Food Lipids: Chemistry, Nutrition, and Biotechnology

2. Gas Chromatography
The GC (or GLC) analysis of lipids has been much studied in the literature. This
method involves partitioning of the components of the lipid mixture in the vapor
state between a mobile gas phase and a stationary nonvolatile liquid phase dispersed
on an inert support.
Analysis of fatty acid composition by GC usually requires derivatization of fatty
acids to increase their volatility. Fatty acid methyl esters (FAME) may be prepared by
different transmethylation techniques and then separated on GC columns and detected
by flame ionization detection (FID). The gas phase for GC is usually nitrogen or
helium for packed columns and helium or hydrogen for capillary columns. The identification
of chromatographic peaks is based on comparison of their retention times
with those of authentic samples. GC analysis of TAG of food lipids may also provide
information about positional distribution of fatty acids in the molecules. Naturally
occurring TAGs that are purified by TLC can then be resolved without derivatization
on the basis of their carbon number or molecular weight using capillary GC equipped
with 8- to 15-m-long columns coated with methylphenyl-, methyl-, or dimethylsilicone
(nonpolar capillary). Use of helium or hydrogen as a carrier gas for separation of TAG
on such columns requires higher temperatures than those employed for separation of
methyl esters. Mono- and diacylglycerols have to be converted to trimethylsilyl (TMS)
or tert-butyldimethylsilyl ethers (TBDMS) for complete resolution [93,94]. A combined
GC and mass spectrometric technique has been applied for determining molecular
species in the glycerol esters. TMS or t-BDMS derivatives of glycerol esters
separated on GC may be subjected to mass spectrometric analysis in order to obtain
information on their molecular structure [95].
3. High-Performance Liquid Chromatography
The HPLC is a highly efficient form of adsorption, partition, or ion exchange LC that
uses a very uniform, finely divided, microspherical (5–10 �m) support of controlled
porosity and degree of hydration. The adsorbent is tightly packed into a stainless steel
column (10–30 cm long, 2–4 mm diameter) and requires a high pressure pump to
obtain an adequate and constant flow of solvent through the column. Elution of the
column may be carried out either isocratically with a solvent mixture of constant
composition, or by gradient elution in which the solvent composition may be varied
linearly or in a stepwise fashion with both binary and ternary solvent systems. The
column eluates are continuously monitored by means of a flow-through detector, which
should be insensitive to solvent flow rate, temperature, and composition [83].
Sample derivatization is employed to facilitate the separation and/or to enhance
the limit of detection for the HPLC analysis. Hydrolysis or saponification is done to
cleave ester linkages and to obtain FFAs for their subsequent analysis. Although
lipids do not possess specific UV absorption peaks, they could be detected in the
region of 203–210 nm due to the presence of double bonds in the fatty acyl groups,
or the functionalities such as carbonyl, carboxyl, phosphate, amino, or quaternary
ammonium groups. However, this low UV range greatly restricts the choice of solvent,
and it is advisable to avoid chloroform–methanol mixtures because they display
a strong absorption below 245 nm. Frequently, diacylglycerols require preparation
of UV-absorbing derivatives (e.g., benzoate, dinitrobenzoates, pentafluorobenzoates,
and TBDMS ethers) for detection. Fatty acids can be analyzed by forming 9-anthryl-
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