SEPARATION OF GLYCOGLYCEROLIPIDS BY
LOW PRESSURE CHROMATOGRAPHY
For the isolation of larger quantities of individual compounds that it is possible by TLC, the lipid extract is first fractionated by preparative column chromatography. Thus, a separation of the whole glycolipids on a silica gel column (elution with pure acetone) allows to remove the bulk of neutral lipids (triglycerides, cholesterol...) and also phospholipids which may co-migrate with glycolipids on TLC plates or columns. To recover the acylated steryl glycosides, an elution with a mixture of chloroform/acetone 92/8, v/v) must be inserted between the chloroform and the acetone elution steps.
This glycolipid fraction is thereafter efficiently separated into its main components by chromatography on a DEAE cellulose column, as previously described.
Silica gel column chromatography can also be used for pre-fractionation with mixtures of chloroform with increasing proportions of methanol or acetone.
We give below an elution scheme used to elute individual glycolipids. The composition of the eluents may vary to some extent and may depend on the lipid load, the hydration state of the support and the size of the column. Usually, the volume of each fraction amounts to about 10 times the volume of the silica gel bed.
1- chloroform : elution of neutral lipids
2- chloroform/acetone 90/10 (v/v) : elution of acetylated steryl glycosides
3- chloroform/acetone 75/25 (v/v) : elution of monogalactosyl diglycerides and cerebrosides
4- chloroform/acetone 40/60 : elution of digalactosyl diglycerides and steryl glycosides (with also cerebrosides)
5- pure acetone : elution of sulfoquinovosyl diglycerides
Most of plant pigments are eliminated by the chloroform wash but some others are eluted with the increasing proportions of acetone and remain difficult to eliminate.
In his famous paper, Lepage M (J Lipid Res 1964, 5, 587) has reported a fractionation scheme including an elution of esterified steryl glycosides with hexane/diethyl ether (3/7, v/v) following elutions with pure hexane and hexane/ether (1/1, v/v). Thereafter, steryl glycosides and glycoglycerolipids may be eluted with acetone/methanol (90/10, v/v).
Other information on the purification of steryl glycosides may be found in another part of this site.