HPLC separation of neutral
lipids
If an HPLC chain and an evapoprative
light-scattering detector are available, the analysis of the neutral lipid fractions
becomes less time consuming and more accurate on a quantitative basis.
One of the more efficient methods allowing the analysis of non-polar lipids in less than
25 min with a binary gradient (El-Hamdy AH et al., J High Resol Chromatogr 1993, 16,
55) is
described below:
Apparatus:
Column: Spherisorb S3CN (100 x 4.6 mm from
Phase Sep).
A dual channel HPLC pump. An evaporative light-scattering detector.
Reagents:
Solvent A: hexane
Solvent B: methyl tert-butyl ether
Procedure:
Solvent B was set at 2% during 3 min and was
increased to 20% over 12 min and to 100% over 5 min and maintained for 10 min. A 10 min
reequilibration phase was observed before the next sample was injected.
EC:
cholesterol esters, TG: triglycerides, CHOL: cholesterol, 1,3- and 1,2-DAG: 1,3- and
1,2-diacylglycerol, MAG: monoacylglycerol
Cholesterol and wax esters are eluted together but well ahead of triglycerides. We
observed that the addition of as little as 0.1 ml acetic acid per liter of mobile phase
improves the retention of free fatty acids.
When compared with pure silica, CN bonded phase is more rapidly equilibrated at the end of
the gradient and allows more regular retention times.
Similar separations are described using silica column and a gradient of
hexane/isopropanol/ethylacetate/formic acid in hexane (Liu J et al JAOCS 1993, 70,
343).
This method was used to determine the content of moglycerides and diglycerides in plant
oils.
