Analysis of yeast cell constitution by thin layer chromatography using diatomaceous earth as a support

Abstract

　Methylated saccharides from yeast cells were analysed by thin layer chromatography (TLC) using diatomaceous earth as a support. Residues from which fatty acids had been extracted for analysis of yeast cell constitution were used as samples. Freeze-dried yeast cells (50 mg) were hydrolysed with 5% methanolic hydrochloride acid, and fatty acid methyl ester was extracted from the hydrolate with n-hexane. Chloride ions were removed from the residual solution with ion exchange resin. The solution was concentrated and used as a sample. Thin layer plates were prepared with mixture of diatomaceous earth (Wako Pure Chemical Indus.) and plaster (9 : 1). A mixture of 67% isopropanol (35 ml) and ethyl acetate (65 ml) was used as the solvent for the one-step development. Anthrone-sulfuric acid reagent was used for detection. The Rf value (×100) of the light yellowish brown spot of xylose was 60. Mannose gave a dark brown major spot of Rf value 47 and a minor spot of Rf value 25, and glucose gave a bluish purple spot of Rf value 40. For galactose, two clear, light yellow spots (Rf value, 25 and 28) and two minor spots (Rf value, 45 and 55) were observed. The presence of minor spots indicating anomers of mannose and galactose did not disturb the detection of major spot of methylated saccharides in mixed solution. Mannose, glucose and galactose were detected in strains of Geotrichum, Kluyveromyces, Pichia and Schizosaccharomyces by this thin layer chromatography. Glucose and galactose were detected in the strain of Cryptococcus and Trichosporon, together with minor spots of xylose. The methylated saccharide mixture could not be separated on a thin layer plate prepared with Kieselgur G of Merck (Germany). This one-step TLC may be suitable to the case of a few samples whose constituent saccharides need to be known immediately, as it needs no multiple development.