Stoichiometry between Humate Unit Molecules and Metal Ions in Supramolecular Assembly Induced by Cu²⁺ and Tb³⁺ Measured by Gel Electrophoresis Techniques

Humic acid (HA), a fraction of humic substances, can strongly complex with metal ions to form a supramolecular assembly via coordination binding and other intermolecular forces. However, determining the supramolecular size distribution and stoichiometry between small HA unit molecules constituting HA supramolecules and metal ions has proven to be challenging. Here, we investigated the changes in the size distributions of HAs induced by Cu²⁺ and Tb³⁺ ions using unique PAGE for the separation and quantification of HA complexes and metal ions bound, followed by UV–vis spectroscopy and excitation–emission matrix–parallel factor analysis. By determining the concentrations of HA and metal ions, it was possible to estimate the stoichiometry of the HA unit molecule to metal ions in supramolecular complexes. It was found that the supramolecular behaviors of Cu²⁺ and Tb³⁺ complexes with HA collected from peat (PAHA) and deep groundwater (HHA) differed. For example, two HHA unit molecules form a supramolecule via cross-linking by a Cu²⁺ ion in the case of Cu²⁺–HHA. Our results suggest that this supramolecular stoichiometry is related to the abundance of sulfur atoms in the elemental composition of HHA. Our experimental results and analysis provide new insights into HA supramolecules formed via metal complexation.