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Kinetic study of the degradation of chlorpyrifos by using a stopped-flow fia system. Semiautomatic determination in commercial formulations
Talanla,Vol. 41, No. 5, pp. 651-657,1994 Copyright0 1994Ebevier8cienczLtd Printedin GreatBritain.All rightsreserved 0039-9140/Ws7.00 + 0.00
Pergamon
KINETIC STUDY OF THE DEGRADATION OF CHLORPYRIFOS BY USING A STOPPED-FLOW FIA SYSTEM. SEMIAUTOMATIC DETERMINATION IN COMMERCIAL FORMULATIONS A. ESPINGBA-MANSILW*,F. SUNAS and A. ZAMORO Department of Analytical Chemistry, University of Extremadura, 06071 Badajoz, Spain (Received 16 June 1993. Revised 15 September 1993. Accepted 30 September 1993) Smanury-A kinetic study of the degradation of chlorpyrifos in an oxidative alkaline medium has been undertaken by using a stopped-flow system. A semiautomatic method for determining chlorpyrifos is proposed for the first time. Linear calibration graphs up to 2 x lo-‘M with a relative standard deviation of 1.8% are obtained. A detection limit of 6.6 x 10Y6Mis reported. Carbaryl, dimethoate and endosulfan have been assayed as interference species. The proposed method has been applied to analyze a commercial formulation.
Chlorpyrifost is the common name accepted for the chemical O,O-diethyl-0-(3,5,6-trichloro-2pyridyl) phosphorotiate. This compound is an organophosphate pesticide exhibiting a broad spectrum of activity as insecticide, reported and reviewed for several authorsi Chlorpyrifos presents a hydrolysis process in alkaline medium and the rate of reaction increases with temperature, presence of metals and elevate pH values. In soil it is initially degraded to 3,5,6-trichloro pyridin-2-01 (TCP) which is subsequently degraded to organ0 chlorine compounds.5 Residues of this pesticide have been evaluated in many substracts. 6-g The classical analytical procedures, widely applied, consisted essentially of extractions with a solvent, water partition and cleanup on a chromatographic column followed by gas chromatographic determination. Several multiresidues methods exist.‘+i2 Recently,13 a comparative study of the different methods for the determination of organochlotine and organophosphorous pesticides in fruit samples has been carried out. A number of solvent extraction systems have been used for multiresidue screening procedures; these mainly include: acetonitrile, acetone, methanol, methylene chloride-methanol, acetone-methanol and *Author to whom correspondence should be addressed. TRegistered as Dursban by Dow Chemical Company. TAL ‘M-c
ethylacetate. In the mentioned paper, capillary gas chromatography was used for identification and quantitation of the individual pesticides in spiked peach samples. The AOAC recommeded method which used acetonitrile or acetonitrile-water,‘4 was modified in 1985 by recommending the use of acteone for extraction of pesticides residues in non-fatty foodsi HPLC techniques are widely employed in the analysis of chlorpyrifos.‘6’g In the AOAC recommended method for formulation analysis an acetonitrile extraction is employed prior to chromatographic separation and small amount of acetic acid is added to mobile phase (acetor&rile-water) to suppress non-reproducible ionization of 3,5,6-trichloro-2-pyridinol, which might otherwise cause interference.20 Recently, HPLC methods have been proposed to analyze chlorpyrifos in production processes” and in water.= Most of papers reported do not incorporate the analysis of metabolites, however, recently, two chromatographic methods for determination of chlorpyrifos and its metabolite (TCP) have been reported by using GC23 and HPLC.” The kinetic methods have been sparingly applied in the analysis of pesticides although inhibition or reactivation processes of the activities of some enzymes due to organophosphorous pesticides have been reported.25*26 A kinetic
651
652
A. ESPINOGA-MANSILLA et al.
no-enzymatic method based in oxidation of o-dianisidine has been proposed for the determination of organophosphorus compounds in water.” The most recent bibliography data apply the classical cholisterase test to water analysis by means of kinetic measurements and computer evaluation of the results. A mixture of paraoxon, carbofuran and chlorpyrifos was calibrated.“’ In this paper a kinetic determination of chlorpyrifos based in the spectrophotometric measure of the degradation-reaction rate in alkaline medium is reported. Although chromatographic methods are the most powerful tool in analysis of pesticides, the following specific advantages in the application of semiautomatic kinetic method can be expected: (a) elimination of some experimental steps prior to the absorbance measurements (e.g. filtration, extraction . . . ), hence considerably simpler and
faster procedures are proposed for routine control, (b) selectivity due to the measure of the evolution of the absorbance with the time reaction instead of the measure of a concrete absorbance value, (c) possibility of no interference of the coloured and/or turbidity background of the samples and (d) possibility of no interference of other absorbent pesticides presenting in the samples if they are exhibiting stability in the chemical reaction conditions established for the proposed method. EXPERIMJZNTAL
Apparatus
A home-made stopped-flow system (Scheme la) was fitted to a Milton Roy Spectronic 3000 Array spectrophotometer (PC-286 microcomputer), equipped with a IO-mm microflow cell (18 ~1). A Milton Roy software was used for all data acquisition, measurement and analysis of the kinetic data. A thermostatically controlled
detector
1
30
T (seg) NaoIi(O.5 M)+&O~(O.5g/L)+EtOh(40%) (a)
Scheme 1. Schematic diagrams used for the determination of chlorpyrifos by (a) stopped-flow system; (b) time program used for the stopped-flow system. Td, delay time; Tm, measurement time.
