用于反相液相色谱法分析疏水蛋白的嵌入式极性C4相的评估

Evaluation of an embedded polar C4 phase for hydrophobic protein analysis by reversed-phase liquid chromatography.

作者信息

Hamada T, Tanaka H, Izumine H, Ohira M

机构信息

GL Sciences Inc, Okajima, Fukushima, Japan.

出版信息

J Chromatogr A. 2004 Jul 16;1043(1):27-32. doi: 10.1016/j.chroma.2004.01.018.

DOI:10.1016/j.chroma.2004.01.018

PMID:15317409

Abstract

A C4 column (InertsilWP300 C4) was developed for a protein analysis in reversed-phase chromatography. Polar groups were embedded on this phase. And this column was evaluated with low concentration of trifluoroacetic acid (TFA) and formic acid. Test C4 phases that controlled carbon loading and commercially available columns were compared with this embedded C4 column. The test column with higher carbon loading showed good separations of proteins at low concentrations of TFA, although hydrophobic proteins were broadened. On the other hand, embedded polar C4 packings showed ideal retention behavior for proteins, which is independent of ion-pairing effects of additives in mobile phase. The reason was concluded to be the shielding effects of polar groups on the sorbents, which reduce silanophilic interactions with polar groups of proteins.

摘要

开发了一种C4柱（InertsilWP300 C4）用于反相色谱中的蛋白质分析。该固定相上嵌入了极性基团。并使用低浓度的三氟乙酸（TFA）和甲酸对该柱进行评估。将控制碳载量的测试C4固定相和市售柱与这种嵌入型C4柱进行比较。碳载量较高的测试柱在低浓度TFA下对蛋白质表现出良好的分离效果，尽管疏水蛋白的峰变宽了。另一方面，嵌入型极性C4填料对蛋白质表现出理想的保留行为，这与流动相中添加剂的离子对效应无关。得出的结论是，吸附剂上极性基团的屏蔽作用减少了与蛋白质极性基团的硅醇基相互作用。