Anal. Chem. 1995,67,2606-2612
Cloud Point Preconcentration and HighlPerformance Liquid Chromatographic Determination of Organophosphorus Pesticides with Dual Electrochemical Detection Cannel0 Garcia Pinto, Jose Luis Wrer P a v h , and Bemardo Moreno Cordem* Depatfamento de Qulmica Anahtica, Nutricibn y Bromatologla, Facultad de Qulmica, Universidad de Salamanca, 37008 Salamanca, Spain
The electrochemical detection of analytes subjected to cloud point preconcentrationopens new perspectives to this methodology, whose major limitation lies in the high absorbance background due to the surfactant. In the present work, dual electrochemicaldetection (reductiveoxidative mode) was used for the liquid chromatographic analysis of organophosphorus pesticides after cloud point preconcentration with the nonionic surfactant Triton X-114. Preconcentrationof as little as 15 mL of sample with a Triton X-114 concentration of 1.0%permits the detection of amounts lower than 0.4 ppb. Organophosphorus pesticides are an important source of environmental contamination owing to their widespread use in agriculture; this involves their later passage to the water table with the ensuing risk for the population. Such a situation has meant that it has become necessary to develop simple and sensitive analytical methods for monitoring these compounds both in water (surface and drinking water) and in different food products commercialized for human consumption. Although the determination of these compounds is usually carried out by gas chromatography,' in recent years the use of high-performance liquid chromatography (HPLC) with both spectrophotometric and electrochemical detection has increased con~iderably.~~~ The presence of nitro and azo groups in the structure of organophosphorus compounds would allow their determination by reductive electrochemical detection as long as the dissolved oxygen is completely eliminated in order to avoid high residual current^.^ This drawback can be readily overcome by oxidative electrochemical detection after transformation, by reduction of the analytes, in derivatives susceptible to later 0xidation.j Recently, a dual electrochemical (reductive-oxidative) detection method has been proposed.6 The low levels at which such substances must be analyzed in most matrices of environmental interest demand a previous step (1) Watts, R R., Ed. Manual ofAnalytica1 Methods for the Analysis of Pesticides in Humans and Environmental Samples; EPA-600/&80-038; U S . Environmental Protection Agency, Environmental Toxicology Division, Health Effects Research Laboratory: Research Triangle Park, NC, 1980. (2) Barcelo, D. Analyst 1991,116, 681-689. (3) Sherma, J. Anal. Chem. 1993,59, 40R-54R (4) Clark, G. J.; Goodin, R. R.; Smiley, J. W. Anal. Chem. 1985,57, 22232228. (5) Lunte, C. E.; Kissinger, P. T.; Shoup, R. E. Anal. Chem. 1985,57, 15411546. (6) Carabias Martinez, R.; Rodriguez Gonzalo, E.; Garay Garcia. F.; Hemandez Mendez, J. J. Chromatogr. 1993,644, 49-58. 2606 Analytical Chemistry, Vol. 67, No. 75,August 7, 7995
involving extraction and cleaning of the extract~.~!8 The preparation and enrichment of pesticide samples are usually carried out by liquid-liquidgJO or solid-liquid6J0-lZ extraction. When solid phases are used, the presence of dissolved organic matter (humic acids) leads to low recovery values of organic species. These lower recoveries from humic acid solutions may be caused by saturation of sorptive sites by the humic material,13 association of the analyte with humic acid strongly retained by the sorbent and not desorbed during solvent e~traction,'~ or lower affinity to the sorbent when associated with humic acid in ~olution.'~J~ This association of humic acids with compounds such as polycyclic aromatic hydrocarbons produces a decrease in the adsorption of these compounds in the containers." Relatively dilute aqueous solutions of many nonionic surfactants have the property of separating into two liquid phases when they are heated above a given temperature, called the cloud point temperature.18-20 One of the phases obtained contains most of the surfactant introduced (referred to as the surfactant-rich phase), while the other (aqueous phase), in equilibrium with the former, contains amounts of surfactant close to the critical micellar concentration (cmc). The mechanism by which phase separation occurs remains to be fully elucidated and has motivated controversy among the different research groups interested in the iss~e.~~-~~ (7) Sharp, G. J.; Brayan, J. G.; Dilli, S.; Haddad, P. R; Desmarchelier, J. M. Analyst 1988,113, 1493-1507. (8) Muir, D. C. G. J. Agric. Food Chem. 1980,28, 714-719. (9) BarcelO, D.; Porte,C.; Cid, J.; AlbaigCs, J. Int. J, Enuiron. Anal. Chem. 1990, 38, 199-209. (10) Bellar, T. A; Budde, W. L. Anal. Chem. 1988,60, 2076-2083. (11) Marvin, C. H.; Brindle, I. D.; Hall, C. D.; Chiba, M. J. Chromatogr. 1990, 503, 167-176. (12) Carabias Martinez, R.; Rodriguez Gonzalo, E.; Amigo Moran, M. J.; Hemandez Mendez, J. J. Chromatogr. 1992,607, 37-45. (13) Pionke, H. B.; Glotfelty, D. E. Water Res. 1989,23, 1301-1307. (14) Pionke, H. B.; Glotfelty, D. E.: Lucas, A D.; Urban, J. B. J. Environ. Qual. 1988,17, 76-84. (15) Hinckley, D. A; Bidleman. T. F. Environ. Sci. Technol. 1989,23, 9951000.
