Question

During a liquid chromatography lab, caffeine and theophylline were separated into an octadecyl silica column (C18) with a mobile phase consisting of a mixture of water and methanol.

Koffein Teofyllin 7 CH3 H3C HC- CH, CH3

What is the elution scheme?

b) Reason with current theory as background what is expected to happen
the retention factors in case:
- the methanol content in the mobile phase is increased?
- the flow rate is increased?
- the column is replaced with one consisting of octyl silica (C8)?
- switching to a normal phase system instead?

Answer 1

Given: An experiment of the separation of mixture of caffeine and theophylline by reverse phase liquid chromatography (HPLC) using octadecyl silica column (C18) with a mobile phase consisting of a mixture of water and methanol.

To determine elution scheme:

After structural analysis, it is confirmed that the caffeine is non polar molecule and theophylline is polar molecule. In the reverse phase liquid chromatography (HPLC) separation the polar molecule elute first followed by non polar molecule. Therefore the given method of separation by HPLC, the theophylline will elute first followed by the elution of caffeine using octadecyl silica column (C18) with a mobile phase consisting of a mixture of water and methanol.

b) Reasons:

1. If the methanol content in the mobile phase is increased:

It will affect the separation and both caffeine and theophylline may elute at same retention time.

2. If the flow rate is increased:

As the increase in flow rate will start elution of both caffeine and theophylline quickly without much difference in the retention time leading to no separation.

3. If the column is replaced with one consisting of octyl silica (C8):

As the column is changed, we need to change the mobile phase because octyl silica (C8) is normal phase liquid chromatography which need non polar solvents systems like ethyl acetate/n-hexane, acetonitrile/ethyl acetate. We need to develop HPLC method like gradient or isocratic mehod using new non polar mobile phase.

3. If switching to a normal phase system instead:

In this normal phase high pressure liquid chromatography (HPLC) , the elution trend would be as the non polar caffeine molecule will elute first followed by the polar theophylline molecule. Also need to develop HPLC method like gradient or isocratic mehod using new non polar mobile phase systems (ethyl acetate/n-hexane, acetonitrile/ethyl acetate, methanol/ethyl acetate)