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Determination and mutagenic activity of nitrogen-containing thiophenic compounds in coal-derived products
Determination and mutagenic activity of nitrogen-containing thiophenic compounds in coal-derived products Masaharu Nishioka, Philip A. Smith, Hirotaka Kudo,“, Daniel R. Muchiri”, LeRoy H. Klemmt
Gary M. Booth, Milton Raymond N. Castle*
L. Lee, and
Departments of Chemistry and Zoology, Brigham Young University, Provo, UT84602, USA * Department of Chemistry, University of South Florida, Tampa, FL 33620, USA t Department of Chemistry, University of Oregon, Eugene, OR 97403, USA (Received 31 July 1985; revised 26 September 7985)
Polycyclic aromatic compounds containing both nitrogen and sulphur heteroatoms in aromatic rings were determined in a coal-derived liquid and a coal tar by capillary column gas chromatography and gas chromatography-mass spectrometry. G.c. with a flame photometric detector was used to analyse the nitrogen-containing polycyclic aromatic compound fraction obtained by adsorption chromatography on neutral alumina. The four possible isomers of the azadibenzothiophenes, and various other azathiophenic compounds, were positively identified by comparison with retention data of newly synthesized standard compounds. The aminodibenzothiophenes were the major nitrogen/sulphur-containing heterocycles in the coal liquid, while the azathiophenic compounds were the major ones in the coal tar. The differences between the two coal-derived products were related to the reaction conditions during their production. The four isomers of the azadibenzothiophenes were assayed for mutagenicity using the Ames test and compared with the mutagenicities previously reported for the aminodibenzothiophenes. It was found that the azadibenzothiophenes were not mutagenic, as opposed to the aminodibenzothiophenes. (Keywords: coal-derived products; mutagenicity;
thiophenic compounds)
Coal-derived liquids are extremely complex mixtures of organic chemicals, the majority of which are polycyclic aromatic compounds (PAC). While the polycyclic
aromatic hydrocarbons (PAH) comprise the largest chemical class fraction of most coal liquids, there are substantial amounts of nitrogen-, sulphur- and oxygencontaining PACs. In most previous studies, PACs containing only one heteroatom were considered. However, evidence has been mounting over the last decade for the existence of PACs containing two heteroatoms in coal-derived products’m5; compounds containing combinations of sulphur, nitrogen and oxygen were tentatively identified by high resolution mass spectrometry. In recent studies, oxygen/sulphur-containing PACs6, nitrogen/sulphur-containing PACs’ and nitrogen/oxyin a solventgen-containing PACs’, were identified refined coal liquid (SRC II heavy distillate) by capillary chromatography (g.c.) and gas column gas chromatography-mass spectrometry (g.c.-m.s.). Newly synthesized standard compounds were used for positive identification of mixture components by comparison of retention data. Also, chemical class fractionation, g.c. on selective stationary phases, derivatization, and g.c. with selective detection were all applied to the detailed characterization of coal-derived products and other similar materials. The major nitrogen/sulphur-containing PACs in the 0016-2361/86/05071 l-04$3.00 sFi‘,1986 Butterworth & Co. (Publishers) Ltd
SRC II heavy distillate were aminodibenzothiophenes and alkylated aminodibenzothiophenes’. These are compounds in which the sulphur heteroatom is part ofthe aromatic ring and the amino-group is substituted on the ring. All isomers of the aminodibenzothiophenes were detected. The four aminodibenzothiophenes were assayed for genotoxicity by two methods: the Ames test and unscheduled DNA synthesis in primary rat hepatocytes. The 2- and 3-aminodibenzothiophenes demonstrated significant genotoxicities in both assays, and 10 to 100 times greater mutagenicity in the Ames test than is demonstrated by benzo[u]pyrene. In contrast to this coal liquid, the major nitrogen/sulphur-containing heterocycles in an anthracene oil and in a coal tar were tentatively identified by Burchill et a1.4,5 to be compounds with both nitrogen and sulphur in the rings. These results have prompted the further investigation of the nitrogen/sulphur-containing PAC in other coal-derived products. In this paper, the analysis of a solvent-refined coal liquid and a coal tar for PACs which contain nitrogen/sulphur heterocycles is described. Capillary column g.c. with sulphur-selective flame photometric detection and mass spectrometry was used to positively identify the components in a fraction containing only compounds which possess nitrogen heteroatoms. Furthermore, the relative abundances of the nitrogen/sulphur-containing PACs in these two samples
FUEL, 1986, Vol 65, May
711
Determination
of nitrogen-containing
thiophenic
compounds:
were compared and discussed with respect to their different process conditions. Pure reference compounds were also synthesized and tested for genotoxicity.
M. Nishioka
et al.
controls as well as positive controls were included with each test. The number of revertants per plate was determined by manual counting (direct or indirect). Indirect counting was performed by counting a 5 cm2 plate area as previously described’.