(16) Johnson, W. E.; Fendinger, N. J.; Plimmer, J. R. Anal. Chem. 1991,63, 1510-1513. (17) Garcia Pinto, C.; Perez Pavon, J. L.; Moreno Cordero, B. Anal. Chem. 1994, 66, 874-881. (18) Watanabe, H. In Solution Behavior of Sutjktants; Mittal, IC L., Fendler, E. F., Eds.; Plenum Press: New York, 1982; Vol. 2, pp 1305-1313. (19) Corti, M.; Minero, C.; Degiorgio, V. J. Phys. Chem. 1984,88, 309-317. (20) Goldstein, R. E. J. Chem. Phys. 1986,84, 3367-3378. (21) Corti, M.; Degiorgio, V.; Hayter, J. B.; Zalauf, M. Chem. Phys. Lett. 1984, 109. 579-583.
0003-2700/95/0367-2606$9.00/0 0 1995 American Chemical Society
The small volume of the surfactant-rich phase obtained (typically between 0.1 and 0.4 mL) permits the design of extraction schemes that are simple, cheap, and of lower toxicity than extraction with organic solvents and that have results similar to those obtained by other separation techniques. This topic has been recently reviewed by Hinze and Pramauro.26 Additionally, the presence of surfactant prevents adsorption of the organic compounds onto the glass containers, and the high contents of dissolved organic matter do not affect the recoveries.17 The cloud point methodology has been successfully used for the preconcentration of species of widely differing character and nature as a previous step to their later determination by HPLC1727-B or FM30 One of the greatest limitations to this methodology is the high absorbance shown by many surfactants in the UV region; in most cases, this prevents their use in a step prior to chromatographic separation when a system of spectrophotometric detection is to be used later unless the mobile phase used contains a high methanol content, in which case elution of the surfactant occurs in a short period of time and does not hinder detection of the a n a l y t e ~ .One ~ ~ possible ~ ~ ~ way of overcoming this problem is to use surfactants that do not absorb at the working wavelengths normally used in chromatography. Thus, Hinze et al.28have used the zwitterionic surfactants 3-(nonyldimethylammonium)propyl sulfate (C9ApSO4) and 3-(decyldimethy1ammonium)propylsulfate (CloApS01). In previous papers, we proposed another, more economic way to overcome this drawback, by using electrochemica127,29 or fluorescent17detection, which, under certain experimental conditions, are transparent to the surfactants usually employed. In this sense, here we propose the use of the cloud point methodology for the preconcentration of the organophosphorus pesticides paraoxon, methylparathion, fenitrothion, and ethylparathion prior to their chromatographic separation by HPLC with dual electrochemical detection. Owing to its physicochemical characteristics, Triton X-114 was chosen as the nonionic surfactant; it has a low cloud point temperature, a higher density of the surfactant-rich phase than that of the aqueous phase (which facilitates phase separation by centrifugation), commercial availability, and a low price, and it lacks electroactive groups in its molecule. The proposed method has been applied to the determination of the above-mentioned pesticides in water from the Tormes River (Salamanca, Spain). EXPERIMENTAL SECTION
Reagents. The nonionic surfactant Triton X-114 was obtained from Fluka and used without further purification. All the organophosphorus pesticides were purchased from Riedel-de Haen (Seelze-Hannover, Germany). The purities of the individual (22) Degiorgio, V.; Piazza, R; Corti. M.; Minero, C. /. Chem. Phys. 1985,82, 1025-1031, (23) Blankschtein, D.;Thurston, G. M.; Benedek, G. B. J Chem. Phys. 1986, 85, 7268-7288. (24) Lindman, B.;Wennerstrom, H. /. Phys. Chem. 1991,95, 6053-6054. (25) Frankewich, R. P.; Hinze, W. L. Anal. Chem. 1994,66, 944-954. (26) Hinze, W. L.; Pramauro, E. CRC Crit. Rev. Anal. Chem. 1993,24, 133177. (27) Moreno Cordero, B.; Perez Pavon, J. L; Garcia Pinto, C.; Fernlndez Laespada, M.E. Tulunta 1993,40, 1703-1710. (28) Saitoh, T.; Hinze, W. L. Anal. Chem. 1991,63, 2520-2525. (29) Garcia Pinto, C.; Perez Pavbn, J. L.; Moreno Cordero, B. Anal. Chem. 1992, 64, 2234-2238. (30) Femindez Laespada, M. E.; Perez Pavbn, J. L.; Moreno Cordero, B. Analyst 1993,118, 209-212.
