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. Three specific detection systems involving a treatment with orcinol, naphthyl ethylenediamine or 5-hydroxy-1-tetralone in a strong acid medium can also be used after a 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).