TLC SEPARATION OF
TLC is a convenient method for purification of glycosphingolipids which were previously fractionated by column chromatography. This technique is also well adapted for the separation of small quantities of purified compounds required for fatty acid or sugar analyses when obtained after removing glycerolipids by mild alkaline hydrolysis. As lipid extracts from animal or plant tissues are complex, many one- and two-dimensional systems are in use, but only one simple mono-dimensional TLC system will be mentioned as example below. Other separation systems are found in several books (Lipid analysis, Christie WW, Pergamon Press, 1982, p. 123) or reviews (Heinz E, Plant glycolipids: structure, isolation and analysis, Advances in lipid methodology – 3, Christie WW Ed, The Oily Press, 1996, pp.247; Schnaar RL et al. Methods in Enzymology 1994, 230, 371).
Glycolipid fractions previously isolated by column chromatography are separated by TLC on silica gel G developed in:
chloroform/methanol/30% ammonia (40/10/1, v/v)
After TLC separation, the localization of glycosphingolipids may be done by non-specific reagents, destructive (charring after sulfuric acid or cupric acetate spray) or non-destructive (primulin spray) or by specific reagents for carbohydrate moieties. Sulphated glycolipids are detected with the cationic dye, Azure A (see below).
Results and comments
Glycolipids containing normal fatty acids migrate (Rf 0.43) ahead of those containing hydroxy fatty acids (Rf 0.33) while sulphated glycolipids migrate poorly (Rf 0.06). The proposed solvent mixture may be changed depending the particular glycolipid under study. Chloroform/methanol/aqueous mixtures ranging from 40/10/1 (v/v) to 25/20/5 (v/v) may be selected.
Sometimes, the separated components appear as double bands, since these molecules may contain long-chain or normal-chain fatty acids. The distinction may be more complicated as compounds may contain different species of long-chain bases (di- or trihydroxy bases).
The reproducibility of the TLC separation of glycolipids with highly polar solvent mixtures was improved using HPTLC plates and a chromatographic tank equipped with a small fan giving optimal saturation conditions (Nores GA et al. J Chromatogr 1994, 686, 155).
When glycolipids containing more than one hexose unit are present, the mixture is resolved by TLC using the Svennerholm’s procedure (Biochim Biophys Acta 1963, 70, 432) with chloroform/methanol/water (65/25/4, v/) as developing solvent. Thus, cerebrosides (monoglycosylceramides) in the fastest band are well separated from di- and triglycosylceramides in decreasing Rf order. These components frequently appear as double bands, since molecules with normal fatty acids move faster than those containing hydroxy fatty acids. Cerebroside sulfate migrates just ahead of diglycosylceramides.
The separation of the monoglycosylceramides according to the sugar species present may be effected on TLC plates previously impregnated with 1.5% Na tetraborate in water and activated 1 h at 110°C. After migration in chloroform/methanol/water (27/7/1, v/v), glucoside-containing lipids migrate ahead of galactoside-containing ones.
Dissolve 2 g of Azure A in 100 ml of 1 mM aqueous sulfuric acid.
Spray the dried chromatogram with the reagent until it appears uniformly dark, then immediately immerse the TLC plate in 40 mM aqueous sulfuric acid/methanol (3/1, v/v). Destain with gentle agitation, changing the solution until the background appears pale blue (about 1 h). Sulphated glycolipids appear dark blue. The minimum detected amount is about 100 pmol of lipid-bound sulphate.
Lyso derivatives of glycosphingolipids must be separated and prepared without alkaline or acid hydrolysis, thus preventing any production from parent compounds.
The extraction step needs a phase separation between water (the upper phase after the Folch extraction) and butanol to recover all the polar lyso molecules.
After a solid phase extraction procedure involving an aminopropyl cartridge (Bodennec J et al., J Lipid Res 2003, 44, 218), a two-dimensional TLC step is necessary to separate the lyso derivatives from their parent compounds. Details of this simple TLC procedure may be found in the report of Bodennec et al. (J Lipid Res 2003, 44, 218).