Food Lipids: Chemistry, Nutrition, and Biotechnology

4. Supercritical Fluid Chromatography (SFC)
Use of SFC for lipid extraction was discussed in a previous section. When CO2 is
compressed at a temperature and pressure above its critical point it does not liquify
but forms a dense gas; thus, as a mobile phase SFC is gaseous and solvating. Such
a dense gas has a number of properties (e.g., relatively high densities and diffusivities)
that make it attractive for use as a mobile phase for LC. SFC with open tubular
columns acts as a substitute for GC, but with the analysis temperature much lower
than that employed in GC.
The temperature and pressure required for SC-CO2 are much lower than that
for HPLC. As CO2 is nonpolar its SFC can dissolve less polar compounds and is
suitable for the analysis of less polar species. To analyze polar components, polar
solvents such as methanol or ethanol may be added to the SFC to cover active sites
(-Si-OH) on the surface of the supporting material and to increase the dissolving
power of the mobile phase. Both packed and capillary columns are used for SFC.
Packing materials developed for HPLC are suitable for SFC packed columns [111].
Similarly, fused silica capillary tubes used for GC are suitable for SFC and stationary
phases may employ dimethylpolysiloxane, methylphenylpolysiloxane, diphenylpolysiloxane,
and cyanopropylpolysiloxane [112].
Capillary SFC with FID or UV has been used for analysis of TAG, FFAs, and
their derivatives [113–115]. SFC argentation chromatography has been used for the
separation of TAG according to the number of double bonds, chain length, and the
nature of the double bonds [116,117]. It is necessary to add acetonitrile as a polar
modifier to CO2 to facilitate elution of TAGs. For improved analytical efficiency of
lipid having a narrow range of unsaturation, mobile phase gradient (e.g., temperature,
pressure and/or density, velocity) can be employed. Detectors for ELSD, FID, UV,
mass spectrometry (MS), and FTIR developed for GC and HPLC are applicable to
SFC. Combination of SFC with supercritical fluid extraction (SFE) has been successful
for analyzing lipids of different food samples. SFE may replace any conventional
lipid extraction method, and the quantification of extracted lipids (instead of
gravimetric analysis) can be performed with a detector (ELSD) that has been directly
connected to the extraction cell. SFE-SFC has also been used to characterize TAG
patterns of seed oils [111,118]. SFC is a viable alternative for reducing any solvent
use in lipid extraction and analysis and has a great potential for further development.
5. Thin-Layer Chromatography
TLC is one of the main analytical tools used for lipid analysis. TLC can be used for
fractionation of complex lipid mixtures, assay of purity, identification, information
on the structure, as well as for monitoring extraction and separation of components
via preparative column chromatography for routine and experimental purposes. The
principles and theory of TLC are based on the difference in the affinity of a component
toward a stationary and a mobile phase. The important components of TLC
are the stationary phase, mobile phase, detection, and quantification [5]. The adsorbent
generally used in TLC for lipid analysis is very fine-grade silica gel and may
contain calcium sulfate as a binder to ensure adhesion to the plate [4]. Alumina and
kieselguhr are also used as stationary phases. These adsorbents can also be modified
by impregnation with other substances so as to achieve the desired separations. The
most popular impregnations are with boric acid or silver nitrate. Silver nitrate–
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.