbook sets would also address the intended chemistry curriculum from a more holistic perspective. Keywords: Intended chemistry curriculum, curriculum orientation, activity for students, visual representation, chem- istry textbook set 1. Introduction 1. 1. Textbooks as Representations of the Intended Chemistry Curriculum The ideas of a curriculum can be manifested by dif- ferent representations of the curriculum,1 such as the in- tended, the implemented, and the attained curriculum.2 The intended curriculum includes the ideal curriculum, which represents the basic philosophy and rationale of a curriculum, and the formal/written curriculum; the writ- ten curriculum represents the intentions as stated in curric- ulum materials such as textbooks.2,3 In Slovenia, textbooks for chemistry as a school subject should be in line with the National Curriculum for Chemistry at certain levels of ed- ucation4,5 and approved by the Council of Experts of the Republic of Slovenia for General Education6 or Vocation- al and Technical Education,7 thus reflecting the ideal and the formal curriculum for chemistry. Textbooks also have a significant impact on implemented and attained curricu- lum,2,3 because they are often used both for teachers’ lesson preparation,8 students’ activities during lessons, homework as well as for students’ independent learning.9–11 1. 2. Curriculum Orientations as a Foundation for the Analysis of the Intended Chemistry Curriculum as Represented by Textbooks Based on a perception of textbooks as a representa- tion of the intended curriculum,2 textbooks can be referred to as a reference point to understanding which curricu- lum orientations are integrated into a particular subject and educational setting and which of them prevails.12 Six basic orientations of the chemistry curriculum have been identified by Eilks and his colleagues13 in relation to the previous research work by De Jong.14 They can be utilised as guiding principles for structuring the curriculum and/ or as designated approaches to teaching particular chem- istry topics.13 The characteristics of each of the curriculum orien- tations are described below: • The chemistry curriculum orientation structure of the discipline emphasises contemporary theories and facts of chemistry and their interrelationships, on which the structure of the curriculum is built. Social or personal 144 Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the issues and technological applications of chemistry are generally not covered (or only for illustration at the end). As such, it provides an excellent foundation for the later academic study of chemistry13 and is a suita- ble approach for a small group of intrinsically motivat- ed15,16 students who have decided to enrol in this study in the future. The structure of the discipline curriculum could be beneficial for teachers in clarifying the main theories of chemistry and their interrelationships.13 However, this approach is not in line with modern educational theory, which emphasises the theories of scientific literacy17 and situated cognition.18 The im- portance of students’ different motivations, interests, and attitudes in teaching and learning chemistry19,20 is neglected. However, modern chemistry curricula are moving towards more holistic approaches that in- tegrate the learning of concepts and theories through different contexts from everyday life, technology, and society.21–24 • The chemistry curriculum orientation history of chem- istry emphasises the content of chemistry as it was gen- erated in history and/or its past development.13 It offers the opportunity to foster an understanding of the nature of science25,26 in general and the nature of chemistry in particular, which is a central element of scientific literacy and is widely regarded as one of the main goals of science and chemistry education.27–29 Benefits also include the potential to improve students’ interest in and attitudes towards chemistry,25,30 to promote higher order learning skills, such as critical thinking and problem solving,31 to improve understanding of the concept of chemistry, and to promote conceptual change.32 In the latter, care must be taken to ensure that students always know which con- cepts are part of history and are no longer used today.13 However, when orientating on the history of chemistry, aspects of the students’ everyday life and society are of- ten not sufficiently taken into account.33 • The chemistry curriculum orientation everyday life, based on the questions of daily life and the chemical knowledge needed to deal with them. Contexts, such as materials used in everyday life, serve as a starting point.13 In most cases, however, the everyday life ori- entation is based on Van Berkel’s curriculum empha- sis34 on fundamental chemistry, which focuses more on learning theoretical concepts and facts than on the relationship between chemistry and technology and its role in societal issues.13 • The environmental orientation of the chemistry cur- riculum focuses on environmental issues, such as acid rain and water pollution and the chemical content behind them. We can assume fundamental chemistry as the curriculum emphasis. However, environmental topics require a more thorough reflection on the inter- relation between science, technology, and society.13 • In contrast, the technology and industry orientation of the chemistry curriculum emphasises chemical tech- nology and developments in industry and the chemical knowledge applied there.13 The teaching and learning of chemistry that incorporates aspects of the chemi- cal industry thus embraces one of the most important features of modern life and its technological achieve- ments.35,36 In doing so, it can provide the opportuni- ty for a broader focus that includes the interaction of chemistry and technology in society.13,35,36 • The socio-scientific orientation of the chemistry curric- ulum emphasises socio-scientific issues13 and focuses on authentic social issues.37 They provide a context for understanding scientific information38 and are not only the starting point of teaching and learning but also the central content.22 They are usually controversial in nature and are intended to be important and engaging for students. They require the use of evidence-based arguments on the one hand and moral reasoning or the evaluation of ethical concerns on the other.38–41 By fostering general education skills in the areas of com- munication and decision-making, the socio-scientific orientation aims to develop students’ scientific literacy and prepare them to become responsible citizens in the future.13,42 This type of orientation also offers opportu- nities to achieve the goals of discipline-oriented educa- tion for sustainable development by using sustainabili- ty-oriented socio-scientific issues.22,43,44 The curriculum orientation everyday life, environ- mental orientation, technology and industry orientation and socio-scientific orientation can be also referred to with- in context-based curricula,33 as they all aim to increase students' interest and motivation in chemistry by linking chemical concepts to real-life contexts and, in such a man- ner make them more relevant for students.33,45,46 1. 3. Activities for Students and Visual Representations in Textbooks as an Essential Part of Developing Chemical Understanding To enhance the teaching and learning of chemistry, significant attention has been devoted to studying stu- dents’ engagement and research on visualisation, particu- larly molecular-level representation.47 Based on research recommendations, efforts are be- ing made to achieve meaningful student engagement in learning, so-called student-centred learning,48 through various types of activities for students, from questions in learning materials49 to practical work in class.47,50,51 One particularly important kind of practical work for chem- istry education is experimental work,52,53 which can take a variety of forms54 and often requires students to make connections between the domain of objects and observa- tions and the domain of ideas in order to develop their scientific knowledge.53 In addition to the acquisition of knowledge, other fundamental goals of experimental work are the development of experimental skills and scientific 145Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the thinking.50,53,55 Learning materials can also contribute to students’ engagement in the learning of chemistry with under- standing,49 whereby realistic, conventional, and hybrid visual representations56 play an important role.11,57 Visual representations can relate to one of the three levels pro- posed by Johnstone58 for representing chemical concepts and processes: macroscopic (observable phenomena), submicroscopic, or particulate (various representations of atomic, molecular and particle structures) and symbolic (mathematical and chemical symbols). Only a few macro- scopic observations can be understood without the use of submicroscopic representations or models.59 Various vis- ualisations are used to help students in linking of the three levels of the concept or process being represented,60–62 since the interpretation of the macroscopic phenomenon at the particulate level is considered crucial to the creation of accurate mental images or internal representations for corresponding phenomena63,64 and, as such, is an impor- tant component of modern chemistry teaching.65 2. The Context and the Purpose of the Study The use of textbooks has been a habitual means of supporting the effective teaching and learning of school subjects in primary and secondary schools, including the school subject chemistry. To support the quality of text- books in chemistry education, much attention has been paid to the analysis of various aspects of the textbook,66,67 for example, the analysis of the learning content,68–70 the visual representations and their integration,9,71,72 and the learning activities.73–75 However, few textbook analyses focus on the aspect that textbooks convey not only explicit information but also hidden ideas, for example, the purpose of learning chemistry subject matter13 and, as such, represent intend- ed chemistry curriculum and direct to its orientation.12 Khaddoor, Al-Amoushab, and Eilks12 examined 10th- grade chemistry textbooks from seven Arab countries and analysed the intended curriculum as presented by them using the theoretical framework of curriculum em- phases34 and orientations of chemistry curricula.13 Based on the methodology of Khaddoor et al.,12 Chen, Chiu and Eilks76 focused on the representation of the intended curriculum in 10th-grade chemistry textbooks from three Chinese communities. Chen, de Goes, Treagust and Ei- lks77 analysed the visual representations of redox reactions in secondary chemistry textbooks from different Chinese communities, focusing on the orientation of the intend- ed curriculum characterised by the contexts proposed for chemistry learning. The same focus was also analysed by authors de Goes, Chen, Nogueira, Fernandez and Eilks,78 with the difference that they focused on Brazilian chemis- try textbooks. In this paper, we seek to provide new insights into the analysis of the intended chemistry curriculum as rep- resented by textbooks, particularly from the perspective of the included activities for students and visual representa- tions in relation to the curriculum orientations. Among textbook components, activities for students and visual representations are namely recognised in the literature as essential to developing students’ deep and coherent under- standing of chemistry47 and have the greatest potential to influence classroom practise.8 This paper focuses on the activities for students and visual representations in Slove- nian chemistry textbooks in relation to the topics of the National Chemistry Curriculum for Primary School,5 and for General Secondary School – Gymnasium,4 which rep- resents the current state of the art for Slovenian primary and secondary school chemistry education. Thereby, it is important to note, that chemistry is an obligatory school subject in Slovenian primary schools in eighth and ninth grades (age 13–15 years) and in general secondary schools (age 15–19 years) in the first, second, and third years, whereas fourth-year students can choose chemistry based on their interests. The following research questions (RQ) were stated: 1st RQ: Which curriculum orientations indicated from the activities for students prevail in the analysed Slovenian chemistry textbook sets for primary school with respect to the curriculum topics? 2nd RQ: Which curriculum orientations indicated from the visual representations prevail in the analysed Slo- venian chemistry textbook sets for primary school with respect to the curriculum topics? 3rd RQ: Which curriculum orientations indicated from the activities for students prevail in the analysed Slovenian chemistry textbook sets for secondary school with respect to the curriculum topics? 4th RQ: Which curriculum orientations indicated from the visual representations prevail in the analysed Slovenian chemistry textbook sets for secondary school with respect to the curriculum topics? 3. Methods 3. 1. Sample To answer the research questions, we focused on textbook sets, specifically chemistry textbooks for primary school (8th and 9th grade; basic compulsory education79) approved by the Council of Experts of the Republic of Slo- venia for General Education and for secondary school (1st, 2nd, and 3rd years; upper secondary general non-compul- sory education – gymnasium79) approved by the Council of Experts of the Republic of Slovenia for Vocational and Technical Education for the 2021/2022 school year, as well as the accompanying workbooks. Due to the large variety of supplementary materials offered by different publishers, no supplementary materials (e.g., recommendations for 146 Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the teachers) were analysed. Only textbook sets in Slovenian were analysed. Textbook sets dealing only with the elective contents of chemistry were not analysed. If a textbook set is available in i- or e-form as well as in printed form, the printed materials for students were analysed. Chemistry textbooks for primary and secondary schools in Slovenia must be written on the basis of the objectives of the National Curriculum for Chemistry at certain levels of education,4,5 which set specific objectives and suggestions for the content for each of the ten topics for primary school and for each of the twelve topics for secondary school (the topics are presented in more detail in section 3.3 Data analysis). Teachers are free to distribute the above curriculum topics in 70 hours in 8th grade and 64 hours in 9th grade in primary school and in 70 hours in 1st year, 70 hours in 2nd year, and 70 hours in 3rd year in secondary school as they see fit. With some publishers, the topics of the National Chemistry Curriculum for Pri- mary School are covered in two different sets of textbooks, namely the 8th-grade textbook set and the 9th-grade text- book set. The same applies to some secondary textbook sets. To overcome this issue, the analysis combined the primary school textbook sets (8th and 9th grade) from the same publisher and the secondary school textbook sets (1st, 2nd, and 3rd year) from the same publisher. Thus, in the analysis of secondary school textbook sets, two text- book sets were excluded whose publishers cover only one of three grades. For a publishing company that offers two Table 1. The list of the analysed textbook sets for primary school Publi­ sher Textbook set title Author(s) Year of publication (Edition) Textbook/ workbook Number of Pages Textbook/ workbook Grade/ Learner’s age Introduction of learning goals at the beginning of chapters Textbook/ workbook Summary of important concepts at the end of chapters Textbook/ workbook DZS Kemija danes 1 Graunar, M., Podlipnik, M., Mirnik, J., Gabrič, A., Glažar, S. A., Slatinek-Žigon, M. (textbook) Graunar, M., Modec. B., Dolenc,D., Gabrič, A., Slatinek Žigon, M. (work- book) 2018 (1st Ed.)/ 2015 (1st Ed.) 160/104 8/13 Yes/No Yes/No Kemija danes 2 Graunar, M., Podlipnik, M., Mirnik, J. (textbook) Dolenc, D., Graunar, M., Modec, B. (workbook) 2016 (1st Ed.)/ 2018 (1st Ed.) 152/96 9/14 Jutro Svet kemije 8, Od atoma do molekule Smrdu, A. 2012 (2nd Ed.)/ 2012 (2nd Ed.) 128/160 8/13 No/Yes Yes/No Svet kemije 9, Od molekule do makromole-kule Smrdu, A. 2013 (2nd Ed.)/ 2018 (2nd Ed.) 128/152 9/14 MK Pogled v kemijo 8 Kornhauser, A., Frazer, M. 2003 (1st Ed.)/ 2004 (1st Ed.) 140/126 8/13 No/No Yes/No Pogled v kemijo 9 Kornhauser, A., Frazer, M. 2005 (1st Ed.)/ 2006 (1st Ed.) 140/115 9/14 Modrijan Moja prva kemija Vrtačnik, M., Wissiak Grm, K. S., Glažar, S. A., Godec, A. 2017 (1st Ed.)/ 2018 (1st Ed.) 239/92 8, 9/13, 14 No/No Yes/No Rokus Klett Peti element 8 Devetak, I., Cvirn Pavlin, T., Jamšek, S. 2017 (1st Ed.)/ 2017 (1st Ed.) 105/71 8/13 Yes/Yes Yes/No Peti element 9 Devetak I., Cvirn Pavlin T., Jamšek S., Vesna, P. Devetak, I.,Cvirn Pavlin, T., Jamšek, S. 2015 (1st Ed.)/ 2012 (1st Ed.) 77/ 79 9/14 Zavod RS za šolstvo Kemija 8, i-učbenik Sajovic, I., Wissiak Grm, K. S., Godec, A., Kralj, B., Smrdu, A., Vrtačnik, M., Glažar, S. 2014 264/0 8/13 Yes Yes Kemija 9, i-učbenik Jamšek, S., Sajovic, I., Wissiak Grm, K. S.., Godec, A., Boh, B., Vrtačnik, M., Glažar, S. 2013 271/0 9/14 147Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the textbooks covering the same curriculum topics for sec- ondary school, the later-released textbook, which also contains a complementary workbook, was chosen. A list of the textbook sets analysed can be found in Table 1 and Table 2. 3. 2 Instruments We employed a rubric, based on the criteria for text- book analysis by Devetak and Vorgrinc,11 for qualitative content analysis of textbook sets in this research. The ru- bric, adapted by Khaddoor, Al-Amoush and Eilks,12 as well as by Chen, Chie and Eliks,76 was used in the analysis and is presented in Table 3. The detailed criteria for the evaluation of the curric- ulum orientations category indicated by the activities for students or visual representations, which are the focus of this paper, are presented in Table 4. To ensure the validity of the rubric, 280 pages of primary school textbook sets and 373 pages of second- ary school textbook sets (10% of all textbook set pages analysed) were analysed by both authors to define the main types of activities for students and the main types of visual representations, and to determine the curric- ulum orientations indicated from the activities for stu- dents and visual representations. The textbook set pages analysed were randomly selected from the textbook sets of all publishers. 47 pages each from the primary school textbook sets of the same publisher and 93 pages from the secondary school textbook sets of the same publish- er were analysed. To reduce the bias associated with us- ing the rubric to categorise activities for students and visual representations, 95% inter-rater reliability of the rubric was determined through discussion and agree- ment. 3. 3 Data Analysis The rubric described in the instruments section was used in the analysis of the general structure, textual ma- Table 2. The list of the analysed textbook sets for secondary school Publisher Textbook set title Author(s) Year of publication (Edition) Textbook/ Workbook Number of Pages Textbook/ workbook Grade/ Learner’s age Introduction of learning goals at the beginning of chapters Textbook/ workbook Summary of important concepts at the end of chapters Textbook/ workbook DZS Kemija za gimnazije 1 Bukovec, N. 2019 (1st Ed.)/ 2011 (1st Ed.) 144/64 1/15 No/Yes Yes/No Kemija za gimnazije 1 Bukovec, N. 20 (1st Ed.)/ 2012 (1st Ed.) 152/72 2/16 No/Yes Yes/No Kemija za gimnazije 2 Graunar, M., Podlipnik, M., Cvirn Pavlin, T. (textbook) Košmrlj, B., Graunar, M (workbooks). 2019 (1st Ed.)/ 2019 (1st Ed.); 2019 (1st) 248/118;118 3/17 No/No Yes/No Jutro Kemija, Snov in spremembe 1 Smrdu, A. 2015 (2nd Ed.)/ 2015 (2nd Ed.) 144/168 1/15 No/Yes Yes/No Kemija, Snov in spremembe 2 Smrdu, A. 2012 (3rd Ed.)/ 2018 (1st Ed.) 152/168 2/16 No/Yes Yes/No Kemija, Snov in spremembe 3 Smrdu, A. 2016 (2rd Ed.)/ 2012 (1st Ed.); 2016 (1st Ed) 184/96;136 3/17 No/Yes Yes/No Modrijan Atomi in molekule Godec, A., Leban, I. (textbook) Cebin, N., Klemenčič, B., Prašnikar, M. (workbook) 2019 (1st Ed.)/ 2012 (1st Ed.) 159/124 1/15 Yes/No Yes/No Kemijske reakcije Godec, A., Leban, I. (textbook) Cebin, N., Klemenčič, B., Prašnikar M. (workbook) 2010 (1st Ed.)/ 2013 (1st Ed.) 174/112 2/16 Yes/No Yes/No Verige in obroči Tršek, Š., Cerkovnik, J. (textbook) Cebin, N., Klemenčič, B., Prašnikar M. (workbook) 2011 (1st Ed.)/ 2015 (1st Ed.) 199/124 3/17 Yes/No Yes/No Zavod RS za šolstvo Kemija 1, i-učbenik Smrdu, A., Zmazek, B., Vrtačnik, M., Glažar, S., Godec, A., Ferk Savec, V. 2014 (1st. Ed.) 296/0 1/15 Yes Yes Kemija 2, i-učbenik Zmazek, B., Smrdu, A., Ferk Savec, V., Glažar, G., Vrtačnik, M. 2014 (1st. Ed.) 245/0 2/16 Yes Yes Kemija 3, i-učbenik Vrtačnik, M., Zmazek, B., Boh, B. 2014 (1st. Ed.) 335/0 3/17 Yes Yes 148 Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the terial, and visual representations of the entire sample of chemistry textbook sets presented in Table 1 and Table 2. Textbook sets were analysed individually. Visual representations that were content-related in a particular area of the textbook set (e.g., submicroscopic representations of modifications of carbon allotropes) and Table 3. The rubric used for analysed textbook sets adapted from Khaddoor, Al-Amoush, and Eilks12 and Chen, Chie, and Eliks.76 G en er al cr ite ri a C at eg o­ ry Subcategories G en er al st ru ct ur e Pa ge s a nd ch ap te rs Number of pages Number of chapter Length of chapters within a specific curriculum topic Te xt ua l m at er ia l A ct iv iti es fo r s tu de nt Number of activities for students Type of activities for students Experimental activities (Demonstrations, Individual students’ experimentations) Other practical activities (Tasks for Internet searches; Project work, building molecular structures etc.) Rating scales related to learning goals Other tasks for repeating and deepening knowledge Curriculum orientations indicated from activities for students Structure of the discipline orientation History of chemistry orientation Everyday life orientation Environmental orientation Technology and industry orientation Socio-scientific orientation In tr od uc tio n an d su m m ar y Presence of introduction of learning goals at the beginning of chapters Presence of summary of important concepts at the end of chapters V is ua l r ep re se nt at io ns V isu al re pr es en ta tio ns (V Rs ) Number of VRs Type of VRs Realistic VRs (Photograph, drawing, video) Conventional VRs (Graph; Flowchart, diagram, map; Table; Pictogram; Molecular structure --Submicroscopic level or Symbolic level or Submicroscopic & symbolic level; Atomic structure; Other) Hybrid VRs (Macroscopic level with molecular structure - Macroscopic, submicroscopic & symbolic level or Macroscopic & submicroscopic level or macroscopic & symbolic level; Other) Curriculum orientations indicated from VRs Structure of the discipline orientation History of chemistry orientation Everyday life orientation Environmental orientation Technology and industry orientation Socio-scientific orientation Table 4. Criteria for the evaluation of the category Curriculum orientations indicated by activities for students or visual representations based on the theoretical framework of Eilks et al.13 and adapted from Khaddoor, Al-Amoush, and Eilks12 and Chen, Chie, and Eliks.76 Category Subcategory Description Curriculum orientations Structure of the discipline orientation The analysed part of the textbook set emphasises the contemporary theories and facts of chemistry and their interrelationships History of chemistry orientation The analysed part of the textbook set emphasises the content of chemistry as it was generated in history and/or its past development. Everyday life orientation The analysed part of the textbook set emphasises the questions from everyday life and the chemical knowledge needed to deal with them. Environmental orientation The analysed part of the textbook set emphasises the environmental issues and chemistry content behind them. Technology and industry orientation The analysed part of the textbook set emphasises chemical technology and developments in industry and the chemical knowledge used in these areas today and in the past. Socio-scientific orientation The analysed part of the textbook set emphasises the socio-scientific issue and concerns to prepare students to become responsible citizens in the future. 149Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the were not specifically separated (e.g., labelled a/b/c) were considered as one visual representation. The analysed aspects of the textbook sets were cate- gorised with regard to the following curriculum topics of the National Chemistry Curriculum for Primary School:5 (1) Chemistry is a World of Matter (orig. Kemija je svet sno- vi); (2) Atom and the Periodic System of Elements (orig. Atom in periodni sistem elementov); (3) Compounds and Bonding (orig. Povezovanje delcev/gradnikov); (4) Chem- ical Reactions (orig. Kemijske reakcije); (5) The Elements in the Periodic Table (orig. Elementi v periodnem sistemu); (6) Acids, Bases and Salts (orig. Kisline, baze in soli); (7) Hydrocarbons and Polymers (orig. Družina ogljikovodik- ov s polimeri); (8) Organic Compounds Containing Oxy- gen (orig. Kisikova družina organskih snovi); (9) Organic Compounds Containing Nitrogen (orig. Dušikova družina organskih spojin), and (10) The Mole (orig. Množina sno- vi) and the following curriculum topics of the National Chemistry Curriculum for Secondary School4: (1) Intro- duction to Safe Experimental Work (orig. Uvod v varno eksperimentalno delo); (2) Building Blocks of Matter (orig. Delci (gradniki) snovi); (3) Compounds and Bonding (orig. Povezovanje delcev (gradnikov)); (4) Amount of Substance and Chemical Equations as Symbolic Representations (orig. Simbolni zapisi in množina snovi); (5) Chemical Re- action as Change of Substance and Energy (orig. Kemijska reakcija kot snovna in energijska sprememba); (6) Alkali Metals and Halogens (orig. Alkalijske kovine in halogeni); (7) Solutions (orig. Raztopine); (8) Chemical Reaction Rates and Equilibrium (orig. Potek kemijskih reakcij); (9) The Elements in the Periodic Table (orig. Elementi v peri- odnem sistemu); (10) Properties of Selected Elements and Compounds in Biological Systems and Modern Technolo- gies (orig. Lastnosti izbranih elementov in spojin bioloških sistemih in sodobnih tehnologijah); (11) Structure and No- menclature of Organic Compounds (orig. Zgradba mol- ekul organskih spojin in njihovo poimenovanje), and (12) Structure and Properties of Organic Compounds (orig. Zgradba in lastnosti organskih spojin). Finally, the types of activities for students, the types of visual representations and the curriculum orientations indicated by them in each of the topics were counted, and the frequencies for each of the textbook sets were calcu- lated. To overcome the variability of textbook sets due to the personal style and opinions of the textbook authors,13 in this article, we use the expression the analysed Slovenian chemistry textbook sets and thereby refer to the calculat- ed average of the data obtained from the textbook sets for each of the curriculum topics. 4. Results and Discussion The results of the analysis of the textbook sets in terms of curriculum orientation indicated by activities for students and visual representations are presented with re- gard to the research questions. The results of other select- ed characteristics of activities for students or visual rep- resentations from the rubric presented in Table 3 can be found in Appendices 1–4. 4. 