Influence of amide linkages on acidity determinations of humic substances: Testing with model-mixtures

A set of experiments, using model compounds, was carried out in order to evaluate the influence of amide linkages on acidity measurements of humic substances (HS). Three benzene–carboxylic acids (2,4-dihydroxybenzoic acid; 3,5-dihydroxybenzoic acid, and phthalic acid) and two peptides (dl-alanyl-dl-alanine, and glycil-l-leucine) were employed to simulate the major acidic functional groups present in humic material. The acidity of the samples was measured, using the Schnitzer and Gupta methods, for each compound separately, as well as for selected mixtures with different compound combinations. General results showed that, in the absence of peptides, phenolic groups with high pKa values are not detected in the barium hydroxide reaction and the data so obtained do not represent the real concentrations of the acidic groups. For the mixtures containing peptides, the presence of amide linkages distorts the results relative to the total acidity (TA) mainly because under the extreme conditions of the reaction with Ba(OH)2 hydrolysis of the peptide occurs, consuming extra hydroxyl groups and increasing artificially the phenolic content. Such a condition is minimized by the reduction of the reaction time.