Column Esterification in the Gas Chromatography of the Desalkyl Metabolites of Methyl Parathion and Methyl Paraoxon P. S . Jaglan, R. B. March, and F. A. Gunther Department of Entomology, University of California, Riverside, Calif. 92502
DIRECT USE of gas chromatography is normally limited to volatile, nonpolar compounds. When the gas chromatographic separation and measurement of nonvolatile, polar, and thermally unstable molecules is desired, it is usual to prepare volatile and thermally stable derivatives or to use carbon skeleton or pyrolysis gas chromatography. In developing gas chromatographic methods for organophosphorus insecticides and their metabolites, the determination of desalkyl metabolites, which have been shown to occur both in insects and in mammals, becomes important ( I , 2). Previous procedures to separate these metabolites include paper chromatography (I+, thin-layer chromatography (3, and ion exchange chromatography ( I , 2, 6), with detection and measurement primarily by radiotracer techniques. No successful gas chromatography of these or of related metabolites has been reported; for example, Giang and Beckman (7) failed to gas chromatograph the desmethyl derivatives of Azodrin (dimethyl phosphate ester of cis-3-hydroxy-N-methyl crotonamide) and Bidrin (dimethyl phosphate ester of cis-3hydroxy-N, N-dimethyl crotonamide) even after esterification of their silver salts with methyl iodide in the fortified samples of lettuce extractives. During the course of an investigation using methyl parathion as the experimental organophosphorus compound, we consistently observed separate peaks for desmethyl methyl parathion and desmethyl methyl paraoxon, on several columns. The retention time of the apparent desmethyl methyl paraoxon was consistently delayed only about 6-10 seconds in comparison to methyl paraoxon and the two peaks were never resolved even with columns of widely differing polarities; separation by low temperature gas chromatography was also unsuccessful. Because the responses of the desmethyl derivatives were much lower than the corresponding triesters, from which they were prepared, it was tempting to dispose of the aberrant chromatographic peaks as impurities. However, even with the most highly purified samples, the same peaks were observed and on-column esterification was therefore suspected. EXPERIMENTAL
Methyl parathion, methyl paraoxon, desmethyl methyl paraoxon sodium salt, and desmethyl methyl parathion sodium salt were prepared ( I , 8) and purified by repeated thinlayer chromatography to single, clean spots. Preparation of Standard Solutions. Standard solutions of methyl parathion and methyl paraoxon were prepared in (1) R. M. Hollingworth, R. L. Metcalf, and T. R. Fukuto, J. Agr. Food Chem., 15, 242 (1967). (2) Ibid., p 250. (3) W. F. Chamberlin,J. Econ. Entomol., 57,119(1964). (4) Zbid., p 329. (5) J. Stenersen, J. Chromatogr., 38,538 (1968). 30, 1622 (1958). (6) F. W. Plapp and J. E. Cassida, ANAL.CHEM. (7) B. Y . Giang and H. F. Beckman, J . Agr. Food Chem., 16, 899 (1968). (8) R. M. Hollingworth, R. L. Metcalf, and T. R. Fukuto, ibid., 15, 235 (1967).
acetone. The sodium salts of the desalkyl compounds were dissolved in equimolar amounts of methanolic HC1 to produce the free acids. Methanolic HCl was prepared by bubbling dry HC1 gas into anhydrous methanol. An approximate 10% stock solution was prepared; normality was determined by titrating with aqueous NaOH solution. Thin-Layer Chromatography. Appropriate amounts of the compounds and their GLC eluates were spotted on 20X 20-cm silica gel plates and developed in ether-hexane (7 :3). The chromatographed sompounds were visualized under ultraviolet light at 2537 A. For the characterization of thionates, a spray of 0.5 % DCQ (2,6-dibromo-benzoquinone-4chloroimide) in cyclohexane was used, producing a red color with thionates (9). Phosphates were visualized with ammonium molybdate in HC1 and HClOd as described by March and Metcalf (IO) based on the method of Hanes and Isherwood (11).
