Evaluation of Column Cleanup for Chlorobenzenes, Polychlorinated

Evaluation of Column Cleanup for Chlorobenzenes, Polychlorinated Biphenyls, Polychlorinated Dibenzo-p-dioxins, and Polychlorinated Dibenzofurans in MM...
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Anal. Chem. 1998, 70, 191-198

Evaluation of Column Cleanup for Chlorobenzenes, Polychlorinated Biphenyls, Polychlorinated Dibenzo-p-dioxins, and Polychlorinated Dibenzofurans in MM5 Flue Gas Analysis Liang K. Tan* and Albert J. Liem

Alberta Research Council Vegreville, P.O. Bag 4000, Vegreville, Alberta, Canada T9C 1T4

The recoveries of chlorobenzenes (CBs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) from the acid-base-on-silica column (ABS), the silver nitrate-on-silica column (SNS), and the alumina column (ALM) were investigated using eluents with various concentrations of dichloromethane in hexane. PCBs recoveries from the ABS and ALM columns are generally increased with increasing number of chloro substituents. However, PCBs recoveries are also complicated by the inductive and resonance contributions due to the different conformation and electron density of the congeners. The recoveries of PCBs with lower number of chloro substituents decreased with increasing silver nitrate content coated on silica. With 2% dichloromethane in hexane (as normally used in MM5 analysis), the recoveries of all CBs and trichloro- to decachlorobiphenyls from ABS, 10% SNS, and ALM columns in series are g80%. However, the 2,4-dichlorobiphenyl is only 16% recovered, and monochlorobiphenyls are not recovered at all. Under these conditions, the recoveries of PCDDs/PCDFs from ABS and 10% SNS columns in series are g80%. With 60% dichloromethane in hexane, PCDDs/PCDFs recoveries from ALM column are g80%. The implication of the selective adsorption of PCBs on the fractionation of CBs/PCBs and PCDDs/PCDFs from these MM5 cleanup columns is discussed. A method known as modified method 5 (MM5) train has been developed for sampling1,2 and analysis3-6 of the semivolatile organic compounds in incineration flue gas. A measured volume of flue gas sample is passed through a cartridge containing XAD-2 (1) Reference Method for Source Testing: Measurement of Releases of Selected Semi-Volatile Organic Compounds from Stationary Source; Report EPS 1/RM/2; Environment Canada, Chemistry Division, River Road Environmental Technology Centre Conservation & Protection: Ottawa, ON, 1989. (2) Modified Method 5 Sampling Train. Test Methods for Evaluating Solid Waste; Method 0010, PB88-239223; U.S. Environmental Protection Agency, Technical Information Service: Springfield, VA, 1986; Vol. II, pp 0010 0010-B-9. (3) Methodology for Organic AnalysissNITEP/Quebec Combustion Tests; Environment Canada, Chemistry Division, River Road Environmental Technology Centre Conservation and Protection: Ottawa, ON, 1987. (4) Chiu, C.; Halman, R.; Li, K.; Thomas, R. S.; Lao, R. C.; Poole, G. Chemosphere 1986, 15, 1091-8. S0003-2700(97)00472-1 CCC: $14.00 Published on Web 01/01/1998

© 1997 American Chemical Society

polystyrene, onto which the organic compounds are adsorbed. The adsorbed compounds are then recovered by solvent extraction, separated by column chromatography cleanup, identified, and measured using gas chromatography with mass spectrometer detector (GC/MS). Chlorobenzenes (CBs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) are among the semivolatile organic compounds specified in the Canadian emission guidelines for incinerators.7 A multiple-step column cleanup procedure has been developed for the purification and enrichment of trace quantities of tetra- to octachlorodibenzo-p-dioxins in fish and particulate samples.8,9 It involves the use of coated and uncoated adsorbents. To remove easily oxidizable species, the sample extract is passed through an acid-base-on-silica column (ABS). Common chemical interferences are removed by passage of the collected fraction from the ABS column through a dual-column system consisting of a top silver nitrate-on-silica column (SNS), draining into a bottom basic alumina column (ALM). The same cleanup procedure has been suggested for the purification and enrichment of the individual CBs/PCBs and PCDDs/PCDFs in the extract of flue gas sample from XAD-2 of the MM5 train.3,4 Fractionation of the CBs/PCBs and PCDDs/PCDFs from the ALM column is carried out using two eluents with different polarity, i.e., with 2 and 50% dichloromethane in hexane, respectively.3-6 Recently, the SNS column has been combined with the ABS column by placing the SNS material in the bottom layer of the ABS column.5,6 The structure-binding relationships for the common receptormediated mechanisms of PCBs have shown that the most active (5) Methodology for Organic AnalysissNITEP/MIDsConnecticut Combustion Test; Environment Canada, Chemistry Division, River Road Environmental Technology Centre Concervation and Protection: Ottawa, ON, 1989; Reference CD891201. (6) Environmental Protection Series: A Method for the Analysis of Polychlorinated Dibenzo-p-Dioxins (PCDDs), Polychlorinated Dibenzofurans (PCDFs) and Polychlorinated Biphenyls (PCBs) in Samples from the Incineration of PCB Waste; Environment Canada, Chemistry Division, River Road Environmental Technology Centre Conservation and Protection: Ottawa, ON, 1990; Reference Method 1/RM/3 (Revised). (7) Operating and Emission Guidelines for Municipal Solid Waste Incinerators; Report CCME-TS/WM-TRE003; Canadian Council of Ministers of the Environment, Minister of Supply and Services Canada: 1989; Catalog No. En 108-3/1-3; ISBN 0-662-56854-0. (8) Lamparski, L. L.; Nestrick, T. J.; Stehl, R. H. Anal. Chem. 1979, 51, 14538. (9) Lamparski, L. L.; Nestrick, T. J. Anal. Chem. 1980, 52, 2045-54.

