Separation properties of novel and commercial polar stationary phases in hydrophilic interaction and reversed-phase liquid chromatography mode

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 CH2-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 Delta H (-4.5 to -14.5 kJ/mol) and (v) the -Delta H 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.