Food Lipids: Chemistry, Nutrition, and Biotechnology

lowing discussion on chemical analysis is based on an understanding of the general
principles of chemical extraction, chromatography, and mass spectrometry. There are
numerous textbooks detailing these principles [5–7].
1. Isolation
Natural waxes are mixtures of long chain apolar compounds found on the surface
of plants and animals. However internal lipids also exist in most organisms. In earlier
times, the plant or animal tissue was dried, whereupon the total lipid material could
be extracted with hexane or chloroform by means of a Soxhlet extractor. The time
of exposure to the organic solvent, particularly chloroform, is kept short to minimize
or avoid the extraction of internal lipids. Because processors are interested in the
surface waxes, it became routine to harvest them by a dipping procedure. For plants
this was usually done in the cold, but occasionally at the boiling point of light
petroleum or by swabbing to remove surface lipids. Chloroform, which has been
widely used, is now known to be toxic; dichloromethane can be substituted. After
removal of the solvent under vacuum, the residue can be weighed. Alternatively, the
efficiency of the extraction can be determined by adding a known quantity of a
standard wax component (not present naturally in the sample) and performing a
quantification based on this component following column chromatography.
2. Separation
The extract of surface lipids contains hydrocarbons, as well as long chain alcohols,
aldehydes and ketones, short chain acid esters of the long chain alcohols, fatty acids,
sterols and sterol esters, and oxygenated forms of these compounds. In most cases
it is necessary to separate the lipid extract into lipid classes prior to the identification
of components. Separation of waxes into their component classes is first achieved
by column chromatography. The extract residue is redissolved in the least polar
solvent possible, usually hexane or light petroleum, and transferred to the chromatographic
column. When the residue is not soluble in hexane or light petroleum, a
hot solution or a more polar solvent, like chloroform of dichloromethane, may be
used to load the column. By gradually increasing the polarity of the eluting solvent,
it is possible to obtain hydrocarbons, esters, aldehydes and ketones, triglycerides,
alcohols, hydroxydiketones, sterols, and fatty acids separately from the column. Most
separations have been achieved on alumina or silica gel. However, Sephadex LH-20
was used to separate the alkanes from Green River Shale. Linde 5-A˚ sieve can
remove the n-alkanes to provide concentrated branched and alicyclic hydrocarbons.
Additionally, silver nitrate can be impregnated into alumina or silica gel columns or
thin-layer chromatography (TLC) plates for separating components according to the
degree of unsaturation.
As the means of further identifying lipids become more sophisticated, it is
possible to obtain a sufficient quantity of the separated wax components by TLC.
One of the major advantages of TLC is that it can be modified very easily, and minor
changes to the system have allowed major changes in separation to be achieved.
Most components of wax esters can be partially or completely separated by TLC on
25 �m silica gel G plates developed in hexane–diethyl ether or benzene–hexane.
The retardation factor (Rf) values of most wax components are listed in Table 1 [8].
If TLC is used, the components must be visualized, and the methods employed
can be either destructive or nondestructive. The commonly used destructive method
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