1. Curriculum Orientations Indicated from the Activities for Students in Analysed Slovenian Chemistry Textbook Sets for Primary School with Respect to the Curriculum Topics (Related to 1st RQ) The average number of different curriculum orienta- tions indicated from the activities for students in analysed Slovenian chemistry textbook sets for primary school is shown in Table 5. Table 5 shows that the largest number of curriculum orientation subcategories with more than 5% of analysed activities for students can be found in the topic ‘Hydrocar- bons and Polymers’ (4 subcategories: Structure of the dis- cipline orientation, Everyday life orientation, Environmen- tal orientation, and Technology and industry orientation), followed by ‘Chemistry is a World of Matter’ (3 subcate- gories: Structure of the discipline orientation, Everyday life orientation, and History of chemistry orientation) and ‘The Elements in the Periodic Table’ (3 subcategories: Structure of the discipline orientation, Everyday life orientation, and Environmental orientation). However, in other curriculum topics, only two subcategories prevail, with more than 5% of the activities for students (2 subcategories: Structure of the discipline orientation and Everyday life orientation). The analysis of the textbook set revealed that within all the topics of the National Chemistry Curriculum for Primary School, with the exception of the topics ‘Organic Compounds Containing Oxygen’ (M = 75.33 activities, FM = 43.93%) and ‘Organic Compounds Containing Nitrogen’ (M = 15.17 activities, FM = 17.71%), more than half of the activities analysed (FM ranges from 53.81% to 87.69%) in- dicate curriculum orientation that can be categorised as Structure of the discipline orientation. The activities that are categorised in this group particularly prevail in the topic ‘Atom and the Periodic System of Elements’ (M = 65.67 activities, FM = 87.69%). The second most frequently used activities within all curriculum topics in the analysed Slovenian chemistry textbook sets indicate a curriculum orientation that can be categorised as Everyday life orientation (FM ranges from 6.87% to 28.17 %). Exceptions are the topics ‘Organic Compounds Containing Oxygen’ and ‘Organic Com- pounds Containing Nitrogen’, for which the Everyday life orientation is used most frequently (M = 97.67 activities, FM = 54.25%; M = 72.67 activities, FM = 81.15%, respec- tively). In contrast, no or very few activities in analysed Slo- venian chemistry textbook sets for primary school within 150 Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the all curriculum topics indicate Socio-scientific orientation (FM ranges from 0.00% to 0.47%). In addition, none or less than 5% of the activities within all curriculum topics indi- cated History of chemistry orientation (FM ranges from 0.00% to 4.25%), with the exception of the topic ‘Chemis- try is a World of Matter’ (M = 7.67 activities, FM = 7.31%), Environmental orientation (FM ranges from 0.00% to 2.29%) with the exception of the topic ‘Hydrocarbons and Polymers’ (M = 12.33 activities, FM = 6.29%) and Technol- ogy and industry orientation (FM ranges from 0.76% to 3.91%) with the exception of the topics ‘The Elements in the Periodic Table’ (M = 14.50 activities, FM = 9.62%) and ‘Hydrocarbons and Polymers’ (M = 7.50 activities, FM = 5.54%). The findings indicate that most activities for students focus on the content of contemporary chemistry theories and facts and their interrelationships and neglect issues related to the individual, society and technology,13 as ac- tivities indicating chemistry curriculum orientation struc- ture of the discipline predominate in most topics of the Na- tional Chemistry Curriculum for Primary School. Such activities mainly encourage students who are intrinsical- ly15 motivated and interested in studying chemistry in the future.13 However, the analysed Slovenian chemistry text- Table 5: The proportion of curriculum orientations indicated from the activities for students within the particular topics of the analysed Slovenian chemistry textbook sets for primary school. The topics of the National Chemistry Curriculum for Primary School (8th and 9th Grade) Curriculum orientations indicated from activities for students Structure of the discipline orientation History of chemistry orientation Everyday life orientation Environmental orientation Technology and industry orientation Socio­scientific orientation MSUM f (%) M[a] Min­max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­max Chemistry is a World of Matter 61.83 33-118 66.52 47.83- 82.52 7.67 0-16 7.32 0.00- 12.63 20.33 8-44 22.12 7.69- 31.88 2.33 0-9 2.29 0.00-6.52 1.17 0-3 1.29 0.00-2.70 0.67 0-4 0.47 0.00-2.80 94.00 55-143 100.00 Atom and the Periodic System of Elements 65.67 37-131 87.69 77.59- 95.62 3.00 0-9 4.25 0.00- 15.52 3.83 0-8 6.87 0.00- 17.02 0.00 0-0 0.00 0.00-0.00 0.83 0-2 1.18 0.00-2.50 0.00 0-0 0.00 0.00-0.00 73.33 47-137 100.00 Compounds and Bonding 49.33 26-78 78.76 44.83- 93.06 0.83 0-4 1.02 0.00- 4.71 10.83 3-32 19.09 3.53- 55.17 0.00 0-0 0.00 0.0-0.00 0.50 0-3 1.14 0.00-6.82 0.00 0-0 0.00 0.00-0.00 61.50 44-85 100.00 Chemical Reactions 51.33 36-67 65.55 46.09- 81.33 1.17 0-5 1.34 0.00- 6.02 24.17 6-58 26.96 12.50- 45.31 2.50 0-9 2.25 0.00-7.03 2.67 0-6 3.91 0.00-12.50 0.00 0-0 0.00 0.00-0.00 81.83 48-128 100.00 The Elements in the Periodic Table 78.83 32-142 53.81 45.16- 63.86 0.83 0-2 0.54 0.00- 1.42 45.67 26-63 34.89 23.85- 47.62 1.50 0-5 1.13 0.00-3.55 14.50 1-34 9.62 1.59-14.68 0.00 0-0 0.00 0.00-0.00 141.33 63-240 100.00 Acids, Bases and Salts 92.33 51-167 70.45 60.40- 83.61 0.33 0-1 0.26 0.00- 0.92 34.83 9-58 26.51 14.75- 38.93 2.33 0-10 1.38 0.00-6.25 1.50 0-3 1.40 0.00-2.75 0.00 0-0 0.00 0.00-0.00 131.33 61-222 100.00 Hydrocarbons and Polymers 104.00 28-276 61.41 51.94- 70.77 2.17 0-7 1.56 0.00- 3.85 35.67 15-56 25.20 14.36- 35.65 12.33 1-39 6.29 1.74- 12.40 7.50 1-16 5.54 0.87-11.54 0.00 0-0 0.00 0.00-0.00 161.67 52-390 100.00 Organic Compounds Containing Oxygen 75.33 43-173 43.93 30.71- 57.55 1.33 0-8 0.60 0.00- 3.62 97.67 38-197 54.25 39.62- 68.57 1.00 0-4 0.46 0.00-1.07 0.83 0-2 0.76 0.00-1.89 0.00 0-0 0.00 0.00-0.00 176.17 83-374 100.00 Organic Compounds Containing Nitrogen 15.17 5-25 17.71 5.00- 28.13 0.50 0-1 0.56 0.00- 1.72 72.67 46-112 81.15 71.88- 94.00 0.17 0-1 0.17 0.00-1.00 0.33 0-1 0.41 0.00-1.54 0.00 0-0 0.00 0.00-0.00 88.83 58-138 100.00 The Mole 34.33 19-50 70.39 54.29- 94.59 0.00 0-0 0.00 0.00- 0.00 15.00 1-37 28.17 2.70- 45.71 0.17 0-1 0.19 0.00-1.14 0.50 0-2 1.24 0.00-4.76 0.00 0-0 0.00 0.00-0.00 50.00 35-88 100.00 [a] M was calculated as the average of the number of identified activities in the textbook sets within the category of specific curriculum orientation and within the specific curriculum topics, thereby min and max represent the minimum and maximum number of identified activities in the textbook sets. [b] fM(%) represents the proportion of M within each curriculum topic, thereby min and max fM(%) represent the minimum and maximum number of identified activities between the textbook sets. 151Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the book sets for primary school also recognise the potential of everyday life as a context for student activities in various curriculum topics that can link chemistry concepts to is- sues in students' daily lives and improve their interest and motivation in chemistry.23,24,33 In the topics ‘Organic Compounds Containing Oxygen’ and ‘Organic Com- pounds Containing Nitrogen’, the everyday life orientation prevails. In the activities for students on the topic ‘Hydro- carbons and Polymers’, the connection of chemical con- cepts with the context of the environment, technology and industry can also be recognised, which indicates the great- est variability in the curriculum orientation of all curricu- lum topics. However, most other topics in the curriculum do not use the potential of linking to everyday life contexts as mentioned above. Furthermore, in most topics, there are no activities that indicate a socio-scientific orientation and focus on socio-scientific issues13 that not only aim to provide a context for understanding chemistry concepts but also encourage students’ development to become re- sponsible citizens in the future.42 The lack of activities rep- resenting socio-scientific orientation indicates a possibly missed opportunity to develop students’ scientific litera- cy17 and to achieve the goals of discipline-oriented educa- tion for sustainable development.22 An unrecognised op- portunity to promote the understanding of the nature of science as an important element of scientific literacy25 in various curriculum topics is also indicated by the absence of activities for students related to the history of chemistry orientation. 4. 2. Curriculum Orientations Indicated from the Visual Representations in Analysed Slovenian Chemistry Textbook Sets for Primary School with Respect to the Curriculum Topics (Related to the 2nd RQ) The average number of different curriculum orienta- tions indicated from the visual representations for stu- dents in the analysed Slovenian chemistry textbook sets in primary school is presented in Table 6. From Table 6, it can be derived that the largest num- ber of subcategories of curriculum orientation, with more than 5% of the analysed visual representations for stu- dents, can be recognised within the topic ‘Chemistry is a World of Matter’ (4 subcategories: Structure of the disci- pline orientation, Everyday life orientation, Technology and industry orientation, and History of chemistry orientation) and ‘Hydrocarbons and Polymers’ (4 subcategories: Struc- ture of the discipline orientation, Everyday life orientation, Technology and industry orientation, and Environmental orientation), followed by ‘Atom and the Periodic Table’ (3 subcategories: Structure of the discipline orientation, Everyday life orientation, and History of chemistry orienta- tion), ‘Chemical Reactions’ (3 subcategories: Structure of the discipline orientation, Everyday life orientation, and Technology and industry orientation) and ‘The Elements in the Periodic Table’ (Structure of the discipline orienta- tion, Everyday life orientation, and Technology and indus- try orientation). However, in the other half of the curricu- lum topics, only two subcategories prevail, with more than 5% of the activities for students (2 subcategories: Structure of the discipline orientation and Everyday life ori- entation). The analysis of the textbook set revealed that the visual representations for students within half of the topics of the National Chemistry Curriculum for Primary School (‘Chemistry is a World of Matter’, ‘Atom and the Periodic System of Elements’, ‘Compounds and Bonding’, ‘Acids, Bases and Salts’, ‘Hydrocarbons and Polymers’) indicate curriculum orientation, which can most often be catego- rised as Structure of the discipline orientation. Whereby the analysed representations represent approximately half or more of all visual representations within a particular cur- riculum topic (M = 38.67 VRs, FM = 47.40%; M = 39.50 VRs, FM = 68.13%; M = 36.00 VRs, FM = 68.60%; M = 42.17 VRs, FM = 57.34%, M = 74.83 VRs, FM = 55.49%, respec- tively). Structure of the discipline orientation represents the second most frequently analysed curriculum orientation, indicated by visual representations within the topics ‘Or- ganic Compounds Containing Oxygen’ (68.17 VRs; 42.39%), ‘Organic Compounds Containing Nitrogen’ (M = 14.00 VRs; FM = 15.64%) and ‘The Mole’ (M = 8.67 VRs; FM =15.64%). For the latter three topics, everyday life ori- entation is the most commonly used curriculum orienta- tion, as indicated by the visual representations analysed, and represents approximately half or more of all visual representations within a given curriculum topic (M = 91.00 VRs, FM = 55.06%; M = 59.17 VRs, FM = 77.88%; M = 14.00 VRs, FM = 60.95%, respectively). Within the other curriculum topics, the subcategory Everyday life orienta- tion represents the second most frequent subcategory of curriculum orientations (FM ranges from 29.74% to 45.23 %) with the exception of the topics ‘Chemical Reactions’ and ‘The Elements in the Periodic Table’, for which the proportion of the subcategory Everyday life orientation (M = 24.00 VRs, FM = 42.06%; M = 42.50 VRs, FM = 45.23%, respectively) is about the same as the proportion of the subcategory Structure of the discipline orientation (M = 25.00 VRs, FM = 45.68%; M = 40.50 VRs, FM = 41.64%, re- spectively). In contrast, no or very few activities in analysed Slo- venian chemistry textbook sets in primary school within all curriculum topics indicate socio-scientific orientation (FM ranges from 0.00% to 0.27%). In addition, none or less than 5% of the activities within all curriculum topics indicates history of chemistry orientation (FM ranges from 0.33% to 3.72%), with the exception of the topics ‘Chem- istry is a World of Matter’ (M = 6.83 VRs, FM=9.09%) and ‘Atom and the Periodic System of Elements’ (M = 8.67 VRs, FM = 14.34%), environmental orientation (FM range 152 Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the from 0.00% to 2.21%), with exception of the topic ‘Hydro- carbons and Polymers’ (M = 9.17 VRs, FM = 7.89%) and technology and industry orientation (FM range from 0.67% to 3.00%), with the exception of the topics ‘Chemistry is a World of Matter’ (M = 5.17 VRs, FM = 5.84%), ‘Chemical Reactions’ (M = 4.17 VRs, FM = 6.93%), ‘The Elements in the Periodic Table’ (M = 10.67 VRs, FM = 10.70%) and ‘Hydrocarbons and Polymers’ (M = 7.50 VRs, FM = 6.97%). The results revealed that in analysed Slovenian chemistry textbook sets for primary school, half of the topics in the National Chemistry Curriculum for Primary School are dominated by visual representations that pres- ent chemical theories and their interconnections to the students without integrating them into different contexts or indicating the structure of the discipline chemistry cur- riculum orientation.13 The prevalence of this type of visual representations neglects the importance of the different Table 6: The proportion of curriculum orientations indicated from the visual representations (VRs) for students within the particular topics of the analysed Slovenian chemistry textbook sets for primary school The topics of the National Chemistry Curriculum for Primary School (8th and 9th Grade) Curriculum orientations indicated from visual representations (VRs) for students Structure of the discipline orientation History of chemistry orientation Everyday life orientation Environmental orientation Technology and industry orientation Socio­scientific orientation MSUM f (%) M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­ max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­ max Chemistry is a World of Matter 38.67 8-61 47.40 21.62- 63.54 6.83 1-11 9.09 1.16- 12.86 25.83 14-36 35.83 20.00- 62.16 1.17 0-2 1.84 0.00-5.41 5.17 0-20 5.84 0.00-21.51 0.00 0-0 0.00 0.00-0.00 77.67 37-96 100.00 Atom and the Periodic System of Elements 39.50 13-71 68.13 44.83- 81.82 8.67 4-21 14.34 11.84- 20.79 7.17 1-14 14.31 2.78- 37.93 0.17 0-1 0.22 0.00-1.32 1.83 0-4 3.00 0.00-5.17 0.00 0-0 0.00 0.00-0.00 57.33 29-101 100.00 Compounds and Bonding 36.00 17-55 68.60 49.02- 82.09 0.17 0-1 0.33 0.00- 1.96 15.67 5-25 29.74 14.93- 49.02 0.00 0-0 0.00 0.00-0.00 0.83 0-3 1.33 0.00-5.00 0.00 0-0 0.00 0.00-0.00 52.67 22-67 100.00 Chemical Reactions 25.00 14-32 45.68 30.77- 68.09 1.67 1-3 3.13 1.54- 6.82 24.00 8-36 42.06 17.02- 55.38 1.33 0-4 2.21 0.00-6.15 4.17 0-11 6.93 0.00-13.92 0.00 0-0 0.00 0.00-0.00 56.17 44-79 100.00 The Elements in the Periodic Table 40.50 22-51 41.64 34.38- 49.49 1.33 0-4 1.50 0.00- 4.26 42.50 31-65 45.23 30.28- 57.81 0.83 0-2 0.93 0.00-1.83 10.67 3-22 10.70 4.55-20.18 0.00 0-0 0.00 0.00-0.00 95.83 64-121 100.00 Acids, Bases and Salts 42.17 35-52 57.34 47.37- 74.47 0.50 0-3 0.69 0.00- 4.17 30.33 12-41 39.19 25.53- 46.05 1.67 0-5 2.09 0.00-6.58 0.67 0-4 0.67 0.00-4.04 0.00 0-0 0.00 0.00-0.00 75.33 47-99 100.00 Hydrocarbons and Polymers 74.83 23-160 55.49 32.86- 65.71 2.33 0-6 1.92 0.00- 4.29 31.83 14-46 27.46 18.78- 41.43 9.17 4-18 7.89 3.05- 12.86 7.50 3-17 6.97 2.14-14.52 0.17 0-1 0.27 0.00-1.61 125.83 62-245 100.00 Organic Compounds Containing Oxygen 68.17 45-95 42.39 34.84- 50.88 1.00 0-3 0.67 0.00- 1.96 91.00 55-155 55.06 43.14- 63.52 1.17 0-3 0.73 0.00-2.26 1.83 0-8 1.15 0.00-5.23 0.00 0-0 0.00 0.00-0.00 163.17 114-244 100.00 Organic Compounds Containing Nitrogen 14.00 0-32 15.64 0.00- 29.36 2.83 0-6 3.72 0.00- 9.38 59.17 48-75 77.88 60.55- 96.00 0.00 0-3 0.00 0.00-0.00 2.50 0-9 2.76 0.00-8.26 0.00 0-0 0.00 0.00-0.00 78.50 50-109 100.00 The Mole 8.67 2-16 32.88 16.67- 47.06 0.83 0-1 3.25 0.00- 5.00 14.00 10-18 60.95 44.12- 83.33 0.33 0-2 0.98 0.00-5.88 0.50 0-1 1.93 0.00-4.55 0.00 0-0 0.00 0.00-0.00 24.33 12-34 100.00 [a] M was calculated as the average of the number of identified visual representations in the textbook sets within the category of specific curriculum orientation and within the specific curriculum topics, thereby min and max represent the minimum and maximum number of identified visual representations in the textbook sets. [b] fM(%) represents the proportion of M within each curriculum topic, thereby min and max fM(%) represent the minimum and maximum number of identified visual representations between the textbook sets. 153Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the motivations, interests and attitudes of students in chem- istry.19,20 In the other half of the curriculum topics (topics ‘Chemical Reactions’, ‘The Elements in the Periodic Table’, ‘Organic Compounds Containing Oxygen’, ‘Organic Com- pounds Containing Nitrogen’ and ‘The Mole’), the visual representations focus almost as often or even more often on the challenges of everyday life and the chemical knowl- edge that is important for dealing with them. In this way, they attempt to increase the students’ interest and motiva- tion for chemistry23,24 and indicate the everyday life chem- istry curriculum orientation. In most cases, however, the focus is on learning the- oretical concepts and facts rather than the relationships between chemistry, technology, and society.13 The greatest diversity of visual representations in terms of curriculum orientation was found in the topic ‘Hydrocarbons and Pol- ymers’, in which visual representations also indicate an en- vironmental curriculum orientation and a technology and industry curriculum orientation, and in the topic ‘Chem- istry is a World of Matter’, in which visual representations also indicate a history of chemistry curriculum orienta- tion and a technology and industry curriculum orienta- tion. Furthermore, in the topics ‘Chemical Reactions’, and ‘The Elements in the Periodic Table’, further visual rep- resentations can be recognised that indicate technology and industry curriculum orientation. In contrast, in most other curriculum topics, the potential of linking chemis- try concepts to real contexts related to the environment, technology and industry, or to the history of chemistry, is rarely used. As with the activities for students, there are no visual representations in most topics that focus on most- ly controversial, engaging social issues that are important to students and that promote general educational skills in terms of communication and decision-making and pre- pare students to take on a responsible role as contributing members of society in the future.39–42 The absence of visual representations representing socio-scientific curriculum orientation indicates a missed opportunity to promote students’ scientific literacy13,42 and provide them with an education geared towards sustainable development.22,43,44 4. 3. Curriculum Orientations Indicated from the Activities for Students in Analysed Slovenian Chemistry Textbook Sets for Secondary School With Respect to the Curriculum Topics (Related to the 3rd RQ) The average number of different curriculum orienta- tions indicated from the activities for students in analysed Slovenian chemistry textbook sets for secondary school is shown in Table 7. Table 7 shows that the largest number of subcatego- ries for curriculum orientation, with more than 5% of the analysed activities for students, can be found in the topic ‘Properties of Selected Elements and Compounds in Bi- ological Systems and Modern Technologies’ (3 subcate- gories: Structure of the discipline orientation, Everyday life orientation, and Technology and industry orientation). In contrast, for the topics ‘Building Blocks of Matter Structure’ and ‘Structure and Nomenclature of Organic Compounds’, there is only one subcategory (Structure of the discipline ori- entation) with more than 5% of the activities for students. The analysis of the secondary textbook sets revealed that within all topics of the National Chemistry Curricu- lum for Secondary School, except for the topic ‘Properties of Selected Elements and Compounds in Biological Sys- tems and Modern Technologies’ (M = 12.75 activities, FM = 31.98%), more than two thirds of the activities analysed (FM ranges from 67.10% to 94.66%) indicate a curriculum orientation that can be categorised as Structure of the dis- cipline orientation. The activities that can be categorised in this group are particularly dominant in the topics ‘Building blocks of matter’ (M = 111.75 activities, FM = 94.66%) and ‘Structure and nomenclature of organic compounds’ (M = 134.50 activities, FM = 94.21%). The second most frequent- ly used activities within all curriculum topics in the typi- cal Slovenian secondary chemistry textbook set indicate a curriculum orientation that can be classified as Everyday life orientation (FM ranges from 2.66% to 28.65%). An ex- ception is the topic ‘Organic Compounds Containing Ox- ygen’, in which the Everyday life orientation is used most frequently (M = 15.75 activities, FM = 43.26%). In contrast, there were no activities in the typical Slo- venian chemistry textbooks within all secondary school curriculum topics that indicated Socio-scientific orienta- tion (M = 0.00 activities, FM = 0.00%). In addition, none or less than 5 % of the activities within all secondary school curriculum topics indicated Technology and industry ori- entation (FM ranges from 0.00% to 2.74%), with the ex- ception of the topic ‘Properties of Selected Elements and Compounds in Biological Systems and Modern Technol- ogies’ (M = 8.50 activities, FM = 22.75%), History of chem- istry orientation (FM ranges from 0.00% to 2.64%) and En- vironmental orientation (FM ranges from 0.00% to 3.36%). The results show that most topics in the National Chemistry Curriculum for Secondary School emphasise the core content of modern chemical theories and facts in the activities for students, while aspects related to the indi- vidual, society and technology are neglected.13 A notable exception is the topic ‘Properties of Selected Elements and Compounds in Biological Systems and Modern Technolo- gies’, whose name inherently signals an integration of re- al-life contexts in order to engage students with different interests and attitudes in the teaching and learning of chemistry.19,20 The lack of use of different contexts in most secondary curriculum topics, and in particular the ab- sence of socio-scientific issues,13 points to the possibility of improving activities to develop both chemical knowl- edge and general education skills for active engagement in social issues in the future.13,42 154 Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the Table 7: The proportion of curriculum orientations indicated from the activities for students within the particular topics of the analysed Slovenian chemistry textbook sets for secondary school The topics of the National Chemistry Curriculum for Secondary School (1st, 2nd and 3th Year) Curriculum orientations indicated from activities for students Structure of the discipline orientation History of chemistry orientation Everyday life orientation Environmental orientation Technology and industry orientation Socio­scientific orientation MSUM f (%) M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­ max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­ max Safe Experimental Work 33.00 20-48 76.48 62.86- 95.45 0.25 0-1 0.40 0.00- 1.59 8.75 2-14 21.69 4.55- 31.43 0.25 0-1 0.71 0.00-2.86 0.25 0-1 0.71 0.00-2.86 0.00 0-0 0.00 0.00-0.00 42.50 8-63 100.00 Building Blocks of Matter 111.75 76-149 94.66 90.63- 99.07 2.00 0-4 1.92 0.00- 3.61 3.25 0-8 2.66 0.00-6.25 0.00 0-0 0.00 0.00-0.00 0.75 0-2 0.77 0.00-2.41 0.00 0-0 0.00 0.00-0.00 117.75 83-153 100.00 Compounds and Bonding 136.25 96-166 81.17 73.85- 89.08 0.50 0-1 0.34 0.00- 0.77 28.75 19-40 17.72 10.92- 23.67 0.00 0-0 0.00 0.00-0.00 1.00 0-4 0.77 0.00-3.08 0.00 0-0 0.00 0.00-0.00 166.50 130-193 100.00 Amount of Substance and Chemical Equations as Symbolic Representa­ tions 93.75 59-135 77.03 71.81- 80.91 1.00 0-2 0.90 0.00- 2.53 27.50 18-50 21.72 18.42- 26.60 0.25 0-1 0.22 0.00-0.88 0.25 0-1 0.13 0.00-0.53 0.00 0-0 0.00 0.00-0.00 122.75 79-188 100.00 Chemical Reaction as Change of Substance and Energy 50.00 31-69 75.83 65.88- 88.46 0.00 0-0 0.00 0.00- 0.00 12.00 3-21 18.45 5.66- 31.91 1.75 0-6 3.36 0.00- 11.32 2.00 0-8 2.35 0.00-9.41 0.00 0-0 0.00 0.00-0.00 65.75 47-85 100.00 Alkali Metals and Halogens 49.00 37-71 85.13 69.81- 92.59 0.25 0-1 0.47 0.00- 1.89 6.75 4-12 12.05 7.41- 22.64 0.25 0-1 0.47 0.00-1.89 1.25 0-3 1.87 0.00-3.77 0.00 0-0 0.00 0.00-0.00 57.50 42-81 100.00 Solutions 92.00 67-131 70.83 54.92- 83.62 0.00 0-0 0.00 0.00- 0.00 35.75 19-54 28.65 15.82- 44.26 0.75 0-2 0.52 0.00-1.27 0.00 0-0 0.00 0.00-0.00 0.00 0-0 0.00 0.00-0.00 128.50 116-158 100.00 Chemical Reaction Rates and Equilibrium 401.75 334-505 89.64 83.71- 93.77 0.50 0-1 0.11 0.00- 0.25 37.75 16-56 8.30 3.99- 12.28 3.00 0-9 0.75 0.00-2.26 5.25 3-7 1.21 0.70-1.75 0.00 0-0 0.00 0.00-0.00 448.25 399-567 100.00 The Elements in the Periodic Table 54.75 31-89 83.94 75.61- 96.55 0.50 0-2 1.22 0.00- 4.88 7.75 1-17 11.19 1.72- 15.45 0.50 0-1 0.91 0.00-1.92 1.75 0-4 2.74 0.00-7.32 0.00 0-0 0.00 0.00-0.00 65.25 41-110 100.00 Properties of Selected Elements and Compounds in Biological Systems and Modern Technologies 12.75 4-24 31.98 12.12- 48.00 0.00 0-0 0.00 0.00- 0.00 15.75 14-19 43.26 30.00- 57.58 0.75 0-2 2.02 0.00-6.06 8.50 4-10 22.75 12.12- 30.30 0.00 0-0 0.00 0.00-0.00 37.75 33-50 100.00 Structure and Nomenclature of Organic Compounds 134.50 70-226 94.21 89.74- 97.84 2.50 0-6 2.64 0.00- 7.69 4.25 2-5 3.15 2.16-4.31 0.00 0-0 0.00 0.00-0.00 0.00 0-0 0.00 0.00-0.00 0.00 0-0 0.00 0.00-0.00 141.25 78-231 100.00 Structure and Properties of Organic Compounds 384.75 290-537 67.10 57.20- 80.15 4.00 0-10 0.79 0.00- 2.08 156.75 121-187 28.53 18.06- 36.88 11.25 9-13 2.06 1.34-2.56 8.00 1-13 1.52 0.15-2.56 0.00 0-0 0.00 0.00-0.00 564.75 481-670 100.00 [a] M was calculated as the average of the number of identified activities in the textbook sets within the category of specific curriculum orientation and within the specific curriculum topics, thereby min and max represent the minimum and maximum number of identified activities in the textbook sets. [b] fM(%) represents the proportion of M within each curriculum topic, thereby min and max fM(%) represent the minimum and maximum number of identified activities between the textbook sets. 155Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the 4. 