Volume 14 (1988): Issue 2 (October 1988)

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.

FT-IR evolved gas analysis, a non-isothermal pyrolysis technique, is used to probe the changes in tobacco leaf composition which occur during flue- and air-curing. A unique perspective of major enzymatic processes and their dependencies on curing method is obtained through simultaneous detection of multiple pyrolytic products and three-dimensional presentation of the product evolution profiles. The conversion of starch to sugars, proteins to amino acids, and the relationships between these processes are explored.

The first part of the paper describes a new method of obtaining reproducible and meaningful headspace profiles of tobacco lamina by using a modified closed loop stripping apparatus. The complex chromatograms are obtained by high-resolution glass capillary gas chromatography. The second part summarizes the results of a chemometric approach to interpret the chromatograms obtained from a series of nine Virginia flue-cured tobaccos from different origins and belonging to different quality groups, each one analysed three times by the method described above. After the elimination of peaks containing redundant information, the resulting data set, consisting of 27 × 17 data points, was analysed to detect natural groupings by using an in-house program (in Basic) for principal component analysis. A subsequent discriminant analysis yielded two discriminant functions capable of separating the nine Virginia tobaccos into three quality groups as defined by a conventional organoleptic analysis carried out by a smoking panel. All the tobaccos could be classified correctly (100 %). A first attempt to classify, by the procedure described above, a group of six Virginia tobaccos whose organoleptic scores were not known, did not yield clearly interpretable results, possibly because the performance of the capillary column used for analysis had slightly deteriorated during the experiment with resultant changes in retention characteristics, which led to wrong identifications of certain peaks.

Watery tobacco extracts were examined with the aid of flow-injection analysis (FIA) for the substances nicotine, nitrate, chloride, ammonium and phosphate. The results proved to be exact and reproducible for the most part and the speed of analysis was between 37 and 113 measurements per hour. Because of its simple modular design the appliance is flexible in its application and is above all suited for the analysis for several components in small series of samples. A special FIA technique (stopped-flow method) enables the undersurface absorption stemming from the sample matrix to be excluded. The solutions used must exhibit uniform fluid composition and the formation of gas bubbles in the analysis system must be excluded.

An in vitro evaluation was made of the measurement of puffing behaviour using an orifice-plate cigarette holder. Four brands of cigarettes were compared ranging in tar yield from 4 to 16 mg, and each was studied with two puff profiles, the “early triangle” and the “square wave” pattern. Two flow rates were used for each profile giving maximum flows of 25.7 and 37.0 ml s^-1(early triangle) and 12.0 and 36.7 (square wave), respectively, and four puff volumes, i.e. 15, 40, 65, and 90 ml for each profile/flow combination. Forty-eight cigarettes of each type were smoked (three for each profile/flow/volume combination). The studies emphasize the need for careful calibration of the alinear flow system and the value of reference volume standards. When calibrated with air, volume measurements on smoke at ambient temperature were overestimated by an average of 1.4 %. An average overestimation of 1.2 % was seen per 1°C rise in temperature. This rise in temperature occurred in the last few puffs, and was mainly influenced by the puff volume. There was much less difference when subdividing the data according to cigarette type, the early triangle or square wave profiles, or the two flow rates. With puff volumes of 15 ml there was virtually no temperature rise, and except at 90 ml the effect was only important in the last 20 % of the smoking period.

Three fungal strains isolated from tobacco were cultured on tobacco water extracts. In these cultures, the mycelium weight was shown to be correlated with the concentration of a steroid, ergosta-5,7,22-trien-3ss-ol [ergosterol]. This steroid is not a tobacco constituent, but tobacco samples where mold or yeast infections had occurred exhibited significant amounts of it. A method is proposed to quantify ergosterol in tobacco samples by reverse-phase high-performance liquid chromatography (HPLC) with UV detection at 282 nm. 7-Dehydrocholesterol can be used as internal standard. When found in a tobacco sample, the ergosterol concentration exhibits a good correlation with that of another related steroid, ergosta-4,6,8(14),22-tetraen-3-one [ETO], for which an HPLC quantification method in tobacco is proposed. Because it is highly fluorescent, ETO is also amenable to a sensitive and quick determination by thin-layer chromatography (TLC). Once produced on tobacco, ergosterol concentration remains stable through storage under normal conditions, and even primary processing does not alter it appreciably. Possible applications of ergosterol analysis to the detection of fungal infections or the monitoring of fungal growth on tobacco are outlined. In addition, TLC estimation of the ETO concentration in a sample may constitute a convenient and fast screening method for fungal infections.