Quantitation of lipids by charring on thin-layer plates and scintillation quenching: Application to ceramide determination

Abstract

A method is described for the quantitative determination of lipids after separation by thin-layer chromatography: the lipid spots are visualized with a charring spray and the darkness of the spots is determined by measuring the quenching produced when the powder is suspended in a scintillation counting gel containing a radioactive sample. A standard curve produced with 2 to 20 [mu]g of ceramide was linear, so only two standard spots (in duplicate) are needed on each thin-layer plate. Using the vertical sample streak technique, one can analyze three unknown samples (in duplicate) on each plate. This technique allows one to use the quantitative thin-layer approach without purchasing a special apparatus for the purpose. As an example of the method, we analyzed tissues for ceramide content, after first removing much polar lipid by a liquid/liquid partitioning technique. This is the most convenient method available for determining this minor, but widely distributed lipid.