Chromatography Forum

Hello,

I'm analyzing coffee and tea samples by reverse phase HPLC (C18 symmetry column). I'm using a gradient method with constant 1% formic acid in water at a constant 10% throughout the gradient run. The mobile phase is MeOH and H2O. 20 to 60% MeOH over 10 mins.

According to literature the components I'm looking for have very variable pka values.

What kind of effect does changing the buffer have on my results. I'm using a diode array detector and a MS detector so I need a volatile buffer.

Compared to literature results, I don't have many resolved peaks. This is perhaps partly due to the length of the run. Perhaps a slower gradient will help, but I'm also not sure what kind of effect the FA buffer has.

Any suggestions would be greatly appreciated!

Thank you.

First, the formic acid provides a suitable environment for the ionization of the analytes in the MS. Second, for LC, the control of pH results in reproducible retention times. Ionizable analytes can change their retention by a factor of 30 going from a completely ionized to a completely non-ionized state. So this is why pH control is important.

To run the formic acid concentration at a constant 0.1% (as you do) is standard practice and works well.

Below is separation of catechins in green tea on Unison UK-C8:
http://www.imtaktusa.com/site_media/fil ... TI145E.pdf

Imtakt used 1% acetic acid as a pH modifier for this separation.