1300
Anal. Chem. 1989, 67, 1300-1302
the mixtures does not occur under normal conditions. (4) The materials that cause the mixtures to deactivate are not only those adsorbed on the bottles, but the constituents of the bottles themselves. When one is using a new vessel, its influence on decomposition rates must be checked before use. LITERATURE CITED (1) Imai, K.; Weinberger, R. TrAC, Trends Anal. Chem. (Pers, Ed.) 1985,
4, 170-175. (2) Honda, K.; Sekino, J.; Imai, K. Anal. Chem. 1983, 55,940-943. (3) Miyaguchi, K.; Honda. K.; Imai, K. J. Chromafogr. 1984, 316,
Iannotta, A. V.: Semsel, A. M.; Clarke, R. A. J , Am. Chem. SOC. 1967,89, 6515-6522. (6) Sigvardson. K. W.; Birks, J. W. Anal. Chem. 1983, 55,432-435. (7) Bansal, N. P.; Doremus, R. H. Handbook of Glass Properties; Academic Press: New York, 1986; Table 3.1. (8) McLellan, G. W.; Shand, E. B. Glass EngineeringHandbook; McGrawHill: New York, 1984; Chapter 3.
Nobuaki Hanaoka Shirnadzu-Kansas Research Laboratory 2095 Constant Avenue Kansas 66046
501-505.
(4) Hanaoka, N.; Givens, R. S.; Schowen, R. L.; Kuwana. T. Anal. Chem. 1988, 60, 2193-2197. (5) Rauhut, M. M.; Bollyky, L. J.; Roberts, B. G.; Loy, M.; Whitman, R . H.;
RECEIVED for review November 14, 1988. Accepted March 10, 1989.
TECHNICAL NOTES Fractionation of Polychlorinated Biphenyls, Polychlorinated Dibenzo-p-dioxins, and Polychlorinated Dibenzofurans on Porous Graphitic Carbon’ Colin S. Creaser* and Ameera Al-Haddad School of Chemical Sciences, University of E a s t Anglia, Norwich NR4 7TJ. U.K Activated carbon (1,2) and Carbopack (3)have been widely used for the separation of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) from polychlorinated biphenyls (PCBs) and other halogenated aromatic compounds. Fractionation of halogenated aromatic compounds on these two types of carbon requires the sample t o be eluted with complex solvent mixtures. Environmental and biological samples cleaned by such carbons typically require elution with cyclohexane/dichloromethane t o elute ortho-chlorine-substitutedPCBs and halogenated pesticides. Non-ortho-chlorine-containing PCBs are then eluted with benzene in ethyl acetate and finally the strongly retained PCDDs and PCDFs are recovered by back flushing the column with toluene. Although many environmental and biological samples have been successfully cleaned u p by activated and Carbopack carbons, the cleanup procedure is lengthy and the inhomogeneity of the active sites on activated carbon results in broad and tailing solute elution profiles. Porous graphitic carbon (PGC) is a novel chromatographic material ( 4 ) consisting of porous carbon spheres whose size can, in principle, be chosen from a few micrometers to a few hundred micrometers. It has a surface area of about 150 m2/g, a mean pore volume of 2.0 cm3/g, and a particle porosity of 70%. PGC is the only carbon that can be used as a packing material for HPLC, because of its strength and ability to withstand the high-pressure gradients used in HPLC and in HPLC slurry packing procedures. The efficiency of PGC is comparable t o that obtained with bonded phase silica gel for many compounds such as methylbenzenes, phenols, ethers, monosubstituted benzenes, amines, and acids ( 4 ) . In this paper, we report the fractionation of chlorinated aromatic compounds including PCBs, PCDFs, PCDDs, and pesticides using porous graphitic carbon. A back-flushing procedure has been used for the cleanup of soil samples for the determination of PCDDs and PCDFs. EXPERIMENTAL SECTION Porous Graphitic Carbon HPLC Column. A 4.7 X 50 mm
