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Anal. Chem. 1980, 52, 2156-2161
Liquid Chromatographic Determination of Selected Carbamate Pesticides in Water with Electrochemical Detection James L. Anderson”’ and David J. Chesney2 Department of Chemistty, North Dakota State University, Fargo, North Dakota 58705
A reverse-phase liquid chromatographic method with electrochemical detection Is described for the trace determination In water of selected examples of the important and ublquitousiy applled carbamate class of agricultural pesticides. The thlnlayer, Kel-F-graphite electrochemical detector is operated in the oxldatlve, constant-potentialarnperornetrlc mode at 1.1 V vs. AglAgCIJ3.5M KCI. Calibration curves are llnear over a wide range of sample concentratlon-typically at least 3 orders of magnitude-with relative standard deviations for repetitive determlnatlons of 1-2% through most of the range. Detection limits in the range of 40-150 pg (slgnalholse ratio 2: 1) have been obtained for test carbamates, corresponding to sample concentrations of 2-7 ppb, using slmple and inexpenslve equipment. Detection llmlts are controlled by “pump” noise resulting from fluctuations In background water oxldation current.
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Pesticides of the carbamate class have become increasingly important in recent years, due to their broad spectrum of biological activity. They are employed as insecticides, fungicides, nematocides, miticides, and molluscicides ( I ) . Presently available analytical methods for carbamate pesticides suffer from a number of difficulties. Improved methods are needed for rapid determination of these pesticides at ultratrace levels. Susceptibility of many carbamates to thermal decomposition complicates gas chromatographic (GC) analysis (2, 3). Detection limits for direct GC determination of underivatized carbamates (2-4) using specially treated column supports typically exceed 10 ng (2, 3 ) . Formation of heatstable derivatives is usually required to minimize degradation of these compounds, allowing GC analysis in the picogram range (I, 2, 5-7). Typical GC derivatization schemes for aromatic carbamates, via hydrolysis to corresponding phenols or amines and reaction with halogen-rich reagents for electron capture detection, are susceptible to interference from adventitious phenols or amines in the sample, requiring careful sample cleanup prior to derivatization and GC analysis ( 2 ) . The gentle conditions available in liquid chromatography (LC) are ideally suited to the analysis of sensitive compounds like the carbamate pesticides. A major potential advantage of LC is the feasibility of direct carbamate analysis without the additional complexity of a derivatization step. An early report on LC carbamate separation demonstrated speed and resolution comparable to GC but 2 to 3 orders of magnitude lower sensitivity with an ultraviolet (UV) detector than attainable with electron capture GC (8). LC procedures have been developed for a wide range of carbamates (2, %19), both direct with UV detection (2, 9-12) and after derivatization with fluorescence detection (13-19). Detection limits in the low nanogram range were reported for most of the carbamates when monitored with state-of-the-art UV detectors at wavelengths of optimum response (typically 190-210 nm) (9). ‘Present address: Department of Chemistry, University of Georgia, Athens, GA 30602. Present address: General Nutrition Corporation, Fargo, ND 58102.
0003-2700/80/0352-2156$01 .OO/O
Extensive tabulations of chromatographic data are available for LC determination of carbamates (9-11). Only slightly better detection limits are obtained by using fluorimetric derivatization and detection, with reported detection limits typically between l and 10 ng for dansyl derivatives formed prior to injection (13, 14) or ranging from 500 pg (19) to 1-10 ng (16,17) for postcolumn derivatization with o-phthaldehyde. A more sensitive detector is clearly needed to make direct LC determination of carbamates competitive with electron capture GC of derivatized species. Electrochemical LC detectors (LCEC) provide a very sensitive, simple, inexpensive, and somewhat selective alternative to conventional UV and fluorescence detectors for LC. Many electroactive organic compounds have minimum detection limits ranging from to 10-l~ mol per injection (2O),making LCEC very applicable for trace analysis. LCEC detection limits often fall in the picogram range attainable with electron capture GC but without the need for prior derivatization. Thus, LCEC offers the potential of greatly simplified analysis. Successful application of LCEC for a wide range of analyses has been reviewed @ I ) , and a bibliography lists over 100 references, most of which have appeared since 1973 (22). Moye (15) noted potential applicability of LCEC for pesticide residue analysis but failed to see any carbamate electroactivity during cyclic voltammetric investigations. It was suggested that low-efficiency reactions could have been obscured by the large background currents. The electrochemical behavior of several carbamate compounds a t negative potentials has been investigated by means of polarography (23, 24) after derivatization via nitration or nitrosation or tensammetry (25). Little oxidative work has been reported, although Lores et al. have demonstrated subnanogram LCEC detection of halogenated anilines which may be formed as hydrolytic metabolites of some carbamate and urea pesticides (26).