653
Kinetic study of the degradation of chlorpyrifos 2.00
spectrum
one spectrum
each 4
one specWt#a
min?
each 2 min.
each
2 min.
C
P
Time (aec)
?
Fig. 1. Evolution of absorption espectrum of chlorpyrifos in (a) absence of hydrogen peroxide, (b) in the presence of 0.1 g/l and (c) 0.8 g/l hydrogen peroxide and (d) kinetic curves obtained at 290 and 328 nm in the presence of 0.5 g/l of hydrogen peroxide.
bath, Selecta Unitronic 320 OR, was used for temperature control. A precision peristaltic pump (Gilson Minipuls-2) and a six-way injection valve with a variable loop (Omnifit) were used. An interface2’ has been built to allow the starting and stopping of the pump, allowing the automation and control of the peristaltic pump through a microcomputer and an adequate BASIC program.2’ The time diagram used for the stopped-flow method is described in Scheme l(b). The delay time (Td) and the measure time (Tm) are controlled by means of input instructions through the internal clock of a microcomputer (Commodore 64). Reagent and chemicals Standard solutions (500 pg/ml) of chlorpyriand dimethoate fos, carbaryl, endosulfan (Chem. Service) were prepared by disolution in ethanol.
All chemicals and solvents used were of analytical reagent grade. The commercial formulation Sadicloato was obtained from SADISA (Spain). Procedure Determination of chlorpyrifos by the stoppedflow system. A 50 ~1 aliquot of a 40% ethanol sample solution containing up to 2.63 pg of chlorpyrifos was injected through the injection valve into the carrier stream (0.5 M NaOH + 40% (v/v) ethanol + 0.55 g/l H201) by using the manifold shown in Scheme l(a). The time program used is shown in Scheme 1(b). The kinetic curve and initial rate measurements were automatically obtained by means of the Milton Roy Software. The reaction was monitor&d at 328 nm and the temperature of the flow cell was kept constant at 45 f 0.1”. Determination of chlorpyrifos in Sadicloato. Sadicloato (SADISA) is a liquid commercial formulation with a theoretical composition:
A.
654
J~PINOWMANSILLA
27.8% (w/v) chlorpyrifos and 22.2% (w/v) dimethoate. A Sadicloato stock solution was prepared by adding 250 ~1 of the formulation (previously homogeneized in an ultrasonic bath) in a loo-ml volumetric flask and diluting to the mark with ethanol. Aliquots (between 0.2 and 1.6 ml) of this stock solution were placed in 25-ml volumetric flasks, suEicient volume of ethanol to give a final ethanoLwater mixture 40 + 60 (v/v) was added and diluted to the mark with water. The samples were then analyzed by the stopped flow proposed method. Conventional calibration graphs (external standard) and the standard addition method were used.
RESULTS AND DISCUSSION
As stated in the literature, Chlorpyrifos, in alkaline medium, exhibits a hydrolysis reaction favoured with temperature, metals and elevate pH values. The rate of the hydrolysis reaction is
Fig. 2. (A) FIA-grams obtained in continuous flow (B) Stopped-flow signals obtained for different Tr values: (a) 12 set; (b) 14 set; (c) 16 set; (d) 18 set; (e) 20 set; (f) 21 sec.
et al.
;’
-2.20
-A
slope = 0.7 (NaOli(> 0.7 M
P
B-2.40
0
Fig. 3. Plot of the logarithm of the reaction rate us the logarithm of concentrations of NaOH for a 4.2 x 10e5A4 chlorpyrifos concentration.
very slow, and the product generated in this reaction shows a bathocromic shifting of the maximum absorption with respect to those of chlorpyrifos. This fact, is also observed when a previous deoxygenation with N, stream, is made. On the other hand, in preliminary studies, it was observed that the rate of reaction is highly increased by the presence of hydrogen peroxide. The absorption spectrum of the generated product, in presence and in absence of hydrogen peroxide, are similar for wavelengths higher than 270 nm. In Fig. 1, the evolution of the absorption spectrum of chlorpyrifos (obtained in a static photometric cell) in absence and in the presence of two different amounts of hydrogen peroxide, are shown. The generated product shows absorption maxima at 321 and 250 nm, and the absorption maxima localized at 290 and 330 nm in the original chlorpyrifos disappear. For higher concentrations of hydrogen peroxide, the maximum located at wavelengths shorter than 280 nm is not observed due to the absorption of the blank of hydrogen peroxide in alkaline medium. Also, it must be noticed that the absorption spectra obtained in both chemical conditions are similiar to those of 3,5,6-trichloro-pyridin-2-01 (TCP). In the Fig. l(d), the kinetic curves of the degradation of chlorpyrifos (1 = 290 nm) and of the formation of TCP (A = 328 nm) are also included. Kinetic behaviour. A stopped-flow system (Scheme la) has been used in the later studies to increase the precision of the initial rate measurements. A kinetic study of the influence of the variables on the rate of reaction was made in order to develop a kinetic method for determi-
Kinetic study of the degradation of chlorpyrifos
nation of chlorpyrifos alone or in mixtures together with other pesticides present in its commercial formulations. The kinetic data were obtained from the initial rate of TCP formation us concentration plots, by measuring the evolution of the absorbance at I = 328 nm with the time. The partial orders for each variable were calculated from the resulting log-log plots. Optimization of the stopped-flow variables. In this study, the variable of the time program, delay time (Td) has been studied. This parameter is considered as the period between the injection of the sample and the starting of the measurement period. Tr, is the time period between the injection of the sample and the stopping of the pump, and Tr’, is the time period between the stopping of the pump and
Time
(s)
-1.60
I3
-2.00 h -i
-2.20
-2
-2.40
V aI ” k 5
-2.60
-2.60
-3.00
I
In rate = 0.55
tL5(
Ha02 - 0.16 D
1
Fig. 4. Influence of hydrogen peroxide concentration: (A) kinetic curves far different hydrogen peroxide concentration (M x LO”): (1) 0.7; (2) 1.4; (3) 2.1; (4) 2.4; (5) 3.5; (6) 4.2; (B) logarithmic plot of rate of reaction us concentration.