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compounds are those substituted on both para and at least two meta positions.10 These are congeners that are approximate isostereomers of 2,3,7,8-tetrachlorodibenzo-p-dioxin in their coplanar conformations.10 The toxicity of 2-chlorobiphenyl to fish and daphnids is intermediate between those of biphenyl and tetrachlorobiphenyl.11 Nevertheless, the structure-activity relationship for neurotoxicity of PCBs indicates that 2,2′-dichlorobiphenyl is the most potent congener of the 43 individual PCB congeners found in accustandards.12 Rainbow trout eggs exposed to 10 µg/L 2,2′-dichlorobiphenyl exhibits 21% incidence of deformations.13 The 2,2′-dichlorobiphenyl also reduces 1-dopa production and inhibits the activity of tyrosine hydroxylase in nonhuman primate brain cells.14 Therefore, it is important to evaluate the recovery values of individual CBs/PCBs from the above-mentioned column cleanup processes, especially those PCB congeners with smaller number of chloro substituents. This paper presents the recovery values of dichloro- to hexachlorobenzenes and monochloro- to decachlorobiphenyls from each of the above-mentioned columns. The effect of the silver nitrate contents (coated on silica) on the recoveries of CBs/PCBs is also included in the present study. In addition, the recovery of PCDDs/PCDFs from the ALM column is discussed in relation to the eluent strength needed to recover the CBs/PCBs from the same column. The recoveries or retention behaviors of individual CB/PCB and PCDD/PCDF from each of the column cleanup methods (in MM5 analysis) have not been reported previously. EXPERIMENTAL SECTION Safety Precautions. PCBs, PCDDs, and PCDFs were not weighed in the authors’ laboratory. All work using these compounds was done with solutions purchased from suppliers mentioned below. Safety glasses and disposable laboratory coats (made of adsorbent paper backed with plastic) and gloves were worn. Benchtops were covered with disposable adsorbent mats backed with plastic. The effluents from the analytical instruments were passed into traps containing activated charcoal. Sample concentrations were carried out in the fumehood. Laboratory coats, gloves, mats, towels, pipets, containers of solution, and waste column packing materials were placed in a plastic bag at the end of each experiment. Sealed bags were incinerated. Chemicals. All solvents (n-hexane, dichloromethane, and toluene) were of distilled-in-glass grade (n-hexane is simply written as “hexane” throughout this paper). Silica (Bio-Sil A, 100-200 mesh, Bio-Rad) was sequentially washed with two portions each of hexane and dichloromethane using a Bu¨chner funnel with suction.3,4,6 The volume of solvent used for each wash was equal to twice the estimated volume of silica. It was dried at 50 °C for 1 h and then conditioned overnight at 225 °C and stored in a bottle with a Teflon cap liner, to be used for that day only. Anhydrous sodium sulfate was prepared in the same manner as the silica.3,4,6 (10) Safe, S. CRC Crit. Rev. Toxicol. 1990, 21, 51-88. (11) Dill, D. C.; Mayes, M. A.; Mendoza, C. G.; Boggs, G. U.; Emmitte, J. A. ASTM Spec. Tech. Publ. 1982, 766 (Aquat. Toxicol. Hazard. Assess.), 24556. (12) Shain, W.; Bush, B.; Seegal, R. Toxicol. Appl. Pharmacol. 1991, 111, 3342. (13) Freitag, D.; Haas-Jobelius, M.; Yin, Y. Toxicol. Environ. Chem. 1991, 3132, 401-8. (14) Seegal, R. F.; Bush, B.; Shain, W. Chemosphere 1991, 23, 1941-9.

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Table 1. CBs/PCBs and PCDDs/PCDFs Used in the Experiment

1,4-dichlorobenzene 1,2-dichlorobenzene 1,3,5-trichlorobenzene 1,2,4-trichlorobenzene 1,2,3-trichlorobenzene 2-chlorobiphenyl 4-chlorobiphenyl 2,4-dichlorobiphenyl 2,4,6-trichlorobiphenyl 2,2′,4,6-tetrachlorobiphenyl 2,2′,3′,4,5-pentachlorobiphenyl 2,3,7,8-tetrachlorodibenzop-dioxin 1,2,3,7,8-pentachlorodibenzop-dioxin 1,2,3,6,7,8-hexachlorodibenzop-dioxin

CBs 1,2,4,5-tetrachlorobenzene 1,2,3,4-tetrachlorobenzene 1,2,3,4,5-pentachlorobenzene hexachlorobenzene PCBs 2,2′,3,4,5,6′-hexachlorobiphenyl 2,2′,3,4,4′,5′,6-heptachlorobiphenyl 2,2′,3,3′,5,5′,6,6′-octachlorobiphenyl 2,2′,3,3′,4,4′,5,6,6′-nonachlorobiphenyl decachlorobiphenyl PCDDs 1,2,3,4,6,7,8-heptachlorodibenzop-dioxin octachlorodibenzo-p-dioxin

PCDFs 2,3,7,8-tetrachlorodibenzofuran 1,2,3,4,6,7,8-heptachlorodibenzofuran 1,2,3,7,8-pentachlorodibenzofuran octachlorodibenzofuran 1,2,3,6,7,8-hexachlorodibenzofuran