This paper is dedicated to the memory of Dr. Roger Beale Homer (1940-1988). 0003-2700/89/0361-1300$01.50/0
porous graphitic carbon column (Hypercarb, Shandon Scientific, Ltd., Cheshire WA7 lPR, England, 7-pm particle size) was used for the fractionation of halogenated aromatic compounds and for the cleanup of soil extracts for the determination of PCDDs and PCDFs. The HPLC equipment consisted of a Waters Model 501 solvent delivery system, Waters Model 455 UV variable wavelength detector, and a Rheodyne Model 7125 syringe loading sample injector with a 100- or 20-pL sample loop. The PGC column was fitted with a 7040 Rheodyne switching valve to enable back flushing of the column. Pesticide grade (BDH) hexane was used as the eluting solvent. The flow rate was 5 mL/min with a back pressure of 1500-2000 psi. The porous graphitic carbon column was conditioned by eluting with about 100 mL of hexane. A mixture of Aroclors (1254 and 1260) in hexane was spiked with a range of PCDDs, PCDFs, pesticides (Figure l),and nonortho PCBs a t 1 yg/g prior to chromatography on PGC. The pesticides tested were parathion, malathion, heptachlor, aldrin, dieldrin, heptaclor epoxide, @-benzenehexachloride (@-BHC), a-benzene hexachloride ((Y BHC), lindane, l,l,l-trichloro-2,2bis(4-chloropheny1)ethane (p,p’-DDT). l,l,l-trichloro-2-(2chlorophenyl)-2-(4-chlorophenyl)ethane (o,p’-DDT), 1,l-dichloro-2,2-bis(4-chlorophenyl)ethylene(p,p’-DDE), 1,l-dichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethylene (o,p’-DDE), 1,l-dichloro-2,2-bis(4-chlorophenyl)ethane ( p , p’-DDD), and 1,ldichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane (o,p’-DDD). The column was eluted first with 100 mL of hexane (PCBs and pesticides fraction), then back flushed with a further 200 mL of the same solvent (PCDDs and PCDFs fraction). The PCBs and pesticides were detected by a UV detection at 245 nm, while the PCDDs and PCDFs were determined by collection of fractions and analysis by gas chromatography-electron capture detection (GC-ECD). PCDD and PCDF recoveries for the PGC cleanup were determined for a selected range of isomers (100 ng each), by comparing the ECD response before and after the PGC fractionation. Commercial PCB mixtures (Aroclor 1242,1254, and 1260)were chromatographed using 8020 (v/v) acetonitrile/water as an eluent, at a flow rate of 1 mL/min and a pressure of ca. 2000 psi. Collection and Cleanup of Soils. Soil samples were collected to a depth of 5 cm, air-dried, and sieved through a 2-mm mesh. The samples (250 g) were spiked with [13Clz]2,3,7,8-TCDDand [13C12]1,2,3,4,6,7,8-H7CDDand then Soxhlet extracted with hexane/acetone (41:59) for 8 h ( 5 ) . The extracted organic phase was applied to a multilayer column (420 mm X 25 mm i.d.1 packed 0 1989 American Chemical Society
ANALYTICAL CHEMISTRY,
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33 18
16 36
1301
50 00
time lmin i Volume of
hexane l m l i
Figure 1. Separation of PCBs and pesticides from PCDDs and PCDFs on porous graphitic carbon (50 X 4.7 mm; eluent, hexane, 5 mL/min). (a) PCBs (Aroclor 1254 + 1260) and pesticides; (b) 3,3',5,5'-TCB and 3,4',5-TCB; (c) 3,3',4,4'-TCB and 3,4,4',5-TCB; (d) 3,3',4,4',5,5'-H&B; (e) 1,2,3,4,6,7,8-H7CDD and Oⅅ (f) 1,2,3,7,8-P5CDD, 1,2,3,7,8P,CDF, 1,3,6,8/ 1,3,7,9-T,CDD, 2,3,7,8-T4CDD, 2,3,7,8-T4CDF, 1,3,7,8-T4CDD, and 1,2,7,8-T4CDD; (9) 1,2,3,4,7,8-H6CDD; (h) 1,2,3,6,7,8-/ 1 ,2,3,7,8,9-H6CDD and 1 ,2,3,4,8,9-H6CDD; (i) 1,2,3,4,6,7,8-H7CDD, and (j)O&DF (0-100 mL UV detection, 245 nm; 100-300 mL GC-ECD analysis of discrete fractions).
