Question

In preparing a hexane-acetone gradient on a normal phase HPLC column, it is desirable to increase or decrease the proportion of hexane as the column is eluted?

Answer 1

Normal-phase liquid chromatography (NP-HPLC), as the name implies, is the original version of HPLC. This means, the stationary phase is more polar than the mobile phase and the interaction between analyte and column has predominantly polar character (hydrogen bonding, π–π or dipole–dipole interactions, etc.). The most commonly used NP stationary phase is silica gel ([SiO₂]_x · [H₂O]_y) or Aluminium oxide. These stationary phases are polar.

In normal phase chromatography – less polar (hydrophobic) compounds elute first, whilst more polar (hydrophilic) compounds elute later.

Normal phase chromatography has been used for the separation of both neutral and ionisable compounds, although neutral sample separations predominate the literature.

This is a liquid-solid chromatography where the stationary phase is polar. For such a gradient, one should start with the less polar solvent such as hexane to elute hydrophobic compounds first and increase the percentage of the more polar solvents (such as methanol) with time in order to force late eluting more polar analytes (more strongly adsorbed) to leave the column.

Answer: It is desirable to decrease the proportion of hexane as the column is eluted.