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General procedures for the
separation of lipid fractions


The procedure we used has proved applicable to most lipid mixtures before further fractionations by HPLC, another low pressure chromatography or thin-layer chromatography.

Apparatus:

Glass columns (inside diameter: 8-10 mm, length: 20 cm) with a stopcock (glass or Teflon) at the bottom to control the solvent flow. Solid phase extraction cartridges (SPE columns) available from various companies (Alltech, Waters, Supelco, Analytichem Int...) may be also used if filled with silica.
Pyrex glass wool.
Glass Pasteur pipettes
Small vials


Reagents:

Silica gel 60 230-400 mesh (Merck)
Chloroform, methanol, acetone


Procedure:

Prepare a slurry of 300 mg silica gel in 2 or 3 ml chloroform and transfer into the column (equipped with a glass wool plug) with the help of a long Pasteur pipette. Open the stopcock to drain slowly the solvent and rinse the column with a portion of chloroform. Allow the solvent level to drop to the top of the gel.
Transfer the lipid sample in chloroform (about 1 ml) to the top of the gel. The amount of lipid that can be practically applied to this type of column is about 10 mg, for higher amounts increase the column size accordingly. A critical study with details on the optimum ratio between lipid mass and sorbent mass has been published (Pernet F et al., J Chromatogr A 2006, 1137, 127).

1- After complete elution of the solvent, add carefully down the sides of the column 10 ml chloroform. This will elute (at about 1 ml/min) neutral lipids (hydrocarbons, pigments, sterols, triglycerides, waxes, fatty acids..).

2- After draining the first solvent, add on the column 15 ml of acetone/methanol (9/1). This will elute glycolipids (cerebrosides, sulfatides, mono- and digalactosyl diglycerides, sterol glycosides) and ceramides.

3- After draining the second solvent, add on the column 10 ml methanol. This will elute all the phospholipids.

Evaporate the three fractions and dissolve the dry lipids with a small volume of chloroform (for neutral lipids) and/or chloroform/methanol (2/1) for all three.


Comments:

If a quantitative recovery of the free fatty acids are required, it is recommended to elute the neutral lipid fraction with 10 ml chloroform containing 1% acetic acid instead of pure chloroform. All other neutral lipids are present in this fraction. 
A fractionation among the sphingolipid pool (with a separation of ceramides) may be carried out using aminopropyl cartridges as described by Bodennec et al.  


Other procedures :

A simple procedure using aminopropyl SPE column (Varian, 2 g) was proposed for the separation of neutral lipids, fatty acids and phospholipids in milk protein concentrates (Vaghela MN et al., JAOCS 1995, 72, 1117). Total lipids (up to 80 mg) were dissolved in about 2 ml of chloroform and applied to the column previously washed with hexane. After draining, the column was eluted with 18 ml of each solvent : chloroform/2-propanol (2/1, v/v), 2% acetic acid in diethyl ether, and methanol, eluting neutral lipids, fatty acids, and phospholipids, respectively. Following that rapid fractionation, an efficient procedure for the separation of several classes of neutral lipids was developed. Neutral lipids were dissolved in 1 ml of hexane and loaded on a 2 g aminopropyl column previously washed with 8 ml of hexane. Cholesterol esters were eluted with 12 ml of hexane, triglycerides with 36 ml of 1% diethyl ether and 10% dichloromethane in hexane, cholesterol with 36 ml of 5% ethyl acetate in hexane, diglycerides with 36 ml of 15% ethyl acetate in hexane, and monoglycerides with 18 ml of chloroform/methanol (2/1, v/v). After removing the solvents under vacuum, lipid fractions were dissolved in a small volume of chloroform for further analysis.
An improvement of the solid phase extraction method by Kaluzny MA et al. (J Lipid Res 1985, 26, 135) using aminopropyl column  was proposed (Ruiz J et al., Anal Chim Acta 2004, 520, 201).
That separation method was shown to be more convenient for separating muscle lipids into major lipid classes and obtaining an accurate fatty acid profile of neutral lipids. 

The review of Ruiz-Gutierrez V et al. (J Chromatogr A 2000, 885, 321-341) would bring further information on the different existing procedures and methodologies developed when SPE is used for lipid separation. The separation of various groups of compounds using a combination of chromatographic supports and solvents are presented.

The review of Panagiotopoulou PM et al. (Grasas y Aceites 2002, 53, 84-95) gives detailed information on the applications of SPE to the analysis of vegetable oils and fats, focusing on sterols, waxes, phospholipids and lipophilic vitamins. Detailed analytical protocols are presented and discussed.

Gangliosides, which are recovered in the upper phase after an extraction according to Folch, were shown to be easily purified using a styrene-divinyl-benzene copolymer as a solid phase sorbent (Popa I et al., J Lipid Res 2002, 43, 1335). The yield of gangliosides was shown to be dependent on various parameters but in optimal conditions, the recovery may be higher than 95%.
Before applying the gangliosides, the copolymer must be preconditioned with methanol / aqueous 0.015 M NaCl (1/1, v/v).  After loading, the columns is washed with distilled water and the gangliosides are then eluted in methanol and chloroform-methanol, 1:1 (v/v). 




Fractionation of all lipid fractions
(including Gangliosides)


When all lipid classes must be investigated, it is convenient to use a procedure modified from the general one in allowing the recovery of non polar as well as the most polar lipids (gangliosides).
The extraction must be complete for all lipid classes. Tissues will be mixed on a vortex in 5 to 10 vol of chloroform/methanol (1/1, v/v) during 30 min. After centrifugation, the pellet is re-extracted with 5 vol of chloroform/methanol (1/2, v/v). After a second centrifugation, the pellet is re-extracted with 5 vol of chloroform/methanol/water (48/35/10, v/v). The three lipid extracts are pooled and dried under nitrogen with warming.
The residue is dissolved in 2.5 ml of chloroform/methanol/water (60/30/4.5, v/v) and applied as previously described to a Sephadex G25 column to purify the extract.
The purified extract is dissolved in 0.5 ml chloroform which is applied to a silica column as previously described for the General procedure.

1-
Non polar lipids are eluted with chloroform.
2-
Phospholipids and glycosphingolipids (except gangliosides) are eluted with:

- 1.5 ml of chloroform/methanol/acetone/acetic acid/water (52/8/8/18/4, v/v) and
- 8 ml of chloroform/methanol (4/1, v/v)

The two extracts are combined, evaporated and dissolved in 1 ml chloroform for further separation, if needed, on another silica column as described for the General procedure.
3-
Gangliosides are eluted successively with:

- 3 ml of chloroform/methanol (2/3, v/v) eluting monosialogangliosides
- 3 ml of chloroform/methanol/water (65/25/4, v/v) eluting disialogangliosides
- 3 ml of chloroform/methanol/water (60/35/8, v/v) eluting polysialogangliosides.


Details can be found in Dreyfus H et al. (Anal Biochem 1997, 249, 67).

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