478
Anal. Chem. 1980, 60, 478-482
Multidimensional Gas Chromatography with Electron Capture Detection for the Determination of Toxic Congeners in Polychlorinated Biphenyl Mixtures Jan C. Duinker,* Detlef E. Schulz, and Gert Petrick Chemistry Department, Institute for Marine Research at the University of Kiel, Dusternbrooker Weg 20, 2300 Kiel, Federal Republic of Germany
A muitldhndonai gas chromatographic technique Is suggested as a tool for effective separation of polychlorinated biphenyl (PCB) congeners which cannot be separated on a single SE 54 or other column. Two capillary columns are artanged In series, such that the second column receives only mall preselected fractions eluting from the flrst column. The technlque offers complete separation and Increased sensttivAy. The possibilities of the technique are demonstrated for toxic PCB congeners which were quantitated accurately for the flrst time in ckphen and Arodor commerclai mlxtures and a seal blubber extract. The relatlve concentrations dlffered consMerably between blubber extract and the commercial mixtures.
We shall demonstrate below that this can be achieved conveniently by a technique using two capillary columns with different stationary phases arranged in series. The second column receives only well-defined, selected fractions of the eluate from the first column. Results for PCB analyses using this technique with flame ionisation detection have been published (IO). The power of the multidimensional GC technique in combination with electron capture detection is shown here for a series of congeners which are toxic, which have not yet been separated on a column of the most commonly used and efficient type (SE 54), and which have been receiving increasing interest in the literature because of their effect on organisms (11,12). It is therefore important to have a method for their accurate analysis in environmental samples.
Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminanta. They are present as complex mixtures of many of the 209 theoretically possible congeners. The introduction of high-resolution gas chromatographic techniques has largely improved the possibilities of analyzing these mixtures accurately. They allow the separation of many individual PCB congeners of interest and permit their determination as well-defined chemical entities rather than as ill-defined mixtures. This has resulted in detailed data for commercial mixtures and environmental samples (e.g., ref 1-3). The data in a recent publication on the chromatographic properties of all 209 congeners are extremely useful for peak identification ( 4 ) . At present not all congeners that occur in a commercial mixture or in an environmental sample can be separated into single peaks with the use of only one GC column. Specific separation problems can, at least in principle, be solved by using several columns with different stationary phases in parallel (5, 6). Attempts to resolve all congeners from neighboring compounds, however, would require parallel injections on many different columns. This would be timeconsuming and cumbersome, because elution patterns would be different on different columns and congeners that are separated on one column may coelute on another. Some problems associated with coeluting congeners have been solved by GC-MS techniques in the case of commercial mixtures (3, 7). The method is less accurate for congeners with low relative contribution to a peak; moreover, it cannot distinguish congeners with the same number of chlorine atoms. The possibilities offered by GC-MS techniques are even more limited in the case of environmental samples because other organic compounds that are present at much higher concentrations than PCB may obscure their signals. Successful application of the method has been reported in some cases, however (8, 9). An appropriate separation from neighboring compounds is a prerequisite for the accurate gas chromatography-electron capture detection (GC-ECD) analysis of any PCB congener.
