Influence of temperature on the retention behaviour of proteins in cation-exchange chromatography

The chromatographic behaviour of several amino acid derivatives, peptides and proteins has been investigated with the so called "tentacle-type" LiChrospher-100 SO3- adsorbent and an analogous poly(2-sulphoethylaspartamide) cation-exchange adsorbent, PolySulphoethyl A. In particular, the dependences of the retention properties of a range of biosolutes on temperature and the chromatographic residence time were evaluated with the objective of gaining further insight into the influence of ligand type and flexibility and the role of solute conformation on the chromatographic behaviour of proteins with these two strong cation-exchange chromatographic adsorbents. The results indicate that significant differences in the chromatographic retention behavior between proteins and low-molecular-mass solutes occur as a function of temperature and the type of co- and counter ions present in the mobile phase with both adsorbents. Moreover, the dependences of the Zc and log Kc values on temperature for most of the proteins examined exhibited significant changes in magnitude between 4 and 75 degrees C, whilst no equivalent changes were evident for low-molecular-mass solutes. With the "tentacle-type" LiChrospher-1000 SO3- adsorbent at higher temperatures, e.g., at 75 degrees C, most of the proteins could still be eluted although several exhibited very large increases in their retention parameters. With the PolySulphoethyl A adsorbent, on the other hand, none of the proteins examined were eluted at 75 degrees C. The results moreover indicate that hydrophobic interactions play an increasingly important role in protein retention with both types of ion-exchange adsorbents at higher temperatures, but are more dominant with the PolySulphoethyl A ligand. In general, the Zc values for the proteins with the "tentacle-type" LiChrospher-1000 SO3- adsorbent were greater than those obtained with the PolySulphoethyl A adsorbent, suggesting that the "tentacular" ligands present on this strong cation-exchange adsorbent interact with protein molecules through larger contact areas. Collectively, these investigations provide further support for the concept that the adsorption behaviour of proteins with the "tentacle-type" LiChrospher-1000 SO3- adsorbent and similar types of "tentacular" ligand systems involves a multilayer dissolution mechanism with the protein interacting with a more diffuse or extended Donnan double layer in the ion-exchange environment, resulting in multi-site binding processes.