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

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... er 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.