Folic Acid by HPLC

[spowers2]

I am seeing intermittent peak splitting of folic acid. I am running with a gradient using 0.025% TFA as my aqueous phase and acetonitrile as my organic phase. Has anyone else encountered this issue with Folic acid and have any remedies? I will also mention that I am diluting the folic acid in water with conc NH4OH added for solubility.

[Andy Alpert]

Folic acid has basic groups in the molecule. These will have an anion as the counterion if they're ionized, and presumably that's true of the folic acid that you're using as a sample or standard. 0.025% of anything is not high enough to reliably convert 100% of the molecules of an ionizable analyte to a form in which they all share the same counterion. Different ion pairs differ in polarity and migrate at different rates in both RPC and HILIC. Try increasing the % TFA in your sample solvent at least. Most of it will probably be titrated by the NH4OH, so just increase the concentration until you get a singlet peak.