EXPERIMENTAL The solvent-refined coal heavy distillate (SRC II HD: 26&45o”C boiling point range) was collected during the processing of a West Virginia coal from the Pittsburgh Seam, and obtained from the Fort Lewis, Washington, pilot plant which was operated by the Pittsburgh & Midway Coal Mining Co. This material is of pilot plant origin and should not necessarily be considered as representative of products that may eventually be produced on a commercial scale. The coal tar was obtained from S.A. Wise (National Bureau of Standards, Washington, DC). The four aminodibenzothiophene isomers and the azathiophenic compounds used in this study were not commercially available and, therefore, had to be synthesized 9,1o in the laboratory. The SRC II HD material and the coal tar were fractionated into chemical classes by adsorption chromatography on neutral alumina and silicic acid according to the procedure of Later et al.“. The third alumina column fraction (A-3) which was composed of the nitrogen-containing PACs, the second silicic acid column fraction (S-2) which was composed of the amino polycyclic aromatic hydrocarbons (APAH), and the third silicic acid column fraction (S-3) which was composed of the tertiary nitrogen-containing PACs were analysed in this study. Portions of the S-2 fractions of both samples were derivatized with pentafluoropropionic anhydride (PFPA) as previously described”. Two Hewlett-Packard Model 5880 gas chromatographs, one equipped with a sulphur-selective flame photometric detector (FPD) and the other with a ‘j3Ni electron capture detector (ECD), were used in this study. Hydrogen (FPD) and helium (ECD) were used as carrier gases at linear velocities of 100 and 50cm s-l, respectively. Semi-quantitation was accomplished by comparing sample component peak areas (FPD) with benzo[b]thiophene and standard those of dibenzothiophene. The capillary columns used in this study were prepared by coating 20 m x 0.3 mm i.d. lengths of fused silica tubing (Hewlett-Packard, Avondale, PA) with SE-54 and a 25% biphenyl polymethylsiloxane stationary phase13 (0.25 pm film thickness). The preparation of these columns was previously described’. A Hewlett-Packard Model 5982A g.c.-m.s. system was used to obtain mass spectral confirmation of identified compounds. The same chromatographic column was used for g.c.-m.s. and the mass spectrometer was operated in the electron impact mode at 70 eV electron energy. The Ames mutagenicity assay was performed as described by Ames et ~1.‘~ with minor modifications. These modifications consisted of using 20 ml of medium and 4 ml of top agar per plate instead of 30 ml and 2 ml per plate, respectively. Salnzoneffa typhimurium strain TA98 (provided by Bruce Ames, Berkeley, CA) was exposed to various concentrations of the compounds in dimethylsulphoxide. The compounds dissolved tested for mutagenicity at 4% S9 rat liver homogenate (Litton Bionetics, Kensington, MD) concentration (0.04ml S9 per ml phosphate buffer solution). Solvent
712
FUEL, 1986, Vol 65, May
RESULTS
AND DISCUSSION
The FPD chromatogram of the SRC II HD A-3 fraction on a 25% biphenyl methylpolysiloxane stationary phase is shown in Figure 1. Retention times were compared with the newly synthesized standard samples. Several small peaks (most likely sulphurcontaining secondary nitrogen heterocycles) were eliminated after acid extraction of the fraction’. A modification of the method described by Later et ~1.~~3~~was used to distinguish the sulphur/tertiary nitrogen-containing PAC and the sulphur/amino-containing PAC is previously reported’. The FPD chromatogram of the coal tar A-3 fraction on the same biphenyl polysiloxane stationary phase is shown in Figure 2. A number of azathiophenic compounds
0 120
10 140
TIM:6min.l 180 TEMPWI
30 220
40 260
Figure 1 FPD chromatogram ofthe SRC II HD A-3 fraction on a 250, biphenyl polysiloxane stationary phase. Conditions: temperature programme from 120 to 265°C at 4’C min-‘, after an initial 2 min isothermal period; hydrogen carrier gas at lOOcms_‘. Peak assignments are listed in Table I
Figure 2 biphenyl programme 4T minat 1OOcm
FPD chromatogram of the coal tar A-3 fraction on a 25”‘b polysiloxane stationary phase. Conditions: temperature from 70 to 120°C at 10°C min-‘, then from 120 to 265°C at ‘, after an initial 2 min isothermal period; hydrogen carrier gas s-‘. Peak assignments are listed in Table I
Determination Table 1 Relative retention coal tar
times and semi-quantitation
of nitrogen-containing
of PACs containing
both sulphur
thiophenic and nitrogen
compounds:
M. Nishioka
et al.
