Reversed-phase chromatography of proteins and peptides on compressed continuous beds
Reversed-phase chromatography of proteins and peptides on compressed continuous beds
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在压缩连续床上进行蛋白质和肽的反相色谱分析
DOI：
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发表时间：
1993
期刊：
影响因子：
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通讯作者：
D. Eaker
中科院分区：
文献类型：
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作者：
S. Hjertén;K. Nakazato;J. Mohammad;D. Eaker
所描述的聚合物床是在水溶液中直接以柱状或批量形式以大簇状小颗粒的形式合成的。因此，省略了在有机溶剂中预先制备珠子的传统的、昂贵的步骤。以哌嗪二丙烯酰胺、甲基丙烯酰胺和烯丙基缩水甘油醚为原料合成了床层。由此获得的环氧活化床可用于非极性配体（如十八醇）或极性富氧物质（如葡聚糖）的共价附着。非极性层用于反相色谱，极性层用于偶联1,2-环氧十八烷。用富含oh的物质涂覆有两个目的：(1)基质变得亲水，减少非特异性相互作用（可以排除改性剂），从而提高分辨率；(2)有许多oh基团可用（例如与环氧化物偶联），这是高配体密度的先决条件。即使在高流速下，蛋白质的分辨率也很高。根据合成床的方法不同，蛋白质的分辨率要么随着流速的增加而增加，要么略有下降。选择合适的温度对cnrr酶解肽的高分辨率非常重要。
SummaryThe polymer beds described are synthesized in aqueous solution directly in the column or batchwise in the form of large clusters of small particles. The conventional, expensive step involving prepreparation of beads in an organic solvent is thus omitted. Beds were synthesized from piperazine diacrylamide, methacrylamide and allyl glycidyl ether. The epoxy-activated beds thus obtained were used for covalent attachment of either nonpolar ligands (e.g. octadecanol) or polar OH-rich substances (e.g. dextran). The non-polar beds were used for reversed-phase chromatography, as were polar ones following coupling with 1,2-epoxyoctadecane. Coating with OH-rich substances serves two purposes: (I) the matrix becomes hydrophilic, decreasing nonspecific interactions (modifiers can be excluded) and hence increases resolution and (II) many—OH groups are available (e.g. for coupling to epoxides), a prerequisite for high ligand density. Resolution of proteins was high even at high flow rates. Depending on the method used for the synthesis of the bed, resolution of proteins either increased with an increase in flow rate or decreased slinghtly. Choice of the correct temperature was very important for high resolution of CNRr-digested peptides.