Concentrated sulfuric acid, concentrated hydrochloric acid, sodium hydroxide, and silver nitrate of certified ACS grade. Forty-four percent H2SO4 on silica3,5,6,8 was prepared by adding 78.6 g of concentrated H2SO4 in small portions to 100 g of freshly prepared silica in a 500 mL glass-stoppered Erlenmeyer flask. Between additions, the mixture was manually shaken until no clumps were observed. It was then stored in a bottle with a Teflonlined screw cap. Thirty-three percent 1 M NaOH on silica3,5,6,8 was prepared by adding 24.6 g of a 1 M NaOH solution to 50 g of freshly prepared silica in the same manner as mentioned above. Ten percent AgNO3 on silica3,5,6,8 was prepared by adding a solution of 5.6 g of AgNO3 in 21.5 mL of water to 50 g of freshly prepared silica in the same manner as mentioned above. After the addition of AgNO3 solution, the material was allowed to stand in the dark for 30 min. The glass stopper was then replaced with an aluminum foil cover. The material in the flask was placed in an oven at 30 °C. Over a 5 h period, the oven temperature was raised to 180 °C, and the flask kept overnight at this temperature. The resulting material was cooled to room temperature and was transferred to an amber glass bottle with a Teflon-lined screw cap, to be used for that day’s experiment only. The same procedure was followed for preparing 0.7, 1.7, 3.3, 6.7, and 20% AgNO3 coated on silica, except the weight of AgNO3 was 0.37, 0.93, 1.87, 3.74, and 11.21 g, respectively. Basic alumina (Alcoa chromatographic grade, 80-200 mesh, Matheson Coleman & Bell) was prepared by heating at 180 °C in a tube in a furnace under nitrogen purge until condensation at the exit port ceased. It was further heated at 350 °C for 3.5 h and then cooled and used immediately in the experiment. Various CB compounds of dichloro- to hexachlorobenzenes (Table 1) were obtained from Aldrich. Stock solutions of 1000 µg/mL in hexane were prepared. A PCBs mixture containing monochloro- to decachlorobiphenyl (their names are listed in Table 1) at 1000 µg/mL each was obtained from Ultra Scientific (Catalog No. RPCBR-1). A working solution of CBs/PCBs

mixture in hexane was prepared at 20 µg/mL each. The internal reference compound was 1,3,5-tribromobenzene (Aldrich), and the stock solution was prepared in the same way as mentioned for CBs. Tetrachloro to octachloro congeners of PCDDs/PCDFs were obtained from Cambridge Isotope Laboratories at either 10 or 50 µg/mL each in nonane (their names are listed in Table 1). A working solution of the PCDDs/PCDFs mixture in toluene was prepared at 1.0 µg/mL each. An internal reference compound for PCDDs/PCDFs measurement was 2,3,7,8-tetrabromodibenzop-dioxin (Ultra Scientific), and the working solution was prepared in the same way as for PCDDs/PCDFs. Three window-defining mixtures obtained from Cambridge Isotope Laboratories were used, i.e., EC-1430 at 2.5 g/mL contained the first- and the lasteluting isomers of monochloro- to decachlorobiphenyls, ED-1732-A at 400 ng/mL contained the first- and the last-eluting isomers of tetrachloro- to heptachlorodibenzo-p-dioxins, and EF-1731-A at 400 ng/mL contained the first- and the last-eluting isomers of tetrachloro- to heptachlorodibenzofurans. Glassware. Glassware was sequentially cleaned with detergent, deionized water, acetone, hexane, and dichloromethane. Pyrex glass wool was rinsed with hexane. All of these were then baked in an oven at 150 °C overnight. Cleanup Columns. Three columns were used similar to the types described in the literature:3,4,9 supermacro (30 cm × 2.2 cm), macro (15 cm × 1.0 cm), and high-aspect (35 cm × 0.8 cm). The two latter columns have receiver heads (15 cm × 2.5 cm) on top. A glass wool plug was inserted in the bottom of each column above the Teflon stopcock. An acid-base-on-silica column (ABS) was prepared by adding the following materials into the supermacrocolumn in the order specified:3,5,6,9 1 g of silica (bottom layer), 2 g of 33% 1 M NaOH on silica, 1 g of silica, 4 g of 44% H2SO4 on silica, 2 g of silica, and 0.5 g of anhydrous Na2SO4 to top off the column. Silver nitrate-on-silica columns (SNS) were prepared by adding 1.5 g of various AgNO3 contents (0.7-20%) on silica (bottom layer) into the macrocolumns and 0.5 g of anhydrous Na2SO4 to top off each of the columns. An alumina column (ALM) was prepared by adding 2.5 g of freshly prepared basic alumina into the high-aspect column. Prewashing for each type of column was done with hexane. Experiment for Column Cleanup Recovery. The emission guidelines for incinerators7 specify CBs/PCBs at 1.0 µg/m3 and total PCDDs/PCDFs at 0.50 ng/m3 (with toxicity equivalency factors of 1-0.001 for the tetrachloro to octachloro congeners). Since the volume of flue gas sampled in MM5 train is about 2.8 m3 (or 2800 L), the anticipated CBs/PCBs and PCDDs/PCDFs in XAD-2 adsorbent of MM5 train are 2.8 µg and 1.4 ng (based on the tetrachloro congener of PCDD/PCDF), respectively. In MM5 analysis, the concentrated extract from XAD-2 is normally divided equally into four portions. Each 25% portion is used for the analysis of chlorophenols, polycyclic aromatic hydrocarbons, and CBs/PCBs/PCDDs/PCDFs and for reserved extract as a contingency measure. Therefore, the anticipated amounts of CBs/ PCBs and PCDDs/PCDFs for column cleanup are only 0.70 µg and 0.35 ng, respectively. If all extract from XAD-2 is used for the analyses of CBs/PCBs/PCDDs/PCDFs, the anticipated amounts of CBs/PCBs and PCDDs/PCDFs for column cleanup are 2.8 µg and 1.4 ng, respectively.

