Wu Junyan, Bicker Wolfgang, Lindner Wolfgang
Christian Doppler Laboratory for Molecular Recognition Materials, Department of Analytical Chemistry and Food Chemistry, University of Vienna, Vienna, Austria.
J Sep Sci. 2008 May;31(9):1492-503. doi: 10.1002/jssc.200800017.
Four novel nonionic polar stationary phases were synthesised by anchoring first 2-mercaptoethanol and 1-thioglycerol, respectively, onto vinylised silica (ME and TG packings) followed by an on-phase oxidation with excess hydrogen peroxide in aqueous medium which yielded sulphoxide analogues of the embedded sulphide groups, i. e. oxidised 2-mercaptoethanol (MEO) and oxidised 1-thioglycerol (TGO) packings. Chromatographic characteristics of these stationary phases were evaluated comparatively to three commercial so-called 'diol' columns. U-shaped response curves of retention factors of adenosine and guanosine with hydro-organic eluents containing 5-95% v/v ACN as well as noticeable CH(2)-increment selectivity demonstrated multimodal separation capabilities of the developed amphiphilic materials, i. e. columns can be operated both in hydrophilic interaction chromatography (HILIC) and in RP mode. Although the selector ligands were physico-chemically related, considerably differing retention and selectivity patterns were observed in the HILIC mode. Thereby the introduction of additional hydroxyl groups in the chromatographic ligand resulted in selectivity increments that were different from those obtained by sulphur oxidation. For example, a set of five vitamins delivered five different elution orders with the overall seven columns. A close examination of HILIC separations of nucleobases and nucleosides on the developed packings revealed that (i) the amount of ACN in the eluent adopts a pivotal role in adjusting retention, (ii) the linearity of the relationship log (retention factor) versus log (volume fraction of water in the eluent) increases with phase polarity in the range of 5-40% v/v water, (iii) the slopes are higher with solutes having more polar interactive sites, (iv) the van't Hoff plots are linear (range 15-45 degrees C) with negative retention enthalpy values DeltaH (-4.5 to -14.5 kJ/mol) and (v) the -DeltaH values tend to be higher with more polar phases and more polar analytes. Based on these data the HILIC retention mechanism is described to be composed of both partitioning and adsorption processes. Distinct types of polar interactive sites in the chromatographic ligands may generate mixed-mode HILIC separation conditions that may additionally be superimposed by surface silanol contributions.
通过分别将2-巯基乙醇和1-硫代甘油首先锚定到乙烯基化硅胶上（ME和TG填料），然后在水介质中用过量过氧化氢进行柱上氧化，合成了四种新型非离子极性固定相，得到嵌入硫化物基团的亚砜类似物，即氧化2-巯基乙醇（MEO）和氧化1-硫代甘油（TGO）填料。将这些固定相的色谱特性与三种商业上所谓的“二醇”柱进行了比较评估。腺苷和鸟苷的保留因子与含有5-95% v/v ACN的水-有机洗脱剂呈现U形响应曲线，以及显著的CH₂增量选择性，证明了所开发的两亲性材料具有多模式分离能力，即这些柱可以在亲水相互作用色谱（HILIC）和反相模式下操作。尽管选择配体在物理化学上相关，但在HILIC模式下观察到了显著不同的保留和选择性模式。因此，在色谱配体中引入额外的羟基导致选择性增加，这与通过硫氧化获得的选择性增加不同。例如，一组五种维生素在总共七根柱上给出了五种不同的洗脱顺序。对所开发填料上的核碱基和核苷的HILIC分离进行仔细研究发现：（i）洗脱剂中ACN的量在调节保留方面起关键作用；（ii）log（保留因子）与log（洗脱剂中水的体积分数）之间关系的线性度在5-40% v/v水的范围内随相极性增加；（iii）对于具有更多极性相互作用位点的溶质，斜率更高；（iv）范特霍夫图是线性的（15-45℃范围），保留焓值ΔH为负（-4.5至-14.5 kJ/mol）；（v）对于极性更强的相和极性更强的分析物，-ΔH值往往更高。基于这些数据，HILIC保留机制被描述为由分配和吸附过程组成。色谱配体中不同类型的极性相互作用位点可能产生混合模式的HILIC分离条件，这些条件可能还会被表面硅醇的贡献叠加。
