A simple modification of a silicic acid lipid fractionation protocol to eliminate free fatty acids from glycolipid and phospholipid fractions

When fractionating natural and standard lipid mixtures according to the most widely used method for the analysis of soil and other environmental materials, free fatty acids (FFAs) were not recovered quantitatively in the fraction expected to contain all simple lipids. Rather than being eluted from activated silicic acid adsorption chromatographic columns with chloroform, FFAs in standard lipid mixtures appeared only in the two subsequent fractions eluted with acetone and methanol, respectively. Substantial quantities of FFAs from cow dung lipid samples were eluted using chloroform as expected but appreciable amounts also eluted in the acetone (glycolipid) fraction. The fatty acid distribution of the methanol (phospholipid) fraction from dung displayed slightly more FFA compositional character than when FFAs were excluded but this influence was not significant (p = 0.08). A simple modification to the silicic acid column technique ensures that FFAs are reproducibly eluted in the chloroform/acetic acid (100:1v/v; simple lipid) fraction. The modification had no deleterious effect upon the elution characteristics of any phospholipid from a variety of headgroup classes tested. The carry-over of FFAs into glycolipid fractions was confirmed by nanospray ionisation mass spectrometry of the intact polar lipid fractions. As far as could be inferred from the distinctive molecular weight distributions of the glycolipid and phospholipid fractions, none of the complex lipids in dung eluted in both fractions.