Abstract (eng)
Four stationary phases having non-charged ligands with hydroxyl as well as sulfide- or sulfoxide-groups bonded to silica were prepared. 2-mercaptoethanol (ME phase) and 1-thioglycerol (TG phase) were immobilized on vinylised silica by radical addition. Subsequent on-phase oxidation of the embedded sulphide groups with hydrogen peroxide delivered sulfoxide-groups (MEO and TGO phases).
These new developed diol-type stationary phases offered HILIC chromatographic selectivity towards the separation of nucleosides, nucleobases and water soluble vitamins. Higher Phase polarity caused a stronger retention of polar compounds and sulfoxide functionality in the ligand was especially useful to change HILIC characterisation of phases. Compared to commercial diol-type HILIC packings complementary selectivity was found which may be an indication for different retention mechanisms being at work. The developed diol-type phases delivered also noticeable CH2-increment selectivity (multimodal separation capability). Silanol activity had definite effects on retention and selectivity in the HILIC elution mode and the extent of response was depending on the type of analytes (acids, bases, non-charged). In the HILIC mode, silanophilic interactions may become the main retention mechanism on silica-based polar phases, especially for charged and chargeable compounds.
The type of protic modifier (water, 1,2-ethanediol, methanol, ethanol) had significant effect on HILIC retention and the polarity of these protic modifiers adopted a primary role in determining the extent of retention and, generally, increased retention was found in the order water < 1,2-ethanediol < methanol < ethanol. Besides the effect on retention, chromatographic selectivity was also affected by the type of alcohol modifier and under isoelutropic conditions compared to an aqueous HILIC eluent the selectivity can be changed up to an inverted elution order. Additionally, the amount of organic solvent, the nature and amount of salt, pH value and column temperature were all effective to optimise HILIC separations.
The retention mechanisms were quite complex in HILIC. It was supposed that the hydrophilic interactions were composed of partitioning and weak adsorption interactions which could be developed by direct solute-ligand interactions, solute-solvent shell on the surface, and/or solute-silanol interactions. For charged packings, electrostatic interactions (attractive or repulsive) can be easily superimposed on the hydrophilic interactions.