title: Development of chromatofocusing techniques employing mixed-mode column packings for protein separations.
abstract: Recent studies reported in the literature using mixed-mode chromatography (MMC) column packings have shown that multiple modes of interactions between the column packing and proteins can be usefully exploited to yield excellent resolution as well as salt-tolerant adsorption of the target protein. In this study, a mixed-mode separation method using commercially available column packings was explored which combines the techniques of hydrophobic-interaction chromatography and chromatofocusing. Two different column packings, one based on mercapto-ethyl-pyridine (MEP) and the other based on hexylamine (HEA) were investigated with regard to their ability to separate proteins when using internally generated, retained pH gradients. The effects of added salt and urea on the behavior of the retained pH gradient and the protein separation achieved when using MMC column packings for chromatofocusing were also investigated. Numerical simulations using methods developed in previous work were shown to agree with experimental results when using reasonable physical parameters. These numerical simulations were also shown to be a useful qualitative method to select the compositions of the starting and elution buffers in order to achieve desired shapes for the pH and ionic strength gradients. The use of the method to fractionate blood serum was explored as a prototype example application.
authors.affiliation: Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, MD 21250, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Guo
authors.affiliation: Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, MD 21250, USA. Electronic address: dfrey1@umbc.edu.
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Frey
mesh_terms: D000327:Adsorption; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002021:Buffers; D002845:Chromatography / Q000191:economics / Q000295:instrumentation / Q000379:methods; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007525:Isoelectric Focusing / Q000191:economics / Q000295:instrumentation / Q000379:methods; D009994:Osmolar Concentration; D011817:Rabbits; D012492:Salts / Q000737:chemistry; D044967:Serum / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D001798:Blood Proteins; D002021:Buffers; D012492:Salts; D014508:Urea
keywords: Blood serum fractionation; Chromatofocusing; Mixed-mode chromatography; Protein separation