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Updated: May 15, 2026

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
Published on: September 21, 2011
Practical investigation of the factors that affect the selectivity in hydrophilic interaction chromatography
Abhinav Kumar1, James C Heaton, David V McCalley
1Centre for Research in Biosciences, University of the West of England, Frenchay, Bristol BS16 1QY, UK.
Abstract:
Retention data for a series of 29 compounds comprising acids, bases and neutrals were obtained on six different hydrophilic interaction (HILIC) columns including bare silica, zwitterionic and those bonded with neutral bonded ligands. The principal aim of the work was to evaluate the effect of various experimental variables such as the nature of the stationary phase, buffer pH, buffer concentration, organic solvent and its concentration, and temperature, in order to determine which factors gave the most effect on the selectivity of the separation. The influence of solute properties on the separation, such as logD values, was also considered. In this way, it was hoped to provide a practical guide to aid in the selection of conditions to achieve a separation in HILIC. The nature of the stationary phase was found to have the greatest effect on selectivity.
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