Peak broadening in protein chromatography with monoliths at very fast separations
Peak broadening in protein chromatography with monoliths at very fast separations
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DOI：
10.1021/ac000688y
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发表时间：
2000-10-15
影响因子：
7.4
通讯作者：
Jungbauer, A
中科院分区：
文献类型：
--
作者：
Hahn, R;Jungbauer, A
整体料是作为连续介质铸造的固定相，其由微孔分支的流动通道交错。使用牛血清白蛋白作为模型蛋白质的脉冲响应实验用于测试基于聚甲基丙烯酸酯的整体材料，导致峰展宽，实际上不受色谱速度的影响。开发了一个经验模型来描述峰展宽，允许用一个术语来解释孔隙对流，并用一个术语来解释孔隙扩散。估计扩散距离低于 10 MI。这对应于用单分散 1-μm 颗粒观察到的值。通过改变检测器的响应时间进行的系统研究表明，无法充分发挥整体的潜力，因为当前可用的色谱系统是数据采集速度和检测器无限长度的虚拟峰展宽的限制因素。液体的惯性以及液体处理和电子控制之间的同步引入了额外的干扰。在尽可能短的响应时间下，可以获得可靠的峰值数据，速度高达 35 厘米/分钟。与传统的填充床相比，连续床的压降要小得多。不同的流动模式和显着减少的涡流可能是高比渗透率的原因。
Monoliths are stationary phases cast as a continuous medium which are interlaced by flow channels ramified with micropores. Pulse response experiments with bovine serum albumin as a model protein were applied for testing polymethacrylate-based monoliths, resulting in peak broadening that practically was not influenced by the chromatographic velocity. An empirical model was developed to describe peak broadening, allowing a term to account for the pore convection and a term for the pore diffusion. A diffusional distance lower than 10 MI was estimated. This corresponds to values observed with monodisperse 1-mu m particles. Systematic investigations by changing the response time of the detector showed that the full potential of the monoliths could not be exploited, since the currently available chromatography systems are the limiting factor regarding the speed of data acquisition and virtual peak broadening by the infinite length of the detector. Inertia of the liquid and synchronization between liquid handling and electronic control introduced an additional disturbance. At the lowest possible response time, reliable peak data could be obtained up to a velocity of 35 cm/min. The pressure drop along the continuous bed was much smaller compared to a conventionally packed bed. Different flow patterns and significantly reduced eddy vortexes may be responsible for the high specific permeability.