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Updated: Jul 31, 2026

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
Published on: September 21, 2011
Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and
R C Chloupek1, W S Hancock, B A Marchylo
1Genentech, Inc., South San Francisco, CA 94080.
Abstract:
Changes in band spacing as a function of temperature and/or gradient steepness were investigated for four peptide or protein samples. Reversed-phase HPLC in a gradient mode was used to separate tryptic digests of tissue plasminogen activator and calmodulin. Additionally, a synthetic peptide mixture and a storage protein sample from wheat were studied. Simultaneous changes in gradient steepness and temperature were found to provide considerable control over band spacing and sample resolution. The effects of temperature and gradient steepness on selectivity in these systems appear to be complementary. Simultaneous optimization of both temperature and gradient steepness thus represents a powerful and convenient means of controlling band spacing and separation. Because of the complexity of these sample chromatograms, computer simulation proved to be a useful tool in both interpreting these experiments and in optimizing final separations.
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