Food Lipids: Chemistry, Nutrition, and Biotechnology

diazomethane (ADAM) derivatives and employing a fluorescence detector. Fatty
acids increase their hydrophobicity when ADAM is bound to the carboxyl group;
thus, the derivatives are retained longer on a reversed phase column than fatty acids
and good separation is obtained [96]. Fluorescence detectors are specific for detection
and quantification of tocopherols and fluorescent derivatives of fatty acids. Evaporative
light-scattering detection (ELSD) and FID have been in wide use for detection
of all types of lipids following HPLC separation [93,97]. These are sometimes referred
to as universal detectors. The principle of ELSD involves evaporation of
mobile phase of the separated lipid fraction by a nebulizer (spray the eluent stream
with a large volume of nitrogen or air) to obtain droplets of solute (lipids). These
solute droplets are directed through a light source (may be a laser light source); the
degree of scattering of the light is proportional to the mass of the solute [98]. The
RI detection may also be used for lipid analysis [99].
Normal phase HPLC also allows the separation of normal chain and hydroxy
fatty acid–containing TAG. Normal phase HPLC on silver ion–loaded anion
exchange columns is currently used to resolve TAG based on their degree of unsaturation
[93]. Reversed phase HPLC (using C18 columns) is also widely applied for
separation and quantification of tri-, di-, and monoacylglycerols.
Since glycerol possesses a prochiral carbon, asymmetrical esterification of the
primary position leads to the formation of enantiomers. Although enantiomeric TAGs
cannot be resolved by normal HPLC, their diastereomeric naphthylethylurethane derivatives
can be separated by HPLC on silica gel [100,101]. Use of a stationary
phase with chiral moieties to separate enantiomers of mono- and diacyl sn-glycerols
after derivatization with 3,5-dinitrophenylurethane (DNPU) by HPLC separation of
enantiomers has been reported [102–104].
Separation of phospholipids is very laborious when TLC is used; however,
HPLC provides a better means of separation and quantification. At present, use of
gradient (binary or tertiary) elution on silica columns is frequently used for separation
of different classes of phospholipids. Several polar solvent systems that are suitable
for such separations are available [105–107]. UV detection, FID, or ELSD is suitable
for phospholipid identification and quantification. Separation of glycolipids can be
achieved using a silica column with a binary gradient (hexane–IPA–2.8 mM ammonium
acetate) [108] or reversed phase C18 (ODS) column [98].
Isocratic, normal phase separation of cholesterol esters, FFAs, and free sterols
is widely used. Simultaneous analysis of nonpolar and polar lipids using HPLC silica
gel column has also been reported. In addition to normal and reversed phase methods,
size exclusion HPLC has been used to separate TAG and other nonpolar lipids. This
has been specifically employed for the analysis of polymerized lipids such as those
generated during deep fat frying [60]. Christopoulou and Perkins [109] have described
separation of monomers, dimers, and trimers of fatty acids in oxidized lipids
using size exclusion column with an RI detector, whereas Burkov and Henderson
[110] have reported the use of a similar column with ELSD to analyze polymers in
autoxidized marine oils.
Micro-HPLC columns have the volume of one-hundredth of that of a conventional
column and benefit from low consumption of material used as stationary phase,
sample and mobile phase, operation at low flow rate, and temperature programming.
It can also be easily coupled with a mass spectrometer and an FTIR detector [96].
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