Electrical double layer chromatography

离子交换色谱（IEC）已广泛用于纯化各种生物分子，如蛋白质和肽。然而，涉及高分辨率IEC分离的机制仍然不清楚。本文研究了蛋白质和离子交换介质表面电荷性质对蛋白质在离子交换介质中的保留和传递现象的影响。作为模型分离系统，采用具有各种IEC介质的β-乳球蛋白A和B（LgA，Lg B）。当移动的相pH接近等电点pI（=5.1-5.2）时，参与保留（吸附）的位点数减少。然而，即使在pH 5.2下，LgA和LgB都保留在阴离子和阳离子交换色谱柱上。在阴离子交换色谱柱中，当pH接近pI时，LgA和LgB的分离度较好， ...更多信息 f吸附位值较小（约2-3）。两种蛋白质在阳离子交换色谱柱上没有分离。讨论了影响分离度和pI附近保留值的因素。洗脱体积显著变化，离子交换容量变化较小。发现必须调节洗脱缓冲液的离子强度以补偿由于离子交换容量变化而引起的洗脱体积的变化。还发现洗脱缓冲液的离子强度和pH是影响洗脱体积的重要变量。在该模型分离系统中，表明pH值应在0.1单位内，离子强度应在0.002 mol/L内，以满足标准（洗脱体积变化5%）。建议获得梯度洗脱数据，以预测逐步洗脱中洗脱体积的变化。通过使用梯度洗脱数据，可以执行过程诊断，并且可以获得关于过程稳定性的重要信息。对大得多的分子如B型肝炎病毒表面抗原进行了类似的实验研究。少

Ion exchange chromatography (IEC) has been widely used for purifying various biomolecules such as proteins and peptides. However, the mechanism involved in high resolution IEC separation is still not clarified. Also it has been suggested that controlling the electrical double-layer may lead to a new efficient separation of proteins by using IEC media.In this paper,the effect of the surface charge properties of both proteins and IEC media on the retention and transport phenomena were investigated. As a model separation system, (-lactoglobulin A and B (LgA, LgB) with various IEC media were employed. The number of sites involved in the retention (adsorption) decreased as the mobile phase pH approached the isoelectric points pI(=5.1-5.2). However, even at pH 5.2 both LgA and LgB were retained on anion- and cation-exchange chromatography columns. The separation (resolution) of LgA and LgB became better when the pH approached the pI in anion exchange chromatography columns where the number o … More f adsorption site values are small (ca. 2-3). The two proteins were not separated for cation exchange chromatography columns. Factors affecting the resolution and the retention near the pI were discussed. The elution volume varied significantly with a small change in the ion-exchange capacity. It was found that the ionic strength of the elution buffer must be adjusted in order to compensate a change in the elution volume due to the ion-exchange capacity variations. The ionic strength and the pH of the elution buffer were also found to be important variables affecting the elution volume. In this model separation system, it was indicated that the pH should be within (0.1 unit and the ionic strength within (0.002 mol/L in order to meet the criteria ((5% elution volume variation). It is recommended that gradient elution data be obtained for predicting elution volume variations in stepwise elution. By using the gradient elution data the process diagnosis can be performed, and the important information on the process stability can be obtained. Similar experimental investigations were done for much larger molecules such as Hepatitis B virus surface antigen. Less