High-performance liquid chromatography of amino acid peptides and proteins - Patt: LXXXIII Evaluation of retention and bandwidth relationships for proteins separated by isocratic anion-exchange chromatography

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Abstract

The retention behaviour of seven globular proteins ranging in molecular weight from 12,000 to 69,000 was investigated using Mono-Q anion-exchange resin as the stationary phase and sodium chloride as the displacer salt. In particular the influence of changes in ionic strength and mobile phase pH on the isocratic retention properties was assessed. Several proteins were found to have significant retention when the pH of the mobile phase was below the reported pl values of the proteins. This behaviour results from the non-uniform charge distribution on the protein surface, which allows interaction with the charged stationary phase even though the protein net charge is equal to or greater than zero. The influence of pH and ionic strength on experimentally observed bandwidths was also investigated. The dependence of the effective reduced plate height on solute capacity factor was found to vary significantly with the mobile phase pH, a behaviour consistent with the interplay of complex multisite binding kinetics. These results provide a basis for further detailed investigations into the mechanism of interaction of proteins not only with charged surfaces associated with adsorptive chromatographic media but also with other macromolecules.

Original languageEnglish
Pages (from-to)769-776
Number of pages8
JournalChromatographia
Volume24
Issue number1
DOIs
Publication statusPublished - Dec 1987

Keywords

  • Anion-exchange chromatography
  • Proteins
  • Retention and bandwidth relationship

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