in an SRC II heavy distillate coal liquid and a
Concentration’
kg g-‘) Peak No. 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19
Relative timeh
Compound” 4-azabenzothiophene 7-azabenzothiophene 6-azabenzothiophene I-azadibenzothiophene 4-azadibenzothiophene 2-azadibenzothiophene 3-azadibenzothiophene Azadibenzothiophene isomers I -azanaphtho[2,1 -b]thiophene Azaphenanthro[4,Wrcd]thiophene isomers 4-aminodibenzothiophene I-aminodibenzothiophene 2-aminodibenzothtophene 3-aminodibenzothiophene C,-Aminodibenzothiophene C,-Aminodibenzothiophene 3-azaphenanthro[9,10-hlthiophene 3-azaphenanthro[2,1-blthiophene 3-azaphenanthro[4,3-blthiophene Azabenzonaphthothiophene isomers C,-Azabenzonaphthothiophene isomers
a Compounds which are not numbered were not found in these coal-derived * Retention relative to naphtho[2,3_b]thiophene ’ Approximate concentration in pg g -i in the crude SRC II heavy distillate d These compounds were detected but not quantified ’ Previously reported in reference 7
including all isomers of the azadibenzothiophenes were positively identified in this study. It was confirmed by comparison of the chromatograms of the A-3 and S-3 fractions that most of the nitrogen/sulphur-containing PAC in this sample were tertiary nitrogen/sulphur heterocycles. Further confirmation of peak identities was obtained by g.c.-m.s. analysis. Table I lists the relative retention times of the PAC containing both sulphur and nitrogen identified in this study, and the results of semiquantitation of a number of these compounds. Tertiary nitrogen heterocycles are more thermally stable than amino-containing PACs, and are major nitrogen-containing PACs in coal-derived products5”5. Azathiophenic compounds were tentatively identified in an anthracene oil and a coal tar using g.c.-m.s. by Burchill et a1.4,5 In the present work, the aminodibenzothiophenes were found to be the major nitrogen/sulphur-containing PAC in the SRC II coal liquid, while the azathiophenic compounds were the major ones in the coal tar. The differences between the two coal-derived products apparently relate to the reaction conditions. In the SRC II process, hydrogen was used in the reaction, and metals contained in the recycled bottoms were utilized as Since amino-PAC in coal liquids result from catalysts”. the ring opening of nitrogen heterocycles by hydrogenation’**r9, aminodibenzothiophenes seem to be more abundant in such products.
On the containing
other PAC
hand, sulphur/tertiary were found to be
nitrogenthe major
retention SRC II HD
0.361 0.361 0.380 0.956 1.04 1.05 1.05
Coal tar n d 0.30 1.7 d d tl tl
0.05
11 0.979
d 1.43 1.47 1.52 1.54
0.33’ 0.32 0.11’ 2.5’ d d
1.74 1.78 1.80
2.0 0.20
li
d
d products and the coal tar
A 4
9
6
I
Figure 3 ECD chromatograms of the PFP derivatized APAH (S-2 fractions) from (A) the SRC II HD and (B) the coal tar on an SE-54 stationary phase. Conditions: temperature programme from 70 to 100°C at 10°C min-‘, then from 100 to 265-C at 4°C mini ‘, after an initial 2 min isothermal period; helium carrier gas at 50 cm s- ‘. Key: (1), I-aminonaphthalene; (2) 2-aminobiphenyl; (3), 2-aminonaphthalene; (4) 3-aminobiphenyl; (5), 4-aminobiphenyl; (6) 4-aminofluorene; (7) laminofluorene; aminoanthracenes/aminophenanthrenes/ (8), aminophenylnaphthalenes; (9), aminopyrene; (lo), aminochrysene
FUEL, 1986,
Vol 65, May
713
Determination Table 2
of nitrogen-containing
Ames mutagenicities
of the isomeric
thiophenic
compounds:
M. Nishioka
et al.
azadibenzothiophenes TA98 revertantsjplate”
Compound
l.Opg
2.0 pg
5.0 ,cg
10.0 ,lg
50.0 pg
100.0pg
I-azadibenzothiophene 2-azadibenzothiophene 3-azadibenzothiophene 4-azadibenzothiophene
7*2 0+3 24+5 7+4
4*2 Ok6 18&10 4+6
18k6 11+4 19+7 9*3
7*2 1*5 30*4 4+5
5i6 0*4 18?7 9*5
5*1 Ok2 5*4 4*3
’ All values are revertants (the average of three Dlates at a eiven concentration the mean of each concentiation. In-all cases, %‘a S9 was-used
sulphur/nitrogen-containing components in the high temperature treated coal tar sample. To investigate the relative abundances of amino-PAC in both products, careful analyses using the sensitive ECD were made. Figure 3 shows the ECD chromatograms of the PFP derivatized S-2 fractions of (a) the SRC II HD and (b) the coal tar, respectively. These chromatograms show only PFP derivatized APAH12s15. Notice that the abundance of the 2-and 3-ring APAH in the SRC II HD is much larger than for the coal tar. For example, the concentrations of 2-aminonaphthalene in the SRC II HD and the coal tar were 60 ppm and 5 ppm, respectively. Since the amino groups are produced in the SRC II 20, APAH and amino/sulphurprocess by hydrogenation containing PACs seem to be relatively abundant in this sample compared with the coal tar. Similarly, the easily desulphurized sulphur heterocycles such as naphtho[2,3blthiophene, and the 4- and 5-ring sulphur heterocycles derived from naphtho[2,3_b]thiophene, were not found in the SRC II sample, while these sulphur heterocycles were present in the coal tar as reported elsewhere2 I. The mutagenicities of all of the isomeric azadibenzothiophenes are listed in Table 2. Although the 2- and 3-aminodibenzothiophenes were strongly mutagenic and their average mutagenic response was 10 to 100 hundred times greater than the average response of However, all azadibenzothiophenes benzo[a]pyrene7. were inactive. Similarly, the azaphenanthrenes, which are structurally similar to the azadibenzothiophenes were inactive or demonstrated only weak mutagenic activities22. Recently, the microbial mutagenicities of numerous isomeric 3-to 5-ring sulphur heterocycles have . The presence of a sulphur been extensively studied 23m26 heteroatom was found to give little effect on mutagenicity. Likewise, compounds containing both nitrogen and sulphur heteroatoms in the rings also demonstrated little mutagenic activity.