standards ranged from 97 to 99%. Standard solution were prepared in HPLC-grade methanol (Carlo Erba, Milan, Italy). All other reagents were of analytical grade. All solvents and analytes were filtered through 0.45pm nylon membrane filters (Millipore), and ultrahigh-quality water obtained from a Elgastat UHQ water purification system was used. Apparatus. A modular component liquid chromatographic system was used consisting of a Spectra Physics SP 8800 ternary pump, an SP 8450 UV detector, an EG&G PAR 400 electrochemical detector, and an SP 4290 integrator. The electrodes were as follows: Ag/AgCl/O.l M KCl reference electrode, gold auxiliary electrode, and a glassy carbon series dual electrode MP 1304. In all experiments, a Rheodyne 7125 injection valve with a 10 pL sample loop was used. The stationary-phase column was 220 x 4.6 mm Spheri 5 ODS from Brownlee. Easyspin Sorval Instruments Du Pont and Kokusan H-103 N centrifuges were also used. Cloud Point Preconcentration. Appropriate aliquots of the cold solution containing the pesticides in the presence of Triton X-114 were kept for 5 min in a thermostated bath at 40 "C. Separation of the two phases was achieved by centrifugation for 5 min at 3500 rpm. Determination of the cloud point temperature and the phase ratio has been described e l ~ e w e r e . ~ ~
Electrochemical Detection and Electrode Pretreatment. The detection was carried out with a dual glassy carbon electrode in the series configuration (reductive-oxidative mode). The working potentials were E1 = -1500 mV (generating electrode) and Ez = +400 mV, the intensity at E2 being the analytical signal. The electrodes were electrochemically pretreated every day as followd E1 was kept for 10 min at -1500 mV and then 10 min at +1500 mV, and Snally it was set at working potential (-1500 mv); meanwhile, E2 was kept for 10 min at +lo00 mV and then 10 min at -lo00 mV and after that another 10 min at +lo00 mV. Finally, it was set at the measuring potential (+400 mV). Liquid ChromatographicAnalysis. After the two phases had been separated, a Hamilton syringe was used to collect 60 pL of the surfactant-rich phase, and 10 pL was injected into the chromatographic system. Separation of the different pesticides was achieved using a mobile phase consisting of a 70:30 (v/v) mixture of methanol/water (flow rate, 1.25 mL/min) to which 0.0025 M acetate buffer @H 4.8) had been added as supporting electrolyte.12 The peak assignments for each pesticide were as follows: peak 1, paraoxon, peak 2, methylparathion, peak 3, fenitrothion, and peak 4, ethylparathion. Extracted Fraction of Pesticides from Spiked Water Samples. The water samples analyzed from the Tormes River were used as representative samples of surface waters in this agricultural area. After collection, the samples were filtered with a 0.45 pm pore size membrane filter to remove suspended particulate matter and were then stored at 4 "C in the dark. Samples were analyzed within 12 h after collection. Aliquots of 15 mL were cloud-point-preconcentratedwith 1.0%Triton X-114, and, after phase separation, 10pL of the surfactant-rich phase was injected into the chromatographic system. RESULTS AND DISCUSSION
ChromatographicBehavior of the Surfactant. When the cloud point methodology is used prior to HPLC analysis, a relatively high amount of surfactant is injected into the chromatographic system. Accordingly, two important factors should be taken into account before fixing the experimental conditions for Analytical Chemistry, Vol. 67,No. 15, August 7, 1995
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m
tm
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c700
a
b
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,700
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Figure 1. Chromatogramsobtained for the injection of a surfactantrich phase after cloud point separation with UV detection at 254 nm (a) and dual electrochemical (€I = -1500 mV and €2 = +400 mV) detection (b) with a methanoVwater (70:30v/v) mobile phase. Chromatographic conditions as described in the text.
