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.
