Use of nile red for the rapid in situ quantitation of lipids on thin-layer chromatograms

Abstract

We describe the use of the fluorescent dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, as a general-purpose reagent for the rapid detection and quantitation of a wide variety of lipids and other hydrophobic compounds separated by thin-layer chromatography. After samples are applied to silica gel plates and chromatographed, the plate is briefly dipped into a nile red solution (8 micrograms/ml of methanol-water 80:20, v/v). Background fluorescence of nile red dye adsorbed to the silica gel is then preferentially destroyed by dipping the plate in a dilute aqueous solution of bleach. After drying, lipid bands are visualized under ultraviolet light. Reflectance fluorometry (Ex: 580 nm; Em: 640 nm) is utilized for in situ quantitative analysis of the fluorescence of the lipids on the nile red-stained plate. Neutral lipids, phospholipids, sphingolipids, and fatty acids can be examined, although the nile red fluorescence intensity varies significantly among the lipid classes. Also, staining is stronger for unsaturated lipids than for saturated lipids. The lower detection limit of the assay is 25-100 ng for cholesterol, cholesteryl esters, triacylglycerols, and phospholipids.