655
Fig. 5. Stopped-flow signal for different concentrations of chlorpyrifos between 1.0 x lo-’ and 1.5 x lo-‘M.
the starting of measurement period. A 40% containing solution chlorpyrifos ethanol (1.0 x 10-4M) has been prepared and aliquots (50 ~1) of this have been inserted in the stream of carrier and reagent composed by 0.5M NaOH, 40% ethanol and 0.5 g/l hydrogen peroxide. After a variable time the continuous flow is stopped and the absorbanetime curves are obtained. In Fig. 2, the FIA-grams curves obtained in stopped- and continuous-flow are shown. In continuous flow, the peak observed permit us to localizate the moment in which the sample is into the flow-cell and it is due to the physical differences between sample and carrier. This phenomenon is frequent when reaction product between the carrier and the sample is monitored. In stopped-flow, Tr, values between 12 and 21 set have been assayed. The slope of the tangent to the absorbance-time curves from the minimum value and the coefficient of linear correlation (r) during 30 set have been calculated for each Tr, and 20 set for Tr (Td = 27 set) is considered as optimum. Optimization of the chemical variables. For these studies, the composition of the sample and carrier were varied simultaneously. The influence of the NaOH concentration was studied in the range 0.08-1.44M. The initial rate remains practically constant with the concentration of NaOH (a 0 partial order with respect to NaOH concentration was obtained up to 0.7M) and increases (a 2/3 partial order was found) for higher values. In Fig. 3, a logarithmic graph of the rate of reaction vs concentration of NaOH, is shown. A 0.5M NaOH concentration was
A.
656
ESPINOSA-MNWLLA et
al.
Table 1. Features of the proposed methods for the determination of chlorpyrifos in Sadicloato by using the standard addition method ~1 of Sadicloato stock solution placed in 25 ml
400
200
Chlorpyrifos Found Added (M) (W 3.30 x IO-’ 0 5.04 x 1o-5 1.74 x 10-S 8.46 x W5 5.22 x 1O-5 13.60 x lo-’ 8.65 x 10-r 15.69 x 1O-5 12.11 x 10-r 0 1.73 x 3.45 x 5.18 x 6.91 x 8.64 x 13.83 x
1o-s 10-5 1O-5 lo-’ lo-’ lo-’
1.45 x 3.00 x 5.45 x 7.00 x 8.82 x 11.18 x 14.54 x
10-5 10-J 10-r 10-r IO-’ 1O-5 10-S
selected as optimum, with the object of obtaining more precision in the rate measurements. Higher concentrations of NaOH give rise to appreciable unstabilities in the stream carrier. The influence of hydrogen peroxide was studied in the range between 0.24 and 1.44 g/l. In Fig. 4, the kinetic curves and a logarithmic graph of the rate of the reaction vs concentration of hydrogen peroxide is represented. The rate of reaction was found to increase with the concentration of hydrogen peroxide, and the linear interval for the measurement decreased for larger concentration of hydrogen peroxide. Efict of the temperature. The effect of the temperature on the reaction rate was examined between 20 and 50”, for samples containing 8 x lo-‘M of chlorpyrifos. Continuous increasing in the rate of reaction with the temperature is observed. A temperature of 45” was chosen as enough for a proper development of the reaction. A plot of the logarithm of the rate constant against the inverse of the absolute temperature allows us to obtain the value of the activation energy, being 4.2 Kcal/mol.K. Table 2. Comparative results for the application of external standard and standard addition methods in the determination of chlorpyrifos in Sadicloato ~1 of Sadicloato stock solution placed in 25-ml 200 400 800 1200 1600
Chlorpyrifos found (%) Standard External addition* standard* 30.0 29.5 30.8 30.8 32.2
Claimed value: 27.8% *Mean of two independent determinations.
