Utilization of immobilized aluminium(III) metal ion affinity chromatography for analysis of humic acids

Immobilized metal ion affinity chromatography (IMAC) method is based on the ability of certain macromolecules to form coordination complexes with metal ions, which have been chelated to a multidentate ligand immobilized onto a support material. In the present work, IMAC was developed for the fractionation of humic acids (HA). Study was focused on the fractionation of the group of aromatic acids or HA by Al-loaded Salicyl IMAC technique. The aromatic acids were selected as potential constituents of the HA. The effect of various pH values (3.9, 6.7 and 8.9, resp.) on the retention of model sample of acids was investigated. The IMAC eluted fractions of acids were separated and determined by reverse-phase high performance liquid chromatography (RP-HPLC) with UV-VIS detection. Finally, IMAC technique was developed for the fractionation HA applying a buffer-based pH gradient (between pH 8.9 and 2.0) for their gradual elution. The HA fractions retained by the Al(III)-IMAC were eluted with mobile phase of decreasing pH value, and the fractions collected were characterized by UV-VIS detection. Based on the results obtained, IMAC appears to be a promising tool for chemical separation of HA. The results will help in the understanding of the nature of HA and their metal binding characterization, and in modelling natural environments. Humic substances (HS) are able due to presence of different combinations of chelating groups to participate in mobilization of elements in soil solutions (8, 13). The complex-forming ability of HS results largely from their content of oxygen-containing functional groups. Soil organic constituents form both soluble P O L I S H J O U R N A L O F S O I L S C I E N C E