Rapid fluorometric micromethod for in situ quantitation of lipids on thin-layer chromatograms

A simple, rapid method is described that is capable of measuring directly and reliably nanomole amounts of triglycerides, free fatty acids, fatty acid esters, free cholesterol, and cholesterol esters. The technique involves (a) chromatography of as little as 120 μg of total extracted lipid on silica gel thin-layer plates incorporating rhodamine 6G, (b) development of the chromatogram in 2 solvent systems in sequence and (c) monitoring the intensity of emitted fluorescence by fluorometry. Standard curves for each of the 5 neutral lipid classes are reproducible over a large range of concentrations. Phospholipid values can be obtained as differences from the total lipid extracted. This microtechnique reduces sample size by 95% from that needed for chemical tests and compares favorably with densitometric methods. The method does not destroy the sample and lends itself easily to other kinds of measurement, such as radioisotope counting and autoradiography, on the same samples which have been quantitated.