Environ. Sci. Technol. 1989, 23, 852-859 McCormick, N. G.; Feeherry, F. E.; Levinson, H. S. App. Environ. Microbiol. 1976, 31, 949. Meyer U. In Microbial Degradation of Xenobiotics and Recalcitrant Compounds. Leisinger, T.; Hutter, R., Cook, A. M., Nuesch, J., Eds.;Academic Press: London, 1981; p 371. Sjoblad, R. D.; Bollag, J.-M. In Soil Biochemistry; Paul, A., Ladd, J. N.; Eds.;Marcel Decker: New York, 1981; Vol. 5, p 113. Reber, H.; Helm, V.; Karanth, N. G. K. Eur. J. Appl. Microbiol. Biotechnol. 1979, 7, 181. Zeyer, J.; Kearney, P. C. Pestic. Biochem. Physiol. 1982, 17, 215. Zeyer, J.; Kearney, P. C. Pestic. Biochem. Physiol. 1982, 17, 224. Zeyer, J.; Wasserfallen, A.; Timmis, K. N. Appl. Environ. Microbiol. 1985, 50, 447. Chisaka, H.; Kearney, P. C. J. Agric. Food Chem. 1970,18, 854. Deuel, L. E.; Brown, K. W.; Turner, F. C.; Westfall, D. G.; Price, J. D. J. Environ. Qual. 1977, 6, 127. Corke, C. T.; Bunce, N. J.; Beaumont, A. L. Merrick, R. L. J. Agric. Food Chem. 1979,27,644. Horowitz, A.; Shelton, D. R.; Cornell, C. P.; Tiedje, J. M. Dev. Ind. Microbiol. 1982, 23,435. Beeman, R. E.; Suflita, J. M. Microb. Ecol. 1987, 14, 39. Dunlap, W. J.; Shew, D. C.; Robertson, J. M.; Toussaint, C. R. In Gas and leachate from landfills; formation, collection, and treatment. Proceedings, Research Symposium, Rutgers University, New Brunswick, NJ, March 1976; EPA-600/9-76-004. Linkfield, T. G.; Sutlita, J. M.; Tiedje, J. M. Characterization of the acclimation period prior to the anaerobic biodegradation of haloaromatic compounds. Submitted. Gibson, S. A.; Suflita, J. M. Appl Environ. Microbiol. 1986, 52, 681. Gibson, S. A.; Suflita, J. M. Abstracts, Annual Meeting,
American Society for Microbiology, Atlanta, GA, 1987; p 299. (40) Bollag, J.-M.; Russel, S. Microbiol. Ecol. 1976, 3, 65. (41) Mogilevich, N. F.; Tashirev, A. B.; Romanova, E. A. Mikrobiologiya 1987,56, 205. (42) Fathepure, B. 2.; Tiedje, J. M.; Boyd, S. A. Appl. Environ. Microbiol. 1988,54, 327. (43) Brown, J. F., Jr.; Wagner, R. E.; Feng, h.; Bedard, D. L.; Brennan, M. J.; Carnahan, J. C.; May, R. J. Environ. Toxicol. Chem. 1987, 6, 579. (44) Dolfing, J.; Tiedje, J. M. Arch. Microbiol. 1988,149, 102. (45) Morrison, R. T.; Boyd, R. N. Organic Chemistry, 3rd ed.; Allyn and Bacon, Inc.: Boston, MA, 1981. (46) Boyd, S. A.; Shelton, D. R. Appl Environ. Microbiol. 1984, 47, 272. (47) Attaway, H. H.; Camper, N. D.; Paynter, M. J. B. Pestic. Biochem. Physiol. 1982, 17, 96. (48) Stepp, T. D.; Camper, N. D.; Paynter, M. J. B. Pestic. Biochem. Physiol. 1985, 23, 256. (49) Wang, C. H.; Broadbent, F. E. J . Environ. Quality 1973, 2, 511. (50) Murthy, N. B. K.; Kaufman, D. D. J . Agric. Food Chem. 1978,26,1151. (51) Aschbacher, P. W.; Feil, V. J. J . Agric. Food Chem. 1983, 31, 1150. (52) Fogel, S.; Lancione, R. L.; Sewall, A. E. Appl. Environ. Microbiol. 1982, 44, 113. (53) Vogel, T. M.; Criddle, C. S.; McCarty, P. L. Environ. Sci. Technol. 1987,21, 722.
