Anal. Chem. 1985, 57,633-639
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Integrated, Multiple-Stage Chromatographic Method for the Separation and Identification of Polycyclic Aromatic Hydrocarbons in Complex Coal Liquids Richard B. Lucke,* Douglas W. Later, Cherylyn W. Wright, Edward K. Chess, a n d Walter C.Weimer Chemical Methods and Kinetics Section, Pacific Northwest Laboratory, Operated by Battelle Memorial Institute, Richland, Washington 99352
A multlple-stage methodology Is described for the separatlon and ldentlfkatlon of neutral polycycllc aromatic hydrocarbons (PAH) In complex coal Ilqulds. Fractional dlstlllatlon followed by adsorption chromatography on neutral alumina wlth subsequent reverse-phase hlgh-performance llquld chromatography were used for the lsolatlon of neutral PAH subfractlons. Capillary column gas chromatography, gas chromatographymass spectrometry, and dkect-probe mass spectromefry were used for the IdentlflCation of sample components. Thls method results In chemlcally less complex subfractions which enable more detalled characterization of both trace level and malor neutral PAH components. The appllcablllty of thls multlple-stage separation method Is demonstrated wlth a solvent refined coal (SRC) I1 material.
In recent years, synfuels have become of increased interegt because of their potential as an alternate energy source. The need to identify potentially toxic and carcinogenic compounds which may be present in alternate fuel materials has resulted in an increased effort to obtain detailed information about their chemical composition as it relates to biological response observed in laboratory test systems. However, synfuels, such as coal-derived liquids, are extremely complex, and the relationships between chemical composition and biological response are not always straightforward. The molecular weight distribution, degree of alkylation and hydrogenation, chemical functionality, and heteroatom content of coal liquids are all variables that contribute to the increased complexity of these materials. This complexity has led to the development of a wide variety of analytical methods aimed at achieving improved separations for chemical and biological characterization of synfuels. Coal liquid fractionation procedures have included fractional distillation (I-4), size-exclusion chromatography &9), solvent extraction (10-15), adsorption chromatography with alumina and/or silica (16-20), normal-phase high-performance liquid chromatography (HPLC) (2I-24), and reverse-phase HPLC (25-28). Analytical methods using combinations of these separation techniques are often required for complete chemical characterization and biological testing of these highly complex mixtures. For example, multidimensional coupledcolumn HPLC techniques that produce discrete fractions have been used to analyze polycyclic aromatic hydrocarbons (PAH) in coal liquids (28, 29). However, coupled-column HPLC requires modified instrumentation and is impractical for the preparative-scale fractionation of material that is often required for biological testing. In this laboratory, a four-step analytical methodology for the separation, identification, and quantitation of neutral PAH and their alkylated analogues in coal liquids has been developed and applied (Figure l). In general, the neutral PAH are the most abundant compound class in coal-derived syn0003-2700/85/0357-0633$0 1.50/0
fuels and have been shown to be primarily responsible for the tumorigenic activity of these materials (30,31). The integrated multiple-stage chromatographic method described in this paper employs fractional distillation followed by adsorption chromatography on neutral alumina, with subsequent reverse-phase HPLC. The combination of these separation techniques produces fractions which are chemically much less complex than the starting crude material and which are well defined in terms of chemical class and subclass. Furthermore, correlation of biological response to chemical constituency is facilitated. Capillary column GC, GC/MS, and direct-probe MS are used for the identification and quantitation of individual PAH components in each fraction. EXPERIMENTAL SECTION Materials. This analytical approach was demonstrated using a solvent refined coal I1 (SRC 11) material from the Fort Lewis, WA, pilot plant. Fractional distillation of