Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1141, Issue 2, Pages 235-243Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2006.12.022
Keywords
HIC adsorbents; octyl; butyl; phenyl; second osmotic virial coefficient; protein solubility; adiabatic compressibility; retention factor
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A general thermodynamic relation was derived to correlate protein solubility to retention in hydrophobic interaction chromatography (HIC). This relation is built on a thermodynamic formulation presented previously by Melander, Horvath and co-workers in the context of the solvophobic theory, but the final result is independent of this model framework. The relation reflects an increase in protein retention in HIC under conditions that promote precipitation or crystallization, consistent with early descriptions of HIC. To examine the contribution of protein solubility to retention in HIC, isocratic elution experiments were per-formed with four different commercially available agarose media and four model proteins (ribonuclease A (RNA), lysozyme (LYS), myoglobin (MYO), and ovalbumin (OVA)). A wide variety of retention trends were observed as a function of protein, adsorbent type, salt type and concentration, and pH. In general, however, the results show that solubility, or its surrogate, the second osmotic virial coefficient, which reflects solution thermodynamic properties, correlates well with HIC retention in many cases; this includes correctly predicting reverse Hofmeister effects, which cannot be explained by retention models based on the solvophobic theory and preferential interaction theory. However, solution properties could not explain retention behavior under some conditions. In those cases, effects such as protein-surface interactions or conformational change could be important determinants of protein adsorption. (c) 2006 Elsevier B.V. All rights reserved.
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