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Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform
Author(s) -
Liu Rangdong,
Pan Hong,
Zheng Yiting,
Hu Jing,
Cheddah Soumia,
Wang Weiwei,
Wang Yan,
Yan Chao
Publication year - 2019
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201900125
Subject(s) - isoelectric focusing , capillary action , chromatography , monolithic hplc column , immobilized ph gradient , glycidyl methacrylate , ethylene glycol , materials science , chemistry , column (typography) , analytical chemistry (journal) , polymer , computer science , polymerization , composite material , telecommunications , biochemistry , organic chemistry , frame (networking) , enzyme
Abstract In the presence of methanol and n ‐decanol as porogens, a partially filled capillary monolithic column was prepared by in situ reaction of glycidyl methacrylate and poly (ethylene glycol) diacrylate. Then, Pharmalyte 3–10 was immobilized on this column in order to obtain a capillary isoelectric focusing (cIEF) column with monolithic immobilized pH gradient (M‐IPG). In addition, an online self‐built platform for protein separation was established on account of the introduction of a cross‐shaped unit and two short‐off valves. In this platform, a cross‐shaped unit was not only used to join the M‐IPG column and a six‐way injection valve (1.5 μL sample loop), but also to supply a volume pool of anode buffer so that the process of injection, focusing and mobilization of samples could be sequentially performed. The short‐off valve in the tee unit or cross‐shaped unit could be used to control the direction of the fluid flow. Using this online cIEF platform and under the optimized conditions, 7‐proteins mixture could be separated and a good linear correlation between pI values and migration times was obtained by the M‐IPG column. Meanwhile, based on the online cIEF platform, human serum proteins and a mixture of Hb A and Hb A 1c have been successfully resolved with the newly developed M‐IPG column.