Asymmetrical Flow Field-Flow Fractionation as a Method to Study the Behavior of Humic Acids in Solution

Asymmetrical flow field-flow fractionation (AsFlFFF) is used to measure the size distributions of humic acids in solution. Shifts in the size distribution are used to study the behavior of these amphiphilic macromolecules in solution with changes in pH, ionic strength, and humic acid concentration. Humic acid concentrations are increased by on-channel focusing of the injected samples. As the concentration is increased, the humic acids aggregate, but the effect varies with solution conditions. As the concentration of calcium chloride is increased, size distributions broaden and become multimodal. The decrease in size is consistent with reports that divalent cations induce conformational changes by the formation of intramolecular bridges between carboxylate moieties; multimodality indicates intermolecular interactions as well. Lowering the pH also promotes intermolecular interactions, with severe aggregation occurring below pH 4. The work demonstrates the utility of AsFlFFF in studies of the hydrodynamic behavior of amphiphilic macromolecules.