SEPARATION OF PHOSPHOLIPIDS
BY COLUMN CHROMATOGRAPHY
Column chromatography with silicic acid is by far the most widely adopted techniques when mg amounts of phospholipids must be prepared. Thus, column chromatography, with adaptation, can be used as a large-scale preparative procedure but of a low resolution. Phospholipids are held by silicic acid in a variety of ways (hydrogen and ionic bonding...) and released by eluting the column with solvents of increasing polarity.
Different brands of silicic acid are available and preliminary assays must be done as results vary, even from one batch to another. We use silica gel G 230-400 mesh from Merck as for the fractionation of lipid classes, but a larger size may be used.
To get optimal results, the degree of hydration of the silicic acid must be held constant, the size of the column must be chosen with a height-to-diameter ratio of about 20. The optimal load is practically of 30 mg of total lipids per g of dry silicic acid and the flow rate is between 1 and 3 ml according to the size of the column.
It is more practical to apply the lipid solution in a small volume of the least polar eluting solvent (acetone after elution of neutral lipids by chloroform) and, after draining, a step-wise elution is processed.
There are many solvent systems which have been described and the choice depends on the objective of the experiment. We give below a classic step-wise elution protocole but it may be adapted by the analyst for its own purpose.
The procedure given bellow is modified from Hanahan DJ (A guide to phospholipid chemistry, Oxford University Press, 1997)
|chloroform/methanol (54/46)||phosphatidylcholine and sphingomyelin|
Approximately 20 column volumes (considering only the silicic acid bed) are used for each elution step.
Phospholipid components are not completely pure, a further fractionation by column chromatography or TLC may be necessary. The purity of each fraction may be rapidly verified by TLC on microplates (HPTLC) as we have previously described.
The use of deactivated silica (20% water, w/w) self-packed in Pasteur pipettes proved to be suitable for the fractionation of food lipids into neutral and polar lipids (Hauff S et al., Anal Chim Acta 2009, 636, 229). Neutral lipids were eluted with cyclohexane/ethyl acetate (1:1, v/v) and phospholpids by methanol.The use of a final column elution with methanol/water (98:2, v/v) was necessary to improve the recovery of phospholipids containing short and medium-chain fatty acids.