the full boiling range material (stage one of the separationmethodology) was performed by Gulf Research and Development Co. (32). A distillate cut with a boiling point range of 8OC-850 O F was used in this investigation. Alumina Adsorption Column Chromatography. The adsorption method used for separating the neutral PAH fraction from coal liquids has been described by Later et al. (16). Briefly, 150-200 mg of the 800-850 OF distillate cut was adsorbed on 3 g of neutral aluminum oxide and packed on top of 6 g of neutral aluminum oxide (Brockman activity I, 80-200 mesh, Fisher No. A 950, Pittsburgh, PA). The alumina was stored in an oven at 160 OC prior to use in the adsorption column chromatographic step. This oven temperature produced an alumina with -1.5% water content (by weight). The details of this alumina standardization technique are available elsewhere (33). Sequential elution with hexane, benzene, and chloroform with 1% ethanol and methanol (spectral grade, Burdick & Jackson, Muskegon, MI) was done to obtain four fractions: aliphatic hydrocarbons (AH), neutral polycyclic aromatic hydrocarbons (PAH), nitrogen-containing polycyclic aromatics compounds (N-PAC),and hydroxy PAH (HPAH), respectively (see Figure 1). The PAH fraction was dried under nitrogen for gravimetric analysis and redissolved in methylene chloride (spectral grade, Burdick & Jackson) for further separation by HPLC. %verse-Phase High-Performance Liquid Chromatography. The PAH fraction was further separated and analyzed by using a Spectra Physics 8100 liquid chromatograph equipped with a Spectra Physics 8110 autosampler and a Waters Assoc. 440 UV absorbance detector fixed at a wavelength of 254 nm. A Spectra Physics 4100 computing integrator was used for data readout. Samples (subfractions) were collected with a modified HewlettPackard 79825 fraction collector. A Supelcosil RP8-DB, 5 pm, 25 cm X 10 mm i.d. reverse-phase column was used for the HPLC separation. The mobile-phase profile consisted of 69% methanol (spectral grade, Burdick & Jackson) in water (doubly distilled, deionized, Milli Q grade) for 40 min followed by a 20-min linear gradient to 79% methanol and a 10-min linear gradient to 100% methanol which was then held for 10 min. Finally, a 12-min period was used to return and equilibrate to initial conditions for repetitive injections. The column temperature was held at 50 "C, with an eluent flow rate of 6 mL/min. The injection volume was 50 pL. Twenty repetitive 0 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985
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Table I. Weight Percent of Fractions from a Full Boiling Range SRC I1 Coal Liquid Obtained from the Different Fractionation Stages of 800-850 "F distillate cut wt %
w t % of full bp SRC I1 material
fraction 800-850 "F SRC I1 distillate cut PAH fraction of 800-850 OF SRC I1 distillate cut HPLC subfraction 1 HPLC subfraction 2 HPLC subfraction 3 HPLC subfraction 4 HPLC subfraction 5 HPLC subfraction 6 HPLC subfraction 7 HPLC subfraction 8
0.28 0.34 0.21 0.32
3.0 1.6 2.9 7.9 9.8 6.0 9.2
HPLC subfraction total
1.46
41.6
FRACTIONATION BY DISTILLATION
STAGE 1
OF
700 750800850
300 I
I
I
I
I
ALUMINA ADSORPTION CHROMATOGRAPHY
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-- - - - - - - - _ REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
STAGE 3
\
HPLC #2 HPLC #4 HPLC #6 HPLC #8
HIGH RESOLUTION GAS CHROMATOGRAPHY DIRECT PROBE M A S S SPECTROMETRY GAS CHROMATOGRAPHYlMASS SPECTROMETRY Flgure 1. Integrated multiple-stage chromatographic scheme for complex coal liquids: aliphatic hydrocarbons (AH), neutral polycyclic aromatlc hydrocarbons(PAH), nRrogen-containing polycyclic aromatic compounds (N-PAC), and hydroxy-PAH (HPAH).
automated injections (at 4 mg/injection) were required to obtain adequate sample material in the eight selected subfractions for chemical characterization by GC, GC/MS, and MS. Sample workup of the HPLC subfractions was accomplished by reducing the methanol/water effluent volume under reduced pressure on a rotary evaporator to near dryness. The samples were then blown to dryness under nitrogen at 50 "C for gravimetric analysis. Gas Chromatography. The PAH fraction and each of the HPLC subfractions were analyzed using an Hewlett-Packard (HP) 5880A gas chromatograph equipped with a flame ionization detector (FID) held at 300 "C and a 25 m X 0.25 mm i.d. DB-5 fused silica column (0.25-wrn film thickness, J & W Scientific, Rancho Cordova, CA). Helium was used as a carrier gas at a linear velocity of 50 cm/s. Samples were injected by the splitless vaporization technique with the injector temperature set at 285 "C. The temperature of the GC oven was linearly programmed from 50 to 100 "C at 10 "C/min and then 100 to 300 "C at 3 "C/min during
