Scientific paper
A Simple Spectrophotometric Determination of Meptyldinocap by its Hydrolysis
Sunita Kurup* and Ajai Kumar Pillai
Department of Chemistry, Govt. V Y T. P.G. Autonomous College, Durg (C.G.), India. 491001 * Corresponding author: E-mail: sunik09@gmail.com Received: 17-06-2012
Abstract
A simple spectrophotometric method is proposed for the determination of meptyldinocap (2,4-dinitro-6-octylphenyl crotonate). The method is based on the hydrolysis of meptyldinocap by hydroxylamine solution in alkaline medium to give 2,4-dinitro-6-octylphenol (2,4-DNOP), having maximum absorption at 380 nm. The reaction is found to be instantaneous in presence of ethanol. Beer's law is valid over the concentration range of 1.2-13 |g mL-1 with molar absorptivity and Sandell's sensitivity of 3.22 x 106 L mol-1cm-1 and 0.0001 |g cm-2 respectively. The limit of detection and quantification were 0.0892 and 0.2703 |g mL-1, respectively. The tolerance limits of interfering ions are discussed. All variables were studied in order to optimize the reaction conditions. The validity of the method was checked by its simultaneous determination in fruits and water samples and the results were statistically compared with those of a reference method by applying the Student's t-test and F-test.
Keywords: Spectrophotometric, Meptyldinocap, Hydroxylamine
1. Introduction
Meptyldinocap also known as "Karathane" is a contact fungicide with protective and curative action, effective in controlling powdery mildew on grapevine, but is also
being developed for use on cucurbits and strawberries.1,2 The maximum residue limit (MRL) of meptyldinocap was 0.05 |g g-1 in mangoes set by the European Union (EC-No 839/2008).3 It is highly toxic to human and warm- blooded animals. It is reported to be carcinogenic, genotoxic and reproductively toxic on long term exposure.4
Due to its wide applicability and high toxicity, various methods have been reported for the determination of meptyldinocap such as HPLC,5-7 GC/IR and GC/MS,8 GC/ECD,9 TLC,10 LC-MS/MS,11 and differential pulse voltametry.12 Spectroscopic methods by diazotization coupling using reagents NEDA13 and by its hydrolysis using ethanolamine14 also have been reported for its determination.
Due to its toxic nature, it is important to develop some more simple and sensitive methods for the determination of meptyldinocap. The proposed method involves hydrolysis of meptyldinocap (2,4-DNOPC) by alkaline hydroxylamine solution to give 2,4-dinitro-6-octylphenol (2,4-DNOP)14-16 having maximum absorbance at 380 nm.
2. Experimental 2. 1. Apparatus
A Systronics Visiscan 167 Spectrophotometer was used for spectral measurements. Systronics pH meter, model 335 was used for pH measurement.
2. 2. Reagents
All the reagents were of analytical grade. Doubly distilled water was used throughout the experiments for preparation and dilution of reagents as well as samples. The experiments were performed using: meptyldinocap (48 % E/C Dow Agro Science Ltd., India), solvents: acetone, ethanol (Merck, Mumbai, India), sodium hydroxide (Merck, Mumbai, India) and hydroxyl ammonium chloride (Merck, Mumbai, India).
Stock solution (1 mg mL-1) of meptyldinocap solution was prepared by dissolving 0.2083g of meptyldinocap in 100 mL acetone and stored in refrigerator. Working standards prepared by appropriate dilution of the stock.
Alkaline hydroxylamine solution was prepared freshly by mixing equal volume of 2 mol L-1 hydroxyl ammonium chloride and 3.5 mol L-1 sodium hydroxide solutions. The solution kept on ice has been found to be stable for ~3 h.
2. 3. Procedure
An aliquot of working standards containing 30 to 325 of meptyldinocap was transferred into a series of 25 mL calibrated flasks, and to each flask 1 mL of 40 % ethanol and 2 mL of alkaline hydroxylamine solution were added, then diluted to mark with distilled water and mixed thoroughly. The yellow color of 2,4-dinitro-6-octylphenol was developed instantaneously at room temperature. The absorbance was measured at 380 nm against reagent blank.