We report here the successful application of an electrochemical detector for the LCEC determination of selected carbamate pesticides in water. The detector is based on a highly organic solvent-resistant Kel-F-graphite (“Kelgraf”) electrode developed in our laboratories (27-29). Many of the carbamates investigated proved to be electroactive within the potential range practically accessible in aqueous systems. Detection limits as low as 40 pg have been achieved after separation by a C18reverse-phase chromatographic column. EXPERIMENTAL SECTION Chemicals. All chemicals used were reagent grade unless otherwise specified. KH2P0, and Na2HP04were recrystallized twice from water. House distilled water was deionized by a Millipore Milli-Q system (Bedford, MA) and further distilled from alkaline permanganate and sulfuric acid t o eliminate organic contaminants. Acetonitrile (MeCN) and methanol (MeOH)were Burdick and Jackson HPLC grade, used without further purification. Pesticide compounds were obtained as analytical reference standards from the US. Environmental Protection Agency. Stock 1 mM solutions of the pesticides in MeOH passed through Millipore 0.22 or 0.45 pm membrane filters before introduction t o the LC system. Equipment. The LC was constructed by using a Milton Roy minipump (Model 396-31), an injection valve (Rheodyne Model 1980 American Chemical Society
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Flgure 2. Reference and auxiliary electrode chamber: (A) standard nylon LC connector: (B) pt counterelectrode; (C) “Thirsty Vycor” isolation frit, secured by shrinkable TeRon tubing: 0)Ag/AgCI reference
Flgure 1. Exploded diagram of electrochemical detector cell: (A) “KeK” cell top; (B) inlet, 1/4-28threaded to fit standard LC connectors: (C) outlet, 1/4-28threaded to fit standard LC connectors: (D) Teflon spacer;
electrode (in 3.5 M KCI): (E) standard taper inner joint; (F) standard taper outer joint: (G) effluent exit.
(E) “Kelgraf”electrode: (F) “Kel-F” electrode sheath, 3/4 in. diameter: (G) movable stage, Plexiglas: (H) milled depression in stage, 3 / 4 in. diameter (to fit “Kel-F” electrode sheath, F): (I) mercury contact to “Kelgraf”electrode: (J) outside copper electrode contact: (K) lower cell body, Plexiglas: (L) elevating screw for mobile stage.
stage (Figure l),which allows easy removal and replacement of electrodes. The center stage is vertically movable by means of a positioning screw. A 1.9 cm circular depression is milled out in the center, ensuring proper positioning of the Kelgraf electrode relative to the eluent inlet and outlet. A 127 p m thick Teflon gasket (Bioanalytical Systems, West Lafayette, IN) serves as a spacer to form the thin-layer channel. No problems were encountered with eluent leakage due to nonplanar electrode surfaces. The center position of the adjusting screw provides equal pressure all around the electrode, enabling a tight gasket seal. A well drilled in the center of the depression provides mercury contact between the electrode and a copper wire running inside the stage for external contact. The Kel-F cell top ensures that only chemically inert material will encounter the flowing eluent. All other cell parts are Plexiglas. The reference electrode and counterelectrode (Ag/AgC1/3.5 M KC1 and Pt, respectively) were positioned downstream in a glass chamber (Figure 2), which was press-fitted while hot into a drilled-nylon 1/4-28 threaded connector which screwed into the cell outlet. Epoxy cement applied to the outside surface of the junction helped to prevent leakage.
