Quantitative determination of thiamine and its phosphate esters by sephadex gel filtration

Abstract

Thiamine, thiamine monosphosphate and thiamine diphosphate are separated and determined quantitatively, after converting them to the thiochromes by the cyanogen bromide method and passing them through a Sephadex G-10 column. When the column is developed by 0.2 M potassium phosphate buffer (pH 6.6), thiochrome diphosphate is eluted at the first peak. This is followed by thiochrome monosphosphate, and finally free thiochrome which is separated into two peaks. The recoveries of the fluorescence for these three compounds are nearly complete within the range of an experimental error at 3 nmoles. The two peaks of free thiochrome give the same fluorescence spectra but are eluted at the same positions on re-chromatography.