In this column cleanup recovery study, an aliquot of CBs/PCBs mixture which contained 1.25 µg of each isomer or congener, or an aliquot of PCDDs/PCDFs mixture which contained 10 ng of each congener, was added on top of each column. Then, in each experiment, the mixture was eluted with eluent of a differing polarity. The eluents of differing polarities were prepared by varying the concentration of dichloromethane in hexane from 0 to 100%, as indicated in the text. The volume of solvent was 65, 45, and 45 mL for ABS column, SNS column, and ALM column, respectively. The collected effluent was rotary evaporated to about 3 mL under reduced pressure and then concentrated under a gentle stream of nitrogen at 40 °C to 1 mL. An aliquot of the corresponding internal reference solution was added to the concentrate prior to GC/MS measurement. Instrumentation. A Varian 6000 gas chromatograph equipped with a splitless injector at 270 °C and a 30 m × 0.25 mm i.d. × 0.25 µm film thickness of 5% phenylmethylpolysiloxane fused capillary (DB5) column was directly coupled to an HP 5970 mass spectrometer ion source, with electron impact in selected ion monitoring mode (SIM). The temperature program for CBs/ PCBs was 70 °C for 4 min, to 180 °C at 10 °C/min, to 240 °C at 3 °C/min, to 285 °C at 10 °C/min, and then held for 10 min. The temperature program for PCDDs/PCDFs was 100 °C for 1 min, to 190 °C at 30 °C/min, to 280 °C at 4 °C/min, and then held for 8.5 min. Parameter settings and the ion masses monitored within each window of the SIM program followed the previously established method.5,6 Quantitation. In this recovery study, quantitation of CBs/ PCBs or PCDDs/PCDFs was based on relative response factors for single calibrations with external standards. The standard solution was a mixture of equivalent spiked solutions with concentrations equal to those added on the cleanup column. RESULTS AND DISCUSSION Recoveries of CBs/PCBs and PCDDs/PCDFs from the Acid-Base-on-Silica Column. All CBs showed high recoveries from the acid-base-on-silica column (ABS) with 0-3% dichloromethane in hexane. The mean recovery values (93-111%) from all CBs at various concentrations of dichloromethane in hexane are shown in Figure 1A. The bars illustrate the 95% confidence intervals. Trichloro- to decachlorobiphenyls were also well recovered from the ABS column at 0-3% dichloromethane in hexane. The mean recovery values of these PCBs (81-117%), with the bars showing the 95% confidence intervals, are plotted against the concentration of dichloromethane in hexane in Figure 1B. However, the recoveries of monochloro- and dichlorobiphenyls were dependent on the concentration of dichloromethane in hexane (Figure 1C). Using hexane, only 2 and 6% of 2-chloroand 4-chlorobiphenyls, respectively, were recovered from this column. The same eluent recovered 40% of 2,4-dichlorobiphenyl. With 0.5% dichloromethane in hexane, total recovery of 2,4dichlorobiphenyl was obtained, whereas only 79 and 53% of 4-chloro- and 2-chlorobiphenyls, respectively, were recovered. Increasing the concentration of dichloromethane to 1% led to a total recovery of 4-chlorobiphenyl, but only 75% of 2-chlorobiphenyl was recovered. For complete recovery of all PCBs, g2% dichloromethane in hexane was required. Obviously, the presence of Analytical Chemistry, Vol. 70, No. 1, January 1, 1998

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Figure 1. CBs/PCBs recoveries from acid-base-on-silica column as a function of the concentration of dichloromethane in hexane. The bars indicate 95% confidence intervals. (A) Mean recoveries of CBs. (B) Mean recoveries of trichloro- to decachlorobiphenyls. (C) Lines a (b), b (×), c (2) represent the data of 2-chloro-, 4-chloro-, and 2,4dichlorobiphenyls, respectively.

dichloromethane in hexane increased the polarity of the eluent and led to the increase of the PCBs recoveries. The above selective recoveries of CBs/PCBs from the ABS column have not been reported earlier. These data indicate that the recoveries of CBs from the ABS column are independent of both the nature of CBs and the concentration of dichloromethane in hexane. PCBs recoveries, however, are dependent on the nature of the individual PCB and the concentration of dichloromethane in hexane. The higher the number of chloro substituents on PCBs, the higher the recoveries or the lower the retention of PCBs on the ABS column. An eluent with higher concentration of dichloromethane in hexane is required in order to recover the PCBs with a lower number of chloro substituents. Also, the retention of monochlorobiphenyl with the chloro substituent in the 2-position is higher than that with the chloro substituent in the 4-position. The trend of retention behaviors from the uncoated-silica column15 was the same as that from the ABS column, which was characterized by the number of chloro substituents on PCBs and the ortho substituent effect. In addition, the extent of PCBs retention is higher on the uncoated silica than on the ABS column.15 For example, 4-chlorobiphenyl was only 5% recovered from the uncoated silica column with 3% dichloromethane in hexane, whereas 1.5% dichloromethane in hexane totally recovered it from the ABS column.15 Since concentrated (32%) H2SO4 or 1 M NaOH solutions were used in the coating of the silica, water was also adsorbed onto the silica surfaces in addition to the acid or base. The silica surfaces that adsorbed water were deactivated and adsorbed CBs/PCBs less effectively. Hence, CBs/PCBs were eluted more easily from the ABS column than from the uncoated silica column. (15) Tan, L. K.; Liem, A. J., unpublished. Complete data for the experiment with uncoated silica column are not presented in this article to avoid an excessive number of pages.

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Figure 2. Mean recoveries of (A) PCDDs and (B) PCDFs from acid-base-on-silica column as a function of the concentration of dichloromethane in hexane. The bars indicate 95% confidence intervals.