Received for review July 19,1988. Accepted February 15,1989. Although the research described in this article has been supported by the U.S. Environmental Protection Agency through assistance agreements No. CR-812808 and CR-813559 to the University of Oklahoma, it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred.
Complete Characterization of Polychlorinated Biphenyl Congeners in Commercial Aroclor and Clophen Mixtures by Multldimenslonal Gas Chromatography-Electron Capture Detection Detlef E. Schulz, Gert Petrlck, and Jan C. Dulnker'
Department of Marine Chemistry, Institute for Marine Research, University of Kiel, Duesternbrooker Weg 20, 2300 Kiel 1, Federal Republic of Germany On the basis of the relative retention times of all 209 possible chlorobiphenyls on a SE-54 column, all congeners present in two series of commercial formulations (Aroclors 1221,1016,1242,1254, and 1260; Clophens A30, A40, A50, and A60) have been identified and quantitated. Complete separation of congeners having identical retention times on SE-54 was obtained with a multidimensional gas chromatographic technique. A total of 132 congeners were found in the series of mixtures at concentrations above 0.05% (w/w), with each congener being present in at least one commercial mixture. For Aroclor and Clophen mixtures having similar overall chlorine contents, a striking qualitative similarity was evident, although quantitative differences were found. The data form the basis for unambiguous analyses of chlorinated biphenyls in environmental samples. A list of the CB congeners that elute as unambiguous single peaks from a SE-54 column is presented. Introduction
Polychlorinated biphenyls (PCBs) are ubiquitous contaminants of the environment (1) in which they occur as complex mixtures of many of the 209 theoretically possible 852
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congeners. The compositions of environmental mixtures differ among sample types; they also differ from those of the commercial products of differing degrees of overall chlorination, e.g., Aroclor 1254, Aroclor 1260, Clophen A50, etc, which are their only sources. Information on individual constituents is therefore essential for understanding the environmental behavior and effects of PCBs. Because of the wide range of (physico-) chemical properties of the various chlorobiphenyls (CBs), information on their distribution can also be used in models for predicting the behavior of other lipophilic organic compounds (2). Several previous attempts have been made to elucidate the composition of technical mixtures (3-11). These analyses are all far from complete for essentially two reasons. All 209 theoretically possible congeners were available as reference compounds in only one case in which the composition of Aroclor 1260 was described (11). In all other cases, the number of congeners available as reference compounds was much less. Moreover, in all cases, the chromatographic resolution between adjacent congeners has been inadequate for their accurate analysis by electron capture detection (ECD), flame ionization detection (FID), and/or mass spectrometry (MS). The relative retention
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Figure 1. ECD chromatograms of Aroclors 1016, 1242, 1254, and 1260 on a single SE-54 column. For chromatographic conditions and equipment 888 text and ref 13. Numbers [denmy the chromatographic domains, defined to include a series of peaks that are not bassllne separated. Corresponding domains In each of the Clophens (Figure 2) and Arociors have the same numbers.