31.0 28.8 -
Recovery (%) 93 97 117 101
Slope (l/mol.min)
Correlation coefficient (r)
1100
0.9930
1100
0.9940
89 115 107 107 112 95
Calibration curves Absorbanc+time stopped-flow signals were recorded for solutions containing different amounts of chlorpyrifos at the experimental conditions indicated above (Fig. 5), and a calibration curve was obtained by use of a rate reaction us concentration plot. The rate reaction was found to be first-order with respect to concentration in the range chlorpyrifos 0.5 x lo-‘-16 x lo-‘M. The correlation coefficient (r) was 0.9990. The slope of the calibration plot was 1100 l/mol mm-‘. The reproducibility of the injection and detection systems were examined by injecting 10 aliquots of a sample containing 1.04 x 10W4M.Values of 1.8 and 2.0% for the relative standard deviations were obtained, respectively. Detection and determination limits of 6.6 x 10P6 and 2.2 x 10mSMwere determined. Kinetic equation The simplified kinetic equation pressed as
can be ex-
lNaOH] < 0.7M-d[chlorpyrifos]/dt = k’[H,0,]1’2[chlorpyrifos] [NaOH] > 0.7M-d[chlorpyrifos]/dt = k”[H202]‘/2[NaOH]2~3[chlorpyrifos] ZnjZuenceof foreign species Several pesticides present in commercial formulations together with chlorpyrifos have been tested as interference species. Dimethoate and endosulfan were tolerated at a, 20: 1 (interference: analyte) iU : M ratio at least. However, carbaryl strongly interfere for 1: 1 M : M ratio. This interference is due to the instantaneously
Kinetic study of the degradation of chlorpyrifos
hydrolysis of carbaryl and the unstability of the generated product in established conditions. APPLICATIONS
The stopped-flow proposed method for determining chlorpyrifos has been tested on a commercial formulation in which chlorpyrifos is formulated together with dimethoate. The slopes obtained in the different calibration curves by application of the standard addition method (Table 1) are identical with the theoretical slope established. In Table 2 the obtained results by application of both proposed methods are summarized. The values found are in agreement with the claimed level of chlorpyrifos. CONCLUSIONS
The proposed method allows to analyze chlorpyrifos in the presence of dimethoate and endosulfan without previous separation procedures. Filtration processes of the commercial samples are unnecessary. Smaller times (about 1 min) that those employed normally in chromatographic process are sufficient to record the total analytical signal. Hence, the method is fast and simple to perform and suitable to apply in routine analysis of commercial formulations. Acknowfedgemenrs-The authors gratefully acknowledge financial support from the DGICYT (project PB91-0856). REFERENCES 1. H. E. Gray, Down ro Earth, 1965, 21, 2. 2. E. E. Kenaga, W. K. Whitney, J. L. Hardy and A. E. Doty, J. .&on. Enromol., 1965, Ss, 1043. 3. N. Zambelli, M. Lodi and A. Koracs; Atti. Giornate Fitopatologiche, Venice, 1971, p. 505. 4. K. N. Komblas; Abstr. VIIth Int. Congr. Pt. Prot. Paris, p. 126, 1970. 5. C. R. Worthing (ed.), The Pesticide Manual. A World Compendium, 8th Ed., The British Crop. Protection Council, 1987.
651
6. W. L. Winterlin, K. Moilanen and W. E. Burgoyne, Down to Earth, 1968, 24, 34. 7. J. H. Wetters, Dow Chemical ACR, 1973, 73-5, 1. 8. M. C. Bowman and M. Beroza, J. Agric. Food Chem., 1967, 16, 651. 9. D. L. Strumble and S. McDonald, J. Encon. Entomol., 1973, 66, 769. 10. Pesticide Analytical Manual, Vol. 1, Food and Drugs Administration, Washington, DC., 1979. 11. M. A. Luke, J. E. Froberg, G. M. Doose and H. T. Masumoto, J. Assoc. Ofl Anal. Chem., 1981,64, 1187. 12. A. Di Muccio, A. M. Cicero, I. Camoni, D. Pontecorvo and R. Dommazco, J. Assoc. 08 Anal. Chem., 1987,70, 106. 13. F. Hembndez, J. M. Grams, J. Beltran and J. Sancho, Cromatographia, 1990, 29,459. 14. O@ial Methodr of Analysis, Sec. 29 “Pesticides and Industrial Chemical Residues”. AOAC, Arlington, VA, 1984. 15. Changes in Methods: J. Assoc. O$. Anal. Chem., 1986, 69, 365. 16. W. A. Saner and J. Giebert; J. Liq. Chromatogr., 1980, 3(1 I), 1753. 17. C. D. Pfeiffer, J. D. Grahan, T. J. Nestrick and B. S. Isenbarger; J. Chromatogr. Sci., 1980, 18, 330. 18. R. J. Bushway, L. B. Perkins and J. M. Kings, J. Assoc. Ofl Chem., 1988, 71(2), 321. 19. J. G. Brayan, P. R. Haddad; J. G. Sharp, S. Dilli and J. M. Desmazchelier, J. Chromatogr., 1988, 447, 249. 20. Oflcial Metho& of Analysis, Sec. 6 “Organophosphorus Pesticides “, pp. 411416. AOAC, Arlington, VA, 1984. 21. S. Husain, P. Nageswara Sarma, G. Y. S. K. Swami and R. Narsimha, J. Chromatogr. 1991, 540(1-2), 331. 22. E. R. Bogus, T. L. Watshke and R. 0. Mumma, J. Agric. Food Chem., 1990, XI(l), 142. 23. C. R. Mourer, G. L. Hall, W. E. Whitead and T. Shibamoto, J. Assoc. 08 Anal. Chem., 1990,73,294. 24. W. J. Allender and J. Kugan, Bull. Environ. Contam. Toxicol., 1991, 46(2), 313. 25. V. Rani, S. Joyce and C. Janaiah, Curr. Sci., 1989, 58(18), 1048. 26. P. J. Grey and R. G. Duggleby, Biochem. J., 1989, 257(2), 419. 27. G. Kh. Shapenova, Sb. Naachun. Tr.-Tashk. Gos. Univ. im. V.I. Lenina, 1980, 622, 68. 28. 0. Schaefer, L. Weil and R. Nessner, Fresenius J. Anal. Chem., 1992, 343(l), 147.