re1 w t % distribution of PAH in HPLC subfraction
3.5 1.7
0.04 0.11
0.06 0.10
49 1.2
2.5 6.1 3.3 6.0 16.1 20.1 12.2 18.7 85.0
the analyses of the SRC I1 800-850 neutral PAH fraction. For the GC analyses of the HPLC subfractions, the oven temperature profile was 50-300 "C at 3 "C/min. Quantitation was based on the average response factors of standard compounds chromatographed at eight concentrations ranging over 2 orders of magnitude as previously described (34, 35). All standard compounds used were either purchased from Aldrich Chemical Co. (Milwaukee, WI) or obtained from private sources. In all cases the purity of the standard reference compounds was >98% as determined by GC. Additionally, the National Bureau of Standards SRM 1647 (priority pollutant PAH) was analyzed as an internal check of the response factors of solutions made in this laboratory. When standard compounds were not available, the response factors for compounds of similar retention time and chemical class were used. The PAH fraction of the 800-850 "F distillate cut was analyzed by an external calibration method only, while the HPLC subfractions were analyzed by an external calibration method using 2-chloroanthracene as an internal standard at a final concentration of 50 pg/mL. All quantitation was based on dilutions of the dried fractions from a single separation at three different concentration levels. The PAH fraction was analyzed at concentrations of 10, 5, and 2.5 mg/mL. The HPLC subfractions were analyzed at 8, 4, and 2 mg/mL. Components in these complex mixtures were identified by absolute retention time, GC/MS, and retention index system (36). Values are reported as the average of the three determinations with appropriate standard deviations of the means. MS Ana GC/MS. Identification of constituents in each HPLC subfraction was achieved using mass spectrometry (MS). Initially, the HPLC subfractions were analyzed by probe inlet MS to determine the major PAH components present and to establish the mass range of each fraction. These analyses were performed at low resolution (1:2000) on a VG Micromass ZAB-1F doublefocusing mass spectrometer operated at 6000-V accelerating potential at a scan rate of 3 s/decade from 50 to 400 m u . The source temperature was held at 250 "C during the analyses with a source trap current of 200 KAfor 70-eV analyses and 50 FA for 10-eV analyses. Data were acquired, stored, and processed on a PDPSA-based VG 2035 data system. HPLC subfractions 1-4 were analyzed by electron impact mass spectrometry using 70-eV ionizing energy. Due to the increased amount of fragmentation observed for the higher alkylated PAH analogues (greater than C,) which eluted predominantly in subfractions 5-8, these HPLC isolateswere analyzed at both 70 eV and at a lower ionizing energy, nominally 10 eV, to reduce the degree of fragmentation observed in the respective spectra and to allow molecular weight determinations of highly alkylated PAH species (alkylated analogues greater than C,) in the HPLC subfractions. A H P 5982.4 GC/MS system interfaced to a H P 5934A data system was used to obtain the mass spectra of the specific PAH compounds in each of the HPLC subfractions. The gas chromatograph was equipped with a 15 m X 0.25 mm i.d. DB-5 (J & W Scientific) fused silica capillary column; the oven temperature was programmed from 50 to 280 "C at 8 OC/min. Both on-column and vaporization injection techniques were used. The mass spectrometer was operated in the electron impact mode at 70-eV
635
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985
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ionizing energy with a 15-mA emission current. Spectra were acquired at a scan rate of 150 amu/s over a mass range of 50-350 m u . Mass spectrometric identifications from the GC/MS data were made by comparison of spectra from either available standard compounds or published spectra from the EPA/NIH Library of Mass Spectra.
