cals, the majority of which are polycyclic aromatic compounds (PAC). While ...
which was composed of the nitrogen-containing PAC, the second silicic acid.
E DETERMINATION OF POLYCYCLIC AROMATIC COMPOUNDS CONTAINING BOTH SULFUR A N D NITROGEN HETEROATOMS I N COAL-DERIVED PRODUCTS
Masaharu N i s h i o k a , P h i l i p A. Smith, Gary M . Booth, and M i l t o n L . Lee Departments of Chemistry and Zoology, Brigham Young U n i v e r s i t y , 84602
Provo, Utah
H i r o t a k a Kudo, Daniel R. M u c hi ri , and Raymond N . Castle Department of Chemistry, U n i v e r s i t y of S o u t h F l o r i d a , Tampa, F l o r i d a
33620
LeRoy, H. Klemm Department of Chemistry, U n i v e r s i t y of Oregon, Eugene, Oregon 97403 Coal-derived p r o d u c t s a r e e xt re m e l y complex m i x t u r e s of o r g a n i c chemic a l s , t h e m a j o r i t y of which a r e p o l y c y c l i c a rom a t i c compounds (PAC). While t h e p o l y c y c l i c a r o m a t i c hydrocarbons (PAH) comprise t h e l a r g e s t chemical c l a s s f r a c t i o n of most c o a l p r o d u c t s , t h e r e a r e s u b s t a n t i a l amounts of n i t r o g e n - , PAC. A f t e r f r a c t i o n a t i o n of t h e n i t r o g e n s u l f u r - , and oxygen-containing c o n t a i n i n g PAC f r a c t i o n , i t was found t h a t t h e amino p o l y c y c l i c a r o m a t i c hydrocarbons ( A P A H ) , which a r e p r e s e n t i n low c o n c e n t r a t i o n s ( u s u a l l y less t h a n 1 % ) , were r e s p o n s i b l e f o r t h e major p a r t of t h e m u t a g e n i c i t y ( 1 - 4 ) . The n i t r o g e n h e t e r o c y c l e s were g e n e r a l l y much less mutagenic t h a n t h e A P A H . T h i s r e s u l t has prompted t h e i n v e s t i g a t i o n of o t h e r p o s s i b l e t r a c e c m p o n e n t s i n c o a l p r o d u c t s , p a r t i c u l a r l y t h e PAC t h a t c o n t a i n two h e t e r o a t o m s , t h a t may be r e s p o n s i b l e f o r some of t h e observed b i o l o g i c a l a c t i v i t y . In t h i s p a p e r , t h e a n a l y s i s of a s o l v e n t - r e f i n e d c o a l l i q u i d a n d a c o a l t a r f o r PAC which c o n t a i n b o t h n i t r o g e n and s u l f u r h e t e r o a t o m s is d e s c r i b e d . C a p i l l a r y column ga s chromatography w i t h s u l f u r - s e l e c t i v e f l a m e p h o t o m e t r i c d e t e c t i o n and mass s p e c t r o m e t r y were used t o i d e n t i f y s e v e r a l new compounds which were p r e v i o u s l y u n r e p o r t e d . Pure r e f e r e n c e compounds were a l s o synthesized and t e s t e d f o r genotoxicity. EXPERIMENTAL
The s o l v e n t - r e f i n e d c o a l heavy d i s t i l l a t e (SRC I1 HD: 260-450°C b o i l i n g p o i n t r a n g e ) was o b t a i n e d from t h e F o r t L e w i s , W a s h i n g t o n , p i l o t p l a n t The c o a l t a r was ( o p e r a t e d by t h e P i t t s b u r g h & Midway Coal Mining C o . ) . o b t a i n e d from S.A. Wise ( N a t i o n a l Bureau of S t a n d a r d s , Washington, D.C.). The f o u r aminodibenzothiophene isomers and a z a t h i o p h e n i c compounds used i n t h i s s t u d y were n o t commercially a v a i l a b l e a n d , t h e r e f o r e , had t o be s y n t h e s i z e d i n our own l a b o r a t o r i e s . The s y n t h e t i c p ro c e d u re s a r e r e p o r t e d e l s e w h e r e ( 5 , 6 ) . The SRC I1 HD m a t e r i a l and c o a l t a r were f r a c t i o n a t e d i n t o c h e m i c a l c l a s s e s by a d s o r p t i o n chromatography on n e u t r a l alumina and s i l i c i c a c i d accor d i n g t o t h e p roc e d u re of L a t e r (7). The t h i r d f r a c t i o n (A-3) which was composed of t h e n i t r o g e n - c o n t a i n i n g P A C , t h e second s i l i c i c a c i d f r a c t i o n (5-2) which was composed of t h e A P A H , and t h e t h i r d s i l i c i c a c i d f r a c t i o n (S-3) which was composed of t h e t e r t i a r y n i t r o g e n - c o n t a i n i n g PAC were an alyz e d i n t h i s s t u d y . Acid e x t r a c t i o n w i t h H2SO4, and d e r i v a t i z a t i o n w i t h p e n t a f l u o r o p r o p i o n i c a n h y d ri d e (PFPA) were p r e v i o u s l y d e s c r i b e d ( 8 ) .