The adsorption of aromatic compounds on uncoated silica is postulated16 as a result of (1) formation of hydrogen bonding between the aromatic compounds and the surface reactive silanol groups (Si-OH) and (2) partial formation of a chemical bond between the electron-donating group in the aromatic compounds and some surface silanol groups. The detailed retention behavior of individual CBs and PCBs on uncoated silica have been reported previously,17,18 where an HPLC technique was used, with uncoated silica as stationary phase and hexane as the mobile phase. This previous work found that retention of CBs/PCBs generally decreased with an increasing number of chloro substituents, and the ortho substitution to the phenyl-phenyl bond (2- and 6-positions) in PCBs promoted retention.17,18 The present work used a more polar eluent than hexane. Therefore, differences in the retention of individual CB was not observed at g1% dichloromethane in hexane. The retention behaviors of PCBs in the present and in the previous works17,18 are in agreement. In general, the present work showed greater retention of PCBs than CBs. This can be attributed to the increase of the ionization probabilities with the greater size of the π-electron system in the biphenyl ring,19 which lead to greater activation of the electron donor groups in the biphenyl ring to the surface silanol groups on the adsorbent. All PCDDs/PCDFs used in this study exhibited good recoveries from the ABS column. Their mean recovery data obtained from the elution of 0-4% dichloromethane in hexane are shown in Figure 2. The bars represent the 95% confidence intervals. Recoveries of CBs/PCBs and PCDDs/PCDFs from the Silver Nitrate-on-Silica Column. All CBs were totally recovered using 0-5% dichloromethane in hexane from the silver nitrateon-silica (SNS) columns packed with 0.7-20% of AgNO3 coated on silica. Using 2% dichloromethane in hexane, Figure 3A shows the mean recoveries of all CBs (81-100%), with the 95% confidence intervals, which are plotted against the AgNO3 content coated on silica. (16) Snyder, L. R. J. Chromatogr. 1966, 25, 274-93. (17) Brinkman, U. A. Th.; De Vries, G. J. Chromatogr. 1979, 169, 167-82. (18) Rekker, R. F.; De Vries, G.; Bijloo, G. J. J. Liq. Chromatogr. 1989, 12, 695728. (19) Deverse, F. T.; King, A. B. J. Chem. Phys. 1964, 41, 3833-8.

Figure 3. Mean recoveries of (A) CBs and (B) tetrachloro- to decachlorobiphenyls with 2% dichloromethane in hexane from silver nitrate-on-silica column as a function of the silver nitrate content coated on silica. The bars indicate 95% confidence intervals.

Figure 4. Recoveries of (A) 2-chlorobiphenyl, (B) 4-chlorobiphenyl, (C) 2,4-dichlorobiphenyl, and (D) 2,4,6-trichlorobiphenyl using various concentrations of dichloromethane in hexane from silver nitrate-onsilica column as a function of the silver nitrate content coated on silica.

With the exception of the 2-chloro-, 4-chloro-, 2,4-dichloro-, 2,4,6-trichloro-, and 2,2′,4,6-tetrachlorobiphenyls, PCBs were well recovered using 0-5% dichloromethane in hexane from 0.7-20% SNS columns. Using 2% dichloromethane in hexane, Figure 3B shows the mean recoveries of the tetra- to decachlorobiphenyls (88-111%), with the 95% confidence intervals, which are plotted against the AgNO3 content coated on silica. The exceptional behaviors of 2-chloro-, 4-chloro-, 2,4-dichloro-, and 2,4,6-trichlorobiphenyls are illustrated in Figure 4A-D, respectively. In these figures, the recoveries of the individual PCB are also plotted as a function of the AgNO3 content coated on silica. Each line in the figure represents the recovery function using an eluent at a given polarity prepared by varying the concentration of dichloromethane in hexane. The recoveries of these PCBs that were obtained from 10% SNS column with 2% dichloromethane in hexane are compared (data with filled

triangles in Figure 4). No 2-chloro- or 4-chlorobiphenyls were detected, yet 16% of 2,4-dichlorobiphenyl and 81% of the 2,4,6trichlorobiphenyl were recovered. When the recoveries of these PCBs from a 3.3% SNS column eluted with 2% dichloromethane in hexane are compared (data with filled triangles in Figure 4), the two monochlorobiphenyls were not recovered, but there were 51 and 92% of 2,4-dichloro- and 2,4,6-trichlorobiphenyls recovered, respectively. From this column, increasing the polarity of the eluent to 4% dichloromethane in hexane (data with squares in Figure 4C) led to 83% recovery of the 2,4-dichlorobiphenyl. However, only 30 and 50% of the 2-chloro- and 4-chlorobiphenyls were recovered with 5% dichloromethane in hexane, respectively (data with squares in Figure 4A and B). For total recovery of 2-chlorobiphenyl with 5% dichloromethane in hexane, the AgNO3 content coated on silica must be e0.7%. The above selective recoveries of CBs/PCBs from SNS column also have not been reported previously. The data demonstrate that the recoveries of CBs from SNS column are independent on the nature of the individual compound, AgNO3 content coated on silica, and polarity of the eluent. However, the recoveries of PCBs are dependent on all three of these factors. Again, the larger the number of the chloro substituents on the PCBs, the greater the recoveries or the smaller the retention of PCBs on SNS, and the lower the polarity of the eluent required to elute them. Also, the retention of monochlorobiphenyl with the chloro substituent in the 4-position was smaller than that with the chloro substituent in the 2-position. A higher AgNO3 content coated on silica reduced the recoveries of PCBs (or increased the retention of PCBs), and a more polar eluent (with higher dichloromethane content in hexane) is required for the elution. It has been postulated20 that the aromatic ring donates electrons to the silver cation, Ag+, in a solution and forms a complex (AgAr+, where Ar is an abbreviation of an aromatic ring). In the present work, the interaction between the π-electrons of the aromatic ring and the Ag+ site on the silica is probably responsible for the retention of CBs/PCBs. The lower retention of CBs as compared to that of PCBs can be attributed to the increase of the ionization probabilities with the greater size of the π-electron system in the biphenyl ring.19 The higher silver nitrate content coated on the silica yields more Ag+ sites on the silica surfaces, resulting in greater PCBs interaction with the active sites, which leads to a higher retention of the PCBs. Again, the same trends of retention behaviors were obtained from the SNS, ABS, and uncoated silica columns, which are characterized by the number of chloro substituents on PCBs and the ortho substituent effect. The extent of CBs/PCBs retention is in the following order: uncoated silica > SNS > ABS. In the preparation of SNS material, although a dilute solution of AgNO3 was used to coat the silica, the subsequent heating removed most of the water adsorbed on some of the silica surfaces. This may account for the greater retention of PCBs in the SNS column than in the ABS column. Figure 5 shows that the recoveries of PCDDs and PCDFs with 2% dichloromethane in hexane were independent on the AgNO3 content coated on silica. However, the mean recovery data were 81-89%, with 11-21% relative standard deviations. (20) Andrews, L. J.; Keefer, R. M. J. Am. Chem. Soc. 1950, 72, 5034-7.