Pergamon
KINETIC STUDY OF THE DEGRADATION OF CHLORPYRIFOS BY USING A STOPPED-FLOW FIA SYSTEM. SEMIAUTOMATIC DETERMINATION IN COMMERCIAL FORMULATIONS A. ESPINGBA-MANSILW*,F. SUNAS and A. ZAMORO Department of Analytical Chemistry, University of Extremadura, 06071 Badajoz, Spain (Received 16 June 1993. Revised 15 September 1993. Accepted 30 September 1993) Smanury-A kinetic study of the degradation of chlorpyrifos in an oxidative alkaline medium has been undertaken by using a stopped-flow system. A semiautomatic method for determining chlorpyrifos is proposed for the first time. Linear calibration graphs up to 2 x lo-‘M with a relative standard deviation of 1.8% are obtained. A detection limit of 6.6 x 10Y6Mis reported. Carbaryl, dimethoate and endosulfan have been assayed as interference species. The proposed method has been applied to analyze a commercial formulation.
Chlorpyrifost is the common name accepted for the chemical O,O-diethyl-0-(3,5,6-trichloro-2pyridyl) phosphorotiate. This compound is an organophosphate pesticide exhibiting a broad spectrum of activity as insecticide, reported and reviewed for several authorsi Chlorpyrifos presents a hydrolysis process in alkaline medium and the rate of reaction increases with temperature, presence of metals and elevate pH values. In soil it is initially degraded to 3,5,6-trichloro pyridin-2-01 (TCP) which is subsequently degraded to organ0 chlorine compounds.5 Residues of this pesticide have been evaluated in many substracts. 6-g The classical analytical procedures, widely applied, consisted essentially of extractions with a solvent, water partition and cleanup on a chromatographic column followed by gas chromatographic determination. Several multiresidues methods exist.‘+i2 Recently,13 a comparative study of the different methods for the determination of organochlotine and organophosphorous pesticides in fruit samples has been carried out. A number of solvent extraction systems have been used for multiresidue screening procedures; these mainly include: acetonitrile, acetone, methanol, methylene chloride-methanol, acetone-methanol and *Author to whom correspondence should be addressed. TRegistered as Dursban by Dow Chemical Company. TAL ‘M-c
ethylacetate. In the mentioned paper, capillary gas chromatography was used for identification and quantitation of the individual pesticides in spiked peach samples. The AOAC recommeded method which used acetonitrile or acetonitrile-water,‘4 was modified in 1985 by recommending the use of acteone for extraction of pesticides residues in non-fatty foodsi HPLC techniques are widely employed in the analysis of chlorpyrifos.‘6’g In the AOAC recommended method for formulation analysis an acetonitrile extraction is employed prior to chromatographic separation and small amount of acetic acid is added to mobile phase (acetor&rile-water) to suppress non-reproducible ionization of 3,5,6-trichloro-2-pyridinol, which might otherwise cause interference.20 Recently, HPLC methods have been proposed to analyze chlorpyrifos in production processes” and in water.= Most of papers reported do not incorporate the analysis of metabolites, however, recently, two chromatographic methods for determination of chlorpyrifos and its metabolite (TCP) have been reported by using GC23 and HPLC.” The kinetic methods have been sparingly applied in the analysis of pesticides although inhibition or reactivation processes of the activities of some enzymes due to organophosphorous pesticides have been reported.25*26 A kinetic
651
652
A. ESPINOGA-MANSILLA et al.
no-enzymatic method based in oxidation of o-dianisidine has been proposed for the determination of organophosphorus compounds in water.” The most recent bibliography data apply the classical cholisterase test to water analysis by means of kinetic measurements and computer evaluation of the results. A mixture of paraoxon, carbofuran and chlorpyrifos was calibrated.“’ In this paper a kinetic determination of chlorpyrifos based in the spectrophotometric measure of the degradation-reaction rate in alkaline medium is reported. Although chromatographic methods are the most powerful tool in analysis of pesticides, the following specific advantages in the application of semiautomatic kinetic method can be expected: (a) elimination of some experimental steps prior to the absorbance measurements (e.g. filtration, extraction . . . ), hence considerably simpler and
faster procedures are proposed for routine control, (b) selectivity due to the measure of the evolution of the absorbance with the time reaction instead of the measure of a concrete absorbance value, (c) possibility of no interference of the coloured and/or turbidity background of the samples and (d) possibility of no interference of other absorbent pesticides presenting in the samples if they are exhibiting stability in the chemical reaction conditions established for the proposed method. EXPERIMJZNTAL
Apparatus
A home-made stopped-flow system (Scheme la) was fitted to a Milton Roy Spectronic 3000 Array spectrophotometer (PC-286 microcomputer), equipped with a IO-mm microflow cell (18 ~1). A Milton Roy software was used for all data acquisition, measurement and analysis of the kinetic data. A thermostatically controlled
detector
1
30
T (seg) NaoIi(O.5 M)+&O~(O.5g/L)+EtOh(40%) (a)
Scheme 1. Schematic diagrams used for the determination of chlorpyrifos by (a) stopped-flow system; (b) time program used for the stopped-flow system. Td, delay time; Tm, measurement time.