4. Curriculum Orientations Indicated from the Visual Representations in Analysed Slovenian Chemistry Textbook Sets for Secondary School with Respect to the Curriculum Topics (Related to 4th RQ) The average number of different curriculum orien- tations indicated from the visual representations for stu- dents in the analysed Slovenian chemistry textbook sets for secondary school is given in Table 8. From Table 8, it can be derived that the largest num- ber of subcategories of curriculum orientation, with more than 5% of the analysed visual representations for students, can be recognised within the topic ‘Chemical Reaction as Change of Substance and Energy’ (4 subcategories: Struc- ture of the discipline orientation, Everyday life orientation, Environmental orientation, and Technology and industry orientation), followed by ‘Safe Experimental Work’, ‘Build- ing Blocks of Matter’, ‘Amount of Substance and Chemical Equations as Symbolic Representations’ (3 subcategories: Structure of the discipline orientation, Everyday life orienta- tion, and History of chemistry orientation) and ‘Solutions’ and ‘Properties of Selected Elements and Compounds in Biological Systems and Modern Technologies’ (3 subcat- egories: Structure of the discipline orientation, Everyday life orientation, and Technology and industry orientation). However, in other six curriculum topics for secondary school, only two subcategories prevail with more than 5% of the activities for students (2 subcategories: Structure of the discipline orientation and Everyday life orientation). The analysis of the secondary textbook sets in relation to the visual representations revealed that the curriculum orientation Structure of the discipline predominates in the topics of the National Chemistry Curriculum for Second- ary School (Fm ranges from 52.95% to 91.87%), with the exception of the topics ‘Solutions’ (M = 20.25 activities, FM = 44.89%), in which about the same number of visual rep- resentations indicate Everyday life orientation (M = 18.75 activities, FM = 44.12%), and ‘Properties of Selected Ele- ments and Compounds in Biological Systems and Modern Technologies’ (M = 7.00 activities, FM = 22.19%), in which the most common curriculum orientation is Everyday life orientation (M = 18.75 activities, FM = 55.34%). Everyday life orientation is the second most common curriculum orientation, as can be found from the analysed visual representations (FM ranges from 7.14% to 44.12%), with the exception of the already discussed topic ‘Prop- erties of Selected Elements and Compounds in Biological Systems and Modern Technologies’ (M = 18.75 activities, FM = 55.34%) and the topic ‘Building Blocks of Matter’ (M = 4.00 activities, FM = 8.86%), with the second most com- mon orientation being History of chemistry orientation (M = 7.50 activities, FM = 13.16%). In contrast, there were no visual representations in the typical Slovenian chemistry textbooks within all sec- ondary school curriculum topics that indicated Socio-sci- entific orientation (M = 0.00 activities, FM = 0.00%). In addition, none or less than 5 % of visual representations within all secondary school curriculum topics indicate History of chemistry orientation (FM ranges from 0.99% to 4.23%), with the exception of ‘Safe Experimental Work’ (M = 1.75 activities, FM = 5.09%), ‘Building Blocks of Matter’ (M = 7.50 activities, FM = 13.16%), and ‘Amount of Sub- stance and Chemical Equations as Symbolic Representa- tions’ (M = 3.75 activities, FM = 9.15%), Environmental orientation (FM ranges from 0.00% to 2.06%), except for the topic ‘Chemical Reaction as Change of Substance and Energy’ (M = 3.50 activities, FM = 10.59%) and Technology and industry orientation (FM ranges from 0.00% to 3.76%), with the exception of the topics ‘Chemical Reaction as Change of Substance and Energy’ (M = 2.50 activities, FM = 6.48%), ‘Solutions’ (M = 3.25 activities, FM = 7.74%) and ‘Properties of Selected Elements and Compounds in Bio- logical Systems and Modern Technologies’ (M = 7.25 ac- tivities, FM = 20.38%). The results indicate that in most topics of the Na- tional Chemistry Curriculum for Secondary School, simi- lar to the activities for students, the visual representations mainly focus on chemical theories, facts, and their inter- relationships.13 In this case, too, the exception is the topic Properties of Selected Elements and Compounds in Bio- logical Systems and Modern Technologies’, and additional- ly the topic ‘Solutions’. However, the visual representations in analysed Slovenian chemistry textbook sets for second- ary school show a greater variety of contexts in some top- ics, which relate not only to questions of everyday life and the chemical knowledge required for this, but in some top- ics also to contexts related to history, the environment and technology, but also there without pronounced socio-sci- entific issues.13,45 It can be derived from Tables 4 to 8 that the num- ber of different curriculum orientations indicated by both the activities and visual representations varies between the textbook sets, with the exception of the activities and visual representations that indicate socio-scientific cur- riculum orientation. This suggests that primary and sec- ondary textbook set authors recognise the potential of each curriculum topic for the use of activities and visual representations that indicate different curriculum orien- tations in different ways. This confirms the influence of textbook set authors’ personal views on the textbook sets as representations of the intended curriculum for chem- istry.12 5. Conclusions Textbook sets are one of the most important teach- ing aids that support the effective teaching and learning of chemistry in primary and secondary schools. They contain various components, with activities for students and visual representations having the greatest potential to influence 156 Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the Table 8: The proportion of curriculum orientations indicated from the visual representations (VRs) for students within the particular topics of the analysed Slovenian chemistry textbook sets for secondary school The topics of the National Chemistry Curriculum for Secondary School (1st, 2nd and 3th Year) Curriculum orientations indicated from visual representations (VRs) for students Structure of the discipline orientation History of chemistry orientation Everyday life orientation Environmental orientation Technology and industry orientation Socio­scientific orientation MSUM f (%) M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­ max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­max M[a] Min­ max fM(%)[b] Min­ max Safe Experimental Work 24.00 17-36 68.63 50.00- 85.00 1.75 1-3 5.09 2.70- 8.82 9.75 2-14 25.61 10.00- 41.18 0.25 0-1 0.68 0.00-2.70 0.00 0-0 0.00 0.00-0.00 0.00 0-0 0.00 0.00-0.00 35.75 20-52 100.00 Building Blocks of Matter 42.75 23-64 75.47 52.17- 86.49 7.50 1-12 13.16 3.57- 26.09 4.00 3-6 8.86 4.05- 14.29 0.00 0-0 0.00 0.00-0.00 1.25 0-4 2.51 0.00-8.70 0.00 0-0 0.00 0.00-0.00 55.50 28-74 100.00 Compounds and Bonding 93.50 62-130 66.54 55.36- 79.47 3.75 1-8 2.44 0.89- 4.19 40.25 26-53 30.63 17.22- 42.86 0.00 0-0 0.00 0.00-0.00 0.50 0-1 0.39 0.00-0.89 0.00 0-0 0.00 0.00-0.00 138.00 98-191 100.00 Amount of Substance and Chemical Equations as Symbolic Representa­ tions 25.75 15-38 61.99 50.00- 78.38 3.75 2-5 9.15 5.41- 11.90 11.00 6-16 27.97 16.22- 38.10 0.00 0-0 0.00 0.00-0.00 0.50 0-2 0.89 0.00-3.57 0.00 0-0 0.00 0.00-0.00 41.00 29-56 100.00 Chemical Reaction as Change of Substance and Energy 19.00 12-27 52.95 37.50- 75.00 0.75 0-2 2.26 0.00- 6.25 10.00 2-16 27.74 5.56- 39.02 3.50 0-9 10.59 0.00- 28.13 2.50 0-6 6.48 0.00-14.63 0.00 0-0 0.00 0.00-0.00 35.75 32-41 100.00 Alkali Metals and Halogens 16.25 8-21 59.06 44.44- 77.78 1.00 1-1 3.94 2.50- 5.56 9.00 3-16 33.28 11.11- 50.00 0.75 0-3 1.88 0.00-7.50 0.50 0-2 1.85 0.00-7.41 0.00 0-0 0.00 0.00-0.00 27.50 18-40 100.00 Solutions 20.25 4-37 44.89 9.52- 67.27 0.50 0-2 1.19 0.00- 4.76 18.75 15-23 44.12 27.27- 54.76 1.00 0-3 2.06 0.00-5.45 3.25 0-13 7.74 0.00-30.95 0.00 0-0 0.00 0.00-0.00 43.75 36-55 100.00 Chemical Reaction Rates and Equilibrium 123.25 93-134 70.11 59.62- 77.78 5.75 3-9 3.25 1.75- 4.62 38.00 27-48 21.78 15.79- 28.85 2.00 0-5 1.23 0.00-3.21 6.25 5-7 3.62 2.56-4.49 0.00 0-0 0.00 0.00-0.00 175.25 156-195 100.00 The Elements in the Periodic Table 29.5 19-42 66.80 51.79- 82.35 2.00 0-4 4.23 0.00- 7.14 11.5 6-23 25.22 14.04- 41.07 0.00 0-0 0.00 0.00-0.00 1.75 0-5 3.76 0.00-8.77 0.00 0-0 0.00 0.00-0.00 44.75 32-57 100.00 Properties of Selected Elements and Compounds in Biological Systems and Modern Technologies 7.00 4-11 22.19 9.52- 39.29 0.75 0-2 2.08 0.00- 4.76 18.75 13-23 55.34 46.43- 65.63 0.00 0-0 0.00 0.00-0.00 7.25 3-13 20.38 10.71- 30.95 0.00 0-0 0.00 0.00-0.00 33.75 28-42 100.00 Structure and Nomenclature of Organic Compounds 163.00 80-277 91.87 85.11- 94.63 1.75 0-4 0.99 0.00- 2.68 11.00 4-18 7.14 2.68- 14.89 0.00 0-0 0.00 0.00-0.00 0.00 0-0 0.00 0.00-0.00 0.00 0-0 0.00 0.00-0.00 175.75 94-297 100.00 Structure and Properties of Organic Compounds 428.00 234-674 62.71 47.27- 76.24 7.00 4-12 1.06 0.64- 1.41 210.75 152-275 33.40 21.04- 46.46 10.25 7-17 1.75 0.79-3.43 6.75 3-10 1.09 0.57-2.02 0.00 0-0 0.00 0.00-0.00 662.75 495-884 100.00 [a] M was calculated as the average of the number of identified visual representations in the textbook sets within the category of specific curriculum orientation and within the specific curriculum topics, thereby min and max represent the minimum and maximum number of identified visual representations in the textbook sets. [b] fM(%) represents the proportion of M within each curriculum topic, thereby min and max fM(%) represent the minimum and maximum number of identified visual representations between the textbook sets. 157Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the teaching practice8 and being essential to the development of students’ knowledge of chemistry.47 As representations of the intended chemistry curriculum2,3 textbook sets can direct to the orientation of the chemistry curriculum.12 Ei- lks and his colleagues13 have defined six basic orientations of the chemistry curriculum, which are guiding principles for structuring the whole curriculum and/or approaches for teaching a particular chemistry subject matter. This paper presents an analysis of the intended chem- istry curriculum in Slovenia, as represented by chemistry textbook sets in primary school (8th and 9th grade) and secondary school (1st, 2nd, and 3rd year), from the perspec- tive of curriculum orientations indicated by the activities for students and visual representations related to the topics of the National Chemistry Curriculum.4,5 Regarding the activities for students and visual rep- resentations in the analysed Slovenian chemistry textbook sets for primary school, the results show the dominance of the chemistry curriculum orientation structure of the discipline and, especially for organic chemistry topics, also the everyday life orientation. The greatest diversity of ac- tivities for students and visual representations in primary school related to curriculum orientation could be found in the topic ‘Hydrocarbons and Polymers’, where the ana- lysed part of the textbook set also indicates environmental orientation and technology and industry orientation. The greatest diversity among the visual representations could be found in the topic ‘Chemistry is a World of Matter’ in which the visual representations also refer to the history of chemistry and the technology and industry orientation. The other curriculum orientations in terms of activities for students and visual representations are less common in most other topics of the National Chemistry Curriculum for Secondary School, with the lack of socio-scientific ori- entation being particularly noticeable. With regard to the activities for students and visual representations in the analysed Slovenian chemistry text- book sets for secondary school, the results indicate that the chemistry curriculum orientation structure of the discipline prevails, and that the everyday life orientation is present. The everyday life orientation is particularly present in the topic ‘Properties of Selected Elements and Compounds in Biological Systems and Modern Technologies’. In the men- tioned topic, it is also possible to find the greatest variety of activities for secondary school students in terms of cur- riculum orientation, with the analysed part of the textbook set also indicating the technology and industry orientation. The greatest diversity among the visual representations could be found in the topic ‘Chemical Reaction as Change of Substance and Energy’ in which the visual representa- tions also refer to the environmental orientation and the technology and industry orientation. As with the analysis at the primary school level, the other curriculum orientations in terms of activities for students and visual representations are relatively rare in most of the other topics of the National Chemistry Curriculum for Secondary School. The findings that the activities for students and the visual representations focus more on learning theoret- ical concepts and facts than on the interaction of chem- istry with technology and society,13 and the lack of use of socio-scientific orientation indicates that the intended chemistry curriculum for primary and secondary school, as represented by the activities and visual representations in the textbook sets, still has much potential to approach modern chemistry curricula that incorporate more holis- tic approaches and integrate the learning of concepts and theories through different contexts from everyday life, technology and society.21-24 They also point to a possibly missed opportunity to develop students’ scientific liter- acy13,17,42 and to achieve the goals of discipline-oriented education for sustainable development,22,43,44 as well as to the possibility of further improving the intended chemis- try curriculum for primary and secondary school as pre- sented in the textbooks. The results of the presented study are particularly important because Slovenia has just started to reform the curricula of all subjects in primary and secondary school, including chemistry. After the implementation of the cur- riculum reform, the existing textbooks will be revised, and it would be beneficial for the students if the results of the study could be taken into account. It is important to note that in our study chosen seg- ments of the textbook sets (the activities for students and visual representations) seem to be a fundamental part of the textbook sets, but we are aware that their ability to ful- ly reveal curriculum orientation is limited.78 Therefore, it would be valuable to consider future research opportuni- ties to analyse the textbook sets also from the perspective of further textbook segments to provide a more holistic insight. 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Hofstein (Eds.): Relevant Chemistry Education, SensePublishers, Rotterdam, Nether- lands, 2015, pp. 1–10. DOI:10.1007/978-94-6300-175-5_1 81. I. Eilks, A. Hofstein, in: K. S. Taber, B. Akpan (Eds.): Science Education, SensePublishers, Rotterdam, Netherlands, 2017, pp. 169–181. DOI:10.1007/978-94-6300-749-8_13 160 Acta Chim. Slov. 2024, 71, 143–160 Hrast and Ferk Savec: Textbook Sets Through the Perspective of the Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Učbeniki imajo osrednjo vlogo pri poučevanju in učenju kemije in predstavljajo predvideni učni načrt za kemijo na nacionalni ravni. Prispevek se osredinja na analizo predvidenega učnega načrta za kemijo, kot ga predstavljajo vizualne reprezentacije in aktivnosti za učence oz. dijake v učbeniških setih v povezavi z vsebinskimi sklopi nacionalnega učne- ga načrta za kemijo za osnovno in srednjo šolo. Analiza, ki je vključevala s strani nacionalnih predstavnikov potrjene učbeniške komplete za šolsko leto 2021/2022, temelji na šestih osnovnih usmeritvah kemijskega učnega načrta, ki jih je opredelil Eilks s sodelavci. Rezultati so pokazali, da v analiziranih slovenskih učbeniških kompletih za kemijo tako za osnovno kot za srednjo šolo pri večini vsebinskih sklopov prevladuje usmerjenost v strukturo discipline, prisotna pa je tudi usmerjenost v vsakdanje življenje. Za namen izboljšanja relevantnosti učbeniških kompletov za učence je potrebno preseči trenutno redko prisotnost usmerjenosti v zgodovino kemije, okolje, tehnologijo in industrijo ter socio-nara- voslovni kontekst, npr. z vključevanjem večje interakcije kemije, tehnologije in družbe. Dragoceno bi bilo, če bi nadaljnje raziskave naslavljale predvideni učni načrt za kemijo tudi z bolj celostnega vidika. 161Acta Chim. Slov. 2024, 71, 161–169 Ahmed et al.: Quality by Design Based Development of Electrospun ... DOI: 10.17344/acsi.2023.8538 Scientific paper Quality by Design Based Development of Electrospun Nanofibrous Solid Dispersion Mats for Oral Delivery of Efavirenz Md. Faseehuddin Ahmed,1,2 Kalpana Swain,1 Satyanarayan Pattnaik1 and Biplab Kumar Dey2,* 1 Talla Padmavathi College of Pharmacy, Warangal, India 2 Faculty of Pharmaceutical Sciences, Assam Down town University, Guwahati, Assam, India * Corresponding author: E-mail: drbiplabdey9@gmail.com Mobile-+91-7386752617 Received: 11-14-2023 Abstract Poor aqueous solubility often results in poor dissolution behavior and, consequently, poor bioavailability for those drugs whose intestinal absorption is dissolution rate limited. It is essential for formulation scientists to identify strategies to improve the solubility and dissolution rate of candidate drugs in order to improve their bioavailability. The present study investigated electrospun polymeric nanofibers for efavirenz (an antiretroviral drug), a Class II drug in the Biopharma- ceutical Classification System. In order to fabricate nanofibers, hydrophilic spinnable polymer like Soluplus was used. Statistical design of experiments was used to optimize electrospinning parameters. Scanning electron microscopy (SEM) studies confirmed the presence of nanofibrous material in the mat. The x-ray diffraction (XRD) and differential scanning calorimetry (DSC) studies advocated the amorphization of efavirenz in the nanofiber samples. The optimized nanofib- er-based platform significantly improved in vitro dissolution of efavirenz (89.0 ± 3.2% in 120 minutes) compared to pure efavirenz crystals (27.3 ± 2.4%). Keywords: Electrospinning; Nanomedicine; Bioavailability; Absorption; Efavirenz 1. Introduction The recent past has witnessed tremendous efforts to develop new chemical entities with promising therapeu- tic efficacy via artificial intelligence tools.1,2 Though the drug candidates exhibited desired therapeutic response during pharmacological screening, many could not reach the market due to poor oral bioavailability which is mostly due to poor aqueous solubility, poor intestinal permeabil- ity, or both.3–7 The effective and optimal therapeutic out- comes of drugs are often related to the availability of the drug in sufficient quantity at the desired site of action. The rate-limiting step for the drugs which exhibit poor soluble but good permeability is the dissolution in biological fluids in vivo. Most of the recently developed drug candidates suffer from the issues of poor solubility and hence the pharmaceutical industry and researchers across the world are seeking a viable solution to this challenge. In this scenario, nanotechnology has offered some wonderful platform technologies for improving the oral bioavailability of drug substances.8–17 Various nanotech- nology-based solutions to improve oral bioavailability, via solubilization of candidate drugs, include nanocrystals, nanomorphs, nanosuspensions, nanocapsules, lipid-based nanoparticles, dendrimers, polymeric nanocarriers, amor- phous solid nanodispersions, nanofibers, etc. Amorphous product development has become a common choice for enhancing the solubility of poorly sol- uble pharmaceutical compounds. In these cases, the crys- talline lattice of the drug substance undergoes disruption, leading to a higher energy state in the amorphous form, thereby improving solubility. The role of polymers in the development of amorphous products includes stabilizing the amorphous system through the prevention of devitrifi- cation and ensuring improved physical stability under var- ious accelerated conditions, such as elevated temperature and relative humidity. In comparison to other document- ed solubilization methods, amorphous solid dispersions are particularly favored for low-solubility drugs. The sus- 162 Acta Chim. Slov. 2024, 71, 161–169 Ahmed et al.: Quality by Design Based Development of Electrospun ... tained supersaturation of these products in the gastroin- testinal tract contributes to the enhanced bioavailability of the drug. Controlled supersaturation allows for increased drug absorption compared to conditions where a saturated solution is maintained. Though solid dispersion technology has tremen- dous capabilities to formulate amorphous drug products, stability issues related to devitrification remain a key challenge. Hot melt extrusion technology as a method of developing amorphous solid dispersion has numerous benefits but limits the processing of thermolabile sub- stances. In this situation, nanofiber technology may be a viable solution to develop amorphous solid dispersions of candidate drugs. Electrospinning is a manufacturing method employed for the creation of extremely fine fib- ers, generally within the nanometer to micrometer scale. This procedure entails applying an electric field to either a polymer solution or melt, leading the material to be pulled into fine fibers due to the influence of electrostatic forces. The outcome is the formation of a nonwoven mat or membrane composed of these fibers.11 Electrospinning generates a potent amorphization effect because the sol- vent evaporates instantly, leading to a solid solution of the drug in the polymer matrix.11,15,18 Due to the uniform dis- tribution of the cargo molecules inside the polymer ma- trix and possible inhibition of molecular mobility leading to impaired devitrification, the amorphous state of the loaded therapeutic ingredient is preserved for longer in the solid dispersions fabricated via electrospinning. Elec- trospinning for the fabrication of drug-loaded nanofibers has been exploited by researchers across the world for multiple purposes including attempts to improve oral bi- oavailability and controlled / sustained drug delivery.19–21 Recently, from our laboratory, we have reported improved in-vitro dissolution and ex-vivo intestinal permeation of ibuprofen (a poorly soluble drug) utilizing the nanofiber technology.15 An amphiphilic polymeric solubilizer, Soluplus® is a polycaprolactam-polyvinylacetate-polyethylene glycol graft copolymer (Figure 1). The creation of solid solutions using Soluplus can significantly increase the solubility of poorly soluble pharmaceuticals in aqueous media because of its outstanding solubilizing properties for Biopharma- ceutical Classification System (BCS) class II drugs.22 The Biopharmaceutics Classification System (BCS) serves as a framework for classifying drugs based on their solubility and permeability characteristics. It consists of four classes: Class I, II, III, and IV. Class I drugs demonstrate both high solubility and permeability, leading to enhanced bioavail- ability. Conversely, Class II drugs have high permeability but low solubility, presenting challenges to bioavailability due to incomplete dissolution. Class III drugs possess high solubility but low permeability, potentially restricting ab- sorption. In contrast, Class IV drugs, characterized by low solubility and permeability, encounter obstacles in both dissolution and absorption processes. Figure 1. Chemical structure of Soluplus. This matrix-forming polymer is quite prevalent, and because they are effective solubilizers, they have long been employed to create solid dispersions. Though, a few at- tempts were reported to prepare Soluplus®-based nanofib- er, an experimentally designed approach for optimization of formulation and process variables is lacking.23 Non-nucleoside reverse transcription inhibi- tors (NNRTIs) have been widely used in antiretroviral cocktails as a part of highly active antiretroviral thera- py (HAART). Efavirenz (EFV) is commonly prescribed NNRTI and has shown significant therapeutic benefits by lowering the viral load in patients with HIV infection (Figure 2).24 However, the drug belongs to BCS class II exhibiting poor aqueous solubility and high intestinal permeability. Subsequently, EFV exhibits a low intrinsic dissolution rate (0.037 mg/cm2/min), and poor oral bi- oavailability (40–50%).24 Such BCS class II drugs where drug dissolution is the rate-limiting step in overall oral drug absorption, strategies to improve aqueous solubility is a promising approach for improvement in oral bioavail- ability.25 Hence, there is a strong motivation to improve the dissolution velocity of EFV. Figure 2. Chemical structure of efavirenz. 163Acta Chim. Slov. 2024, 71, 161–169 Ahmed et al.: Quality by Design Based Development of Electrospun ... An electrospinning approach was explored in the present study to amorphize EFV to improve its aqueous solubility and oral bioavailability. Nanofibers were charac- terized by scanning electron microscopy (SEM), X-ray dif- fraction (XRD), and differential scanning calorimeter (DSC). The results showed that EFV was successfully amorphized and converted into nanofibers. 2. Materials and Methods 2. 1. Materials Efavirenz (EFV) was obtained as a gift sample from Cipla Ltd (Mumbai, India). Soluplus® (polyvinyl caprol- actam-polyvinyl acetate-polyethylene glycol graft co-pol- ymer; CAS Number-402932-23-4) was obtained as a gift sample from BASF Corporation, New Jersey. Ethanol (an- alytical grade) was obtained from Sigma-Aldrich Corp (India). All other chemicals used were of analytical grade and procured locally. 1. 2. Development of Nanofibers 2. 2. 1. Design of Experiments (DoE) Statistical design of experiment (DoE) was used for the development of electrospun nanofibers.26–29 Diverse factors which include electrospinning setup variables, working fluid variables, and ambient variables, influence the electrospinning process for the fabrication of poly- meric nanofibers.11 Hence, there is a need for optimiza- tion of these variables during development. A response surface randomized Box-Behnken quadratic design with 17 runs was deployed for product development. Three im- portant variables i.e., DC voltage, flow rate, and polymer concentration, are studied at three levels and are coded as –1(low), 0(moderate), and +1(high). The DC voltage was varied at 10kV (–1), 14kV (0), and 18 kV (+1). The flow rate was varied at 0.4 ml/h (–1), 0.8 ml/h (0), and 1.2 ml/h (+1). The Soluplus concentration was also varied at three levels i.e., 45% w/w (–1), 50 % w/w (0), and 55 % w/w (+1). The cumulative percent of drug released was measured for each run as a dependent response. Design-Expert® soft- ware (Version-13; Stat-Ease, Inc., Minneapolis) was used to generate and process the design. The nanofiber formu- lation with maximum drug dissolution (at 120 minutes) was selected as the optimized product for further charac- terization. 2. 2. 2. Fabrication of Nanofibers Electrospinning equipment (Super ES 2; E-Spin Na- notech, India) was used for the preparation of the nanofib- er mats. The electrospinning setup includes a high-voltage DC supply (up to 50kV), and a syringe pump to control the volumetric flow rate of the working solution with the option to control the temperature and humidity of the electrospinning chamber. The electrospinning solution consisted of Soluplus® with efavirenz in ethanol (Table 1). The concentration of EFV was fixed for all the runs, but the concentration of Soluplus® varied from 45 to 55% w/w. The stated amount of EFV was initially dissolved in ethanol and subsequently, the drug was dissolved in the polymer solution. According to the Box-Behnken design, electrospinning was carried out at an applied DC voltage of 10, 14, or 18 kV with a volumetric flow rate of 0.4, 0.8, or 1.2 ml/h and spinneret to collector distance of 12 cm (Table 1). The application of high voltages, usually within the kilovolt range, carries the potential for electrical shock hazards. Consequently, measures were taken to guaran- tee the proper grounding of equipment before initiating operations, and strict adherence to safety protocols, in- cluding the use of suitable personal protective equip- ment (PPE), was maintained when working with high voltages. 2. 3. Drug Entrapment Efficiency (EE) The efficiency of efavirenz entrapment in the fabri- cated nanofiber mats was assessed as follows. EE (%) = (EFV content measured in the sample/Ac- tual amount of EFV added) × 100 % All tests were repeated in triplicate and the mean is reported. Dissolving the generated fibers in ethanol al- lowed the amount of efavirenz in them to be determined. To assess the amount of efavirenz in each sample, the solu- tions were spectrophotometrically analyzed at 248 nm.30 Table 1. Experimental runs for fabrication of efavirenz loaded na- nofibers following Box-Behnken design. Voltage (kV): 10 (–1), 14 (0), 18 (+1); Flow Rate (ml/h): 0.4 (–1), 0.8 (0); 1.2 (+1); Soluplus conc (% w/w): 45 (–1), 50 (0), 55 (+1). The numbers inside the pa- rentheses (–1, 0, +1) indicate the levels of the variable (low, moder- ate, and high). Run Voltage Flow Rate Soluplus Cumulative (kV) (ml/h) Conc (%) Percent Drug Released (CPD) (%) F1 0 (14) –1(0.4) 1(55) 79.28 F2 0(14) 0(0.8) 0(50) 89.45 F3 –1(10) –1(0.4) 0(50) 75.22 F4 1(18) 0(0.8) –1(45) 82.43 F5 –1(10) 0(0.8) 1(55) 65.74 F6 –1(10) 1(1.2) 0(50) 65.82 F7 0(14) 1(1.2) –1(45) 80.16 F8 1(18) 1(1.2) 0(50) 69.84 F9 0(14) 1(1.2) 1(55) 78.52 F10 –1(10) 0(0.8) –1(45) 77.59 F11 1(18) 0(0.8) 1(55) 64.52 F12 0(14) –1(0.4) –1(45) 82.18 F13 1(18) –1(0.4) 0(50) 64.13 164 Acta Chim. Slov. 2024, 71, 161–169 Ahmed et al.: Quality by Design Based Development of Electrospun ... 2.4. In Vitro Dissolution Studies As part of the dissolution study, samples of naive efa- virenz (100 mg) or nanofiber samples equivalent to 100 mg EFV were loaded into hard gelatin capsules and tied to pad- dles. We used 900 ml of 0.2% sodium lauryl sulfate (SLS) in 0.1 N HCl as dissolution media and the study was carried out under sink conditions. Spectrophotometric analyses of the samples at 248 nm were performed at predetermined intervals for 120 minutes following any removal of the solu- tion, filtering, and subsequently analyzing the data using the UV-160 (Shimadzu, Japan) spectrophotometer. It was necessary to replace the same amount of medium at the same temperature to maintain the sink condition. At least three repetitions were used to calculate the average of the experimental points. The nanofiber sample that demon- strated the highest drug release underwent additional char- acterization using Scanning electron microscopy, differen- tial scanning calorimetry, and x-ray diffractometry. 2. 5. Scanning Electron Microscopy (SEM) Using the S-3700N scanning electron microscope (Hitachi, Okinawa, Japan), fiber morphology and diameter were examined. A sputter coater was used to coat samples with gold at 20 nm under vacuum. An acceleration voltage of 5 kV was used for all micrographs. Everhart-Thornley detectors were used to detect secondary electrons. An ad- equate number of measurement points in the image were manually determined to assess the diameter of the nano- fibers after a careful calibration of the instrument for size determination. 2. 6. X-ray Diffraction Studies The X-ray diffractometer uses X-rays to fire at the samples and then analyzes diffracted patterns. It is possi- ble to determine the crystallinity of samples by measuring their diffracted patterns. Diffractometer parameters such as voltage, current, and angular range are optimized to ac- curately assess crystallinity. A Panalytical X'Pert Pro X-ray diffractometer (Model: Panalytical, X'Pert Pro, UK) was used to evaluate the crystallinity of the optimized nanofib- er sample and native EFV sample using nickel-filtered Cu Ka radiation (k = 1.54 A). During the measurement, the voltage and current were 35 kV and 30 mA, respectively, and were smoothed to 95. Measurements were carried out in the angular range from 6° to 50° (2θ) using step sizes 0.02 and 0.01s per step. 2. 