Fe³⁺‐immobilized nanoparticle‐modified capillary for capillary electrophoretic separation of phosphoproteins and non‐phosphoproteins

Abstract

A fused-silica capillary modified with Fe³⁺-immobilized magnetic nanoparticles (Fe³⁺-IMAN) has been investigated for the capillary electrophoretic (CE) separation of phosphoproteins and non-phosphoproteins. The Fe³⁺-IMAN capillary was achieved by covalently immobilising epoxy-based magnetic silica nanoparticles (160 nm) on the prederivatized 3-aminopropyl-trimethoxysilane (APTMS) fused-silica capillary (75 μm id), followed by disodium iminodiacetate and Fe³⁺. The buildup process was examined by measuring the streaming potentials of the bare capillary, APTMS capillary, epoxy-based nanoparticle capillary and Fe³⁺-IMAN capillary by varying the buffer pH. An inverted fluorescence microscope was used to determine the surface features of the Fe³⁺-IMAN capillary derivatized with morin. Further experimental results confirmed that Fe³⁺-IMAN bonded on the inner wall of the APTMS capillary could provide sufficient solute-bonded phase interactions to allow for the CE separation of phosphoproteins and non-phosphoproteins at concentration levels down to 50 μg/mL. The highest number of theoretical plates obtained was about 233,000/m, and the relative standard deviation (RSD) for migration times was <2.57% for eight consecutive runs, respectively. Additionally, the Fe³⁺-IMAN modifing method was also applied to the analyses of bovine milk proteins. With simplicity, high resolving power, and high repeatability, the proposed method has shown great potential for phosphoproteomics applications.

Poly(norepinephrine)‐coated open tubular column for the separation of proteins and recombination human erythropoietin by capillary electrochromatography

“…The difference in the electrophoretic mobilities of proteins and PNE coating combined to play roles for the separation of these proteins. In comparison with the previous work aiming at the separation of proteins by OT‐CEC , the PNE‐coated column showed good separation ability for acidic and basic proteins. More importantly, the column can resolve the glycoforms of rhEPO.…”

“…To improve the reproducibility, the preconditioning of the capillary should be performed. Open tubular capillary electrochromatography (OT‐CEC), combining the high separation efficiency of CE and high selectivity of LC, has demonstrated superiority in proteins separation due to the advantages including simple column preparation, no bubble formation, and simple instrumental handling . More importantly, the coatings in CEC can effectively restrain the adsorption of proteins on the inner surface of columns and improve the resolution of proteins separation .…”

Carboxyl modified magnetic nanoparticles coated open tubular column for capillary electrochromatographic separation of biomolecules

“…The as-prepared OT columns were successfully applied for separation of amino acids, dipeptides and proteins based on the electrophoretic and chromatographic mechanisms. In comparison with other OT columns which aimed at separation of proteins [3,8,[42][43][44][45][46], the procedure for preparation of PDDA@Fe 3 O 4 -COOH MNPs coated OT column was simpler and milder. The Fe 3 O 4 -COOH MNPs coating containing abundant carboxyl groups and Lewis acid sites effectively improved the separation selectivity and restrained the adsorption of proteins on the capillary wall.…”

“…Capillary electrochromatography (CEC) combining high selectivity of high-performance liquid chromatography and high efficiency of capillary electrophoresis has been widely applied in the analysis of biomolecules including amino acids [1], peptides [2] and proteins [3]. As the core of CEC, various forms of columns including packed, monolithic, and open tubular (OT) columns are commonly used.…”

Polydopamine-coated open tubular column for the separation of proteins by capillary electrochromatography

“…Up to now, HPLC and CE have been utilized for the separation of proteins . In HPLC, RP and exclusion chromatography were the most used chromatographic modes for protein separation.…”