An immobilized metal-ion affinity chromatography (IMAC) method has been developed and validated for the separation of copper complexing ligands from soil solution. We first investigated the retention behavior of simple model ligands on the IMAC column and found that the ability to form ternary complexes of the structure Cu-IDA-ligand was the dominant factor influencing ligand retention on the IMAC column. The logK value of the Cu-complex was found to have only a minor influence on the retention. Legends containing only carboxylic acid functional groups were not retained on the column. To optimize reproducibility and quantitative recovery of copper ligands from soil solution, different composition and pH values of eluting buffer were tested. Soil solution chromatograms exhibited one non-retained fraction and two retained fractions. The elution times of the retained fractions were characteristic of peptides and proteins (first peak) and for compounds containing aromatic amines (second peak). The results show that IMAC is an effective tool for the fractionation of copper complexing ligands that are capable of forming ternary complexes.