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Figure 5. Mean recoveries of (A) PCDDs and (B) PCDFs using 2% dichloromethane in hexane from silver nitrate-on-silica column as a function of the silver nitrate content coated on silica. The bars indicate 95% confidence intervals.

Figure 6. CBs recoveries from alumina column as a function of the concentration of dichloromethane in hexane. (A) Dots (b) and circles (O) represent data of 1,2-dichloro- and 1,4-dichlorobenzenes, respectively. (B) Lines a (O), b (0), and c (b) represent data of 1,3,5trichloro-, 1,2,3-trichloro-, and 1,2,4-trichlorobenzenes, respectively. (C) Lines a (O) and b (b) represent 1,2,4,5-tetrachloro- and 1,2,3,4tetrachlorobenzenes, respectively. (D) Data with (O) and (b) represent pentachloro- and hexachlorobenzenes, respectively.

Recoveries of CBs/PCBs and PCDDs/PCDFs from the Alumina Column. Figure 6 illustrates the recoveries of CBs from the alumina column (ALM) as a function of the concentration of dichloromethane in hexane. Hexane eluted only e5% of the 1,2-dichloro- and 1,4-dichlorobenzenes (Figure 6A). Increasing the concentration of dichloromethane in hexane led to an increase in the recoveries of these compounds, but the data were scattered. Above 1% dichloromethane in hexane, recoveries of these compounds reached a total. With g2% dichloromethane in hexane, complete and reproducible recoveries were obtained. In contrast, 1,3,5-trichlorobenzene (circles in Figure 6B) was eluted with hexane (88% recovery). However, increasing the concentration of dichloromethane in hexane did not improve its recovery nor the scatter of the data (88 ( 8%). Low recoveries were obtained for 1,2,3-trichloro- and 1,2,4-trichlorobenzenes (Figure 6B) when eluted with e0.5% dichloromethane in hexane. Reproducible 196

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recoveries of the former (94 ( 3%) and the latter (90 ( 3%) trichlorobenzenes were achieved using g1% dichloromethane in hexane. Similar recovery behavior was observed for 1,2,3,4tetrachlorobenzene (98 ( 2%), as indicated in Figure 6C. Although the recovery of 1,2,4,5-tetrachlorobenzene reached a maximum with g1% dichloromethane in hexane, the values were scattered (83 ( 9%) and did not achieve 100% recovery with 1-5% dichloromethane in hexane. Pentachloro- (100 ( 3%) and hexachlorobenzenes (101 ( 3%) achieved reproducible and total recoveries with g0.5% dichloromethane in hexane (Figure 6D). Observation of the recovery data eluted with 0.25% dichloromethane in hexane (Figure 6) shows the following order of CBs retention (the numbers in parentheses are the percent recoveries of the mentioned CB): 1,2,3-trichloro- (18%) > 1,2,3,4-tetrachloro(32%) > 1,2-dichloro- (72%) ≈ 1,4-dichloro- (80%) ≈ 1,2,4-trichloro(78%) > pentachloro- (84%) ≈ hexachloro- (82%) > 1,2,4,5tetrachloro- (78%, maximum recovery) > 1,3,5-trichloro- (90%). Snyder21 has postulated that the specific adsorption between a substituted aromatic compound and alumina is due to an electrostatic interaction of the substituent with a positive field perpendicular to the alumina surface. Rekker et al.18 described the adsorption as the ionic interaction between the π-electrons (affected by both the resonance and inductive contributions) of the aromatic ring with the Al3+ sites on the alumina surface. The present results indicate that the elution of CBs is dependent on the nature of the individual CB and the polarity of the eluent. The position of the chloro substituents exert a marked influence on the CBs elution. The electron-withdrawing effect of the chloro substituents in CBs affects the relative electron density of the aromatic ring. In the 1,3,5-trichlorobenzene, all chloro substituents are meta to one another and lead to the stabilization of π-electrons, which, in turn, deactivate the interaction between this CB and the Al3+ site of the adsorbent. Therefore, 1,3,5trichlorobenzene is the least retained. An analogous explanation can be applied to 1,2,4,5-tetrachlorobenzene, where the chloro substituents, in either positions 1 and 5 or positions 2 and 4, are meta to one another. This suggests that 1,2,4,5-tetrachlorobenzene should have the lowest retention of all the tetrachlorobenzenes. In 1,2,3-trichloro- and 1,2,3,4-tetrachlorobenzenes, one to the next chloro substituents are in the ortho position. This configuration causes higher retention on alumina adsorbent. The retention of 1,2-dichlorobenzene is only slightly larger than that of 1,4-dichlorobenzene, as indicated by only 8% difference in their recoveries. o-Dichlorobenzene is unique in permitting steric interaction of the two chloro substituents. The retention of dichlorobenzenes in the present result is in the order of ortho ≈ para. This is in agreement with the result of Snyder22 but is in disagreement with the result of Brinkman and De Vries,17 where the retention order is ortho > para. The above results from ALM column are distinct from those obtained using uncoated silica, ABS, or SNS columns. The elution of CBs from the latter columns is independent of the nature of the individual CB, the polarity of the eluent, and the AgNO3 content coated on silica. This indicates that CBs are more strongly adsorbed on alumina than on silica or coated silica. Previous works also reported that the capacity factor for alumina as (21) Snyder, L. R. Principles of Adsorption Chromatography; Marcel Dekker: New York, 1968; Chapter 3. (22) Snyder, L. R. J. Chromatogr. 1965, 20, 463-95.

