Retention model of protein for mixed‐mode interaction mechanism in ion exchange and hydrophobic interaction chromatography

Abstract

AbstractA unified retention equation of proteins was proved to be valid for a mixed‐mode interaction mechanism in ion exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC). The reason to form a “U” shape retention curve of proteins in both HIC and IEC was explained and the concentration range of the strongest elution ability for the mobile phase was determined with this equation. The parameters in this equation could be used to characterize the difference for either HIC or IEC adsorbents and tbe changes in tbe molecular conformation of proteins. With the parameters in this equation, the contributions of salt and water in tbe mobile phase to the protein retention in HIC and IEC were discussed, respectively. In addition, the comparison between the unified equation and Melander's three‐parameter equation for mixed‐mode interaction chromatography was also investigated and better results were obtained in former equation.