HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF AMINO-ACIDS, PEPTIDES AND PROTEINS .93. INFLUENCE OF TEMPERATURE ON THE RETENTION BEHAVIOR OF PROTEINS IN CATION-EXCHANGE CHROMATOGRAPHY

The chromatographic behaviour of several amino acid derivatives, pepti des and proteins has been investigated with the so called ''tentacle-t ype'' LiChrospher-100 SO3- adsorbent and an analogous poly(2-sulphoeth ylaspartamide) cation-exchange adsorbent, PolySulphoethyl A. In partic ular, the dependences of the retention properties of a range of biosol utes on temperature and the chromatographic residence time were evalua ted with the objective of gaining further insight into the influence o f ligand type and flexibility and the role of solute conformation on t he chromatographic behaviour of proteins with these two strong cation- exchange chromatographic adsorbents. The results indicate that signifi cant differences in the chromatographic retention behaviour between pr oteins and low-molecular-mass solutes occur as a function of temperatu re and the type of co- and counter ions present in the mobile phase wi th both adsorbents. Moreover, the dependences of the Z(c) and log K-c values on temperature for most of the proteins examined exhibited sign ificant changes in magnitude between 4 and 75 degrees C, whilst no equ ivalent changes were evident for low-molecular-mass solutes. With the ''tentacle-type'' LiChrospher-1000 SO3- adsorbent at higher temperatur es, e.g., at 75 degrees C, most of the proteins could still be eluted although several exhibited very large increases in their retention par ameters. With the PolySulphoethyl A adsorbent, on the other hand, none of the proteins examined were eluted at 75 degrees C. The results mor eover indicate that hydrophobic interactions play an increasingly impo rtant role in protein retention with both types of ion-exchange adsorb ents at higher temperatures, but are more dominant with the PolySulpho ethyl A ligand. In general, the Z(c) values for the proteins with the ''tentacle-type'' LiChrospher-1000 SO3- adsorbent were greater than th ose obtained with the PolySulphoethyl A adsorbent, suggesting that the ''tentacular'' ligands present on this strong cation-exchange adsorbe nt interact with protein molecules through larger contact areas. Colle ctively, these investigations provide further support for the concept that the adsorption behaviour of proteins with the ''tentacle-type'' L iChrospher-1000 SO3- adsorbent and similar types of ''tentacular'' lig and systems involves a multilayer dissolution mechanism with the prote in interacting with a more diffuse or extended Donnan double layer in the ion-exchange environment, resulting in multi-site binding processe s.