Chromatography Forum

I am trying to measure reaction kinetics using a RP-HPLC to quantitate reactants and products. I want to make sure that the lost UV signal is not due to injection conditions, so an internal standard that will survive my reaction would be ideal. My current method is using a 0 to 90% water methanol gradient on a C-18 column and my interest molecules elute late in the run (30-80%MeOH).

This molecule needs:
1. Stability (the reaction is light driven with a 150W tungsten bulb at 20deg C. in the absence of oxogen) This molecule shouldn’t react.
2. Detectability. I have a PDA that runs a 3D time/intensity/wavelength from 220 to 600nm. My current molecules have a absorbance from 245 to 270nm and 400 to 500nm. So this internal std should absorb out side of those ranges (cannot harvest light at 450nm!) OR elute early (highly polar)

Any suggestions? Should I just start trying different molecules? The two suggestions that I have had are benzoic acid and Cytidine monophosphate, any thoughts?

Can´t you add the internal st. just after you stop the reaction?

I was hoping to reduce the error by preping all of the samples with the same buffer containing internal standard. That way there is no sample to sample differences because of pipetting/dilution. I am not sure it will solve my variance problem.

Without a detailed protocol it is not possible to comment further.