Characterization of gangliosides by two-dimensional high-performance thin-layer chromatography

A method for the qualitative and quantitative analysis of complex mixtures of gangliosides by computer-assisted image densitometry is reported. Gangliosides were applied to silica gel high-performance thin-layer plates and developed in two dimensions using one of several solvent systems. The plates were developed with a resorcinol reagent and the gangliosides were visualized as bluish-purple bands or spots. The plates were scanned with a Biolmage Visage 110 image analyzer and saved to disk. The densities of the spots were determined and the quantities of gangliosides determined by comparison with an internal standard, GM4. Two-dimensional chromatography using chloroform-methanol-0.2% CaC12 (50:40:10) in the first dimension and n-propanol-ammonia(aq.) (6:3) in the second dimension gave linear and reproducible results when analyzed by image densitometry. Gangliosides isolated from human plasma were resolved with the above method. We suggest that the use of image densitometry improves the accuracy of quantitative analyses of complex mixtures of gangliosides by thin-layer chromatography.