Cholinesterase Inhibition. Cholinesterase inhibition of the compounds and their eluates was determined by the automated method of Ott and Gunther (12) as modified by Ott (13). Method of GLC Analysis. The GLC analyses were carried out on a Hewlett-Packard 402 high-efficiency gas chromatograph modified for thermionic detection with a KC1 burner pellet. Five per cent Apiezon L coated on Gas-Chrom Q, 80/100 mesh, in a 610 X 4-mm i.d. borosilicate glass column was used. The temperatures and flow rates are given at appropriate places below.
RESULTS AND DISCUSSION The retention behavior of methyl parathion, methyl paraoxon, and their desalkyl derivatives is shown in Figure 1. Although the retentions of methyl paraoxon and desmethyl methyl paraoxon are apparently different, the peaks were not resolved. Efforts to resolve them on 1-20% QF-1, SE-30, OV-1, OV-3, OV-17, DC-200, DC-710, and DEGS loaded columns from 1-6 feet in length and by temperature programming were also unsuccessful and the retention pattern was unchanged. The peak of desmethyl methyl paraoxon was broader than that of methyl paraoxon and became even broader and flatter at lower temperatures. The gas chromatographic peak response of this desalkyl compound changed from hour to hour and from day to day; its peak appeared only when relatively large amounts were injected, no peak being observed below 100 ng. The peak of desmethyl methyl parathion was broader than that of methyl parathion and the response was higher than that of desmethyl methyl paraoxon, as little as 10 ng being detectable. Instead of appearing near methyl parathion, the peak of this desmethyl analog was delayed.
(9) J. J. Menn, W. R. Erwin, and H. T . Gordon, ibid., 5,601 (1957). (10) R. B. March and R. L. Metcalf, ibid., 2,732 (1954). ( 1 1 ) C. S. Hanes and F. A. Isherwood, Nature, 164, 1107 (1949). (12) D. E. Ott and F. A. Gunther, J . Assoc. Ofic.Anal. Chem., 51, 697 (1968). (13) D. E. Ott, J. Agr. Food Chem., 16, 874 (1968). VOL. 41, NO. 12, OCTOBER 1969
1671
1
2 3 Minutes
4
Figure 1. Chromatogram of methyl parathion and metabolites on 5% Apiezon L on GasChrom Q, 80/100 mesh, 610 X 4 mm i.d. glass column; temperature of column, injector and detector 190, 210, and 200 "C, respectively; flow rates of nitrogen, hydrogen, and air 40, 20 and 300 ml/min, respectively Thin-layer chromatography of the reference compounds and their GLC eluates is shown in Table I. Reference desmethyl methyl paraoxon remained at the origin while its GLC eluate had an R, identical to that of methyl paraoxon. The desmethyl methyl parathion GLC eluate had an R, very near to methyl paraoxon while the reference compound remained at the origin. The DCQ reagent gave a red color for the reference desmethyl methyl parathion spot, but not for that of its GLC eluate, indicating that the eluate was an isomerized analog. Gas chromatography of the S-methyl isomeride of desmethyl methyl parathion resulted in a peak that had the same retention time as that of desmethyl methyl parathion, but the response was poor. When the reference desalkyl compounds were gas chromatographed in methanol, there was little or no response, but addition of equimolar methanolic HCl resulted in a 100-fold in-
Table I. Thin-Layer Chromatography of Methyl Parathion and Its Metabolites. Compound Color Rf Methyl parathion 0.56 Red with DCQ Red with DCQ Methyl parathion GLC eluate 0.56 Methyl paraoxon Blue with molybdate 0.21 Blue with molybdate Methyl paraoxon GLC eluate 0.21 Red with DCQ Desmethyl methyl parathion 0.00 Yellow, not red with Desmethyl methyl parathion 0.23 GLC eluate DCQ Blue with molybdate Desmethyl methyl paraoxon 0.00 Blue with molybdate 0.21 Desmethyl methyl paraoxon GLC eluate See text for details. (1
1672
ANALYTICAL CHEMISTRY
1
2
3
4 5 6 7
8
Automated sample number