from the top with the following: anhydrous sodium sulfate; sulfuric acid on silica (l:l, w/w), sodium bicarbonate/anhydrous sodium sulfate (1:9 w/w); silica; anhydrous sodium sulfate. The eluate from the multilayer column was concentrated and applied to a Florisil column (150 mm X 4 mm i.d.) activated at 130 "C. The column was eluted with 7 mL of hexane followed by 40 mL of 2% dichloromethane in hexane and 40 mL of dichloromethane. Dodecane (20 pL) was added to the PCDD/PCDF fraction and the dichloromethane was removed with a stream of dry nitrogen (5). The concentrated extract in dodecane was diluted to 80 pL with hexane and injected into the PGC column by use of a 100-pL sample loop. The column was eluted with 100 mL of hexane, then back flushed with 200 mL of the same solvent. The 200-mL fraction was reconcentrated after the addition of 20 pL of dodecane and analyzed for PCDDs and PCDFs by gas chromatographymass spectrometry (GC-MS). Gas chromatography-mass spectrometry analysis was carried out on a Kratos MS25 spectrometer interfaced to a Sigma 3B gas chromatograph or a Hewlett-Packard 5970B spectrometer interfaced to a Hewlett-Packard 5890A gas chromatograph. The hexa-, hepta-, and octa-PCDDs and -PCDFs were chromatographed on a 50 m X 0.2 mm i.d. BP-5 column, while the tetraand pentadioxin and -furan isomers were separated using a multidimensional GC-GC-MS procedure on a 5 m x 0.2 mm i.d. BP-5 (film thickness 0.25 pm) column connected to a 50 m X 0.2 mm i.d. CP-Si1 88 (film thickness 0.2 pm) column (5).
RESULTS AND DISCUSSION The use of activated carbon and Carbopack in the separation of PCDDs and PCDFs from PCBs and other halogenated compounds has been reported by several workers (1-3). This separation method, although efficient, requires the use of several solvents. In addition, since activated carbon is characterized by a broad spectrum of adsorptive sites, solute elution profiles are usually broad and tailing, and batch to batch differences have been observed. An efficient fractionation of several classes of halogenated aromatic compounds is possible by HPLC on porous graphitic carbon eluted with hexane. Figure 1shows the separation of pesticides, PCBs, PCDDs, and PCDFs on this stationary phase using hexane as a solvent. Pesticides and ortho- (2,2',6 or 6')-substituted PCBs elute as a sharp peak in the first 10 mL of the eluting solvent. An improved separation of the PCB isomers on porous graphitic carbon can be achieved by elution with acetonitrile/water 80:20 (v/v) (Figure 2). The reverse-phase separation of PCBs on this column is similar to
Figure 2. Elution of PCBs (mixture of Aroclor 1242, 1254 and 1260) on PGC (column, 50 X 4.7 mm, 7 pm; eluent, acetonitriidwater 80/20 (v/v); 1 mL/min).
