Quantitative estimation of sugars in blood and urine by paper chromatography using direct densitometry

Abstract

A system of quantitative paper chromatography is described in which compounds are directly scanned on the paper after a locating colour reaction. Sources of error arising at different stages are analysed and controlled by modifications in technique. Reproducibility is improved by using paper with uniform solvent flow characteristics, and the colour reaction controlled by choice of optimal reagent concentration, a modified dipping technique and rotation of the chromatogram during heating in the hot-air oven. After paper clarification by oiling, spots are sanned transversely, across the development axis, by double reflectance densitometry using a Joyce-Loebl Chromoscan, and calculations are based on comparison of peak areas. Most of the principles involved are generally appliable to paper chromatography. Sugar estimations in plasma and urine have a coefficient of variation between ± 2.0 and 3.5% when applications around 20 μg are used without replication. Sugar recoveries from urine are between 99 and 108% provided the desalting resin is correctly prepared; but from plasma and haemolysed whole blood recovery is higher due to a factor arising at the stage of deproteinization. The method has been used to estimate stachyose, raffinose, melibiose, lactose, lactulose, palatinose, sucrose, galactose, glucose, fructose, mannose, fucose, xylose, xylublose and 3-O-methyl glucose, either singly or in various combinations, in concentrations down to 1 mg/100 ml. The advantages of the method, in relation to other available techniques, are discussed.