minus the average of four solvent controls) + the standard
This work was supported by the Department of Energy, Office of Health and Environmental Research, Contract No. DE-AC02-79EV10237.
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FUEL, 1986, Vol 65, May
from
REFERENCES
7
8 9 10 II 12 13
14 15 16 17 18 19 20 21 22
23 24
ACKNOWLEDGEMENT
deviation
25 26
Mckay, J. F., Weber, J. H. and Latham, D. R. Anul. C’hm. 1976, 48, 891 Paudler, W. W. and Cheplen, M. Furl 1979, 58, 775 Hunt, D. F. and Shabanowitz, J. Anul. Chem. 1982, 54. 574 Burchill, P., Herod, A. A. and Pritchard, E. J. Chrorntrroyr. 1982, 242, 65 Burchill, P., Herod, A. A. and Pritchard, E. far/ 1983, 62, 20 Nishioka, M., Lee, M. L., Kudo, H., Muchiri, D. R., Baldwin, L. J.. Pakray, S., Stuart, J. G. and Castle, R. N. Anul. Chem. 1985, 57. 1327 Nishioka, M.,Campbell, R. M., West, W. R., Smith, P. A., Booth, G. M., Lee, M. L., Kudo, H. and Castle, R. N. Anul. Chern. 1985. 57, 1868 Nishioka, M., Campbell, R. M., Lee, M. L.. Muchiri, D. R., Stuart, J. G. and Castle, R. N. Amrl. Chem 1985, 57. 2211 Kudo, H., Castle, R. N. and Lee, M. L. J. Hetrrocyclic Chem. 1985, 22, 211 Castle, R. N. (unpublished results) Later, D. W., Lee, M. L., Bartle, K. D., Kong. R. C. and Vassilaros, D. L. Anul. Chrm. 1981, 53, 1612 Later, D. W., Lee, M. L. and Wilson, B. W. Anul. Chem. 1982,54, II7 Kuei, J. C.,Shelton, J. I.,Castle, L. W., Kong, R. C., Richter, B. E., Bradshaw, J. S. and Lee, M. L. J. High. Resole. Ckromatoyr./Chromutoyr. Commun. 1984, 7, 13 Ames, B. N., McCann, J. M. and Yamasaki, E. Mut. Rex 1975,31, 347 Later, D. W., Andros, T. G. and Lee, M. L. And. C’hem. 1983,55, 2126 Flinn, R. A., Larson, 0. A. and Beuther. H. Hydrocarbon Prows & Prtrol. Rc$ner. 1963, 42(9), 129 Schmid, B. K. and Jackson, D. M. 85th National Meeting of AIchE, Philadelphia, PA, 448 June 1978 Shabatai, J., Veluswamy, L. and Oblad, A. G. Amer. Chon. Sot. Dir. Fue( Chern., Prepr. 1978, 23(l), 1 I4 Burchill, P., Herod, A. A. and Prichard. E. J. Chromutogr. 1982, 246. 271 Nishioka, M. and Lee. M. L., in preparation Nishioka, M., Lee, M. L. and Castle, R. N. Fuel (submitted) Later, D. W. ‘Handbook of Polycyclic Aromatic Hydrocarbons’, (Vol. 2) Eds. A. Bjorseth and T. Ramdahl; Marcel Dekker: New York, NY (in press) Pelroy, R. A., Stewart, D. L., Tominaga. Y., Iwao, M., Castle, R. N. and Lee, M. L. Muttrrion Rrs. 1983, 117, 31 Eastmond, D. A., Booth. G. M. and Lee, M. L. Arch. Etxirorl. Conram. Toxicol. 1984, 13, 105 McFall. T., Booth, G. M.. Lee, M. L.. Tominaga, Y.. Pratap. R.. Tedjamulia, M. and Castle. R. N. MutcrtionRex 1984, 135, 97 Pelroy, R. A,Stewart, D. L., Lee, M. L., Pratap, R.. Tominaga. Y., Iwao, M., Thompson, R. D. and Castle, R. N. Mutation Rex (in press)
Gary M. Booth, Milton Raymond N. Castle*
L. Lee, and
Departments of Chemistry and Zoology, Brigham Young University, Provo, UT84602, USA * Department of Chemistry, University of South Florida, Tampa, FL 33620, USA t Department of Chemistry, University of Oregon, Eugene, OR 97403, USA (Received 31 July 1985; revised 26 September 7985)
Polycyclic aromatic compounds containing both nitrogen and sulphur heteroatoms in aromatic rings were determined in a coal-derived liquid and a coal tar by capillary column gas chromatography and gas chromatography-mass spectrometry. G.c. with a flame photometric detector was used to analyse the nitrogen-containing polycyclic aromatic compound fraction obtained by adsorption chromatography on neutral alumina. The four possible isomers of the azadibenzothiophenes, and various other azathiophenic compounds, were positively identified by comparison with retention data of newly synthesized standard compounds. The aminodibenzothiophenes were the major nitrogen/sulphur-containing heterocycles in the coal liquid, while the azathiophenic compounds were the major ones in the coal tar. The differences between the two coal-derived products were related to the reaction conditions during their production. The four isomers of the azadibenzothiophenes were assayed for mutagenicity using the Ames test and compared with the mutagenicities previously reported for the aminodibenzothiophenes. It was found that the azadibenzothiophenes were not mutagenic, as opposed to the aminodibenzothiophenes. (Keywords: coal-derived products; mutagenicity;