7. Differential Scanning Calorimetry Differential scanning calorimetry (DSC 3+, Mettler Toledo) was used to study the thermal behavior of native EFV and drug-loaded optimized nanofibers. Through this technique, the researchers were able to measure the amount of heat released or absorbed when the nanofibers underwent a physical or chemical change. It allowed them to determine how the nanofibers would respond to chang- es in temperature and characterize their thermal behav- ior. Approximately 5 mg of powdered sample was placed in an aluminum pan (40 μL standard aluminum crucible with pierced lid), making sure that the crucible base and the pan surface were uniformly in contact. The pan was sealed and heated to 200 degrees Celsius at a temperature ramp rate of 10 degrees Celsius per minute under nitrogen gas (40 ml/min). A five-minute equilibration period was followed by each measurement of samples at 30 °C. For each peak, Mettler Toledo software calculated the transi- tion temperatures and enthalpies. 2. Results and Discussions 2. 1. Design of Experiments (DoE) Quality by Design (QbD), is a systematic approach to pharmaceutical development that is used to ensure the quality of pharmaceutical products. It is a proactive ap- proach that focuses on building quality into the product from the beginning rather than relying on quality testing at the end of the manufacturing process. DoE (Design of Experiments) is a statistical method used in QbD to systematically explore and optimize process parameters and their interactions. By using DOE, manufacturers can identify the most critical process parameters and their optimal settings to achieve the desired quality objectives. The principles of DoE have been widely deployed by phar- maceutical researchers for the development and optimi- zation of drug products.28,29 The present study adopted a Box-Behnken study design and the data were fitted to a quadratic model. The critical electrospinning variables like DC voltage, working fluid flow rate, and Soluplus con- centration was chosen and varied at three levels to assess their influence on the dependent response (Table 1). The sequential model sum of squares selects the highest-order polynomial where the terms are significant and the mod- el is not aliased. 29 Here the cubic model was aliased and hence quadratic model was selected. Further, the model summary statistics indicate that the quadratic model was suitable for analyzing the dependent response. Interested readers are invited to refer to the supplementary material available in Appendix 1 for the sequential model sum of squares and model summary statistics information. The equation in coded values generated for the quad- ratic model is as follows. Cumulative percent drug released (CPD) = +89.45– 0.4312A –0.8087 B –4.29 C+3.78 AB –1.52 AC+0.3150 BC –14.08 A²–6.62 B² –2.80 C² The coded factors A, B, and C represent DC voltage, flow rate, and Soluplus concentration, respectively. The equation in terms of coded factors can be used to make predictions about the response for given levels of each fac- 165Acta Chim. Slov. 2024, 71, 161–169 Ahmed et al.: Quality by Design Based Development of Electrospun ... tor. By default, the high levels of the factors are coded as +1 and the low levels are coded as –1. To assess the significance of the model terms, an analysis of variance (ANOVA) was performed. The F-Val- ues and p-values were monitored for the purpose of iden- tifying the significant model terms (Table 2). The Model F-value of 11.18 implies the model is significant. There is only a 0.22% chance that an F-value this large could oc- cur due to noise. p-values less than 0.0500 indicate model terms are significant. In this case, C, A², B² are significant model terms (Table 2). Values greater than 0.1000 indicate the model terms are not significant.25 The coded equation is useful for identifying the relative impact of the factors by comparing the factor coefficients. The factor coefficients are presented in Table 3. The coefficient estimate repre- sents the expected change in response per unit change in factor value when all remaining factors are held constant. The intercept in an orthogonal design is the overall average response of all the runs. The coefficients are adjustments around that average based on the factor settings. The high- er the absolute value of the coefficient estimate the high- er the influence of the factor on the dependent response. In the present case, the highest estimate was observed for Soluplus concentration followed by flow rate and voltage indicating the predominant impact of Soluplus concentra- tion on the dependent response, i.e., cumulative percent drug released (Table 3). This is also evident from the 3d surface plot represented in Fig 3. 3. 2. Drug Entrapment Efficiency The loading of drugs into drug delivery systems is one of the most important factors to consider when eval- uating the suitability of drug carrier systems. In addition to blending (in which the drug is dissolved or dispersed in a polymer solution), surface modification (in which the drug is conjugated to the nanofiber surface), coaxial pro- cessing (co-electrospinning of a drug solution as the core and a polymer solution as the sheath), etc, there are several other ways in which drugs can be loaded into polymeric nanofibers.11,31 For the loading of efavirenz in the present study, the blending method was used. The efavirenz load- ing efficiency in the fabricated electrospun nanofibers was as high as 98.4 ± 4.2% w/w. Table 2. ANOVA for Quadratic model. Source Sum of Squares df Mean Square F-value p-value Model 1349.89 9 149.99 11.18 0.0022 A-Voltage 1.49 1 1.49 0.1109 0.7489 B-Flow rate 5.23 1 5.23 0.3901 0.5521 C-Soluplus Conc 147.06 1 147.06 10.96 0.0129 AB 57.08 1 57.08 4.26 0.0780 AC 9.18 1 9.18 0.6845 0.4353 BC 0.3969 1 0.3969 0.0296 0.8683 A² 834.87 1 834.87 62.24 < 0.0001 B² 184.31 1 184.31 13.74 0.0076 C² 32.98 1 32.98 2.46 0.1609 Residual 93.89 7 13.41 Lack of Fit 93.89 3 31.30 Pure Error 0.0000 4 0.0000 Cor Total 1443.79 16 Table 3. Coefficient estimates in Terms of Coded Factors. Factor Coefficient df Standard 95 % 95 % VIF Estimate Error CI Low CI High Intercept 89.45 1 1.64 85.58 93.32 A-Voltage –0.4312 1 1.29 –3.49 2.63 1.0000 B-Flow rate –0.8087 1 1.29 –3.87 2.25 1.0000 C-Soluplus Conc –4.29 1 1.29 –7.35 –1.23 1.0000 AB 3.78 1 1.83 –0.5526 8.11 1.0000 AC –1.52 1 1.83 –5.85 2.82 1.0000 BC 0.3150 1 1.83 –4.02 4.65 1.0000 A² –14.08 1 1.78 –18.30 –9.86 1.01 B² –6.62 1 1.78 –10.84 –2.40 1.01 C² –2.80 1 1.78 –7.02 1.42 1.01 166 Acta Chim. Slov. 2024, 71, 161–169 Ahmed et al.: Quality by Design Based Development of Electrospun ... Figure 3. Three dimensional response plot of the experimental runs. 3. 3. In Vitro Dissolution Studies In the case of BCS II drug candidates such as efa- virenz, dissolution is the rate-limiting step in oral absorp- tion. An effective oral delivery strategy would be to improve the dissolution of the drug in such a situation. In this study, raw efavirenz (EFV) dissolution was found to be slow and incomplete (27.3 ± 2.4%). Compared with native raw efa- virenz (EFV), the dissolution profiles of the studied samples (Figs. 4 and 5) revealed that the nanofiber samples released the drug significantly faster (p < 0.05) than native raw efa- virenz (EFV). The formulation F2 (EFV-NF) released the highest percentage of efavirenz (89.0 ± 3.2%) from the na- nofiber samples which may be due to the amorphous state of the drug.6 We previously reported an improvement in dissolution when co-processing with hydrophilic polymers such as hydroxypropyl methylcellulose and polyvinyl pyrro- lidone.32 The optimized formulation F2 (EFV-NF) was fur- ther subjected to other instrumental characterization. 3. 4. Scanning Electron Microscopic Investigation An electron microscope is a powerful tool for ob- serving the morphology and structure of a sample at a very high resolution. By coating the sample with gold, the sput- ter coater prevents charge build-up on the sample, which can distort images. This gold layer acts as a conductive lay- er on the sample. Focus depth and resolution are affected by the acceleration voltage used in the micrographs. Sec- ondary electrons are detected by the Everhart-Thornley detector, which improves contrast and resolution. SEM confirmed the presence of nanofibers (Fig. 6). Inspect- ing the nanofibers (optimized nanofiber formulation i.e. EFV-NF and polymer-only nanofiber) with SEM revealed that their diameter ranged within 250–275 nm. The diam- eter of the nanofibers, particularly for EFV-NF, remained largely unaffected by drug loading. However, SEM analysis was not conducted for the other nanofiber formulations, preventing the reporting of additional findings for those variants. No crystals of the drug were visible on the SEM images of efavirenz-loaded nanofibers (D), indicating that the loaded efavirenz was dispersed molecularly in the pol- ymer matrices. However, scanning electron microscopy (SEM) images of pure efavirenz showed a clear crystalline structure. 3. 5. X-ray Diffraction Studies The prepared samples were analyzed by X-ray dif- fraction to assess whether any polymorphic transitions took place in efavirenz when formulated as nanofibers (EFV-NF). The X-ray diffraction patterns of native efa- virenz, as a physical mixture with the matrix-forming polymer, and efavirenz-loaded nanofibers are depicted in Fig. 7. The XRD pattern of efavirenz alone (EFV) ex- hibited high-intensity peaks at diffraction angles 6.12°, 10.41°, 12.28°, 13.25°, 14.21°, 16.90°, 21.24°, and 24.90° (2θ) which revealed its crystalline nature. In contrast, efa- Figure 4. The dissolution profile of the nanofiber samples from the experimental runs. Figure 5. The dissolution profile of the naïve efavirenz (EFV), the phys- ical mixture (PM), and the optimized nanofiber sample (EFV-NF). 167Acta Chim. Slov. 2024, 71, 161–169 Ahmed et al.: Quality by Design Based Development of Electrospun ... virenz-loaded nanofibers showed broad and diffuse maxi- ma, which may be attributed to efavirenz's amorphization here (EFV-NF). Efavirenz (PM) samples prepared by mix- ing efavirenz with Soluplus also retained their crystalline properties. Drug substances in the amorphous state pos- sess many advantages over their crystalline counterparts, including improved solubility, wettability, and dissolution rate.30,31 3. 6. Differential Scanning Calorimetry (DSC) As can be seen from Fig. 8, the DSC thermograms of samples correlate well with the XRD results. A sharp endothermic peak was identified for efavirenz alone (EFV) at 139.85 °C, the melting point of the drug, demonstrating its crystalline nature.33 The physical mixture sample (PM) retained efavirenz's melting endotherm in the DSC stud- ies. A small peak was observed in the nanofiber samples (EFV-NF) in association with the melting of the efavirenz, indicating its significant amorphization in the nanofiber samples (EFV-NF).34 Thus, DSC studies agreed with the XRD analysis. This confirmed the amorphization of efa- virenz in the electrospun nanofibers. The nanofibers thus presented an improved solubility and dissolution rate of efavirenz as observed in the in vitro studies. 4. Conclusion QbD principles were efficiently applied to the develop- ment of efavirenz-loaded nanofibrous mats with enhanced dissolution. DSC and XRD results indicate the presence of efavirenz in an amorphous state in the nanofiber matrix. It Figure 7. X-ray diffraction patterns of the studied samples indicat- ed negligible or no crystallinity in the efavirenz-loaded nanofiber sample (EFV-NF). The raw efavirenz (EFV) and physical mixture (PM) samples retained the crystalline peaks. Figure 6. Scanning electron microscopic images of various samples. (A) native efavirenz, (B) Soluplus as received, (C) physical mixture of efavirenz and Soluplus, (D) efavirenz-loaded Soluplus nanofibers, and (E) Soluplus nanofibers. 168 Acta Chim. Slov. 2024, 71, 161–169 Ahmed et al.: Quality by Design Based Development of Electrospun ... is possible that Soluplus's antinucleating properties are re- sponsible for the absence of crystalline efavirenz traces in the nanofiber samples. Further, a long-term stability study is planned to assess the shelf-life and storage conditions of the efavirenz-loaded nanofibrous mats. Additional preclinical studies are warranted based on the findings of the study. It appears that the nanofiber matrix provides a safe and effec- tive environment for the delivery of amorphous efavirenz to the body. There is still a need for further research to assess its efficacy and safety in clinical trials. Conflict of Interest The authors declare no conflict of interest. The au- thors alone are responsible for the content and writing of the article. Acknowledgment The authors would like to acknowledge the finan- cial support of All India Council for Technical Education (AICTE), New Delhi, India, under Research Promotion Scheme [File No. 8-27/FDC/RPS (POLICY 1)/2019-20]. 5. References 1. D. Paul, G. Sanap, S. Shenoy, D. Kalyane, K. Kalia, R. K. Teka- de, Drug Discov. Today 2021, 26, 80–93. DOI:10.1016/j.drudis.2020.10.010 2. L. Tripathi, P. Kumar, K. Swain, S. Pattnaik, in Drug Design Using Machine Learning, ed. by Inamuddin, Tariq Altalhi, Jor- ddy N. Cruz, Moamen Salah El-Deen Refat, John Wiley & Sons, Ltd, New Jersey, 2022, pp. 143–164. DOI:10.1002/9781394167258.ch5 3. S. Mallick, S. Pattnaik, K. Swain, P. K. De, Drug Dev. Ind. Pharm. 2007, 33, 865–873. DOI:10.1080/03639040701429333 4. S. Pattnaik, K. Swain, J. V. Rao, V. Talla, K. B. Prusty, S. K. Subudhi, RSC Adv. 2015, 5, 74720–74725. DOI:10.1039/C5RA13038G 5. S. Mallick, S. Pattnaik, K. 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Moura Ramos, M. F. M. Piedade, H. P. Diogo, M. T. Vici- osa, J. Pharm. Sci. 2019, 108, 1254–1263. DOI:10.1016/j.xphs.2018.10.050 34. Z. M. M. Lavra, D. Pereira de Santana, M. I. Ré, Drug Dev. Ind. Pharm. 2017, 43, 42–54. DOI:10.1080/03639045.2016.1205598 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Slaba vodotopnost pogosto povzroči slabo raztapljanje in posledično slabo biološko uporabnost zdravil, katerih absorp- cija v črevesju je omejena s hitrostjo raztapljanja. Farmacevtski tehnologi morajo opredeliti strategije za izboljšanje to- pnosti in hitrosti raztapljanja kandidatnih zdravil, da bi izboljšali njihovo biološko uporabnost. V pričujoči raziskavi so proučevali elektrostatsko sukanje polimernih nanovlaken z efavirenzom (protiretrovirusno zdravilo), zdravilo razreda II po biofarmacevtskem klasifikacijskem sistemu. Za izdelavo nanovlaken je bil uporabljen hidrofilni polimer, ki ga je mogoče elektrostatsko sukati, kot je Soluplus. Za optimizacijo parametrov elektrostatskega sukanja je bila uporabljena statistična zasnova eksperimentov. Študije s vrstično elektronsko mikroskopijo (SEM) so potrdile prisotnost nanovlaken. Študije rentgenske difrakcije (XRD) in diferenčne dinamične kalorimetrije (DSC) so potrdile amorfizacijo efavirenza v vzorcih nanovlaken. Optimizirana platforma na osnovi nanovlaken je v primerjavi s čistimi kristali efavirenza (27,3 ± 2,4 %) znatno izboljšala in vitro raztapljanje efavirenza (89,0 ± 3,2 % v 120 minutah). 170 Acta Chim. Slov. 2024, 71, 170–178 Petrović et al.: QSAR Modeling of Sphingomyelin Synthase 2 Inhibitors ... DOI: 10.17344/acsi.2023.8566 Scientific paper QSAR Modeling of Sphingomyelin Synthase 2 Inhibitors for Their Potential as Anti-Atherosclerotic Agents Dejan Petrović,1,2 Marina Deljanin Ilić,1,2 Dejan Simonović,2 Zoran Marčetić,3 Milovan Stojanović,1,2 Sanja Stojanović,2 Nebojša Arsić,4 Dušan Sokolović4 and Aleksandar M. Veselinović5* 1 Faculty of Medicine, University of Niš, Niš, Serbia 2 Institute for Treatment and Rehabilitation, Niška Banja, Serbia 3 Medical faculty, University of Pristina, Kosovska Mitrovica, Serbia 4 Health Center Medveđa, Medveđa, Serbia 5 Faculty of Medicine, University of Niš, Department of Biohemistry, Niš, Serbia 6 Faculty of Medicine, University of Niš, Department of Chemistry, Niš, Serbi * Corresponding author: E-mail: aveselinovic@medfak.ni.ac.rs Fax: +381 18 4238770; Phone: +381 18 4570029 Received: 11-30-2023 Abstract Sphingomyelin synthase 2 (SMS2) has emerged as a promising target for atherosclerosis threatment. However, the avail- ability of selective SMS2 inhibitors and their associated pharmacological properties remains limited. This research pa- per explores various QSAR modeling techniques applied to a range of compounds acting as SMS2 inhibitors. Multiple distinct QSAR modeling methodologies were employed, including conformation-independent, GA-MLR and 3D based QSAR modeling, and their mutual correlations were investigated, Various statistical methods were applied to assess the quality, robustness, and predictive capacity of these developed models, yielding favorable results. Furthermore, molec- ular fragments derived from SMILES notation descriptors, which account for the observed changes in the evaluated activity, were defined. The methodology presented in this research holds potential for identifying novel agents for ather- osclerosis treatment by targeting sphingomyelin synthase 2. Keywords: Sphingomyelin synthase 2, atherosclerosis, QSAR, Molecular modeling, Drug design 1. Introduction Sphingomyelin (SM) is a major phospholipid in the circulatory system, and scientific literature indicates that human plasma SM levels are an independent risk factor for coronary heart disease.1–3 Moreover, in patients with acute coronary syndrome, the measurement of human plasma SM levels can serve as a valuable prognostic tool.3 Studies have demonstrated that control mice exhibit ap- proximately one-fourth the plasma SM levels compared to apoE KO mice, and this increase in plasma SM levels may be associated with the development of atherosclerosis in these animals.4,5 Additionally, SM has been shown to have significant effects on the metabolism of apoB-containing lipoproteins, and a deficiency in SM could potentially re- duce the atherogenic properties of the mice.6,7 Inhibition of serine palmitoyltransferase (SPT), the initial enzyme involved in sphingomyelin (SM) biosynthe- sis, has been shown to reduce SM levels in mouse models.8,9 However, it is worth noting that this approach may lead to various off-target side effects because the entire de novo synthesis pathway of sphingolipids can be affected by the in- hibition of SPT. As an alternative strategy to lower SM levels, inhibiting sphingomyelin synthase (SMS) is considered. Scientific literature suggests that the overexpres- sion of sphingomyelin synthase (SMS) promotes the ac- 171Acta Chim. Slov. 2024, 71, 170–178 Petrović et al.: QSAR Modeling of Sphingomyelin Synthase 2 Inhibitors ... cumulation of atherogenic lipoproteins and increases the atherogenic potential. Conversely, in a mouse model, the alleviation of atherosclerosis is linked to the reduction of sphingomyelin (SM) accumulation due to SMS defi- ciency.10–13 Based on these findings, SMS2 emerges as a potential therapeutic target for atherosclerosis, and the development of future anti-atherosclerotic drugs may be connected to the use of selective SMS2 inhibitors. How- ever, it's important to note that the limited number of reported SMS2 inhibitors is partly attributed to experi- mental challenges, hindering their exploration as potential anti-atherosclerotic agents. The process of drug discovery and development is often exceptionally time-consuming, as it relies on various time and resource constraints. To address this challenge, chemoinformatic studies are employed. Chemoinformat- ics, which involves in silico methods, offers a wide range of applications, including the identification of novel lead compounds and the optimization of the pharmacological activity or pharmacokinetic properties of existing chemical compounds with known biological activities.14,15 Among the various chemoinformatic methods, Quantitative Struc- ture-Activity Relationship (QSAR) has emerged as the most prominent and widely used approach. In contempo- rary QSAR studies, models are constructed by employing diverse molecular descriptors derived from specific mole- cule structures, each with its own strengths and limitations. These models are then expressed as mathematical equa- tions that establish a relationship between the biological activities of the studied molecules and their chemical char- acteristics, as represented by the molecular descriptors.16–18 In this research, a variety of in silico methods were employed to identify new compounds with the potential to inhibit sphingomyelin synthase 2 (SMS2). The study de- veloped QSAR models based on the following approaches: conformation-independent molecular descriptors, utiliz- ing both SMILES notation and local graph invariants, in conjunction with the Monte Carlo optimization method; 2D molecular descriptors, with the aid of a genetic algo- rithm and multiple linear regression; and 3D field contri- bution. One of the primary objectives of the study was to identify molecular fragments or structural features that lead to SMS2 inhibition effects and to assess the correla- tions between these different methods. The research suc- cessfully identified fragments present in small molecules that are relevant to ligand-receptor interactions, which can potentially be applied in the design and development of anti-atherosclerotic agents. 2. Materials and Methods In this study, a dataset comprising 51 molecules known to exhibit inhibitory effects on SMS2 was collect- ed from the scientific literature.19,20 The general chemi- cal structures of these molecules are illustrated in Figure 1. The activities of these molecules, quantified as pIC50 values, were used as the dependent variables in the anal- ysis. The SMILES notation for all the molecules used in the study, along with their corresponding pIC50 values, is provided in Table S1 within the Supplementary Material. To ensure the robustness of the analysis, the dataset was randomly divided into three sets: a training set consisting of 38 compounds (75%) and a test set comprising 13 com- pounds (25%). The normality of the activity distribution for all the dataset splits was assessed following the meth- odology described in a published reference.21 Figure 1. General chemical structures of used molecules for QSAR models development. 2. 1. QSAR Modeling Utilizing the Monte Carlo Optimization Method The Monte Carlo optimization method was em- ployed to develop a conformation-independent QSAR model using a hybrid approach that incorporates both molecular graph and SMILES notation-based descriptors. The molecular graph-based descriptors included local graph invariants based on fundamental graph concepts like paths and walks, with their detailed mathematical definitions available in the literature.22 The optimal top- ological descriptors from the molecular graph-based ap- proach comprised Morgan extended connectivity indices of increasing orders (EC0), valence shells of range 2 and 3 (s2, s3), path numbers of length 2 and 3 (p2, p3), the count of carbon atom neighbors (Number Of Carbon), and the count of non-carbon atom neighbors (Number of Non Carbon). In contrast, SMILES notation-based mo- lecular descriptors offer a mechanistic interpretation, as they are related to molecular fragments. The numerical value of each SMILES notation descriptor for a molecule contributes to the molecule's correlation weight (DCW). This DCW is mathematically defined as the sum of all the defined SMILES descriptor correlation weights (CW), in accordance with Equation 1. DCW(T,Nepoch) = zCW(ATOMPAIR) + xCW(NOSP) + yCW(BOND) + tCW(HALO) + rCW(HARD) + αΣCW(Sk) + (1) βΣCW(SSk) + γΣCW(SSSk) 172 Acta Chim. Slov. 2024, 71, 170–178 Petrović et al.: QSAR Modeling of Sphingomyelin Synthase 2 Inhibitors ... In Equation 1, the variables z, x, y, t, α, β and γ de- note either the value 1 (indicating "yes") or 0 (indicating "no"). These values determine whether the correspond- ing SMILES descriptor is utilized in the model's develop- ment. The symbol Sk specifies the SMILES atom with one SMILES notation symbol (or two inseparable ones) and is linked to the local descriptors. are additionally construct- ed as linear combinations of two and three SMILES atoms, represented by the SSk and SSSk symbols, respectively. The second category of optimal descriptors in accordance with SMILES notation is the global descriptor, which pertains to the overall characteristics of the studied molecule. The study utilized the following global SMILES notation-based descriptors: ATOMPAIR, HALO, BOND, NOSP and HARD, all defined based on the methodology published in reference.23 The development of the QSAR model in this study involved a combination of both SMILES notation (both local and global) and local graph invariant descrip- tors. This approach enabled the calculation of the DCW for the molecules as per Equation 2. DCW(T,Nepoch) = ΣCW(Sk) + ΣCW(SSk) + ΣCW(SSSk) + ΣCW(EC0k) + ΣCW(PT2k) + ΣCW(PT3k) + ΣCW(VS2k) + ΣCW(VS3k) + (2) ΣCW(NNCk) In addition to the previously defined symbols Sk, SSk and SSSk, Equation 2 incorporates the following symbols: The Morgan connectivity index of zero order (the hydro- gen-suppressed graph was used in this research) – EC0k, paths of length of 2 and 3 – PT2k and PT3k, valence shell 2 and 3 – VS2k, and VS3k, and Nearest Neighbors – NNCk.22 The molecular descriptors mentioned above were all com- puted using the CORAL software (CORrelation and Log- ic), which can be accessed at http://www.insilico.eu/coral. Once an optimal descriptor is identified through the appli- cation of the Monte Carlo method, each descriptor is as- signed a numerical value known as the correlation weight (CW). The Monte Carlo method accomplishes this by generating suitable random numbers and observing how this fractional number corresponds to a specific proper- ty or properties. The CW value is then randomly assigned to the descriptors based on the SMILES notation for each individual Monte Carlo run and for a specified endpoint. The optimization process of the Monte Carlo method involves performing numerical calculations to determine the correlation weights that yield the maximum correla- tion coefficient value between the optimal descriptor and a given endpoint. When utilizing this method for creating a QSAR model, it's essential to consider two key parame- ters. Threshold is a coefficient used to categorize a range of molecular features, which encompass both SMILES-based indices and SMILES-based molecular fragments. These features are derived from SMILES notation and sorted into two categories: a) active ones (in this case, the modeling process involves the correlation weight); and b) rare ones (in this case, the modeling process omits the correlation weight). The process is executed as follows: If a particular mo- lecular feature (X) extracted from the SMILES notation of molecules in the training set occurs fewer than T times, then the molecule descriptor X is excluded from the model construction. Consequently, the numerical value for this feature (the correlation weight of X, CW(X)) is set to zero, categorizing it as "rare." All other molecular features that occur more frequently are considered "active" and can be employed in the model-building process. Nepoch, repre- senting the epoch number in Monte Carlo optimization, is crucial for achieving the highest statistical quality within the training set. When an unlimited number of epochs is employed, the training set attains the maximum correla- tion coefficient through the mentioned Monte Carlo op- timization. However, it's important to note that the maxi- mum correlation coefficient between the endpoint for the external test set and the optimal descriptor is achieved with a specific, finite number of epochs. The calculations favor this specific epoch number, as it offers excellent pre- dictive potential for the obtained model, provided that the number of epochs reaches this value. However, it's worth noting that an increase in the threshold (T) results in a de- crease in the correlation coefficient within the training set. Nonetheless, it is important to highlight that there exists a threshold value that maximizes the correlation coefficient of the test set. From a practical perspective, the mentioned threshold is the preferred choice. Furthermore, defining optimal values for both the threshold (T) and the Mon- te Carlo optimization epoch number (Nepoch), is essential for constructing a robust QSAR model. This construction involves the utilization of both SMILES notation and opti- mal descriptors based on the molecular graph, as outlined in reference.23 Monte Carlo method simulations are carried out using iterative algorithms to uncover the distribution of an unknown probabilistic entity. In the Monte Carlo op- timization process, the epoch number is still a part of the equation for a specific target function within the training set. The initial step involves setting the CW (SA) for each SMILES SA attribute, with all CW values commencing at 1±0.01×Rnd (where Rnd is a random value generator with a range between 0 and 1). The usual sequential order of attribute numbers is replaced with a random sequence. The subsequent step involves evaluating the initial value of the target function and making further adjustments to the correlation weights. After this, the relevant steps must be reiterated in the Monte Carlo optimization process for all the non-rare attributes, as specified in references.23,24 The linear regression approach is used to compute the QSAR model (utilizing the training set) as indicated in Equation 3. This is achieved when the numerical data regarding the correlation weights are derived from the model, leading to favorable statistical results for the test set. In this specif- ic study, the search for the optimal combination of T and 173Acta Chim. Slov. 2024, 71, 170–178 Petrović et al.: QSAR Modeling of Sphingomyelin Synthase 2 Inhibitors ... Nepoch was carried out within the ranges of 1–5 for T and 0–50 for Nepoch. Ac = C0 + C1×DCW(T,Nepoch) (3) 2. 