3. Result and Discussion
The hydrolysis of meptyldinocap by alkali to 2,4-dinitro-6-octylphenol is a slow process; it takes several hours depending upon the strength of alkali and the temperature. In the reported method11 meptyldinocap (2,4-dinitro-6-octylphenyl crotonate) was heated at 60 °C in an ultrasonic bath for 1 h to liberate 2,4-DNOP. In the present work 2,4-DNOPC was reacted with alkaline hydroxy-lamine solution with instantaneous development of colored species, i.e. alkaline hydroxylamine solution hydrolyses 2,4-DNOPC to 2,4-DNOP which gives characteristic absorption at 380 nm at room temperature (Scheme 1). The
action rate and absorbance was obtained at 2 mol L-1 hydroxylamine further increase in the concentration has no change in absorbance (Fig. 1).
Fig. 1: Effect of concentration on the sensitivity. (a) hydroxylamine, (b) NaOH; Conditions: meptyldinocap: 8 |g mL-1; 40 % EtOH; temperature: 25 °C
Meptyldinocap	Hydroxylamine
Scheme 1: Reaction of meptyldinocap with alkaline hydroxylamine
reaction rate is increased when the reaction was performed in the presence of ethanol. The intensity and stability of colored species depends on the concentration of reagent.
3. 1. Effect of Sodium Hydroxide Concentration
The effect of sodium hydroxide concentration on the absorbance value was investigated in the concentration range of 0.5 mol L-1 to 5 mol L-1. Maximum absorbance was obtained at 3.5 mol L-1 further increase in the concentration has no change in absorbance (Fig. 1).
3. 2. Effect of Hydroxylamine Concentration
The effect of hydroxylamine concentration on the absorbance value and reaction rate was studied in the concentration range of 0.5 mol L-1 to 4 mol L-1. Maximum re-
3. 3. Effect of pH
The effect of pH on the hydrolysis of meptyldinocap was studied in the range of 6 to 12. It has been found that the absorbance and reaction rate of the system increased till pH 10 and remained almost the same in the range 10 to 12 (Fig. 2).
3. 4. Effect of Time and Temperature
The kinetic characteristics of the proposed method were studied. After addition of alkaline hydroxylamine solution in the presence of ethanol, the color intensity of the detection system was recorded as a function of reaction time (Fig. 3). From the figure 3, it can be seen that there was a marked increase in the color intensity within few seconds of addition of reagent and it reaches maximum in about 3 min, after which it remains almost constant. To obtain high sensitivity and reproducible results, a
Fig. 2: Effect of pH on the sensitivity. Conditions: hydroxylamine: 2 mol L-1; NaOH: 3.5 mol L-1; 40 % EtOH; temperature: 25 °C
5 min reaction time was selected for further experiments. Further increase in the time there was no change in ab-sorbance. Hence, a 5 min reaction time at room temperature was sufficient for routine analysis. It was also observed that there was no appreciable change in the ab-sorbance if the order of addition of reagents were varied.
Fig. 3: Kinetic behavior of the proposed reaction. (a) alkaline hydroxylamine, (b) alkaline hydroxylamine + meptyldinocap (12 |g mL-1), (c) alkaline hydroxylamine + meptyldinocap (12 |g mL-1) +40 % EtOH
3. 5. Effect of Ionic Strength
The effect of ionic strength on the sensitivity of reaction was studied. The ionic strength was varied from 0.02 to 0.40 mol L-1 using sodium chloride solution. The
results showed that this factor had no effect on the sensitivity of reaction up to 0.05 mol L-1. A further increase of ionic strength caused a decrease in the sensitivity of reaction.
