A simple, specific spray for the detection of phospholipids on thin-layer chromatograms

Abstract

A chemical reagent spray specific for phosphate esters, based on molybdenum blue, gives an instantaneous, specific reaction with phospholipids on silica gel or alumina thin layer chromatography (TLC) plates. The reagent is prepared as two solutions: (I) add 40.11 g of MoO 3 to 1 liter of 25 N H 2 SO 4 and boil gently until dissolved, (II) add 1.78 g of powdered molybdenum to 500 ml of the first solution and boil gently for 15 minutes. The greenish yellow spray consists of equal parts of solutions I and II with two volumes of water. It is stable for months. Plates sprayed lightly show phosphate esters as blue spots on a white or light blue-grey background. The spots intensify on standing but are obscured by a darkening background after several hours. Detection limit is 0.005 mumole of phosphatidyl ethanolamine and phosphatidyl choline. The spray can be used with Rhodamine 6G spray (universal lipids) and ninhydrin (amino groups); a sequential procedure of Rhodamine 6G, ninhydrin, and molybdenum spray is described in detail. The sensitivity of the molybdenum spray is halved when used after other sprays. Some of the compounds giving positive and negative reactions are listed in the article.