2. QSAR Modeling Using Genetic Algorithm in Conjunction with Multiple Linear Regression In this section, 2D descriptors were calculated using PaDEL.25 Descriptors with low variance were eliminated from the initial descriptor pool, and further reduction of descriptors was conducted based on filtering using high pairwise correlation coefficients. The QSARINS program (QSAR-INSUBRIA) available at www.qsar.it was em- ployed for various descriptor reductions and for the de- velopment of QSAR models.26,27 After reducing the num- ber of descriptors, they were scaled, and suitable QSAR models were created using the genetic algorithm (GA) optimization method, following the same molecule split- ting approach as used in conformation-independent mod- eling.28,29 Within the QSARINS program, the genetic al- gorithm (GA) is combined with multiple linear regression (MLR) as the fitness evaluator.30,31 For the development of QSAR models, the following parameters were adjusted ac- cording to the total number of features in the model: the number of variables in GA optimization was set to 4, the number of GA iterations (generations per size) was set to 500, the population size (the number of models on which GA evolves) was set to 10, and random mutations for gen- erating a diverse pool of descriptors (mutation rate) were set at a 20% mutation rate. 2. 3. 3D Field-based QSAR Model Before creating the 3D-based QSAR model, geome- try optimization was performed on all the molecules using the MMFF94 force field, utilizing Marvin sketch software (Marvin 6.1.0, 2013, ChemAxon). The split that yielded the highest r2 for the conformation-independent model was employed to divide the molecules into the training and test sets for QSAR model development. The following parame- ters were utilized in model construction: a maximum of 6 PLS (Partial Least Squares) factors, steric and electrostatic force fields limited to 30.0 kcal/mol, a grid spacing of 1.0 Å with a 3.0 Å extension beyond the training set limits, and elimination of all variables with a standard deviation less than 0.01. The primary software used for developing the 3D field-based QSAR model was Schrodinger Maestro Version 11.5.011. 2. 4. Validation of the Developed QSAR Models Various validation metrics were employed to assess the quality of the developed conformation-independent and 2D-based QSAR models. These metrics included the determination of the squared correlation coefficient. (r2), the root-mean-squared-error (RMSE), leave-one-out and leave-many-out cross-validation coefficients, the F-value, the mean absolute error (MAE), and y-scrambling, as refer- enced.32–35 To further validate the developed QSAR mod- els, the following statistical metrics were employed: Rm2 and MAE-based metrics, the correlation coefficient (CCC), and the index of the ideality of correlation (IIC), as described.36 The applicability domain (AD) is a pivotal aspect of any QSAR model and must be established before utilizing the model.37,38 In this study, a literature-derived AD method was employed to define applicability domains for confor- mation-independent QSAR models.39 It is essential to de- fine the applicability domain (AD) for prediction purposes before making use of any QSAR model. Furthermore, es- tablishing the applicability domain (AD) is an essential and integral component of a pertinent, sturdy, trustworthy, and valid QSAR model. In this study, the AD for the developed QSAR models was determined by examining the "statisti- cal defects" of conformation-independent molecular de- scriptors, specifically d(A), which had been previously em- ployed in the construction of QSAR models.23,24,36 These calculations were carried out using the CORAL software, following the procedures outlined in Equation 4. (4) In the equation above, P(Atrain) and P(Acalib) denote the probabilities of a conformation-independent attribute or descriptor (A) in the training and test sets, respective- ly. Meanwhile, N(Atrain) and N(Acalib) represent the frequency of occurrence of a conformation-independent attribute or descriptor (A) in the training set and the test set, respectively. The statistical SMILES defect (D) is the cumulative sum of the defects, d(A), of all the attributes found in the SMILES notation of the molecules. It is com- puted according to Equation 5. (5) A molecule is labeled as an outlier if it falls outside the defined applicability domain (AD), which happens when its D exceeds 2 times Dav, where Dav represents the average D calculated for the relevant set (whether it's the training or test set) in which the molecule is located. The AD for the GA-MLR QSAR models was established using a distance-based approach, and the outliers were detected using the Williams plot, which plots standardized residu- als against leverages. 3. Results and Discussion Table 1 provides the numerical values of all the met- rics utilized to assess the quality of the developed confor- 174 Acta Chim. Slov. 2024, 71, 170–178 Petrović et al.: QSAR Modeling of Sphingomyelin Synthase 2 Inhibitors ... mation-independent QSAR models created through the Monte Carlo optimization method. The results indicate that the Monte Carlo optimization method yielded QSAR models with strong predictive capabilities and satisfactory reproducibility. Based on the applied metrics, the most fa- vorable QSAR model was achieved with the second split, featuring a T value of 4 and an Nepoch of 15. No outliers were identified, as the methodology applied for the ap- plicability domain (AD) indicated that all molecules fell within the defined AD. Figure 2 illustrates a graphical representation of the best-performing QSAR model (the one with the highest obtained r2 value) for all three splits in the best Monte Carlo optimization run. The concord- ance correlation coefficient (CCC) was employed to vali- date the QSAR models obtained, particularly with respect to their reproducibility. The results indicated that all the models exhibited high reproducibility. Additionally, the results for the MAE-based metric were noted as "GOOD," further confirming the validity of the developed QSAR model. Table 1. The statistical quality of the developed conformational-independent QSAR models for sphingomyelin synthase 2 inhibition Training set Test set r2 CCC IIC q2 s MAE F r2 CCC IIC q2 s MAE F Split 1 1 run 0.9087 0.9522 0.8579 0.8959 0.363 0.308 358 0.8907 0.9364 0.9438 0.8453 0.368 0.324 90 2 run 0.8742 0.9329 0.8415 0.8597 0.426 0.346 250 0.8711 0.9175 0.9333 0.8071 0.395 0.306 74 3 run 0.8934 0.9437 0.7652 0.8776 0.392 0.32 302 0.8763 0.9292 0.9361 0.8133 0.395 0.309 78 Av 0.8921 0.9429 0.8215 0.8777 0.394 0.325 303 0.8794 0.9277 0.9377 0.8219 0.386 0.313 81 Split 2 1 run 0.9098 0.9528 0.8584 0.8958 0.341 0.277 363 0.9496 0.9415 0.9744 0.9315 0.401 0.311 207 2 run 0.9152 0.9557 0.8581 0.9026 0.331 0.273 388 0.9433 0.9518 0.9712 0.9151 0.372 0.313 183 3 run 0.9092 0.9525 0.8582 0.8949 0.342 0.272 361 0.9427 0.9396 0.9708 0.9196 0.408 0.338 181 Av 0.9114 0.9537 0.8582 0.8978 0.338 0.274 371 0.9452 0.9443 0.9721 0.9221 0.394 0.321 190 Split 3 1 run 0.9203 0.9585 0.7766 0.9084 0.337 0.272 416 0.8612 0.9223 0.928 0.8226 0.431 0.303 68 2 run 0.8927 0.9433 0.7649 0.8797 0.391 0.296 300 0.8526 0.9213 0.9232 0.8076 0.433 0.304 64 3 run 0.8706 0.9308 0.8398 0.8526 0.429 0.342 242 0.8622 0.9246 0.9284 0.8173 0.411 0.277 69 Av 0.8945 0.9442 0.7938 0.8802 0.386 0.303 319 0.8587 0.9227 0.9265 0.8185 0.425 0.295 67 r2 – Correlation coefficient; CCC – Concordance correlation coefficient; IIC – Index of ideality of correlation; q2 – Cross-validated correlation coefficient; s – Standard error of estimation; MAE – Mean absolute error; F – Fischer ratio; Av – Average value for statistical parameters obtained from three independent Monte Carlo optimization runs Figure 2. Above) Graphical presentation of the best Monte Carlo optimization runs (the highest value for r2) for the developed QSAR models; Bel- low) Diff. – Difference between experimental and calculated values for pIC50. 175Acta Chim. Slov. 2024, 71, 170–178 Petrović et al.: QSAR Modeling of Sphingomyelin Synthase 2 Inhibitors ... The robustness of the developed QSAR models was assessed using Y-randomization, where Y values were shuffled in 1000 trials for ten separate runs. The outcomes, as presented in Table S2, suggest that the developed QSAR models do not rely on accidental correlations. The final assessment of the quality of the developed QSAR models was conducted using the calculated index of the ideality of correlation (IIC), and the results strongly suggest that the developed QSAR models possess a high predictive po- tential. The mathematical formulations for the top-perform- ing QSAR models, as determined by the test set r2 values for all the splits, are provided in Equations 6–8. Split 1: pIC50 = –1.1653(±0.0716) + 0.0290(±0.0003)×DCW(3,7) (6) Split 2: pIC50 = –1.6041(±0.0810) + 0.0400(± 0.0005)×DCW(4,15) (7) Split 3: pIC50 = –1.8678(±0.0950) + 0.0359(±0.0005)×DCW(2,7) (8) The equations (Eq. 6–8) show that for split 1, the preferred values for T and Nepoch are 3 and 7, respective- ly. For split 2, the preferred values are 4 for T and 15 for Nepoch, while for split 3, the preferred values are 2 for T and 7 for Nepoch. Equation 9 represents the mathematical equation that characterizes the developed QSAR models generated through GA-MLR modeling for all the splits. A graphical representation of this equation is provided in the supplementary material. The numerical values for all the calculated statistical parameters suggest that the developed QSAR models exhibit satisfactory predictive potential and robustness in terms of prediction. The sta- tistical parameters used for the fitting criteria were as follows: R2 : 0.9543; R2adj: 0.9472; R2-R2adj : 0.0071; LOF : 0.1176; Kxx : 0.5102; ΔK : 0.0554; RMSE : 0.2526; MAE : 0.1882; RSS : 2.4253; CCC tr: 0.9766; s: 0.2753; F: 134 The statistical parameters used for internal validation criteria were as follows: Q2loo : 0.9341; R2-Q2loo : 0.0202; RMSE: 0.3035; MAE : 0.2257; PRESS : 3.5003; CCC : 0.9664; Q2LMO : 0.9234. The statistical parameters used for ex- ternal validation criteria were as follows: RMSE: 0.6506; MAE: 0.5620; PRESS: 5.5020; R2 : 0.6316; CCC : 0.7916; r2m aver.: 0.6047; Δr2m : 0.0353. The model development included the consideration of the following molecular de- scriptor: Eta_D_beta_A, which represents the ETA aver- age measure of electronic features; C-040 – Atom-centred fragments R-C(=X)-X / R-C#X / X=C=X; SsssCH – Sum of sssCH E-states; SaaN – Sum of aaN E-states; MLogP – Mannhold LogP. pIC50 = 3.4370 + 3.3715×Eta_D_beta_A – 1.4345×C-040 + 1.5324×SsssCH + (9) 0.2257×SaaN + 0.4852× MLogP The 3D QSAR model exhibited a test set correlation coefficient of 0.9392, with a standard deviation of 0.2967. Additionally, the training set correlation coefficient for the 3D QSAR model was 0.6843, with a standard deviation of 0.2824. These values collectively indicate that the mod- el demonstrates good predictability. The results derived from the 3D QSAR model provide the following Gaussian field fraction contributions: 0.4240 for steric interactions, 0.0825 for electrostatic interactions, 0.2815 for hydropho- bic interactions, 0.1971 for hydrogen bond acceptor inter- Figure 3. 3D QSAR model fields (fields are shown as surfaces). A) Steric – favourable regions (green); B) Hydrophobic – favoured (yellow) and disfavoured (white); C) Electrostatic – favoured electropositive (blue) and disfavoured electronegative (red); D) Hydrogen bond acceptor – favoured (red) and disfavoured (magenta); E) Hydrogen bond donor – favoured (purple) and disfavoured (cyan). 176 Acta Chim. Slov. 2024, 71, 170–178 Petrović et al.: QSAR Modeling of Sphingomyelin Synthase 2 Inhibitors ... actions, and 0.0149 for hydrogen bond donor interactions. These results suggest that steric interactions, followed by hydrophobic interactions, exert the most significant in- fluence on the studied activity, particularly with regard to the increase in the size of substituent groups. In contrast, electrostatic and hydrogen bond donor interactions have the least impact. The surfaces representing the fields ob- tained for the developed 3D QSAR model are depicted in Figure 3. One of the main objectives of this research was to identify the molecular fragments defined as optimal de- scriptors in the SMILES notation that have both positive and negative impacts on the studied activity, as refer- enced.23,24,40–43 The comprehensive list of calculated mo- lecular descriptors, based on both the SMILES notation and the molecular graph, can be found in Table S3 (Sup- plementary material). For clarity, an example of the calcu- lation for the molecule's summarized correlation weight (DCW) and the studied activity (pIC50) is provided in Ta- ble 2, with the molecular graph-based descriptors omitted to facilitate interpretation. Additionally, a graphical rep- resentation of the molecular fragments for the same mole- cule is presented in Figure 4. Based on the results obtained from QSAR mode- ling, the SMILES notation reveals the following molecular fragments that influence pIC50 activity: “C............” – car- bon atom or a methyl group; “O............” – oxygen atom or hydroxyl group; “C...C.......” – representing two connect- ed carbon atoms or an ethyl group; “c...........”, “c...1.......”, “c...c.......”, “c...1...c...”, "c...c...1...", and “c...c...c...” – one ar- omatic carbon atom, two or three linear combinations of aromatic carbon atoms; “O...C...” – referring to a methoxy group or two connected carbon and oxygen atoms; “c...1... (...”, “c...(...1...”, “c...(...C...”, “c...C...(...”, “c...1...C...” – linked to the addition of at least one methyl group to benzene, result- ing in branching; “(...(.......”, “(...........”, “(...C...(...” SMILES Table 2. The example of DCW(4,15) calculation SMILES notation: CN(C(=O)c1c(OCCCC2CCCCC2)c2ccccc2n(c1=O)C)Cc1cc(cc(c1)C(F)(F)F)C(F)(F)F DCW = 113.53444 pIC50(calc.) = 3.7003 SA(CW) CW SA(CW) CW SA(CW) CW SA(CW) CW 10011001000 –0.9 2...n...(... 0.7295 c...c...(... 0.4785 N...(...C... 0.1565 (...(....... –0.5175 BOND10000 2.2636 C...C....... 0.4516 n...(...c... 0.0245 (........... –0.2838 C...(....... –0.5648 c...c....... 0.0705 N........... –0.7298 (...C...(... –0.9619 c...(....... 0.1721 c...C....... 0.1702 n........... 0.0083 (...F...(... 0.3981 C...(...=... –0.8874 C...c...1... 0.311 n...2....... –0.2463 ++++F---B2== 0.9554 C...(...1... 0.4141 c...c...1... 0.0902 n...2...c... –0.8469 ++++F---N=== 2.0554 c...(...2... –4.0999 C...C...2... –0.5719 N...C....... 0.238 ++++F---O=== 2.0323 C...(...C... 0.0284 c...c...2... –1.4034 Nmax.1...... 2.2076 ++++N---B2== 2.413 c...(...c... 0.4344 C...C...C... –2.8767 NOSP110000 6.3387 ++++N---O=== 3.2561 c...(...O... 0.1071 c...c...c... 0.1149 O...(....... –0.9913 ++++O---B2== –1.8357 C........... 0.0043 C...N...(... –0.9921 O...(...C... 0.675 =...(....... 0.667 c........... 0.0275 C...O...(... –0.7001 O........... 0.1213 =........... 0.4955 c...1...(... 0.2445 Cmax.2...... –1.702 O...=...(... –0.6294 =...1....... 0.4017 c...1....... 0.192 F...(...(... –0.8584 O...=....... –0.8534 =...O...(... 0.4016 c...1...=... –0.8758 F...(....... 0.1322 O...=...1... 0.0153 1...(....... 0.1372 c...1...c... 0.3067 F...(...C... –0.8886 O...C....... 0.3938 1........... 0.3516 C...2...(... –0.7831 F...(...F... –0.7542 O...C...C... 0.3251 1...c...(... 0.0674 C...2....... –0.8551 F........... 0.0899 Omax.3...... 6.8713 2...(....... –4.0883 c...2....... –2.6633 HALO100000 –0.7419 Smax.0...... 4.2841 2........... –0.7638 c...2...c... –1.7803 N...(....... –0.5548 2...c...(... 0.296 c...C...(... –0.0875 n...(....... 0.6401 Figure 4. Molecular fragments contribution to sphingomyelin syn- thase 2 inhibition (green – increase, red – decrease). 177Acta Chim. Slov. 2024, 71, 170–178 Petrović et al.: QSAR Modeling of Sphingomyelin Synthase 2 Inhibitors ... notation fragment associated with molecular branching: “F...........”, “c...F.......”, “F...c...1...” SMILES notation fragments associated with the addition of a fluorine atom to the ben- zene ring. "N.........""— representing a nitrogen atom with a negative impact on studied activity, but N......."— denot- ing a nitrogen atom involved in molecular branching has a positive impact. Similar to the aromatic carbon, the aro- matic nitrogen atom, indicated by the "n..........." molecular descriptor, also exerts a positive influence on the studied activity. "N...C..."— the primary amine group contributes positively, while secondary and tertiary amines, indicated with branching as "C...N...", have a negative impact. "=....." – a double bond exerts a positive influence, but the double bond with the oxygen atom, represented as "O..=..," nega- tively affects the studied activity. The presence of one ring, whether aromatic or aliphatic, positively impacts the stud- ied activity. This molecular feature is defined by the follow- ing molecular descriptors: “1...........”, “c...1.......”, “c...c...1...”, "C...(...1..."). Nevertheless, a further increase in the num- ber of rings, whether aromatic or aliphatic, has a nega- tive impact on the studied activity: “c...2.......”, "c...(...2..."), “c...2...c...”, “c...c...2...”, “2...........”, “C...2.......”, “C...C...2...”. Molecular branching as a feature and molecular branch- ing with involved carbon atoms defined as “(...........”, “(... (.......”, “C...(.......”, "(...C...(...")" have a negative impact on the studied activity. Both fluorine atoms ("F." and molecular branching involving fluorine atoms “F...(...(...”, “(...F...(...” and “F...(...F...” positively affect the studied activity. 4. Conclusion The primary objective of this study was to create re- liable QSAR models that demonstrate strong predictabili- ty, assessed using a range of statistical parameters, for the inhibition of sphingomyelin synthase 2. The Monte Carlo optimization method was employed to compute confor- mation-independent QSAR models. These models were built using optimal descriptors derived from both a local graph and SMILES notation invariants. A QSAR model was constructed using a genetic algorithm in conjunction with multiple linear regression, utilizing an extensive set of 2D molecule descriptors. The assessment of the robustness and predictive capability of these developed QSAR models was achieved through the application of various statistical techniques. The numerical values derived to validate the developed QSAR models demonstrate their high applica- bility. A field-based contribution approach was employed to establish the 3D QSAR model, and the results obtained revealed that the steric and hydrophobic parameters had the most significant impact on the inhibition activity. Molecular fragments, employed as SMILES notation frag- ments in QSAR modeling, with both positive and negative effects on sphingomyelin synthase 2 inhibition were iden- tified through the Monte Carlo optimization method. The methodology outlined in this study can be adapted to dis- cover novel therapeutics for the treatment of atherosclero- sis by targeting the inhibition of sphingomyelin synthase 2. Funding: This work is supported by the Ministry of Education and Science, the Republic of Serbia and the Faculty of Medicine, University of Niš, Republic of Ser- bia (project No. 70). 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Perić, S. Mladenović, A. M. Veselinović. Acta Chim. Slov. 2023, 70, 318–326. DOI: 10.17344/acsi.2023.8081 42. S. Ahmadi, S. Lotfi, S. Afshari, P. Kumar, E Ghasemi, SAR QSAR Environ. Res. 2021, 32, 1013–1031. DOI: 10.1080/1062936X.2021.2003429 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Sfingomielin sintaza 2 (SMS2) se je izkazala kot obetavna trača cza zdravljenje ateroskleroze. Kljub temu pa je dostopnost selektivnih zaviralcev SMS2 in njihove povezane farmakološke lastnosti omejena. Ta članek raziskuje različne tehnike modeliranja, osnovane na kvantitativnem razmerju med strukturo in delovanjem (QSAR), ki so bile uporabljene na različnih spojinah, ki delujejo kot inhibitorji SMS2. Uporabili smo različne metodologije modeliranja QSAR, vključno s konformacijsko neodvisnim modeliranjem, GA-MLR in 3D modeliranjem QSAR, proučili pa smo tudi korelacije med njimi. Za oceno kakovosti, robustnosti in napovedne sposobnosti napravljenih modelov smo uporabili različne statis- tične metode, pri čemer smo dosegli dobre rezultate. Poleg tega smo določili molekularne fragmente, pridobljene iz SMILES notacije deskriptorjev, ki upoštevajo opažene spremembe v ocenjeni aktivnosti. Metodologija, predstavljena v tej raziskavi, ima potencial za identifikacijo novih učinkovin za zdravljenje ateroskleroze z usmerjanjem na SMS2. S1Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti DRUŠTVENE VESTI IN DRUGE AKTIVNOSTI SOCIETY NEWS, ANNOUNCEMENTS, ACTIVITIES Vsebina Doktorska in magistrska dela, diplome v letu 2023 ............................................................ S3 Koledar važnejših znanstvenih srečanj s področja kemije in kemijske tehnologije ........ S33 Navodila za avtorje .................................................................................................................. S36 Contents Doctoral theses, master degree theses, and diplomas in 2023 ........................................... S3 Scientific meetings – Chemistry and chemical engineering ............................................... S33 Instructions for authors .......................................................................................................... S36 S2 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti S3Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti UNIVERZA V LJUBLJANI FAKULTETA ZA KEMIJO IN KEMIJSKO TEHNOLOGIJO 1. januar – 31. december 2023 DOKTORATI DOKTORSKI ŠTUDIJSKI PROGRAM KEMIJSKE ZNANOSTI KEMIJA Matjaž SIMONČIČ Mentor: prof. dr. Miha Lukšič VLOGA SOTOPLJENCEV PRI KOMPLEKSACIJI GLOBULARNIH PROTEINOV S SINTETIČNIMI POLIELEKTROLITI V VODNIH RAZTOPINAH Datum zagovora: 31. 3. 2023 Aleksandra KULJANIN Mentorica: doc. dr. Nataša Gros Somentor: prof. dr. Uroš Lotrič VIRTUALNI INSTRUMENTI ZA PRETOČNE IN DISKRETNE ANALIZNE METODE Datum zagovora: 17. 4. 2023 Tadej ŽUMBAR Mentorica: prof. dr. Nataša Novak Tušar RAZVOJ KOMPOZITNIH KATALIZATORJEV ZA RAZGRADNJO LAHKOHLAPNIH ORGANSKIH ONESNAŽIL V ZRAKU Datum zagovora: 9. 6. 2023 Ema GRIČAR Mentor: prof. dr. Mitja Kolar Somentor: izr. prof. dr. Boštjan Genorio RAZVOJ IN UPORABA ELEKTROKEMIJSKIH SENZORJEV NA OSNOVI GRAFENSKIH DERIVATOV Datum zagovora: 26. 9. 2023 Luka CIBER Mentor: prof. dr. Uroš Grošelj SINTEZA IN VREDNOTENJE ENDIAMINSKIH IN ENAMINONSKIH ASIMETRIČNIH BIFUNKCIONALNIH ORGANOKATALIZATORJEV NA OSNOVI PRIVILEGIRANIH KIRALNIH SKELETOV Datum zagovora: 26. 9. 2023 Blaž ZDOVC Mentorica: znan. svet. dr. Ema Žagar OPREDELITEV KOMPLEKSNIH POLIMEROV S TEKOČINSKIMI SEPARACIJSKIMI TEHNIKAMI Datum zagovora: 17. 10. 2023 Tia KRISTIAN TAJNŠEK Mentor: viš. znan. sod. dr. 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STOPNJE – KEMIJA Matic DOKL Mentor: prof. dr. Igor Plazl Somentor: prof. dr. Tomaž Urbič SIMULACIJA DVOKOMPONENTNIH SISTEMOV Z UPORABO MREŽNE BOLTZMANNOVE METODE Datum zagovora: 20. 2. 2023 Blaž TOPLAK Mentor: prof. dr. Mitja Kolar Somentor: znan. sod. dr. Ivan Jerman OPTIMIZACIJA PEROVSKITNIH SONČNIH CELIC BREZ PLASTI ZA PRENOS PRAZNIN Datum zagovora: 28. 2. 2023 Jona ŽOHAR Mentor: prof. dr. Matevž Pompe UPORABA PRINCIPA UPRAVLJANJA KVALITETE PRI RAZVOJU ANALIZNEGA POSTOPKA ZA DOLOČEVANJE GLIKANOV Datum zagovora: 21. 3. 2023 Tadej KLOBUČAR Mentorica: doc. dr. Nataša Gros VREDNOTENJE KAKOVOSTI EPRUVET ZA VAKUUMSKI ODVZEM KRVI ZA KOAGULACIJSKE DOLOČITVE Datum zagovora: 24. 3. 2023 Luka SETNIKAR Mentor: prof. dr. Franc Perdih LUMINISCENČNE LASTNOSTI CINKOVIH KOORDINACIJSKIH SPOJIN Z INDOL-3-PROPIONATNIM IN INDOL-3-ACETATNIM LIGANDOM Z NEVTRALNIMI DUŠIKOVIMI LIGANDI Datum zagovora: 28. 3. 2023 Urša VONČINA Mentor: prof. dr. Bogdan Štefane FOTOINDUCIRANE PRETVORBE NEKATERIH ENINONSKIH DERIVATOV Datum zagovora: 19. 4. 2023 Katja GABROVEC Mentor: prof. dr. Jernej Iskra DERIVATI EMODINA KOT FOTOKATALIZATORJI ZA REDUKCIJE ARIL HALIDOV IN OKSIDACIJE SULFIDOV Datum zagovora: 24. 4. 2023 Zala IVANČIČ Mentorica: znan. sod. dr. Vesna Glavnik Somentorica: prof. dr. Helena Prosen RAZVOJ IN OPTIMIZACIJA RAZLIČNIH POSTOPKOV EKSTRAKCIJ BIOAKTIVNIH SPOJIN IZ LISTOV JAPONSKEGA DRESNIKA (FALLOPIA JAPONICA HOUTT. ) Datum zagovora: 25. 5. 2023 Monika VIDMAR Mentor: izr. prof. dr. Blaž Likozar Somentorica: prof. dr. Irena Kralj Cigić PRIMERJAVA RAZLIČNIH VREDNOTENJ ZGRADBE LIGNINA KOT OPREDELITVE KINETIČNIH SKUPKOV Datum zagovora: 30. 5. 2023 Sebastian PLEŠKO Mentor: doc. dr. Črtomir Podlipnik ISKANJE LIGANDOV ZA BIOLOŠKE TARČE S POMOČJO ALGORITMOV IN SKUPNOSTNE ZNANOSTI Datum zagovora: 31. 5. 2023 Anže HUBMAN Mentor: prof. dr. Tomaž Urbič Somentor: izr. prof. Franci Merzel MODELIRANJE ADSORPCIJE VODE V ALUMINOFOSFATU TIPA LTA Datum zagovora: 27. 6. 2023 Anja KOROŠEC Mentorica: prof. dr. Irena Kralj Cigić Sprememba arome sirov med skladiščenjem Datum zagovora: 29. 6. 2023 Ana ŠIŠKO Mentorica: prof. dr. Irena Kralj Cigić Somentorica: znan. sod. dr. Jasna Malešič STABILNOST PAPIRJA V PRISOTNOSTI PIGMENTA VERDIGRIS Datum zagovora: 29. 6. 2023 David RIBAR Mentorica: prof. dr. Irena Kralj Cigić RAZISKAVA TEMELJNEGA RETENCIJSKEGA MEHANIZMA REVERZNO-FAZNE TEKOČINSKE KROMATOGRAFIJE Datum zagovora: 6. 7. 2023 Ana BRODNIK Mentor: prof. dr. Janez Košmrlj SINTEZA DIFENILACETILENSKIH DERIVATOV Z ZANIMIVIMI OPTIČNIMI LASTNOSTMI Datum zagovora: 17. 8. 2023 Špela POK Mentorica: prof. dr. Helena Prosen RAZVOJ ANALIZNIH METOD ZA SPREMLJANJE FOTORAZGRADNIH PRODUKTOV ADSORBIRANIH ONESNAŽEVAL NA MIKROPLASTIKI Datum zagovora: 18. 8. 2023 Tomaž MRŽLJAK Mentorica: prof. dr. Irena Kralj Cigić KARAKTERIZACIJA SORBENTOV NA OSNOVI EMISIJ HOS Datum zagovora: 24. 8. 2023 S6 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Miha HOTKO Mentor: prof. dr. Miran Gaberšček AKTIVNOST NI KATALIZATORJEV PRI ELEKTROKEMIJSKI REAKCIJI RAZVOJA KISIKA (OER) Datum zagovora: 24. 8. 2023 Jasmin RAČIĆ Mentorica: prof. dr. Helena Prosen Spremljanje razgradnih produktov, nastalih z uporabo naprednih oksidacijskih procesov na izbranih onesnaževalih Datum zagovora: 25. 8. 2023 Blagoj MUKAETOV Mentor: prof. dr. Janez Košmrlj SINTEZA IN OPTIČNE LASTNOSTI IZBRANIH 2-FENILNAFTALENSKIH DERIVATOV Datum zagovora: 28. 8. 2023 Patrik ZAVRŠNIK Mentor: prof. dr. Mitja Kolar OPTIMIZACIJA KEMIJSKO MODIFICIRANE IONOSELEKTIVNE ELEKTRODE IZ OGLJIKOVE PASTE ZA POTENCIOMETRIČNO DOLOČANJE MAPROTILIN HIDROKLORIDA Datum zagovora: 29. 8. 2023 Kaja PROSENAK Mentor: prof. dr. Uroš Grošelj (+)-IZOKAMFOLENSKA KISLINA – NERAZISKAN SUBSTRAT ZA SINTEZO POTENCIALNIH DIŠAV Datum zagovora: 29. 8. 2023 Kris ANTOLINC Mentor: prof. dr. Jurij Svete FOTOREDOKS ARILACIJE 4-OKSO-4H-PIRIDINO[1,2-A] PIRIMIDIN-3-DIAZONIJEVEGA TETRAFLUOROBORATA Datum zagovora: 29. 8. 2023 Jan ŠEGINA Mentor: prof. dr. Uroš Grošelj SINTEZA PREKURZORJEV N-HETEROCIKLIČNIH KARBENOV NA OSNOVI KAFRE Datum zagovora: 31. 8. 2023 Jan DEŽAN Mentorica: izr. prof. dr. Amalija Golobič Somentorica: prof. dr. Nataša Zabukovec Logar PRIPRAVA MODIFICIRANIH ZEOLITNIH IMIDAZOLATNIH OGRODIJ S POSINTEZNO IZMENJAVO LIGANDOV Datum zagovora: 4. 9. 2023 Anže BRUS Mentor: prof. dr. Iztok Turel SINTEZA KOMPLEKSOV ZLATA Z DERIVATI PIRITIONA Datum zagovora: 4. 9. 2023 Klara KLEMENČIČ Mentor: prof. dr. Uroš Grošelj SINTEZA IN KATALITSKA AKTIVNOST 1,2-BENZENDIAMINSKIH ORGANOKATALIZATORJEV NA OSNOVI (1R,2R)-2-(PIPERIDIN-1-IL)CIKLOHEKSAN- 1-AMINA Datum zagovora: 4. 9. 2023 Petra KREMŽAR Mentor: izr. prof. dr. 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Igor Plazl NAPOVEDOVANJE HITROSTNIH PROFILOV ZA DVOFAZNI SISTEM V MIKROKANALU ZA SEGMENTIRAN TOK Z UPORABO METODE KONČNIH ELEMENTOV Datum zagovora: 6. 6. 2023 Nik SMRKOLJ Mentor: prof. dr. Marjan Marinšek Optimizacija procesa sintranja steatitne keramike Datum zagovora: 22. 6. 2023 Gašper TEGELJ Mentor: prof. dr. Igor Plazl OPTIMIZACIJA PROCESNIH SPREMENLJIVK PRI AKTIVACIJI REOLOŠKEGA ADITIVA V 2K PUR PREMAZIH Datum zagovora: 23. 6. 