Urban Jirí, Jandera Pavel, Kucerová Zdenka, van Straten Marion A, Claessens Henk A
Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Nám. Cs. Legií 565, CZ-53210 Pardubice, Czech Republic.
J Chromatogr A. 2007 Oct 5;1167(1):63-75. doi: 10.1016/j.chroma.2007.08.027. Epub 2007 Aug 17.
The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample elution. Hydrodynamic properties, efficiency and gradient elution of proteins were investigated on five commercial C18 columns with wide-pore totally porous particles, with superficially porous layer particles, non-porous particles and a silica-based monolithic bed. The efficiency in the terms of reduced plate height is higher for low-molecular ethylbenzene than for proteins, but depends on the character of the pores in the individual columns tested. The superficially porous Poroshell and the non-porous Micra columns provide the best efficiency for proteins at high mobile phase flow rates, probably because of similar pore architecture in the stationary phase. The Zorbax column with similar pore architecture as the Poroshell active layer, i.e. narrow pore distribution of wider pores shows better efficiency than the packed column with narrow pores and broad pore distribution. The monolithic column shows lower efficiency for proteins at high flow rates, but it performs better than the broad-pore distribution totally porous particulate columns. Different pore architecture affects also the retention and selectivity for proteins on the individual columns. The retention times on all columns can be predicted using the model for reversed-phase gradient elution developed originally for low-molecular compounds. Consideration of the limited pore volume accessible to the biopolymers has negligible effect on the prediction of retention on the columns packed with non-porous or superficially porous particles, but improves the accuracy of the predicted data for the totally porous columns with broad pore distribution.
反相液相色谱中固定相的类型对样品洗脱有显著影响。在五种商业C18柱上研究了蛋白质的流体动力学性质、柱效和梯度洗脱，这些柱子分别填充有宽孔全多孔颗粒、表面多孔层颗粒、无孔颗粒以及硅胶整体柱。以折合塔板高度衡量的柱效，低分子乙苯高于蛋白质，但取决于所测试的各柱子的孔特性。表面多孔的Poroshell柱和无孔的Micra柱在高流动相流速下对蛋白质具有最佳柱效，这可能是因为固定相具有相似的孔结构。与Poroshell活性层具有相似孔结构的Zorbax柱，即宽孔的窄孔分布，比具有窄孔和宽孔分布的填充柱表现出更好的柱效。整体柱在高流速下对蛋白质的柱效较低，但比宽孔分布的全多孔颗粒柱表现更好。不同的孔结构也会影响各柱子上蛋白质的保留和选择性。所有柱子上的保留时间都可以使用最初为低分子化合物开发的反相梯度洗脱模型进行预测。考虑生物聚合物可利用的有限孔体积，对预测无孔或表面多孔颗粒填充柱上的保留影响可忽略不计，但能提高宽孔分布全多孔柱预测数据的准确性。
