Food Lipids: Chemistry, Nutrition, and Biotechnology

may be packed or bound to a column, as is the case in GC and HPLC, or bound to
a glass plate as in the case of thin-layer chromatography (TLC). The mobile phase
in gas chromatography is usually an inert gas such as helium or nitrogen. For HPLC
or TLC, the mobile phase may be an aqueous or organic solvent. Intact triacylglycerols
or fatty acids can be separated, but many chromatography applications consist
of separating the methyl ester or other derivatives of individual fatty acids.
A. Gas Chromatography
The most significant occurrence in the chromatography of lipids has been the development
of gas chromatography. Fatty acid methyl esters, including saturates and
unsaturates, were first successfully separated in 1956 with a 4-foot-long column
packed with Apiezon M vacuum grease [52]. Separations were demonstrated based
on chain length, degree of unsaturation, and cis or trans geometric configuration.
1. Separations Using Packed Columns
In general, retention times of fatty acid derivatives on nonpolar columns are based
on volatility and, therefore, separation occurs primarily by carbon chain length. Retention
times of fatty acid derivatives on polar columns are mainly determined on
the basis of polarity and chain length. These columns, therefore, are more effective
at resolving unsaturated fatty acids with different degrees of unsaturation. The chromatographic
characteristics of several packed columns (Silar 10C, Silar 9CP, SP
2340, and OV-275), made available in the mid-1970s as a result of the then recently
developed silicone stationary phases, were compared [53]. The development of these
highly polar, temperature-resistant columns made it possible to separate the geometric
isomers of fatty acids. A column packed with 12% Silar 10C as the stationary
phase was able to baseline separate the all-cis from the all-trans components of
octadecadiethylenic fatty acid methyl esters. All eight of the geometric isomers of
linolenic acid were also partially resolved. However, it was not possible to effectively
separate individual positional cis and trans isomers using a 20-foot-long packed
column [48]. This drawback of packed columns was due largely to practical considerations
that limited separation efficiencies, even with highly selective stationary
phases [54]. Although larger numbers of theoretical plates could be produced, the
need to compensate for back pressure prevented the operation of these columns at
more than 6000 theoretical plates. However, a packed column was successfully used
to determine trans concentration in a collaborative study [55]. This study concluded
that GC analysis using an OV-275 packed column was as effective as IR spectroscopy
in quantitating total trans unsaturation.
2. Separations Using Capillary Columns
The development of flexible fused silica columns in the early 1980s [56] led to the
popularity of capillary columns, which dramatically increase the number of effective
plates, thus improving column separation efficiencies. The number of effective plates
could be raised from 40,000 to 250,000 by increasing the column length from 15 m
to 100 m.
The increased availability of long capillary columns with a variety of diameters
and stationary phases has decreased the use of packed columns in the last 10 years.
As Figure 3 indicates, several monoene, diene, triene, and conjugated fatty acid
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.