2023 Tia HAFNER Mentor: prof. dr. Aleš Podgornik DOLOČEVANJE UČINKOVITOSTI ODSTRANJEVANJA NEČISTOČ KROMATOGRAFSKIH STOPENJ V PROCESU IZOLACIJE IN ČIŠČENJA REKOMBINANTNEGA FUZIJSKEGA PROTEINA Datum zagovora: 23. 6. 2023 Mark STARIN Mentorica: izr. prof. dr. Gabriela Kalčikova VPLIV MIKROPLASTIKE NA UČINKOVITOST RASTLINSKE ČISTILNE NAPRAVE Datum zagovora: 3. 7. 2023 Maja GABRIČ Mentorica: izr. prof. dr. Gabriela Kalčikova ODSTRANJEVANJE MIKROPLASTIKE IZ KOMUNALNE ODPADNE VODE Z RASTLINSKO ČISTILNO NAPRAVO Datum zagovora: 6. 7. 2023 Leja PLEŠKO Mentorica: izr. prof. dr. Gabriela Kalčikova INTERAKCIJE MIKROPLASTIKE Z VIRUSI Datum zagovora: 7. 7. 2024 Doroteja KRAJNC Mentor: prof. dr. Aleš Podgornik VPLIV UČINKOVANJA BAKTERIOFAGOV NA STAFILOKOKE V RAZLIČNIH FIZIOLOŠKIH STANJIH Datum zagovora: 28. 8. 2023 Jošt OBLAK Mentor: izr. prof. dr. Blaž Likozar Somentor: prof. dr. Igor Plazl KINETIKA HIDROGENACIJE 2-METILKINOLINA KOT N-HETEROCIKLIČNEGA PREDSTAVNIKA TEKOČIH ORGANSKIH NOSILCEV VODIKA Datum zagovora: 4. 9. 2023 Anej BLAŽIČ Mentorica: izr. prof. dr. Gabriela Kalčikova ŠTUDIJA ADSORPCIJE MIKROPLASTIKE IZ POLIETILENA NA PLAVAJOČO RASTLINO Datum zagovora: 6. 9. 2023 Uroš KARLIČ Mentor: prof. dr. Igor Plazl NAČRTOVANJE IN TESTIRANJE MIKROFLUIDNE NAPRAVE ZA LOČEVANJE DELCEV RAZLIČNIH VELIKOSTI Datum zagovora: 7. 9. 2023 Jošt OBLAK Mentor: prof. dr. Miran Gaberšček Somentor: prof. dr. Andraž Legat SPREMLJANJE KOROZIJE JEKLA V BETONU Z ELEKTROKEMIJSKO IMPEDANČNO SPEKTROSKOPIJO Datum zagovora: 7. 9. 2023 Nikola POLJANEC Mentor: prof. dr. Aleš Podgornik MEHANSKE LASTNOSTI POROZNIH METAKRILATNIH POLIMEROV Datum zagovora: 12. 9. 2023 Peter GARTNAR Mentorica: doc. dr. Lidija Slemenik Perše VPLIV S SREBROM OPLAŠČENIH BAKRENIH LUSK NA IZDELAVO IN FIZIKALNE LASTNOSTI VISOKOPREVODNIH POLIMERNIH KOMPOZITOV Datum zagovora: 19. 9. 2023 Alexander Marjan MULEC Mentor: izr. prof. dr. Aleš Ručigaj VPLIV ADSORPCIJE NA SPROŠČANJE PROTEINOV IZ ALGINATNIH IN NANOCELULOZNIH HIDROGELOV Datum zagovora: 20. 9. 2023 Sebastijan RAJŠTER Mentor: prof. dr. Igor Plazl PROCESNA INTENZIFIKACIJA KATALITSKIH REAKCIJ V DVOFAZNEM SISTEMU Datum zagovora: 21. 9. 2023 Andrej ŽERJAL Mentor: prof. dr. Igor Plazl Katalitska hidrogenacija levulinske kisline v mikroreaktorju med dvema ploščama Datum zagovora: 21. 9. 2023 Alen NAVODNIK Mentor: izr. prof. dr. Blaž Likozar Somentor: prof. dr. Igor Plazl VPLIV REAKCIJSKIH POGOJEV TER STRUKTURE CU/ AL2O3 KATALIZATORJA NA PRETVORBO CO2 IN H2 V CO Datum zagovora: 22. 9. 2023 Andreja Kavčnik Mentor: prof. dr. Igor Plazl Somentorica: doc. dr. Tina Trdan Lušin PROUČEVANJE HITROSTI RAZPADA ZDRAVILNIH UČINKOVIN ZA ZDRAVLJENJE PREHLADNIH OBOLENJ V NAPITKIH PRIPRAVLJENIH ZA PERORALNO UPORABO. Datum zagovora: 25. 9. 2023 S9Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Nejc LAPAJNE Mentor: doc. dr. Matjaž Spreitzer Somentor: prof. dr. Matjaž Krajnc NANOSTRUKTURIRANE TANKE PLASTI TIO2 ZA ELEKTROKEMIJSKO CEPITEV VODE Datum zagovora: 25. 9. 2023 Marko TRAVICA Mentor: prof. dr. Aleš Podgornik PRIPRAVA IN KARAKTERIZACIJA SREBROVIH POLIHIPE NOSILCEV Datum zagovora: 25. 9. 2023 Luka TACER Mentorica: prof. dr. Polona Žnidaršič Plazl IMOBILIZACIJA ENCIMOV NA MAGNETNE MIKRODELCE ZA IZVEDBO KONTINUIRNE TRANSAMINACIJE V MIKROREAKTORJU Z UPORABO EVTEKTIČNIH TOPIL Datum zagovora: 25. 9. 2023 Zarja MEDVED Mentorica: prof. dr. Polona Žnidaršič Plazl Somentor: doc. dr. Iztok Jože Košir EKSTRAKCIJA HMELJNIH KOMPONENT MED HLADNIM HMELJENJEM LEŽAK PIVA Datum zagovora: 25. 9. 2023 Lan Julij ZADRAVEC Mentorica: prof. dr. Polona Žnidaršič Plazl RAZVOJ MIKROPRETOČNIH SISTEMOV ZA GOJENJE BIOFILMOV BAKTERIJE BACILLUS SUBTILIS Datum zagovora: 25. 9. 2023 Urška KOVAČIČ Mentor: prof. dr. Marjan Marinšek IN VITRO ACELULARNO RAZTAPLJANJE VLAKEN MINERALNE VOLNE V RAZLIČNIH SIMULIRANIH PLJUČNIH TEKOČINAH Datum zagovora: 26. 9. 2023 Nina KUKOVIČIČ Mentorica: prof. dr. Andreja Žgajnar Gotvajn VPLIV PVC MIKROPLASTIKE NA PROIZVODNJO BIOPLINA IZ PREDOBDELANEGA ODPADNEGA BLATA Datum zagovora: 26. 9. 2023 Mia Henjak Mentorica: izr. prof. dr. Gabriela Kalčikova Staranje in vplivi mikroplastike iz avtomobilskih pnevmatik v vodnem okolju Datum zagovora: 26. 9. 2023 Ana Kristina KLANČIČ Mentor: doc. dr. Rok Ambrožič MIKROFLUIDNI SISTEM ZA KONTINUIRNO RAZGRADNJO ORGANSKIH ONESNAŽEVAL Datum zagovora: 26. 10. 2023 Sara OMERZEL Mentorica: prof. dr. Polona Žnidaršič Plazl IMOBILIZACIJA AMIN TRANSAMINAZE NA FUNKCIONALIZIRANE SILIKATNE NANODELCE PREKO HEKSAHISTIDINSKEGA OZNAČEVALCA Datum zagovora: 29. 11. 2023 Mejrema NUHANOVIĆ Mentor: prof. dr. Igor Plazl RAZVOJ MATEMATIČNEGA MODELA ZA OPTIMIZACIJO PROCESNIH PARAMETROV PRI ELEKTROKEMIJSKEM PROCESU PROIZVODNJE VODIKOVEGA PEROKSIDA V MIKROREAKTORSKEM SISTEMU MED DVEMA PLOŠČAMA Datum zagovora: 14. 12. 2023 Katja TRILER Mentorica: prof. dr. Polona Žnidaršič Plazl IMOBILIZACIJA AMIN TRANSAMINAZE N-HIS6-ATA-V1 NA FUNKCIONALIZIRANE SILIKATNE DELCE Datum zagovora: 27. 12. 2023 MAGISTRSKI ŠTUDIJSKI PROGRAM 2. STOPNJE – BIOKEMIJA Doroteja ARMIČ Mentorica: izr. prof. dr. Marina Klemenčič IZRAŽANJE IN BIOKEMIJSKA KARAKTERIZACIJA VAKUOLNEGA PROCESIVNEGA ENCIMA IZ ENOCELIČNE ALGE CHLAMYDOMONAS REINHARDTII Datum zagovora: 6. 1. 2023 Urša LOVŠE Mentor: viš. znan. sod. dr. Aleš Lapanje Somentor: prof. dr. Marko Dolinar OPTIMIZACIJA IZOLACIJE BAKTERIJSKE GENOMSKE DNA ZA SEKVENCIRANJE S TEHNOLOGIJO NANOPORE IN ANALIZA ZAPOREDIJ IZBRANIH SEVOV Datum zagovora: 17. 2. 2023 Nika MIKULIČ VERNIK Mentor: prof. dr. Marko Novinec KARAKTERIZACIJA INTERAKCIJE MED DERIVATOM PIRAZOLA IN L-TREONIN DEHIDROGENAZO IN SUKCINAT DEHIDROGENAZO BAKTERIJE ESCHERICHIA COLI. Datum zagovora: 22. 2. 2023 Mateja ŽVIPELJ Mentor: prof. dr. Roman Jerala Somentor: prof. dr. Marko Dolinar UPORABA ALTERNATIVNEGA IZREZOVANJA ZA NADZOR IZRAŽANJA PROTEINOV Datum zagovora: 28. 2. 2023 S10 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Saša SLABE Mentorica: doc. dr. Tadeja Režen UGOTAVLJANJE VPLIVA IZBRANIH KROŽNIH RNA NA CIRKADIANI RITEM CELIC Datum zagovora: 2. 3. 2023 Katja DOLENC Mentorica: znan. sod. dr. Maja Marušič Somentor: doc. dr. Gregor Gunčar INTERAKCIJE PEPTIDOV DOMENE SUD M NSP3 VIRUSA SARS-COV-2 S 3’-NEPREVEDENIMI REGIJAMI ČLOVEŠKE MRNA Datum zagovora: 17. 3. 2023 Klementina POLANEC Mentorica: doc. dr. Vera Župunski ANALIZA TARČ ORF1P, ZAZNANIH Z BIOTINSKO IDENTIFIKACIJO, IN OPTIMIZACIJA TESTA RETROTRANSPOZICIJE Z VTRNA TER YRNA Datum zagovora: 30. 5. 2023 Sara LAZNIK Mentor: prof. dr. Janez Plavec STRUKTURNA ŠTUDIJA G-KVADRUPLEKSA DOLGE NEKODIRAJOČE RNA PSEVDOGENA REG1CP Datum zagovora: 20. 6. 2023 Tina KOLENC MILAVEC Mentorica: doc. dr. Barbara Breznik VPLIV CELIC ŽILJA NA ODPORNOST CELIC GLIOBLASTOMA NA KEMOTERAPIJO Datum zagovora: 21. 6. 2023 Nina VARDA Mentorica: izr. prof. dr. Mojca Benčina Somentor: doc. dr. Aljaž Gaber SINTEZNOBIOLOŠKA ORODJA V PODPORO ULTRAZVOČNI STIMULACIJI CELIC Datum zagovora: 3. 7. 2023 Tina LOGONDER Mentor: doc. dr. Aljaž Gaber VZPOSTAVITEV METODE OBNOVITVE FLUORESCENCE BODIPY ZA DOLOČANJE KOLIČINE LIPIDOV V OLEOGENI KVASOVKI YARROWIA LIPOLYTICA IN KARAKTERIZACIJA SEVOV S SPREMENJENIM METABOLIZMOM LIPIDOV Datum zagovora: 3. 7. 2023 Matija RUPARČIČ Mentor: prof. dr. Marko Dolinar EKSPERIMENTALNA KARAKTERIZACIJA ŠESTIH KANDIDATNIH SISTEMOV TOKSIN-ANTITOKSIN TIPA I CIANOBAKTERIJE MICROCYSTIS AERUGINOSA PCC 7806 S POUDARKOM NA MSOT1/MSOA1 Datum zagovora: 5. 9. 2023 Vesna PODGRAJŠEK Mentor: prof. dr. Marko Dolinar PREPOZNAVANJE PARAZITSKE GLIVE BRINOVEGA ŠČETINCA (GYMNOSPORANGIUM CLAVARIIFORME) NA SEKUNDARNEM GOSTITELJU ENOVRATEM GLOGU (CRATAEGUS MONOGYNA) NA OSNOVI ČRTNE KODE DNA Datum zagovora: 6. 9. 2023 Luka GREGORIČ Mentorica: prof. dr. Helena Prosen DOLOČANJE PROTEINOV Z UV SPEKTROMETRIJO Z VARIABILNO OPTIČNO POTJO Datum zagovora: 6. 9. 2023 Urška ZAGORC Mentorica: prof. dr. Ksenija Kogej STABILNOST UMETNIH IN NARAVNIH VEZIKLOV Datum zagovora: 7. 9. 2023 Luka GNIDOVEC Mentorica: doc. dr. Vera Župunski KARAKTERIZACIJA VEZAVE PROTEINA HNRNP H1 NA HEKSANUKLEOTIDNE PONOVITVE GGGGCC Datum zagovora: 8. 9. 2023 Eva KEBER Mentorica: izr. prof. dr. Nataša Debeljak IZBOR KONTROLNIH CELIČNIH LINIJ ZA ANALIZO IZRAŽANJA KANABINOIDNIH RECEPTORJEV PRI RAKU DOJKE Datum zagovora: 14. 9. 2023 Anže KARLEK Mentorica: prof. dr. Boris Rogelj IDENTIFIKACIJA MEHANIZMA NASTANKA STRESNIH GRANUL V CELICAH PO OBDELAVI S HLADNO ATMOSFERSKO PLAZMO Datum zagovora: 25. 9. 2023 Urška FAJDIGA Mentor: prof. dr. Rok Romih IZRAŽANJE ENDOTELIJSKE SINTAZE DUŠIKOVEGA OKSIDA IN KAVEOLINA 1 V NORMALNEM IN VNETEM SEČNEM MEHURJU MIŠI Datum zagovora: 28. 9. 2023 Sara JEREB Mentor: dr. Toni Petan Somentorica: doc. dr. Vera Župunski VLOGA LIPIDNIH KAPLJIC PRI OBRAMBI CELIC RAKA DOJKE PRED FEROPTOZO Datum zagovora: 12. 10. 2023 Rok FERENC Mentorica: prof. dr. Kristina Gruden ANALIZA VPLIVA PROTEINA HCPRO VIRUSA Y KROMPIRJA NA TRANSKRIPCIJSKI FAKTOR PTI5 V TOBAKU NICOTIANA BENTHAMIANA IN PROIZVODNJA DVOVERIŽNE RNA ZA UPORABO KOT PESTICID PROTI KOLORADSKEMU HROŠČU Datum zagovora: 13. 10. 2023 Meta KODRIČ Mentor: dr. Duško Lainšček Somentorica: doc. dr. Ajda Taler-Verčič UPORABA SISTEMA CCEXO-STREPTAVIDIN ZA TARČNO INTEGRACIJO ZAPISA ZA CD19-CAR V GENOM LIMFOCITOV T Datum zagovora: 26. 10. 2023 S11Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Barbara JAKLIČ Mentorica: dr. Anna Coll URAVNAVANJE IZRAŽANJA NEKATERIH PEROKSIDAZ S TRANSKRIPCIJSKIMI FAKTORJI TGA PRI ODZIVU KROMPIRJA NA VIRUSNO OKUŽBO Datum zagovora: 3. 11. 2023 Neža PAVKO Mentor: znan. sod. dr. Helena Gradišar Somentor: doc. dr. Gregor Gunčar TVORBA KOMPLEKSNIH STRUKTUR IZ TETRAEDRSKIH PROTEINSKIH NANOKLETK Z UPORABO SISTEMA SPYCATCHER/SPYTAG Datum zagovora: 6. 11. 2023 Kim GLAVIČ Mentorica: dr. Iva Hafner Bratkovič Somentorca: doc. dr. Vera Župunski VLOGA NABITIH SEGMENTOV PRI KANONIČNI AKTIVACIJI INFLAMASOMA NLRP3 Datum zagovora: 7. 11. 2023 Dunia SAHIR Mentorica: prof. dr. Kristina Djinović Carugo STRUKTURNE OSNOVE INHIBICIJE KALCINEVRINA S FATZ-1: POSLEDICE PRI PREOBLIKOVANJU MIŠIČNIH VLAKEN Datum zagovora: 22. 11. 2023 Nika VEGELJ Mentor: doc. dr. Aljaž Gaber PRIPRAVA REKOMBINANTNIH OGRODNIH PROTEINOV Β-KATENIN UNIČEVALNEGA KOMPLEKSA IN REKONSTRUKCIJA NJIHOVIH BIOMOLEKULARNIH KONDENZATOV Datum zagovora: 12. 12. 2023 Eva GARTNER Mentorica: doc. dr. Helena Motaln Somentor: prof. dr. Boris Rogelj VPLIV FOSFORILACIJE NA ZNOTRAJCELIČNO RAZPOREJANJE PROTEINA FUS V DIFERENCIRANIH CELICAH SH-SY5Y FLPIN Datum zagovora: 18. 12. 2023 Nina LUKANČIČ Mentor: izr. prof. dr. Zdenko Časar Somentor: izr. prof. dr. Miha Pavšič VPLIV PROCESNIH PARAMETROV NA TVORBO LIPIDNIH NANODELCEV Z VGRAJENO ZDRAVILNO UČINKOVINO NA OSNOVI SIRNK Datum zagovora: 20. 12. 2023 MAGISTRSKI ŠTUDIJSKI PROGRAM 2. STOPNJE – KEMIJSKO IZOBRAŽEVANJE Maja GLOBOČNIK Mentorica: prof. dr. Helena Prosen DOLOČANJE NEONIKOTINOIDNIH PESTICIDOV V PELODU Datum zagovora: 12. 5. 2023 Anja SEVER Mentorica: izr. prof. dr. Barbara Modec PRODUKTI REAKCIJE MED NITRILOM IN AMINOM V PRISOTNOSTI CINKOVEGA(II) SULFATA(VI) Datum zagovora: 14. 6. 2023 Leon ŽAGAR Mentor: prof. dr. Miha Lukšič UPORABA PREPROSTE OPTOELEKTRONSKE NAPRAVE ZA PRIKAZ ELEKTROKROMIZMA V ŠOLI Datum zagovora: 7. 7. 2023 Marko CUJNIK Mentor: doc. dr. Krištof Kranjc ORGANSKE DUŠIKOVE SPOJINE IN SPOZNAVANJE KEMIJSKIH PROCESOV PRI DELOVANJU FRIZERSKIH PREPARATOV Datum zagovora: 31. 8. 2023 Petra ŠPORAR Mentor: prof. dr. Marjan Jereb NEKATERE PRETVORBE FRIEDEL-CRAFTSOVEGA TIPA Datum zagovora: 6. 9. 2023 Natalija SITNIKOVA Mentor: doc. dr. Krištof Kranjc RAZVOJ TEČAJA ΜMOOC O UPORABI INFORMACIJSKO- KOMUNIKACIJSKE TEHNOLOGIJE PRI IZVAJANJU PROJEKTNEGA IN PROBLEMSKEGA UČENJA ZA IZOBRAŽEVANJE UČITELJEV KEMIJE NA VISOKOŠOLSKI STOPNJI IZOBRAŽEVANJA Datum zagovora: 28. 9. 2023 S12 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti MAGISTRSKI ŠTUDIJSKI PROGRAM 2. STOPNJE – TEHNIŠKA VARNOST Jure SELAN Mentor: prof. dr. Simon Schnabl DROGE IN ŠTUDENTSKO DELO Datum zagovora: 3. 3. 2023 Jan ŠPINDLER Mentor: prof. dr. Simon Schnabl PRIMERJAVA IN VPLIV RAZLIČNIH DEJAVNIKOV NA EVAKUACIJSKI ČAS S POMOČJO PROGRAMA PATHFINDER Datum zagovora: 16. 6. 2023 Timotej GRČAR Mentorica: doc. dr. Barbara Novosel PRAŠNE EKSPLOZIJE ALUMINIJEVEGA PRAHU Datum zagovora: 27. 6. 2023 Mirna ŠAFRANKO Mentor: doc. dr. Domen Kušar MODELIRANJE EVAKUACIJE V OBSTOJEČI VEČSTANOVANJSKI STAVBI Datum zagovora: 6. 10. 2023 Rok BRULC Mentor: prof. dr. Simon Schnabl POŽARNA VARNOST ŠPORTNE DVORANE STOPIČE Datum zagovora: 27. 10. 2023 Haris HAJRLAHOVIĆ Mentorica: doc. dr. Barbara Novosel Somentor: prof. dr. Mitja Kolar DOLOČEVANJE IZBRANIH TEŽKIH KOVIN TER DRUGIH KEMIJSKIH PARAMETROV V POVRŠINSKI VODI POTOKA DRTIJŠČICE IN OCENA NJIHOVIH VPLIVOV NA ZDRAVJE LJUDI Datum zagovora: 6. 12. 2023 Ana LOBNIK Mentor: prof. dr. Simon Schnabl DOLOČITEV EKSPLOZIJSKIH PARAMETROV MLETE IN INSTANT KAVE Datum zagovora: 21. 12. 2023 S13Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti DIPLOME – UNIVERZITETNI ŠTUDIJ KEMIJA – 1. STOPNJA Mark KRŽIŠNIK Mentor: izr. prof. dr. Drago Kočar DOLOČANJE VSEBNOSTI BIOGENIH KOVIN V PIVU Datum zagovora: 28. 2. 2023 Katja SMREKAR Mentorica: doc. dr. Nataša Gros ANALIZNA UPORABA LABORATORIJA V BRIZGI Datum zagovora: 19. 5. 2023 Manca ŠINCEK Mentorica: doc. dr. Saša Petriček KOMPLEKSI PREHODNIH KOVIN 4. PERIODE Z 2-AMINO-3-HIDROKSIPIRIDINOM. Datum zagovora: 21. 6. 2023 Lana JAMNIK Mentor: prof. dr. Uroš Grošelj SINTEZA (R)-4-(2-BROMOETIL)-1,5,5- TRIMETILCIKLOPENT-1-ENA Datum zagovora: 22. 6. 2023 Aleksandar TRAJKOVSKI Mentor: prof. dr. Jurij Svete SINTEZA IN PRETVORBE ETIL 3-AMINO-4-OXO-4H- KINOLIZIN-1-KARBOKSILATA Datum zagovora: 26. 6. 2023 Patricia SADAR JOVIĆ Mentorica: doc. dr. Nataša Čelan Korošin VPLIV ZAVIRALCEV GORENJA NA TERMIČNO STABILNOST IZBRANIH MATERIALOV Datum zagovora: 29. 6. 2023 Lara Maruša ŠTIH Mentor: prof. dr. Jernej Iskra HALOGENIRANJE KVERCETINA Datum zagovora: 30. 6. 2023 Nejc GODEC Mentorica: prof. dr. Romana Cerc Korošec PRIPRAVA IN PREUČEVANJE FOTOKATALIZATORJA COOX NA RAZLIČNIH NOSILCIH Datum zagovora: 3. 7. 2023 Jan MATOH Mentorica: prof. dr. Irena Kralj Cigić DOLOČANJE ACESULFAMA K V PREHRANSKIH DODATKIH Datum zagovora: 7. 7. 2023 Nejc FLAJŠAR Mentor: prof. dr. Tomaž Urbič RAČUNALNIŠKE SIMULACIJE PREPROSTIH MODELOV VODE Datum zagovora: 18. 8. 2023 Timotej ŠUMAN Mentor: prof. dr. Jurij Svete SINTEZA IN PRETVORBE 3-AMINO-4-OKSO-4H- KINOLIZIN-1-KARBONITRILA Datum zagovora: 21. 8. 2023 Tajda KLEMEN Mentor: prof. dr. Bogdan Štefane SINTEZA SUBSTITUIRANIH PIRAZOLIDINONOV KOT PREKURZORJEV ZA AZOMETIN IMINE Datum zagovora: 21. 8. 2023 Ana HOČEVAR Mentorica: prof. dr. Irena Kralj Cigić OPTIMIZACIJA EKSTRAKCIJE ANTIOKSIDANTOV IZ PVC Datum zagovora: 24. 8. 2023 Sara KOTNIK Mentor: prof. dr. Matija Strlič MERJENJE EMISIJ KOROZIVNIH HLAPNIH ORGANSKIH SNOVI IZ TESNILNIH MAS Datum zagovora: 24. 8. 2023 Nejc CVETKOVIĆ Mentor: prof. dr. Franc Požgan 2-BROMOPIRIDINI KOT SINTONI ZA PRIPRAVO PIRIDIL PIRIDONOV Datum zagovora: 24. 8. 2023 Lena GROŠELJ Mentor: prof. dr. Franc Požgan KATALITSKA FUNKCIONALIZACIJA C-H VEZI 2-FENILIMIDAZOLA Datum zagovora: 24. 8. 2023 Aljaž FLIS Mentor: prof. dr. Uroš Grošelj SINTEZA IN NADALJNJE PRETVORBE ALIFATSKIH AMIDOV KETOPINSKE KISLINE Datum zagovora: 29. 8. 2023 Rok RUTAR Mentor: prof. dr. Tomaž Urbič SIMULACIJA MOLEKULSKE DINAMIKE ZA SISTEM DELCEV S POTENCIALOM Z VEČ KARAKTERISTIČNIMI DOLŽINAMI Datum zagovora: 29. 8. 2023 Boštjan ADAMLJE Mentor: prof. dr. Uroš Grošelj SINTEZA 2-IZOBUTIL-3-METOKSIPIRAZINA Datum zagovora: 29. 8. 2023 Blaž ANTONIN Mentor: doc. dr. Jakob Kljun SINTEZA IN KARAKTERIZACIJA BAKROVIH(I) KOMPLEKSOV S KELATNIMI 2-(METILTIO)PIRIMIDINI Datum zagovora: 29. 8. 2023 S14 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Nina TEŽAK Mentorica: prof. dr. Helena Prosen RAZVOJ DISPERZIVNE MIKROEKSTRAKCIJE NA TRDNO FAZO ZA IZBRANA OKOLJSKA ONESNAŽEVALA Datum zagovora: 29. 8. 2023 Adam MODIC Mentor: izr. prof. dr. Drago Kočar RAZVOJ SPEKTROSKOPSKE METODE ZA MERJENJE KONCENTRACIJE VODIKOVEGA PEROKSIDA V PLINSKI FAZI Datum zagovora: 29. 8. 2023 Hana Marija ŽIBERT Mentor: prof. dr. Mitja Kolar DOLOČEVANJE JODA V SLINI Z ICP-MS Datum zagovora: 29. 8. 2023 Vid ZEMLJARIČ Mentorica: doc. dr. Marta Počkaj PRIPRAVA IN KARAKTERIZACIJA KOORDINACIJSKIH SPOJIN CINKA S PROPANDIOJSKO KISLINO Datum zagovora: 30. 8. 2023 Eva Lea BOKAL Mentor: doc. dr. Andrej Pevec SINTEZE KOORDINACIJSKIH SPOJIN Z 2-KLOROPIRIDINOM Datum zagovora: 30. 8. 2023 Katja STRAŽIŠČAR Mentor: prof. dr. Uroš Grošelj EPOKSIDACIJA IN NADALJNJE PRETVORBE WEINREBOVEGA AMIDA (+)-IZOKAMFOLENSKE KISLINE Datum zagovora: 31. 8. 2023 Blaž OMAHEN Mentor: prof. dr. Jernej Iskra ŠTUDIJA NUKLEOFILNEGA FLUORIRANJA ALKOHOLOV Z IMIDAZOLNIM TIPOM REAGENTA Datum zagovora: 1. 9. 2023 Dominik KUŠAR Mentor: doc. dr. Krištof Kranjc 3-ACILAMINO-2H-PIRAN-2-ONI IN NJIHOVI DERIVATI KOT DIENI ZA [4+2] CIKOADICIJE Z RAZLIČNIMI CIKLIČNIMI ELEKTRONSKO SIROMAŠNIMI ALKENI Datum zagovora: 1. 9. 2023 Zala HRIBERŠEK Mentorica: doc. dr. Nataša Gros DELOVNE ZNAČILNOSTI MODULARNEGA MOLEKULARNEGA FLUORESCENČNEGA SPEKTROMETRA Datum zagovora: 1. 9. 2023 Jerneja BURG Mentor: prof. dr. Anton Meden RENTGENSKA PRAŠKOVNA DIFRAKCIJA MINERALNIH GNOJIL Datum zagovora: 1. 9. 2023 Bor KOLAR BAČNIK Mentor: doc. dr. Krištof Kranjc SINTEZA 2H-PIRAN-2-ONA IZ AKTIVIRANEGA KETONA Z ZAŠČITENO AMINSKO SKUPINO KOT INTERMEDIATA NA POTI DO DVOJNIH SUKCINANHIDRIDNIH BICIKLO[2. 2. 2]OKTENOV IN NJIHOVIH HIDRAZONOV Datum zagovora: 1. 9. 2023 Tej KOLAR Mentor: prof. dr. Uroš Grošelj ORGANOKATALIZIRANA ADICIJA TETRONSKE IN TETRAMSKE KISLINE NA MICHAELOV AKCEPTOR NA OSNOVI PIRAZOLONA. Datum zagovora: 4. 9. 2023 Luka ZOBEC Mentor: doc. dr. Krištof Kranjc SINTEZE 5,6-DISUBSTITUIRANIH 3-BENZOILAMINO-2H- PIRAN-2-ONOV, PRETVORBE V 3-ACILAMINO DERIVATE TER NADALJNJE CIKLOADICIJE DO BICIKLO[2. 2. 2] OKTENOV Datum zagovora: 4. 9. 2023 Matevž POLAK Mentorica: prof. dr. Helena Prosen OPTIMIZACIJA EKSTRAKCIJE ZA ALKILFENOLE IZ VODNIH VZORCEV Datum zagovora: 4. 9. 2023 Iva EDROVSKA Mentor: prof. dr. Jurij Svete SINTEZA IN PRETVORBE ETIL (E)-3-(FENILAMINO) AKRILATA Datum zagovora: 4. 9. 2023 Ivana OSTOJIĆ Mentor: prof. dr. Bogdan Štefane SINTEZA NEKATERIH 2-ARIL-1,2-DIHIDRO-3H-PIRAZOL- 3-ONOV Datum zagovora: 4. 9. 2023 Fedja ŠTRUKELJ KUČAN Mentorica: prof. dr. Irena Kralj Cigić OPTIMIZACIJA METODE PRIPRAVE VZORCA Z MIKROVALOVNIM RAZKROJEM ZA DOLOČITEV VEČELEMENTNE SESTAVE V VZORCIH INSEKTOV Z ICP-MS Datum zagovora: 4. 9. 2023 Nika RUPNIK Mentor: prof. dr. Janez Košmrlj PRIPRAVA IZBRANIH 2-NAFTILAMINOV Z BUCHERERJEVO REAKCIJO Datum zagovora: 5. 9. 2023 Ruta KOPRIVEC Mentor: prof. dr. Marjan Jereb TRIFLUOROMETILTIOLIRANJE AMINOV Datum zagovora: 6. 9. 2023 Špela MAKUC Mentor: doc. dr. Martin Gazvoda HIDROARILIRANJE STIRENOV Z AROMATSKIMI ALDEHIDI Z UPORABO NIKLJEVEGA KATALIZATORJA Datum zagovora: 6. 9. 2023 S15Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Ana JERE Mentor: prof. dr. Jernej Iskra NUKLEOFILNO FLUORIRANJE SEKUNDARNIH ALKOHOLOV Z DIETILAMINOŽVEPLOVIM TRIFLUORIDOM Datum zagovora: 6. 9. 2023 Špela POPOVIČ Mentor: doc. dr. San Hadži DOLOČANJE TERMODINAMSKE STABILNOSTI RIBOZA- FOSFAT PIROFOSFOKINAZE IZ ESCHERICHIE COLI Datum zagovora: 7. 9. 2023 Dominik DOLINAR Mentor: prof. dr. Franc Perdih OPTIMIZACIJA SINTEZE METIL 6-(HIDROKSIMETIL) PIKOLINATA ZA PRIPRAVO LIGANDOV Datum zagovora: 7. 9. 2023 Dylan Joseph SAMUEL Mentorica: doc. dr. Nataša Gros DOLOČANJE GLICEROLA Z MOLEKULARNO FLOURESCENČNO SPEKTROMETRIJO Datum zagovora: 7. 9. 2023 Maruša ŠČULIJA Mentor: prof. dr. Marjan Jereb FUNKCIONALIZACIJA AMINOV Z N-TRIFLUOROMETILTIOSAHARINOM Datum zagovora: 7. 9. 2023 Eva KOLENC Mentor: doc. dr. Gregor Marolt RAZVOJ IN OPTIMIZACIJA IZOLACIJE KORDICEPINA IZ EKSTRAKTOV GLIVE CORDYCEPS MILITARIS Datum zagovora: 7. 9. 2023 Blaž FRELIH Mentor: prof. dr. Jurij Svete SINTEZA IN PRETVORBE 6-SUBSTITUIRANIH-4,5- DIHIDROPIRIDAZIN-3(2H)-ONOV Datum zagovora: 7. 9. 2023 Anja ŠVAJGER Mentor: prof. dr. Bogdan Štefane SINTEZA 1,4-DIENOV IZ ALIL BROMIDA TER TERMINALNIH ALKINOV Datum zagovora: 7. 9. 2023 Brina BASTIČ Mentorica: prof. dr. Barbara Hribar Lee Vpliv Dodatka Soli Na Stabilnost Koloidnih Suspenzij Nanodelcev Datum zagovora: 7. 9. 2023 David URBANČIČ Mentor: prof. dr. Jurij Svete SINTEZA IN PRETVORBE (E)-3-(FENILAMINO)-1-(4- KLOROFENIL)PROP-2-EN-1-ONA Datum zagovora: 8. 9. 2023 Luka ANDRIJAŠIČ Mentor: doc. dr. Martin Gazvoda AROMATSKI ALDEHIDI KOT SUBSTRATI PRI Z NIKLJEM- KATALIZIRANEM HIDROARILIRANJU STIRENOV Datum zagovora: 8. 9. 2023 Tina ŠEGINA Mentor: doc. dr. Gregor Marolt UPORABA ANALIZNIH METOD V KOMBINACIJI Z METODO GLAVNIH OSI PRI DOLOČEVANJU LASTNOSTI VINA Datum zagovora: 8. 9. 2023 Luka RAČIČ Mentor: prof. dr. Franc Perdih REAKCIJE L-TIROKSINA Z NIKOTINAMIDOM IN 3-HIDROKSIPIRIDINOM Datum zagovora: 8. 9. 2023 Andraž ČERNOGA Mentor: prof. dr. Jernej Iskra REVERZIBILNE INTERAKCIJE KARBOKSILNIH KISLIN Z AMINIRANIM STEKLOM Datum zagovora: 15. 9. 2023 Špela NOVAK Mentorica: prof. dr. Irena Kralj Cigić KARAKTERIZACIJA VEZIV V REALNIH VZORCIH UMETNIŠKIH BARV Datum zagovora: 25. 9. 2023 Nika FRELIH Mentor: izr. prof. dr. Drago Kočar VPLIV STRESNIH POGOJEV NA RAZGRADNJO PARACETAMOLA V ZDRAVILIH Datum zagovora: 4. 12. 2023 KEMIJSKO INŽENIRSTVO – 1. STOPNJA Zala ZIBELNIK Mentor: doc. dr. Rok Ambrožič UPORABA MIKROREAKTORSKE TEHNOLOGIJE ZA SINTEZO BIOKOMPATIBILNIH FILMOV/HIDROGELOV Z ANTIMIKROBNIM DELOVANJEM Datum zagovora: 3. 3. 2024 Matija MARINIČ Mentor: izr. prof. dr. Aleš Ručigaj VPLIV ULTRAZVOČNEGA SONICIRANJA NA ZAMREŽEVANJE TEMPO MODIFICIRANE NANOCELULOZE IN NJENIH MEŠANIC Datum zagovora: 11. 4. 2023 Špela POLAK Mentor: prof. dr. Marjan Marinšek OGNJEODPORNI MATERIALI ZA STEKLARSKE PEČI Datum zagovora: 1. 6. 2023 . Iva KLOFUTAR Mentor: izr. prof. dr. Blaž Likozar PRIMERJAVA UČINKOVITOSTI KATALIZATORJEV PRI RADIOLIZI AMONIJAKA, METANOLA IN OGLJIKOVEGA DIOKSIDA Datum zagovora: 29. 6. 2023 S16 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Simon VELEČIČ Mentor: izr. prof. dr. Aleš Ručigaj HIDROGELI NA OSNOVI KATEHOL-KOVINSKEGA IONA S SPOSOBNOSTJO SAMOCELJENJA Datum zagovora: 5. 7. 2023 Tamara KLEMENČIČ Mentor: izr. prof. dr. Boštjan Genorio SINTEZA GRAFENA NA PLATINSKEM NOSILCU S KEMIJSKIM NAPAREVANJEM IZ PARNE FAZE METANA IN NJEGOVA UPORABA ZA REAKCIJO REDUKCIJE KISIKA Datum zagovora: 7. 7. 2023 Luka RAZBORŠEK Mentor: izr. prof. dr. Boštjan Genorio SINTEZA GRAFENA Z METODO KEMIJSKEGA NAPAREVANJA IZ PARNE FAZE NA NIKLJEVEM SUBSTRATU Datum zagovora: 7. 7. 2023 Žan ADAM Mentor: izr. prof. dr. Boštjan Genorio ZORENJE AKTIVNEGA MATERIALA V SVINČENO- KISLINSKIH ZAGONSKIH AKUMULATORJIH Datum zagovora: 21. 8. 2023 Tjaša JECL Mentor: prof. dr. Marjan Marinšek MODERNI BETONI Z DODATKOM FAZNO SPREMENLJIVIH MATERIALOV Datum zagovora: 21. 8. 2023 Leja TRATAR Mentorica: prof. dr. Andreja Žgajnar Gotvajn ČIŠČENJE INDUSTRIJSKIH ODPADNIH VOD Z AKTIVNIM OGLJEM Datum zagovora: 22. 8. 2023 Filip Jakob NOVAK Mentor: prof. dr. Miran Gaberšček ANALIZA MEHANIZMA REAKCIJE RAZVOJA KISIKA PRI ELEKTROLIZI VODE Datum zagovora: 24. 8. 2023 Ina ROJKO Mentor: prof. dr. Igor Plazl VPLIVNI PARAMETRI NA RAZVOJ POTEKA ZGOŠČEVANJA SUSPENZIJE SADRE Datum zagovora: 25. 8. 2023 Anže PREGRAD Mentor: izr. prof. dr. Boštjan Genorio POSTAVITEV SISTEMA ZA BLISKOVITO JOULE-OVO SEGRETJE OGLJIČNIH MATERIALOV Datum zagovora: 25. 8. 2023 Mario KRIŽNAR Mentor: prof. dr. Igor Plazl PROCES KARBONIZACIJE Z DIMNIMI PLINI V GLOBOKO EVTEKTIČNIH TOPILIH Datum zagovora: 28. 8. 2023 Špela BLAZNIK Mentor: prof. dr. Aleš Podgornik SPREMLJANJE VPLIVA SREBROVIH NANOPLOŠČIC NA BAKTERIJE IN BAKTERIOFAGE TER FLUORESCENTNO BARVANJE BAKTERIOFAGOV Datum zagovora: 28. 8. 2023 Lan ČUČEK MERŠOL Mentorica: izr. prof. dr. Gabriela Kalčikova ŠTUDIJ VEZAVE MIKROPLASTIKE IZ AVTOMOBILSKIH PNEVMATIK NA RASTLINSKO BIOMASO V VODNEM OKOLJU Datum zagovora: 28. 8. 2023 Sara SOVDAT Mentorica: izr. prof. dr. Gabriela Kalčikova VPLIV SUROVE IN STARANE MIKROPLASTIKE NA VODNE ORGANIZME Datum zagovora: 29. 8. 2023 Jan VIDERGAR Mentor: izr. prof. dr. Aleš Ručigaj LASTNOSTI BIOAKTIVNIH POLIMEROV ZA REGENERACIJO KOSTNINE Datum zagovora: 29. 8. 2023 Maja GORENC Mentorica: prof. dr. Polona Žnidaršič Plazl PREGLED VPELJAVE MIKROREAKTORJEV V PROIZVODNJO FARMACEVTSKIH UČINKOVIN Datum zagovora: 29. 8. 2023 Sara PERŠA Mentorica: izr. prof. dr. Gabriela Kalčikova INTERAKCIJE VODOTOPNIH POLIMEROV Z AKTIVNIM BLATOM Datum zagovora: 29. 8. 2023 Lenart MRZELJ Mentor: izr. prof. dr. Aleš Ručigaj UPORABA 3D STRUKTURIRANIH PREVODNIH HIDROGELOV V SISTEMIH ZA SHRANJEVANJE ENERGIJE Datum zagovora: 29. 8. 2023 Maša KAMBIČ Mentorica: izr. prof. dr. Gabriela Kalčikova VPLIV MIKROPLASTIKE IZ AVTOMOBILSKIH PNEVMATIK NA MALO VODNO LEČO atum zagovora: 29. 8. 2023 Anja PERGAR Mentor: prof. dr. Marjan Marinšek SAMOOBNOVLJIVI ENCIMSKI GRADBENI MATERIALI Datum zagovora: 30. 8. 2023 Jaša KONJAR Mentorica: prof. dr. Urška Šebenik DODATEK GRAFEN OKSIDA V POLISAHARIDNE HIDROGELE ZA IZBOLJŠANJE ADSORPTIVNIH SPOSOBNOSTI Datum zagovora: 5. 9. 2023 S17Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Alma Dizdarević Mentorica: prof. dr. Urška Šebenik Somentor: viš. pred. dr. Branko Alič ZAMREŽENJE NANOCELULOZE ZA PRIPRAVO HIDROGELOV Datum zagovora: 5. 9. 2023 Janja SLOVŠA Mentor: izr. prof. dr. Blaž Likozar ELEKTRIFIKACIJA PRI KATALITSKEM POSTOPKU EPOKSIDACIJE Datum zagovora: 5. 9. 2023 Luka MALIĆ Mentor: doc. dr. Tilen Kopač ZAMREŽEVANJE BIOPOLIMEROV ZA NAČRTOVANJE HIDROGELOV, UPORABNIH V BIOMEDICINSKIH APLIKACIJAH Datum zagovora: 5. 9. 2023 Jaka JANEŽIČ Mentorica: prof. dr. Andreja Žgajnar Gotvajn ODSTRANJEVANJE FENOLOV IN NJIHOVIH SPOJIN IZ INDUSTRIJSKIH ODPADNIH VOD Datum zagovora: 6. 9. 2023 Luka KOSTANJŠEK Mentor: prof. dr. Igor Plazl VPLIV RAZLIČNIH ADSORBENTOV NA UČINKOVITOST ADSORPCIJSKEGA SUŠILNIKA Datum zagovora: 6. 9. 2023 Luka FERJANČIČ Mentor: prof. dr. Marko Hočevar IZDELAVA VODNIH TURBIN Z METODAMI 3D TISKA Datum zagovora: 6. 9. 2023 Niko KUČIŠ Mentor: prof. dr. Aleš Podgornik VISOKOPOROZNI PRETOČNI FUNKCIONALNI POLIMERI Datum zagovora: 6. 9. 2023 Leon MISLEJ Mentorica: prof. dr. Andreja Žgajnar Gotvajn ODSTRANJEVANJE STABILNEGA ANTIBIOTIKA GENTAMICIN SULFATA IZ MODELNE ODPADNE VODE Z OZONACIJO Datum zagovora: 6. 9. 2023 Lora ŠTRAVS Mentor: prof. dr. Aleš Podgornik PRIMERJAVA UČINKOVITOSTI LEPLJENJA RAZLIČNIH LEPIL Datum zagovora: 6. 9. 2023 Katja GRUDEN Mentor: prof. dr. Miran Gaberšček BATERIJSKI SISTEMI KOVINA-ZRAK Datum zagovora: 7. 9. 2023 Rok KOZAMERNIK Mentorica: prof. dr. Polona Žnidaršič Plazl IMOBILIZACIJA ENCIMOV NA NANOMATERIALE V MIKROREAKTORJIH Datum zagovora: 7. 9. 2023 Nika HRIBERNIK Mentor: doc. dr. Rok Ambrožič VPLIV FORMULACIJE IN VRSTE ELEKTRODE NA ELEKTRODEPOZICIJO HITOZANA Datum zagovora: 7. 9. 2023 Leila Lea GREGORN Mentor: prof. dr. Igor Plazl TRENDI IN IZZIVI KOMUNALNIH ČISTILNIH NAPRAV Datum zagovora: 7. 9. 2023 Manja PLANINC Mentor: doc. dr. Tilen Kopač NAČRTOVANJE HIDROGELOV ZA UPORABO V FORENZIKI Datum zagovora: 7. 9. 2023 Ajda NOVAK Mentor: prof. dr. Matevž Dular RAZVOJ METODE ZA DOLOČEVANJE BARVIL S SPEKTROFOTOMETROM Datum zagovora: 7. 9. 2023 Jan LESKOVAR Mentorica: doc. dr. Tina Skalar OKOLJSKO SPREJEMLJIVEJŠI NAČINI MODIFIKACIJE LESA ZA IZBOLJŠANJE MEHANSKIH LASTNOSTI IN POŽARNE ODPORNOSTI Datum zagovora: 7. 9. 2023 Martina POTOČNIK Mentor: doc. dr. Rok Ambrožič RAZVOJ IN DIZAJN MIKRO-STRUKTURIRANIH SEPARACIJSKIH NAPRAV – MINIATURIZACIJA KONTINUIRNE DESTILACIJE Datum zagovora: 7. 9. 2023 Nina PUGELJ Mentor: prof. dr. Marjan Marinšek VISOKOENTROPIJSKE ZLITINE Datum zagovora: 7. 9. 2023 Maritn JAZBEC Mentorica: prof. dr. Polona Žnidaršič Plazl UPORABA HIDROGELOV ZA VZGOJO MATIČNIH CELIC Datum zagovora: 8. 9. 2023 Tina ČERNEJŠEK Mentorica: prof. dr. Polona Žnidaršič Plazl BIOSENZORJI NA OSNOVI GLUKOZA OKSIDAZE Datum zagovora: 8. 9. 2023 Helena POTOČNIK Mentor: doc. dr. Rok Ambrožič VPLIV PH VREDNOSTI NA LASTNOSTI HIDROGELOV, PRIPRAVLJENIH Z DODATKOM EVTEKTIČNIH TOPIL Datum zagovora: 8. 9. 2023 Andraž VERCE Mentorica: prof. dr. Polona Žnidaršič Plazl ENOSTOPENJSKO ČIŠČENJE IN IMOBILIZACIJA ENCIMOV Datum zagovora: 8. 9. 2023 S18 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Brin ŠULIGOJ Mentorica: doc. dr. Tina Skalarx KATALIZATORJI NA OSNOVI MATERIALOV Z VISOKO ENTROPIJO Datum zagovora: 15. 9. 2023 Natan VOVK Mentor: izr. prof. dr. Boštjan Genorio OPTIMIZACIJA PROCESA MOKREGA MLETJA OGLJIČNEGA NOSILCA KATALIZATORJA ZA UPORABO V GORIVNIH CELICAH Datum zagovora: 22. 9. 2023 Tjaša NOSE Mentor: prof. dr. Matevž Dular DINAMIKA KAVITACIJSKEGA MEHURČKA MED DVEMA PROSTIMA POVRŠINAMA Datum zagovora: 26. 9. 2023 Ana VALENČIČ Mentorica: doc. dr. Tina Skalar UTRJEVANJE HISTORIČNIH MATERIALOV Z UTRJEVALCI NA OSNOVI KALCIJEVEGA HIDROKSIDA IN KALCIJEVEGA ACETOACETATA Datum zagovora: 27. 9. 2023 Timotej ZGONIK Mentor: doc. dr. San Hadži ANALIZA ENERGIJSKO POMEMBNIH INTERAKCIJ V KOMPLEKSIH NANOTELO-ANTIGEN Datum zagovora: 31. 3. 2023 Zala PERKO Mentor: prof. dr. Marko Novinec PROTIMIKROBNO DELOVANJE IN ISKANJE PROTEINSKIH TARČ IZBRANIH DERIVATOV HIDROKSINAFTOJSKE KISLINE Datum zagovora: 11. 5. 2023 Urša ŠTEFAN Mentor: prof. dr. Janez Plavec ŠESTKVARTETNI G-KVADRUPLEKSI KOT NOVA OBLIKA ŠTIRIVIJAČNE DNA Datum zagovora: 20. 6. 2023 Neža LESKOVAR Mentor: doc. dr. Jernej Ogorevc ONESPOSOBITEV TLR10 V CELICAH A549 Z UPORABO TEHNOLOGIJE CRISPR/CAS9 Datum zagovora: 22. 6. 2023 Rahela PETROVČIČ Mentor: prof. dr. Marko Novinec OVREDNOTENJE PROTIBAKTERIJSKIH LASTNOSTI NEKATERIH DERIVATOV FENIDONA (1-FENILPIRAZOLIDIN-3-ON) Datum zagovora: 3. 