3. 6. Spectral Characteristics and Analytical Parameters
Fig. 4: Calibration line for determination of meptyldinocap. Conditions: hydroxylamine: 2 mol L-1; NaOH: 3.5 mol L-1; 40 % EtOH; temperature: 25 °C
The absorption spectra of final colored product showed a maximum absorbance at 380 nm. The reagent blank has negligible absorbance at this wavelength. Beer's law was obeyed over the concentration range of 1.2-13 ^g mL-1 for meptyldinocap (Fig. 4). The molar absorptivity and Sandell's sensitivity17 of meptyldinocap is given in Table 1. The slope, intercept, and the correlation coefficient were calculated by least square regression analysis (Table 1). The standard deviation17 and relative standard deviation17 for seven replicate determinations of 8 ^g mL-1 showed that the precision was good for the method (Table 1). Limit of quantification (LOQ) is evaluated by the relation 10 o/s and the limit of detection is by 3 o/s, where o is standard deviation of the blank with respect to water and s is the slope of the calibration curve.
In order to evaluate the analytical applicability of the proposed method to the quantification of meptyldino-cap in fruits and water samples, the results obtained by the proposed method was compared to those of the reference method13 by applying Student's t-test17 for accuracy and F-test17 for precision. The results (Table 1) show that the Student's t-test and F-values at 95 % confidence level are
less than the theoretical values, which confirmed that there is a good agreement between the results obtained by the proposed methods and the reference method with respect to accuracy and precision.
Table 1: Analytical and method validation parameters
Parameter	Dinocap
^max (nm)	380
Limit of Beer's Law (|g mL 1)	1.2-13
Molar absorptivity (Lmol-1 cm-1)	3.2 x106
Sandell's sensitivity (|g cm-2)	0.0001
Limit of detection (|g mL-1)	0.089
Limit of quantification (|g mL-1)	0.270
Regression equation(y = bx + a)*	
Correlation coefficient(r)	0.999
Slope ( b )	0.062
Intercept (a)	0.002
Standard deviation(±)	0.004
Relative standard deviation	0.76 %
t**	0.683
F**	1.147
* Concentration in |g mL-1; ** The F and t value refer to comparison of the proposed method with the reported method. Theoretical F value at 95 % confidence level is 5.05; Theoretical t value at 95 % confidence level is 2.228.
3. 7. Interference Studies
In order to evaluate the suitability of the proposed method for the determination of meptyldinocap, in various environmental matrices, the effect of common interfering ions and pesticides on the reaction was studied by adding known amount of these compounds and ions to a standard meptyldinocap solution (8 |g mL-1) and analyzed, the tolerance limit is given in Table 2.
Table 2: The effect of foreign species on reaction Interfering ions and pesticides Tolerance limit (^g mL-1)a
carbaryl , propoxur
Cd2+,Mg2+, Ca2+,Sn2+, CO32-
Fe2+ b, NH4+
Captan
moncozeb
so42-,ch3coo-
Cr3+, Na+, K+, Mn2+. Al3+ Atrazine, pyrethroid pesticide
NO0
1000 500 450 450 400 250 200 100
a Causing(±)2 % variation in absorbance value; b masking by the addition of 2 mL of 0.4 mol L-1 EDTA
3. 8. Performance of Proposed Method and Statistical Comparison With Reported Methods
The proposed method was applied for the determination of meptyldinocap by spiking fruits and water sam-
ples with a known quantity of meptyldinocap and recovery studies carried out. The results obtained by the proposed method were confirmed by reported method.13 It is evident from Table 3 that there is no significant difference in the results between the spiked amounts and those determined by the proposed and reported method, indicating that the proposed method is an accurate and precise as the reported method.
4. Application
4. 1. Determination of Meptyldinocap in Water
To check the applicability of the method, the determination of meptyldinocap in polluted water from agriculture fields was carried out. The samples were found to be free from meptyldinocap. Therefore, known amount of meptyldinocap were added to polluted water samples. Aliquot of spiked water samples was extracted with 2 x 20 mL of benzene from 100 mL of sample. The benzene extract was evaporated off under reduced pressure to obtain the solid residue containing meptyldinocap. This residue was dissolved in ethanol and analyzed by the proposed as well as the reported method13 and the result is shown in Table 3.