Figure 7. PCBs recoveries from alumina column as a function of the concentration of dichloromethane in hexane. (A) Lines a (O) and b (b) represent 4-chloro- and 2-chlorobiphenyls, respectively. (B) Lines a (b) and b (O) represent 2,4-dichloro- and 2,4,6-trichlorobiphenyls, respectively. (C) Lines a (b) and b (O) represent 2,2′,3′,4,5pentachloro- and 2,2′,4,6-tetrachlorobiphenyls, respectively. (D) Lines a (0), b (b), and c (O) represent 2,2′,3,4,5,6′-hexachloro-, 2,2′,3,4,4′,5′,6-heptachloro-, and 2,2′,3,3′,5,5′,6,6′-octachlorobiphenyls, respectively. (E) Data with (O) and (b) represent 2,2′,3,3′,4,4′,5,6,6′nonachloro- and decachlorobiphenyls, respectively.

adsorbent is higher than that for silica17,18 and that the adsorption energies of halogenated derivatives are stronger on alumina than on silica.16 Figure 7 illustrates the recoveries of PCBs from the ALM column as a function of the concentration of dichloromethane (05%) in hexane. Hexane recovered 1 and 5% of 2,2′,3,3′,4,4′,5,6,6′nonachloro- and decachlorobiphenyls, respectively, but it could not recover the remaining PCBs used in this study. Storr-Hansen et al.23 studied the retentions of 34 PCBs congeners and found that 4-chloro-, 4,4′-dichloro-, 3,4,4′-trichloro-, and 3,4,4′,5-tetrachlorobiphenyls are not eluted at all with hexane from an ALM column. They propose that PCB congeners that are able to obtain a planar conformation are much more strongly retained than the remaining congeners.23 In the present study, 0.5% dichloromethane in hexane led to the recoveries of 4, 84, and 98% of 2,2′,3,4,5,6′-hexa-, 2,2′,3,4,4′,5′,6hepta-, and 2,2′,3,3′,5,5′,6,6′-octachlorobiphenyls, respectively. Increasing the concentration of dichloromethane to 0.75% in hexane led to the recoveries of 5, 8, 9, 18, 44, 48, and 98% of 2,4dichloro-, 2,2′,3′,4,5-pentachloro-, 2-chloro-, 2,2′,3,4,5,6′-hexachloro-, 2,2′,4,6-tetrachloro-, 2,4,6-trichloro-, and 2,2′,3,4,4′,5′,6-heptachlorobiphenyls. Under these conditions, 4-chlorobiphenyl was not recovered at all. The order of retention of PCBs under study is, therefore, as follows: 4-chloro- > 2,4-dichloro- > 2,2′,3′,4,5pentachloro- ≈ 2-chloro- > 2,2′,3,4,5,6′-hexachloro- > 2,2′,4,6tetrachloro- ≈ 2,4,6-trichloro- > 2,2′,3,4,4′,5′,6-heptachloro- > 2,2′,3,3′,5,5′,6,6′-octachloro- > 2,2′,3,3′,4,4′5,6,6′-nonachloro- > decachlorobiphenyls. The extreme case was 4-chlorobiphenyl, with a recovery of only 7%, even though an eluent containing 5% dichloromethane (23) Storr-Hansen, E.; Cleemann, M.; Cederberg, T.; Jansson, B. Chemosphere 1992, 24, 323-33.

Figure 8. PCDDs/PCDFs recoveries from alumina column as a function of the concentration of dichloromethane in hexane. Lines a (O), b (b), and c (4) represent 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and 1,2,3,7,8-pentachlorodibenzop-dioxin, respectively.

in hexane was used. In comparison, its ortho isomer, 2-chlorobiphenyl, was completely recovered (103 ( 8%) with g2% dichloromethane in hexane. In addition, the recoveries of 2,4dichlorobiphenyl and 2,2′,3′,4,5-pentachlorobiphenyl did not exceed the mean value of 66 ( 9% and 87 ( 10%, respectively. These incomplete recoveries of some PCBs with dichloromethane in hexane from the ALM column have not been reported previously. In the previous MM5 method development,2-6 the recoveries of CBs/PCBs from this column have not been stated. In HPLC experiments,16-18,21,22 the incomplete recoveries of some PCBs are not reported because the elution results from the column were not compared to the standards that were not passed through the column. However, the recovery of 2,2′,4,6-tetrachlorobiphenyl from the present work was complete (95 ( 5%). The remaining PCBs were also completely recovered, with mean values of 100104% and relative standard deviation of 3%. Clearly, the elution of PCBs is dependent on the nature of the individual PCB and the polarity of the eluent. There is a general tendency for greater retention with decreasing number of chloro substituents on PCBs, and hence higher polarity eluent is required for good recovery. However, the positions of the chloro substituents also play an important role in the retention of PCBs on alumina, as readily observed for the much higher retention of parasubstituted 4-chlorobiphenyl than of ortho-substituted 2-chlorobiphenyl. This behavior is anomalous to that resulting from the uncoated silica,15 ABS (Figure 1C), and SNS (Figure 4A,B) columns, where 4-chlorobiphenyl has a lower retention than the 2-chlorobiphenyl. Other reseachers17,18 conducting experiments with various PCBs using HPLC technique with uncoated-silica or alumina stationary phase and hexane mobile phase, have also reported the anomalous retention orders of ortho > para > meta on silica, and para > meta . ortho on alumina. The typical recoveries of 2,3,7,8-tetrachlorodibenzo-p-dioxin (circles), 2,3,7,8-tetrachlorodibenzofuran (dots), and 1,2,3,7,8pentachlorodibenzo-p-dioxin (triangles) from the ALM column as a function of dichloromethane concentration in hexane are presented in Figure 8. Other PCDDs/PCDFs elution behaviors were similar and are not included in this figure to avoid overcrowding. The 2,3,7,8-tetrachlorodibenzofuran began to desorb from the ALM column at about 4-8% dichloromethane in Analytical Chemistry, Vol. 70, No. 1, January 1, 1998

