Multidimensional Capillary Gas Chromatography of Polychlorinated Biphenyl Marker Compounds Russell M. Kinghorn and Philip J. Marriott* Department of Applied Chemistry, RMIT, GPO Box 2476V, Melbourne 3001, Australia

Mark Cumbers SGE International Pty. Ltd., 7 Argent Place, Ringwood 3 134, Australia

Key Words: Multidimensional capillary chromatography Polychlorinated biphenyls PCBs Congener

Summary

Multidimensional chromatography was used to resolve the specific chlorobiphenyl (CB) congeners 28,S2,101,118,138,153, and 180 in technical aroclor standards. Single column analysis of polychlori- nated biphenyls (PCBs) results in co-elution of key congeners with other components in the mixture; therefore using two columns of different selectivity was necessary to enhance the resolution achiev- able on just one column. The HT8 column (8% phenylpolycarbo- rane-siloxane phase) has been reported to have specific selectivity characteristics for improved PCB separation. When coupled with a BPXS column (5 % phenylpolysiloxane-silphenylene phase), it has been shown here to provide unambiguous identification of 7 marker compounds which are used to monitor PCB occurrence and distri- bution. A11 seven marker CBs are present in aroclor 1254, and by adjusting the size of the heartcut window, it was possible to obtain resolution of the marker congeners from other congeners. Single column analysis is unable to achieve this result. This offers an alternative to GC-MS analysis.

1 Introduction

The analysis of PCB concentration and distribution in the envi- ronment has, until recently, been expressed relative to technical aroclor and clophen standards. However, GC elution patterns of environmental samples seldom represent any particular technical standard, due to metabolic changes that take place during transi- tion from one animal species to another in the food chain or other selective environmental physicochemical effects. Therefore, specific CB congener analysis is becoming increasingly popular, with studies concentrating on specific congener movement and occurrence in the environment. This approach acknowledges that congeners have differing toxicities and abundances 11-51,

Out of the total 209 possible CB congeners, over 160 have been reported in technical standards and environmental samples [6]. Many regulatory bodies have chosen 7 CB congeners as marker compounds to monitor occurrence and distribution in the envi- ronment [7] (this list may be further expanded in the future). These are IUPAC [8] congener numbers 28 (2,4,4‘-trichloro- biphenyl), 52 (2,5,2’,5’-tetrachlorobiphenyl), 101 (2,4,5,2’,5’- pentachlorobiphenyl), 1 18 (2,4,5,3’,4‘-pentachlorobiphenyl), 1 38 (2,3,4,2’,4‘,5’-hexachlorobiphenyl), 153 (2,4,5,2’,4‘,5’-hex- achlorobiphenyl) and I80 (2,3,4,5,2’,4‘,5’-heptachlorobiphenyl). These particular congeners were chosen due to their high con- centrations in the environment and technical standards. At pre- sent, no single capillary column can adequately resolve the 7 marker congeners without co-elution from other CBs, therefore

the 7 marker congeners are regularly being reported as the sum of their own contribution plus any contribution of a co-eluting congener to each particular peak.

Commercially available columns that claim to have some CB selectivity include stationary phases such as 50% dioctyldi- methylsiloxane, 10% methyl-octadecylpolysiloxane, 15% diphenyl dimethylsiloxane and the work by Mullin et al. [9] who synthesized and determined relative retention times of all 209 CBs on a 5% diphenyl-1% vinyl dimethylsiloxane stationary phase (SE-54). This column has therefore been the column of choice in PCB analysis for some time. However over recent years, literature reports of retention data for all 209 CBs has increased, with for instance results from 25 different stationary phases listed in one international study 11 01. It was concluded that the DB-XLB (J&W Scientific, Folsom, USA) phase results in the least number of co-elutions when studying all 209 CBs; however, this phase does not necessarily give the optimum separation for the 7 marker CBs chosen to be monitored.

