Food Lipids: Chemistry, Nutrition, and Biotechnology

impregnated (argentation) TLC may be used to separate TAGs or fatty acid methyl
esters according to the number of double bonds and also by virtue of the geometry
(e.g., cis or trans) and position of the double bonds in the alkyl chain. The silver
ion forms a reversible complex with the � electrons of the double bond of unsaturated
fatty acids, thereby decreasing their mobility [119]. Structural isomers of TAGs
(due to their fatty acid constituents) may also be separated on this type of TLC plate
[97]. Impregnation of the TLC plates with boric acid (3%, w/v) prevents isomerization
of mono- and diacylglycerols while separating neutral lipids [83]. Boric acid
complexes with vicinal hydroxyl groups and leads to slower migration of these compounds
[119].
Samples of lipid extracts are applied as discrete spots or as narrow streaks,
1.5–2 cm from the bottom of the plate. The plate is then developed in a chamber
containing the developing solvent or a solvent mixture. The solvent moves up the
plate by capillary action, taking the various components with it at different rates,
depending on their polarity and how tightly they might be held by the adsorbent.
The plate is removed from the developing chamber when the solvent approaches the
top of it and then dried in the air or under a flow of nitrogen. Solvents with low
boiling point, viscosity, and toxicity are suitable for TLC application. A low boiling
point helps in the quick evaporation of the solvent from the surface layer, and low
viscosity facilitates faster movement of the solvent during development. The selection
of a suitable solvent is very important for good separation of the lipid classes.
Several solvent systems may be used to resolve individual lipid classes, as exemplified
in Table 3.
The location of the corresponding lipid spots on a developed plate has to be
detected prior to their isolation. Detection of the spots may be done using a reagent
directly on the plate. This reagent could be specific to certain functional groups of
the lipid molecules or may be a nonspecific reagent that renders all lipids visible.
There are nondestructive chemical reagents, such as 2�,7�-dichlorofluorescin in 95%
methanol (1%, w/v), iodine, rhodamine 6G, and water, which allow recovery of lipids
after detection. Lipids exhibit a yellow color and in the presence rhodamine 6G
(0.01%, w/v) produce pink spots under UV light. These chemicals may also be used
as a nondestructive spray for preparative TLC. When water is used separated lipids
may appear as white spots in a translucent background and can easily be distinguished.
Developed plates may also be subjected to saturated iodine vapor in a
chamber and this may produce brown spots due to the reaction of iodine with unsaturated
bonds of the lipid molecules. However, unsaturated lipids may form artifacts
with iodine, if sufficient time is allowed. The destructive methods include spraying
of the plate with sulfuric acid (50%, v/v) and drying at 180�C for 1 hour to make
lipids visible as black deposits of carbon [4]. Potassium dichromate (5%, w/v) in
40% (v/v) sulfuric acid also works in a similar manner to sulfuric acid spray. Molybdophosphoric
acid (5%, w/v) in ethanol turns lipids into blue then black when
heated at 120�C for 1 hour. Coomassie blue (0.03% in 20% methanol) turns lipid
into blue spots on white background. Examples of specific reagents that react selectively
with specific functional groups include FeCl 3 to detect cholesterol and cholesterol
esters, ninhydrin for choline-containing phospholipids, and orcinol or naphthol/H
2SO 4 for glycolipids [123]. Some lipids contain chromophores and can be
visualized directly under UV or visible light without staining.
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.