et.
93
A Hewlett-Packard Model 5880 g a s chromatograph equipped w i t h a s u l f u r s e l e c t i v e f l a m e p h o t o m e t r i c d e t e c t o r (FPD), and o p e r a t e d i n t h e s p l i t l e s s Hydrogen i n j e c t i o n mode was used t o o b t a i n chromatographic r e t e n t i o n d a t a . The c a p i l l a r y was used a s t h e c a r r i e r g a s a t a l i n e a r v e l o c i t y of 100 em s-1. column used i n t h i s s t u d y was prepared by c o a t i n g a 20 m x 0.31 mm i . d . l e n g t h of f u s e d s i l i c a t u b i n g (Hewlett-Packard, Avondale, PA) w i t h a 25% biphenyl p o ly me t h y l s i l o x a n e s t a t i o n a r y phase (0.25 um f i l m t h i c k n e s s ) ( 9 ) . A HewlettPackard Model 5982A GC-MS system was used t o o b t a i n mass s p e c t r a l confirma t i o n of i d e n t i f i e d compounds. The same chromatographic column was used a s f o r g a s chromatography, and t h e mass s p e c t r o m e t e r was o p e r a t e d i n t h e e l e c t r o n i m p a c t mode a t 70 eV e l e c t r o n energy. The A m e s m u t a g e n i c i t y a s s a y was performed a s d e s c r i b e d by Ames e t a l . ( 1 0 ) w i t h minor m o d i f i c a t i o n s .
RESULTS A N D DISCUSSION The chromatogram of t h e SRC I1 H D A-3 f r a c t i o n o n t h e 2 5 % b i p h e n y l R e t e n t i o n times m eth y l p o l y s i l o x a n e s t a t i o n a r y phase i s shown i n F i g u r e 1 . were compared w i t h t h e newly s y n t h e s i z e d s t a n d a r d samples, and t h e 1 - t o 4-aminodibenzothiophenes and v a r i o u s a z a t h i o p h e n i c compounds were i d e n t i f i e d . S e v e r a l s m a l l peaks (most l i k e l y s u l f u r - c o n t a i n i n g secondary n i t r o g e n h e t e r o cycles) were e l i m i n a t e d a f t e r a c i d e x t r a c t i o n of t h e f r a c t i o n . A m o d i f i c a t i o n of t h e method d e s c r i b e d by L a t e r et a l . ( 1 1 ) was u s e d t o d i s t i n g u i s h t h e s u l f u r / t e r t i a r y n i t r o g e n - c o n t a i n i n g PAH a n d s u l f u r / a m i n o - c o n t a i n i n g P A H . Comparison of t h e chromatogram of t h e PFP d e r i v a t i v e s w i t h F i g u r e 1 showed t h a t some of t h e peaks s h i f t e d i n r e t e n t i o n t i m e a f t e r d e r i v a t i z a t i o n . R eten t i o n times f o r t h e PFP amide d e r i v a t i v e s d e c r e a s e d because t h e p o l a r amino groups were blocked b y t h e PFP g ro u p s , c a u s i n g l e s s d i p o l e / i n d u c e d d i p o l e i n t e r a c t i o n s w i t h t h e p o l a r i z a b l e b i p h e ny l s t a t i o n a r y phase. I d e n t i f i c a t i o n of t h e f o u r aminodi benzothiophene isomers was confirmed by comparing t h e r e t e n t i o n times of t h e P F P -de ri va t i z e d s t a n d a r d s w i t h t h e r e t e n t i o n times of t h e s h i f t e d peaks i n t h e chromatogram of t h e P F P - d e r i v a t i z e d A-3 