Anal. Chem. 1980, 52, 2227-2229
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Column Chromatography Separation of Polychlorinated Biphenyls from Dichlorodiphenyltrichloroethane and Metabolites Larry L. Needham,” Ann L. Smrek, Susan L. Head, Virlyn W. Burse, and John A. Liddle Center for Disease Control, Public Health Service,
US.Department of
Gas chromatographic determinations of organochlorine insectides or polychlorinated biphenyls (PCBs) in biological material are often complicated by their simultaneous presence in the eluates from the column chromatography steps. For example, failure to separate dichlorodiphenyltrichloroethane (DDT) or dichlorodiphenyldichloroethylene (DDE) from PCBs greatly magnifies the problem because the response of the electron capture detector to DDT and DDE is 20-30 times that of an equal weight of Aroclor 1254 ( I ) . Recently, we devised a gas chromatography/electron capture detector procedure to analyze serum for PCBs (Aroclor 1260) in the presence of the ortho, para and para,para isomers of DDT and its metabolites, DDE and dichlorodiphenyldichloroethane (DDD). In attempts to accomplish this separation, residue chemists have tried various column adsorbents, such as silicic acid ( 2 ) ,Florisil ( 3 ) ,and silica gel ( 4 ) . They have also tried chemical reactions such as oxidation ( 5 ) ,alkaline hydrolysis (6) of some of the chlorinated hydrocarbons, and perchlorination ( 7 ) of the PCBs in order to aid in the interpretation of the resulting chromatogram. All of these methods have noticeable shortcomings. We successfully accomplished the required separation by using a column of 10% silver nitrate on silica gel. Chemists of Dow Chemical Co. recently developed this adsorption column for the determination of 2,3,7,8-tetrachlorodibenzo-p-dioxin in fish (8). Somewhat analogous to this is the application of transistion-metal complex formation in gas chromatography (9). We describe the use of the silver nitrate on silica gel column to separate PCBs from DDT and metabolites and compare it to two other methods. EXPERIMENTAL SECTION Apparatus. The gas chromatograph/data system was the Perkin-Elmer Sigma I. The gas chromatography contained a constant-current63Nidetector and was fitted with a Perkin-Elmer 4990 automatic sampler. The 1.83 m by 4 mm (i.d.) glass column was packed with 3% SE-30 on 80/100 Supelcoport. The glass chromatography columns containing the silver nitrate on silica adsorbent were 170 mm X 7 mm, with a 50-mL reservoir. Reagents. 10% AgNO, on Silica. The silica gel, Woelm 1OC-200 mesh, was treated with silver nitrate as described in the literature (8). All solvents were Burdick and Jackson, distilledin-glass quality. The silver nitrate was ACS grade from Fisher Scientific Co. Procedure. Sample Preparation and Cleanup. One milliliter of methanol was added to 2 mL of serum in a 16 X 125 mm culture tube with a poly(tetrafluoroethy1ene)-lined cap. After this had been mixed briefly by vortex, 5 mL of hexane was added, and the tube was rotated at 30 rpm for 10 min on a rotary mixed. After being centrifuged, the extract was transferred to another culture tube. The extraction steps were repeated twice. The combined extracts were reduced to approximately 1 mL by using a warm water bath (40 “C) and a gentle stream of nitrogen. A silanized glass wool plug, 17 mm of hexane-washed anhydrous sodium sulfate, 77 mm (1.4 g) of 10% silver nitrate on silica gel, and 17 mm of the sodium sulfate were added to the chromatography column. After a prewash of the column contents with 10 mL of hexane, the extract was pipetted onto the head of the column. The culture tube was rinsed with 2 mL of hexane, and this was added to the column. Additional hexane was added and the first 30 mL of eluate was collected. The eluate was reduced in volume as above to 2 mL and pipetted into autosampler vials from which 5pL was injected into the gas chromatograph operated at the following conditions: temperature of injector, column, and detector
Health and Human Services, Atlanta, Georgia 30333
were 250, 195, and 330 O C , respectively; nitrogen column flow and detector makeup flow were 15 and 20 mL/min, respectively.
