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Updated: Aug 26, 2026

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
Published on: September 21, 2011
Hydrophobic and cation exchange mechanisms in the retention of basic compounds in a polymeric column
Rebeca Ruiz1, Ma José Ruiz-Angel, Ma Celia García-Alvarez-Coque
1Departament de Química Analítica, Universitat de Barcelona, Diagonal 647, 08028 Barcelona, Spain.
Abstract:
A cation exchange retention mechanism concomitant with the well-known hydrophobic partition mechanism in a polymeric column has been observed and investigated. This exchange process is attributed to ionization of some acidic sites present in the polymer column at basic mobile phase pH values. Several drugs of different basicity have been chromatographed on a polymeric PLRP-S column with methanol-water and acetonitrile-water mobile phases. The cation exchange between the protonated basic drug and the buffer cations (Na+, K+ and BuNH4+) is observed at the pH range where the protonated drug and the ionized sites of the column coexist. This process produces a shift of the retention versus pH plot of the base to pH values lower than those expected from the pKa of the base as well as a maximum in the plot at basic pH values. These effects are more pronounced for acetonitrile-water mobile phases.
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