Mixed-mode chromatography integrated with high-performance liquid chromatography for protein analysis and separation: Using bovine serum albumin and lysozyme as the model target

• Published in: Journal of separation science Vol. 39; no. 10; pp. 1900 - 1907
• Main Authors: Xia, Hai-Feng, Don, Bin-Bin, Zheng, Meng-Jie
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 01.05.2016; Wiley Subscription Services, Inc
• Subjects: Acetonitriles - chemistry; Animals; Cattle; Chromatography; Chromatography, High Pressure Liquid; High performance liquid chromatography; Hydrogen-Ion Concentration; Lysozyme; Mixed-mode chromatography; Models, Molecular; Muramidase - analysis; Muramidase - metabolism; Protein separation; Proteins; Resolution; Retention; Separation; Serum albumin; Serum Albumin, Bovine - analysis; Silicon Dioxide - chemistry; Sodium Chloride - chemistry; Strategy; Protein separation; Mixed-mode chromatography; Retention; Resolution
• ISSN: 1615-9306; 1615-9314
• DOI: 10.1002/jssc.201501394

A type of mixed‐mode chromatography was integrated with high‐performance liquid chromatography for protein analysis and separation. The chromatographic behavior was tested using bovine serum albumin and lysozyme as model proteins. For the mixed‐mode column, the silica beads were activated with γ‐(2,3‐epoxypropoxy)‐propytrimethoxysilane and coupled with 4‐mercaptopyridine as the functional ligand. The effects of pH, salt, and the organic solvent conditions of the mobile phase on the retention behavior were studied, which provided valuable clues for separation strategy. When eluted with a suitable pH gradient, salt concentration gradient, and acetonitrile content gradient, the separation behavior of bovine serum albumin and lysozyme could be controlled by altering the conditions of the mobile phase. The results indicated this type of chromatography might be a useful method for protein analysis and separation.