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hexane, while the 2,3,7,8-tetrachlorodibenzo-p-dioxin started to desorb at slightly higher eluent polarity, i.e., about 12% dichloromethane in hexane. The other higher chlorinated congeners of PCDDs/PCDFs began to desorb from the ALM column at about 2-4% dichloromethane in hexane. Other researchers also observed that the 2,3,7,8-tetrachloro congeners were the latesteluting congeners among all of the tetrachloro dibenzo-p-dioxins and dibenzofurans from a basic ALM column.24 Increasing the concentration of dichloromethane in hexane led to the increase in PCDDs/PCDFs recoveries. With 50% dichloromethane in hexane, 72 and 79% of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-dibenzofuran were recovered, respectively. Under these conditions, pentachloro to octachloro congeners of PCDDs/ PCDFs gave better recoveries (103 ( 11%) than the tetrachloro congeners. An eluent containing 60% dichloromethane in hexane increased the tetrachlorodibenzo-p-dioxin recovery to 80%. Column Cleanup in MM5 Analysis. The column cleanup procedures in MM5 analysis employed the above-mentioned ABS, 10% SNS, and ALM columns.3,4 Elution of CBs/PCBs and PCDDs/PCDFs was done using 2% dichloromethane in hexane from the ABS column.3,4 The eluent was concentrated and then was passed through the 10% SNS column that was placed on top of the ALM column.3,4 The CBs/PCBs and PCDDs/PCDFs were also eluted with 2% dichloromethane in hexane from the 10% SNS column.3,4 The CBs/PCBs and PCDDs/PCDFs in the ALM column were then separated by eluting first the CBs/PCBs fraction with 2% dichloromethane in hexane, followed by the PCDDs/PCDFs fraction with 50% dichloromethane in hexane.3,4 All CBs were satisfactorily recovered (>80%) from all types of columns (Figures 1A, 3A, and 6A-D). All PCBs were satisfactorily (>80%) recovered from the ABS column (Figure 1B,C). Although the trichloro- to decachlorobiphenyls were satisfactorily recovered from the 10% SNS column, the recovery of 2,4dichlorobiphenyl was low. In addition, both 2-chloro- and 4-chlorobiphenyls were not recovered at all (Figures 3B and 4A-D). All PCBs were recovered (>80%) from the ALM column, with the exception of 2,4-dichlorobiphenyl (66% recovery) and 4-chlorobiphenyl (0% recovery) (Figure 7). Therefore, if the flue gas sample contains 2-chloro- and 4-chlorobiphenyls, the MM5 column cleanup procedures would yield false negative results, simply because they are not recoverable with 2% dichloromethane in hexane. In addition, the 2,4dichlorobiphenyl in the sample would be reported at a much lower concentration if the measurement were done by comparison with a standard mixture of PCBs solution. Therefore, the quantitation of PCBs is suggested to be done by comparison with the standard mixture that has been passed through the column cleanup procedures in the same manner as that of the sample. Increasing the dichloromethane content in hexane will lead to better recovery of these PCBs; however, it cannot be done, because PCDDs/PCDFs will then be eluted from the ALM column

(Figure 8), and the separation of CBs/PCBs from PCDDs/PCDFs will then fail. PCDDs/PCDFs were satisfactorily (>80%) recovered with 2% dichloromethane in hexane from ABS and 10% SNS columns (Figures 2A,B and 5A,B, respectively). Their recoveries with 50% dichloromethane in hexane from the alumina column are also acceptable (72% to complete recoveries). CONCLUSIONS CBs and PCDDs/PCDFs were well recovered from the cleanup columns used in MM5 analysis. The monochlorobiphenyls cannot be determined on the basis of the reference MM5 method due to their retention behavior on the cleanup columns. The 2,4dichlorobiphenyl is partially recovered from the cleanup columns. It would have been underestimated when quantitation were done by comparison with the standard mixture. Therefore, the quantitation of the organic compounds in MM5 sample is suggested to be done by comparison with the standard mixture that has been passed through the column cleanup procedures in the same manner as that of the sample. The possible relationships between retention and structure have been examined in the present work with 11 PCBs and in the earlier works with 99 PCBs congeners.17,18,23 Although there is a general tendency for regulation by the number of chloro substituents in PCBs, there are additional factors that cause deviations from the general retention behavior. Inductive and resonance effects, polarizability, and steric hindrance of the PCBs are possible factors. The mode of interaction of these factors is complex and not well understood for predicting the recoveries of the 209 PCBs congeners from the cleanup columns. The incomplete combustion of chlorinated organic waste may generate any of the PCBs congeners. Although the most active PCBs are congeners that are approximate isostereomers of 2,3,7,8tetrachlorodibenzo-p-dioxin (based on the structure-binding relationships for the common receptor-mediated mechanisms), other structural features may also be toxic from a different mechanistic point of view. They should be included in the analysis not only for concerns about their toxicity but also for the inventory of pollutants in the environment. The assumption that all congeners and isomers are recovered to the same degree from the cleanup columns would lead to false analytical results. Therefore, knowledge of the recovery of all PCBs from the cleanup columns is important. ACKNOWLEDGMENT The authors gratefully recognize the technical assistance of D. Kavich and J. P. Dach. The authors also acknowledge meaningful discussion with R. D. Smillie, F. Skinner, and A. Kharrat. Received for review May 7, 1997. Accepted October 21, 1997.X AC9704725

(24) Swerev, M.; Ballschmiter, K. Anal. Chem. 1987, 59, 2536-8.

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Abstract published in Advance ACS Abstracts, December 1, 1997.