title: Preparation and characterization of a novel dual-retention mechanism mixed-mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes.
abstract: A novel dual-retention mechanism mixed-mode stationary phase based on silica gel functionalized with PEG 400 and succinic anhydride as the ligand was prepared and characterized by infrared spectra and elemental analysis. Because of the ligand containing PEG 400 and carboxyl function groups, it displayed hydrophobic interaction chromatography (HIC) characteristic in a high-salt-concentration mobile phase, and weak cation exchange chromatography (WCX) characteristic in a low-salt-concentration mobile phase. As a result, it can be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase was evaluated under both HIC and WCX modes with protein standards, and its performance was comparable to that of conventional ion-exchange chromatography and HIC columns. The results indicated that the novel dual-retention mechanism column, in many cases, could replace two individual WCX and HIC columns as a '2D column'. In addition, the mixed retention mechanism of proteins on this '2D column' was investigated with stoichiometric displacement theory for retention of solute in liquid chromatography in detail in order to understand why the dual-retention mechanism column has high resolution and selectivity for protein separation under WCX and HIC modes, respectively. Based on this '2D column', a new 2DLC technology with a single column was developed. It is very important in proteome research and recombinant protein drug production to save column expense and simplify the processes in biotechnology.
authors.affiliation: Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Institute of Modern Separation Science, Key Lab of Modern Separation Science in Shaanxi Province, Northwest University, Xi'an, 710069, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Song
authors.forename: Jianshan
authors.initials: J
authors.lastname: Wang
authors.forename: Kailou
authors.initials: K
authors.lastname: Zhao
authors.forename: Quan
authors.initials: Q
authors.lastname: Bai
mesh_terms: D000327:Adsorption; D002852:Chromatography, Ion Exchange / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D013387:Succinic Anhydrides / Q000737:chemistry*
chemical_list: D011994:Recombinant Proteins; D013387:Succinic Anhydrides; D011092:Polyethylene Glycols; C031801:succinic anhydride
keywords: hydrophobic interaction chromatography; ion-exchange chromatography; mixed-mode chromatography; stationary phase; two-dimensional liquid chromatography