that normally obtained on an ODS column (4.6 X 250 mm) except that the retention times of PCBs on the latter are much shorter than those on the PGC column. Non-ortho-substituted PCBs have larger retention volumes on PGC than the ortho-substituted isomers, requiring u p to 100 mL of hexane for the higher chlorinated isomers to elute (Figure 1). This greater retention is attributed to the ability of the non-ortho-substituted PCBs to assume a coplanar configuration more readily than the ortho-substituted isomers, allowing a stronger interaction with the planar PGC surface ( 4 ) . In addition to coplanarity, the selectivity of PGC, is governed by the number of electronegative substituents on the biphenyl skeleton, as is the case for activated carbon (2). Hexachlorobiphenyls thus have retention times longer than those of penta- and tetrachlorobiphenyls. The need for a simple analytical method for the isolation of non-ortho PCBs in Aroclor mixtures stems from evidence that a large part of Aroclor toxicity is associated with the small amounts of non-ortho-chlorine-substitutedbiphenyls present in the Aroclor mixtures. It has been reported (6)that exposure of mice and guinea pigs to non-ortho-chlorine-substituted 3,4,3',4'-tetrachlorobiphenyl (TCB) and 3,4,5,3',4',5'-hexachlorobiphenyl (HCB) resulted in an intoxication syndrome similar to that produced by the tetrachlorodibenzo-p-dioxins (TCDDs). The ability of porous graphitic carbon to separate chlorinated aromatics on the basis of molecular planarity provides a highly efficient and straightforward method for the direct isolation of non-ortho-chlorine substituted PCBs from Aroclor mixtures. A detailed assessment of the recoveries of PCBs and pesticides and the applicability of PGC for the determination of these analytes in real sample matrices are currently under investigation. A further advantage of porous graphitic carbon over activated carbon (PX-21) (2) is the use of a single eluting solvent. A stepwise gradient of cyclohexane/dichloromethane, benzene in ethyl acetate, and toluene is typically used to fractionate halogenated aromatic compounds on activated carbon, while hexane alone can be used with PGC to obtain a similar separation. The PCB peaks obtained with the PGC column are sharper than those obtained with the activated carbon, because of the homogeneous nature of the active sites of PGC compared with those of activated carbon. Porous graphitic carbon has a strong affinity for PCDDs and PCDFs because of their coplanar structures. Even strong solvents such as hexane and toluene failed to elute these compounds from the column with reasonable retention volumes. However, by back flushing the PGC column with 200 mL of hexane, these compounds elute in a broad peak with little discrimination between PCDDs and PCDFs or different degrees of chlorination (Figure 1). Smaller amounts of toluene can be used instead of hexane to back flush the column.
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ANALYTICAL CHEMISTRY, VOL.
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Table I. Recovery Data for Some PCDDs and PCDFs PCDDs/PCDFs isomers 2,3,7,8-TCDD 1,3,6,8-/1,3,7,9-TCDD 1,3,6,8-TCDD 1,2,7,8-TCDD 2,3,7,8-TCDF
av 90 recovery
coeff of var (100%)'
54 56 64 58 53 63 58
3.8 5.2
1,2,3,6,7,8-/1,2,3,7,8-H&DD 1,2,3,4,8,9-H&DD 1,2,3,4,7,8-H&DF 1,2,3,4,6,7,8-H7CDD 1,2,3,4,6,7,8-H7CDF
85 81 86 76 76
5.7 6.2 3.9 4.2 4.7
1,2,3,4,6,7,8,9-OCDD 1,2,3,4,6,7,8,9-OCDF
74 71
5.7 5.2
For five redicate samdes.
-
concn
determined
4.2 3.9 4.9 3.5 3.8
1,2,3,7,8-PCDD 1,2,3,7,8-PCDF
I15 L , 01
Table 11. Comparison of PCDD and PCDF Concentrations before and after PGC Cleanup
PCDDs/PCDFs
congeners" TCDD PCDD H&DD H7CDD OCDD H&DF H&DF OCDF
before PGC cleanuD. ng kg"'
concn after PGC cleanup, ng kg-I
NQb ND' 26 49 183
11
18 11
ND' 30 48 157 30 14
14
11
"Totals for each congener groups. bNot quantified due to interferences. cNot detected (detection limit 1 ng kg-').