653
Kinetic study of the degradation of chlorpyrifos 2.00
spectrum
one spectrum
each 4
one specWt#a
min?
each 2 min.
each
2 min.
C
P
Time (aec)
?
Fig. 1. Evolution of absorption espectrum of chlorpyrifos in (a) absence of hydrogen peroxide, (b) in the presence of 0.1 g/l and (c) 0.8 g/l hydrogen peroxide and (d) kinetic curves obtained at 290 and 328 nm in the presence of 0.5 g/l of hydrogen peroxide.
bath, Selecta Unitronic 320 OR, was used for temperature control. A precision peristaltic pump (Gilson Minipuls-2) and a six-way injection valve with a variable loop (Omnifit) were used. An interface2’ has been built to allow the starting and stopping of the pump, allowing the automation and control of the peristaltic pump through a microcomputer and an adequate BASIC program.2’ The time diagram used for the stopped-flow method is described in Scheme l(b). The delay time (Td) and the measure time (Tm) are controlled by means of input instructions through the internal clock of a microcomputer (Commodore 64). Reagent and chemicals Standard solutions (500 pg/ml) of chlorpyriand dimethoate fos, carbaryl, endosulfan (Chem. Service) were prepared by disolution in ethanol.
All chemicals and solvents used were of analytical reagent grade. The commercial formulation Sadicloato was obtained from SADISA (Spain). Procedure Determination of chlorpyrifos by the stoppedflow system. A 50 ~1 aliquot of a 40% ethanol sample solution containing up to 2.63 pg of chlorpyrifos was injected through the injection valve into the carrier stream (0.5 M NaOH + 40% (v/v) ethanol + 0.55 g/l H201) by using the manifold shown in Scheme l(a). The time program used is shown in Scheme 1(b). The kinetic curve and initial rate measurements were automatically obtained by means of the Milton Roy Software. The reaction was monitor&d at 328 nm and the temperature of the flow cell was kept constant at 45 f 0.1”. Determination of chlorpyrifos in Sadicloato. Sadicloato (SADISA) is a liquid commercial formulation with a theoretical composition:
A.
654
J~PINOWMANSILLA
27.8% (w/v) chlorpyrifos and 22.2% (w/v) dimethoate. A Sadicloato stock solution was prepared by adding 250 ~1 of the formulation (previously homogeneized in an ultrasonic bath) in a loo-ml volumetric flask and diluting to the mark with ethanol. Aliquots (between 0.2 and 1.6 ml) of this stock solution were placed in 25-ml volumetric flasks, suEicient volume of ethanol to give a final ethanoLwater mixture 40 + 60 (v/v) was added and diluted to the mark with water. The samples were then analyzed by the stopped flow proposed method. Conventional calibration graphs (external standard) and the standard addition method were used.
RESULTS AND DISCUSSION
As stated in the literature, Chlorpyrifos, in alkaline medium, exhibits a hydrolysis reaction favoured with temperature, metals and elevate pH values. The rate of the hydrolysis reaction is
Fig. 2. (A) FIA-grams obtained in continuous flow (B) Stopped-flow signals obtained for different Tr values: (a) 12 set; (b) 14 set; (c) 16 set; (d) 18 set; (e) 20 set; (f) 21 sec.
et al.
;’
-2.20
-A
slope = 0.7 (NaOli(> 0.7 M
P
B-2.40
0
Fig. 3. Plot of the logarithm of the reaction rate us the logarithm of concentrations of NaOH for a 4.2 x 10e5A4 chlorpyrifos concentration.
very slow, and the product generated in this reaction shows a bathocromic shifting of the maximum absorption with respect to those of chlorpyrifos. This fact, is also observed when a previous deoxygenation with N, stream, is made. On the other hand, in preliminary studies, it was observed that the rate of reaction is highly increased by the presence of hydrogen peroxide. The absorption spectrum of the generated product, in presence and in absence of hydrogen peroxide, are similar for wavelengths higher than 270 nm. In Fig. 1, the evolution of the absorption spectrum of chlorpyrifos (obtained in a static photometric cell) in absence and in the presence of two different amounts of hydrogen peroxide, are shown. The generated product shows absorption maxima at 321 and 250 nm, and the absorption maxima localized at 290 and 330 nm in the original chlorpyrifos disappear. For higher concentrations of hydrogen peroxide, the maximum located at wavelengths shorter than 280 nm is not observed due to the absorption of the blank of hydrogen peroxide in alkaline medium. Also, it must be noticed that the absorption spectra obtained in both chemical conditions are similiar to those of 3,5,6-trichloro-pyridin-2-01 (TCP). In the Fig. l(d), the kinetic curves of the degradation of chlorpyrifos (1 = 290 nm) and of the formation of TCP (A = 328 nm) are also included. Kinetic behaviour. A stopped-flow system (Scheme la) has been used in the later studies to increase the precision of the initial rate measurements. A kinetic study of the influence of the variables on the rate of reaction was made in order to develop a kinetic method for determi-
Kinetic study of the degradation of chlorpyrifos
nation of chlorpyrifos alone or in mixtures together with other pesticides present in its commercial formulations. The kinetic data were obtained from the initial rate of TCP formation us concentration plots, by measuring the evolution of the absorbance at I = 328 nm with the time. The partial orders for each variable were calculated from the resulting log-log plots. Optimization of the stopped-flow variables. In this study, the variable of the time program, delay time (Td) has been studied. This parameter is considered as the period between the injection of the sample and the starting of the measurement period. Tr, is the time period between the injection of the sample and the stopping of the pump, and Tr’, is the time period between the stopping of the pump and
Time
(s)
-1.60
I3
-2.00 h -i
-2.20
-2
-2.40
V aI ” k 5
-2.60
-2.60
-3.00
I
In rate = 0.55
tL5(
Ha02 - 0.16 D
1
Fig. 4. Influence of hydrogen peroxide concentration: (A) kinetic curves far different hydrogen peroxide concentration (M x LO”): (1) 0.7; (2) 1.4; (3) 2.1; (4) 2.4; (5) 3.5; (6) 4.2; (B) logarithmic plot of rate of reaction us concentration.
