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SOLVENT FRACTIONATION


This is the simplest way and sometimes the most efficient for separating a group of lipids of interest when the others are without value. This approach depends on the differential solubility of lipids in organic solvents.

Separation of polar lipids:

Acetone precipitation is currently used to separate in one step polar lipids (phospholipids and glycolipids) from all neutral or non-polar lipids (triglycerides, cholesterol, some pigments..).

Procedure:

- Evaporate under nitrogen an aliquot of lipid extract and add about 20-30 volumes of acetone.
- Mix by vortexing one min. and leave on ice one hour.
- Centrifuge and collect the supernatant.
- Repeat the same procedure and mix the acetone extracts.
- The pellet, rich in phospholipids and glycolipids, is dried under nitrogen and vacuum, weighed and re-dissolved in chloroform/methanol mixture for further analysis.
- The acetone extract can be used to analyze glycerides, sterols, sterol esters, carotenoids, lipid soluble vitamins. It may contain also monogalactoside diglycerides and sterol glycosides.

It was recently shown that the purification of phosphatidylcholine from a lipid extract can be rapidly made by mixing 2 hours at room temperature the dried lipids with 5 volumes of acetone, filtration of the suspension and leaving the acetone extract 5 hours at -20C. The rapid filtration of the mixture gives a precipitate of phospholipids enriched in phosphatidylcholine (Baudimant G, Thesis, Paris 1996). Further trials are needed to define the yield of the different phospholipid classes according to the used lipid extract.

A simple procedure, similar to the previously described, was reported for enriching the phospholipid content in commercial soybean lecithin (dried gums) (Vandana V et al., JAOCS 2001, 78, 555). Briefly, lecithin (50 g) was dissolved in acetone (20 ml) and slowly added to chilled acetone (230 ml). The content was kept in a refrigerator for 60 min at 4.5C and centrifuged. The acetone layer, containing neutral lipids, was decanted, and the acetone-insoluble material was extracted with chilled acetone (2 x 50 ml) followed by centrifugation. The acetone insolubles (light yellow precipitate) are enriched in phospholipids. In adding that fraction to a determined amount of crude lecithin, fractions with higher phospholipid contents were prepared. Commercial lecithin can thus be enriched to any required percentage of phospholipids for various applications.

Instead of precipitation or direct extraction of tissue lipids with selected solvents, a less common practice consists in a partition of lipid classes, previously extracted, between different immiscible solvents, this procedure is known as:

solvent partition
or
countercurrent distribution

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