title: Displacement chromatography of proteins using a retained pH front in a hydrophobic charge induction chromatography column.
abstract: The chromatographic separation of two proteins into a displacement train of two adjoined rectangular bands was accomplished using a novel method for hydrophobic charge induction chromatography (HCIC) which employs a self-sharpening pH front as the displacer. This method exploits the fact that protein elution in HCIC is promoted by a pH change, but is relatively independent of salt effects, so that a retained pH front can be used in place of a traditional displacer in displacement chromatography. The retained pH front was produced using the two adsorbed buffering species tricine and acetic acid. The separation of lysozyme and α-chymotrypsinogen A into adjoined, rectangular bands was accomplished with overall recoveries based on the total mass injected greater than 90 and 70%, respectively. The addition of urea to the buffer system increased the sharpness of the pH front by 36% while the yields of lysozyme and α-chymotrypsinogen A based on the total mass eluted increased from 76% to 99% and from 37% to 85%, respectively, when the purities of both proteins in their product fractions were fixed at 85%. The results demonstrate that the method developed in this study is a useful variant of HCIC and is also a useful alternative to other displacement chromatography methods.
authors.affiliation: Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, MD 21250, USA.
authors.forename: N D S
authors.initials: ND
authors.lastname: Pinto
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; D002853:Chromatography, Liquid; D002919:Chymotrypsinogen / Q000032:analysis / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D059625:Liquid-Liquid Extraction / Q000379:methods*; D009113:Muramidase / Q000032:analysis / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D018892:Proton-Motive Force
chemical_list: D011506:Proteins; D002919:Chymotrypsinogen; D009113:Muramidase
keywords: Chromatofocusing; Displacement chromatography; Hydrophobic charge induction chromatography; MEP HyperCel; Proteins