Chromatographic Behavior of Uric Acid and Methyl Uric Acids on a Diol Column in HILIC

Abstract

The retention behavior of uric acid (UA) and methyl uric acids (MUAs) on a diol column in hydrophilic interaction chromatography (HILIC) is presented. The effects of chromatographic parameters such as organic solvent content and salt concentration in the mobile phase, and column temperature were systematically investigated using a design of experiment (DOE). The results of the DOE revealed that the content of organic solvent, acetonitrile (ACN), had the most influence on the retention of the analytes. The effect of salt concentration indicated the possibility of electrostatic repulsion between the negatively charged analytes and the deprotonized residual silanol groups on the surface of the stationary phase. Van’t Hoff plots were constructed to further study the effects of column temperature on the retention of the compounds on the diol phase. Nonlinear van’t Hoff plots were observed for all the analytes in the studied temperature range (0–50 °C) signifying that the retention of UAs and MUAs on the diol column is governed by mixed retention mechanisms. Retention prediction models derived by multiple linear regression (MLR) showed that the retention of the analytes is dependent on the percentage of ACN in the mobile phase and two solute-related descriptors namely ovality (Ov) and the lowest unoccupied molecular orbital (LUMO). The incorporation of Ov, a shape parameter, in the MLR equation signified the importance of the shape/size of the compounds on the observed retention on the diol phase.