30] Separation of carotenoids on lipophilic sephadex

Abstract

This chapter discusses the separation of carotenoids on lipophilic Sephadex. For the study of biogenesis of carotenoids using radioisotopes in vivo , it is necessary to remove steroids from carotenoids. Because both the groups have a common precursor, mevalonic acid, the radioactivity incorporated into steroids may interfere with the precise radioassay for carotenoids. This chapter discusses the liquid-gel chromatographic separation of carotenes from sterols on Sephadex LH-20. In this study, columns of Sephadex LH-20 are prepared by using the solvent system chloroform–methanol- n -hexane (65:5:30) and the column size 1.5 × 140 cm for a long column or 1 × l0 cm for a short column. Samples are dissolved in a small volume of the same solvent. Elution is carried out at room temperature. Carotenoids are determined spectrophotometrically. Squalene and squalane are analyzed by gas-liquid chromatography. The present method has an advantage that not only sterols but also squalene can be separated from carotenes. The results demonstrate that Sephadex LH-20 chromatography with nonpolar solvent systems, such as chloroform-methanol- n -hexane, is one of the convenient methods to separate sterols from carotenes. The method is expected to greatly aid in obtaining a sterol-free carotene preparation from various biological materials.