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

Song, Chao; Wang, Jianshan; Zhao, Tianshan; Bai, Qingshun

doi:10.1002/bmc.2988

Cited by 12 publications

(1 citation statement)

References 43 publications

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“…A dual‐function stationary phase which combines both strong cation exchange and hydrophobic interaction chromatography characteristics was prepared for separation of proteins . A mixed‐mode stationary phase functionalized with PEG 400 and succinic anhydride as the ligand was used for proteins . Chromatofocusing techniques employing mixed‐mode column packings for protein separations were developed .…”

Section: Introductionmentioning

confidence: 99%

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

Ali

Kim

et al. 2019

J of Separation Science

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A phase with both hydrophobic and hydrophilic functionalities has been synthesized by modification of ground silica monolith particles with C18 and 1‐[3‐(trimethoxysilyl)propyl] urea ligands. A series of phases was prepared by changing the ratio of the two ligands to determine the optimal ratio in view of separation efficiency. The resultant optimized stationary phase was packed in narrow‐bore glass‐lined stainless‐steel columns (1 × 300 mm and 2.1 × 100 mm) and used for the separation of synthetic peptides and proteins. The average numbers of theoretical plates (N) of 52 100/column (174 000/m, 5.75 µm plate height) and 35 500/column (118 000/m, 8.47 µm plate height) were achieved with the 300 mm column at a flow rate of 25 µL/min (0.86 mm/s) in 60:40 v/v acetonitrile/30 mM aqueous ammonium formate for the mixture of peptides (Thr‐Tyr‐Ser, Val‐Ala‐Pro‐Gly, angiotensin I, isotocin, and bradykinin) and for the mixture of proteins (myoglobin, human serum albumin, and insulin), respectively. Fast analysis of the peptides and proteins was also carried out at a flow rate of 0.9 mL/min (6.88 mm/s) with the 100 mm column and all the analytes were eluted within 2 min with good separation efficiency.

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Section: Introductionmentioning

confidence: 99%

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

Ali

Kim

et al. 2019

J of Separation Science

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Preparation of a novel weak cation exchange/hydrophobic interaction chromatography dual-function polymer-based stationary phase for protein separation using “thiol–ene click chemistry”

et al. 2014

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A novel dual-function mixed-mode stationary phase based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) microspheres was synthesized by thiol-ene click chemistry and characterized by infrared spectroscopy and elemental analysis. The new system displays both hydrophobic interaction chromatography (HIC) character in a high salt concentration mobile phase, and weak cation exchange (WCX) chromatography character in a low salt concentration mobile phase. It can be used to separate proteins in both ion-exchange chromatography (IEC) mode and HIC mode. The resolution and selectivity of the stationary phase were evaluated in both HIC mode and IEC mode using protein standards. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. Protein mass and bioactivity recoveries of more than 96% can be achieved in both HIC mode and IEC mode using this column. The results indicate that the novel dual-function mixed-mode column in many cases can replace the use of two individual WCX and HIC columns. In addition, the effects on protein separation of different ligand structures in the dual-function stationary phase and the pH of the mobile phase used were also investigated in detail. The results show that electrostatic interaction of the ligand with proteins must match the hydrophobicity of the ligand, which is an important factor to prepare the dual-function stationary phase. On the basis of this dual-function mixed-mode chromatography column, a new two-dimensional liquid chromatography technology with a single column system was also developed in this study, and was used to renature and purify recombinant human interferon-γ from inclusion bodies. The mass recovery, purity, and specific bioactivity obtained for the purified recombinant human interferon-γ were 87.2%, 92.4%, and 2.8 × 10(7) IU/mg, respectively, in IEC mode, and 83.4%, 95.2%, and 4.3 × 10(7) IU/mg, respectively, in HIC mode. The results indicate that the dual-function mixed-mode stationary phase prepared in this study may aid in the development of new two-dimensional liquid chromatography technology with a single column for intensive proteomic studies, and it may also find applications in recombinant protein drug production since it can save column costs and simplify the biotechnological processes.

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Preparation and evaluation of a molybdenum disulfide quantum dots embedded C18 mixed-mode chromatographic stationary phase

et al. 2020

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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

Cited by 12 publications

References 43 publications

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

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

Preparation of a novel weak cation exchange/hydrophobic interaction chromatography dual-function polymer-based stationary phase for protein separation using “thiol–ene click chemistry”

Preparation and evaluation of a molybdenum disulfide quantum dots embedded C18 mixed-mode chromatographic stationary phase

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