A Comprehensive Study to Protein Retention in Hydrophobic Interaction Chromatography

Sebastiaan Eeltink, Xiaodong Liu, Ken Cook

Research output: Chapter in Book/Report/Conference proceedingMeeting abstract (Book)

Abstract

Hydrophobic interaction chromatography (HIC) capitalizes on the interaction between hydrophobic patches of proteins and weakly hydrophobic ligands attached to the stationary phase. The separation is typically performed using aqueous (non-denaturing) buffer systems at pH = 7 and applying a linear salt
gradient starting at relatively high salt concentration. Although HIC shows great potential for native biomolecule separations, maintaining protein 3D conformation, the effects of operating conditions such as mobile phase composition (type and concentration of salts and the possibility to add organic modifiers), and stationary-phase chemistry on retention are sti
ll not fully understood.

The performance of four commercially-available HIC columns was assessed for protein HIC separations and the effects of eluent type and concentration on protein retention was investigated using different kosmotropic/chaotropic salt systems. The addition of a kosmotropic salt to the mobile phase leads to
exclusion water molecules from the protein and resin surface, thereby promoting hydrophobic interactions. Furthermore, the elution conditions selected may affect protein 3D conformation, and hence affect the accessible hydrophobic surface.

Finally, the possibilities and applicability of two retention-time models have been assessed enhance method development. In an attempt to making MD development more user-friendly, model parameters were assessed based on two gradient scouting runs. To improve the prediction accuracy, also
possibilities to model HIC protein retention with a non-linear
retention time model was investigated.
Original languageEnglish
Title of host publicationEighth International Symposium on the Separation and Characterization of Natural and Synthetic Macromolecules - Programme
Publication statusPublished - Feb 2017
EventEighth International Symposium on the Separation and Characterization of Natural and Synthetic Macromolecules - Rhône Congress Centre, Amsterdam, Netherlands
Duration: 1 Feb 20173 Feb 2017
http://www.scm-8.nl/index.html

Conference

ConferenceEighth International Symposium on the Separation and Characterization of Natural and Synthetic Macromolecules
Abbreviated titleSCM-8
CountryNetherlands
CityAmsterdam
Period1/02/173/02/17
Internet address

Keywords

  • Protein Retention
  • HIC
  • Hydrophobic Interaction Chromatography

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