Chromatographic Concentration of Polynuclear Aromatic Hydrocarbons of Tobacco Smoke M. E. Snook, W. J. Chamberlain, R.
F. Severson, and 0. T. Chortyk
Tobacco Laboratory, Agricultural Research Service, USDA, P.0. Box 5677, Athens, GA 30604
Polynuclear aromatic hydrocarbons (PAH) have been extensively studied in air pollutants, coal tars, gasoline engine exhausts, various thermal decomposition products, and in numerous other sources. They have been identified by thin-layer, paper, gas, and liquid chromatography, IR and UV spectrometry, fluorescence, spectrophotofluorimetry, mass spectrometry, and combinations of these techniques. T h e extensive research efforts were prompted by t h e fact t h a t selected PAH have shown high tumorigenic activity ( I , 2 ) and constitute health hazards for man (3). T h e presence of PAH has also been confirmed in cigarette smoke condensate, which is also known t o possess considerable tumorigenic properties ( 4 , 5 ) . More than 90 PAH have been reported t o occur in cigarette smoke condensate (CSC) (6). However, because of the highly complex nature of CSC and t h e fact t h a t many PAH occur therein in only minor amounts, one reviewer (6) stated, "In many reported cases, identification of PAH must be considered tentative, if not questionable." Today, the combined analytical powers of gas chromatography and mass spectrometry have eliminated many problems of identification of trace constituents. However, the partial separation of the PAH, prior t o gas chromatography, from t h e other thousand or more smoke constituents still remains. Our interests in the PAH of tobacco smoke stem from our fractionation and bioassay studies of CSC (7, 8). Previous work in this laboratory on fractionation of CSC has resulted in t h e separation of several active fractions, including a neutral subfraction (F-20, Figure l),which contained 0.32% of the original weight of crude CSC, but still possessed high tumorigenic activity (7, 8). This subfraction and similar PAH-enriched fractions contained benzo[a]pyrene (BaP) and other PAH, and the activities of these subfractions were attributed to the PAH (9). However, the presence of large amounts of interfering material has deterred the complete analysis and identification of the PAH in F-20. For example, another PAH-enriched fraction was also shown t o contain pesticide residues and nitrogen heterocyclic compounds (IO). A quantitative determination for B a P in such PAH fractions has been performed, using the radio-labeled compound (11). One preliminary separation and concentration of the PAH, contained in a neutral CSC fraction, was based on gel filtration chromatography (12). Other techniques have involved column chromatography on alumina (13, 14). Recently, two other reports have appeared on the application of gel filtration chromatography t o the separation of the P A H of CSC (15, 16). However, these experiments utilized crude CSC resulting in gel fractions t h a t were still contaminated with other CSC components: the final PAH fraction contained 0.95% of the CSC weight (16), three times the 0.32% of our F-20 fraction. We have developed a chromatographic procedure (Figure 1) which efficiently isolates the P A H in CSC, in order t h a t individual PAH can be separated and identified by gas chromatography. T h e advantages of this method, for obtaining a PAH-enriched fraction, over previously reported procedures are detailed. This method should be applicable to t h e separation and identification of PAH in other complex organic mixtures,
GC
1
GC-MS
.
F-20 (032%)
PAH Separation
PAH-Enriched
ond Iden t i l i
F. Severson, and 0. T. Chortyk
Tobacco Laboratory, Agricultural Research Service, USDA, P.0. Box 5677, Athens, GA 30604
Polynuclear aromatic hydrocarbons (PAH) have been extensively studied in air pollutants, coal tars, gasoline engine exhausts, various thermal decomposition products, and in numerous other sources. They have been identified by thin-layer, paper, gas, and liquid chromatography, IR and UV spectrometry, fluorescence, spectrophotofluorimetry, mass spectrometry, and combinations of these techniques. T h e extensive research efforts were prompted by t h e fact t h a t selected PAH have shown high tumorigenic activity ( I , 2 ) and constitute health hazards for man (3). T h e presence of PAH has also been confirmed in cigarette smoke condensate, which is also known t o possess considerable tumorigenic properties ( 4 , 5 ) . More than 90 PAH have been reported t o occur in cigarette smoke condensate (CSC) (6). However, because of the highly complex nature of CSC and t h e fact t h a t many PAH occur therein in only minor amounts, one reviewer (6) stated, "In many reported cases, identification of PAH must be considered tentative, if not questionable." Today, the combined analytical powers of gas chromatography and mass spectrometry have eliminated many problems of identification of trace constituents. However, the partial separation of the PAH, prior t o gas chromatography, from t h e other thousand or more smoke constituents still remains. Our interests in the PAH of tobacco smoke stem from our fractionation and bioassay studies of CSC (7, 8). Previous work in this laboratory on fractionation of CSC has resulted in t h e separation of several active fractions, including a neutral subfraction (F-20, Figure l),which contained 0.32% of the original weight of crude CSC, but still possessed high tumorigenic activity (7, 8). This subfraction and similar PAH-enriched fractions contained benzo[a]pyrene (BaP) and other PAH, and the activities of these subfractions were attributed to the PAH (9). However, the presence of large amounts of interfering material has deterred the complete analysis and identification of the PAH in F-20. For example, another PAH-enriched fraction was also shown t o contain pesticide residues and nitrogen heterocyclic compounds (IO). A quantitative determination for B a P in such PAH fractions has been performed, using the radio-labeled compound (11). One preliminary separation and concentration of the PAH, contained in a neutral CSC fraction, was based on gel filtration chromatography (12). Other techniques have involved column chromatography on alumina (13, 14). Recently, two other reports have appeared on the application of gel filtration chromatography t o the separation of the P A H of CSC (15, 16). However, these experiments utilized crude CSC resulting in gel fractions t h a t were still contaminated with other CSC components: the final PAH fraction contained 0.95% of the CSC weight (16), three times the 0.32% of our F-20 fraction. We have developed a chromatographic procedure (Figure 1) which efficiently isolates the P A H in CSC, in order t h a t individual PAH can be separated and identified by gas chromatography. T h e advantages of this method, for obtaining a PAH-enriched fraction, over previously reported procedures are detailed. This method should be applicable to t h e separation and identification of PAH in other complex organic mixtures,
GC
1
GC-MS
.
F-20 (032%)
PAH Separation
PAH-Enriched
ond Iden t i l i
