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Updated: Nov 17, 2025

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing IEF Method
Published on: June 14, 2020
pH-dependent selective separation of acidic and basic proteins using quaternary ammoniation functionalized
Jianzhong Wang1, Jianshan Wang1, Xiaohui Ning1
1Key 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.
Abstract:
In this paper, a cysteine-functionalized zwitterionic stationary phase (Cys-silica) was prepared based on the "thiol-ene" click chemistry between cysteine and vinyl-functionalized silica, and was further modified with bromoethane, 1-bromooctane and 1-bromooctadecane, respectively, to obtain a series of quaternary ammoniation-functionalized stationary phases (Cys-silica-Cn, n = 2, 8 and 18). These zwitterionic stationary phases were regarded as reversed-phase/ion-exchange (RP/IEC) mixed-mode chromatography (MMC) stationary phases for protein separation. The retention behaviors of proteins on these zwitterionic stationary phases were carefully investigated. The results indicated that the retentions of acidic and basic proteins on these zwitterinonic stationary phases were significantly influenced by the acetonitrile and salt concentrations, pH of mobile phase as well as the hydrophobicity of the ligand. The separation selectivity of proteins on these zwitterionic stationary phases strongly depended on the pH value of mobile phase. The baseline separation of 6 kinds of basic proteins can be achieved at pH 8.0 using Cys-silica-C2 or Cys-silica-C8 column, and 5 kinds of acidic proteins can also be separated completely at pH 4.0 with Cys-silica-C2 column. Moreover, owing to the quaternary ammoniation-functionalization on Cys-silica by using appropriately hydrophobic bromoalkanes, the selectivity and separation efficiency of proteins can be enhanced greatly. As a result, the acidic and basic proteins can be separated completely step by step from the complex sample by adjusting pH of mobile phase using a single Cys-silica-C2 column, which illustrates that the cysteine-functionalized zwitterionic stationary phase has a great potential for protein separation.
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