elution windows for these species, although in all cases the quality of the mass chromatograms was improved. In the case of the TCDDs, the PGC has removed major interferences within the elution window for these isomers, allowing the quantitative determination of the TCDDs present (Table 11). This quantitation was not possible without chromatography on PGC. Similarly, for the H6CDFs, a reassessment of the concentration was possible because of improvements in the quality of the corresponding mass chromatogram. The PGC column was back flushed with 300 mL of hexane between samples. Blanks analyzed by GC-MS after the flushing procedure showed no sign of cross-contamination and the retention volumes of PCDDs and PCDFs did not vary with time even after the cleanup of many soil extracts. The potential of porous graphitic carbon as an HPLC adsorbent for the isolation of halogenated aromatic compounds has been demonstrated by the fractionation of PCBs, chlorinated pesticides, PCDDs, and PCDFs. The elution procedures described are now used routinely in this laboratory for the determination of PCDDs and PCDFs in soil extracts showing significant interferences after preliminary cleanup, because of the reproducible behavior of PGC compared to activated carbons and the convenience of a single solvent elution method.
j W L -(J . . .. . L 17 41
20 23
time i m i n I
.
20 0
26 0 time lmin I
Flgure 3. GC-MS mass chromatograms of T,CDDs and H,CDDs (a) before and (b) after the PGC cleanup, PCDD isomers are indicated as
0.
However, we prefer to use hexane because of its availability in grades suitable for GC-MS analysis, its lower boiling point, and the simplicity associated with the use of a single solvent throughout the fractionation process. The average recoveries of some PCDD and PCDF isomers were measured by using a series of standards. The results shown in Table I demonstrate recoveries in the range 53-86%, for tetra- to octa-PCDD and -PCDF isomers, with an average standard deviation of 4.6%. These recoveries compare well with those obtained by using activated carbon (7). Figure 3 shows the GC-MS mass chromatograms for TCDD ( m / z 322) and H,CDD ( m / z 390) obtained for a typical soil extract before (Figure 3a) and after (Figure 3b) cleanup by PGC. Soil sample extracts were chromatographed on PGC by using the procedure summarized in Figure 1. In both cases, the same preliminary pretreatment was applied to the extracts, since the relatively low capacity of PGC necessitated the removal of the bulk of coextracted organics, which would otherwise overload the PGC column. Significant improvements in the mass chromatograms are observed for both m / z 322 and m / z 390 as a result of the removal of unidentified interferences. The ions at m / z 320 and 388 showed similar improvements. Table I1 lists the quantitative data for PCDDs and PCDFs, before and after chromatography on PGC. Most of the congener groups (e.g. H,CDD) give similar results, because of the absence of major interferences within the
ACKNOWLEDGMENT We thank J. H. Knox and Chromatographite, Ltd., for the gift of a sample of PGC, A. R. Fernandes for GC-MS analyses, and S. J. Harrad for assistance with the cleanup of soil samples. LITERATURE CITED (1) Smith, L.; Stalling, D.; Johnson, J. Anal. Chem. 1985, 56, 2830-1842. (2) Stalling, D. L.; Smith, L.;Petty, J. Measurements of Organic Pollutants in Water and Waste Water; ASTM STP 686; Van Hall, C. E., Ed., American Society for Testing and Materials: Philadelphia: PA, 1979, pp 302-323. (3) Stanley, J.; Sack, T. Protocol for the Analysis of 2,3,7,8 Tetrachlorodibenzo -p -dioxin by High Resolution Gas Chromatographyf High Reso lution Mass Spectrometry; U.S. Government Printing Office: Washington, DC, 1986; EPA6001/4-96-004. (4) Knox, J. H.; Kaur, B. J . Chromstogr. 1966, 352. 3-25. (5) Creaser, C. S.;Fernandes, A,; AI-Haddad, A,; Harrad, S.; Homer, R.; Skett, P.;Cox, E. Chemosphere, in press. (6) Poland, A,; Glover, E. Mol. Pharmacol. 1977, 73, 924-938. (7) Smith, L. Anal. Chem. 1981, 53, 2152-2154.
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RECEIVED for review July 26,1988. Accepted January 26,1989.
