Food Lipids: Chemistry, Nutrition, and Biotechnology

violet chromophore. Application of ultraviolet detection was limited to wavelength
around 210 nm. Some components with isolated double bonds and carbonyl group
(e.g., esters, aldehydes, ketones) can be detected in this wavelength. Hamilton and
coworkers have examined an alternative detection system, infrared detection at 5.74
�m, which allowed the hydrocarbon components to be detected [9]. While the sensitivity
of this method of detection could not match that of ultraviolet detection, it
has merit for use in the preparative mode, where it is feasible to allow the whole
output from the column to flow through the detector. The third useful mode for
HPLC is mass spectrometry. The coupling of HPLC and MS makes this form of
chromatography a very important analytical technique.
3. Analysis
When individual classes of waxes have been isolated, the identity of each must be
determined. Because of the complex composition of these materials, combined analytical
approaches (e.g., GC-MS) have been used to analyze individual wax classes.
Mass spectrometry is a major analytical method for the analysis of this class of
compounds. With the electron impact–mass spectrometry (EI-MS), the wax molecules
tend to give cleavage fragments rather than parent ions. Thus, soft (chemical)
ionization (CI), and fast atom bombardment (FAB) have been frequently used to give
additional information for wax analysis.
In GC-MS analysis, the hydrocarbon fraction and many components of the wax
ester fraction can be analyzed directly, while long chain alcohols, the aldehydes, and
fatty acids are often analyzed as their acetate esters of alcohols, dimethylhydrazones
of aldehydes, and methyl ethers of fatty acids. The analysis of wax esters after
hydrolysis and derivatization will provide additional information on high molecular
weight esters. For example, the chain branching of a certain component might be
primarily examined with respect to its unusual retention time on GC analysis, then
determined by converting to the corresponding hydrocarbon through the reduction
of its iodide intermediate with LiAID4 (the functional group end is labeled by the
deuterium atom). A similar approach is to convert the alcohol of the target component
to an alkyl chloride via methanesulfonyl chloride. This method labels the functional
end with a chlorine atom, and its mass spectra are easily interpreted because of the
chlorine isotopes. As mentioned above, unsaturated hydrocarbons can be separated
from saturated hydrocarbons and unsaturated isomers by column chromatography or
TLC with silver nitrate silica gel or alumina gel media. The position and number of
double bonds affect the volatility of the hydrocarbons, thereby altering their retention
in GC and HPLC analysis. The location of a double bond is based on the mass
spectra of their derivatives, using either positive or negative CI.
D. Biosynthesis of Natural Waxes
Epicuticular waxes (from the outermost layer of plant and insect cuticles) comprise
very long chain nonpolar lipid molecules that are soluble in organic solvents. In
many cases this lipid layer may contain proteins and pigments, and great variability
in molecular architecture is possible, depending on the chemical composition of the
wax and on environmental factors [10,11].
A variety of waxes can be found in the cuticle. On the outer surface of plants
these intracuticular waxes entrap cutin, which is an insoluble lipid polymer of hy-
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.