Preparation of a weak anion exchange/hydrophobic interaction dual‐function mixed‐mode chromatography stationary phase for protein separation using click chemistry

Abstract

In this study, 3-diethylamino-1-propyne was covalently bonded to the azide-silica by a click reaction to obtain a novel dual-function mixed-mode chromatography stationary phase for protein separation with a ligand containing tertiary amine and two ethyl groups capable of electrostatic and hydrophobic interaction functionalities, which can display hydrophobic interaction chromatography character in a high-salt-concentration mobile phase and weak anion exchange character in a low-salt-concentration mobile phase employed for protein separation. As a result, it can be employed to separate proteins with weak anion exchange and hydrophobic interaction modes, respectively. The resolution and selectivity of the stationary phase were evaluated in both hydrophobic interaction and ion exchange modes with standard proteins, respectively, which can be comparable to that of conventional weak anion exchange and hydrophobic interaction chromatography columns. Therefore, the synthesized weak anion exchange/hydrophobic interaction dual-function mixed-mode chromatography column can be used to replace two corresponding conventional weak anion exchange and hydrophobic interaction chromatography columns to separate proteins. Based on this mixed-mode chromatography stationary phase, a new off-line two-dimensional liquid chromatography technology using only a single dual-function mixed-mode chromatography column was developed. Nine kinds of tested proteins can be separated completely using the developed method within 2.0 h.

An optimized mixed‐mode stationary phase based on silica monolith particles for the separation of peptides and proteins in high‐performance liquid chromatography

“…Chromatofocusing techniques employing mixed‐mode column packings for protein separations were developed . Another dual‐function mixed‐mode chromatography stationary phase was prepared for protein separation . The separation of oxidized species was carried out by the mixed mode of size exclusion and hydrophobic interaction between the stationary phase (Sepax Zenix SEC‐300MK) and antibodies .…”

Preparation of mixed-mode stationary phase for separation of peptides and proteins in high performance liquid chromatography

“…Mixed-mode chromatography which offers multimodal interaction could be an alternative to RP-LC for the separation of peptides, proteins and other complex mixtures. Several mixed-mode stationary phases have been prepared and the columns packed with these stationary phases have been used for peptides and proteins separation 17 – 21 . Mixed-mode stationary phases (WAX/RPLC, HILIC/RPLC, polar embedded/RPLC) are suitable for peptides and proteins separation owing to the presence of both polar and non-polar groups 22 – 28 .…”

Performance and preparation of cation‐exchange chromatographic stationary phases without negatively charged groups for protein separation

“…Since these ligands contain both ion exchange and hydrophobic functional groups, majorly two kinds of interaction between solutes and IEC/HIC MMC stationary phases take place in the separation process. We chose these ligands that contain ion‐exchange and hydrophobic groups purposely when preparing the IEC/HIC dual‐function MMC medium, and the results were satisfactory .…”