The aim of these studies was to investigate the use of an optimized potentiometric system for the determination of the acid−base dissociation constants of immobilized chelating ligands and the stability constants of the derived metal ion complexes used in the immobilized metal ion affinity chromatographic (IMAC) analysis and purification of peptides and proteins. In particular, potentiometric comparison of the immobilized iminodiacetic acid (IDA) ligand system with a range of hard metal ions, such as Fe3+, Al3+, Yb3+ or Ca2+, and the borderline metal ion Cu2+, has been determined with the commonly used chromatographic support material, Sepharose CL-4B. The values of these derived constants have been compared to the values of the corresponding constants for the free chelating ligand and their metal ion complexes in solution. In addition, the same potentiometric methods have been employed for the determination of the acid−base dissociation constants and stability constants of the derived metal ion complexes for the tridentate ligand, O−phosphoserine (OPS). Besides the participation of the metal ion−ligand complexes of the type ML and ML2, the results indicate the participation of hydrolytic complexes of the type M(OH) m L n with some of these IMAC systems. The availability of this information on the physicochemical characteristics of the free and immobilized metal ion chelate complexes should facilitate the interpretation of the binding behavior with peptides and proteins observed with IMAC adsorbents under a variety of adsorption and elution conditions.