Fractionation and Characterization of Humic Substances in Nutritional Supplements Using Immobilized Metal Affinity Chromatography

This work presents the study of the immobilized metal affinity chromatography method for the analysis of humic substances in a few food supplements. Separation mechanisms of the immobilized metal affinity chromatography method are based on the affinity of functional groups of humic substances to metal ions. The immobilized metal affinity chromatography system employed an Iontosorb Salicyl sorbent with immobilized aluminum ions. A step-wise gradient was used for elution, consisting of two mobile phases of acetate buffer solution at varying pH values and a mixture of sulfuric acid and sodium sulfate. The change in concentration and volume of Al(III) ions and pH of the eluent have been studied for sorption characteristics and immobilized metal affinity chromatography separation. The chosen value was 0.20 mmol of aluminum per 1 g of the chelating sorbent at pH 7.0. The immobilized metal affinity chromatography system has been studied by using a pair of substances with different chelating properties (phenol and salicylic acid) and a mixture of eleven substances, which represented a simplified model structure of humic substances. The results of these studies have been applied to the fractionation of humic substance standards and to the characterization of humic substances in food supplements containing humic substances by the immobilized metal affinity chromatography system. To better characterize particular humic substances, the immobilized metal affinity chromatography fractions were further analyzed by size-exclusion chromatography. Based on the results obtained, the immobilized metal affinity chromatography method appears to be a promising tool for the analysis of humic substances, and it may help in the characterization of their structure and understanding their interactions with metal ions.