f r a c t i o n . A chromatogram of t h e S-2 APAH f r a c t i o n of t h e SRC I1 H D showed only aminodibenzothiophenes and a l k y l a t e d aminodibenzothiophenes. The c h r o m a t o g ra m of t h e c o a l t a r A-3 f r a c t i o n o n t h e 25% biphenyl m eth y l p o l y s i l o x a n e s t a t i o n a r y phase is shown i n F i g u r e 2. R e t e n t i o n times of compounds i n b o t h t h e A-3 and S-3 f r a c t i o n s were compared w i t h t h e r e f e r e n c e samples, and 3 i s o m e r s of t h e azabenzothiophenes and a l l i s o m e r s o f t h e azad ib e n z o t h i o p h e n e s were i d e n t i f i e d . I n c o n t r a s t t o t h e SRC I1 H D , a z a t h i o phenic compounds were major components and aminodibenzothiophenes were n o t found F u r t h e r c o n f i r m a t i o n of peak i d e n t i t i e s was o b t a i n e d by GC-MS a n a l y s i s . Tab le 1 l i s t s t h e r e l a t i v e r e t e n t i o n times of t h e PAC c o n t a i n i n g both s u l f u r and n i t r o g e n i d e n t i f i e d i n t h i s s t ud y and t h e r e s u l t of s e m i - q u a n t i t a t i o n of s e v e r a l of t h e s e compounds i n t h e samples. T e r t i a r y n i t r o g e n h e t e r o c y c l e s a r e more t h e r m a l l y s t a b l e t h a n aminoc o n t a i n i n g PAC, and w e major n i t r o g e n - c o n t a i n i n g PAC i n c o a l - d e r i v e d p r o d u c t s (1 2 ) . Azathiophenic compounds were t e n t a t i v e l y i d e n t i f i e d i n a n a n t h r a c e n e O i l and c o a l t a r u s i n g GC-MS by B u r c h i l l e t a 1 . ( 1 2 , 1 3 ) . However, t h e aminodibenzothiophenes were t h e major nitrogen/sulfur-containing heterocycles i n t h i s Coal l i q u i d , w h i l e t h e a z a t h i o p h e n i c compounds were t h e major ones i n t h e c o a l t a r . The d i f f e r e n c e between t h e two c oa l - d e r i v e d p r o d u c t s a p p a r e n t l y r e l a t e s t o t h e r e a c t i o n c o n d i t i o n s . I n t h e SRC I1 p r o c e s s , hydrogen was used
.
94
1 IME Im1n.l
0
2.0
10
120
140
180
TEMPI% I
30
40
220
260
Figure 1. FPD chromatogram of the SRC I1 HD A-3 fraction on a 25% biphenyl polysiloxane stationary phase. Conditions: temperature program from 120°C to 265'C at 4°C min-I, after an initial 2-min isothermal period; hydrogen carrier gas at 100 cm s - 1 . Peak assignments are listed in Table 1.
JL 0
70
10 120
TIMElmln.1 20 160 TEMPI*cl
30
40 240
265
Figure 2. FPD chromatogram of the coal tar A-3 fraction on a 25% biphenyl polysiloxane stationary phase. Conditions: temperature program from 70 "c to 120°C at 1 0 ° C min'l, then from 120°C to 265°C at 4 ° C min-1, after an initial 2-min isothermal period; hydrogen carrier gas at 100 cm s - 1 . Peak assignments are listed in Table 1.
95
Table 1.
Relative retention times and semi-quantitation (selected examples) of PAC containing both sulfur and nitrogen in an SRC I1 heavy distillate coal liquid and a coal tar.
Peak No.