the chromatographic determination of the analytes under study: the time necessary for the surfactant injected to be eluted and the response of the detection system. The elution time depends on the composition of the mobile phase; in view of the hydrophobic nature of Triton X-114, this value will increase as the content of water in the mobile phase increases. Figure l a shows a chromatogram, recorded with spectrophotometric detection at 254 nm, obtained when 10p L of the surfactantrich phase containing only Triton X-114 was injected into the chromatographic system and 70:30 (v/v) methanol/water was used as the mobile phase. Figure l b shows the chromatogram obtained, with dual electrochemical detection, when a surfactant-rich phase was eluted under the same experimental conditions as those under which the pesticides studied are separated and detected. The Triton X-114 does not show an appreciable electrochemical signal during the first 20 min, even though, during that time, an important part of it has passed through the detector. Elution of the greater part of the surfactant occurs in a broad peak between 15 and 35 min, although the lower molecular weight fractions are eluted previously. The spectrophotometric signal of the surfactant prevents quantification of the analytes studied. When electrochemical detection with a single glassy carbon electrode is performed, the signal afforded by a surfactant-rich phase depends strongly on the anode potential applied.29 However, there are no studies on the electrochemical response of the surfactant-rich phase in a dual electrode system. Figure 2 (upper part) shows the chromatograms obtained on eluting a surfactantrich phase with a mobile phase of 100% methanol and varying the electrode potential, maintaining constant the potential applied to the generator electrode. It may be seen that the surfactant signal increases in magnitude when potentials above $600 mV 2608 Analytical Chemistry, Vol. 67,No. 75,August 7, 7995
I
16 min
*
Figure 2. Dual electrochemical detection (upper, El = -1000 mV and applied potentials €2 on the peaks) and chromatograms (lower, €2 = +700 mV and applied potentials €1 on the peaks) obtained for the injection of a surfactant-rich phase after cloud point separation. Chromatographic conditions as described in the text.
are applied. At lower potentials, Triton X-114 does not afford a signal that might interfere in the detection of the analytes. Similarly, in the chromatograms corresponding to a surfactantrich phase, recorded by applying a fixed measurement potential (Ez= +700 mV) and varying the potential applied to the generator electrode (Figure 2, lower part), the surfactant signal is seen to increase with the increase in the cathode potential applied to this electrode. Because the surfactant does not have electroactive groups in its structure, these electrochemical signals could be due to impurities in the Triton X-114 itself, arising in its synthesis; these can be detected directly or after reduction on the working electrodes. The experiments conducted point to the need to choose the electrode potentials and the composition of the mobile phase very carefully if satisfactory results are to be obtained. The chromatographic behavior of the surfactant permits determination of the pesticides studied (which elute at times lower than 12 min) with dual electrochemical detection. However, total elution of the surfactant does not occur until 35 min have elapsed. To remove the Triton X-114 remaining in the stationary phase after the analytes had been eluted, a washing cycle with 100% methanol over 5 min was performed after each injection. With the washing step, it was possible to reduce the analysis time to 20 min, obtaining better reproducibility in the chromatographic process without having to pretreat the electrode between each inje~tion.2~ Stability of the Analytical Signal. The loss of sensitivity of solid electrodes owing to the adsorption of analytes or their electrodic reaction products is a well-known phenomenon which may be affected by passage of high surfactant concentrations across the electrode. On preconcentrating solutions with an initial pesticide concentration around 85 ppb (Figure 3a) after only seven injections of the surfactant-rich phase containing the preconcentrated pesticides, a loss in signal, ranging between 30% in the case of
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injection number Figure 3. Reproducibility of response with electrode pretreatment. Signals for initial pesticide concentrations of (a) 85 and (b) 19 ppb. Chromatographic conditions as described in the Experimental Section. Peak assignment: 1, paraoxon; 2,methylparathion; 3, fenitrothion; and 4, ethylparathion. Table 1. Enhancement Factors.
