A mixture of two drugs and their metabolites were analysed by reverse phase HPLC on a 50 mm ODS column by isocratic elution using a mobile phase of 50% methanol / 50% aqueous 1% acetic acid delivered at a flow rate of 1.5 mL/min. The column dead volume was measured as 0.30 mL and a column plate height (H) of 0.02 mm was determined.

Calculate the elution time in minutes of an unretained solute (t_m) and the average linear velocity (u_x, mm/s) of the mobile phas

The mobile phase in the above analysis is 50% methanol / 50% aqueous 1% acetic acid. Firstly, it is changed to 40% methanol / 60% water (with 1% acetic acid) and then, secondly, to 50% acetonitrile / 50% water (with 1% acetic acid) instead of the original mobile phase in a series of additional chromatography experiments. All other experimental conditions remain the same. Explain (3-6 sentences using key words to describe the chemistry) the effect that each change will have on:

1. the chemical nature of the mobile phase,

2. interaction of the different solute types with the stationary phase, and

3. the retention times of the two peaks due to drugs A & B in each instance