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The retention mechanism of polar compounds in hydrophilic interaction chromatography (HILIC) was investigated in order to analyse many polar compounds in pharmaceutical and biological samples. HILIC uses a polar stationary phase in conjunction with an aqueous mobile phase containing a high concentration of acetonitrile (60- 95%). The use of bare silica, silica based bonded phases and polymer based bonded phases were investigated for the separation of catecholamines (dopamine, epinephrine and norepinephrine) in HILIC and per aqueous liquid chromatography (PALC) mode. A bonded phase column provided complete resolution of catecholamines in comparison to a bare silica column. The retention mechanisms for these polar basic compounds were found to be ionic interaction, partitioning and adsorption. The use of a polymer based ZIC-HILIC column proved that some of the ionic interaction (which is less on a silica based ZIC-HILIC column) is due to sulfonic groups on the column. The HILIC contribution is higher at higher buffer concentration on both polymer and silica based columns. High buffer concentration reduces ionic interaction but also may absorb more water resulting in a thicker aqueous layer on the column surface for solute partitioning. An overloading study of various basic and a quaternary ammonium compound was performed in the HILIC mode with respect to various pH and % ACN in the mobile phase using different colunms including bare silica (Atlantis HILIC, Zorbax Rx SIL, Ascentis HILIC) and bonded phase (ZIC-HILIC, Diol), The results of the study were complex and the results difficult to interpret. However, it was possible to draw some general conclusion. In general the loading capacity of the bare silica and bonded phase column was of the same order. However, there were variations in the comparative loading capacities with pH. The partitioning mechanism was investigated in the context of the octanol-water distribution coefficient Dow of the solute, A good correlation (R) was achieved between log D and the retention factor (k) of 29 compounds (acidic, basic, quaternary ammonium and neutral) on six different HILIC columns comprising bare silica, amide, dial. zwitterionic and a mixed-mode dial/hydrophobic phase. The correlation of k with log D was found to be improved for each column when the aqueous content of the mobile phase was increased from 5- 15% and by changing the pH from 6.0 to 3.0. It is possible that adsorption on to the polar groups of the stationary phase at low water content and ionic interact ion at higher pH (ionised silica) are competing mechanisms. The effect of pH, % ACN, temperature and stationary phase on selectivity was studied by comparing the correlation of plots of k using one condition vs a second condition. The higher the deviation from linearity, the greater is the effect of the condition change on selectivity. Changing the stationary phase was shown to be a powerful tool in influencing selectivity. HILIC was successfully used for bioanalytical purposes for the analysis of catecholamines in urine using electrochemical detection and gives a very good result in order to analyse polar antibiotics in human serum using mass spectrometry detection. Further, HILIC can be used for the analysis of other polar metabolites in biological samples for drug discovery and metabolomics study |
