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Updated: May 18, 2026

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
Published on: September 21, 2011
A simple graphical representation of selectivity in hydrophilic interaction liquid chromatography
Mohammed E A Ibrahim1, Yang Liu, Charles A Lucy
1Department of Chemistry, University of Alberta, Gunning/Lemieux Chemistry Centre, Edmonton, Alberta T6G 2G2, Canada.
Abstract:
This paper uses the HILIC selectivity data of Dinh et al. (J. Chromatogr. A 1218 (2011) 5880) to yield simple and easy to understand plots analogous to Neue plots for selectivity in HILIC. The plots categorize 21 previously studied HILIC phases (data from Dinh et al.), 8 additional HILIC columns and 4 reversed phase columns (our data) using selected probes for specific interactions. The relative retention of cytosine vs. uracil is used to probe the "hydrophilicity" of the HILIC phases; adenosine vs. adenine is used to probe the ability of the stationary phase to participate in hydrogen bonding; and benzyltrimethylammonium (BTMA) vs. cytosine is used to probe the cation exchange and anion exchange character of the column. Plots of kBTMA/kcytosine vs. kcytosine/kuracil successfully classify silica, amide, zwitterionic, diol and reverse phase columns in terms of their HILIC behavior. Polymeric columns including polymer substrate and polymer coated columns show low ion exchange character, but vary widely in their hydrophilicity. Alternatively a HILIC-Phase Selectivity Chart, in analogy to the Neue plot, is constructed by plotting log(kBTMA/kcytosine) vs. log(kcytosine). This plot enables classification of HILIC columns that will yield similar or significantly different separations.
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