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SEPARATION OF GLYCOGLYCEROLIPIDS BY TLC


TLC is a convenient method for purification of glycolipids which were previously fractionated by column chromatography. This technique is also well adapted for the separation of small quantities of glycolipids required for fatty acid or sugar analyses. As lipid extracts from plant tissues are complex, many one- and two-dimensional systems are in use, but only two simple TLC systems will be mentioned as examples below. Other separation systems are found in several books (Lipid analysis, Christie WW, Pergamon Press, 1982) or reviews (Heinz E, Plant glycolipids: structure, isolation and analysis, Advances in lipid methodology - 3, Christie WW Ed, The Oily Press, 1996, pp.211-332).

Procedure

Glycolipid fractions are separated  by TLC on silica gel G developed in:
 
- chloroform/methanol/30% ammonia (60/35/5, v/v)
or  chloroform/methanol/30% ammonia (40/10/1, v/v)
- chloroform/acetone/water (30/60/2, v/v)

After TLC separation, the localization of glycolipids may be done by non-specific reagents, destructive (charring after sulfuric acid or cupric acetate spray) or non-destructive (primuline spray) and by specific reagents for carbohydrate moieties.  

Chloroform/methanol/ammonia

Chloroform/acetone/water

1: non-polar lipids and acyl MGDG, 2: 6-O-acyl SG, 3: MGDG, 4: SG, 5: DGDG, 6: SQDG, a: non polar lipids, b: acyl MGDG.

DGDG: digalactosyl diglycerides, MGDG: monogalactosyl diglycerides, , SG: steryl glycosides, SQDG: sulfoquinovosyl diglycerides

As these solvents are also used to separate glycosphingolipids, some confusion may arise if some of them are present. Thus, cerebrosides with normal fatty acids run close to steryl glycosides and those with hydroxylated fatty acids run slower but ahead of DGDG. Sulfatides remain close to the start line. Some distinction may be established doing TLC on intact fractions and on aliquots treated by a mild saponification. The spots determined as containing sugars and which are absent after this treatment may be suspected to contain glycoglycerolipids or acylated compounds.

A characteristic of these separations is that MGDG, DGDG and SQDG may all separate into more or less well resolved spots, according to their fatty acid composition, those with longer carbon chain moving higher than those with shorter chains.

The acylated forms of glycoglycerolipids may be separated by TLC using silica gel plates and a two-dimensional elution system (chloroform / methanol / water, 65/25/4 by volume for the first dimension and chloroform / acetone / methanol/ acetic acid / water, 10/4/2/2/1). A schematic picture of separation of acylated and non-acylated glycoglycerolipids may be found in the paper of Kim YH et al. (Lipids 1999, 34, 847).

A critical point is the recovery of these polar glycolipids. The extraction from the silica gel scrapings is better made by shaking for 5 min the scrapings with a mixture of 4 volumes of chloroform/methanol (2/1, v/v) with 1 volume of aqueous sodium chloride solution (9 g/l). After centrifugation, the lower phase contains the polar lipids with a good recovery.  

The difficulties of recoveries were largely resolved in a method introduced for the transfer of separated lipids from a high-performance TLC plate onto a polyvinylidene difluoride membrane, this method was designated as TLCBlot (Far-Eastern blot) (Taki T et al., Anal Biochem 1994, 223, 232). Lipid bands that had been transferred onto the membrane were cut and introduced onto the target stage for secondary ion MS (Taki T et al., Anal. Biochem 1995, 225, 24
). A higher sensitivity has been obtained by analysis of the transfer membranes with matrix-assisted laser desorption/ionization quadripole ion trap time-of-flight imaging mass spectrometry (Goto-Inoue N et al., J Chromatogr B 2008, 870, 74). Teh method gives details on the ceramide structure and the limit of detection was about 1 pmol of the ganglioside GM1, a value ten times lower than the value in conventional reports.



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