High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

Lydia Kisley, Jixin Chen, Andrea P. Mansur, Sergio Dominguez-Medina, Eliona Kulla, Marci K. Kang, Bo Shuang, Katerina Kourentzi, Mohan Vivekanandan Poongavanam, Sagar Dhamane, Richard C. Willson, Christy F. Landes

Research output: Contribution to journalArticlepeer-review

39 Scopus citations


The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.

Original languageEnglish (US)
Pages (from-to)135-142
Number of pages8
JournalJournal of Chromatography A
StatePublished - May 23 2014


  • Bioseparations
  • Heterogeneity
  • Ion-exchange
  • Optical nanoscopy

ASJC Scopus subject areas

  • Analytical Chemistry
  • Organic Chemistry
  • Biochemistry


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