655
Fig. 5. Stopped-flow signal for different concentrations of chlorpyrifos between 1.0 x lo-’ and 1.5 x lo-‘M.
the starting of measurement period. A 40% containing solution chlorpyrifos ethanol (1.0 x 10-4M) has been prepared and aliquots (50 ~1) of this have been inserted in the stream of carrier and reagent composed by 0.5M NaOH, 40% ethanol and 0.5 g/l hydrogen peroxide. After a variable time the continuous flow is stopped and the absorbanetime curves are obtained. In Fig. 2, the FIA-grams curves obtained in stopped- and continuous-flow are shown. In continuous flow, the peak observed permit us to localizate the moment in which the sample is into the flow-cell and it is due to the physical differences between sample and carrier. This phenomenon is frequent when reaction product between the carrier and the sample is monitored. In stopped-flow, Tr, values between 12 and 21 set have been assayed. The slope of the tangent to the absorbance-time curves from the minimum value and the coefficient of linear correlation (r) during 30 set have been calculated for each Tr, and 20 set for Tr (Td = 27 set) is considered as optimum. Optimization of the chemical variables. For these studies, the composition of the sample and carrier were varied simultaneously. The influence of the NaOH concentration was studied in the range 0.08-1.44M. The initial rate remains practically constant with the concentration of NaOH (a 0 partial order with respect to NaOH concentration was obtained up to 0.7M) and increases (a 2/3 partial order was found) for higher values. In Fig. 3, a logarithmic graph of the rate of reaction vs concentration of NaOH, is shown. A 0.5M NaOH concentration was
A.
656
ESPINOSA-MNWLLA et
al.
Table 1. Features of the proposed methods for the determination of chlorpyrifos in Sadicloato by using the standard addition method ~1 of Sadicloato stock solution placed in 25 ml
400
200
Chlorpyrifos Found Added (M) (W 3.30 x IO-’ 0 5.04 x 1o-5 1.74 x 10-S 8.46 x W5 5.22 x 1O-5 13.60 x lo-’ 8.65 x 10-r 15.69 x 1O-5 12.11 x 10-r 0 1.73 x 3.45 x 5.18 x 6.91 x 8.64 x 13.83 x
1o-s 10-5 1O-5 lo-’ lo-’ lo-’
1.45 x 3.00 x 5.45 x 7.00 x 8.82 x 11.18 x 14.54 x
10-5 10-J 10-r 10-r IO-’ 1O-5 10-S
selected as optimum, with the object of obtaining more precision in the rate measurements. Higher concentrations of NaOH give rise to appreciable unstabilities in the stream carrier. The influence of hydrogen peroxide was studied in the range between 0.24 and 1.44 g/l. In Fig. 4, the kinetic curves and a logarithmic graph of the rate of the reaction vs concentration of hydrogen peroxide is represented. The rate of reaction was found to increase with the concentration of hydrogen peroxide, and the linear interval for the measurement decreased for larger concentration of hydrogen peroxide. Efict of the temperature. The effect of the temperature on the reaction rate was examined between 20 and 50”, for samples containing 8 x lo-‘M of chlorpyrifos. Continuous increasing in the rate of reaction with the temperature is observed. A temperature of 45” was chosen as enough for a proper development of the reaction. A plot of the logarithm of the rate constant against the inverse of the absolute temperature allows us to obtain the value of the activation energy, being 4.2 Kcal/mol.K. Table 2. Comparative results for the application of external standard and standard addition methods in the determination of chlorpyrifos in Sadicloato ~1 of Sadicloato stock solution placed in 25-ml 200 400 800 1200 1600
Chlorpyrifos found (%) Standard External addition* standard* 30.0 29.5 30.8 30.8 32.2
Claimed value: 27.8% *Mean of two independent determinations.