RESULTS AND DISCUSSION Fractional distillation (stage one of Figure 1) provided gross fractionation of components in coal-derived process materials
according to boiling point, which correlates roughly to molecular weight. Previous studies have demonstrated that high-boiling distillate fractions are generally responsible for the majority of the biological activity of coal liquids (1-4). The chemical class separation of polycyclic aromatic compounds (PAC) by alumina adsorption column chromatography (stage two of Figure 1)has been reported for several coal-derived materials (16,33,37). Collection of a discrete PAH fraction from the SRC I1 800-850 OF distillate cut was achieved by
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alumina adsorption chromatography and used for HPLC subfractionation. The PAH fraction of the 800-850 OF coal liquid distillate cut obtained from the adsorption chromatographic step required an additional separation by reverse-phase HPLC for two reasons. First, the PAH fraction of this 50 OF distillate cut was still extremely complex, even after chemical class fractination. This is demonstrated by the complexity of the GC trace and mass spectrum in Figure 2A. Inspection of the gas chromatogram revealed the presence of many closely eluting components and lack of base-line resolution. In addition, the mass spectrum indicated that components of a wide mass range were present. Clearly, further separation was required to facilitate chemical analysis and to allow adequate separation of minor constituents that may be important in determining biological response. Second, certain PAH fractions isolated from coal liquids with constituents that have more than three aromatic rings have been shown to be potential carcinogens and mutagens (30,31,38);however, relatively little is known about the synergistic and/or antagonistic effects of complex mixtures on the biological expression of individual PAH. Hence, separation of the neutral PAH fraction into well-characterized subfractions would permit investigation of such effects. Biological testing of the subfractions using the microbial mutagenicity and initiation/ promotion mouse skin tumorigenicity assays could provide valuable insights for understanding the matrix effects of complex, coal-derived mixtures on carcinogenesis and mutagenesis. Reverse-phase HPLC was chosen for further separation of the PAH fraction (stage three of Figure 1)because of its ability to resolve the nonpolar PAH and to separate the parent and alkylated PAH (25,29,39).Initially, 14 HPLC subfractions were arbitrarily designated and collected for GC/MS analysis to determine the proper selection of HPLC cut points for preparation of subfractions of this material for biological testing. Subsequently, eight subfractions (shown in Figure 2B) were selected so that the parent and alkylated PAH components of interest would generally be grouped in separate subfractions. The selection process for designating the HPLC subfraction cut points was based solely on results from GC and GC/MS analyses; preselected standard reference compounds were not used to define subfraction parameters. The eight HPLC subfractions chosen provided an optimum number of fractions in terms of effort required for chemical analysis and information obtained from correlated chemical/biological investigations. The relative and normalized weight contributions of each HPLC subfraction as determined by gravimetric measurements are shown in Table I. It is apparent that the subfractions are greatly simplified compared to the full boiling point range material. For example, when normalized, HPLC subfraction 1constituted only 0.04% of the full boiling range crude material from which the distillate cuts were obtained. Almost 80% of the overall weight of the in3tial PAH fraction was isolated in HPLC cuts 5-8. The average recovery for 10 replicate runs in the alumina adsorption step was 95% (by weight) of the initial quantity of crude distillate cut introduced onto the column; 85% average total recovery was achieved for the HPLC fractions in 20 replications. Figure 3 shows the capillary gas chromatograms and the direct-probe mass spectra insets for three of the HPLC subfractions; Table I1 lists the identification and quantitation of the specific components in each of the eight HPLC subfractions. Comparison of the GC traces of the HPLC subfractions with that of the initial PAH fraction in Figure 2A reveals that the complexity of the HPLC cuts is indeed greatly reduced. HPLC subfractions 1and 2 showed base-line GC resolution
838
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985 100
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for most components while GC analyses of HPLC subfractions 5-8 showed increased complexity and decreased resolution due to the high degree of alkylation and the increased number of possible isomers (Figure 3). Nonalkylated PAH were the major components in subfractions 1-3 while the alkylated PAH are the major components in the remaining five subfractions. The six isomers of methylchrysene were major
OF
