The nitrogen analogues of polynuclear aromatic hydrocarbons (aza-arenes) were isolated and identified in a basic fraction of cigarette smoke condensate. Silicic acid chromatography removed the predominant nicotine alkaloids, while gel chromatography on Bio-Beads S-X12 in benzene effectively separated the aza-arenes from interfering aliphatic compounds. In addition, the gel columns separated the aza-arenes by ring number and degree of alkylation on the basis of an adsorption-type mechanism. These gel characteristics facilitated the identifications of a large number of isomeric aza-arenes. Compounds identified included 2-vinylpyridine, 3-vinylpyridine and 2-phenylpyridine as well as quinoline, isoquinoline, 4-azafluorene, benzoquinolines, benzoisoquinolines, 1-azafluoranthene, 7-azafluoranthene, 4-aza-pyrene, 7- azaindole, pyrroloquinoline and their mono-, di- and trimethyl derivatives. All eight possible isomers of benzoquinoline and benzoisoquinoline were found, four of which are being reported for the first time. Evidence was also found for the probable presence of 5,6-benzo-7-azaindole.
A new chromatographic method utilizing the hydrogen bonding properties of Sephadex LH-20 gel in methanol / chloroform, was developed for the isolation of phenolic acids. This method was applied to the characterization of phenolic acids in flue-cured tobacco. Gel chromatography successfully isolated and concentrated the phenolic acids from other acidic substances and allowed identification and quantitation of these compounds by GC and GC-MS, as their trimethylsilyl derivatives. Compounds identified included: cis- and trans-isomers of p-coumaric, ferulic, caffeic, and sinapic acids; o-, m-, and p-hydroxybenzoic acids; o-hydroxyphenylacetic acid; 2,5- and 3,4-dihydroxybenzoic acids; and 2,3-, 2,5- and 3,4-dihydroxybenzaldehydes. Caffeic acid was the major compound, probably arising from the degradation of the chlorogenic acid of leaf. Aliphatic acids having two or more carboxyl groups were also isolated and included the following: malonic, succinic, fumaric, malic, and citric acids. A dihydroxycinnamaldehyde and a dihydroxynaphthoic acid are reported in tobacco leaf for the first time.