Food Lipids: Chemistry, Nutrition, and Biotechnology

Table 1 TLC Separation of Wax Components on Silica Gel: R f Values for Common
Wax Components
Component
Solvent systems a
A B C D E F G H
Hydrocarbon 0.95 0.96 0.95 0.85 0.83 0.95 0.85
Squalene 0.80
Trialkylglyceryl ethers 0.90
Steryl esters 0.90 0.95 0.57
Wax esters 0.90 0.82 0.84 0.71 0.65 0.91 0.75
�-Diketones 0.75 0.54
Monoketones 0.53
Fatty acid methyl esters 0.65 0.47 0.75
Aldehydes 0.55 0.65 0.47 0.66
Triterpenyl acetates 0.53
Secondary alcohols 0.36
Triacylglycerols 0.35 0.61 0.37
Free fatty acids 0.18 0.00 0.00 0.35 0.20
Triterpenols 0.22
Primary alcohols 0.15 0.14 0.16 0.09 0.15 0.21 0.19
Sterols 0.10 0.16 0.10 0.12
Hydroxy-�-diketones 0.09 0.04
Triterpenoid acid 0.05
a A, petroleum ether (b.p. 60–70�C)–diethyl ether–glacial acetic acid (90:10:1, v/v); B, benzene;
C, chloroform containing 1% ethanol; D, petroleum ether (b.p. 40–60�C)–diethyl ether (80:20, v/v);
E, chloroform containing 1% ethanol; F, hexane–heptane–diethyl ether–glacial acetic acid (63:18.5:18.5,
v/v) to 2 cm from top, then full development with carbon tetrachloride; G (1) petroleum ether–diethyl
ether–glacial acetic acid (80:20:1, v/v); (2) petroleum ether; H, benzene–chloroform (70:30 v/v).
is to spray TLC plates with sulfuric or molybdic acid in ethanol and heat them. This
technique is very sensitive, but it destroys the compounds and does not work well
with free acids. Iodine vapors will cause a colored band to appear, particularly with
unsaturated compounds, and is widely used to both locate and quantify the lipids.
Since the iodine can evaporate from the plate readily after removal from iodine
chamber, the components usually remain unchanged. Iodine vapor is one of the ideal
visualization media in the isolation of lipid classes from TLC plates. Commercial
TLC plates with fluorescent indicators are available as well, and bands can be visualized
under UV light. However, if it is necessary to use solvents more polar than
diethyl ether to extract polar components from the matrix, the fluorescent indicators
may also be extracted, and these additives will interfere with subsequent analyses.
To isolate lipid classes from TLC plates after a nondestructive method of visualization,
the silica gel can be scraped into a champagne funnel and eluted with
an appropriate solvent. Or, the gel can be scraped into a test tube and the apolar
lipid extracted with diethyl ether by vortexing, centrifuging, and decanting off the
ether. Polar lipids are extracted in the same manner, using a more polar solvent such
as chloroform and/or methanol. High performance liquid chromatography has been
used in the separation and analysis of natural waxes, but its application was halted
by the lack of a suitable detector, since most wax components have no useful ultra-
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