compound
0.01
enhancementfactor
paraoxon
*"! 1
28 38
methylparathion fenitrothion ethylparathion
50 54
Ratio of peak intensity of preconcentrated sample to that obtained without any preconcentration step.
paraoxon and 18%for ethylparathion, is observed. However, when the pesticide concentration is reduced to 19 ppb (Figure 3b), no appreciable decrease in the signal is seen. The precision of the analytical response for 10 successive injections of the surfactantrich phase, expressed as their relative standard deviation, ranged between 2.1 and 3.2%. The decrease in electrode performance depends on the concentration of pesticide injected. The presence of Triton X-114 does not affect the response of the electrode, and therefore, for the analyte concentrations at which one works in the analysis of residues, the daily electrochemical pretreatment described in the Experimental Section affords reproducible results with no additional pretreatment between each injection. Electrochemical Response of Pesticides in the Presence of Triton X- 114. The presence of surfactants is known to modify the electric bilayer of the electrode-solution interphase, possibly altering the rate and mechanism of charge and mass transfer; this is due to the greater or lesser adsorption of the surfactant onto the surface of the electrode, which depends on the potential applied, the reaction medium, and the nature of the surfactant. Although the presence of surfactant generally decreases current intensity and distorts the I-V curve, in some cases when the charge of the surfactant is opposite to that of the electroactive species, this may increase the rate of charge transfer and increase the current i n t e n ~ i t y . ~ l - ~ Table 1 shows the increase in signal obtained when 15 mL of a sample containing 1.0%of Triton X-114 (phase ratio of 40) is ~~
~
(31) Kaifer, A. E.; Bard, A. J. I. Phys. Chem. 1985,89, 4876-4880. (32) Zhang, H.; Rusling, J. F. Tuluntu 1993,40,741-747. (33) Marino, A; Brajter-Toth, A. Anal. Chem. 1993,65, 370-374. (34) Jaramillo, A: Marino, A.; Brajter-Toth, A. Anal. Chem. 1993,65, 34413446.
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Figure 4. Chromatograms of (a) a surfactant-rich phase with UV detection and (b) a solution of preconcentrated pesticides with
electrochemical detection. Chromatographicconditions as described in the Experimental Section. Peak assignment as in Figure 3. subjected to the preconcentration process. It may be seen that for the pesticides paraoxon and methylparathion, the enhancement factor is lower than the phase ratio. However, in the cases of the pesticides fenitrothion and ethylparathion, the enhancement factor is greater. This suggests the existence of an additional sensitization effect to the cloud point preconcentration process. This increase in sensitivity can be attributed to modifications occurring in the microenvironment of the analytes when they reach the detector in the presence of the surfactant. Figure 4 shows the chromatograms corresponding to a surfactant-rich phase with spectrophotometric detection at 260 nm (a) and to a solution of pesticides preconcentrated with electrochemical detection (b). Fenitrothion and ethylparathion coelute with different fractions of the surfactant, which may mod& the electrochemical response of these compounds and produce the aforementioned Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
2609
-1
2
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3
3 10
0
20
30
40
9
60
[Triton X-114]/% Figure 5. Peak area values versus Triton X-114 concentration for solutions of pesticides without preconcentration. Samples: 63 ppb paraoxon, 63 ppb methylparathion, 39 ppb fenitrothion, and 61 ppb
ethylparathion. Chromatographic conditions a s described in the Experimental Section. Peak assignment as in Figure 3.
A/106
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Timdmin Figure 7. Chromatograms obtained for the injection of a surfactantrich phase of (a) 0.25 and (b) 2.0% Triton X-114 solutions after cloud
point preconcentration. Concentration of pesticides, chromatographic conditions as described in the text. Peak assignment as in Figure 3. Table 2. Fraction Extracted from Pesticides
fraction extracted (%)
0 0.0
0.5
1.0
1.5
Triton X-114 concn (%) 0.13 0.53 1.0 2.0 2.7 3.3
paraoxon
methylparathion
fenitrothion
eth 1 paratiion
21 45 63 77 81 85
73 89 100 100 100 100
80 100 100 100 100 100
83 100 100 100 100 100
2.0
[Trlton X-l14]/ % Figure 6. Current intensity versus Triton X-114 concentration. Samples: 21 ppb paraoxon, 20 ppb methylparathion, 21 ppb fenitrothion, and 19 ppb ethylparathion. Chromatographic conditions as described in the text. Peak assignment as in Figure 3.