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components in the neutral PAH fraction and were detected in HPLC subfractions 1-5. Minor components that were not identified in the PAH fraction were separated and could readily be identified in the HPLC subfractions. For example, the sulfur-containing PAH which often require oxidation, further separation, and reduction before identification (40) or sulfur specific detection
ANALYTICAL CHEMISTRY, VOL. 57,
are now directly identified without undergoing these procedures. The benzonaphthothiophenes were well resolved in HPLC subfraction 1,and the methylbenzonaphthothiophenes were identified in HPLC subfraction 4. Since only selected components were identified and quantified, the sum total of the concentrations of components in Table I1 does not account for the total weight found in the HPLC subfractions. When comparing the quantitative composition of the PAH fraction before and after HPLC separation (last two column8 in Table 11), quantitative agreement was observed in some cases, although discrepancies in concentration are noticeable. The differences can be partly explained by errors in integration due to differences in the base-line definition of unresolved components in the more complex neutral PAH fraction vs. the less complex HPLC subfractions. Furthermore, the combined HPLC subfraction component concentrations were only corrected by using the 85% recovery obtained from gravimetric analysis of the HPLC subfractions and not from the recovery of individual compounds spiked into the complex mixture. It is also possible that coeluting components were present in the GC analysis of the neutral PAH fraction prior to HPLC separation. This separation method is of interest from the biological testing standpoint for two reasons. First, it isolates discrete chemical fractions in which the parent PAH are separated from their alkylated species. Also, i t isolates many of the putative carcinogens such as benzo[a]pyrene, 5-methylchrysene, and 7,12-dimethylbenz[a]anthracene.The biological responses of the individual HPLC subfractions compared to that of the unfractionated neutral PAH fraction may give valuable information in understanding the effect of the complex sample matrix on the biological expression of PAH components in a coal-derived process material. The integrated multiple-stage chromatographic methodology presented and discussed in this paper has been applied to the separation and analyses of a wide variety of coal liquefaction process materials including SRC I1 fKKl-850 O F and 850 O F plus distillate cuts, 650-700, 700-750, and >BOO O F distillate cuts from the EDS process, and an integrated twostage liquefaction (ITSL) 850 O F plus distillate cut. Results from the detailed chemical analyses of the SRC I1 800-850 O F distillate were presented in this paper. Results of the biological testing of the HPLC subfractions will appear in a subsequent paper. T o date, this multiple-stage separation scheme has only been applied for analysis of the PAH fraction but could easily be extended for analysis of the AH, N-PAC, and HPAC fractions from the alumina column chromatographic separation. Registry No. 1-Methylpyrene, 2381-21-7; methylbenzo[b]naphthofuran, 93755-97-6; benzo[b]naphtbo[2,1-d]thiophene, 239-35-0; benzo[b]naphtho[l,2-d]thiophene, 205-43-6;benzo[b]naphtho[2,3-d]thiophene,243-46-9; benz[a]anthracene, 56-55-3; benz[ blanthracene, 92-24-0; methylbenzo[b]naphthothiophene, 67526-85-6; 3-methylchrysene, 3351-31-3; 5-methylchrysene7 3697-24-3; methylchrysene, 41637-90-5; benzofluoranthene, 56832-73-6; benzo[k]fluoranthene, 207-08-9; benzo[e]pyrene, 192-97-2;benzo[a]pyrene, 50-32-8; perylene, 198-55-0;benzo[e]pyrene, 192-97-2; benzo[a]pyrene, 50-32-8; perylene, 198-55-0.
LITERATURE CITED (1) Wilson, E. W.; Wllley, C.; Later, D. W.; Lee, M. L. Fuel 1982, 61, 473-477. (2) WHson. B. W.; Pelroy, R. A.; Mahlum, D. D. In "Chemical Characterlzatlon and Genotoxlc Potential Related to Boiling Point for Fractionally
NO.3, MARCH 1985
639
Distilled SRC-I Coal Liquids"; NTIS: Springfield, VA, 1982; PNL-4277. Later, D. W.; Wright, C. W.; Wilson, B. W. Prepr. Pap.-Am. Chem. SOC..Dlv. FuelChem. 1983, 2 8 , 273-284. Pelroy, R. A.; Wilson, B. W. In "FractionalDlstlllatlon as a Strategy for Reducing the Genotoxic Potential of SpC I1 Coal Liquids"; NTIS:
Springfield, VA, 1981; PNL-3787. Streull, C. A. J . Chromatogr. 1971, 5 6 , 225-229. Jones, A. R.; Geurln, M. R.; Clark, B. R. Anal. Chem. 1977, 4 9 , 1766-1771.
Rao, T. K.; Allen. B. E.; Ramey. D. W.; Epler, J. L.; Rubin, I. B.; Guerin,
R. M.; Clark. 8. R. Mutat. Res. 1981, 8 5 , 29-39. Ho, C.-h.; Ma, C. Y.; Clark, B. R.; Guerin, M. R.; Rao, T. K.; Epler, J. L. Envlron. Res. 1980, 2 2 , 412-422. Toste, A. P.; Sklarew, D. S.; Pelroy, R. A. J . Chromatogr. 1882, 249, 267-282. Mlma, M. J.; Schultz. H.; McKlnstry, W. E. In. "Analytical Methods for Coal and Coal Products"; Karr, C, Ed.; Academic Press: New york, 1978; Vol. 1, Chapter 19, p 557. Burke, F. P.; Wlnschel, R. A.; Wooton, D. L. Fuel 1979, 5 8 , 539-541. Schwager, I.; Yen, T. F. Fuel 1978, 5 7 , 100-104. Steffgen, F. W.; Schroeder, K. T.; Bockrath, B. C. Anal. Chem. 1979, 51, 1164-1167. Boduszynskl, M. M.; Hurtublse, R. J.; Silver, H. F. Anal. Chem. 1982, 5 4 , 372-375. Schweighardt,F. K.; Thames, B. M. Anal. Chem. 1978, 5 0 , 372-375.
Later, D. W.; Lee, M. L.; Bartle, K. D.; Kong, R. C.; Vassllaros, D. L. Anal. Chem. 1981, 5 3 , 1612-1620.
