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Updated: Mar 18, 2026

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
Published on: September 21, 2011
The hydrophilicity vs. ion interaction selectivity plot revisited: The effect of mobile phase pH and buffer
Chad D Iverson1, Xinyun Gu1, Charles A Lucy1
1Department of Chemistry, University of Alberta, Gunning/Lemieux Chemistry Centre, Edmonton, Alberta T6G 2G2, Canada.
Abstract:
This work systematically investigates the selectivity changes on many HILIC phases from w(w)pH 3.7-6.8, at 5 and 25mM buffer concentrations. Hydrophilicity (kcytosine/kuracil) vs. ion interaction (kBTMA/kuracil) selectivity plots developed by Ibrahim et al. (J. Chromatogr. A 1260 (2012) 126-131) are used to investigate the effect of mobile phase changes on the selectivity of 18 HILIC columns from various classes. "Selectivity change plots" focus on the change in hydrophilicity and ion interaction that the columns exhibit upon changing mobile phase conditions. In general, the selectivity behavior of most HILIC columns is dominated by silanol activity. Minimal changes in selectivity are observed upon changing pH between w(w)pH 5 and 6.8. However, a reduction in ionic interaction is observed when the buffer concentration is increased at w(w)pH≥5.0 due to ionic shielding. Reduction of the w(w)pH to<5.0 results in decreasing cation exchange activity due to silanol protonation. Under all eluent conditions, the majority of phases show little change in their hydrophilicity.
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