Analysis of hydrophobic charge induction displacement chromatography by visualization with confocal laser scanning microscopy
Analysis of hydrophobic charge induction displacement chromatography by visualization with confocal laser scanning microscopy
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DOI：
10.1016/j.seppur.2011.09.002
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发表时间：
2011-10
影响因子：
8.6
通讯作者：
Guofeng Zhao;Lin Zhang;S. Bai;Y. Sun
中科院分区：
文献类型：
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作者：
Guofeng Zhao;Lin Zhang;S. Bai;Y. Sun
位移色谱法是一种容量大、分辨率高的分离技术。传质动力学是决定置换色谱分离效率的重要因素，但对置换过程的动力学，特别是在颗粒水平上的动力学研究很少。本文采用搅拌批实验和共聚焦激光扫描显微镜（CLSM）在线可视化技术，研究了疏水电荷感应位移色谱中蛋白质和置换剂吸附动力学、蛋白质置换和吸附剂再生动力学。搅拌批实验表明，小分子取代剂比蛋白质具有更高的有效扩散率和传质率。对于这项工作中使用的置换系统，置换速率取决于置换剂的传质和吸附速率。通过用异硫氰酸荧光素（FITC）标记蛋白质并使用荧光置换剂罗丹明6G，可以直接可视化置换过程。CLSM得到的浓度曲线清楚地揭示了置换剂的高颗粒内传质率和随后的高置换率。CLSM的观察结果与搅拌批过程的结果基本一致。然而，尽管CLSM的直接可视化为疏水电荷感应位移色谱的微观过程提供了有价值的信息，但仍需要努力开发一种改进的位移模型系统（蛋白质和位移剂），以提供更准确和定量的位移过程信息。
Displacement chromatography is a separation technique with high capacity and high resolving power. Mass transfer kinetics is an essential factor that determines the separation efficiency of displacement chromatography, but few studies have been reported on the kinetics of the displacement process, especially at the particle level. In this work, the kinetics of protein and displacer adsorptions, protein displacement and adsorbent regeneration in hydrophobic charge induction displacement chromatography were studied by both stirred-batch experiments and online visualization with confocal laser scanning microscopy (CLSM). Stirred-batch experiments showed that small-molecule displacers had higher effective diffusivities and mass transfer rates than proteins. For the displacement systems used in this work, the rate of displacement was dependent on the mass transfer and adsorption rate of the displacer. Direct visualization of the displacement process was achieved by labeling the protein with fluorescein isothiocyanate (FITC) and by using a fluorescent displacer, rhodamine 6G. The concentration profiles obtained by CLSM clearly revealed the higher intraparticle mass transfer rate of the displacer and subsequent high rate of displacement. The observations by CLSM were mostly consistent with the results of the stirred-batch processes. However, although the direct visualizations by CLSM has provided valuable information of the microscopic processes in hydrophobic charge induction displacement chromatography, efforts are still needed to develop an improved displacement model system (protein and displacer) that can provide more accurate and quantitative information of displacement processes.