thiophenic compounds)
Coal-derived liquids are extremely complex mixtures of organic chemicals, the majority of which are polycyclic aromatic compounds (PAC). While the polycyclic
aromatic hydrocarbons (PAH) comprise the largest chemical class fraction of most coal liquids, there are substantial amounts of nitrogen-, sulphur- and oxygencontaining PACs. In most previous studies, PACs containing only one heteroatom were considered. However, evidence has been mounting over the last decade for the existence of PACs containing two heteroatoms in coal-derived products’m5; compounds containing combinations of sulphur, nitrogen and oxygen were tentatively identified by high resolution mass spectrometry. In recent studies, oxygen/sulphur-containing PACs6, nitrogen/sulphur-containing PACs’ and nitrogen/oxyin a solventgen-containing PACs’, were identified refined coal liquid (SRC II heavy distillate) by capillary chromatography (g.c.) and gas column gas chromatography-mass spectrometry (g.c.-m.s.). Newly synthesized standard compounds were used for positive identification of mixture components by comparison of retention data. Also, chemical class fractionation, g.c. on selective stationary phases, derivatization, and g.c. with selective detection were all applied to the detailed characterization of coal-derived products and other similar materials. The major nitrogen/sulphur-containing PACs in the 0016-2361/86/05071 l-04$3.00 sFi‘,1986 Butterworth & Co. (Publishers) Ltd
SRC II heavy distillate were aminodibenzothiophenes and alkylated aminodibenzothiophenes’. These are compounds in which the sulphur heteroatom is part ofthe aromatic ring and the amino-group is substituted on the ring. All isomers of the aminodibenzothiophenes were detected. The four aminodibenzothiophenes were assayed for genotoxicity by two methods: the Ames test and unscheduled DNA synthesis in primary rat hepatocytes. The 2- and 3-aminodibenzothiophenes demonstrated significant genotoxicities in both assays, and 10 to 100 times greater mutagenicity in the Ames test than is demonstrated by benzo[u]pyrene. In contrast to this coal liquid, the major nitrogen/sulphur-containing heterocycles in an anthracene oil and in a coal tar were tentatively identified by Burchill et a1.4,5 to be compounds with both nitrogen and sulphur in the rings. These results have prompted the further investigation of the nitrogen/sulphur-containing PAC in other coal-derived products. In this paper, the analysis of a solvent-refined coal liquid and a coal tar for PACs which contain nitrogen/sulphur heterocycles is described. Capillary column g.c. with sulphur-selective flame photometric detection and mass spectrometry was used to positively identify the components in a fraction containing only compounds which possess nitrogen heteroatoms. Furthermore, the relative abundances of the nitrogen/sulphur-containing PACs in these two samples
FUEL, 1986, Vol 65, May
711
Determination
of nitrogen-containing
thiophenic
compounds:
were compared and discussed with respect to their different process conditions. Pure reference compounds were also synthesized and tested for genotoxicity.
M. Nishioka
et al.
controls as well as positive controls were included with each test. The number of revertants per plate was determined by manual counting (direct or indirect). Indirect counting was performed by counting a 5 cm2 plate area as previously described’.
