Electrical double layer chromatography

离子交换色谱法已广泛应用于蛋白质、多肽等多种生物分子的纯化。然而，高分辨率IEC分离的机理尚不清楚。本文还指出，控制电双层可能会导致一种新的高效分离蛋白质的方法。本文研究了蛋白质和IEC介质的表面电荷性质对保留和传输现象的影响。采用（-乳球蛋白a和B） （LgA, LgB）与各种IEC介质作为模型分离系统。当流动相pH接近等电点pI（=5.1-5.2）时，参与保留（吸附）的位点数量减少。然而，即使在pH 5.2时，LgA和LgB在阴离子和阳离子交换色谱柱上仍被保留。在阴离子交换色谱柱中，当pH值接近pI时，LgA和LgB的分离（分离）效果较好，且吸附位点数较小（约2-3个）。阳离子交换色谱柱未分离这两种蛋白。讨论了影响分辨率和pI附近保留率的因素。洗脱体积变化显著，离子交换容量变化不大。研究发现，必须调整洗脱缓冲液的离子强度，以补偿因离子交换容量变化而引起的洗脱体积变化。离子强度和洗脱缓冲液的pH值也是影响洗脱体积的重要因素。在该模型分离体系中，pH值应在0.1单位以内，离子强度应在0.002 mol/L以内，以满足（5%洗脱体积变化）的要求。建议采用梯度洗脱数据来预测逐步洗脱的洗脱体积变化。利用梯度洗脱数据可以进行工艺诊断，获得工艺稳定性的重要信息。对乙肝病毒表面抗原等更大的分子也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