Solvent fractionation

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 -20°C. 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 4.5°C 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.

Two techniques are used:

SOLVENT PARTITION

COUNTERCURRENT DISTRIBUTION

$$$$$$$$$$$

 

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.

A simple technique has been proposed also to extract and fractionate lipid mixtures using a single-step procedure (Vale G et al., JLR 2019, 60, 694). The extraction technique is composed of one aqueous and two organic phases. The upper organic phase is enriched in neutral lipids (triacylglycerols and cholesterol esters) while the middle organic phase contains the major phospholipids. The procedure its a valuable tool for fatty acid profiling by GLC combined with MS.  

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.

A review of the general techniques concerning high speed countercurrent chromatography may be consulted for further developments (Khan BM et al., J Liquid Chromatography related Technol 2018, 41, 629).