Isolation and Identification of the Wax Esters from the Cuticular Waxes of Green Tobacco Leaf

Abstract

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.

Cistus ladanifer L. Shrub is Rich in Saturated and Branched Chain Fatty Acids and their Concentration Increases in the Mediterranean Dry Season

“…Tsydendambaev et al [33] identified eight iso-BCFA (iso-15:0, -16:0, -17:0, -18:0, -20:0, -22:0, -24:0, and -26:0) on the green above-ground parts of four alpine plants, and Radunz [34] identified considerable quantities of iso-12:0, -14:0, -16:0, -18:0, and -22:0 BCFA in petals and chlorophyll-deficient leaves (averaging 9.0 and 4.2 % of total FA, respectively) of Antirrhinum majus and Nicotiana tabacum plants. In addition, BCFA have been identified in epi-cuticular waxes of several plants [35,36].…”

Structural characterization of wax esters by electron ionization mass spectrometry

“…The moderately abundant peaks (10-18%) one mass unit lower ( m/z 270 in WE 14:0-17:0) were radical cations [RCOOH] +• rationalized by a single hydrogen rearrangement ( 16,18 ). The C-C bond ␣ -cleavage at the carbonyl group provided small (4-10%) fragments [R'OCO] + ( m/z 241 in WE 14:0-17:0), which were practical for the confi rmation of the alcohol chain length ( 16,19 + ( m/z 61) ( 27 ). Their intensities ranged from 4-10% and 7-22%, respectively.…”

“…However, radicals were eliminated from [R'−H] +• and [R'-C 2 H 5 ] +• ions yielding fragments indicative of the branching position. For instance, they appeared hydrogen transfer (16)(17)(18)(19). The moderately abundant peaks (10-18%) one mass unit lower ( m/z 270 in WE 14:0-17:0) were radical cations [RCOOH] +• rationalized by a single hydrogen rearrangement ( 16,18 ).…”

“…The EI spectra are explained perhaps in all of the textbooks on organic mass spectrometry, but the discussion is mostly limited to methyl or ethyl esters. The EI mass spectra of saturated straight-chain WEs has been studied for more than 40 years using both low-and high-resolution measurements and deuterium-labeling experiments (16)(17)(18)(19)(20)(21)(22). The spectra interpretation is usually straightforward in terms of the determination of the molecular weight and acid/alcohol chain length.…”

Changes in Cuticular Compounds of Developing Pecan Leaves

“…Wax esters were present in trace amounts of up to 0.2 µg·cm -2 and ranged from C 35 to C 52 compounds, with the predominant wax ester being the C 35 compound. The composition of the nonpolar or hydrocarbonwax ester fraction resembled paraffinic hydrocarbons and wax esters observed for other plants, such as tobacco (Arrendale et al, 1988). The hydrocarbon-wax ester data for the resistant 'Sumner' were very similar to those for < Elliott' and, thus, are not presented.…”