Figure 2. Cholinesterase inhibition by desalkyl compounds and their GLC eluates, determined by an automated ChE method (12, IS) crease in peak height. Elevation of the temperature of the flash heater resulted in a further increase in response and the peaks became sharp and symmetrical. The use of ethanolic HCI resulted in much diminished peaks, and with isopropanolic HC1 there was no response at all. With desmethyl methyl paraoxon, the reaction efficiency paralleled the concentration of the reactants, the response averaging 40-50 of theoretical with pg-range injections. At ng concentrations, the response decreased to 10-20%, and there was no response below 100 ng. With desmethyl methyl parathion the response was nearly equivalent to that of the parent triester at all concentrations. The cholinesterase inhibition by desalkyl GLC eluates is shown in Figure 2. Both eluates were potent cholinesterase inhibitors, whereas the original compounds were inactive. These results in toto strongly suggest on-column methyl esterification of methanolic HCl solutions of the desalkyl derivatives of methyl parathion and methyl paraoxon, and probably some ethyl esterification from ethanolic HCl. Because the response of desmethyl methyl paraoxon was relatively low, alternative derivative methods were investigated. With diazomethane (14), excellent off-column esterification of desmethyl methyl paraoxon was obtained. The esterified derivative produced a single GLC peak identical to methyl paraoxon and the response was so much increased that less than 10 ng could be detected. However, gas chromatography of the reaction products of diazomethane with desmethyl methyl parathion resulted in two peaks at relative retention times of 1.0 and 1.6. The peak at the former retention time, coincident with methyl parathion, constituted (14) H. Schlenk and J. L. Gellerman, ANAL. CHEM.,32, 1412 (1960).
only 10% of the injected material. The peak at retention time 1.6 was the major peak, and was the same as that observed with methanolic HCI. When the S-methyl isomeride of desmethyl methyl parathion was reacted with diazomethane and the product was analyzed, it produced a single peak at 1.6. Thus an attack on sulfur is apparently favored by the esterifying reagent although under the conditions of reaction with desmethyl methyl parathion, the free hydroxyl group is also esterified. Further experimental details on the esterification of desalkyl derivatives of methyl parathion and methyl paraoxon
and its application to a GLC method for the determination of methyl parathion and methyl paraoxon and their desalkyl and desaryl metabolites will be published elsewhere. RECEIVED for review June 30, 1969. Accepted July 30, 1969. Taken in part from the Ph.D. dissertation of the senior author, June 1969. This work was partially supported by Public Health Service Research Grant CC-00038, by the United States Department of Interior, Office of Water Resources Research, and by the University of California, Water Resources Center.
Determination of Vanadium by Controlled-Potential Coulometry L. P. Rigdon and J. E. Harrar Chemistry Department, Lawrence Radiation Laboratory, University of California, Liaermore, Calif. 94550 FORTHE DETERMINATION of vanadium, an accurate controlledpotential coulometric procedure would offer several advantages over the commonly used volumetric methods ( I ) , especially those requiring unstable titrants. In addition, the technique generally provides a superior means for the oxidation-state analysis of mixtures. Although the possibility of a controlled-potential coulometric determination with a mercury pool working electrode and various valence states is clearly evident from previous electrochemical work, particularly that of Meites and associates (2-4), the methods chosen for development here are based on the use of a platinum electrode and the reactions of the strongly oxidizing V(V)-V(1V) couple. These are the most commonly encountered species in metallurgical samples, and fewer interferences are expected at the anodic potentials involved. Also, platinum working-electrode cells are more convenient to use, once electrode pretreatment problems have been solved. Several investigators (5-10) have shown that the V(1V)V(V) system is totally irreversible at platinum electrodes in