RESULTS A N D D I S C U S S I O N For the quantitation of the PCBs, we adapted the method of Webb-McCall (10) to a microprocessor controlled data system. In this method, all retention times are reported relative to p,p’-DDE X 100. Since some PCB congeners have been reported to be more toxic than others (11),we sought to determine not only the recovery of total PCBs from serum but also the recovery of the PCBs in each of the chromatographic peaks of Aroclor 1260. For this experiment, ten samples from a bovine serum pool that had been fortified with Aroclor 1260 (41 ppb), o,p’-DDT (10 ppm), p,p’-DDT (21 ppb), o,p’-DDD (137 ppb), p,p’-DDD 11 ppb), o,p’-DDE (31 ppb), and p,p’-DDE (378 ppb) were carried through the reported procedure. Bovine serum that had been fortified with DDT and metabolites, but not with PCBs, was also analyzed. The resulting chromatograms showed no evidence of DDT or its metabolites a t detection limits of less than 1 ppb. The recovery data for total PCBs (94.9%) and for each PCB peak are given in Table I. In the determinations of in vivo samples that have an Aroclor 1260 pattern, we have found the relative order of concentration of PCBs to be in peaks 146, 174,125,280, and 203. T h e mean recovery for each of these peaks in the in vitro pool was 88% or higher. In the actual case study for which this PCB method was developed, the serum samples were first analyzed for DDT, DDE, and DDD. As part of our quality assurance program, an in vivo pool was prepared from serum drawn from 11 participants in this study. Unknown to the analysts, one sample of this pool was used in the 31 analytical runs of 20 samples per run. The mean and standard deviation in parts per billion for o,p’-DDE, p,p’-DDE, o,p’-DDD, p,p’-DDD, o,p’-DDT, and p,p’-DDT were 17.7 and 1.6, 236 and 14.7, 1.9 and 0.1, 2.8 and 0.2, 7.3 and 0.5, and 12.0 and 1.0, respectively. This same pool, along with two bovine serum pools fortified with PCBs and DDT (and metabolites) and one bovine serum pool fortified only with DDT (and metabolites), were analyzed for PCBs in 23 analytical runs of 24 samples per run;precision data for these four pools, denoted as A, B, C, and D, respectively, are given in Table 11. Their gas chromatograms gave no indication of DDT or metabolites. This again points to the efficiency of this silver nitrate silica based adsorbent. In OUT laboratory, we have analyzed a large number of serum samples for PCBs, using 3% deactivated silica gel as the adsorbent (12),and, more recently, we have used silica gel prepared according to the method of Picer and Ahel (13). In experiments designed to compare the three column chromatographic procedures, we extracted 4-mL bovine serum samples and then concentrated these extracts. The concentrated extracts were fortified with Aroclor 1.254 and nine chlorinated hydrocarbons, which consisted of hexachlorobenzene, hexachlorocyclohexane, oxychlordane, heptachlorepoxide, transnonachlor, p,p’-DDE, dieldrin, o,p’-DDT, and p,p’-DDT. These chlorinated hydrocarbons were added to bovine serum at levels found in human serum of residents in the USA, and at 10 times those levels (14). By use of the Picer/Ahel approach, hexachlorobenzene, trans-nonachlor, and p,p’-DDE were eluted with the PCBs; however, only p,p’-DDE (24-4470
This article not subject to U.S. Copyright. Published 1980 by the American Chemical Society
2228
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
Table 11. Between-Run Estimates of Precision for PCB Analyses
no. of Lnalyses mean ( X i n ppb) std d e v ( S i n p p b )
RSD ( % )
pool
pool
A
B
23 43.9 3.2 7.3
23 11.2 0.9 8.0
I
53
pool C
pool
23 72.3 5.6
23 2.3 0.4 17.4
7.7
I
1
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165
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Flgure 1. GC chromatograms, electron capture detection, of 921 pg of Aroclor 1260 (solid line) and the following amounts (pg) of commonly occurring chlorinated hydrocarbons (broken lines): hexachlorobenzene (6); &hexachlorocyclohexane(24); oxychlordane (12); heptachlorepoxide (12); trans-nonachlor (15); p,p'-DDE (12); dieldrin (12); o,p'-DOT (36); and p,p'-DDT (37.5). Attenuation of 8.
coeluted) interfered with the PCB determination. The 3% deactivated silica gel allowed hexachlorobenzene and larger amounts of o,p'-DDT, p,p'-DDT, and p,p'-DDE (76-83%) to elute with the PCBs. The silver nitrate/silica gel column allowed hexachlorobenzene and a small amount of trans-nonachlor (4-1170)to elute with the PCBs; neither of these chlorinated hydrocarbons interfered with the determination of Aroclor 1254 or 1260 (Figure 1). We found that the DDT isomers were converted to the respective DDE, which can be eluted from the silver nitrate/silica gel column with 5% ethyl ether in hexane. The need for using this proposed method in the analysis for PCBs depends upon the Aroclor being analyzed, the degree of metabolism, and/or elimination of certain PCBs, and on the relative amounts of DDT and metabolites. Figure 2 shows a chromatogram of Aroclor 1260 superimposed on that of DDT and metabolites. Depending on the amounts of PCBs and DDTs, we have found o,p'-DDE to interfere with peak 84,
2229
Anal. Chem. 1980, 52, 2229-2230
The method described herein has been shown to yield high recoveries of PCBs, with no interference from chlorinated hydrocarbons normally found in human serum. We have examined many other proposed clean-up procedures for this analysis and have not found any that meet these criteria. More time may be required to prepare the column adsorbent by this procedure than by other procedures; however, we have found very good reproducibility among batches of our prepared silver nitrate on silica gel. We have routinely detected samples containing PCBs a t less than 5 ppb. ACKNOWLEDGMENT We thank Monsanto Chemical Company for supplying the Aroclor 1260, Lot AK3, and the U. S. Environmental Protection Agency for supplying standards of the chlorinated hydrocarbon insecticides. The technical assistance of Margaret Korver and Chester Lepeza is also acknowledged. LITERATURE CITED
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Figure 2. GC chromatograms, electron capture detection, of Aroclor 1260 (solid line) concentration equivalent to 3 0 p p b and o,p'-DDE, p,p'-ODE, o,p'-DDD, p,p'DDD, o,p'-DDT, and p,p'-DDT (broken lines) concentrations equivalent to 29,49,29,29,29, and 29 ppb, respectively. Attenutation of 8.
