Tandem separation schemes for preparative high-performance liquid chromatography of proteins.
Tandem separation schemes for preparative high-performance liquid chromatography of proteins.
复制标题
用于蛋白质制备型高效液相色谱的串联分离方案。
DOI：
10.1016/s0021-9673(00)94780-0
复制
发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Horváth,C
中科院分区：
文献类型：
--
作者：
Lee,AL;Liao,AW;Horváth,C
蛋白质混合物的制备层析是通过串联分离方案进行的，包括正面层析，然后逐步解吸或置换。通过这种方式，在分析性高效液相色谱中通常使用柱和仪器，可以提纯蛋白质的数量与通常由制备规模系统分离的蛋白质的量相似。将β-乳球蛋白A和B的混合物装载到阴离子交换柱上，在此过程中，很大一部分较少保留的β-乳球蛋白B以纯形式被回收。然后用载体冲洗柱，然后通过单步解吸、两步解吸或置换对结合在固定相上的物质进行随后的解吸。对于这种混合物，最后两种方法在单位柱体积获得的产物量方面产生了大致相同的结果。虽然分步解吸是一种比置换更简单的技术，但后者是分离具有相似吸附行为的组分所必需的。在另一组实验中，通过对人类生长激素进行热处理而获得的蛋白质混合物在反相柱上进行了分级。在用前向色谱柱加载后，将主要产物的很大一部分从柱上保留的其他组分中分离出来，然后采用四个解吸步骤来回收单独的组分。这些分离方案为蛋白质的制备层析提供了一种方法，该方法在柱负载能力、低流动相消耗、同时分离和浓缩以及对痕量组分的浓缩方面优于传统的线性洗脱。
Preparative chromatography of protein mixtures was carried out by tandem separation schemes involving frontal chromatography followed by stepwise desorption or displacement. In this way, with columns and instruments generally employed in analytical high-performance liquid chromatography, proteins could be purified in quantities similar to those typically separated by a preparative-scale system. A mixture of β-lactoglobulin A and B was loaded onto an anion-exchange column, and, in the process, a large fraction of the less-retained β-lactoglobulin B was recovered in pure form. The column was then flushed with the carrier, and subsequent desorption of the substances bound on the stationary phase was carried out by single-step desorption, two-step desorption, or displacement. With this mixture, the last two methods yielded approximately the same results in terms of the amount of product obtained per unit column volume. Whereas stepwise desorption is a simpler technique than displacement, the latter is required for the separation of components having similar adsorption behavior. In another set of experiments, a protein mixture obtained by heat treatment of human growth hormone was fractionated on a reversed-phase column. After loading the column by frontal chromatography, which separated a large fraction of the main product from the other components retained by the column, four desorption steps were applied to recover the individual components. These separation schemes offer an approach to preparative chromatography of proteins that is superior to conventional linear elution in terms of column load capacity, low mobile phase consumption, simultaneous separation and concentration, as well as enrichment of trace components.
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影响因子：
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作者：
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DOI：
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发表时间：
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期刊：
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DOI：
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发表时间：
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期刊：
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影响因子：
--
作者：
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通讯作者：
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影响因子：
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