Department of Analytical Chemistry, University of Vienna, Waehringer Strasse 38, A-1090 Vienna, Austria.
J Chromatogr A. 2011 Feb 18;1218(7):882-95. doi: 10.1016/j.chroma.2010.10.073. Epub 2010 Oct 23.
The separation properties of five silica packings bonded with 1-[3-(trimethoxysilyl)propyl]urea in the range of 0-3.67 μmol m⁻² were investigated in the hydrophilic interaction chromatography (HILIC) elution mode. An increase of the ligand surface density promoted retention of non-charged polar compounds and even more so for acids. An opposite trend was observed for bases, while the amphoteric compound tyrosine exhibited a U-shaped response profile. An overall partitioning retention mechanism was incompatible with these observations; rather, the substantial involvement of adsorptive interactions was implicated. Support for the latter was provided by column-specific changes in analyte retention and concomitant selectivity effects due to variations of salt concentration, type of salt, pH value, organic modifier content, and column temperature. Silica was more selective for separating compounds differing in charge state (e.g. tyramine vs. 4-hydroxybenzoic acid), while in cases where structural differences of solutes resided in non-charged polar groups (e.g. tyramine vs. 5-hydroxydopamine, nucleoside vs. nucleobase) more selective separations were obtained on bonded phases. Hierarchical cluster analysis of the home-made urea-type and three commercial amide-type bonded packings evinced considerable differences in separation properties. The present data emphasise that the role of the packing material under HILIC elution conditions is hardly just the polar support for a dynamic coating with a water-enriched layer. Three major retention mechanisms are claimed to be relevant on bare silica and the urea-type bonded packings: (i) HILIC-type partitioning, (ii) HILIC-type weak adsorption such as hydrogen bonding between solutes and ligands or solutes and silanols (potentially influenced by individual degrees of solvation, salt bridging, etc.), (iii) strong electrostatic (ionic) solute-silanol interactions (attractive/repulsive). Even when non-charged polar bonded phases are used, solute-silanol interactions should not be discounted, which makes them a prime parameter to be characterised by HILIC column tests. Multi/mixed-mode type separations seem to be common under HILIC elution conditions, associated with a great deal of selectivity increments. They are accessible and controllable by a careful choice of the type of packing, the mobile phase composition, and the temperature.
在亲水作用色谱（HILIC）洗脱模式下，研究了五种键合 1-[3-(三甲氧基硅基)丙基]脲的硅胶固定相在 0-3.67 μmol m⁻²范围内的分离性能。配体表面密度的增加促进了非带电极性化合物的保留，对酸的保留作用更大。对于碱则观察到相反的趋势，而两性化合物酪氨酸呈现出 U 形响应轮廓。整体分配保留机制与这些观察结果不一致；相反，吸附相互作用的大量参与被暗示。这一点得到了以下支持：由于盐浓度、盐的类型、pH 值、有机改性剂含量和柱温的变化，会引起分析物保留和伴随的选择性效应的柱特异性变化。硅胶对分离电荷状态不同的化合物（例如酪胺与 4-羟基苯甲酸）更具选择性，而对于溶质结构差异在于非带电极性基团（例如酪胺与 5-羟多巴胺、核苷与碱基）的情况，在键合相上获得了更具选择性的分离。对自制脲型和三种商业酰胺型键合固定相的层次聚类分析表明，分离性能存在很大差异。本研究强调，在 HILIC 洗脱条件下，填充材料的作用不仅仅是极性支持，还可以形成富含水的动态涂层。据称，在裸露的硅胶和脲型键合固定相上有三种主要的保留机制：（i）HILIC 型分配；（ii）HILIC 型弱吸附，例如溶质与配体或溶质与硅醇之间的氢键（可能受溶剂化程度、盐桥接等的影响）；（iii）强静电（离子）溶质-硅醇相互作用（吸引/排斥）。即使使用非带电极性键合相，也不应忽视溶质-硅醇相互作用，因为它们是通过 HILIC 柱测试来表征的关键参数。在 HILIC 洗脱条件下，似乎经常出现多/混合模式类型分离，与大量选择性增加有关。通过仔细选择填充类型、流动相组成和温度，可以实现和控制这种分离。
