TLC SEPARATION OF
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).
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.
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.
ON TLC PLATES
Glycoglycerolipids may be located on TLC plates first by non-specific techniques such as primulin spray combined with the relative Rf of the spots on the plates but another reagent is needed to identify accurately the sugar components.
While primuline detection is non-destructive, the detection of sugars is destructive and no fatty acid composition can be determined. Four specific detection systems involving a treatment with orcinol, naphthyl ethylenediamine, 5-hydroxy-1-tetralone or thymol in a strong acid medium may also be used instead of or after primuline spray.
1 – Orcinol reaction
Dissolve 20 mg of orcinol in 10 ml of 70% sulfuric acid (70 ml of concentrated sulfuric acid diluted to 100 ml with water). This solution can be kept several days if kept at 4°C.
Prepare the sulfuric acid dilution with care in cooling the vial with crushed ice.
The plates are sprayed (slightly wetted) with the orcinol solution in an efficient fume hood and heated at 100°C in an oven for 10-15 min.
Spots containing sugars appear pink-violet on a white background. The detection limit is about 0.5 nmol of total sugar per spot, thus, as little as 0.5-1 mg of glycolipids can thus be detected.
2 – Naphthyl ethylenediamine reaction
Prepare a solution of 13 mM N-(1-naphthyl) ethylenediamine (34 mg in 10 ml) in methanol/conc. sulfuric acid (97/3, v/v).
Spray the dried plates until wetted with the reagent and heat at 125°C for 10 min. Glycolipids appear pink on a white background but the color fades rapidly. The detection limit is about 100 pmol of sugar in the spots (0.1 mg of lipid).
3 – Hydroxytetralone reaction
Dissolve 10 mg of the reagent in 80% sulfuric acid. Keep the solution at 4°C.
The plates are sprayed with the reagent solution and heated at 120°C for 10 min. The fluorescent intensities of glycolipids on the plate are determined at an excitation wavelength of 470 nm with an optical cut-off filter (500 nm). Glycolipids give yellow spots easily distinguishable from the light blue spots of phospholipids. The responses are linear over the concentration range 10 to 100 pmol and a detection limit of about 3 pmol of glycolipid was reported. We use the method with Whatman TLC plates but other brands are usable (Baker, Macherey-Nagel) (Watanabe K et al., J Lipid Res 1995, 36, 1848).
4 – Thymol-sulfuric acid reagent
This reagent has been used for the colorimetric detection of carbohydrates. The thymol-sulphuric acid reagent, developed by Hans Molisch in 1886, was initially used for the detection of carbohydrates. Interestingly, the derivatization of galactosyl group from galactolipids is not affected by acyl glycerol, acyl chains and galactose residues. Thus, it has been used to follow accurately the hydrolysis of galactosylglycerides, quantifying in one step the initial substrate (MGDG) and all generated galactosylated products (Sahaka M et al., J Chromatogr B 2021, 1173, 122674).
Preparation of the thymol-sulfuric acid reagent: One gram of thymol is dissolved in 190 mL of ethanol. Ten millilitres of 96% sulfuric acid were then added gradually maintaining the vial in a cold water bath.
Plates are dried at room temperature, dipped in the thymol-sulfuric acid reagent, dried at room temperature and placed in oven at 100°C for 10 min. Absorbances of revealed spots and bands are measured with a dedicated device at both 510 and 366 nm. The quantification is carried out using calibration curves established from MGDG, DGDG or galactose standards spotted on each plate. Technical details may be found in the authors paper.