EXPERIMENTAL SECTION Multidimensional GC was carried out with a Siemens Sichromat-2 gas chromatograph equipped with two independent ovens and two 63Nielectron capture detectors (the main and the monitor detector). Most of the eluate of the first column (which is located in oven 1)passes through the monitor detector. After the so-called live T-piece is activated, a selected part of the eluate can be diverted through the second column equipped with the main detector. The response of the monitor detector shows the usual ECD gas chromatogram of the sample, except for one or more interruptions, reflecting the cuts (Figures l b and 2b,e). The response of the main detector (Figures ICand 2c,d,f)reflects the separation achieved by the second column of only the few compounds which were cut from the eluate of the first column. Principles of the method and technical details of the equipment have been described elsewhere (13, 14). An essential aspect is the valveless, pneumatic control system to regulate the flow of sample molecules. They can only be transported to the monitor detector or to the second column, depending on pressure conditions in the live T-peice. In no way can they get lost, get trapped by dead volume, or escape from the system undetected by one of the ECD's. The system is optimized by regulating two gas flows in such a way that maximum and zero signals are obtained on the monitor and main detectors, respectively, when the flow is directed through the monitor detector (cut off) and maximum and zero signals are obtained on the main and monitor detector, respectively, when the cut is on. These conditions were fulfilled in our experiments (Figures lb,c and 2b,e). This is the basis for quantitative transfer of all components in a specific cut. We applied the technique with the following conditions: first column, 25-m fused silica SE 54 (0.25 pm), 0.32 mm i.d. (ICT, Frankfurt, West Germany); second column, 30-m fused silica OV-210 (0.25 1.1bar on the monitor pm), 0.32 mm i.d. (ICT); gas pressure (H2), detector and 0.6 bar on the main detector. Both ECDs were kept at 300 O C . The standard PTV injection system (programmable temperature evaporator) was modified to allow flash evaporation on-column injection. Temperature programming conditions were fist column from 140 to 250 "C at 4 "C min-' and second column until 20 min after injection at 160 O C and then temperature increase to 230 "C at 4 "C min-I. Aroclor 1221,1242,1248, 1254, and 1260 were obtained from Alltech Associates, Inc., (lot 6373) and Clophen A30, A40, A50, and A60 reference solutions were the same as in ref 3. PCB
0003-2700/88/0360-0478$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988 110
37 42
59
44 72
479
t1
Clophen A 4 0
52
149 118
42 44
4’
87
59
-
j’u cut
Figure 1. (a) Chromatogram of Clophen A40 on the first column (SE 54) as detected by the monitor ECD. (b) Detail of the chromatogram recorded with the monitor ECD. The peak contalning congeners 37, 42,59, and 72 coelutlng from the Rrst column has been cut from this eclateto passthrolrghtheseccndcoknm. (c)Chromatogram recorded with the main ECD, reflecting the composition of the peak cut in b. PCB congeners are numbered according to ref 2.
congeners were seleded on the basis of their toxicity. Quantitative structure-activity relationships indicate that isostereomers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are the most toxic onea exhibiting similar toxic properties (11,12). It is hypothesized that their toxicity is related to the presence of 4,4’ and/or 3,3’ 5,5’ chlorine substitution as well as to the possibility of the two benzene rings to attain a coplanar configuration when either one or both ortho positions are not substituted with chlorine. A total of 13 congeners satisfies this condition (Table I). The congeners were obtained from Promochem (West Germany) or Ultra-science (USA). Those unavailable were synthesized by us according to ref 4. A systematic numbering system (2) is used for congener identification. The purity of all compounds was at least 98%. Seal blubber originated from an animal found dead in the Dutch Wadden Sea (15).
RESULTS AND DISCUSSION The complete ECD chromatograms of Clophen A40 and A50, as detected by the monitor detector, are represented in Figures l a and 2a. Some peaks are labeled in terms of the contributing congeners in order to facilitate qualitative comparison between the chromatograms. More details on the identity of peaks eluting from the SE 54 column have been given before (3).The regionswhere chromatographic cuts have been made are given in more detail in Figures l b and 2b,e.
I 1
\cut 1 v
110
110 ~
\
77
I
cut 2 v
153
Figure 2. (a) Chromatogram of Clophen A50 on the first column (SE 54) as detected with the monitor ECD. (b and e) Details of the chromatograms recorded with the monitor ECD. The peaks labeled 110, 77, and 153, 132, 105, eluting from the first column, have been cut consecutively from this eluate to pass through the second column. (c and f) Chromatograms recorded with the ECD reflecting the compositions of the peaks cut In b and e. The position where congener 77 would have eluted if present is indicated by an arrow. (d) Signal of congeners 110 and 77 on the main ECD after cutting the corresponding peak from the first column after inJection of a synthetic mixture of these congeners. Numbering of PCB congeners is as in Figure 1.