4. 2. Determination of Meptyldinocap in Fruits
Fruits samples were collected from fields were meptyldinocap was sprayed. These samples were washed well with 2x20 mL of ethanol. The washings were analyzed by the proposed method. The fruits were found to have negligible amount of meptyldinocap. Therefore known amount of meptyldinocap was spiked to 25 g of mangoes and grapes and left for 24 h. The mango and grapes sample were chopped and macerated with 50 mL of acetone: methanol: 4 N HCl (100:10:5; v/v/v). The sample was centrifuged, and the supernatants were transferred to a clean bottle via a glass wool. An aliquot 30 mL of acetone: methanol: 4 N HCl (100:10:5; v/v/v) was added to the residue and macerated as previously. The sample was centrifuged, and the supernatants were combined. The total volume was adjusted to 100 mL using the extraction solvent. An aliquot of supernatants was evaporated off under reduced pressure and then residue is dissolved in ethanol and analyzed by proposed as well as the reported method13 and the result are shown in Table 3.
5. Conclusion
The proposed method is simple, rapid and sensitive as compared with other spectrophotometry method for
Table 3: Determination of meptyldinocap in various samples
Proposed method	Reported method
Sample	Meptyldinocap added (^g)	Total meptyldinocap recovery founda (MS)	SDb	%	Total meptyldinocap recovery founda (Mg)	SDb	%
Water							
	50	47.40	± 0.03	94.80	46.20	± 0.04	92.40
	100	96.74	± 0.02	96.74	94.80	± 0.03	94.80
	200	198.80	± 0.02	99.40	196.68	± 0.03	98.34
Mangoes	50	47.87	± 0.04	95.74	47.16	± 0.04	94.32
	100	98.70	± 0.03	98.70	97.52	± 0.03	97.52
	200	199.20	± 0.03	99.60	197.60	± 0.03	98.80
Grapes	50	47.81	± 0.03	95.62	46.76	± 0.03	93.72
	100	97.82	± 0.03	97.82	96.80	± 0.03	96.80
	200	198.01	± 0.04	99.01	197.40	± 0.04	98.70
a Mean of three observations. b Standard deviation. c The reported method involves the reduction of nitro group of meptyldinocap to amine and coupled with NEDA by diazotization coupling.
determination of meptyldinocap. The color development was rapid and involves less stringent control of experimental conditions including duration of time analysis and pH control, which make this method easy and more practical as far as the procedure is concerned. In addition, the proposed method neither involves solvent extraction nor employs sophisticated instruments. The statistical parameter and recovery study data clearly indicates the precision and accuracy of the method. Thus, it can be concluded that this method can be used as a chemical sensor for determination of meptyldinocap in water and fruits samples.
6. Acknowledgement
The authors are thankful to DST-FIST for instrumental support and Department of chemistry, Govt. V. Y. T. P. G. Autonomous College Durg, for providing laboratory facilities and granting permission to carry out the research work.
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Povzetek
Predlagamo preprosto spektrofotometrično metodo za določitev meptildinokapa (2,4-dinitro-6-oktilfenil krotonat). Metoda je osnovana na hidrolizi meptildinokapa z raztopino hidroksilamina v alkalnem, pri čemer nastane 2,4-dinitro-6-oktilfenol (2,4-DNOP), ki ima absorpcijski maksimum pri 380 nm. Reakcija poteče takoj v prisotnosti etanola. Beerov zakon velja v koncentracijskem območju 1,2-13 |g mL-1, molska absorptivnost je 3,22 x106 L mol-1 cm 1 in Sandellova občutljivost je 0,0001 |g cm-2. Meja zaznave je 0,0892 |g mL-1, meja določanja pa 0,2703 |g mL-1. Razpravljamo tudi o mejah tolerance za moteče ione.
Preučevali smo vse spremenljivke z namenom optimizacije reakcijskih pogojev. Uspešnost metode smo preverili s hkratno določitvijo v vzorcih sadja in vode, rezultate pa smo statistično primerjali z rezultati referenčne metode; uporabili smo Študentov t test in F test.