Fractionation and characterization of fulvic acid by immobilized metal ion affinity chromatography Original Research Article

Immobilized metal ion affinity chromatography (IMAC) has been used to separate humic substances (HS) based on their affinity for metal ions. The work was carried out with a stream fulvic acid (FA) as a representative HS. The effect of pH and ionic strength on the retention of HS was investigated, and comparison of different metal ions (Cu2+, Ni2+, Co2+ and Cd2+) for IMAC of HS was examined. pH dependence of retained HS and retention volume, and salt-promoted complexation were found; these results are similar to those reported for some proteins using IMAC in previous studies. The HS binding capacity and retention volume in four metal ions studied for IMAC were in the order: Cu2+>Ni2+>Co2+>Cd2+, which agrees with the Irving–Williams series. The HS retained on the Cu2+-IMAC was eluted with eluent of decreasing pH value, and the fractions collected were characterized by UV–VIS and fluorescence spectra, and fluorescence quenching titration. The results show that HS fractions eluted with lower pH eluent have a higher conditional stability constant (log KCu–HS′) for copper(II), red-shifted maximum excitation (Ex) and emission (Em) fluorescence wavelengths (shifted towards longer wavelengths), lower fluorescence (Flu)/absorbance (Abs) ratios, spectral slope and E265 nm/E365 nm, indicating that the stronger affinity HS fractions may have relatively higher molecular size and a higher proportion of compounds absorbing and fluorescing at longer wavelength. Based on the results obtained, IMAC appears to be a promising tool for chemical separation of HS. The results will help in the understanding of the nature of HS and their metal binding characterization, and in modelling natural environments.