The response of the main detector to these cuts is represented in Figures ICand 2c,f. In cases where a candidate congener (on the basis of the retention times of all 209 congeners ( 4 ) ) was not detected by the main detector, a mixture containing this congener was analyzed (Figure 2d); in the case of no. 77 in the seal sample, this congener was analyzed before and after standard addition of no. 77 and 110 to the sample (Figure 34. Standard solutions of individual congeners and synthetic mixtures were used to identify and quantitate peaks in the signal of the main detector. The more effective separation offered by the use of two columns in series in comparison with SE 54 only is summarized in Table I. It appears that con-
480
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988 132 105
Table I. Retention Data for Toxic PCB Congeners (*) and Closely Eluting Congeners on a SE 54 Column (Monitor Detector) and on the Main Detector (OV-210 Column) after Cutting the Appropriate Fraction from the SE 54 Columna
PCB n0.O
structureb
monitor detector tpc min min
min
tRve
AtR!
main detector min
37* 42 59
3,4,4’ 2,2’,3,4‘ 2,3,3’,6
18.23 18.22 18.22
0.01 0.01
26.26 25.44 25.30
0.82 0.96
17* 110
3,3’,4,4’ 2,3,3’,4’,6
23.24 23.27
0.03
31.56 30.79
0.77
81*
3,4,4‘,5
22.75
30.72
118* 123* 149
2,3’,4,4’,5 2’,3,4,4’,5 2,2’,3,4’,5’,6
24.54 24.45 24.44
32.21 32.00 31.56
114*
2,3,4,4‘,5
25.07
105* 132 153
2,3,3’,4,4’ 2,2’,3,3’,4,6’ 2,2’,4,4’,5,5’
25.83 25.72 25.68
0.11 0.15
33.72 33.14 32.96
0.58 0.76
126* 129 178
3,3’,4,4’,5 2,2’,3,3’,4,5 2,2’,3,3’,5’,6
26.81 26.80 26.85
0.01 0.04
37.98 37.13 36.49
0.85 1.49
167*
2,3‘,4,4’,5,5’
27.38
156* 171 202
2,3,3’,4,4’,5 2,2’,3,3’,4,4’,6 2,2’,3,3‘,5,5‘,6,6’
29.53 29.50 29.49
0.03 0.04
37.18 36.58 35.88
0.60 1.30
157* 173 200
2,3,3’,4,4’,5’ 2,2’,3,3’,4,5,6 2,2’,3,3’,4,5‘,6,6’
29.70 29.68 29.72
0.02 0.02
39.94 38.91 38.22
0.03 1.72
169*
3,3’,4,4’,5,5’
31.33
39.47
189*
2,3,3’,4,4’,5,5’
34.16
41.35
0.09 0.10
Seal blubber
U
standard addition
110
I
0.21 0.65
j
153
a
n
‘132
I
32.54
35.60
Systematic number of PCB congener (*: toxic congener). *Chlorinesubstitution pattern. Retention time (tR in minutes) on first column. dDifference in retention times on first column between closely eluting congeners. eRetention time on first and second column (main detector). f Difference in retention time upon elution from second column. geners that are separated on an SE 54 column by only 0.01-0.1 min elute from the second column with peak separations of 0.5-1 min. Thus, each of the presently discussed cluster of Congeners, poorly separated on the SE 54 column, can be completely separated into its constituents on the second column. With the present approach, concentrations of all congeners in the Clophen and Aroclor mixtures were determined. These are summarized in Table 11. The concentration of each congener differs between the commercial mixtures of different overall chlorine content in a systematic way for Clophen and Aroclor mixtures (Table 11). Because the candidate congeners which may appear in a narrow chromatographic range are known ( 4 ) and increased separation necessarily results in more reliable quantitation, the present results are more accurate than those obtained on congeners eluting as ill-separated clusters from a single (SE 54) column, mainly because the composition of the clusters thus remained uncertain. Although coeluting congeners with different chlorine numbers can be and have been distinguished by GC-MS techniques, their estimation is less accurate, especially for congeners with low relative abundance. GC-MS techniques cannot, however, distinguish coeluting congeners with the same chlorine numbers. The following example illustrates the potentialities of the present technique. For
Flgure 3. (a) Chromatogram of a seal blubber extract on the monitor ECD. (b) Chromatogram recorded with the main ECD, reflecting the cornpositlon of the peak labeled 110, 77 (in Figure 3a) from the eluate of the first column (u = unknown). (c) Chromatogram recorded with the main ECD, reflecting the composition of the peak labeled 110, 77 (In Figwe 3a),after standard addition of 110 and 77. (d) Chromatogram recorded with the main ECD, reflecting the composition of the peak labeled 105, 132, 153 (in Figure 3a). Numbering of PCB congeners is as in Figure 1.