31.0 28.8 -
Recovery (%) 93 97 117 101
Slope (l/mol.min)
Correlation coefficient (r)
1100
0.9930
1100
0.9940
89 115 107 107 112 95
Calibration curves Absorbanc+time stopped-flow signals were recorded for solutions containing different amounts of chlorpyrifos at the experimental conditions indicated above (Fig. 5), and a calibration curve was obtained by use of a rate reaction us concentration plot. The rate reaction was found to be first-order with respect to concentration in the range chlorpyrifos 0.5 x lo-‘-16 x lo-‘M. The correlation coefficient (r) was 0.9990. The slope of the calibration plot was 1100 l/mol mm-‘. The reproducibility of the injection and detection systems were examined by injecting 10 aliquots of a sample containing 1.04 x 10W4M.Values of 1.8 and 2.0% for the relative standard deviations were obtained, respectively. Detection and determination limits of 6.6 x 10P6 and 2.2 x 10mSMwere determined. Kinetic equation The simplified kinetic equation pressed as
can be ex-
lNaOH] < 0.7M-d[chlorpyrifos]/dt = k’[H,0,]1’2[chlorpyrifos] [NaOH] > 0.7M-d[chlorpyrifos]/dt = k”[H202]‘/2[NaOH]2~3[chlorpyrifos] ZnjZuenceof foreign species Several pesticides present in commercial formulations together with chlorpyrifos have been tested as interference species. Dimethoate and endosulfan were tolerated at a, 20: 1 (interference: analyte) iU : M ratio at least. However, carbaryl strongly interfere for 1: 1 M : M ratio. This interference is due to the instantaneously
Kinetic study of the degradation of chlorpyrifos
hydrolysis of carbaryl and the unstability of the generated product in established conditions. APPLICATIONS
The stopped-flow proposed method for determining chlorpyrifos has been tested on a commercial formulation in which chlorpyrifos is formulated together with dimethoate. The slopes obtained in the different calibration curves by application of the standard addition method (Table 1) are identical with the theoretical slope established. In Table 2 the obtained results by application of both proposed methods are summarized. The values found are in agreement with the claimed level of chlorpyrifos. CONCLUSIONS
The proposed method allows to analyze chlorpyrifos in the presence of dimethoate and endosulfan without previous separation procedures. Filtration processes of the commercial samples are unnecessary. Smaller times (about 1 min) that those employed normally in chromatographic process are sufficient to record the total analytical signal. Hence, the method is fast and simple to perform and suitable to apply in routine analysis of commercial formulations. Acknowfedgemenrs-The authors gratefully acknowledge financial support from the DGICYT (project PB91-0856). REFERENCES 1. H. E. Gray, Down ro Earth, 1965, 21, 2. 2. E. E. Kenaga, W. K. Whitney, J. L. Hardy and A. E. Doty, J. .&on. Enromol., 1965, Ss, 1043. 3. N. Zambelli, M. Lodi and A. Koracs; Atti. Giornate Fitopatologiche, Venice, 1971, p. 505. 4. K. N. Komblas; Abstr. VIIth Int. Congr. Pt. Prot. Paris, p. 126, 1970. 5. C. R. Worthing (ed.), The Pesticide Manual. A World Compendium, 8th Ed., The British Crop. Protection Council, 1987.
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6. W. L. Winterlin, K. Moilanen and W. E. Burgoyne, Down to Earth, 1968, 24, 34. 7. J. H. Wetters, Dow Chemical ACR, 1973, 73-5, 1. 8. M. C. Bowman and M. Beroza, J. Agric. Food Chem., 1967, 16, 651. 9. D. L. Strumble and S. McDonald, J. Encon. Entomol., 1973, 66, 769. 10. Pesticide Analytical Manual, Vol. 1, Food and Drugs Administration, Washington, DC., 1979. 11. M. A. Luke, J. E. Froberg, G. M. Doose and H. T. Masumoto, J. Assoc. Ofl Anal. Chem., 1981,64, 1187. 12. A. Di Muccio, A. M. Cicero, I. Camoni, D. Pontecorvo and R. Dommazco, J. Assoc. 08 Anal. Chem., 1987,70, 106. 13. F. Hembndez, J. M. Grams, J. Beltran and J. Sancho, Cromatographia, 1990, 29,459. 14. O@ial Methodr of Analysis, Sec. 29 “Pesticides and Industrial Chemical Residues”. AOAC, Arlington, VA, 1984. 15. Changes in Methods: J. Assoc. O$. Anal. Chem., 1986, 69, 365. 16. W. A. Saner and J. Giebert; J. Liq. Chromatogr., 1980, 3(1 I), 1753. 17. C. D. Pfeiffer, J. D. Grahan, T. J. Nestrick and B. S. Isenbarger; J. Chromatogr. Sci., 1980, 18, 330. 18. R. J. Bushway, L. B. Perkins and J. M. Kings, J. Assoc. Ofl Chem., 1988, 71(2), 321. 19. J. G. Brayan, P. R. Haddad; J. G. Sharp, S. Dilli and J. M. Desmazchelier, J. Chromatogr., 1988, 447, 249. 20. Oflcial Metho& of Analysis, Sec. 6 “Organophosphorus Pesticides “, pp. 411416. AOAC, Arlington, VA, 1984. 21. S. Husain, P. Nageswara Sarma, G. Y. S. K. Swami and R. Narsimha, J. Chromatogr. 1991, 540(1-2), 331. 22. E. R. Bogus, T. L. Watshke and R. 0. Mumma, J. Agric. Food Chem., 1990, XI(l), 142. 23. C. R. Mourer, G. L. Hall, W. E. Whitead and T. Shibamoto, J. Assoc. 08 Anal. Chem., 1990,73,294. 24. W. J. Allender and J. Kugan, Bull. Environ. Contam. Toxicol., 1991, 46(2), 313. 25. V. Rani, S. Joyce and C. Janaiah, Curr. Sci., 1989, 58(18), 1048. 26. P. J. Grey and R. G. Duggleby, Biochem. J., 1989, 257(2), 419. 27. G. Kh. Shapenova, Sb. Naachun. Tr.-Tashk. Gos. Univ. im. V.I. Lenina, 1980, 622, 68. 28. 0. Schaefer, L. Weil and R. Nessner, Fresenius J. Anal. Chem., 1992, 343(l), 147.