CBs are non-polar compounds, and therefore only weakly inter- act with the stationary phases indicated above [6], therefore separation will be largely based on volatility differences rather than structural effects. By introducing a highly polar group or a non-polar long chain aliphatic moiety in the stationary phase, the interaction between the CB and stationary phase is much stronger causing significant changes in the retention of some CBs. How- ever, such phases have alow chemical and thermal stability which limits their maximum operating temperatures. Stationary phases based on carborane-polydimethylsiloxane copolymers do pos- sess favourable interaction properties, and have high thermal stability. By introducing a carborane unit into the backbone of the stationary phase, the increased freedom of rotation observed for non ortho-substituted CBs, apparently allows the coplanar configuration of the CB to have a strong interaction with the carborane group [ 1 I]. The carborane-based polymer has been promoted as a new capillary column coating by SGEInternational (Ringwood, Australia), and developed specifically for PCB analysis.

This column, the HT8, has subsequently been identified by Larsen et al. 16, I I ] as one of the best PCB columns commercially available for either congener specific analysis or rapid aroclor screening. Other columns are also reported to give comparable CB resolution [4, 6, 11-13], but cannot provide unambiguous analysis of all 7 marker CBs without mass spectrometric (MS)

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detection. Larsen et al. [6,1 I ] used a S O m HT8 column with H2 carrier gas to resolve 6 of 7 marker CBs, and was able to distin- guish all 7 using MS detection. The same column also distin- guished all 36 of the McFarland and Clarke congeners [ 141 with MS detection, with the DB-XLB column reported to distinguish 31 of 36 congeners over a 2 h run time [ 151. These results are not achievable with ECD detection, therefore it is necessary to apply more powerful resolving techniques in order to fully resolve all 7 CBs when MS detection is not available. The more expensive GC-MS instrument is widely used for many environmental ap- plications including CB congener analysis [5,16-221, but cannot distinguish between congeners of the same chlorine number or those which differ by two chlorines, and therefore has limitations in CB analysis if the desired congener co-elutes with another indistinguishable congener. ECD is by far the most sensitive and popular detection system used for CB analysis [.5, 17, 20, 21, 23-27], and since i t does not provide mass discrimination, the effective resolution of each desired congener from other inter- fering congeners is still required.

With the use of a 25m x 0.22mm i.d. x 0.25 pm df HT8 column, 3 of the 7 congener marker compounds co-elute with other CBs. Multidimensional chromatography (MDC) is a technique that has the potential to resolve the 7 CBs without the need for MS detection, and to date, has been used with vaned success [5, 23, 28-34]. The use of a liquid crystal analytical column by de Boer and Dao [30] revealed impressive results for the resolution of the marker CBs. However with a maximum operating temperature of 250 "C and significant column bleed above 200 "C, along with a different temperature program for each marker congener, this approach is not practical. Sippola et al. [33] successfully resolved CBs 77, 8 1, 105, 126, and 169 using an ECD as the monitor detector and a MS analytical detector, while Schultz et al. [34] completely characterized CB congeners in commercial Aroclor and Clophen standards.

In this work, two capillary columns are chosen for their combined ability to resolve the seven marker CBs from technical aroclor standards. Using the highly selective HT8 phase, and another phase of different selectivity (BPXS, 5% phenylpolysiloxane-sil- phenylene phase), co-eluting CBs on one column can be heartcut to the analytical column in order to resolve the congener marker compounds. This paper will outline the approach used to achieve this.

2 Experimental

2.1 Gas Chromatography Equipment and Columns

A Shimadzu model GC- 17AAF gas chromatograph (Shimadzu, Kyoto, Japan) was used for all analyses, fitted with an AOC- 17 autoinjector and ECD (63Ni, 370MBq) detector. Shimadzu ClassGC software was used for instrument control and data ac- quisition via the Shimadzu CBM- 101 Communications Bus Module. Capillary columns used were supplied by SGE Interna- tional (SGE International Pty. Ltd, Ringwood, Australia) and included BPX5 (5% phenylpolysiloxane-silphenylene phase), BPX35 (35% phenylpolysiloxane-silphenylene phase) and HT8 (8% phenylpolycarborane-siloxane phase), all of dimensions 25 ni x 0.22 mm i.d., with a 0.25 pm film thickness. All gases used were of high purity (99.99%), and an oxytrap was used in the carrier gas line. All chromatograms reproduced were con-

verted into ASCII and imported into the Origin v4.0 scientific and technical graphics program.