sensitivity effect. This sensitization is less important for paraoxon and methylparathion since they elute over shorter times than Triton X-114. Figure 5 shows the variation in the analytical signal (quantified as the area of the chromatographic peak) corresponding to the pesticides, without preconcentration, in the presence of different concentrations of surfactant. An inverted bell shape response is seen with a maximum for values close to 20% this increase in signal could be attributed to the stabilization of some of the intermediates participating in the electrodic process or to hydrophobic interactions between the surfactant temporarily adsorbed onto the electrpde and the pesticide, which could lead to a slight increase in the mass transfer rate. Cloud Point Preconcentration and Liquid Chromatographic Analysis. The amount of surfactant used in the preconcentration process determines the volume of the surfactantrich phase obtained, and its choice depends on the sensitivity one wishes to achieve and the minimum volume of this phase required for chromatographic analysis. Figure 6 shows the relationship between the analytical signal and the concentration of Triton X-114when 15 mL of a sample containing the pesticides studied and 10 pL of the surfactant-rich phases obtained are injected into the chromatographic system. 2610 Analytical Chemistry, Vol. 67,No. 15, August 1, 1995
The chromatograms shown in Figure 7 correspond to the preconcentration of solutions of 21 ppb of paraoxon, 20 ppb of methylparathion, 21 ppb of fenitrothion, and 19 ppb of ethylparathion with Triton X-114concentrations of 0.25 (a) and 2.0% (b), respectively. The increase in surfactant concentration leads to a decrease in the analytic signal owing to the decrease in the phase ratio. The fraction extracted from each of the pesticides depends on the hydrophobicity of the pesticide and on the amount of Triton X-114 used in the preconcentration step. The values of the fractions extracted for different surfactant concentrations Fable 2) show that by using 1.0%Triton X-114,it is possible to achieve almost complete extraction for methylparathion, fenitrothion, and ethylparathion. However, total extraction of paraoxon would be achieved with surfactant concentrations above 3.5%,leading to an important loss of sensitivity. Calibration graphs were constructed for 15 mL samples with 1.0%Triton X-114.Under these experimental conditions, the phase ratio was 40. The volume of surfactant-rich phase obtained was sufficient to carry out at least 2 injections/sample, although lower surfactant concentrations would yield greater sensitivity. In all cases, linear relationships were obtained between current intensity and the concentration of the analytes studied. Table 3 shows the parameters of the least-squares fittings, the detection limits (calculated as twice the noise), and the relative standard deviation for 10 samples to which the complete procedure (cloud point preconcentration and chromatographic separation) was applied.
Table 3. Analytical Characteristics of the Method.
compound
range @pb)
paraoxon methylparathion fenitrothion ethylparathion
0.99-60 0.97-58 0.80-47 0.96-58
slope (2.09 i.0.09) (4.45 & 0.02) (5.55 f 0.02) (4.12 i 0.02)
intercept 105 105 105 105
(5 f 1) (2 & 2) (1 f 1) (1 2)
105 105 105 105
12
RSDb (%)
LODc @pb)
0.9986 0.9990 0.9993 0.9990
4.2 (4.7) 4.0 (4.5) 3.5 (3.6) 4.6 (4.1)
0.35 0.21 0.18 0.33
a Samples, 15 mL, with ~ . W Triton O X-114; duplicate injection. Values in parentheses are the compound concentrations for which RSD was obtained. LOD, limit of detection (calculated as twice the noise).
2
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Figure 8. Chromatograms obtained for the injection of pesticides before (left) and after (right) cloud point preconcentration of 200 mL of the sample with 0.25% Triton X-114. Concentration of pesticides, chromatographic conditionsas described in the text. Peak assignment as in Figure 3. Table 4. Limits of Detection
c 0
compound
without preconcentration
preconcentratedn
paraoxon methylparathion fenitrothion ethylparathion
10 9.5 8.9 19
0.08 0.04 0.03 0.06
a
Samples, 200 mL, with 0.25%Triton X-114.
