Abstract High-performance immobilized metal ion affinity chromatography (HP-IMAC) was used to fractionate humic substances (HS) based on their affinity for the immobilized copper(II) ion using acidic and glycine eluents. The work was carried out with two naturally occurring aqueous fulvic acids and commercially available Suwannee River fulvic acid. The IMAC-fractionated HS were then characterized by reversed-phase high-performance liquid chromatography (RP-HPLC) and size exclusion chromatography. The results showed that the affinity HS fraction eluted first using an acidic pH=2 eluent exhibited a relatively high hydrophilic character, whereas the fraction eluted later using a glycine eluent exhibited both a higher hydrophobic character and larger molecular size. On the other hand, the HS fraction with no affinity for the immobilized copper had low molecular size. The affinity of the HS fraction for copper(II) increased with increasing molecular weight. Based on the composite results of three different HS, it is evident that strong relationships exist between affinity, molecular weight, and hydrophilic/hydrophobic properties during the HP-IMAC fractionation. The results presented here have significance for understanding the nature of chemical interactions at the molecular level between dissolved organic matter and trace metals. IMAC, coupled with other liquid chromatographic strategies, is a promising tool for chemical fractionation and characterization of HS.