A Colorimetric Method for the Determination of Sugars

Abstract

VOLUMETRIC procedures have been used for the quantitative detennination of sugars after separation by partition chromatography 1 • 2 • It has been our experience that these methods not only require considerable skill, but also they are lengthy and sensitive to slight variation of the conditions. We have therefore attempted to develop a simple quantitative colorimetric procedure. , Preliminary experiments showed that the anthrone 3 and the 0(-naphthol sulphonate• reagents give good .results with pure sggar solutions, but the presence of only traces of solvents such as butanol, phenol and propionic acid used in the chromatographic separation of the sugars rendered them useless.It was then discovered that phenol itself in the presence of sulphuric acid provides a simple rapid method for the quantitative colorimetric determination of ketoses and aldoses and their methyl derivatives on a sub-micro scale. The method, applicable to all carbohydrates with either a free or potential reducing group, is particularly useful for determining sugars which have been separated by partition chromatography using phenol-water as the solvent. The orange-yellow colour, produced by adding sulphuric acid (5 ml.) to the sugar solution (2 ml.) containing phenol, is permanent; its optical density (measured at 490 mµ for hexoses and hexuronic acids and their derivatives and at 475 mµ for pentoses and their derivatives) when referred to a standard curve gives the concentration of the sugar.This phenol-sulphuric acid reagent, which has enabled us to determine the composition of polysaccharides and their methyl derivatives on as little as 1 mgm. of material, thus offers an additional method for end-group analysis of polysaccharides 5 • The reaction is not limited to phenol, for certain amines such as N(l-naphthyl) ethylene diamine can replace phenol in the above reaction.In conjunction with the above colorimetric method, it has been found convenient to extract the sugars from the strips of paper cut from the chromatogram by simply immersing them in water.Since the extraction is carried out at room temperature, there is no danger of decomposing the sugars•. Substances in filter paper which interfere with the sugar analysis can be largely removed by three or four 'chromatographic' washings with water; washing with. dilute sodium hydroxide does not seem to improve the paper.Further details of this work.. and its application to the detennination of the structure of various polysaccharides will appear elsewhere.

Biofilm formation by zygomycetes: quantification, structure and matrix composition

“…Protein concentration was determined in the water-soluble fraction by the bicinchoninic acid method (Smith et al, 1985). Total carbohydrates in the water-soluble fraction were determined by the phenol/sulphuric acid method (Dubois et al, 1951). Glucose was quantified in the water-soluble fraction after acid hydrolysis by using a glucose oxidase kit (Sigma-Aldrich) according to the manufacturer's instructions.…”

Structural and physicochemical properties of granular starches after treatment with debranching enzyme

“…The hydrolysis rate was measured by total carbohydrate values in the supernatant through the phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956;Huang, Fu, He, Luo, Yu, & Li, 2010). The total carbohydrate value was calculated as follows.…”

Purification and Properties of β-Galactosidases fromBacillus circulans

“…16 ) The carbohydrate content of p-galactosidase was determined by the phenol-sulfuric acid method. 14 ) RESULTS AND DISCUSSION…”