Figure 2. ECD chromatograms of Clophens A30, A40, A50, and A60 on a single SE-54 column. For chromatographic conditlons and equipment see text and ref 13. Numbers klentlfy the chromatographic domains, defined to include peaks that are not base-line separated. Cortespondingdomains in each of the Aroclors (Figure 1) and Clophens have the same numbers.
times of all congeners on one particular column type (i.e., SE-54) have been published (12). In addition, multidimensional gas chromatography (MDGC), involving columns of different polarities arranged in series, has been shown to allow the separation of individual CBs having identical retention times on a SE-54 capillary column; for instance,all toxic congeners in Clophen mixtures have been determined by this technique (13). The present report describes a complete qualitative and quantitative analysis of Aroclor and Clophen technical mixtures by MDGCECD based on the use of pure standards.
and 2). Eighty-seven chromatographic domains were identified, each of which contained either one well-resolved peak or a cluster of unresolved peaks. Each domain is essentially base-line separated from neighboring domains (Figures 1and 2). Throughout the commercial mixtures, retention times are characteristic of each domain; only small variations occur for clusters of peaks, depending on the relative contributions of individual congeners. Each domain has been given a number (1-87) in order to facilitate comparison among commercial mixtures and permit a systematic description of the compositions of each domain in the various mixtures. In some cases, where peaks are nearly base-line separated, these have still been considered as one domain, e.g., peak no. 47 and 52. In our recent publication on the separation of the toxic congeners
Methods and Materials
ECD chromatograms of commercial mixtures were obtained with a 50-m SE-54 fused silica column (Figures 1
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with this technique, CB no. 114 was reported to be well separated (13). In the present paper we have applied more stringent criteria, resulting in an apparent discrepancy with the earlier paper. Information from previous GC-ECD and GC-?VIS analyses of Clophens with 102 individual congeners as reference compounds (8) has been most valuable. Also, the availability of the relative retention times (RRT) on SE-54 of all 209 congeners, the identity of which had been verified by mass spectrometric and nuclear magnetic resonance techniques (12),was essential for the identification of the potential constituents of each domain. The actual retention times on our SE-54 column were determined for each of these congeners (obtained either commercially or synthesized by us) and compared with the expected values (8). The retention times on SE-54 werr also the basis for the determination of optimum cut conditions (cut-on/cut-off times) for each chromatographic domain in the MDGC-ECD mode. For instance, for domain no. 14, candidate congeners were CB no. 20, 21, 33, and 53 and for domain no. 3 the only candidate was CB no. 6 (the numbering system was taken from ref 6). MDGC-ECD was applied to all domains including, for security reasons, those in which only one candidate congener exists. The MDGC-ECD technique involves the use of two columns of different polarities in series, each in a separate temperature-controlled oven. The eluate of the first column is carried either through the monitor ECD, producing the usual ECD chromatogram, or through the second column and the main ECD. The extreme usefulness of the technique, in both a qualitative and a quantitative sense, lies in the fact that a valveless pneumatic system involving a live T-piece allows a preselected small fraction to be cut from the eluate of the first column and to be transferred quantitatively and reproducibly to the second column (14). The chromatogram recorded by the main ECD shows only a few peaks reflecting the congeners included in that cut (Figure 3b,c) with the otherwise usual ECD chromatogram recorded by the monitor detector being interrupted during the cut time (Figure 3a). The identity of the congeners potentially included in the cut is known from the retention time on the SE-54 column. This allows them to be identified on the basis of retention time data on the two columns in series and to be quantitated by analyzing, under identical cut conditions, quantitative mixtures of congeners with the use of SE-54 as the first column and OV-210, or (3-87, as the second. Cuts were made for each domain in all Aroclors and Clophens and in synthetic mixtures of the corresponding individual congeners. For calibration purposes, all congeners were also injected as single compounds and analyzed under identical MDGC-ECD conditions. It appeared that, in all cases, baseline separation was achieved after elution from the second column. In most cases OV-210 was appropriate. However, for a few pairs of early-eluting congeners, a nonpolar C-87 column was required to achieve complete separation. Chromatographic conditions were as follows: multidimensional GC-gCD was carried out with a Siemens Sichromat-2 gas chromatograph equipped with two independent ovens and two "i electron capture detectors; first column, 25-m SE-54 (0.25 pm), 0.32 mm i.d. ICT, Frankfurt, FRG); second column, 30-m fused silica OV(0.25 pm), 0.32 mm i.d. (ICT); gas pressure (HJ, 1.1bar on the first column and 0.6 bar on the second column. Temperature-programming conditions: first column 140-250 "C at 4 "C min-' and second column until 20 min after injection at 160 "C and then temperature increase 854
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Figure 3. (a) Chromatogram of Arocior 1254 recorded by the monitoring ECD in the MDGC mode. First column, SE-54; second column, OV-210. For conditions, see text. Three consecutive cuts in a single chromatographic run invoking domains with no.32,46, and 54 (Flgure 1) are indicated in the chromatogram, as identified by cut 1, cut 2, and cut 3. (b) Chromatograms recorded by the main ECD in the MDGC mode, reflecting the compositions of the domains, cut from Aroclor 1254 (Figure 3a). Congener identification by numbers as in Table I. (c) Chromatograms recorded by the main ECD of standard solutions including ail possible congeners in the relevant domains, under the same cut conditions as in Figure 3b. These conditions exclude no. 93. Congener identification by numbers as in Table I.
