Electrical double layer chromatography

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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