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SOLVENT PARTITION

This procedure is an efficient preliminary step in fractionation of lipid extracts rich in glycerides (seed oils, adipose tissue, cosmetic cream) or in pigments (plant lipids).
It can be used to enrich the lipid extract in phospholipids, glycosphingolipids, or gangliosides.

Purification of phospholipids

We propose below a reliable procedure for the isolation of phospholipids from animal or vegetal oils (Galanos DS et al., J Lipid Res 1962, 3, 134).

Apparatus:


Separatory funnels with Teflon stopcock (total volume: 100 to 200 ml for a 10 g lipid sample) or centrifuge tubes.

Reagents:

Partition 87% aqueous ethanol with hexane or petroleum ether (1/1, v/v), after mixing collect the upper phase (solvent A) and the lower phase (solvent B).

Procedure:

1- dissolve 10 g of lipid sample in 45 ml of solvent A and add to 15 ml of solvent B in a first separatory funnel or centrifuge tube.
2- shake for 2 min, allow to settle or centrifuge at low speed for 5 minutes.
3- collect the lower phase (15 ml) in a second funnel or centrifuge tube containing 45 ml of solvent A. Shake for 2 min and centrifuge.
4- Withdraw the lower phase to an evaporation flask, add 15 ml of fresh solvent B to the first separation device, shake 2 min, transfer the lower phase to the second device with the 45 ml of solvent A, shake and centrifuge.
5- Repeat the procedure 4 to 6 times.
6- The combined extracts (4 or 6 x 15 ml of solvent B) contain polar lipids (phospholipids and glycolipids).
7- evaporate the hexane phases, the dry extract contains non polar lipids (mainly triglycerides).


Comments:

To get reproducible results, it is important to operate the whole procedure at a constant temperature.

Purification of glycosphingolipids

Sphingolipids were efficiently separated from neutral lipids in soybean extracts by solvent partition. The method was based on a concentration of glycolipids in the 87% ethanol phase after equilibration with petroleum ether (Gutierez E et al., JAOCS 2004, 81, 737).
Briefly, 87%ethanol was added to petroleum ether where lipids were dissolved and the funnel was shaken thoroughly. The equilibrated lower ethanol phase was transferred to a second funnel containing petroleum ether. The equilibrated lower ethanol phase was transferred to a flask to complete one cycle of extraction. To begin another cycle, 87% ethanol was added to the first funnel and the ethanol layer was transferred to the second funnel containing petroleum ether. Eight cycles were needed to complete one extraction. The recovery of cerebrosides was said to be about 93%, better than the recovery obtained with other classical procedures. It must be noticed that ceramides were severely lost along that procedure. 

Purification of gangliosides

A simple method for the purification of gangliosides was described (Ladisch S et al., Anal Biochem 1985, 146, 220). 
The dried total lipid extract is partitioned in the mixture diisopropyl ether / 1-butanol / 50 mM aqueous NaCl (6/4/5, v/v). Gangliosides are concentrated in the lower aqueous phase. That phase is then freed of salts and other impurities by gel filtration. 

Countercurrent distribution

 

A combination of high-speed counter-current chromatography and preparative HPLC was used for preparing the galactolipids from pumpkin (Cucurbita moschata) (Du Q et al.,J Chromatogr A 2009, 1216, 4176). The solvent systems were light petroleum/dichloromethane/methanol/n-propanol. water (1:5:6:1:4, v/v) or light petroleum/dichloromethane/methanol/n-propanol.water (3:5:6:1:4, v/v) with aqueous upper phase as the stationary phase.

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