Tandem columns and mixed-bed columns in high-performance liquid chromatography of proteins.
Tandem columns and mixed-bed columns in high-performance liquid chromatography of proteins.
复制标题
DOI：
10.1016/0021-9673(86)80079-6
复制
发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Z. Rassi;Csaba. Horvath
中科院分区：
文献类型：
--
作者：
Z. Rassi;Csaba. Horvath
通过使用一个阳离子和阴离子交换柱串联，在pH 7.0的盐梯度增加的情况下，在一次色谱运行中分离了酸性和碱性蛋白质的混合物。发现柱的序列顺序会影响色谱结果，这种影响归因于穿越第一根离子交换柱时盐梯度剖面的改变。单柱填充阳离子和阴离子交换剂的二元混合物，得到与串联柱相似的色谱结果，因此提供了在单次色谱运行中分离酸性和碱性蛋白质的替代方法。在两种离子交换剂的混合物中加入轻度疏水固定相，得到三元混合相。随着盐梯度的增加，该色谱柱可作为碱性蛋白质分离的阳离子交换剂，或作为酸性蛋白质分离的阴离子交换剂。此外，它还可以用作盐梯度增加的“双极”静电相互作用柱和盐梯度减少的疏水相互作用柱，用于在单次色谱运行中分离两种类型的蛋白质。混合床色谱柱中使用的固定相由相同的二氧化硅载体制备，即。，它们具有相同的颗粒和孔隙尺寸、密度和孔隙体积。除了它们在分析应用中的明显优势外，适当的混合固定相都对待分离组分具有保留性质，预计在制备色谱中也很有用，可以在不损失分离能力的情况下为给定的分离问题“定制”柱选择性。
By using a cation- and an anion-exchange column in series, mixtures of acidic and basic proteins were separated in a single chromatographic run with increasing salt gradient at pH 7.0. The serial order of the columns was found to affect the chromatographic results, and the effect was attributed to alteration of the salt gradient profile upon traversing the first ion-exchange column. Single columns, packed with a binary mixture of a cation and an anion exchanger gave similar chromatographic results as the tandem columns and thus offered an alternative approach to the separation of both acidic and basic proteins in a single chromatographic run. A ternary mixed phase was obtained by adding a mildly hydrophobic stationary phase to the mixture of the two ion exchangers. This column could be used with increasing salt gradient as a cation exchanger for the separation of basic proteins, or as an anion exchanger for the separation of acidic proteins. Furthermore, it could be used as a “bipolar” electrostatic-interaction column with increasing salt gradient and as a hydrophobic-interaction column with decreasing salt gradient for the separation of both types of proteins in a single chromatographic run. The constituent stationary phases used in the mixed-bed columns were prepared from the same silica support,i.e., they had the same particle and pore dimensions, density, and pore volume. Besides their obvious advantages in analytical applications, appropriate mixed stationary phases, all having retentive properties for the components to be separated, are expected to be useful also in preparative chromatography to “tailor” column selectivity for a given separation problem without loss of separating capacity.