These detection limits. can be improved considerably by varying the volume of sample and the amount of surfactant with which the preconcentration step is carried out. Figure 8 (left part) shows the chromatograms corresponding to the elution of an aqueous solution of non-preconcentrated pesticides and the surfactant-rich phase obtained after preconcentrating 200 mL of the previous solution with 0.25% Triton X-114 (right part). The pesticide concentrations were as follows: 10 ppb of paraoxon, 9.5 ppb of methylparathion, 8.0 ppb of fenitrothion, and 10 ppb of ethylparathion. The phase ratio under these experimental conditions was 160. Table 4 shows the detection limits (twice the noise) corresponding to samples without preconcentration and after preconcentration under these experimental conditions. Preconcentration of Pesticides in River Water. In order to check the usefulness of the cloud point methodology for the preconcentration and chromatographic separation of the pesticides
'
1
4
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8 Timdmin
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Figure 9. Chromatograms obtained for the injection of the surfactant-rich phases of (a) a river water sample and (b) a spiked river water sample after cloud point preconcentration. Chromatographic conditions as described in the text. Peak assignment as in Figure 3.
studied, the proposed method was applied to the determination of these analytes in river water samples from different points of the Tormes River. To do so, aliquots of 15 mL of each of the samples were firstly analyzed according to the proposed procedure in order to provide ambient-level pesticide concentrations. No peaks corresponding to the species studied were detected in any of the samples analyzed (the chromatogram of one sample is shown in Figure 9a). The samples were then fortifed at concentration levels between 1.5 and 10 pg/L of each pesticide. The fractions extracted li-om this matrix ranged from 99 to 101%. Figure 9b shows the chromatogram corresponding to a sample spiked with 1.0 ppb of paraoxon, 1.9 ppb of methylparathion, 1.6 ppb of fenitrothion, and 1.9 ppb of ethylparathion. These results show that the cloud point preconcentration method can be applied to the determination of organophosphorus pesticides in river water. This methodology could also be used in the determination of these analytes in drinking water, in which the maximum concentration permitted by the European Union is lower than 0.1 ppb/individual substance.35 In this case, it would Analytical Chemisfy, Vol. 67,No. 75,August 7, 7995 2611
be necessary to preconcentrate a larger volume of sample with a suitable amount of surfactant (see Table 4). CONCLUSIONS
The micelle-mediated methodology has been applied to the preconcentration of organophosphorus pesticides prior to their separation by chromatography. The mobile phase employed for the elution of the compounds assayed prevents the spectrophotometric detection of these compounds owing to the signal produced by Triton X-114. However, the use of dual electrochemical detection permits suitable detection and quantification of the organophosphorus pesticides paraoxon, methylparathion, fenitrothion, and ethylparathion. Injection of high surfactant concentrations does not appreciably affect the response of the electrode, and no electrochemical pretreatment is necessary between injections. Additionally, the injection of the surfactant-rich phase, containing the preconcentrated pesticides, into the chromatographic system elicits an additional increase in the sensitivity of the electrochemical detection of fenitrothion and ethylparathion owing to the joint elution of these compounds with small fractions of surfactant. The preconcentration of as little as 15 mL of sample with a Triton X-114 concentration of 1.0%affords detection limits lower (35) EEC Drinking Water Guideline 80/778/EEC. OflJ. Eur. Commun. 1980, Aug 30, L229/11-29.
2612 Analytical Chemistry, Val. 67,No. 15, August 1, 1995
than 0.5 ppb. This sensitivity can be enhanced by preconcentrating a larger volume of sample or by using lower surfactant concentrations. The results obtained in the analysis of river water samples show that the method is valid for the determination of organophosphorus pesticide in river water. Although the organophosphorus pesticides paraoxon, methylparathion, fenitrothion, and ethylparathion were used in this work, the proposed method should afford similar results when used on compounds with similar characteristics. ACKNOWLEWMENT
The authors wish to thank Dr. Carabias Martinez and Dr. Rodriguez Gonzalo for useful information and helpful discussions on electrochemical detection. C.G.P. acknowledges financial support by the Spanish Government (PFPI). This work was supported by DGICYT (Project PB91-185) and the Consejeria de Cultura y Turismo de la Junta de Castilla y Leon (Project S A W 93).
Received for review February 17, 1995. Accepted May 3,
1995.B AC950178H ~~~~
@Abstractpublished in Advance ACS Abstracts, June 15, 1995.
