Detailed insights into the retention mechanism of caffeine metabolites on the amide stationary phase in hydrophilic interaction chromatography

• A very detailed study successfully substantiated some proposals and theories previously reported in the literature.
• The thermodynamic and kinetic studies were conducted under different mobile phase conditions at multiple acetonitrile levels.
• This is the first study to investigate the thermodynamic and kinetic behavior of the polar phases operated in the RPLC mode.

The amide phase was investigated using a wide range of acetonitrile content in the mobile phase in both the HILIC and RPLC modes. Using caffeine metabolites as the model compounds, the retention, thermodynamic and kinetic data was obtained under various mobile phase conditions and supported the previous postulation that there might be a transition of the predominant retention mechanism in relation to the acetonitrile content in HILIC. On the amide phase, hydrophilic partitioning seemed to be the predominant retention mechanism below 85% acetonitrile; and a different retention mechanism (presumably surface adsorption) made more and more significant contributions to the overall retention when the acetonitrile content reached above 85%. This study also provided more direct evidences to explain the effect of salt concentration on the retention of non-charged solutes in HILIC. In addition, the retention, thermodynamic and kinetic data suggest that the amide phase behaved very differently from the conventional C18 phase in the RPLC mode.