A bright and a Burley tobacco were grown at four fertilization rates and each tobacco was then both flue-cured and air-cured. Levels of alkaloids and nitrosamines were found to increase with increasing fertilization levels. Levels of alkaloids, N-nitrosonornicotine (NNN), and other tobacco-specific nitrosamines (TSNA) were consistently higher in the Burley tobacco than in the bright tobacco, regardless of curing method. In comparing the effects of curing, it was found that NNN and total TSNA levels were higher in the midrib than in the lamina of the air-cured samples, while just the opposite was found for the flue-cured samples. Flue-curing bright tobacco produced three times the level of TSNA vs air-curing the same tobacco. On the other hand, flue-curing Burley tobacco reduced the alkaloids, but greatly increased the TSNA in the lamina. As midribs from the air-cured Burley leaves had three times the TSNA concentration of the lamina, the use of air-cured midribs in tobacco products should be avoided. It was concluded that lower fertilization levels and careful manipulations of curing parameters could lower nitrosamine levels in cured tobacco.
Recent studies on the pyrogenesis of tobacco smoke constituents have been reviewed. Where appropriate, representative studies on pyrolyses of compounds reportedly present in tobacco leaf have been included. Various experimental conditions such as temperature, gaseous environment, and thermal stability of precursor were considered in light of current understanding of the smoking process. Attempts to alter the chemical constituents of smoke by use of additives have been discussed. Finally, various smoke constituents have been correlated with pyrolyses of leaf constituents in further hopes of better understanding the complex processes that produce tobacco smoke
A rapid, semi-micropyrolysis technique was developed and applied to materials representative of tobacco cell-wall constituents and sucrose. Glass capillary gas chromatography - mass spectrometry was used to separate and identify the major semi-volatile pyrolyzate components. Cellulose and dextrin produced a pattern of furan and cyclic ketones of potential importance to flavour and aroma of tobacco smoke. Sucrose pyrolysis resulted in the formation of substantial amounts of 2-furaldehyde and lesser quantities of substituted furans. The cell-wall biopolymer lignin was a source of phenols, but contributed little to the compounds produced in the thermal breakdown of carbohydrates.
Paraffins of tobacco Ieaf were separated by column chromatography on silicic acid. Leaf paraffins were fractionated from other wax constituents by chromatography in a definite sample to substrate to solvent ratio. The developed method was used to evaluate the transfer of paraffins and neophytadiene from leaf to smoke in a reference cigarette. Gas chromatographic separations were performed on a high-temperature liquid phase. Gas chromatography in conjunction with mass spectrometry was used to determine the paraffin composition of a representative flue-cured tobacco, a reference cigarette tobacco, and smoke condensate. It was concluded that paraffins were probably transferred to smoke relatively unchanged, while neophytadiene underwent some pyrolytic decomposition.
RF Arrendale, RF Severson, OT Chortyk and MG Stephenson
Recent studies in our laboratory on the cuticular chemicals of green tobacco leaf have revealed the presence of wax esters, composed of fatty acids bound to fatty alcohols. Cuticular components of young green NC 2326 tobacco leaves were removed with methylene chloride, and partitioned between hexane and 80 % MeOH-H2O. The hexane-soluble fraction, which contained wax esters, paraffinic hydrocarbons, and fatty alcohols, was separated by silicic acid column chromatography, and the resulting wax ester fraction was further purified by lipophilic gel chromatography. Initial analyses of the wax ester fraction by capillary gas chromatography [GC] and capillary GC / MS, on a short fused silica [FS] SE-54 capillary column, indicated the presence of C30 - C52 wax esters. Application of the cold on-column injection technique and use of immobilized stationary phase, FS SE-54 capillary columns greatly improved the GC separation of the complex wax ester fraction and permitted the identification of individual wax ester isomers. Identification of wax ester isomers by GC/MS relied upon the presence of a molecular ion and ions characteristic of the acid and alcohol moieties. For the acid portion, these ions included the acid MW + 1 a.m.u. and MW - 17 a.m.u. ions, while for the alcohol, they were the alcohol MW - 18 a.m.u. and MW + 27 a.m.u. ions. Saponification of the wax ester fraction and subsequent analyses of the alcohols (as trimethylsilyl ethers) and acids (as methyl esters) revealed extensive iso- and anteiso-methyl branching of the acid moieties. The wax ester isomers with iso- and anteiso- methyl-branched acid moieties were separated from each other and from the normal straight-chain isomers by capillary GC and were identified by GC/MS, based upon characteristic ions resulting from the losses of the iso-branched (MW - 43 a.m.u.) and anteiso-branched (MW - 57 a.m.u.) groups from the molecular ion and from the acid moiety. One hundred and seventy individual wax esters were identified.
R.F. Severson, K.L. McDuffie, R.F. Arrendale and O.T. Chortyk
A rapid method for the analysis of aliphatic hydrocarbons and neophytadiene in cured tobacco was developed. Briefly, ground tobacco was extracted for 15 min with methylene chloride in a flask placed in an ultrasonic vibration bath, 3-Methyltricosane was added as the internal standard. The solution was filtered and the solvent removed. The sample was redissolved in hexane and chromatographed on a small silicic acid column. The hydrocarbonneophytadiene fraction that eluted with hexane was reduced in volume and analysed on a SE-54 wall-coated glass capillary column, which resolved and permitted quantitation of the normal, iso-methyl- branched, and anteiso-methyl-branched hydrocarbons. The analytical data obtained by this method were equal to those obtained by the conventional Soxhlet extraction procedure. Based on experience in our laboratory, analysis time was reduced about fivefold and isomeric hydrocarbons were resolved by capillary gas chromatography.
M. E. Snook, R. F. Severson, H. C. Higman, R. F. Arrendale and O.T. Chortyk
A neutral fraction of cigarette smoke condensate, which had shown biological activity and was known to contain polynuclear aromatic hydrocarbons (PAH), was fractionated by analytical gel filtration chromatography. These gel fractions were subjected to gas chromatographic separation and their components were identified by relative GC retention times, UV spectra, and mass spectral data. More than 300 PAH, ranging from indene to the dimethylbenzopyrenes, were characterized. This method of isolation has yielded fractions which were more amenable to definitive identifications. The criteria used for identification are tabulated for all the identified PAH compounds.
A gel filtration chromatography method was developed for the isolation and concentration of the high molecular weight polynuclear aromatic hydrocarbons (PAH) contained in the most biologically active fraction of cigarette smoke condensate (CSC). The unusually complex mixture of large PAH found in CSC necessitated the use of preparative gas chromatography followed by high-pressure liquid chromatography to achieve separation and identification. Mass spectral, ultra-violet absorption, and chromatographic retention data were needed for the comprehensive identification of the large molecular weight PAH components of CSC. The majority of the more than 200 isolated compounds were identified. Compounds newly identified in CSC included 3,4-dimethylenepyrene, 3,4-trimethylenepyrene, cyclopenta(c,-d)pyrene, 4,5-methylenetriphenylene, benzo[b]perylene, and several dibenzofluoranthenes.