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:
Column: Spherisorb S3CN (100 x 4.6 mm from Phase Sep).
A dual channel HPLC pump. An evaporative light-scattering detector.
Solvent A: hexane
Solvent B: methyl tert-butyl ether
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.