Utilization of immobilized aluminium (III) metal ion affinity chromatography for analysis of humic acid
2009
Halko, R., Comenius Univ., Bratislava (Slovak Republic) | Neuročný, T., Comenius Univ., Bratislava (Slovak Republic) | Hutta, M., Comenius Univ., Bratislava (Slovak Republic)
Immobilized metal ion affinity chromatography (IMAC) method is based on the ability of certain macromolecules to form coordination complexes with metal ions, which were chelated to a multidentate ligand immobilized onto a support material. In the present work, IMAC was developed for the fractionation of humic acid (HA). Iontosorb Salicyl has been used as the chelating resin containing salicylic acid bound via azo group in side chains of modified bead-form cellulose. Sorption characteristics of Al (III) ions at this chelating sorbent were determined at different pH values. Simple photometric detection at 590 nm was used for monitoring aluminium as SPADNS-Al (III) chelate in eluent during breakthrough experiments. Resulting sorption capacity is the highest at pH 5.5 giving value 36 micromol of aluminium per 1 g of sorbent. Furher study was focused on the fractionation of the group of aromatic acids or HA by Al-loaded Salicyl IMAC technique. 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 obtained results, 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 modeling natural environments.
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