EXPERIMENTAL The solvent-refined coal heavy distillate (SRC II HD: 26&45o”C boiling point range) was collected during the processing of a West Virginia coal from the Pittsburgh Seam, and obtained from the Fort Lewis, Washington, pilot plant which was operated by the Pittsburgh & Midway Coal Mining Co. This material is of pilot plant origin and should not necessarily be considered as representative of products that may eventually be produced on a commercial scale. The coal tar was obtained from S.A. Wise (National Bureau of Standards, Washington, DC). The four aminodibenzothiophene isomers and the azathiophenic compounds used in this study were not commercially available and, therefore, had to be synthesized 9,1o in the laboratory. The SRC II HD material and the coal tar were fractionated into chemical classes by adsorption chromatography on neutral alumina and silicic acid according to the procedure of Later et al.“. The third alumina column fraction (A-3) which was composed of the nitrogen-containing PACs, the second silicic acid column fraction (S-2) which was composed of the amino polycyclic aromatic hydrocarbons (APAH), and the third silicic acid column fraction (S-3) which was composed of the tertiary nitrogen-containing PACs were analysed in this study. Portions of the S-2 fractions of both samples were derivatized with pentafluoropropionic anhydride (PFPA) as previously described”. Two Hewlett-Packard Model 5880 gas chromatographs, one equipped with a sulphur-selective flame photometric detector (FPD) and the other with a ‘j3Ni electron capture detector (ECD), were used in this study. Hydrogen (FPD) and helium (ECD) were used as carrier gases at linear velocities of 100 and 50cm s-l, respectively. Semi-quantitation was accomplished by comparing sample component peak areas (FPD) with benzo[b]thiophene and standard those of dibenzothiophene. The capillary columns used in this study were prepared by coating 20 m x 0.3 mm i.d. lengths of fused silica tubing (Hewlett-Packard, Avondale, PA) with SE-54 and a 25% biphenyl polymethylsiloxane stationary phase13 (0.25 pm film thickness). The preparation of these columns was previously described’. A Hewlett-Packard Model 5982A g.c.-m.s. system was used to obtain mass spectral confirmation of identified compounds. The same chromatographic column was used for g.c.-m.s. and the mass spectrometer was operated in the electron impact mode at 70 eV electron energy. The Ames mutagenicity assay was performed as described by Ames et ~1.‘~ with minor modifications. These modifications consisted of using 20 ml of medium and 4 ml of top agar per plate instead of 30 ml and 2 ml per plate, respectively. Salnzoneffa typhimurium strain TA98 (provided by Bruce Ames, Berkeley, CA) was exposed to various concentrations of the compounds in dimethylsulphoxide. The compounds dissolved tested for mutagenicity at 4% S9 rat liver homogenate (Litton Bionetics, Kensington, MD) concentration (0.04ml S9 per ml phosphate buffer solution). Solvent
712
FUEL, 1986, Vol 65, May
RESULTS
AND DISCUSSION
The FPD chromatogram of the SRC II HD A-3 fraction on a 25% biphenyl methylpolysiloxane stationary phase is shown in Figure 1. Retention times were compared with the newly synthesized standard samples. Several small peaks (most likely sulphurcontaining secondary nitrogen heterocycles) were eliminated after acid extraction of the fraction’. A modification of the method described by Later et ~1.~~3~~was used to distinguish the sulphur/tertiary nitrogen-containing PAC and the sulphur/amino-containing PAC is previously reported’. The FPD chromatogram of the coal tar A-3 fraction on the same biphenyl polysiloxane stationary phase is shown in Figure 2. A number of azathiophenic compounds
0 120
10 140
TIM:6min.l 180 TEMPWI
30 220
40 260
Figure 1 FPD chromatogram ofthe SRC II HD A-3 fraction on a 250, biphenyl polysiloxane stationary phase. Conditions: temperature programme from 120 to 265°C at 4’C min-‘, after an initial 2 min isothermal period; hydrogen carrier gas at lOOcms_‘. Peak assignments are listed in Table I
Figure 2 biphenyl programme 4T minat 1OOcm
FPD chromatogram of the coal tar A-3 fraction on a 25”‘b polysiloxane stationary phase. Conditions: temperature from 70 to 120°C at 10°C min-‘, then from 120 to 265°C at ‘, after an initial 2 min isothermal period; hydrogen carrier gas s-‘. Peak assignments are listed in Table I
Determination Table 1 Relative retention coal tar
times and semi-quantitation
of nitrogen-containing
of PACs containing
both sulphur
thiophenic and nitrogen
compounds:
M. Nishioka
et al.
