Cation-exchange chromatography is one of the principal techniques used for the
separation and purification of proteins. In addition to electrostatic interactions, protein
retention is often strongly influenced by the chemical nature of the ion-exchange ligand
and by the properties of the column matrix.
The aim of this study was to compare the performance of three different cation-exchange
monolithic columns, CIM SO3, CIM SO4 1 and CIM SO4 2, which differed in the type
of ion-exchange group, ligand density and matrix composition. The study focused on how
these differences affect the adsorption mechanism of two model proteins, Cytochrome c
and Lysozyme. The chromatographic behavior of the proteins was first investigated using
linear salt-gradient elution. The ion-exchange process was subsequently characterized
using the GH-IR model. This was followed by a thermodynamic analysis of adsorption
based on the nonlinear Van ‘t Hoff equation, first applied to basic amino acids and then
to both model proteins. Finally, the observed trends in thermodynamic parameters were
further evaluated by isothermal titration calorimetry. The results clearly demonstrated
differences in the adsorption mechanisms of the model proteins and, consequently, in the
performance of the investigated ion-exchange columns. Among the studied supports,
CIM SO4 2 showed the most distinct behavior and, compared with the other two columns,
indicated a multimodal character.
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