Yamamoto S, Watler P K, Feng D, Kaltenbrunner O
Department of Chemical Engineering, Yamaguchi University, Tokiwadai, Ube, Japan.
J Chromatogr A. 1999 Aug 6;852(1):37-41. doi: 10.1016/s0021-9673(99)00592-0.
Very fine separation of proteins by stepwise elution ion-exchange chromatography is very often a unstable process. To characterize the unstability of such processes the elution volume variations were examined by the model equation which contained the ion-exchange capacity and the number of adsorption sites. The data needed for the model calculation were obtained from gradient elution experiments. As a model separation system stepwise elution of a model protein (beta-lactoglobulin) near the isoelectric point on a weak cation-exchange chromatography column was chosen. 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.
通过分步洗脱离子交换色谱法对蛋白质进行非常精细的分离往往是一个不稳定的过程。为了表征此类过程的不稳定性，通过包含离子交换容量和吸附位点数量的模型方程来研究洗脱体积的变化。模型计算所需的数据来自梯度洗脱实验。作为一个模型分离系统，选择了在弱阳离子交换色谱柱上在等电点附近对模型蛋白质（β-乳球蛋白）进行分步洗脱。洗脱体积会随着离子交换容量的微小变化而显著变化。研究发现，必须调整洗脱缓冲液的离子强度，以补偿由于离子交换容量变化而导致的洗脱体积变化。还发现洗脱缓冲液的离子强度和pH值是影响洗脱体积的重要变量。在这个模型分离系统中，结果表明，为了满足标准（洗脱体积变化±5%），pH值应在±0.1单位以内，离子强度应在±0.002 mol/l以内。建议获取梯度洗脱数据以预测分步洗脱中的洗脱体积变化。通过使用梯度洗脱数据，可以进行过程诊断，并获得有关过程稳定性的重要信息。