For relative retention studies, helium was used as the carrier gas at 20 psi head pressure; the injector and detector temperatures were both 330 "C with a split (30:l) injection mode and the make up gas to the detector was 5% CH4 in Argon at 24mL/min, with range at 10 and current 1mA. The column temperature was maintained at 70' C for 1 min, programmed at 20 "/min to 140 "C, then programmed at 3 "/min to 296 "C and held for 3.5 min (total run time 60 min).

0

2.2 Multidimensional Analysis

The Shimadzu model GC- 17AAF gas chromatograph was fitted with a SGE MDS-2000 multidimensional system. The Commu- nications Bus Module CBM-101 was modified to acquire from two channels, FID being the monitor detector and ECD the analytical detector. A SGE BPX5 capillary column was used for the pre-column, and a HT8 for the analytical column. Column dimensions were identical to that above. Helium was used as the carrier gas at 28.5 psi pre-column pressure and 19.Opsi mid-point restrictor pressure. Injector and detector temperature were as above, with make up flows of 25 mL/min and 30 mLlmin N2 for the FID and ECD respectively. The ECD was operated at range 10' and current 0.05 mA. The column temperature was main- tained at 70' C for 1 min, programmed at 20 "/min to 140 "C, then programmed at 3 "/min to 296 "C. After 3.5 min, the column was cooled to 70 "C at -20 "/min, held for 3.7 min then pro- grammed at 20 "/min to 140 "C, and 3 "/min to 296 "C and held for a further 3.5 min (total run time 134 min). Cryogenic cooling was used to focus heartcut fractions at the head of the analytical column; the liquid C02 (technical grade) cryogenic coolant flow was turned off at 74.0 min, prior to elution of the analytical column.

2.3 Chemical Standards

Individual CB standards were provided by SGE International as 30 vials each containing 6 or 7 congeners, and in total included all 209 congeners at 0.35 mg/L in iso-octane. Each standard contained congener 209 as an internal reference. Technical aro- clor standards 1242, 1254, and 1260 present at 100 mgfL in n-hexane were purchased from Polyscience (Polyscience, Illi- nois, USA) and were used as received.

3 Results and Discussion

All 209 possible CB congeners were chromatographed on the stationary phase types BPX5, BPX35, and HT8. In this study, columns of identical dimensions were used so that the stationary phase type was the only variable in the retention time studies on all 209 CBs. A compilation of retention times, peak widths and peak resolutions was tabulated, along with resolutions between the target marker congeners and those peaks neighboring each marker. The study protocol involved single GC injections; the t~ of CB209, together with a reference mix injected 6 times peri- odically throughout the whole analysis, was used to monitor reproducibility. The complete analysis (54 h) was carried out continuously for each column studied, in order to maintain analy- sis condition reproducibility. Typical reproducibility of retention data for CB209 on the BPXS column were: average t~ = 47.10min,s.d.=0.011 minandr.s.d.=0.02%.

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MDGC of Polychlorinated Biphenyl Marker Compounds

Table 1 presents a sample of the summary data used to evaluate different column combinations for their ability to resolve marker congeners from other congeners. Table 1 shows that there are two peaks of concern neighboring CB52 on the BPXS column, namely CBs 43 and 48. Whilst there may be other congeners which co-elute or overlap with CB52 on the HT8 column, they do not need to be considered in this table since they do not co-elute with CB52 on the BPX5 pre-column, nor do they exist in aroclors 1242, 1254 or 1260, so will not be heartcut to the HT8 during MDC operation. From the 6 possible column combinations and carrying out the full interpretation as above [36], it was deter- mined that using a BPXS as the monitor column and a HT8 as the analytical column in the multidimensional mode would result in the best possible resolution of the 7 marker CBs from interfer- ing congeners.