in an SRC II heavy distillate coal liquid and a
Concentration’
kg g-‘) Peak No. 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19
Relative timeh
Compound” 4-azabenzothiophene 7-azabenzothiophene 6-azabenzothiophene I-azadibenzothiophene 4-azadibenzothiophene 2-azadibenzothiophene 3-azadibenzothiophene Azadibenzothiophene isomers I -azanaphtho[2,1 -b]thiophene Azaphenanthro[4,Wrcd]thiophene isomers 4-aminodibenzothiophene I-aminodibenzothiophene 2-aminodibenzothtophene 3-aminodibenzothiophene C,-Aminodibenzothiophene C,-Aminodibenzothiophene 3-azaphenanthro[9,10-hlthiophene 3-azaphenanthro[2,1-blthiophene 3-azaphenanthro[4,3-blthiophene Azabenzonaphthothiophene isomers C,-Azabenzonaphthothiophene isomers
a Compounds which are not numbered were not found in these coal-derived * Retention relative to naphtho[2,3_b]thiophene ’ Approximate concentration in pg g -i in the crude SRC II heavy distillate d These compounds were detected but not quantified ’ Previously reported in reference 7
including all isomers of the azadibenzothiophenes were positively identified in this study. It was confirmed by comparison of the chromatograms of the A-3 and S-3 fractions that most of the nitrogen/sulphur-containing PAC in this sample were tertiary nitrogen/sulphur heterocycles. Further confirmation of peak identities was obtained by g.c.-m.s. analysis. Table I lists the relative retention times of the PAC containing both sulphur and nitrogen identified in this study, and the results of semiquantitation of a number of these compounds. Tertiary nitrogen heterocycles are more thermally stable than amino-containing PACs, and are major nitrogen-containing PACs in coal-derived products5”5. Azathiophenic compounds were tentatively identified in an anthracene oil and a coal tar using g.c.-m.s. by Burchill et a1.4,5 In the present work, the aminodibenzothiophenes were found to be the major nitrogen/sulphur-containing PAC in the SRC II coal liquid, while the azathiophenic compounds were the major ones in the coal tar. The differences between the two coal-derived products apparently relate to the reaction conditions. In the SRC II process, hydrogen was used in the reaction, and metals contained in the recycled bottoms were utilized as Since amino-PAC in coal liquids result from catalysts”. the ring opening of nitrogen heterocycles by hydrogenation’**r9, aminodibenzothiophenes seem to be more abundant in such products.
On the containing
other PAC
hand, sulphur/tertiary were found to be
nitrogenthe major
retention SRC II HD
0.361 0.361 0.380 0.956 1.04 1.05 1.05
Coal tar n d 0.30 1.7 d d tl tl
0.05
11 0.979
d 1.43 1.47 1.52 1.54
0.33’ 0.32 0.11’ 2.5’ d d
1.74 1.78 1.80
2.0 0.20
li
d
d products and the coal tar
A 4
9
6
I
Figure 3 ECD chromatograms of the PFP derivatized APAH (S-2 fractions) from (A) the SRC II HD and (B) the coal tar on an SE-54 stationary phase. Conditions: temperature programme from 70 to 100°C at 10°C min-‘, then from 100 to 265-C at 4°C mini ‘, after an initial 2 min isothermal period; helium carrier gas at 50 cm s- ‘. Key: (1), I-aminonaphthalene; (2) 2-aminobiphenyl; (3), 2-aminonaphthalene; (4) 3-aminobiphenyl; (5), 4-aminobiphenyl; (6) 4-aminofluorene; (7) laminofluorene; aminoanthracenes/aminophenanthrenes/ (8), aminophenylnaphthalenes; (9), aminopyrene; (lo), aminochrysene
FUEL, 1986,
Vol 65, May
713
Determination Table 2
of nitrogen-containing
Ames mutagenicities
of the isomeric
thiophenic
compounds:
M. Nishioka
et al.
azadibenzothiophenes TA98 revertantsjplate”
Compound
l.Opg
2.0 pg
5.0 ,cg
10.0 ,lg
50.0 pg
100.0pg
I-azadibenzothiophene 2-azadibenzothiophene 3-azadibenzothiophene 4-azadibenzothiophene
7*2 0+3 24+5 7+4
4*2 Ok6 18&10 4+6
18k6 11+4 19+7 9*3
7*2 1*5 30*4 4+5
5i6 0*4 18?7 9*5
5*1 Ok2 5*4 4*3
’ All values are revertants (the average of three Dlates at a eiven concentration the mean of each concentiation. In-all cases, %‘a S9 was-used
sulphur/nitrogen-containing components in the high temperature treated coal tar sample. To investigate the relative abundances of amino-PAC in both products, careful analyses using the sensitive ECD were made. Figure 3 shows the ECD chromatograms of the PFP derivatized S-2 fractions of (a) the SRC II HD and (b) the coal tar, respectively. These chromatograms show only PFP derivatized APAH12s15. Notice that the abundance of the 2-and 3-ring APAH in the SRC II HD is much larger than for the coal tar. For example, the concentrations of 2-aminonaphthalene in the SRC II HD and the coal tar were 60 ppm and 5 ppm, respectively. Since the amino groups are produced in the SRC II 20, APAH and amino/sulphurprocess by hydrogenation containing PACs seem to be relatively abundant in this sample compared with the coal tar. Similarly, the easily desulphurized sulphur heterocycles such as naphtho[2,3blthiophene, and the 4- and 5-ring sulphur heterocycles derived from naphtho[2,3_b]thiophene, were not found in the SRC II sample, while these sulphur heterocycles were present in the coal tar as reported elsewhere2 I. The mutagenicities of all of the isomeric azadibenzothiophenes are listed in Table 2. Although the 2- and 3-aminodibenzothiophenes were strongly mutagenic and their average mutagenic response was 10 to 100 hundred times greater than the average response of However, all azadibenzothiophenes benzo[a]pyrene7. were inactive. Similarly, the azaphenanthrenes, which are structurally similar to the azadibenzothiophenes were inactive or demonstrated only weak mutagenic activities22. Recently, the microbial mutagenicities of numerous isomeric 3-to 5-ring sulphur heterocycles have . The presence of a sulphur been extensively studied 23m26 heteroatom was found to give little effect on mutagenicity. Likewise, compounds containing both nitrogen and sulphur heteroatoms in the rings also demonstrated little mutagenic activity.
