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.
Procedure 1
Reagents:
DEAE cellulose from Sigma or Whatman (fine for
chromatography), 1 N HCl - 0.1 N KOH, ammonium acetate, acetic acid, methanol, chloroform
Procedure:
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:
Lipid elution
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).
Procedure 2
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).
Reagents:
AG4X-X4 acetate resin (Bio-Rad), 6 ml glass syringe, chloroform, methanol,
acetic acid, ammonia, triethylamine, isopropanol, hexane
Procedure:
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 redissolveld in
isopropanol/hexane/water (3/1/1, v/v).