Table 1. Predicted resolution data for the BPXS pre-column and HT8 analytical column for the 7 marker congeners.

Target CB Nearby CB

28 31 52 43

48 101 113

99 84

118 134 138 IS8

160 163 164

153 132 I05

1 so 191

of different aroclors to be deduced. Aroclor 1242 contains four of the marker congeners, CB28, CB52, CB 101, and CB 1 18, with the first 3 co-eluting with another CB on the HT8 phase. In the multidimensional mode, all 4 marker CBs have been completely resolved, with the analytical channel response shown in Figure 1. (Note that in Figures 1 and 3 the reported retention time consists of the total time arising from column 1 elution +cooling + column 2 elution.) Since CB 11 8 is already adequately resolved on the first column, it may be analyzed on the monitor channel, so need not be heartcut on to the analytical column. However if the analytical channel is to be used for quantitation, CB 11 8 can be readily included in the heartcut process.

BPXS HT8

0.06 R 0.3 1 I .20 1.41 R 0.18 1.01 1.23 R I .26 R 1.05 R 0.32 0.93 0.33 0.44 0.59 0 6 7 0.70 0.74 0.48 R I .41 R 1.26 R

R denotes resolution greater than 1.5 Note: Only those nedrhy CBE with Rs les\ than I .5 and that are present in either aroclor 1242, 1254, or 1260 are included in the BPXS data column. The table shows, for example, that CB31 is not well resolved from CB2X on the BPXS column (R, = 0.06) but is resolved on the HT8 column when the composite CB28/3 I peak is heancut to the HT8 column.

Using the BPX5 as the pre-column, a total of 26 CBs have a resolution less than 1.5 with the seven marker compounds when all 209 CBs are considered. Assuming that these peaks are selec- tively heartcut onto the HT8 analytical column, and considering only the congeners that exist in the aroclors under study, only 5 of these result in a resolution less than 1.5, as shown in Table 1. On the other hand, a combination of BPX35 and HT8 in a similar comparison gives 32 CBs co-eluting with the marker CBs on BPX35, which is reduced to 9 when selectively heartcut to HT8. This result shows the BPXS and HT8 combination as the better choice to resolve the 7 marker congeners.

Evaluating the multidimensional system with technical aroclor standards enables a worst possible scenario for congener separa- tion to be investigated as CBs not present in such mixtures are seldom encountered in environmental samples. In these studies, the marker CBs were analyzed separately, and their retentions on the analytical column determined after the heartcut process. This confirmed the retention times of each marker CB and al- lowed their identification in the aroclor mixtures. This work and literature reviews [e .g . 6,28,30, 34, 351 allows the composition

28

31 I

I I 1

5 99 102 105

Retention T ime (min) Figure 1. Analytical channel response for heartcut fractions from aroclor 1242 analysed on the HT8 column after the BPXS pre-column. Three heartcut events are used. For GC conditions refer to Section 2.2.

Aroclor 1254 contains all 7 marker congeners, with CBs 28 and 180 present at low abundances. On the HT8 phase, CB52, CB101, and CB153 co-elute with CB69, CB150, and CB122 respectively, but these interfering congeners are well separated on BPXS so are excluded by the heartcut window. CB138 and CB 180 are only partially resolved from neighboring congeners. Figure 2 illustrates the six heartcuts required, excluding CB 1 18 which, as above, is adequately resolved on the BPXS phase. As shown, an FID monitor detector gives adequate sensitivity in this system since relatively high concentration aroclor standards were used for evaluation. If environmental samples were to be analysed, a twin ECD system would be needed to achieve the necessary sensitivity.

