Retention behaviour of ceramides in sub-critical fluid chromatography in comparison with non-aqueous reversed-phase liquid chromatography

This study was devoted to the development of an analytical method for ceram ide analysis in packed subcritical fluid chromatography (pSubFC). Monofunct ional grafted silica support was found to be more suitable for ceramide ana lysis. Five Kromasil columns were coupled and the parameters, temperature, pressure and percentage of organic modifier in CO2 were optimised, consider ing selectivity and analysis time. The final conditions were 31 degrees C, 6% of methanol (MeOH) and 13 MPa. In these conditions the selectivity for s tructural differences (methylene group, unsaturation or two different bases ) were studied. As classically observed, the methylene selectivity decrease d with the increase of the eluotropic strength. Moreover, unlike in non-aqu eous reversed-phase liquid chromatography (NARP-LC), adding a further unsat uration and two further methylene groups on ceramide results to an increase of retention in pSubFC. Moreover, this last technique allowed to separate ceramides with the same total number of carbons containing unsaturated fatt y acids, when the distribution of carbon number of the two chain is very di fferent. These results had enabled to plot retention chart in order to pred ict ceramide structure in view to identify additional ceramide. This retent ion chart was finally compared with the one already obtained in NARP-LC. (C ) 2000 Elsevier Science B.V. All rights reserved.