Protein separation by open tubular capillary electrochromatography employing a capillary coated with phenylalanine functionalized tentacle-type polymer under both cathodic and anodic electroosmotic flows.
Protein separation by open tubular capillary electrochromatography employing a capillary coated with phenylalanine functionalized tentacle-type polymer under both cathodic and anodic electroosmotic flows.
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DOI：
10.1016/j.chroma.2007.12.083
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发表时间：
2008-03
期刊：
影响因子：
--
通讯作者：
Liang Xu;Y. Sun
中科院分区：
文献类型：
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作者：
Liang Xu;Y. Sun
本工作采用苯丙氨酸（Phe）功能化的触角型聚合物涂层毛细管柱，通过开管毛细管电色谱（OTCEC）分离蛋白质。采用50μm内径的硅烷化熔融石英毛细管柱制备了触角型固定相。通过甲基丙烯酸缩水甘油酯接枝聚合和随后的Phe官能化。由于Phe的两性官能团键合在触角型聚合物固定相上，通过改变移动的相的pH值，在制备的柱中的蛋白质分离可以在阴极和阳极电渗流（electrosomal flow，简称electrosomal flow，简称electrosomal flow）下进行。模型蛋白质包括核糖核酸酶A（RNase A）、肌红蛋白、转铁蛋白、胰岛素在阴极电泳下基线分离，移动的相为pH 8.8。比较了4种蛋白质在OTCEC和毛细管区带电泳条件下的分离结果，表明4种蛋白质在制备的柱中的迁移行为是色谱保留和电泳迁移相互作用的结果。此外，在酸性运行缓冲液（pH 2.5）中，RNase A、细胞色素c（Cyt-c）和溶菌酶（Lys）三种碱性蛋白质在阳极氧化条件下得到了完全分离。洗脱顺序与蛋白质的等电点值相同（RNase A<Cyt-c<Lys）。此外，本工作中所用蛋白质的迁移时间在反复使用中是稳定的，蛋白质的柱效在13，000 ~ 182，000板/m之间。
The use of a phenylalanine (Phe) functionalized tentacle-type polymer coated capillary column for protein separation by open tubular capillary electrochromatography (OTCEC) was demonstrated in this work. The tentacle-type stationary phase was prepared from silanized fused-silica capillaries of 50μm I.D. by glycidyl methacrylate graft polymerization and subsequent Phe functionalization. Due to the amphoteric functional groups of the Phe bonded on the tentacle-type polymer stationary phase, protein separation in the prepared column can be performed under both cathodic and anodic electroosmotic flow (EOF) by varying the pH values of the mobile phase. Model proteins including ribonuclease A (RNase A), myoglobin, transferrin, insulin were baseline separated under cathodic EOF with a mobile phase of pH 8.8. Comparison between the separation result of the four proteins under conditions of OTCEC and capillary zone electrophoresis indicates that the migration behavior of the four proteins in the prepared column was the result of the interplay of chromatographic retention and electrophoretic migration. Besides, three basic proteins including RNase A, cytochrome c (Cyt-c) and lysozyme (Lys) were fully resolved under anodic EOF with an acidic running buffer (pH 2.5). The elution order was the same as the isoelectric point values of the proteins (RNase A<Cyt-c<Lys). Moreover, it was proved that the migration times of all the proteins used in this work were stable in repeated uses of the column, and the column efficiency of proteins was in the range from 13,000 to 182,000plates/m.