minus the average of four solvent controls) + the standard
This work was supported by the Department of Energy, Office of Health and Environmental Research, Contract No. DE-AC02-79EV10237.
714
FUEL, 1986, Vol 65, May
from
REFERENCES
7
8 9 10 II 12 13
14 15 16 17 18 19 20 21 22
23 24
ACKNOWLEDGEMENT
deviation
25 26
Mckay, J. F., Weber, J. H. and Latham, D. R. Anul. C’hm. 1976, 48, 891 Paudler, W. W. and Cheplen, M. Furl 1979, 58, 775 Hunt, D. F. and Shabanowitz, J. Anul. Chem. 1982, 54. 574 Burchill, P., Herod, A. A. and Pritchard, E. J. Chrorntrroyr. 1982, 242, 65 Burchill, P., Herod, A. A. and Pritchard, E. far/ 1983, 62, 20 Nishioka, M., Lee, M. L., Kudo, H., Muchiri, D. R., Baldwin, L. J.. Pakray, S., Stuart, J. G. and Castle, R. N. Anul. Chem. 1985, 57. 1327 Nishioka, M.,Campbell, R. M., West, W. R., Smith, P. A., Booth, G. M., Lee, M. L., Kudo, H. and Castle, R. N. Anul. Chern. 1985. 57, 1868 Nishioka, M., Campbell, R. M., Lee, M. L.. Muchiri, D. R., Stuart, J. G. and Castle, R. N. Amrl. Chem 1985, 57. 2211 Kudo, H., Castle, R. N. and Lee, M. L. J. Hetrrocyclic Chem. 1985, 22, 211 Castle, R. N. (unpublished results) Later, D. W., Lee, M. L., Bartle, K. D., Kong. R. C. and Vassilaros, D. L. Anul. Chrm. 1981, 53, 1612 Later, D. W., Lee, M. L. and Wilson, B. W. Anul. Chem. 1982,54, II7 Kuei, J. C.,Shelton, J. I.,Castle, L. W., Kong, R. C., Richter, B. E., Bradshaw, J. S. and Lee, M. L. J. High. Resole. Ckromatoyr./Chromutoyr. Commun. 1984, 7, 13 Ames, B. N., McCann, J. M. and Yamasaki, E. Mut. Rex 1975,31, 347 Later, D. W., Andros, T. G. and Lee, M. L. And. C’hem. 1983,55, 2126 Flinn, R. A., Larson, 0. A. and Beuther. H. Hydrocarbon Prows & Prtrol. Rc$ner. 1963, 42(9), 129 Schmid, B. K. and Jackson, D. M. 85th National Meeting of AIchE, Philadelphia, PA, 448 June 1978 Shabatai, J., Veluswamy, L. and Oblad, A. G. Amer. Chon. Sot. Dir. Fue( Chern., Prepr. 1978, 23(l), 1 I4 Burchill, P., Herod, A. A. and Prichard. E. J. Chromutogr. 1982, 246. 271 Nishioka, M. and Lee. M. L., in preparation Nishioka, M., Lee, M. L. and Castle, R. N. Fuel (submitted) Later, D. W. ‘Handbook of Polycyclic Aromatic Hydrocarbons’, (Vol. 2) Eds. A. Bjorseth and T. Ramdahl; Marcel Dekker: New York, NY (in press) Pelroy, R. A., Stewart, D. L., Tominaga. Y., Iwao, M., Castle, R. N. and Lee, M. L. Muttrrion Rrs. 1983, 117, 31 Eastmond, D. A., Booth. G. M. and Lee, M. L. Arch. Etxirorl. Conram. Toxicol. 1984, 13, 105 McFall. T., Booth, G. M.. Lee, M. L.. Tominaga, Y.. Pratap. R.. Tedjamulia, M. and Castle. R. N. MutcrtionRex 1984, 135, 97 Pelroy, R. A,Stewart, D. L., Lee, M. L., Pratap, R.. Tominaga. Y., Iwao, M., Thompson, R. D. and Castle, R. N. Mutation Rex (in press)