Callen, R. B.; Simpson, C. A.; Bendoraitls, J. G.; Votz, S. E. Chem. Prod. Res. Dev. 1976, 15, 222-233.
Ind, €ng.
Boduszynskl, R. J.; Hurtublse, R. J.; Sliver, H. F. Anal. Chem. 1982, 5 4 , 375-381.
Leary, J. A.; Lafluer, A. L.; Longwell, J. P.; Peters, W. A.; Kruzel, E. L.; Blemann, K. In "Polynuclear Aromatic Hydrocarbons: Formation, M a tabollsm, and Measurement"; Cook, M., Dennis, A. J., Eds.; Battelle Press: Columbus, OH, 1983; pp 799-808. Farcaslu, M. Fuel 1977. 5 6 , 9-14. Toste, A. P.; Sklarew, D. S.; Pelroy, R. A. In "Advance Techniques In Synthetic Fuels Analysls"; Wright, C. W., Welmer, W. C., Felix, D. W., Eds.; NTIS: Springfleld, VA, 1983; Chapter 5, pp 74-94. Wise, S. A.; Chesler, S. N.; Hertz, H. S.; Hllpert, L. R.; May, W. E. Anal. Chem. 1977, 49, 2306-2310. S.; Brown, J. M.; Chesler, S. N.; Guenther, F. R.; Hllpert, L. R.; May, W. E.; Parrls, R. M.; Wise, S. A. Anal. Chem. 1980, 52, 1650-1657. Tomkins, B. A.; Grlest, W. H.; Caton, J. E.; Reagan, R . R. I n
Hertz, H.
"Polynuclear Aromatic Hydrocarbons: Physical and Blologlcal Chemlstry"; Cooke, M., Dennis, A. J., Fisher, G. L., Eds.; Battelle Press: Columbus, OH, 1982; pp 813-824. Burke, F. P.; Wlnschel, R. A.; Pochapsky, T. C. Prepr. Pap.-Am. Chem. SOC.,Dlv. FuelChem. 1981, 6 0 , 562-572. J . Chromatogr. Scl. 1978, 16, 289-229. Chmlelowlec, J. Anal. Chem. 1983, 5 5 , 2367-2374. Ogan, K.; Katz, E. Anal. Chem. 1982, 5 4 , 169-173. Sonnefeld, W. J.; Zoller, W. H.; May, W. E.; Wise, S. A. Anal. Chem. 1982, 5 4 , 723-727. Mahlum, D. D. J . Appl. Toxicol. 1983, 3 , 1, 31-33. Later, D. W.; Pelroy, R. A.; Mahlum, D. D.; Wright, C. W.; Lee, M. L.;
Dark, W. A.; McFadden, W. H.
Welmer, W. C.; Wilson, B. W. In "Polynuclear Aromatic Hydrocarbons: Seventh International Symposium on Formation, Metabolism, and Measurement";Cooke, M. W., Dennis, A. J., Eds.; Battelle Press: Columbus, OH, 1982; pp 771-783. Gary, J. A. "Solvent Refined Coal (SRC) Process: Selected Physical, Chemical and Thermodynamic Properties of Narrow Boiling Range Coal Llqulds for the SRC I1 Process"; NTIS: Springfield, VA, 1981. Later, D. W.; Lee, M. L. In "Advanced Techniques for Synthetic Fuels Analysis"; Wright, C. W., Weimer, W. C., Felix, W. D., Eds.; NTIS: Springfield, VA, 1983; Chapter 4, pp 44-73. Wilson, 8. W.; Pelroy, R. A.; Mahlum, D. D.; Frazler, M. E.; Later, D.
W.; Wright, C. W. Fuel 1984, 63, 46-55. Wright, C. W. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1984, 7 , 83-88.
Vassllaros, D. L.; Kong. R. C.; Later, D. W.; Lee, M. L. J . Chromatogr. 1982, 252, 1-20.
Later, D. W.; Lee, M. L.; Wilson, B. W. Anal. Chem. 1982, 5 4 , 117-123.
Mahlum, D. D.; Wright, C. W.; Chess, E. K.; Wilson, B. W. Cancer Res. 1984, 44, 5176-5181.
May, W. E.; Wise, S. A. Anal. Chem. 1984, 5 6 , 225-232. Wllley, C.; Iwao, M.; Castle, R. N.; Lee, M. L. Anal. Chem. 1981, 5 3 , 400-407.
RECEIVEDfor review July 2,1984. Accepted October 10,1984. This work was supported by the Department of Energy under Contract DE-AC06-76RLO.