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MDGC of Polychlorinated Biphenyl Marker Compounds

150 I i 8 1601138

I

CUTS 1 and 2 CUT 3 CUTS 4 , 5 AND 6 Retention Time

ECD Analytical Channel Responses Figure 2. Monitor (FID) and analytical (ECD) channel responses for PCB congeners in aroclor 1254 showing selection of the six heartcut events. Condi- tions as in Figure I .

Table 2 lists the resolution obtainable for analysis of aroclor 12.54 from the multidimensional system compared to that of the single 2.5m HT8 column. Six of the seven marker CBs are resolved, with CB138 showing no improvement in resolution. This par- ticular congener has been receiving increasing attention due to the difficulty in resolving it from closely eluting congeners CB160,CB163, andCB164, andnearbycongenerCB1.58.Larsen et al. 161 reported almost baseline resolution of CB138 using a S0m HT8 column and H2 as the carrier gas. The reason for MDC being unable to improve resolution for CB138 is that both the BPXS and HT8 column do not give adequate separation of CB I 38 from CB160, CB163, CB164, and CB1.58. Thus the heartcut event is not able to successfully exclude these interfering conge- ners from being transferred to the HT8 analytical column without sacrificing or excluding some of the CB 138 marker under inves- tigation. Figure 2 demonstrates that CB163 and CB164 can be removed from the heartcut window, however total transfer and resolution of CB 138 is not achieved due to the interfering con- geners CB160 and CB158. In Frame’s recent study [3.5] CB138 was unresolved from CB 163 and CB 164 on a 30 m DB 1 column, and so the peak was reported as the total from all contributing congeners; also CB 1.58 was reported as a separate peak, however its resolution from the CB138 peak appears to be about 0.9. The solution to improve resolution in the MDC experiment in the present case will be to use a longer (higher efficiency) analytical column, however it is more difficult to balance the pressure

switching system of the MDS-2000 if columns of significantly different dimensions are utilized. Interestingly, on the BPXS column CB132 elutes after CB1.53, but on the HT8 column CB 132 elutes before CB 1.53, thus exhibiting the different selec- tivities of these two columns.

Table 2. Resolution comparison for aroclor 1254

Marker CB Resolution on single MDC Resolution BPXYHT8 HT8 column

28

52

101

118

153

138

180

resolved resolved

co-elutes resolved

co-elutes resolved

resolved resolved

co-elutes resolved

0.80 0.80

1.2 resolved

Aroclor 1260 contains five of the marker congeners, with CB 10 1, CB138, CB1.53, CB180 all present at high concentrations, and a low abundance of CB 1 18. The multidimensional system success- fully resolved all the marker congeners except CB 138, which again proved to be difficult. GC-MS cannot be utilized to distin- guish CB 138 as the4 co-eluting CBs are all hexachlorobiphenyls. A chromatogram of the heartcut fractions is shown in Figure 3.

a, v) c 0 n v) a, E

153 I 138

179 180

105 110 115 120

Retention time (min)

Figure 3. Analytical channel response for heartcut fractions from aroclor 1260, with five heartcut events. CB118 has now been included in the heartcuts. Conditions as in Figure 1.

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4 Conclusion

MDC with a column combination of BPXS and HT8 offers a route to routine specific congener analysis of complex PCB samples, providing resolution not possible with traditional single column chromatographic techniques. In addition, it enables con- gener analysis through chromatographic peak separation rather than methods requiring additional mass selectivity, such as single column GC-MS analysis. The analysis time (134 min per run) is the only drawback in this multidimensional analysis in the pro- cedure reported. An alternative approach to reducing the overall time to under 60 min is currently being addressed [36] and will be the subject of a further communication.

Acknowledgments The authors wish to acknowledge provision of the GC-17A instrument by Shimadzu Oceania, and SGE International for the MDS-2000 unit. Mr Paul Momson (RMIT) is thanked for his technical support.

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Ms received: February I , 1996; accepted: September 24, 1996

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