7. 2023 Aljaž SIMONIČ Mentor: izr. prof. dr. Miha Pavšič MOLEKULSKA DINAMIKA PROTEINA EPCAM IZ RAZLIČNIH VRST IN NJEGOVA INTERAKCIJA S PARALOGNIM PROTEINOM TROP2 Datum zagovora: 5. 7. 2023 Nuša KOS THALER Mentor: prof. dr. Janez Plavec RNA STIKALO Z METILTRANSFERAZNO AKTIVNOSTJO Datum zagovora: 17. 8. 2023 Anja MOŠKRIČ Mentor: prof. dr. Janez Plavec ŠTUDIJA Z GVANINI BOGATEGA ZAPOREDJA PROMOTORSKE REGIJE GENA SOST, POVEZANEGA Z METABOLIZMOM KOSTI Datum zagovora: 22. 8. 2023 Ema KAVČIČ Mentor: prof. dr. Marko Dolinar RAZVOJ POSTOPKA ZA ANALIZO POLIMORFNIH REGIJ V GENOMU RIŽA Datum zagovora: 30. 8. 2023 Nuša BRDNIK Mentor: izr. prof. dr. Sergej Pirkmajer VPLIV DIFERENCIACIJE SKELETNOMIŠIČNIH CELIC NA IZRAŽANJE RECEPTORJEV ZA CITOKINE IZ DRUŽINE IL-6 Datum zagovora: 30. 8. 2023 Teja SPRUK Mentor: izr. prof. dr. Sergej Pirkmajer VPLIV DIFERENCIACIJE SKELETNOMIŠIČNIH CELIC NA IZRAŽANJE CITOKINOV IZ DRUŽINE IL-6 Datum zagovora: 30. 8. 2023 Bor KRAJNIK Mentor: prof. dr. Marko Dolinar EKSPERIMENTI ZA PRIPRAVO NOVEGA VEKTORJA ZA KLONIRANJE PCR-PRODUKTOV Z UPORABO SISTEMA TOKSIN-ANTITOKSIN IPF_1065/1067 CIANOBAKTERIJE MICROCYSTIS AERUGINOSA PCC 7806SL Datum zagovora: 1. 9. 2023 Ana PERVANJA Mentorica: doc. dr. Vera Župunski KLONIRANJE IN IZRAŽANJE PROTEINA SFPQ Datum zagovora: 1. 9. 2023 Vanja IVOŠEVIĆ Mentorica: doc. dr. Vera Župunski Kloniranje smernih in protismernih ponovitev GGGGCC v vektor pRINT in njihova detekcija v sesalskih celičnih kulturah Datum zagovora: 1. 9. 2023 Žan ŽNIDAR Mentor: prof. dr. Uroš Petrovič VZPOSTAVITEV MODELNEGA SISTEMA ZA PROIZVODNJO NANOTELES V KVASOVKI SACCHAROMYCES CEREVISIAE Datum zagovora: 4. 9. 2023 Rebeka JERINA Mentor: doc. dr. Aljaž Gaber PRIPRAVA REKOMBINANTNEGA ČLOVEŠKEGA Β-KATENINA V FUZIJI S FLUORESCENČNIMI PROTEINI NA N- IN C-KONCU Datum zagovora: 4. 9. 2023 S19Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Eva VENE Mentorica: prof. dr. Nina Gunde Cimerman PROUČEVANJE RAZNOLIKOSTI SEVOV VRSTE HORTAEA WERNECKII RAZLIČNIH PLOIDNOSTI V ODVISNOSTI OD TEMPERATURE IN KONCENTRACIJE NACL Datum zagovora: 5. 9. 2023 Martin STANONIK Mentorica: doc. dr. Nada Žnidaršič ULTRASTRUKTURA HITINSKIH MATRIKSOV V ČREVESU PLODOVE VINSKE MUŠICE (DROSOPHILA SUZUKII) IN KOLORADSKEGA HROŠČA (LEPTINOTARSA DECEMLINEATA) Datum zagovora: 5. 9. 2023 Klara RAZBORŠEK Mentor: prof. dr. Boris Rogelj Molekulsko kloniranje in priprava proteina parapeg HNRNPK v bakteriji Escherichia coli Datum zagovora: 5. 9. 2023 Ela KOVAČ Mentorica: prof. dr. Ksenija Kogej PRIPRAVA IN KARAKTERIZACIJA LIPOSOMOV V MEŠANICAH SOJIN LECITIN-VODA-GLICEROL Z DODATKOM IZVLEČKOV IZ CHIINIH IN KONOPLJINIH SEMEN Datum zagovora: 5. 9. 2023 Tina ZAJEC HUDNIK Mentor: prof. ddr. Boris Turk PRIMERJAVA DVEH BAKTERIJSKIH EKSPRESIJSKIH SISTEMOV ZA PRIPRAVO MIŠJEGA KATEPSINA L Datum zagovora: 5. 9. 2023 Kostadin MITKOV Mentor: doc. dr. Aljaž Gaber Izražanje In Izolacija Rekombinantne Človeške Kinaze CK1α Datum zagovora: 5. 9. 2023 Hana GLAVNIK Mentor: prof. ddr. Boris Turk AKTIVNOST GRANCIMA B V KO-KULTURI PRIMARNIH NARAVNIH CELIC UBIJALK IN RAKAVIH CELIC Datum zagovora: 5. 9. 2023 Luka HAFNER Mentor: prof. dr. Uroš Petrovič OPTIMIZACIJA METOD TRANSFORMACIJE ZA HIERARHIČNO SESTAVLJANJE DNA Datum zagovora: 6. 9. 2023 Jakob TOMŠIČ Mentor: prof. dr. Marko Dolinar POSKUS DIREKTNEGA DOLOČANJA NUKLEOTIDNEGA ZAPOREDJA POMNOŽKOV ZA DOLOČEVANJE ČRTNE KODE DNA ŠKORPIJONOV Datum zagovora: 6. 9. 2023 Jan KOGOVŠEK Mentor: izr. prof. dr. Drago Kočar VALIDACIJA METODE ZA DOLOČANJE OGLJIKOVIH HIDRATOV S FLUORESCENČNO DETEKCIJO Z UPORABO PRISTOPA »QUALITY BY DESIGN« Datum zagovora: 6. 9. 2023 Maja DEUTSCH Mentor: prof. dr. Rok Romih IZRAŽANJE MEHANORECEPTORJEV PIEZO V UROTELIJU MED OBNOVO PO KEMIJSKO IZZVANEM CISTITISU Datum zagovora: 6. 9. 2023 Ena KARTAL Mentorica: doc. dr. Ajda Taler-Verčič PRIPRAVA IN IZOLACIJA REKOMBINANTNEGA PROTEINA BETA KARBOANHIDRAZE RASTLINE ARABIDOPSIS THALIANA Datum zagovora: 6. 9. 2023 Marko KOVAČIĆ Mentor: doc. dr. Aljaž Gaber MOLEKULSKO KLONIRANJE, IZRAŽANJE IN IZOLACIJA REKOMBINANTNE ČLOVEŠKE KINAZE GSK3Β Datum zagovora: 6. 9. 2023 Mateja MILOŠEVIĆ Mentor: doc. dr. San Hadži UPORABA METODE SCANLAG ZA SPREMLJANJE RASTI BAKTERIJSKIH KOLONIJ Datum zagovora: 6. 9. 2023 Jan TREBUŠAK Mentor: doc. dr. Martin Gazvoda IZRAŽANJE PROTEINOV Z NENARAVNO AMINOKISLINO Datum zagovora: 6. 9. 2023 Tina JAVERŠEK Mentorica: izr. prof. dr. Nina Vardjan VZDRAŽNOST ASTROCITOV PO SOČASNI AKTIVACIJI Z AGONISTI ADRENERGIČNIH IN PURINERGIČNIH RECEPTORJEV Datum zagovora: 6. 9. 2023 Mark LOBOREC Mentor: izr. prof. dr. Miha Pavšič IZRAŽANJE KONFORMACIJSKO ZAKLENJENEGA ALFA- AKTININA Datum zagovora: 7. 9. 2023 Klara AŽBE Mentorica: izr. prof. dr. Katarina Černe OPTIMIZACIJA PROTOKOLA ZA DOLOČANJE ATP7A NA CELIČNI LINIJI RAKA JAJČNIKOV S POMOČJO PRETOČNE CITOMETRIJE. Datum zagovora: 7. 9. 2023 Ana MAUČEC Mentor: izr. prof. dr. Miha Pavšič PRIPRAVA HETERODIMERA EKTODOMEN PROTEINOV EPCAM IN TROP2 Datum zagovora: 7. 9. 2023 Gašper STRUNA Mentor: izr. prof. dr. Miha Pavšič PRIPRAVA PROTEINOV WNT IN TEST INTERAKCIJE WNT:EPCAM Datum zagovora: 7. 9. 2023 S20 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Luka STANKOVIĆ Mentor: prof. dr. Marjan Jereb PRETVORBA AROMATSKIH SPOJIN V KETONE NA KLASIČEN IN TRAJNOSTNI NAČIN Datum zagovora: 8. 9. 2023 Miha RAZDEVŠEK Mentor: prof. dr. Marko Novinec PROTEINSKI INŽENIRING DIMERNIH OBLIK ČLOVEŠKEGA KATEPSINA B S KOMBINACIJO RACIONALNEGA PRISTOPA IN NAKLJUČNE MUTAGENEZE Datum zagovora: 8. 9. 2023 David VALTE Mentor: prof. dr. Janez Plavec ŠTUDIJ VPLIVA DVOVALENTNIH KATIONOV NA STRUKTURNE LASTNOSTI DNA S PENTANUKLEOTIDNIMI PONOVITVAMI D(ATTTC)3 Datum zagovora: 8. 9. 2023 Špela SOTLAR Mentor: doc. dr. Gregor Gunčar Kloniranje in priprava proteina Obg iz bakterije Neisseria gonorrhoeae Datum zagovora: 8. 9. 2023 Alliana KOLAR Mentorica: doc. dr. Ajda Taler-Verčič IZRAŽANJE IN IZOLACIJA RASTLINSKIH ENCIMOV PORA IN CYP18-3 Datum zagovora: 8. 9. 2023 Katja RESNIK Mentorica: doc. dr. Tadeja Režen VPLIV KROŽNIH RNA NA IZRAŽANJE GENOV CIRKADIANEGA RITMA V CELIČNI LINIJI U2OS Datum zagovora: 26. 10. 2023 Špela RAPUŠ Mentor: prof. dr. Boris Rogelj IZRAŽANJE IN IZOLACIJA PROTEINA FENILALANIN- TRNA SINTETAZE Datum zagovora: 1. 12. 2023 KEMIJSKA TEHNOLOGIJA – 1. STOPNJA Lea REBERNIK Mentorica: doc. dr. Sabina Huč ANALIZA TEHNIČNIH ZAHTEV ZA PROSTORE, V KATERIH LAHKO NASTANE POVIŠAN TLAK Datum zagovora: 19. 1. 2023 Nuša BENJE Mentorica: prof. dr. Marija Bešter-Rogač MERILA ZA TOPLOTNE OBREMENITVE V SLOVENIJI Datum zagovora: 4. 7. 2023 Jan ANTOLIN Mentorica: doc. dr. Barbara Novosel NADZOR RABE PSIHOAKTIVNIH SNOVI PRI RAZISKOVALNEM DELU Datum zagovora: 4. 9. 2023 Urška RUSTAN Mentorica: doc. dr. Barbara Novosel ZAGOTAVLJANJE VARNEGA IN ZDRAVEGA DELA V KEMIJSKIH LABORATORIJIH Datum zagovora: 5. 9. 2023 Primož RAJŠP Mentor: prof. dr. Simon Schnabl VPLIV METEOROLOŠKIH PARAMETROV NA POJAVNOST IN INTENZITETO POŽAROV V NARAVNEM OKOLJU NA OBMOČJU MOL Datum zagovora: 6. 9. 2023 Žiga MLAKAR Mentor: prof. dr. Matija Tomšič USPOSABLJANJE DELAVCEV ZA VARNO DELO V PODJETJU IZ PANOGE TLAČNEGA LITJA IN ORODJARSTVA Datum zagovora: 6. 9. 2023 David FRANCA Mentorica: prof. dr. Marija Bešter-Rogač PRIMERJAVA PREZRAČEVALNIH SISTEMOV V JAVNIH STAVBAH Datum zagovora: 7. 9. 2023 Lejla VELIĆ Mentorica: doc. dr. Barbara Novosel OBVLADOVANJE SAMOSEGREVAJOČIH IN SAMOREAKTIVNIH NEVARNIH SNOVI Datum zagovora: 7. 9. 2023 Tjaša VRBINC Mentor: prof. dr. Simon Schnabl VARNOST PRI DELU NA DALJAVO Z VIDIKA ELEKTRIKE Datum zagovora: 8. 9. 2023 Jan BEVEC Mentor: prof. dr. Simon Schnabl SAMOVŽIG SENENE KRME V RAZSUTEM STANJU Datum zagovora: 6. 10. 2023 Nika PRELESNIK Mentorica: doc. dr. Barbara Novosel DOLOČITEV MINIMALNE VŽIGNE ENERGIJE CINKOVEGA IN ŽELEZOVEGA PRAHU Datum zagovora: 6. 10. 2023 Matej VIDMAR Mentor: prof. dr. Simon Schnabl POŽARI NA OBJEKTIH V SLOVENIJI OD LETA 2010 DO 2021 Datum zagovora: 20. 10. 2023 S21Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Valentina KOLIGAR Mentorica: doc. dr. Klementina Zupan POZITIVNE IN NEGATIVNE PLATI DELA OD DOMA MED EPIDEMIJO Datum zagovora: 25. 10. 2023 DIPLOME – UNIVERZITETNI ŠTUDIJ KEMIJSKA TEHNOLOGIJA – 1. STOPNJA Iris SAKSIDA Mentor: doc. dr. Črtomir Podlipnik PREGLED INHIBITORJEV GLAVNE PROTEAZE VIRUSA SARS-COV-2 Datum zagovora: 5. 1. 2023 Sara KUŽNER Mentor: doc. dr. Gregor Marolt EKSTRAKCIJA IN DOLOČEVANJE FITINSKE KISLINE TER OSTALIH INOZITOL FOSFATOV V HRANI Z IONSKO KROMATOGRAFIJO Datum zagovora: 31. 1. 2023 Karin VOLT Mentorica: doc. dr. Saša Petriček HIDROKSIPIRIDINIJEVE SOLI – IONSKE TEKOČINE Datum zagovora: 21. 2. 2023 Klara VOLOVEC Mentor: izr. prof. dr. Drago Kočar DOLOČANJE 5-HIDROKSIMETILFURFURALA V MEDU S TEKOČINSKO KROMATOGRAFIJO VISOKE LOČLJIVOSTI Datum zagovora: 29. 3. 2023 Laura ERČULJ Mentor: viš. pred. dr. Branko Alič VPLIV PROCESNIH POGOJEV NA PENJENJE PLASTISOLOV S KEMIJSKIMI PENILCI Datum zagovora: 19. 4. 2023 Eva MUŠIČ Mentor: doc. dr. Bojan Kozlevčar SINTEZA IN KARAKTERIZACIJA KOORDINACIJSKIH SPOJIN S FENOKSIOCETNO KISLINO Datum zagovora: 19. 4. 2023 Erik ČREŠNOVAR Mentorica: izr. prof. dr. Barbara Modec DVOJEDRNE KOORDINACIJSKE SPOJINE BAKRA(II) Z ALKOHOLAMINI Datum zagovora: 25. 4. 2023 Kristijan FRLAN Mentor: viš. pred. dr. Branko Alič 3D TISKANJE UV ZAMREŽLJIVIH SMOL Datum zagovora: 8. 5. 2023 Jure CANKAR Mentor: prof. dr. Franc Požgan TIOFEN KOT TEHNOLOŠKO POMEMBNA ORGANSKA MOLEKULA ALI KOT POLUTANT Datum zagovora: 9. 5. 2023 Lucija KASTELIC Mentorica: izr. prof. dr. Barbara Modec SINTEZE KOORDINACIJSKIH SPOJIN CINKA(II) ALI BAKRA(II) IZ VODNE RAZTOPINE AMONIAKA Datum zagovora: 27. 6. 2023 Daša TRIVUNČEVIĆ Mentor: prof. dr. Matija Strlič VPLIV SESTAVIN SIMULIRANE PLJUČNE TEKOČINE PRI IN VITRO TESTIH TOPNOSTI DELCEV STEKLA Datum zagovora: 7. 7. 2023 Tina SELIČ Mentor: prof. dr. Mitja Kolar VALIDACIJA POSTOPKA DOLOČITVE TOTALNEGA OGLJIKA V TRDNIH VZORCIH ILMENITA IN TITANOVE ŽLINDRE Datum zagovora: 7. 7. 2023 Klara ŠVEGELJ Mentorica: prof. dr. Marija Bešter-Rogač EVTEKTIČNA TOPILA L-MENTOLA IN OKTANOJSKE KISLINE Datum zagovora: 7. 7. 2023 Sara CIMERMANČIČ Mentorica: prof. dr. Barbara Hribar Lee DOLOČANJE VISKOZNOSTI GELOV Z ROTACIJSKIM VISKOZIMETROM Datum zagovora: 17. 8. 2023 Miha KRIŠTOF Mentorica: doc. dr. Nataša Čelan Korošin DOLOČITEV AKTIVACIJSKE ENERGIJE RAZPADA SREBROVIH(I) KOORDINACIJSKIH SPOJIN Z LUTIDINI S TEHNIKO TERMOGRAVIMETRIČNE ANALIZE Datum zagovora: 22. 8. 2023 Manca PASAR Mentor: doc. dr. Bojan Kozlevčar SPOJINE BAKRA, KOBALTA IN CINKA Z IZONIKOTINAMIDOM IN 5-SULFOSALICILNO KISLINO Datum zagovora: 25. 8. 2023 S22 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Karin BOJC Mentor: prof. dr. Iztok Turel OPTIMIZACIJA SINTEZE DERIVATOV PIRITIONA IN NJIHOVIH ORGANORUTENIJEVIH KOORDINACIJSKIH SPOJIN Datum zagovora: 29. 8. 2023 Nastja ŠKUFCA Mentor: prof. dr. Mitja Kolar DOLOČANJE VODIKOVEGA PEROKSIDA V ALKALNEM MEDIJU Datum zagovora: 29. 8. 2023 Nika BRCAR Mentorica: prof. dr. Romana Cerc Korošec DOLOČEVANJE DELEŽA BARVILA KREZOLA, VEZANEGA NA MEZOPOROZEN SIO2, Z METODAMI TERMIČNE ANALIZE Datum zagovora: 29. 8. 2023 Laura TROBEVŠEK Mentorica: izr. prof. dr. Barbara Modec PRIPRAVA IN REAKCIJE IZBRANIH CINKOVIH(II) KOMPLEKSOV Z AMONIAKOM Datum zagovora: 30. 8. 2023 Andraž LUKŠIČ Mentor: doc. dr. Andrej Pevec KINALDINSKA KISLINA KOT KATION V NEKATERIH FLUORIDOMETALATNIH SOLEH Datum zagovora: 30. 8. 2023 Natalija TOMAŽIN Mentorica: prof. dr. Helena Prosen OPTIMIZACIJA EKSTRAKCIJE IZBRANIH ONESNAŽEVAL Z MIKROPLASTIKE V VODNIH VZORCIH Datum zagovora: 30. 8. 2023 Tisa GOLOB Mentor: doc. dr. Andrej Pevec STRUKTURNA PRIMERJAVA FENANTROLINIJEVIH HEKSAFLUORIDOFOSFATOV Datum zagovora: 30. 8. 2023 Karmen RABZELJ Mentor: prof. dr. Miran Gaberšček VPLIV CELULOZE NA STARANJE LITIJEVIH ELEKTROD Datum zagovora: 30. 8. 2023 Alja CRNKIĆ Mentor: doc. dr. Bojan Šarac SPEKTROSKOPSKE RAZISKAVE VEZANJA LIGANDOV NA DNK Datum zagovora: 31. 8. 2023 Laura MARTINČIČ Mentor: doc. dr. Bojan Šarac SPEKTROSKOPSKE RAZISKAVE KEMIJSKE DENATURACIJE PROTEINOV Datum zagovora: 31. 8. 2023 Nataša BARTOL Mentorica: doc. dr. Marta Počkaj IDENTIFIKACIJA KRISTALNIH FAZ V PERORALNIH PRAŠKIH Z RENTGENSKO PRAŠKOVNO DIFRAKCIJO Datum zagovora: 1. 9. 2023 Anja POLJANŠEK Mentorica: prof. dr. Helena Prosen RAZVOJ EKSTRAKCIJE NA TRDNO FAZO Z NEPOLARNIMI INTERAKCIJAMI ZA IZBRANA ONESNAŽEVALA Datum zagovora: 1. 9. 2023 Laura DRAME Mentor: prof. dr. Anton Meden Rentgenska Praškovna Difrakcija Različnih Vrst Cementa Datum zagovora: 1. 9. 2023 Tjaša HABINC Mentor: izr. prof. dr. Drago Kočar DOLOČEVANJE METILSULFONILMETANA V PREHRANSKIH DOPOLNILIH S PLINSKO KROMATOGRAFIJO Datum zagovora: 4. 9. 2023 Natalija PAPEŽ Mentorica: doc. dr. Saša Petriček KOBALTOVE SPOJINE S PIRAZINOJSKO KISLINO Datum zagovora: 4. 9. 2023 Sara PIRC Mentorica: doc. dr. Nataša Čelan Korošin DOLOČITEV IN IZRIS EVTEKTIČNEGA FAZNEGA DIAGRAMA MED AMONIJEVIM NITRATOM IN NATRIJEVIM NITRATOM S POMOČJO DSC ANALIZE, OPTIMIZACIJA PRIPRAVE VZORCA Datum zagovora: 4. 9. 2023 Eva BRAČUN Mentorica: doc. dr. Lidija Slemenik Perše REOLOŠKE LASTNOSTI MASLA Datum zagovora: 5. 9. 2023 Tomaž MEGLAJ Mentor: doc. dr. Jakob Kljun SINTEZA IN KARAKTERIZACIJA SREBROVIH KOMPLEKSOV S 2,5,5-TRIMETIL-1,2,4-TRIAZOLIDIN-3- TIONOM Datum zagovora: 5. 9. 2023 Leja BELE Mentor: prof. dr. Anton Meden KVALITATIVNA FAZNA ANALIZA ZOBNIH PAST Z RENTGENSKO PRAŠKOVNO DIFRAKCIJO Datum zagovora: 5. 9. 2023 Špela ŠTANTE Mentor: doc. dr. Bojan Kozlevčar HIDROKSIPIRIDINSKI KOMPLEKSI BAKROVIH(II) 5-SULFOSALICILATOV Datum zagovora: 5. 9. 2023 S23Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Maja KURENT Mentorica: doc. dr. Nataša Čelan Korošin DOLOČANJE OKSIDACIJSKIH LASTNOSTI JEDILNIH OLJ IN MAŠČOB Z METODAMI TERMIČNE ANALIZE Datum zagovora: 5. 9. 2023 Klara DERGANC Mentorica: doc. dr. Saša Petriček CINKOVE SPOJINE S PIRAZINOJSKO KISLINO Datum zagovora: 5. 9. 2023 Hana BRADAČ Mentorica: izr. prof. dr. Amalija Golobič MINERALNA SESTAVA KAMNIN IN PRSTI S PODROČJA IVANČNE GORICE Datum zagovora: 6. 9. 2023 Matic JERE Mentor: doc. dr. Janez Cerkovnik ŠTUDIJ FORMULACIJ VODIKOVEGA PEROKSIDA Z MLEČNO IN DODECILBENZENSULFONSKO KISLINO Datum zagovora: 6. 9. 2023 Jaka ČRNIČ Mentor: prof. dr. Franc Perdih KARAKTERIZACIJA SOLI DIATRIZOJIČNE KISLINE Datum zagovora: 6. 9. 2023 Mateja ZOTLER Mentor: doc. dr. Gregor Marolt EKSTRAKCIJA ZDRAVILNE UČINKOVINE KORDICEPIN IZ GLIVE CORDYCEPS MILITARIS IN NJEGOVO DOLOČEVANJE Z UPORABO VISOKOLOČLJIVOSTNE TEKOČINSKE KROMATOGRAFIJE Datum zagovora: 7. 9. 2023 Nik KERČMAR Mentor: izr. prof. dr. Janez Cerar ODVISNOST FIZIKALNO-KEMIJSKIH LASTNOSTI KONCENTRIRANIH VODNIH RAZTOPIN GLOBOKO EVTEKTIČNIH TOPIL OD NAČINA NJIHOVE PRIPRAVE Datum zagovora: 7. 9. 2023 Miha TOMŠIČ Mentor: doc. dr. Črtomir Podlipnik UPORABA TRIDIMENZIONALNEGA TISKA V KEMIJI Datum zagovora: 7. 9. 2023 Tim FORTUNA Mentor: doc. dr. Gregor Marolt DOLOČEVANJE FITINSKE KISLINE IN FOSFATOV V EKSTRAKTIH FERMENTIRANE MOKE Datum zagovora: 8. 9. 2023 Ana STRAJNAR Mentor: doc. dr. Bojan Kozlevčar BAKROVE SPOJINE S 5-SULFOSALICILNO KISLINO IN AMIDNIMI DERIVATI PIRIDINA Datum zagovora: 14. 9. 2023 Sanja ŽVEGLIČ Mentor: prof. dr. Franc Perdih SINTEZA CINKOVIH KOMPLEKSOV Z DIPIKOLINSKO KISLINO Datum zagovora: 25. 9. 2023 Maruša TURK Mentorica: izr. prof. dr. Amalija Golobič ANALIZA MODELIRNE MASE IN PLASTELINA Z RENTGENSKO PRAŠKOVNO DIFRAKCIJO Datum zagovora: 26. 9. 2023 Kristina TRUGAR Mentor: doc. dr. Andrej Pevec SINTEZA IN KARAKTERIZACIJA NEKATERIH HALOPIRIDINIJEVIH HAKSAFLUORIDOTITANATOV Datum zagovora: 29. 9. 2023 Špela ŠOLAJA Mentor: prof. dr. Mitja Kolar PRIMERJAVA IN LASTNOSTI KOMERCIALNIH FLUORIDNIH IONOSELEKTIVNIH ELEKTROD Datum zagovora: 4. 10. 2023 Lara VRANIČAR Mentor: prof. dr. Mitja Kolar RAZVOJ IN VALIDACIJA METODE ZA DOLOČANJE RESORCINOLA Datum zagovora: 4. 10. 2023 Ana VIDIC Mentorica: izr. prof. dr. Barbara Modec Somentorica: asist. dr. Nina Podjed NASTANEK ACETAMIDINA V PRISOTNOSTI CINKOVIH(II) KOMPLEKSOV Datum zagovora: 24. 10. 2023 Lara HENČIČ Mentorica: prof. dr. Urška Lavrenčič Štangar FOTOKATALITSKE LASTNOSTI PROZORNIH TANKIH PLASTI DOPIRANEGA TIO2 Z VANADIJEM V SILIKATNEM VEZIVU Datum zagovora: 23. 11. 2023 Špela ŠOLAJA Mentor: prof. dr. Mitja Kolar PRIMERJAVA IN LASTNOSTI KOMERCIALNIH FLUORIDNIH IONOSELEKTIVNIH ELEKTROD Datum zagovora: 4. 10. 2023 Jan GROŠELJ Mentor: prof. dr. Mitja Kolar DOLOČANJE KALIJA V RASTLINSKIH VZORCIH Datum zagovora: 5. 12. 2023 Jaka ROŽEJ Mentor: prof. dr. Iztok Turel PRIPRAVA BAKROVIH KOORDINACIJSKIH SPOJIN IZBRANIH PIRITIONOV Datum zagovora: 22. 12. 2023 S24 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti UNIVERZA V MARIBORU FAKULTETA ZA KEMIJO IN KEMIJSKO TEHNOLOGIJO 1. januar – 31. december 2022 DOKTORATI DOKTORSKI ŠTUDIJ – 3. STOPNJA Katja BIZAJ Mentor: prof. dr. Željko Knez Somentorica: prof. dr. Mojca Škerget EKSTRAKCIJA HMELJA (HUMULUS LUPULUS L. ) S SUB- IN SUPERKRITIČNIMI FLUIDI Datum zagovora: 21. 2. 2023 Staša JURGEC Mentor: prof. dr. Uroš Potočnik Somentor: prof. dr. Željko Knez MOLEKULARNI IN CELIČNI ODZIV NA OKSIDATIVNI STRES, HIPOKSIJO IN NARAVNE ANTIOKSIDANTE, PRIDOBLJENE S KONVENCIONALNO IN SUPERKRITIČNO EKSTRAKCIJO, PRI AKUTNI IN KRONIČNI MIELOIČNI LEVKEMIJI IN VITRO Datum zagovora: 27. 6. 2023 Anja PFEIFER Mentor: prof. dr. Mojca Škerget PRIPRAVA IN UPORABA ADSORBENTA IZ RDEČE SADRE ZA ČIŠČENJE ODPADNIH VOD Datum zagovora: 13. 7. 2023 Sanja POTRČ Mentor: prof. dr. Zdravko Kravanja M SINTEZA TRAJNOSTNIH IN REGENERATIVNIH OSKRBOVALNIH MREŽ NA OSNOVI MATEMATIČNEGA PROGRAMIRANJA ZA POSTOPNO DOSEGANJE OGLJIČNE NEVTRALNOSTI ODELIRANJE IN VEČNAMENSKA OPTIMIZACIJA PRIDOBIVANJA ENERGIJE IN KORISTNIH PRODUKTOV IZ ORGANSKIH ODPADKOV NA OSNOVI ANAEROBNE RAZGRADNJE Datum zagovora: 27. 9. 2023 Ksenija RUTNIK Mentor: dr. Iztok Jože Košir DOLOČANJE SPREMEMB V KEMIJSKI SESTAVI HMELJA PRI RAZLIČNIH POGOJIH SKLADIŠČENJA TER VREDNOTENJE VPLIVA POSTARANEGA HMELJA NA AROMO IN GRENČICO PIVA Datum zagovora: 14. 11. 2023 Stanko KRAMER Mentor: prof. dr. Peter Krajnc PRIPRAVA SINTETIČNIH IN NARAVNIH POROZNIH POLIMEROV IZ VEČFAZNIH MEDIJEV Datum zagovora: 23. 11. 2023 MAGISTERIJI MAGISTRSKI ŠTUDIJ – 2. STOPNJA Noemi SEP Mentorica: izr. prof. dr. Lidija Čuček Somentorica: prof. dr. Lidija Fras Zemljič Sometorica: asist. dr. Olivija Plohl FRAGMENTACIJA PLASTIČNIH MATERIALOV V RAZLIČNIH VODNIH OKOLJIH Datum zagovora: 15. 2. 2023 Andrej ZIDARIČ Mentorica: izr. prof. dr. Lidija Čuček Somentorica: asist. Monika Dokl Somentor: Rok Gomilšek OPTIMIZACIJA GEOTERMALNE ELEKTRARNE V EL SALVADORJU Z OZIROM NA TERMODINAMIKO IN RUDARJENJE BITCOINA Datum zagovora: 15. 2. 2023 Urška BRENCE Mentorica: izr. prof. dr. Lidija Čuček Somentor: dr. Miha Grilc Somentorica: dr. Annamaria Vujanović Somentorica: dr. Edita Jasiukaityte Grojzdek FRAKCIONACIJA LIGNOCELULOZNE BIOMASE TER NJENA PRETVORBA V VREDNE PRODUKTE Datum zagovora: 23. 2. 2023 Nikolina CETIN Mentorica: doc. ddr. Andreja Nemet Somentor: prof. dr. Zdravko Kravanja Somentor: dr. Elvis Ahmetović PRIMERJAVA ZAPOREDNE IN SIMULTANE SINTEZE TOPLOTNO INTEGRIRANIH VODNIH OMREŽIJ Datum zagovora: 22. 3. 2023 S25Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Jernej JELENKO Mentorica: prof. dr. Marjana Simonič Somentorica: izr. prof. dr. Julija Volmajer Valh Somentorica: dr. Matejka Turel ANALIZA BIORAZGRADNJE ZAŠČITNIH MASK ZA PREPREČEVANJE ŠIRJENJA VIRUSA SARS-COV-2 V VODNIH MEDIJIH Datum zagovora: 22. 3. 2023 Ela LIKOVNIK Mentor: izr. prof. dr. Matjaž Finšgar Somentor: asist. David Majer DOLOČEVANJE TEŽKIH KOVIN V PIJAČAH Z ICP-OES Datum zagovora: 22. 3. 2023 Monika OSTROŠKO Mentorica: doc. dr. Petra Kotnik Somentorica: izr. prof. dr. Maša Knez Marevci Somentorica: asist. dr. Taja Žitek Makoter DOLOČANJE FIZIKALNO KEMIJSKIH LASTNOSTI DOSTAVNIH SISTEMOV S KONTROLIRANIM SPROŠČANJEM NARAVNIH UČINKOVIN Datum zagovora: 22. 3. 2023 Žiga ŠROT Mentorica: prof. dr. Marjana Simonič Somentorica: izr. prof. dr. Julija Volmajer Valh Somentorica: dr. Matejka Turel BIORAZGRADNJE ZAŠČITNIH MASK (COVID-19) NA OSNOVI RESPIROMETRIJSKIH MERITEV V KOMPOSTU Datum zagovora: 22. 3. 2023 Ana GARMUT Mentorica: doc. dr. Petra Kotnik Somentorica: izr. prof. dr. Maša Knez Marevci PREUČEVANJE FIZIKALNO-KEMIJSKIH LASTNOSTI NEKATERIH FARMACEVTSKIH UČINKOVIN Datum zagovora: 19. 4. 2023 Špela HABJANIČ Mentorica: prof. dr. Andreja Goršek Somentorica: izr. prof. dr. Darja Pečar VPLIV VSEBNOSTI CO 2 IN PROCESNIH POGOJEV NA POTEK FERMENTACIJE IN SENZORIČNE LASTNOSTI KOMBUČE Datum zagovora: 19. 4. 2023 Mojca HRAŠ Mentorica: prof. dr. Mojca Škerget Somentorica: dr. Majda Hadolin PRIDOBIVANJE PRODUKTOV IZ PEGASTEGA BADLJA (SYLIBUM MARIANUM) Datum zagovora: 19. 4. 2023 Ivan KONJEVIĆ Mentor: izr. prof. dr. Matjaž Finšgar Somentor: dr. Samo Hočevar VPLIV RAZLIČNIH METOD PRIPRAVE POVRŠINE SPE NA ADSORPCIJO IZBRANIH PROTEINOV ZA ZASNOVO BIOSENZORJEV Datum zagovora: 19. 4. 2023 Marjetka KOUTER Mentorica: doc. dr. Petra Kotnik Somentorica: izr. prof. dr. Maša Knez Marevci DOLOČITEV PIROLIZIDINSKIH ALKALOIDOV V MEDU Datum zagovora: 19. 4. 2023 Adriana KRALJ Mentor: prof. dr. Uroš Potočnik Somentor: dr. Boris Gole VPLIV IZRAŽANJA MMD NA ODZIVNOST MONOCITOV IN MAKROFAGOV NA ADALIMUMAB IN VITRO Datum zagovora: 19. 4. 2023 Aljaž ŠPORIN Mentor: izr. prof. dr. Matjaž Finšgar Somentor: asist. David Majer Validacija metod za ovrednotenje prenosa analita avtomatskega sistema dissoBOT za teste raztapljanja farmacevtskih učinkovin in optimizacija cikla čiščenja Datum zagovora: 19. 4. 2023 Žan SMREKAR Mentor: prof. dr. Urban Bren Somentor: izr. prof. dr. Marko Jukić ŠTUDIJ VEZAVE PEPTIDOV NA FC REGIJE PROTITELES Datum zagovora: 23. 5. 2023 Anja COPOT Mentorica: doc. dr. Petra Kotnik Somentorica: izr. prof. dr. Maša Knez Marevci IZOLACIJA TRIGONELINA IZ NARAVNIH MATERIALOV IN NJEGOVA ANTIOKSIDATIVNA AKTIVNOST Datum zagovora: 21. 6. 2023 Tjaša HEDŽET Mentorica: prof. dr. Mojca Škerget Somentor: Hrvoje Ćurić OPTIMIZACIJA PROCESA EKSTRAKCIJE KLOROFILA IZ RASTLINE ALFALFA (MEDICAGO SATIVA) Datum zagovora: 21. 6. 2023 Živa NEKREP Mentorica: doc. dr. Petra Kotnik Somentorica: izr. prof. dr. MAŠA KNEZ MAREVC IZBIRA METODE ZA DOLOČEVANJE ANTIOKSIDATIVNE AKTIVNOSTI BIOLOŠKO AKTIVNIH KOMPONENT Datum zagovora: 21. 6. 2023 Andreja BEŽAN Mentorica: doc. dr. Petra Kotnik Somentorica: izr. prof. dr. Maša Knez Marevci VSEBNOST FLAVONOIDNIH KOMPONENT V POSUŠENIH OLUPKIH MANDARIN IN NJIHOVA ANTIOKSIDATIVNA AKTIVNOST Datum zagovora: 5. 7. 2023 Matija ZRIMŠEK Mentorica: doc. ddr. Andreja Nemet Somentor: doc. dr. Miloš Bogataj PRIMERJAVA OPTIMIRANJA V GAMSU TER NA KVANTNEM RAČUNALNIKU Datum zagovora: 5. 7. 2023 S26 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Anže NOVAK Mentor: izr. prof. dr. Matjaž Finšgar Somentorica: asist. dr. Tanja Vrabelj UPORABA RAZLIČNIH NAČINOV MERJENJA S TEHNIKO MIKROSKOPIJE NA ATOMSKO SILO Datum zagovora: 11. 7. 2023 Tjaša SKARLOVNIK Mentor: prof. dr. Urban Bren Somentor: doc. dr. Gregor Hostnik Somentor: dr. Andraž Lamut MERITVE OSMOLARNOSTI IZOTONIČNIH NAPITKOV ZA HIDRACIJO TELESA PRI ŠPORTNI AKTIVNOSTI Datum zagovora: 11. 7. 2023 Sven GRUBER Mentor: prof. dr. Darko Goričanec Somentorica: izr. prof. dr. Danijela Urbancl INOVATIVEN ENERGETSKI SISTEM UPORABE OBNOVLJIVIH VIROV ENERGIJE ZA VISOKOTEMPERATURNO OGREVANJE Datum zagovora: 31. 8. 2023 Rok KRAMBERGER Mentor: prof. dr. Darko Goričanec Somentorica: izr. prof. dr. Danijela Urbancl PROCES PROIZVODNJE TEKOČEGA ZRAKA Z IZRABO KRIOGENE ENERGIJE UPLINJANJA UTEKOČINJENEGA ZEMELJSKEGA PLINA Datum zagovora: 31. 8. 2023 Klemen ROLA Mentorica: izr. prof. dr. Danijela Urbancl Somentor: prof. dr. Darko Goričanec IZRABA OBNOVLJIVIH VIROV ENERGIJE ZA PROIZVODNJO SINTETIČNEGA METANA Datum zagovora: 31. 8. 2023 Nika ATELŠEK HOZJAN Mentor: prof. dr. Zoran Novak Somentorica: asist. dr. Gabrijela Horvat RAZVOJ MATERIALOV Z AKTIVNIM KISIKOM ZA HITREJŠE CELJENJE RAN Datum zagovora: 1. 9. 2023 Sara KARLOVŠEK Mentor: izr. prof. dr. Maša Knez Marevci Somentorica: asist. dr. Taja Žitek Makoter EKSTRAKCIJA OLJ IZ SEMEN INDUSTRIJSKE KONOPLJE IN KARAKTERIZACIJA BIOLOŠKO AKTIVNIH KOMPONENT Datum zagovora: 1. 9. 2023 Tinkara OŠLOVNIK Mentorica: prof. dr. Zorka Novak Pintarič Somentor: asist. Jan Drofenik KVANTITATIVNO SPREMLJANJE NAPREDKA H KROŽNEMU GOSPODARSTVU Datum zagovora: 1. 9. 2023 Rok PUČNIK Mentorica: izr. prof. dr. Lidija Čuček Somentorica: dr. Annamaria Vujanović Somentorica: dr. Filipa Alexandra Andre Vicente KROŽNO MODRO BIOGOSPODARSTVO ZA VALORIZACIJO ODPADKOV IZ LUPIN KOZIC: OCENA VPLIVOV NA OKOLJE Datum zagovora: 1. 9. 2023 Jan GIMPELJ Mentor: doc. dr. Miloš Bogataj Somentor: Aleš Cvik RAZVOJ IN IMPLEMENTACIJA SIMULACIJSKEGA MODELA REAKTORJA ZA FISHER-TROPSCHEVO SINTEZO ZA ZELENI PREHOD V AVL CRUISE TM M Datum zagovora: 8. 9. 2023 Vajna JAKOVLJEVIĆ Mentorica: doc. dr. Maša Islamčević Razboršek Somentorica: Pija Rep DOLOČANJE TEŽKIH KOVIN Z METODO ICP-MS V VZORCIH NAJBOLJ PRODAJANIH PREHRANSKIH DODATKOV V ČASU EPIDEMIJE COVID-19 IN OCENA KONTAMINACIJE Datum zagovora: 8. 9. 2023 Ana PERPAR Mentor: doc. dr. Sebastijan Kovačič Somentor: dr. Gregor Žerjav FOTOKATALITSKA OKSIDACIJA ONESNAŽIL V ODPADNIH VODAH Z UPORABO VIDNE SVETLOBE IN FOTOKATALIZATORJEV NA OSNOVI TIO 2 Datum zagovora: 8. 9. 2023 Dea SIMONIČ Mentor: prof. dr. Uroš Potočnik Somentorica: prof. dr. Darja Arko Sometorica: Maja Petek PROTEOMSKA ANALIZA MONONUKLEARNIH CELIC PERIFERNE KRVI PRI RAKU DOJKE Datum zagovora: 8. 9. 2023 Urška VTIČ Mentorica: prof. dr. Mojca Škerget Somentorica: asist. dr. Maja Čolnik HIDROTERMIČNO RECIKLIRANJE VOLNENIH TEKSTILNIH ODPADKOV Datum zagovora: 8. 9. 2023 Marcel ŽAFRAN Mentor: izr. prof. dr. Matjaž Finšgar Somentor: dr. Gregor Žerjav SINTEZA IN UPORABA FOTOKATALIZATORJEV V NAPREDNIH OKSIDACIJSKIH POSTOPKIH ZA OKSIDATIVNO RAZGRADNJO ORGANSKIH ONESNAŽIL V ODPADNIH VODAH Datum zagovora: 8. 9. 2023 Nejc BRUNČEK Mentorica: izr. prof. dr. Maša Knez Marevci Somentorica: asist. dr. Milica Pantić Somentor: dr. Andrej Golle PROTIGLIVNI UČINEK EKSTRAKTOV ČESNA (ALLIUM SATIVUM) TER NJIHOVA FORMULACIJA Datum zagovora: 21. 9. 2023 S27Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Maja GRAČNER Mentorica: prof. dr. Mojca Škerget Somentorica: asist. dr. Maja Čolnik SEPARACIJA IN KARAKTERIZACIJA VREDNIH SPOJIN IZ ZUNANJE ARAŠIDOVE (ARACHIS HYPOGAEA L. ) LUPINE Z VODO PRI PODKRITIČNIH POGOJIH Datum zagovora: 21. 9. 2023 Eva KROPUŠEK Mentor: izr. prof. dr. Matjaž Finšgar Somentor: asist. David Majer Somentor: Matej Birk PRIMERJAVA ANALITSKIH TEHNIK ZA DOLOČEVANJE IN SPREMLJANJE NABITIH OBLIK TERAPEVTSKIH PROTEINOV Datum zagovora: 21. 9. 2023 Kaja MAKOTER Mentorica: prof. dr. Mojca Škerget Somentorica: asist. dr. Maja Čolnik RECIKLIRANJE ODPADNIH PLASTIČNIH MAS S HIDROTERMIČNIMI POSTOPKI Datum zagovora: 21. 9. 2023 Andraž OŠTIR Mentor: prof. dr. Peter Krajnc Somentor: prof. dr. Lukas J. Gooben Svetlobno Inducirana S Paladijem Katalizirana Buchwald- Hartwig Aminacija Datum zagovora: 21. 9. 2023 Adam BRUMEN Mentorica: prof. dr. Maja Leitgeb Somentorica: asist. dr. Katja Vasić IMOBILIZACIJA ß-LAKTAMAZE Datum zagovora: 26. 9. 2023 Matic KOŠIR Mentor: prof. dr. Uroš Potočnik Somentor: dr. Rok Gaber FENOTIPIZACIJA SESALSKE CELIČNE LINIJE CHO V BIOPROCESU Z DOHRANJEVANJEM PO INTENZIVIRANEM IN KLASIČNEM POSTOPKU Datum zagovora: 26. 9. 2023 Klemen GRADIŠNIK Mentorica: prof. dr. Mojca Škerget Somentorica: asist. dr. Maja Čolnik Somentorica: dr. Mojca Poberžnik IZOLACIJA KERATINA IZ ODPADNE BIOMASE Z ALKALNO HIDROLIZO Datum zagovora: 18. 10. 2023 Nina BELINA Mentorica: izr. prof. dr. Darja Pečar Somentorica: prof. dr. Andreja Goršek Somentorica: doc. dr. Lucija Črepinšek Lipuš VPLIV MAGNETNEGA POLJA NA POTEK ENCIMSKO KATALIZIRANE REAKCIJE Datum zagovora: 22. 11. 2023 Urban GSELMAN Mentor: prof. dr. Darko Goričanec Somentorica: izr. prof. dr. Danijela Urbancl Somentorica: dr. Mojca Božič PROIZVODNJA ELEKTRIČNE ENERGIJE Z GEOTERMIČNO GRAVITACIJSKO TOPLOTNO CEVJO Datum zagovora: 22. 11. 2023 Zala SERIANZ Mentor: prof. dr. Urban Bren Somentorica: asist. dr. Anja Kolarič ISKANJE NOVIH ZAVIRALCEV, KI PREPREČUJEJO VEZAVO SARS-COV-2 NA NEUROPILIN 1 Datum zagovora: 22. 11. 2023 Nejc ARH Mentorica: doc. ddr. Andreja Nemet Somentorica: asist. dr. Sanja Potrč OPTIMIRANJE Z UPORABO MATLABA V POVEZAVI Z ASPEN PLUS SIMULATORJEM Datum zagovora: 20. 12. 2023 Gal BJELOVUČIĆ Mentor: izr. prof. dr. Matjaž Finšgar Somentor: dr. Roman Kranvogl UPORABA UHPLC-HRMS ZA DOLOČANJE ORGANSKIH SPOJIN V VZORCIH PSIHOAKTIVNIH SNOVI IN PREHRANSKIH DOPOLNIL Datum zagovora: 20. 12. 2023 DIPLOME – UNIVERZITETNI ŠTUDIJ UNIVERZITETNI ŠTUDIJ – 1. STOPNJA Eva ZAJŠEK Mentor: prof. dr. Uroš Potočnik Somentorica: dr. Maya Petek OPTIMIZACIJA PRIPRAVE PROTEINOV KRVNE PLAZME Z RAZGRADNJO NA PEPTIDE V RAZTOPINI ZA PROTEOMSKO ANALIZO Z MASNO SPEKTROMETRIJO Datum zagovora: 22. 3. 2023 Katja ZORKO Mentor: prof. dr. Uroš Potočnik Somentorica: dr. Maya Petek UPORABA SELEKTIVNIH MEMBRAN ZA PRIPRAVO PROTEINSKIH VZORCEV KRVNE PLAZME ZA DOLOČANJE CITOKINOV Z MASNO SPEKTROMETRIJO Datum zagovora: 22. 3. 2023 S28 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Barbara ODER Mentorica: prof. dr. Marjana Simonič Somentorica: doc. dr. Maša Islamčević Razboršek ANALIZA FLUORIDNIH, KLORIDNIH IN SULFATNIH ANIONOV Z IONSKO KROMATOGRAFIJO IN PRIMERJAVA VSEBNOSTI V VODI IN ČAJIH Datum zagovora: 30. 3. 