instance, tetrachloro congeners no. 42, 59, and 72 have practically the same retention time on SE 54 as the toxic trichloro congener no. 37 (Table I and ref 4). The presence of trichloro- and tetrachlorocongeners in the corresponding peak in the commercial mixtures was established by GC-MS (3). No. 37 is the only trichloro candidate congener (4). Which one of the tetrachloro congeners is also present cannot be estabiished with the use of an SE 54 column alone. The peak had been thought to consist of 37 and 42 in ref 3 and 7. The peak, however, appears to consist of three contributors, i.e. 37,42, and 59. Injection of standard solution of no. 72 showed its absence in the commercial mixtures. The data on the other clusters of congeners that elute very closely spaced from an SE 54 column show interesting details. For instance, in contrast to earlier findings ( 3 ) ,we show the presence of no. 123 (identified as a toxic congener (11, 12)) in the cluster 149, 118, and 123 (4). The concentrations of the various congeners in the seal sample are included in Table 11. The identification and quantitation of toxic congeners in marine mammals are of particular ecological significance because of their posible role in the decreased reproduction success observed in some populations (16,17). Extremely high concentrations of PCBs have been reported for mammal tissues in the Western European Wadden Sea (15-17). Although the latter papers contain either explicit or implicit data on toxic congeners in these organisms, the analyses are based on assumptions as to which congeners contribute to unresolved GC-ECD peaks. It should be mentioned that the ECD chromatograms of mammal tissues from the Wadden Sea obtained on an SE 54 column are very similar to those of Clophen A60 or Aroclor 1260 (15). A striking result of the present data is the extremely low concentration level of most toxic congeners with high degree of chlorination relative to their contributions in Clophen A60 or Aroclor 1260, e.g. no. 118,156,169, and 189; the levels of no. 37, 81, 114, and 123 in seal blubber were below
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988
481
Table 11. Contribution of PCB Congeners in Clophen and Aroclor Mixtures (as Weight Percentages, %) and in Seal Blubber Extract (ne*& wet weight)#
in Aroclor
in Clophen A50 %,
A30 %
A40 %
37* 42 59
0.40 0.76 0.39
0.17 1.79 0.64
77* 110
0.39 0.42
0.66 2.91
81*
0.15
-
118* 1231 149
0.42 0.55 0.03
2.47 0.56 0.18
114*
0.06
0.13
-
1051 132 153
0.49 0.21 0.55
1.43 0.60 1.15
1.90 2.57 4.17
0.12 4.52 11.43
-
-
-
-
0.08 0.83 0.19
0.46 1.19 1.27
0.05
0.45
126* 129 178
-
A60 %
1221 %
1242 %
1248 %
0.05 0.13 0.02
0.30 0.92 0.38
0.32 1.93 0.30
0.13
-
-
-
6.27
2.15
0.40 0.43
0.50 1.70
0.30 1.25
-
-
-
-
-
1.74
0.45
-
-
1.80
8.57
0.92
-
-
-
12.60 0.93 5.50
-
1254 %
-
-
1260 %
-
in seal blubber npg-l wet weight