p,p'-DDE and o,p'-DDD with peaks 98, 104, and 117, p,p'DDD with peak 125, and p,p'-DDT with peaks 160 and 174. In our study of approximately 500 individuals in an Alabama community, the highest concentrations (in ppb) found in serum for o,p'-DDE, p,p'-DDE, o,p'-DDD, p,p'-DDD, o,p'DDT, and p,p'-DDT were 83, 2450, 26, 96, 61, and 102, respectively. -The highest PCB (as Aroclor 1260) observed was 155 ppb. Since other Aroclors, such as 1254,1248,1242, etc., contain PCBs that have similar GC retention times as DDT and metabolites, their determinations would also be subject to interference from DDT and metabolites.
"U.S. Food and Drug Administration Pesticide Analytical Manual"; Washington, DC, 1968, Vol. 1. Armour, J. A.; Burke, J. A. J. J . Assoc. Off. Anal. Chem. 1970, 5 3 , 761-768. Reynold, L. Bull. Environ. Contam. Toxicoi. 1080, 4 , 128-143. Snyder, D.; Reinert, R. Bull. Environ. Contam. Toxicoi. 1071, 6 , 385-390. Mulher, D. M.; Cromattie, E.; Reichel, W. I-.; Bellsle, A. A. J. Assoc. Off. Anal. Chem. 1970, 5 4 , 548-550. Young, S. V.; Burke, J. A. Bull. Environ. Contam. Toxicoi. 1972, 7 , 160-167. Armour, J. A. J. Assoc. Off. Anal. Chem. 1972, 56, 987-993. Lamparski. L. L.; Nestrick, T. J.: Stehl, R. H. Anal. Chem. 1079, 5 1 , 1453-1458. Szezepaniak, W.; Nawrocki, J.; Wasiak, IN. Chromatographis 1979, 12. 559-564. Webb, R. G.;McCall, A. C. J . Chromatogr. Sci. 1073, 1 7 , 366-373. Goldstein, J. A,; Hickman. P.; Bergman, ii.; McKinney, J. D.: Walker, M. P. Chem. Bioi. Interact. 1977, 17, 69-87. Burse, V. W.; Needham, L. L.; Liddle, J. A,; Bayse, D. D. Clin. Chem. (Winston-Salem, N.C.)1978, 2 4 . 1030. Picer, M.; Ahei, M. J . Chromatogr. 1978, 150, 119-127. Kutz, Frederick W., personal communication, U.S. Environmental Prctection Agency, Washington, DC, Dec '12, 1976.
RECEIVED for review April 21,1980. Accepted August 4,1980. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.
Chlorine Determination in Chlorine-Nitrogen Mixtures by Ultraviolet Spectrophotometry Raul G. Coltters Universidad Sim6n Bohar, Departamento de Ciencias de Materiales, Laboratorio ' I E " , Secci6n de Metalurgia Extractiva, P.O. Box 80659, Caracas 1080. Venezuela
A method has been developed for rapid and continuous measurement of chlorine concentration in chlorine-nitrogen mixtures. The technique resulted from a study on the kinetics of chlorination of tin-plated scrap as a source of tin for the manufacture of stannous chloride. When any of the proposed chlorination processes (1-4) are examined in detail, it is immediately apparent that the nature of the chlorination reaction is not understood. Clearly some progress might be possible in the development of chlorination technique if additional information on the behavior of individual reactants were available. Accordingly, in this study, the kinetics of chlorination with chlorine-nitrogen mixtures was studied by monitoring the chlorine concentration in the reactive mixtures, during its consumption due to the chemical reaction. Because the change of mixture chlorine concentration is rapid during the reaction, periodic sample collection and 0003-2700/80/0352-2229$01 .OO/O
subsequent gravimetric analysis were not convenient. The rapid method reported here takes advantage of the fact that in chlorine-nitrogen mixtures, chlorine is the UV absorbing species. This allows the direct and continuous determination of it with adequate precision. This is a useful technique with very practical applications. EXPERIMENTAL SECTION Apparatus. The cell assembly is schematically shown in Figure 1. It was made of a Beckman standard silica cell. Spectral scans were obtained with a Beckman UV recording spectrophotometer, Model 25K. Instrument calibration was checked by using holmium oxide glass and aqueous solutions of potassium dichromate and nitrate ( 5 ) . Reagents. Commercial nitrogen and chlorine were used throughout. Both gases were purified before being used. Nitrogen was passed through two Drierite columns to absorb moisture and 0 1980 American Chemical Society
