Separation of Ceramides Species on Borate and Silver Nitrate TLC Plates
Ordinary silica gel TLC plates can be impregnated with sodium borate for the separation of ceramides. This is particularly suitable for the separation of ce-ramides containing saturated sphingoid base (sphinga-nine) from ceramide containing monounsaturated sphingoid base, such as sphingosine [3]. Resolution of such ceramide species is not really possible on ordinary or arsenite-impregnated silica gel plates. Boration of the TLC plate can circumvent such a challenge, because resolution of ceramides according to the presence or not of the sphingoid 4-5 trans double bond is efficiently achieved [3]. Moreover, the effect of borate as a complexing agent on fatty acid unsaturation was shown not to be significant, so that groups of ceramides could be separated with respect to the unsaturation of their sphingoid bases but not to the unsaturation of the N-linked fatty acids. This chromatographic tool has been used to study the bioconversion of dihydroce-ramide to ceramide [8].
The separation of ceramide species according to fatty acid unsaturation is, however, possible by using silver-ion-containing silica layers. In such conditions, ceramide species will migrate faster when decreasing the fatty acid unsaturation degree. Moreover, the degree of unsaturation of the sphingoid base is of significant importance, because the Rf of ceramide species will decrease by increasing the unsaturation of the sphingoid base. This can result in difficulties for the accurate separation and identification of some ceramide species differing in unsaturation of sphingoid bases or fatty acids [3]. Although such a separation of ceramide molecular species is possible, it is, in fact, seldom used. When a detailed analysis is needed, TLC shows its limits, as other analytical tools give more information. Gas chromatography or HPLC, either alone or coupled with mass spectrometry, are the best tools for the determination of ceramide molecular
& u species. However, TLC represents a reliable chromatographic technique which can be efficiently used for ce-ramide separation and purification into groups of compounds of relative structural homology. This is often based on the presence of different hydroxyl groups at different locations of the ceramide molecules. The separation scheme can be efficiently performed by running ceramide samples in a single direction on ordinary silica gel or arsenite-impregnated silica gel TLC plates. Poten-tiation of this separation scheme could be accomplished by running the plate in a second direction, perpendicular to the first one, after boration of the migration field so that further separation of sphinganine-containing ce-ramides from sphingosine-containing ceramides can simultaneously be achieved with the separation of groups with different hydroxyls. Such preparative and analytical use makes TLC an interesting tool for the investigation of ceramide biochemistry.
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