Analytical Evaluation of Sodium Tetraborate Effects on the Colorimetric Quantification of Total Uronic Acids

Uronic acids are key structural components of plant and algal polysaccharides, and their quantification is central to the carbohydrate analysis. The widely used colorimetric assay by Blumenkrantz and Asboe-Hansen relies on acid-catalyzed degradation of uronic acids and the subsequent reaction with 3-phenylphenol to form a magenta xanthylium chromophore. Although the addition of borate is known to enhance color stability and sensitivity, its molecular role has remained unclear. Here, the effect of borate on the assay was systematically investigated by using photometric analysis, NMR spectroscopy, and UHPLC-DAD/MS. Two main effects were identified: enhanced formation of the chromophore precursor 5-formyl-2-furancarboxylic acid and increased stability of the xanthylium chromophore, resulting in a higher absorbance and improved reproducibility. NMR analysis showed no significant borate-induced shift in the acid-lactone equilibrium of uronic acids under strongly acidic conditions. Instead, chromophore stabilization is attributed to increased sulfonation of 3-phenylphenol, which improves charge compensation and spectral stability.