The use of iodine staining for the quantitative analysis of lipids separated by thin layer chromatography

Abstract

The method described for quantitative estimation of lipids separated on thin layer chromatography plates exploits the observation that most lipids can be stained by iodine vapor and that, in “controlled” conditions, the intensity of this staining is proportional to the actual amount of lipid in the spot. The method consists of i) exposing the developed plate to iodine vapor; ii) spraying it with a suitable solvent to prevent halogen evaporation; iii) collecting the stained lipids by scraping the spots off the plate; and iv) determining by a rate-sensing method the absorbed iodine. The final determination is performed by measuring spectrophotometrically (at 410 nm) the rate of decolorization of a solution of Ce(IV) by As(III) in strong acidic conditions. The reaction rate, which is positively related to the concentration of iodine, is derived from the slope of the absorbance change plotted vs time.

Providing that standards and samples are stained simultaneously, a quantitative estimation of lipid components of a mixture is possible in a reasonable time with excellent accuracy and reproducibility. In our hands, the method has been successfully applied to several common phospholipids, long chain fatty acids, cholesterol and deoxycholate, and triacylglycerols, in the range of 5–60 μg.