70-10) with a 20-pL sample loop for sample introduction, and a glass column (25 cm length, 2 mm. i.d.). The column was packed with pellicular Bondapak CISCorasil(37-50 pm diameter; Waters Associates) by the tap-fill method suggested by Snyder and Kirkland (30). All eluent connections were made with 1.6 mm 0.d. Teflon tubing (Altex). A loo0 psi pressure gauge (Matheson) and a glass bourdon tube (2 mm. i.d., 35 cm length) in a tee configuration were used to dampen flow fluctuations. The potentiostat was constructed by using National Semiconductor LF 356H operational amplifers, in a standard differential-controller configuration, with provision for background current offset at the recorder (31). The potentiostat was powered by a Boston Tech (Coral Gables, FL) f15 V power supply. Cyclic voltammetric voltage waveforms were supplied by a Princeton Applied Research (Princeton,NJ) Model 175 universal programmer. The voltammetric scans were recorded on a Hewlett-Packard (Palo Alto, CA) Model 7035B X-Y recorder. The voltammetric solutions were thoroughly deoxygenatedby bubbling with nitrogen made oxygen-freeby passage through a vanadium(I1) scrubber. The LC eluents were vacuum degassed prior to each day’s use. Applied potentials were monitored by a Data Precision (Danvers, MA) Model 245 digital voltmeter. A Houston Instruments (Austin, TX) Omniscribe B-5000 dual-pen strip chart recorder was used to monitor the detector output. Electrodes. The fabrication of the Kelgraf working electrode was reported previously (27,28). The working electrode for the thin-layer detector was formed in the shape of a thin pellet (1.9 cm diameter, 0.32 cm thick) in a locally constructed die. After manual surfacing with progressively finer grit sandpaper, the electrodes were polished to a smooth, shiny finish with 1 p m alumina (Buehler Micropolish) on kitten-ear polishing cloth (Buehler) mounted on locally constructed lapping wheels. A 25% Kelgraf voltammetric electrode (25% carbon, 75% Kel-F w/w) and platinum spiral counterelectrode were used with potentials referred to Ag/AgC1/3.5 M KC1. Cell Design. The thin-layer detector cell, of design similar to that of Kissinger and Adams (32),is constructed with a movable
RESULTS AND DISCUSSION Cyclic Voltammetry. The presence of a substituted aromatic amine, or an aromatically substituted carbamate oxygen or nitrogen moiety, suggested t h a t many carbamate pesticides might be oxidized at positive potentials, by analogy to the behavior of various substituted phenolic and aromatic amine (33)species. This investigation concentrated, therefore, on the oxidative detection of carbamates and related aromatic amine compounds. Oxidative cyclic voltammetric (CV) scans were initiated from initial potentials near 0 V t o switching potentials near +1.3 V. Oxidative peak potentials and conditions are reported for eight carbamate and three aromatic amine pesticides in Table I. CV scans for Aminocarb and Carbendazim are shown in Figures 3 and 4, respectively. Voltammetric investigations on the compounds listed were performed in mixtures of aqueous phosphate buffer with MeCN and/or MeOH of composition similar to expected reverse-phase chromatographic eluents. All solutions contained 10% MeOH, due to a 1O:l dilution from the 1 m M stock solution to the voltam-
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A g AgCl
(solution = aqueous phosphate buffer, pH 6/30% CH&N/lO% MeOH, electrode = Kelgraf, polished, 0.32 cm2 area, scan rate = 50 mV/s): (a) blank; (b) ca. 0.2 mM Aminocarb. Flgure 3. Cyclic voltammogram-Aminocarb
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Flgure 4. Cyclic v o ~ m m o g r a m - C a r b e n z i ~ ~ ~ as ~ iino Figure ns 3: (a) blank; (b) ca. 0.2 mM Carbendazim.
metric working concentration of 0.1 mM. Most carbamates investigated could be oxidized at practically attainable potentials in these media. The available potential range was limited by a sharp increase in the background current a t ca. +1.3 V vs. Ag/AgCl, associated with the oxidation of water. Most of the compounds under study exhibited oxidation processes a t potentials near the positive limit of this potential “window”. In most cases, oxidation waves were not well separated from the solvent oxidation wave. BPMC showed no electroactivity at all under the conditions investigated. Carbaryl exhibited no electroactivity a t attainable potentials in mixed aqueous pH 6 buffer/MeCN/MeOH solution but developed an oxidative wave (at E = +1.2 V) poorly resolved from the solvent oxidation wave, in 90% pH 7.6 aqueous buffer/lO% MeOH solution. The ill-defined oxidative wave exhibited by Desmedipham in the aqueous buffer/MeCN/MeOH solution resolved itself into two distinct waves in the 90% aqueous buffer/lO% MeOH solution. While the initial oxidative wave of Phenmedipham was well-defined, it too resolved into two waves upon oxidation in 90% aqueous buffer/lO% MeOH. Both Desmedipham and Phenmedipham exhibit a reductive wave on the reverse scan which could represent a reduction of a chemical product of the oxidation, an adsorbed electroactive species, or both. Chlorpropham and Dichloran also exhibit this type of reductive wave. Further work will continue to investigate the nature of these processes. A number of carbamates suffered severe electrode fouling problems a t the relatively high concentrations (0.1 mM or ca. 20 ppm) used during initial voltammetric studies. Electrodes were resurfaced on O / O and 4/0 sandpaper before each voltammetric scan for Chlorpropham, Desmedipham, and Phenmedipham. Roughened electrodes yielded substantially higher background currents than smoothly polished electrodes but permitted more reliable determination of the electroactivity of these compounds. Roughening also helped to resolve the
oxidative wave for Carbaryl. Electrode fouling was not anticipated to be a problem in the LCEC detector, since the concentrations of these analytes would be much smaller (
