THE ANALYSIS OF HEXURONIC ACIDS IN BIOLOGICAL MATERIALS BY GAS–LIQUID PARTITION CHROMATOGRAPHY

Abstract

A new method for the determination and characterization of hexuronic acids is presented. Hexuronic acid salts are quantitatively reduced by aqueous sodium borohydride to the corresponding aldonic acids which are subsequently converted to their 1,4-lactones. Treatment of the aldono-1,4-lactones with hexamethyldisilazane and trimethylchlorosilane in pyridine solution results in their rapid conversion to the 2,3,5,6-tetra-O-trimethylsilyl-aldono-1,4-lactones, which are sufficiently stable and volatile to allow their separation and quantitative analysis to be made by gas–liquid partition chromatography (g.l.p.c.).The hexuronic acids present in polysaccharides have been identified by acid hydrolysis of the polymers in the presence of platinum and hydrogen, which results in the conversion of the liberated hexuronic acids to their acid-stable aldonic acid derivatives. The O-trimethylsilylated products of the hydrolytic and reductive procedure were analyzed by gas–liquid partition chromatography. The 2,3,5,6-tetra-O-trimethylsilyl-aldono-1,4-lactones arising from the original hexuronic acid residues in the polysaccharides were readily identified. Preparative scale gas chromatography yielded samples of the individual 2,3,5,6-tetra-O-trimethylsilyl-aldono-1,4-lactones, which had characteristic specific optical rotations, infrared spectra, and g.l.p.c. retention times, and which, when they were refluxed with aqueous methanol, gave the unsubstituted parent aldonic acids. This latter procedure affords a microscale method for the identification of hexuronic acids present in polysaccharides.