to 230 "C at 4 "C m i d (OV-210) or second column until 20 min after injection at 190 OC and then temperature increase to 220 "C a t 4 "C min-' (C-87). Materials. The following criteria were applied for the selection of commercial mixtures. The Clophens A30, A40, A50, and A60 were selected because of our earlier analyses (8); we also selected from the series of Aroclors three mixtures with comparable overall chlorine contents (Aroclors 1242,1254,and 1260) and a fourth mixture (Aroclor 1016)having roughly the same chlorine content as Clophen A30 and Aroclor 1242 but with a reduced content of penta-, hexa-, and heptachlor0 congeners (15). Aroclor 1221 was included for analysis of the monochloro congeners. The
Aroclors were obtained from the Environmental Protection Agency: Aroclor 1016, CAS 12674-11-2; Aroclor 1221, CAS 111-04-28-2;Aroclor 1242, CAS 53496-21-9; Aroclor 1254, CAS 11097-69-1;Aroclor 1260, CAS 11096-82-5. Clophens were the same as in our earlier paper on the toxic congeners (13).We attempted to obtain individual congeners in the form of pure compounds. Many are gradually becoming commercially available in sufficiently pure form for reference purposes. We obtained them from Promochem (FRG) and CIL (GB), but several were still not available commercially. We therefore synthesized them according to ref 12. The reference materials were also checked by GC-FID. In some cases, several strong peaks were observed, even in the commercially obtained so-called “pure” standards. In such cases, these materials were rejected and replaced.
Results and Discussion The extreme sensitivity of the multidimensional GCECD method allows the accurate determination of trace constituents, even if they coelute with a major component from the first column (13). For instance, we detected congener no. 105 at a concentration level of 0.1% in the most chlorinated commercial mixtures containing 10% of the major congener no. 153. Congeners present at concentrations of 0.01% can still be identified and quantitated by MDGC-ECD even in the presence of other coeluting congeners. Since, in the ECD chromatogram obtained with a normal single SE-54 column any congener a t such low (0.01%) concentrations will give hardly any detectable signal, we have taken the 0.05%concentration in the commercial mixtures as the limit for the presence or absence of any individual congener. In total, 132 congeners were identified on this basis in the technical mixtures. This number includes the three monochloro congeners (CB no. 1-3) in Aroclor 1221. These three compounds, contributing as much as 50% to Aroclor 1221 (in agreement with ref 151, are well separated on a SE-54 column, appearing earlier than peak no. 1 in our figures. Those 129 congeners that were identified and quantitated in the commercial mixtures are listed in Table I by their IUPAC numbers (second column) and structures (third column). Concentrations of each congener in the mixtures are given in columns 4-7 for the Clophens and in columns 8-11 for the Aroclors. In the last column we have listed all congeners with concentration levels