2023 Nika FEKONJA Mentorica: izr. prof. dr. Danijela Urbancl Somentorica: prof. dr. Marjana Simonič ANALIZA OBRATOVANJA IN BLATA MALIH KOMUNALNIH ČISTILNIH NAPRAV Datum zagovora: 5. 7. 2023 Jure KONJAR Mentorica: izr. prof. dr. Darja Pečar Somentorica: prof. dr. Andreja Goršek PRIMERJAVA METOD KONSTRUIRANJA FAZNIH DIAGRAMOV TRDNO-TEKOČE BINARNIH ZMESI ORGANSKIH SPOJIN Datum zagovora: 29. 8. 2023 Neja MENCIGER KOCBEK Mentorica: prof. dr. Andreja Goršek Somentorica: izr. prof. dr. Darja Pečar VPLIV DODATKA KOVINSKIH NANODELCEV NA POTEK ANAEROBNE DIGESTIJE Datum zagovora: 29. 8. 2023 Matej ZAZIJAL Mentorica: izr. prof. dr. Danijela Urbancl Somentorica: doc. dr. Aleksandra Petrovič PRIMERJAVA LASTNOSTI PRODUKTOV PRIDOBLJENIH S HIDROTERMALNO KARBONIZACIJO IN TOREFIKACIJO Datum zagovora: 31. 8. 2023 Karin BAJDE Mentorica: izr. prof. dr. Maša Knez Marevci Somentorica: asist. dr. Taja Žitek Makoter OPTIMIZACIJA POSTOPKA SUPERKRITIČNE EKSTRAKCIJE SLADKEGA PELINA (ARTEMISIA ANNUA L. ) S PROGRAMOM DESIGN EXPERT Datum zagovora: 1. 9. 2023 Miha BERK BEVC Mentor: prof. dr. Zoran Novak Somentorica: asist. dr. Milica Pantić PRIPRAVA KOMPOZITOV IZ POLISAHARIDNIH AEROGELOV IN SUPERKRITIČNIH PEN ZA POTREBE TKIVNEGA INŽENIRSTVA Datum zagovora: 1. 9. 2023 Liza CURK Mentorica: izr. prof. dr. Maša Knez Marevci Somentorica: doc. dr. Petra Kotnik IZOLACIJA ETERIČNIH OLJ IZ POPROVE METE (MENTHA PIPERITA) Datum zagovora: 1. 9. 2023 Tilen FARKAŠ Mentorica: prof. dr. Marjana Simonič Somentorica: doc. dr. Aleksandra Petrovič- RAZVOJ TEHNOLOŠKEGA POSTOPKA ZA ODSTRANJEVANJE MIKROONESNAŽIL NA VIRU PITNE VODE Datum zagovora: 1. 9. 2023 Kaja GAJŠT Mentorica: izr. prof. dr. Mateja Primožič Somentorica: prof. dr. Maja Leitgeb Somentorica: asist. dr. Katja Vasić AKTIVNOST INTRACELULARNIH ENCIMOV IZ GOB Z ANTIKANCEROGENIM DELOVANJEM Datum zagovora: 1. 9. 2023 Kaja KOGAL Mentor: prof. dr. Peter Krajnc Somentorica: dr. Amadeja Koler SINTEZA IN KARAKTERIZACIJA AMINO FUNKCIONALIZIRANEGA AKRILAMIDA KOT PREKURZORJA ZA INTELIGENTNE POLIMERE Datum zagovora: 1. 9. 2023 Doroteja KOVAČ Mentorica: prof. dr. Mojca Škerget Somentorica: asist. dr. Maja Čolnik SEPARACIJA VREDNIH SPOJIN IZ KORUZNIH STORŽEV Z UPORABO EKSTRAKCIJE S PODKRITIČNO VODO Datum zagovora: 1. 9. 2023 Neva KOVAČ Mentorica: izr. prof. dr. Mateja Primožič Somentorica: prof. dr. Maja Leitgeb Somentorica: asist. Nika Kučuk LIPOSOMI KOT NOSILCI BIOAKTIVNIH UČINKOVIN Datum zagovora: 1. 9. 2023 Eva PATIK Mentorica: prof. dr. Regina Fuchs Godec Somentor: prof. dr. Urban Bren KOMERCIALNA KURKUMA KOT INHIBITOR KOROZIJSKIH PROCESOV Datum zagovora: 1. 9. 2023 Anže PEGAN Mentor: izr. prof. dr. Matjaž Kristl TERMOGRAVIMETRIČNA, ULTIMATIVNA IN PROKSIMATIVNA ANALIZA PLASTIČNIH MATERIALOV Datum zagovora: 1. 9. 2023 Tine PIGAC Mentorica: doc. ddr. Andreja Nemet Somentor: doc. dr. Miloš Bogataj OPTIMIZACIJA ALTERNATIVNEGA ENERGETSKEGA SISTEMA Z VKLJUČEVANJEM PLINASTIH GORIV Datum zagovora: 1. 9. 2023 Armina RAHMANOVIĆ Mentorica: izr. prof. dr. Maša Knez Marevci Somentorica: sist. dr. Taja Žitek Makoter OPTIMIZACIJA POSTOPKA EKSTRAKCIJE FENOLNIH SPOJIN IZ NAVADNEGA OŽEPKA (HYSSOPUS OFFICINALIS) Datum zagovora: 1. 9. 2023 S29Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Ana RAJH Mentorica: izr. prof. dr. Maša Knez Marevci Somentorica: sist. dr. Taja Žitek Makoter OPTIMIZACIJA POSTOPKA EKSTRAKCIJE SLADKEGA PELINA (ARTEMISIA ANNUA L. ) ZA IZOLACIJO ANTIOKSIDATIVNIH IN PROTIRAKAVIH UČINKOVIN Datum zagovora: 1. 9. 2023 Tajda SENEKOVIČ Mentorica: prof. dr. Maja Leitgeb Sometorica: asist. dr. Katja Vasić Somentorica: izr. prof. dr. Mateja Primožič IMOBILIZIRANA Β-LAKTAMAZA ZA ČIŠČENJE ODPADNIH VOD Datum zagovora: 1. 9. 2023 Vitan ŠLAMBERGER Mentor: prof. dr. Peter Krajnc Somentori asist. Stanko Kramer HIPERZAMREŽENJE POLIHIPE MATERIALOV Z UPORABO TIOL-EN KLIK KEMIJE Datum zagovora: 1. 9. 2023 Sanja TOPLAK Mentorica: izr. prof. dr. Maša Knez Marevci Somentorica: asist. dr. Taja Žitek Makoter VPLIV OPTIMIZIRANEGA EKSTRAKTA CVETNEGA PRAHU NA METABOLNO AKTIVNOST MELANOMSKIH CELIC WM-266-4 Datum zagovora: 1. 9. 2023 Neža ZANJKOVIČ Mentor: izr. prof. dr. Matjaž Kristl Somentorica: doc. dr. Janja Stergar MEHANOKEMIJSKA SINTEZA NANODELCEV TERNARNIH KADMIJEVIH HALKOGENIDOV Datum zagovora: 1. 9. 2023 Chiara ŽELEZNIK Mentorica: prof. dr. Mojca Škerget Somentorica: asist. dr. Maja Čolnik HIDROTERMIČNO UPLINJANJE LIGNOCELULOZNE BIOMASE Datum zagovora: 1. 9. 2023 Jan ČOKOLIČ Mentor: prof. dr. Urban Bren Somentor: dr. Matja Zalar EKSPERIMENTALNA ANALIZA VEZAVE POMOŽNIH SNOVI NA ZDRAVILNO UČINKOVINO Datum zagovora: 8. 9. 2023 Katja FINGUŠT Mentorica: izr. prof. dr. Maša Knez Marevci Somentorica: asist. dr. Darija Cör Andrejč VPLIV PROCESNIH PARAMETROV NA KAKOVOST IN OBSTOJNOST EKSTRAKTA AMERIŠKEGA SLAMNIKA (ECHINACEA PURPUREA) Datum zagovora: 8. 9. 2023 Tadej JERŠIČ Mentor: doc. dr. Sebastijan Kovačič Somentor: prof. dr. Zoran Novak SINTEZA Π-KONJUGIRANIH POLIMERNIH PEN Z RAZLIČNIMI REAKCIJAMI KONDENZACIJE Datum zagovora: 8. 9. 2023 Tinkara KOVAČIČ Mentorica: doc. dr. Janja Stergar Somentorica: doc. dr. Irena Ban SINTEZA FECU MAGNETNIH NANODELCEV S PLANETARNIM MIKROMLINOM ZA UPORABO V BIOMEDICINSKIH APLIKACIJAH Datum zagovora: 8. 9. 2023 Ana Katarina KOVAČIČ Mentorica: prof. dr. Mojca Škerget Somentorica: asist. dr. Maja Čolnik IZOLACIJA KERATINA IZ ODPADNE BIOMASE S KISLINSKO HIDROLIZO Datum zagovora: 8. 9. 2023 Marijana KRSTIĆ Mentor: prof. dr. Urban Bren Somentorica: prof. dr. Aleksandra Lobnik Somentorica: Valeriia Sliesarenko OPTIČNA DETEKCIJA STRESNIH PARAMETROV Datum zagovora: 8. 9. 2023 Jure MARTINUZZI Mentorica: izr. prof. dr. Maša Knez Marevci Somentorica: doc. dr. Petra Kotnik PRIMERJAVA NEKATERIH KEMIJSKIH LASTNOSTI RAZLIČNIH VRST MEDU Datum zagovora: 8. 9. 2023 Marko PAVLOVIĆ Mentorica: izr. prof. dr. Darja Pečar Somentorica: prof. dr. Andreja Goršek SINTEZA HETEROGENEGA KATALIZATORJA ZA KISLINSKO HIDROLIZO PET Datum zagovora: 8. 9. 2023 Žiga ŠKRINJARIČ Mentorica: doc. dr. Muzafera Paljevac Somentor: prof. dr. Peter Krajnc RAZVOJ SINTEZNE METODE ZA PRIPRAVO BIARILOV Z ULLMANNOVO REAKCIJO Datum zagovora: 8. 9. 2023 Jure ŠUSTER Mentor: prof. dr. Uroš Potočnik Somentor: dr. Tomaž Büdefeld Somentor: doc. dr. Boštjan Lanišnik VLOGA DOLGE NEKODIRAJOČE RNA INC-FANCI-2 PRI RAKU GLAVE IN VRATU Datum zagovora: 8. 9. 2023 Stela TASHKOVA Mentor: prof. dr. Urban Bren Somentor: asist. dr. Žiga Zebec OPTIMIZACIJA POGOJEV ZA ENCIMATSKO RAZGRADNJO VLAKNATIH BOMBAŽNIH TEKSTILNIH ODPADKOV Datum zagovora: 8. 9. 2023 S30 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Amanda ŽIŽEK Mentorica: doc. dr. Janja Stergar Somentorica: doc. dr. Irena Ban SINTEZA FUNKCIONALIZIRANIH MAGNETNIH NANODELCEV Z MIKROVALOVNO PEČICO ZA UPORABO V BIOMEDICINI Datum zagovora: 8. 9. 2023 Aljaž KNEZ Mentor: prof. dr. Urban Bren Somentor: izr. prof. dr. Marko Jukić ANALIZA GRUČ KEMIJSKEGA PROSTORA PROTIBAKTERIJSKIH UČINKOVIN Datum zagovora: 21. 9. 2023 Petra MUNĐAR Mentor: doc. dr. Miloš Bogataj Somentorica: doc. ddr. Andreja Nemet RAZVOJ SUROGATNIH ALGEBRSKIH MODELOV KINETIČNIH REAKTORJEV Datum zagovora: 21. 9. 2023 Karin TURNER Mentorica: doc. dr. Irena Ban Somentorica: doc. dr. Janja Stergar SINTEZA MAGNETNIH NANODELCEV ZA UPORABO V BIOMEDICINI Datum zagovora: 21. 9. 2023 Melani VENGUST Mentorica: izr. prof. dr. Maša Knez Marevci Somentorica: asist. dr. Taja Žitek Makoter IZOLACIJA KAPSAICINIDOV IZ RAZLIČNIH VRST ČILI PAPRIK Datum zagovora: 21. 9. 2023 DIPLOME – VISOKOŠOLSKI STROKOVNI ŠTUDIJ VISOKOŠOLSKI STROKOVNI ŠTUDIJ – 1. STOPNJA Maša KRAKAR Mentor: prof. dr. Zoran Novak Somentorica: dr. Gabrijela Horvat Somentor: dr. Boštjan Jerman MIGRACIJA VODE V ZMESI FARMACEVTSKE UČINKOVINE IN POMOŽNIH SNOVI Datum zagovora: 22. 3. 2023 Manca POTOČNIK Mentor: dr. Blaž Likozar Somentor: dr. Matej Huš KINETIČNO MODELIRANJE EPOKSIDACIJE ETILENA NA SREBROVIH KATALIZATORJIH Datum zagovora: 30. 3. 2023 Alen KOSTEVC Mentor: prof. dr. Urban Bren Somentor: dr. Tomaž Mohorič ADSORPCIJA POLIAKRILNE KISLINE (Paa) NA POVRŠINO KALCIJEVEGA KARBONATA Datum zagovora: 23. 5. 2023 Tajda FLIS Mentorica: doc. dr. Mojca Slemnik Somentorica: doc. dr. Janja Stergar KOROZIJA KOVINSKIH IMPLANTATOV V UMETNI SLINI S SIMULACIJO DODATKA ŽELODČNE KISLINE Datum zagovora: 1. 9. 2023 Sara GRM Mentorica: prof. dr. Marjana Simonič Somentor: dr. Andrej Horvat ŠTUDIJA MOŽNOSTI UPORABE ZEOLITOV ZA FIKSIRANJE KOVINSKIH IONOV V SEDIMENTIH Datum zagovora: 1. 9. 2023 Lea KAISERSBERGER Mentorica: prof. dr. Marjana Simonič Somentorica: doc. dr. Aleksandra Petrovič Somentor: dr. Andrej Horvat MOŽNOST UPORABE ZEOLITOV PRI ODSTRANJEVANJU IZBRANIH KOVINSKIH IONOV IZ VODE Datum zagovora: 1. 9. 2023 Patricija ZAVRŠKI Mentorica: prof. dr. Marjana Simonič Somentorica: doc. dr. Aleksandra Petrovič MODIFIKACIJA HIDRO-OGLJA PRIDOBLJENJEGA S HIDROTERMALNO KARBONIZACIJO ZA UPORABO V PROCESU ADSORPCIJE Datum zagovora: 1. 9. 2023 Karin JAKOPIČ Mentorica: izr. prof. dr. Maša Knez Marevci Somentorja: asist. Vesna Postružnik VPLIV POSTOPKA PREDOBDELAVE MATERIALA NA VSEBNOST VITAMINOV V EKSTRAKTIH CVETNEGA PRAHU Datum zagovora: 8. 9. 2023 S31Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Neja SAVEC Mentorica: red. prof. dr. Maja Leitgeb Somentorica: doc. dr. Mateja Primožič Somentorica: asist. NIKA KUČUK FORMULACIJA NANOLIPIDNIH VEZIKLOV ZA KOZMETIČNO INDUSTRIJO Datum zagovora: 8. 9. 2023 Benjamin STRADAR Mentor: izr. prof. dr. Marko Jukić Somentor: prof. dr. Urban Bren NAČRTOVANJE PENTAPEPTIDOV ZA VEZAVO NA FC REGIJO PROTITELES Datum zagovora: 21. 9. 2023 Karin GOLE Mentorica: doc. dr. Maša Islamčević Razboršek Somentor: dr. Miha Ocvirk OPTIMIZACIJA IN VALIDACIJA METODE ZA DOLOČANJE SESTAVE ETERIČNIH OLJ KONOPLJE Datum zagovora: 26. 9. 2023 Urban KOLER Mentorica: izr. prof. dr. Darja Pečar Somentorica: prof. dr. Andreja Goršek SINTEZA IN KARAKTERIZACIJA KATALIZATORJEV ZA DEGRADACIJO POLIMEROV Datum zagovora: 18. 10. 2023 S32 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti UNIVERZA V NOVI GORICI FAKULTETA ZA ZNANOSTI O OKOLJU 1. januar – 31. december 2023 MAGISTRSKI ŠTUDIJ MAGISTRSKI ŠTUDIJSKI PROGRAM OKOLJE – 2. STOPNJA Manuel PERSOGLIA Mentor: prof. dr. Anton Brancelj SPREMLJANJE SEZONSKE DINAMIKE BENTOŠKIH ORGANIZMOV V DVEH VISOKOGORSKIH JEZERIH V JULIJSKIH ALPAH Datum zagovora: 29. 8. 2023 Irma HOSTNIK Mentorica: dr. Manca Kovač Viršek PRISOTNOST MIKROPLASTIKE IN NJENA PESTROST V VODNEM STOLPCU IN ŠKOLJKAH KLAPAVICAH (MYTILUS GALLOPROVINCIALIS) IZ ŠKOLJČIŠČ SLOVENSKEGA MORJA Datum zagovora: 6. 10. 2023 DIPLOME UNIVERZITETNI ŠTUDIJSKI PROGRAM OKOLJE – 1. STOPNJA DIPLOMSKI SEMINARJI: Miroslav ŠTRBAC Datum diplomiranja: 5. 7. 2023 Katarina ERKER Datum diplomiranja: 5. 7. 2023 Kenan KAPETANOVIĆ Datum diplomiranja: 5. 7. 2023 Matej POGORELC Datum diplomiranja: 5. 7. 2023 Tjaša RUTAR Datum diplomiranja: 5. 7. 2023 Nina ŽVAB PERNAT Datum diplomiranja: 5. 7. 2023 Blaž BOHINC Datum diplomiranja: 5. 7. 2023 Gaja RAMIĆ Datum diplomiranja: 5. 7. 2023 Lucijan Danijel ZGONIK Datum diplomiranja: 5. 7. 2023 Patrik CINGERLI Datum diplomiranja: 12. 9. 2023 Hena ZUKIĆ Datum diplomiranja: 14. 9. 2023 S33Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti 2024 May 2024 19 – 22 INTERNATIONAL CONFERENCE ON BIOMASS – ICONBM2024 Palermo, Italy Information: https://www.aidic.it/iconbm2024/ 26 – 29 INTERNATIONAL SCHOOL OF PROCESS CHEMISTRY 2024 (ISPROCHEM 2024) Gargnano, Italy Information: http://www.isprochem.unimi.it/ 27 – 31 POLY-CHAR 2024 – POLYMERS FOR OUR FUTURE Madrid, Spain Information: https://www.poly-char2024.org June 2024 2 – 7 8TH EUCHEMS CONFERENCE ON NITROGEN LIGANDS (NLIGANDS’2024) Padova, Italy Information: https://photocat24.com/ 3 – 7 INTERNATIONAL SCHOOL IN PHOTO AND BIOCATALYSIS (PHOTOCAT24) Cassis, France Information: https://www.cinam.univ-mrs.fr/site/NLigands2024/ 17 – 21 12TH EUROPEAN CONFERENCE ON SOLAR CHEMISTRY AND PHOTOCATALYSIS: ENERGY AND ENVIRONMENTAL APPLICATIONS Belfast, United Kingdom Information: https://www.ulster.ac.uk/conference/spea12 24 – 28 85TH PRAGUE MEETING ON MACROMOLECULES – POLYMERS FOR SUSTAINABLE FUTURE Prague, Czech Republic Information: https://www.imc.cas.cz/sympo/85pmm/ 27 – 30 5TH INTERNATIONAL CONGRESS OF CHEMISTS AND CHEMICAL ENGINEERS OF BOSNIA AND HERZEGOVINA Sarajevo, Bosnia and Herzegovina Information: https://icccebih.dktks.ba/ 30 – 3 19TH INTERNATIONAL BIOTECHNOLOGY SYMPOSIUM Rotterdam, Netherlands Information: https://www.ecb2024.com/ KOLEDAR VAŽNEJŠIH ZNANSTVENIH SREČANJ S PODROČJA KEMIJE IN KEMIJSKE TEHNOLOGIJE SCIENTIFIC MEETINGS – CHEMISTRY AND CHEMICAL ENGINEERING S34 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti 30 – 5 XVI POSTGRADUATE SUMMER SCHOOL ON GREEN CHEMISTRY Venezia, Italy Information: https://www.greenchemistry.school/ July 2024 2 – 5 7TH INTERNATIONAL CONGRESS CHEMISTRY FOR CULTURAL HERITAGE 2024 (CHEMCH 2024) Bratislava, Slovakia Information: https://chemch2024.educell.sk/ 7 – 11 9TH EUCHEMS CHEMISTRY CONGRESS (ECC9) Dublin, Ireland Information: https://euchems2024.org/ 7 – 10 BALTICUM ORGANICUM SYNTHETICUM 2024 (BOS2024) Riga, Latvia Information: https://boschem.eu/ S35Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti S36 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Sub mis sions Submission to ACSi is made with the implicit under- standing 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 else- where. 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The legend should be inside the graph area. The style of all graphs should be the same. Figures and illustrations should be of sufcient 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 refer- ees, ideally as JPEGs. High­resolution figures (in JPEG, TIFF, or EPS format) might be required if the paper is accepted for publication. Tab les should be prepared in the Word file of the pa- per as usual Word tables. The captions should appear above the table and should be self-explanatory. Re fe ren ces should be numbered and ordered se- quentially as they appear in the text, likewise meth- ods, tables, figure captions. When cited in the text, reference numbers should be superscripted, follow- ing punctuation marks. It is the sole responsibility 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. Whi te, Ac ta Chim. Slov. 2008, 55, 1055–1059. 2. M. F. Kem me re, T. F. Keu rent jes, in: S. P. Nu nes, K. V. Pei ne mann (Ed.): Mem bra ne Tech no logy in the Che mi cal In du stry, Wi ley­VCH, Wein heim, Ger­ many, 2008, pp. 229–255. 3. J. Le vec, Ar ran ge ment and pro cess for oxi di zing an aqu e ous me dium, US Pa tent Num ber 5,928,521, da te of pa tent July 27, 1999. 4. L. A. Bur sill, J. M. Tho mas, in: R. Ser sa le, C. Col le la, R. Aiel lo (Eds.), Re cent Pro gress Re port and Dis cus­ sions: 5th In ter na tional Zeo li te Con fe ren ce, Na ples, Italy, 1980, Gia ni ni, Na ples, 1981, pp. 25–30. 5. J. Sze gez di, F. Csiz ma dia, Pre dic tion of dis so cia tion con stant using mi cro con stants, http://www. che­ ma xon.com/conf/Pre dic tion_of_dis so cia tion _con­ stant_using_mi cro co nstants.pdf, (as ses sed: March 31, 2008) Titles of journals should be abbreviated according to Chemical Abstracts Service Source Index (CASSI). Spe cial No tes • Com ple te cha rac te ri za tion, inc lu ding cry stal struc tu re, should be gi ven when the synthe sis of new com pounds in cry stal form is re por ted. • Nu me ri cal da ta should be re por ted with the num ber of sig ni fi cant di gits cor res pon ding to the mag ni tu de of ex pe ri men tal un cer tainty. • The SI system of units and IUPAC re com men­ da tions for nomenclature, symbols and abbrevia- tions should be followed closely. Additionally, the authors should follow the general guidelines when citing spectral and analytical data, and depositing crystallographic data. • Cha rac ters 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 ru les and re com men da tions of the IUBMB and the In ter na tio nal Union of Pure and Ap plied Che mi stry (IUPAC) should be used for abbreviation of chemical names, nomenclature of chemical com- pounds, enzyme nomenclature, isotopic compounds, optically active isomers, and spectroscopic data. • A conf ict of in te rest occurs when an individual (author, reviewer, editor) or its organization is in- S38 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti volved in multiple interests, one of which could pos- sibly corrupt the motivation for an act in the other. Financial relationships are the most easily identifi- able conflicts of interest, while conflicts can occur also as personal relationships, academic competi- tion, etc. The Edi 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 aut hors 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 fund- ing support should be mentioned. The statement of disclosure must be provided as Comments to Editor during the submission process. • Pub lis hed sta te ment of In for med Con sent. Research described in papers submitted to ACSi must adhere to the principles of the Declaration of Helsinki (http://www.wma.net/e/po licy/ b3.htm). These studies must be approved by an appropriate institutional review board or commit- tee, and informed consent must be obtained from subjects. The Methods section of the paper must include: 1) a statement of protocol approval from an institutional review board or committee and 2), a statement that informed consent was obtained from the human subjects or their representatives. • Pub lis hed Sta te ment of Hu man and Ani mal Rights.When reporting experiments on human subjects, authors should indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and na- tional) and with the Helsinki Declaration of 1975, as revised in 2008. 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 ap- proved 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. • To avoid conflict of interest between authors and referees we expect that not more than one referee is from the same country as the corresponding au- thor(s), however, not from the same institution. • Con tri bu tions aut ho red by Slo ve nian scien tists are evaluated by non-Slovenian referees. • Pa pers des cri bing mi cro wa ve­as si sted reac­ tions performed in domestic microwave ovens are not considered for publication in Acta Chimica Slovenica. • Ma nus cripts that are not pre pa red and sub mit­ ted in ac cord with the in struc tions for aut hors are not con si de red for pub li ca tion. Ap pen di ces Authors are encouraged to make use of supporting in- formation for publication, which is supplementary ma- terial (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 particular- ly 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 to be accessible to readers without difculty. All files of supplementary materials are loaded separately during the submission process as supplementary files. Pro po sed Co ver Pic tu re and Grap hi cal Ab stract Image Grap hi cal con tent: an ideally full-colour illustration of resolution 300 dpi from the manuscript must be proposed with the submission. Graphical abstract pic- tures are printed in size 6.5 x 4 cm (hence minimal resolution of 770 x 470 pixels). Cover picture is print- ed in size 11 x 9.5 cm (hence minimal resolution of 1300 x 1130 pixels) Authors are encouraged to submit illustrations as can- didates for the journal Cover Picture*. The illustration must be related to the subject matter of the paper. Usually both proposed cover picture and graphical ab- stract are the same, but authors may provide different pictures as well. * The authors will be asked to contribute to the costs of the cover picture production. Sta te ment of no velty Statement of novelty is provided in a Word file and submitted as a supplementary file in step 4 of sub- mission process. Authors should in no more than 100 words emphasize the scientific novelty of the present- ed research. Do not repeat for this purpose the con- tent of your abstract. List of sug ge sted re vie wers List of suggested reviewers is a Word file submitted as a supplementary file in step 4 of submission pro- cess. Authors should propose the names, full afliation (department, institution, city and country) and e­mail addresses of five potential referees. Field of expertise and at least two references relevant to the scientif- ic field of the submitted manuscript must be provid- ed for each of the suggested reviewers. The referees should be knowledgeable about the subject but have no close connection with any of the authors. In addi- tion, referees should be from institutions other than (and countries other than) those of any of the authors. Authors declare no conflict of interest with suggested reviewers. Authors declare that suggested reviewers are experts in the field of submitted manuscript. How to Sub mit Users registered in the role of author can start sub- mission by choosing USER HOME link on the top of the page, then choosing the role of the Author and follow the relevant link for starting the submission process. Prior to submission we strongly recommend that you familiarize yourself with the ACSi style by browsing the journal, particularly if you have not submitted to the ACSi before or recently. S39Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti Cor res pon den ce All correspondence with the ACSi editor regarding the paper goes through this web site and emails. Emails are sent and recorded in the web site database. In the correspondence with the editorial ofce please provide ID number of your manuscript. All emails you receive from the system contain relevant links. Please do not answer the emails directly but use the embed­ ded links in the emails for carrying out relevant actions. Alternatively, you can carry out all the ac- tions and correspondence through the online system by logging in and selecting relevant options. Proofs Proofs will be dispatched via e-mail and corrections should be returned to the editor by e­mail as quick- ly as possible, normally within 48 hours of receipt. Typing errors should be corrected; other changes of contents will be treated as new submissions. Sub mis sion Pre pa ra tion Chec klist As part of the submission process, authors are required to check off their submission’s compliance with all of the following items, and submissions may be returned to authors that do not adhere to these guidelines. 1. The submission has not been previously published, nor is it under consideration for publication in any other journal (or an explanation has been provid- ed in Comments to the Editor). 2. All the listed authors have agreed on the content and the corresponding (submitting) author is re- sponsible for having ensured that this agreement has been reached. 3. The submission files are in the correct format: manuscript is created in MS Word but will be sub­ mitted in PDF (for reviewers) as well as in orig- inal MS Word format (as a supplementary file for technical editing); diagrams and graphs are cre- ated in Excel and saved in one of the file formats: TIFF, EPS or JPG; illustrations are also saved in one of these formats. The preferred position of graphic files in a document is to embed them close to the place where they are mentioned in the text (See Author guidelines for details). 4. The ma nus cript has been exa mi ned for spel ling and gram mar (spell chec ked). 5. The tit le (ma xi mum 150 cha rac ters) briefly ex­ plains the con tents of the ma nus cript. 6. Full names (first and last) of all authors together with the afliation address are provided. Name of author(s) denoted as the corresponding author(s), together with their e­mail address, full postal ad- dress and telephone/fax numbers are given. 7. The ab stract sta tes the ob jec ti ve and conc lu­ sions of the re search con ci sely in no mo re than 150 words. 8. Keywords (minimum three, maximum six) are provided. 9. Sta te ment of no velty (maximum 100 words) clearly explaining new findings reported in the manuscript should be prepared as a separate Word file. 10. The text adheres to the stylistic and bibliographic requirements outlined in the Aut hor gui de li nes. 11. Text in normal style is set to single column, 1.5 line spacing, and 12 pt. Times New Roman font is recommended. All tables, figures and illustrations have appropriate captions and are placed within the text at the appropriate points. 12. Mathematical and chemical equations are provided in separate lines and numbered (Arabic numbers) consecutively in parenthesis at the end of the line. All equation numbers are (if necessary) appropri- ately included in the text. Corresponding numbers are checked. 13. Tables, Figures, illustrations, are prepared in cor- rect format and resolution (see Aut hor gui de li­ nes). 14. The let te ring used in the fi gu res and graphs do not vary greatly in si ze. The re com men ded let te ring si ze is 8 point Arial. 15. Separate files for each figure and illustration are prepared. The names (numbers) of the separate files are the same as they appear in the text. All the figure files are packed for uploading in a single ZIP file. 16. Aut hors ha ve read spe cial no tes and ha ve ac cor- dingly pre pa red their ma nus cript (if ne ces sary). 17. Re fe ren ces in the text and in the Re fe ren ces are cor rectly ci ted. (see Aut hor gui de li nes). All ref- erences mentioned in the Reference list are cited in the text, and vice versa. 18. Permission has been obtained for use of copy- righted material from other sources (including the Web). 19. The names, full afliation (department, institution, city and country), e­mail addresses and referenc- es of five potential referees from institutions other than (and countries other than) those of any of the authors are prepared in the word file. At least two relevant references (important recent papers with high impact factor, head positions of departments, labs, research groups, etc.) for each suggested re- viewer must be provided. Authors declare no con- flict of interest with suggested reviewers. Authors declare that suggested reviewers are experts in the field of submitted manuscript. 20. Full-colour illustration or graph from the manu- script is proposed for graphical abstract. 21. Ap pen di ces (if appropriate) as supplementary material are prepared and will be submitted at the same time as the manuscript. Pri vacy Sta te ment The na mes and email ad dres ses en te red in this journal si te will be used exc lu si vely for the sta ted pur po ses of this jour nal and will not be ma de avai lab le for any ot­ her pur po se or to any ot her party. ISSN: 1580­3155 S40 Acta Chim. Slov. 2024, 71, (1), Supplement Društvene vesti in druge aktivnosti 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/ European Science Foundation www.esf.org European Federation of Chemical Engineering https://efce.info/ International Union of Pure and Applied Chemistry https://iupac.org/ Brussels News Updates http://www.euchems.eu/newsletters/ Novice europske zveze kemijskih društev EuChemS najdete na: Koristni naslovi B R E ZP LA Č N O POSEBNA PONUDBA Telefon: +386 (0)1 24 182 09 Email: office-si@donaulab.com Izkoristite ponudbo! Skenirajte QR kodo Naročite rotavapor z vakuumskim kontrolerjem in črpalko Prejmite pretočni hladilnik BREZPLAČNO www.helios-group.eu Znanje, kreativnost zaposlenih in inovacije so ključnega pomena v okolju, kjer nastajajo pametni premazi skupine KANSAI HELIOS. Z rešitvami, ki zadostijo široki paleti potreb, kontinuiranim razvojem ter s kakovostnimi izdelki, Helios predstavlja evropski center za inovacije in poslovni razvoj skupine Kansai Paint. Razvoj in inovacije za globalno uspešnost 1. Sevelius H et al. Bioavailability of Naproxen Sodium and Its Relationship to Clinical Analgesic Effects. Br J Clin Pharmacol 1980; 10: 259–63. www.nalgesin.si Pred uporabo natančno preberite navodilo! O tveganju in neželenih učinkih se posvetujte z zdravnikom ali s farmacevtom. Ali ste vedeli, da deluje do 12 ur? Hitro opravi z bolečino. Učinkuje v 15 minutah. (1) N al ge si n S v se b uj e na tr ije v na p ro ks en at . 444402-2023 Nalgesin S Ad 205x276 ACSi SI.indd 1 27. 10. 2023 11:17:35 Hajdrihova 19, 1000 Ljubljana Slovenia www.ki.si Basic and applied research in materials, life sciences, biotechnology, chemical engineering, structural and theoretical chemistry, analytical chemistry and environmental protection. In line with EU research and innovation priorities: nanotechnology, genomics and biotechnology for health, sustainable development, climate change, energy efficiency and food quality and safety. We expand knowledge and technology transfer to domestic and foreign chemical, automotive and nanobiotechnology industries. We are aware of the power of youth, so we transfer our knowledge to younger generations and offer many opportunities for cooperation. contact: mladi@ki.si research EXCELENCE 4 n Year 2024, Vol. 71, No. 1 ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChim A cta C him ica Slovenica 71/2024 Pages 1–178 Pages 1–178 n Year 2024, Vol. 71, No. 1 http://acta.chem-soc.si 1 71/2024 1 ISSN 1580-3155 In 2023, 46 study programmes offering environmental sciences with diverse chemistry content were identified in Slovenia, comprising ten in secondary education, ten in short-cycle higher vocational education, nine in bachelor’s programmes, 11 in master’s programmes, and six in doctoral programmes. Environmental Education Programmes