A comprehensive study to protein retention in hydrophobic interaction chromatography

Martyna Baca, Jelle De Vos, Gilles Bruylants, Kristin Bartik, Xiaodong Liu, Ken Cook, Sebastiaan Eeltink

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19 Citations (Scopus)


The effect of different kosmotropic/chaotropic salt systems on retention characteristics of intact proteins has been examined in hydrophobic interaction chromatography (HIC). The performance was assessed using different column chemistries, i.e., polyalkylamide, alkylamine incorporating hydrophobic moieties, and a butyl chemistry. Selectivity in HIC is mainly governed by the salt concentration and by the molal surface tension increment of the salt. Typically, a linear relationship between the natural logarithm of the retention factor and the salt concentration is obtained. Using a 250 mm long column packed with 5 mu m polyalkylamide functionalized silica particles and applying a 30 min linear salt gradient, a peak capacity of 78 was achieved, allowing the baseline separation of seven intact proteins. The hydrophobicity index appeared to be a good indicator to predict the elution order of intact proteins in HIC mode. Furthermore, the effect of adding additives in the mobile phase, such as calcium chloride (stabilizing the 3D conformation of a-lactalbumin) and isopropanol, on retention properties has been assessed. Results indicate that HIC retention is also governed by conformational in the proteins which affect the number of accessible hydrophobic moieties. (C) 2016 Elsevier B.V. All rights reserved.
Original languageEnglish
Pages (from-to)182-188
Number of pages7
JournalJournal of chromatography. B
Issue numberSI
Publication statusPublished - 1 Oct 2016


  • Salting-out chromatography
  • Hofmeister
  • Hydrophobicity
  • Monoclonal antibodies
  • Conformational change
  • Practical Method Development
  • Selectivity
  • Mobile-Phase
  • Separation
  • Precipitation
  • Optimization
  • Biologics
  • Behavior

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