Food Lipids: Chemistry, Nutrition, and Biotechnology

C. High-Performance Liquid Chromatography
For most applications, HPLC does not exhibit the separation efficiencies offered by
long capillary GC columns. It does, however, offer several advantages. For example,
the mild conditions used in HPLC work enable heat-sensitive components to be
separated, a feature that reduces the possibility of isomerization taking place during
the analysis of unsaturated fatty acids. Another advantage is that fatty acid fractions
can be collected and analyzed further using hyphenated techniques such as GC-FTIR
and GC-MS.
A variety of detectors are used to identify lipid solutes as they elute from HPLC
columns. The most common HPLC detector has been ultraviolet-based equipment
that allows one to monitor the column effluent at about 200 nm. The lack of strong
absorbing lipid chromophores limits the sensitivity of this detection method unless
fatty acid derivatives that absorb strongly in the UV range are prepared. Refractive
index (RI) detectors are also commonly used but cannot be paired with solvent
gradients and are sensitive to temperature fluctuations. Evaporative light-scattering
detectors (ELSD) can be used with solvent gradients, are at least as sensitive as the
best RI detectors, and can be used for quantitation. The advantages of these and
other detectors useful for HPLC analysis have been reviewed [82].
1. Reversed Phase HPLC
Fatty acids and fatty acid methyl esters were successfully separated on Vydac reversed
phase columns in an early study [83]. In another study, a Bondapack C18
reversed phase column was used with a methanol–water mobile phase to separate
methyl elaidate and oleate [84]. Five fractions from hydrogenated menhaden oil,
containing saturated fatty acids as well as cis and trans monoene isomers, were
collected with two C18 preparative HPLC reversed phase columns (Waters, 5.7 cm
� 30 cm) connected in series [85].
2. Silver Ion HPLC
The many advantages of using argentation HPLC columns over TLC plates [84]
include reproducible separation of analytes, column reusability, short run times, and
high recoveries. Until recently, a lack of stable columns with controlled silver levels
limited the use of silver ion HPLC separations [71]. The silver ions would bleed
from the silica adsorbent, cause unpredictable results, shorten the column life, and
hinder the reproducibility of the results. A more successful argentation HPLC procedure
was developed by linking the silver ions via ionic bonds to a silica–phenylsulfonic
acid matrix [86]. This column gave excellent reproducible separations for
triacylglycerols, fatty acids, and their positional and geometric isomers [87]. This
column was also used for the separation, collection, and quantification of all eight
geometric isomers of linolenic acid phenacyl esters, with a mobile phase ranging
from 5% methanol in dichloromethane to a 50:50 solvent mixture [88]. Figure 5
shows the excellent resolution obtained for 18:3 isomers using the silver ion HPLC
column.
Commercial silver ion HPLC columns were recently introduced and should
dramatically increase the use of this technique. These columns have been reported
to give satisfactory results for up to one year [71]. A commercially available (Chrompack
Ltd.) argentation HPLC column with an acetonitrile–hexane mobile phase was
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