Open tubular capillary column for the separation of cytochrome C tryptic digest in capillary electrochromatography

Abstract

A silica capillary of 50 μm internal diameter and 500 mm length (416 mm effective length) was chemically modified with 4-(trifluoromethoxy) phenyl isocyanate in the presence of dibutyl tin dichloride as catalyst. Sodium diethyl dithiocarbamate was reacted with the terminal halogen of the bound ligand to incorporate the initiator moiety, and in situ polymerization was performed using a monomer mixture of styrene, N-phenylacrylamide, and methacrylic acid. The resultant open tubular capillary column immobilized with the copolymer layer was used for the separation of tryptic digest of cytochrome C in capillary electrochromatography. The sample was well eluted and separated into many components. The elution patterns of tryptic digest of cytochrome C were studied with respect to pH and water content in the mobile phase. This preliminary study demonstrates that open tubular capillary electrochromatography columns with a modified copolymer layer composed of proper nonpolar and polar units fabricated by reversible addition-fragmentation transfer polymerization can be useful as separation media for proteomic analysis.

High Efficiency Robust Open Tubular Capillary Electrochromatography Column for the Separation of Peptides

“…The analytical power of LC/MS could be improved by development of the LC stationary phases of better selectivity, resolution, and separation efficiency . Useful prevailing chromatographic columns for separation of peptides are monolith columns, ultra‐high performance liquid chromatography (UPLC) columns with sub‐3 μm porous particles or fused core particles along with various CEC columns including open tubular capillary electrochromatography (OT‐CEC) columns . CEC columns as well as LC columns may be used in multidimensional separation …”

“…Readers are recommended to view the literature and the references quoted there for detailed information of RAFT polymerization. The monomer mixture of this study was modified to a different formulation from those of Refs . and to fit the purpose of this study.…”

“…The formulations of the polymerization mixtures of current study and those of Refs. and are as follows.…”

“…The mixture of Ref. is composed of 0.65 mL styrene, 0.3 mL MAA, 70 mg N ‐phenyl acrylamide, and 2 mL toluene. The mixture of Ref.…”

Surface-Confined Aqueous Reversible Addition–Fragmentation Chain Transfer (SCARAFT) Polymerization Method for Preparation of Coated Capillary Leads to over 10 000 Peptides Identified from 25 ng HeLa Digest by Using Capillary Zone Electrophoresis-Tandem Mass Spectrometry

“…17–19 A ligand with a terminal halogen (4-chloromethylphenyl isocyanate), or 4-(trifluoromethoxy) phenyl isocyanate was bound to the inner surface of a pretreated silica capillary in the presence of dibutyltin dichloride as a catalyst through an isocyanate-hydroxyl reaction. 17–18 Attachment of initiator (sodium diethyl dithiocarbamate) to the bound ligand was carried out and followed by in situ polymerization. The resulting capillary showed good separation performance for derivatized saccharide isomers and tryptic digest of cytochrome C in capillary electrochromatography with UV detection.…”