Food Lipids: Chemistry, Nutrition, and Biotechnology

ments, etc.) primarily involves chromatographic and spectroscopic methods. Usually
a combination of separation techniques is used to achieve a high degree of purity of
respective lipid components and this could be analytical (for quantitation) or
preparative.
The first step in the analyses involves separation of lipids into their various
polarity components. It may simply separate the lipid into its polar and nonpolar
fractions or may entail analysis of TAG, FFAs, sterols, steryl esters, glycolipids and
phospholipids. Traditionally, liquid–liquid extraction, thin-layer chromatography
(TLC), or liquid–solid column chromatography have been used for fractionation,
cleaning, and concentration of lipid extracts. The most commonly used chromatographic
techniques for lipid analysis include column chromatography, gas chromatography
(GC), high-performance liquid chromatography (HPLC), supercritical
fluid chromatography (SFC), and TLC. Applications of these techniques for the analysis
of food lipids are discussed below.
1. Column Chromatography
Lipid extracts are usually fractionated by column chromatography on a preparative
scale before subjecting them to detailed analysis. Solid–liquid (adsorption), liquid–
liquid (partition), and ion exchange chromatography are among the widely used
methods of lipid fractionation. In solid–liquid chromatography, separation is based
on partitioning and/or adsorption of the lipid components between solid and liquid
(mobile) phases. Elution of the desired lipid class is achieved by varying the polarity
and strength of the mobile phase. Common stationary phases for column chromatography
are silica, alumina, and ion exchange resins, whereas the preferred column
materials for lipid analysis are silicic acid as well as florisil (magnesium silicate).
Low pressure column chromatography using 50- to 500-mesh adsorbents has
been used commonly for separation of different lipid classes. The main parameters
involved in column chromatography include weight of the adsorbent, conditioning
of the adsorbent (moisture content), and column size. It is generally accepted that
long narrow columns give the best resolution, but large-diameter columns increase
sample capacity. For convenience, diameters over 5 cm and heights over 45 cm are
not recommended for typical laboratory use [4,82].
In adsorption chromatography, compounds are bound to the solid adsorbent by
polar, ionic, and, to a lesser extent, nonpolar or van der Waals forces. Therefore,
separation of lipid components takes place according to the relative polarities of the
individual components, which are determined by the number and type of nonpolar
hydrophobic groups. In general, elution of the column with solvents with increasing
polarity separates the lipid mixture according to increasing polarity of its components
in the following order: saturated hydrocarbons, unsaturated hydrocarbons, wax esters,
steryl esters, long chain aldehydes, triacylglycerols, long chain alcohols, FFAs,
quinones, sterols, diacylglycerols, monoacylglycerols, cerebrosides, glycosyl diacylglycerols,
sulfolipids, acidic glycerophosphatides, phosphatidylethanolamine, lysophosphatidylethanolamine,
phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine
[83]. The procedure applicable to most lipid mixtures is eluting on a silicic
acid column with the solvent sequence of chloroform, acetone, and methanol to
separate into neutral lipids, glycolipids, and phospholipids, respectively [4,83].
The ion exchange columns carry ionic groups bind to the opposite charge of
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.