noncomplexing, acid media. Clearly defined limiting currents are difficult to obtain for the oxidation process, and the electrode surface condition markedly influences the reduction reaction (7, 8, 10); thus voltammetric analysis would be difficult. Preliminary work in this laboratory, however, indicated that both oxidation and reduction processes could form the basis of a useful coulometric analysis procedure. Therefore, both were examined further (in phosphoric acid media) with respect to current efficiency, the effects of interferences, and the influence of electrode pretreatment. Electrode surface effects are advantageously of less importance in the technique of controlled-potential coulometry than in voltammetry. (1) H. R. Grady in “Treatise on Analytical Chemistry,” Part 11, Vol. 8, 1. M. Kolthoff and P. J. Elving, Eds., Interscience, New York, N. Y., 1963, pp 222-31. (2) L. Meites and S. A. Moros, ANAL.CHEM., 31, 23 (1959). (3) Y. Israel and L. Meites, J. Eleclroanal. Chem., 8,99 (1964). (4) “Handbook of Analytical Chemistry,” L. Meites, Ed., McGraw-Hill,New York, N. Y.. 1963. Sec. 5. u 200. (5) F. Foerster and F. Bottcher, 2.’ Phy~.’?hem. (Leipzig), 151,
321 (1930). (6) 0.A. Songina, Zmod. Lab., 20, 531 (1954). (7) D. G. Davis, Tuhnta, 3, 335 (1960). (8) F. C. Anson and D. M. King, ANAL. CHEM.. 34. 362 (1962). (9) V. A. Mirkin and M. T. Kozlovskii, J . Anal. Chem. USSR, 17, 698 (1962). (IO) D. Cozzi, G. Ciantelli,and G. Raspi, Ric. Sci,7(11), 589 (1964).
EXPERIMENTAL Apparatus. The cell assembly and instrumentation for voltammetry and coulometry, along with their general operation, have been described previously (11).. Reagents. Solutions of vanadium were prepared from Alfa Inorganics VOS04.xHzO and NaV03.xHz0, and from Materials Research Corp. Marz grade vanadium metal. Approximately 1-gram portions of the metal were dissolved in 5 ml of concentrated HzS04,5 ml of concentrated H N 0 3 , and 25 ml of HzO, and then fumed. The solutions were diluted to volume in certified flasks to give concentrations of approximately 10 mg per ml. Calibrated micropipets were used to take aliquots of the stock solutions for coulometric analysis. Vanadium solutions were standardized for V(1V) by titration with K M n 0 4 ( I ) , which in turn was standardized against NBS As203. Vanadium(V) was titrated with ferrous ion using sodium diphenylamine sulfonate indicator and K2Cr207 as the primary standard (1). The vanadium metal was assayed by both titration methods; the vanadium was reduced to the tetravalent state with SO2 or Na2S03for the oxidimetric titration and oxidized to the pentavalent state with (NH4)&08 for the reductimetric titration (1). The results of the titrations of the metal solutions yielded a value for the assay of 99.70% with a standard deviation of 0.15%. The impurities in the vanadium metal were determined by emission spectrography, spark source mass spectrography, vacuum techniques, and by atomic absorption spectrophotometry, with the following results in ppm: C, 87; H, 77; N, 380: 0, 272; Si, 650; and other metallic impurities, 540. The total impurity level from these data is 0.207& which provides adequate confirmation of the vanadium assay results. The accuracies of the coulometric methods for total vanadium were therefore based on the vanadium metal titrimetric assay values. Linde cryogenic oxygen of 99.6% purity was used for the oxidation of sulfite. For the voltammetric measurements, solutions of V(V) were deoxygenated with high purity nitrogen. Deionized water was used for the preparation of solutions. All other chemicals were reagent grade or of comparable purity. Procedure. SAMPLEDISSOLUTION AND PRETREATMENT. Dissolve 1 gram vanadium metal or alloy in an oxidizing and H2S04. If mineral acid mixture, preferably “ 0 3 silica is present, it may be removed by treatment with HF (1). If manganese is present, H3P04 should not be used for the dissolution, because formation of the Mn(II1) pyrophosphate
(11) J. E. Harrar and L. P. Rigdon, ANAL.CHEM., 41, 758 (1969). VOL. 41, NO. 12, OCTOBER 1969
1673
