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.