Jandera P, Bocian S, Molíková M, Buszewski B
Department of Analytical Chemistry, University of Pardubice, Nám. Cs. legií 565, CZ-532 10 Pardubice, Czech Republic.
J Chromatogr A. 2009 Jan 9;1216(2):237-48. doi: 10.1016/j.chroma.2008.11.055. Epub 2008 Nov 27.
We investigated the effects of the concentration of naphthalene sulphonic acids (NSAs) as anionic test compounds in the injected sample and of the salt additives to the mobile phase on ion-exclusion. The retention behaviour of NSAs sensitively reflects even minor changes in the ionic and hydrophobic interactions and can be useful for predicting the effects of the stationary phases in reversed-phase chromatography of polar and ionic compounds, both small ones and biopolymers, e.g., oligonucleotides. We studied chromatographic properties of several stationary phases intended for separations in aqueous mobile phases: a C18 column end-capped with polar hydrophilic groups, a densely bonded C8 column doubly end-capped with short alkyl groups, a short alkyl stationary phase designed to keep full pore accessibility in highly-aqueous mobile phases and a Bidentate column with "bridged" C18 groups attached to the silica hydride support. The chemistry and pore structure of various types of column packing materials and of the salt additives to the mobile phase affect the proportion of the pore volume non-accessible to anions due to ion-exclusion and consequently the peak asymmetry and hydrophobic selectivity in reversed-phase chromatography of organic acids. We also addressed the problems connected with the determination of column hold-up volume in aqueous mobile phases. The accessibility of the stationary phase for anionic compounds in contact with the sample zone is affected by ion-exclusion due to repulsive interactions with the negatively charged surface in the pores of the stationary phase. The accessible part of the stationary phase increases and consequently the migration velocity along the column decreases with increasing concentration of the sample in the zone moving along the column. Because of a limited access to the stationary phase, its capacity can be easily overloaded. The combination of the column overload and ion-exclusion effects may result in fronting or tailing peak asymmetry. To explain this behaviour, we proposed a modified Langmuir model, respecting the variation of the column capacity due to the effects of sample concentration on ion-exclusion.
我们研究了作为阴离子测试化合物的萘磺酸（NSAs）在进样样品中的浓度以及流动相中的盐添加剂对离子排斥的影响。NSAs的保留行为能灵敏地反映出离子和疏水相互作用中哪怕是微小的变化，对于预测极性和离子化合物（包括小分子和生物聚合物，如寡核苷酸）在反相色谱中固定相的作用可能很有用。我们研究了几种用于在水性流动相中分离的固定相的色谱性质：一种用极性亲水基团封端的C18柱、一种用短烷基双封端的密集键合C8柱、一种设计用于在高水性流动相中保持全孔可及性的短烷基固定相以及一种在硅氢化物载体上连接有“桥连”C18基团的双齿柱。各种类型的柱填充材料的化学性质和孔结构以及流动相中的盐添加剂会影响由于离子排斥而导致阴离子无法进入的孔体积比例，从而影响有机酸反相色谱中的峰不对称性和疏水选择性。我们还探讨了与测定水性流动相中柱保留体积相关的问题。与样品区接触的阴离子化合物对固定相的可及性会受到离子排斥的影响，这是由于与固定相孔中带负电荷的表面存在排斥相互作用。随着沿柱移动的区域中样品浓度的增加，固定相的可及部分增加，因此沿柱的迁移速度降低。由于对固定相的可及性有限，其容量很容易过载。柱过载和离子排斥效应的结合可能导致峰前沿或峰拖尾的不对称性。为了解释这种行为，我们提出了一个修正的朗缪尔模型，该模型考虑了样品浓度对离子排斥的影响导致的柱容量变化。