Compounda
Relative Retention Timeb
Concentrationc (ug/g) S R C I1
1 3 2
4 6 7 5
-a 9
11 12 13 10
14 15
16
-
17
-
18 19
4-azabenzothiophene 6-azabenzothiophene 7-azabenzothiophene 1-azadibenzothiophene 2-azadibenzothiophene 3-azadibenzothiophene 4-azadibenzothiophene azadibenzothiophene isomers l-azanaphtho[2,1-k]thiophene azaphenanthro[4,5-b,c,dlthiophene isomers 1-aminodibenzothiophene 2-aminodibenzothiophene 3-aminodibenzothiophene 4-aminodibenzothiophene C1 aminodibenzothiophene C2 aminodibenzothiophene 3-azaphenanthro[9,10-k]thiophene 3-azaphenanthro[2,1-k]thiophene 3-azaphenanthro[4,3-k]thiophene 1-azaphenanthroClt2-L]thiophene 5-azabenzo[b_]naphtho[lt 2-glthiophene azabenzonaphthothiophene isomers C1 azabenzonaphthothiophene isomers
HD Coal Tar
0.361 0.380 0.361 0.956 1.05 1.05 1.04
0.18
1.7
-
0.979
1.47 1 .52 1.54 1.43
-
0.32 0.11 2.5 0.33
1.74 1.78
1.80 -
-
d d
acornpounds which are not numbered were not found in these coal-derived products. bRetention relative to naphto[2,3-k]thiophene. CApproximate concentration in ug/g in the crude SRC I1 heavy distillate and the coal tar. dCompound did not elute under the chromatographic conditions used. in the reaction, and metals contained in the recycle oil were utilized as catalysts ( 1 4). Therefore, aminodibenzothiophenes seem to be more abundant than azathiophenic compounds in the coal liquid because of hydrogenation in the process. On the other hand, sulfur/tertiary nitrogen-containing PAC were thought to be major components in the high-temperature treated coal tar Sa.InPle. Similarly, the easily desulfurized sulfur heterocycles such as naphtho[2,3-blthiophene, 4- and 5-ring sulfur heterocycles derived from naphtho[2,3-blthiophene were not found in SRC I1 sample, while these sulfur heterocycles were present in the same coal tar as reported elsewhere (15). The mutagenicities of all isomers of the aminodibenzothiophenes and the
96
1
T a b l e 2.
Ames m u t a g e n i c i t y of t h e aminodibenzothiophenes and azadibenzothiophenes.
Compound
re v/ &
1-aminodibenzothiophene 0% S9b 4% S9d 2- a m i nodi benzo t h i ophene 0% S9b 4% s 9 e 3- ami nodi benzo t hiophene 0% S9b 4% S9f 4-aminodi benzothiophene 0% S9b 4% S9d 1 - a z a d i benzothiophene 4% s 9 g 2-azadi benzothiophene 4% S9h 3-azadi benzothiophene 4% S9h 4-azadi benzothiophene 4% s 9 g
C
1.94
x
3.43
x
R2
--
2.36
0.96
9.07 104
0.98 0.97
4.74 103
0.90 0.97
--
C
--
C
--
C
--
C
--
C
--
C
aLinear r e s p o n s e r e g i o n used t o c a l c u l a t e dose r e s p o n s e by linear regression curve f i t t i n g . bSolvent c o n t r o l v a l u e (0% S 9 ): 2124 CResponse < 2X s o l v e n t c o n t r o l v a l u e s dSolvent c o n t r o l v a l u e (4% S 9 ): 3855 eSolvent c o n t r o l v a l u e ( 4 % S 9 ) : 2954 fSolvent control value (4% S9): 3 1 ~ 4 g S o l v e n t c o n t r o l v a l u e (4% S 9 ): 3852 hSolvent c o n t r o l v a l u e (4% S9): 4626 azadibenzothiophenes a r e l i s t e d i n T a b l e 2. The 2- and 3-aminodibenzothiophenes a r e s t r o n g l y mutagenic, and t h e i r a v e r a g e mutagenic r e s p o n s e was t e n t o one hundred t i m e s g r e a t e r t h a n t h e average r e s p o n s e of benzo[a_]pyrene (200-300 r e v e r t a n t s / p l a t e a t 4 ug and 4% S9) ( 1 6 ) . A l l isomers of t h e azadibenzothiophenes were i n a c t i v e . A comparison of t h e s t r u c t u r e s of aminodibenzothiophenes t o t h e aminophenanthrenes r e v e a l s a g e o m e t r i c s i m i l a r i t y between them. The 2-, 3-, and 9-aminophenanthrenes demonstrated t h e h i g h e s t mutaThe 3 - a m i n o p h e n a n t h r e n e g e n i c i t y of t h e aminophenanthrene isomers ( 1 7 ) . isomer had t h e s t r o n g e s t m u t a g e n i c i t y (30,300 r e v l u g a v e r a g e r e s p o n s e ) , and t h i s s t r u c t u r e is analogous t o 2-aminodibenzothiophene w h i c h i s t h e most a c t i v e of t h e aminodibenzothiophene i s ome rs .
97
ACKNOWLEDGMENT T h i s work was s u p p o r t e d by t h e Department of Energy, O f f i c e o f H e a l t h and Environmental R e s e a r c h , C o n t r a c t No. DE-AC02-79EV10237.
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