Chromatography Forum

Hi, everyone!
Recently I am developing a isocratic elution mehod for a diastereomer. There are two peaks could be separated on ZORBAX SB-CN column, but peak shape isn't very well. The strange thing is peak area percent changed with the ratio of mobile phase.
One is 0.2% H3PO4 in H2O : ACN = 70:30. Resolution between two peaks is about 1.6 and tailing factor is about 1.8. Peak area percent of two peaks is about 70%:30%.
The other is 0.2% H3PO4 in H2O : ACN = 72:28.Resolution between two peaks is about 1.8. Peak area percent of two peaks is about 80%:20%.
Could you tell me what is the reason?

Unless there is some effect of pH on signal strength my guess would be that it is an effect of different automatic integration of the two peaks - you will get very different results if you drop a perpendicular to the baseline between the peaks than if you run the baseline valley to valley for instance.

Seeing a chromatogramwould help.

Peter

i update the chaomatogram:
mobile phase: 0.2% H3PO4 in H2O : ACN = 70:30

mobile phase: 0.2% H3PO4 in H2O : ACN = 72:28

We don't have the structures in front of us, of course, but in some molecules it wouldn't be so farfetched to imagine that UV response has this kind of finicky dependence on pH / solvent composition which is different for two diastereomers.

Do you have enough of these compounds to take a full UV/Vis spectrum of each, under both solvent conditions? Or can your LC's detector do that during a chromatography run? If you are not measuring close to the peak absorbance, then you are in a region where a slight change in wavelength (and conversely a slight change in the absorption spectrum at constant wavelength) can have a significant effect on results.

How repeatable is this?

Thank you very much for your helpful suggestion!