Separation of lipids by ion-exchange chromatography is based on the ionic groups present in the molecule but some other groups such as hydroxyl groups exert an influence.
Non-ionic, acidic and zwitterionic lipids are separated on several ion-exchange materials: diethylaminoethyl cellulose (DEAE), triethylaminoethyl cellulose (TEAE) or ion exchange resins. DEAE is the most frequently used to separate lipid classes (Rouser et al in “Liquid chromatographic analyses, 1976, Dekker, vol 3, 713). Carboxymethyl cellulose is also used for preparative separation of phospholipid classes.
Here, we describe two reliable procedures to purify and separate the glycolipid fraction in vegetal and animal extracts. They are particularly useful to separate charged from neutral glycolipids.
DEAE cellulose from Sigma or Whatman (fine for chromatography), 1 N HCl – 0.1 N KOH, ammonium acetate, acetic acid, methanol, chloroform
Preparation of the DEAE cellulose
Wash in a Becher vessel the cellulose powder with 1 N HCl (3 volumes) during 5 min under slight agitation and wash with water until neutrality. Remove the fine particles after some minutes settling.
Wash similarly with 0.1 N KOH and rinse with water.
Wash with 3 volumes pure acetic acid, change and keep overnight in acetic acid.
Pour a portion of cellulose suspension into a glass column (as for silica gel): a bed height of 8 cm (1 cm diameter) is convenient for up to 20 mg lipids.
Wash the column with:
– 5 volumes of methanol (30 ml/g cellulose)
– 3 volumes of chloroform/methanol (1/1, v/v) (20 ml/g)
– 5 volumes of chloroform (30 ml/g)
The glycolipid fraction isolated on a silica gel column is evaporated and dissolved in chloroform with the help of a short sonication.
The sample is applied on the cellulose column and eluted slowly (no more than 2 ml/min) with the solvent sequence below:
1- Elute with 20 volumes of chloroform, this isolates monogalactosyl diglycerides
2- Elute with 10 volumes of chloroform/methanol (95/5, v/v), this isolates sterylglycosides and cerebrosides, if present
3- elute with 10 volumes of chloroform/methanol (90/10, v/v), this isolates digalactosyl diglycerides
4- elute with 10 volumes of chloroform/methanol (4/1, v/v) containing 2% of aqueous ammonia (concentrated solution) and 50 mM ammonium acetate (0.38 mg/10 ml), this isolates sulpholipids (cerebroside sulfate, sulfoglycosyl diglyceride, sterol sulfate).
Salts in the last column fraction are easily removed by evaporation followed by a Folch wash (dissolve in a known volume of the mixture chloroform/methanol/water, 8/4/3).
This procedure was developed to purify phosphoinositol-containing sphingolipids from yeast (Wells GB et al., J Bacteriol 1996, 178, 6223) and later adapted for the separation of major plant sphingolipid classes (Markham JE et al., J Biol Chem 2006, 281, 22684).
AG4X-X4 acetate resin (Bio-Rad), 6 ml glass syringe, chloroform, methanol, acetic acid, ammonia, triethylamine, isopropanol, hexane
The lipid extract is applied to 2 ml of resin supported in a 6 ml syringe with upper and lower Teflon frit and allowed to flow by gravity. The column is washed with chloroform/methanol/water (16/16/5, v/v) until the eluate runs clear. This fraction contains neutral lipids which after drying are redissolved in a known volume of chloroform/acetic acid (99/1). The charged lipids are eluted with 6 ml of chloroform/methanol/water/ammonia (16/16/5/1, v/v) containing 0.1% triethylamine. After drying, these lipids are redissolved in isopropanol/hexane/water (3/1/1, v/v).
A method utilizing strong anion exchanger cartridges was developed to separate gangliosides (Kato T et al., J Oleo Sci 2008, 57, 397). Total lipids are rapidly separated into neutral and acidic lipids, neutral lipids passed through the SAX cartridge while acidic lipids adsorbed onto the cartridge. Gangliosides were eluted by increasing the concentration of ammonium acetate. The gangliosides yield of this procedure as higher than 95%.