Capillary electrophoresis in the analysis of humic substances facts and artifacts

Humic substances, extracted as mixtures from soil and surface waters according to their solubility in acids and bases, are relatively high- molecular-mass polyelectrolytes containing aromatic, aliphatic and heterocyclic subunits. The degree of ionization of their phenolic and carboxylic groups is governed by the capillary electrophoresis (CE) buffer pH. In CE, fulvic acids exhibit a consistent and characteristic set of sharp peaks (phenolic acids), extending from a humic 'hump' whose average electrophoretic mobility (AEM) depends on humic structure and buffer composition; humic acids give only the 'hump'. Special attention must be given to the interpretation of CE electropherograms when fingerprinting humic substances with borate buffers because observed peaks do not necessarily indicate distinct humic fractions, but may be artifacts caused by the interaction of borate ions with 1,2- and 1,3-diols present in the humic mixtures. Depending on the molarity of borate ions in the separation buffer, humic acids exhibit electropherograms with sharp peaks extending from the 'humic hump' and corresponding to borate complexes. The potential of capillary zone electrophoresis for the comparison of electropherogram patterns is illustrated for the Suwannee River reference humic substances extracted according to the recommendations of the IHSS compared with a fraction of the same source concentrated with the more recent reverse osmosis (RO) technique. The Suwannee River RO fraction behaved like the extracted Suwannee River humic acid fraction under these experimental conditions.