Chromatography Forum

Thanks for looking any input would be appriciated

As stated Im having issues with repeatability of standards for a Vitamin C assay. My sequence consists of three injections of standard followed by three injections of sample and finished with three more standard injections. Im getting %RSD of 7-15% for the standards with the area counts increasing over time. I got individual vials for each injection (eliminated evaporation as cause). Filtered with individual syringe filters for each vial (eliminates filter build up or blow-through). I read on here about Ascorbic acid reacting with the glass, how plausible is this theory?
Any other ideas would be appricated.

Assay parameters
mobile: 10:90 ACN: H2SO4 4mM
column: bio-rad aminex HPX-87H
runtime: 30 min
diluent 0.025% m-phosphoric acid
wavelength:210nm
inj volume: 30 ul

There have been some weird stories about ascorbic acid, but reacting with glass? How would that increase the peaks?
I don´t have a catalog of columns in my head, is this an ion exchange method? If so, why would one use low pH mobile phase?

Yes it is an Ion exchange but I am new to this form of chromatography but from what I can gather it is very much pH dependant. so could a change in the pH of the mobile be responsible for increased adsorbtion? Would the pH change that much through the course of a batch run ~5hrs?

I have done quite a bit work with ascorbic, either derivatized (nitrophenyl osazones, didn´t like it as it was messy, produced equilibrating isomers) and C-18 RP at low pH. At higher pH one gets ascorbate anions which have a strong tendency to be oxidized via catalyzed reaction with atmospheric oxygen. I suspect that you don´t have a robust system and are initially loosing ascorbic acid (too few ascorbate ions). If you go to higher pH you will have more ascorbate ions, but may get oxydation. This is theoretical, maybe someone with experience has found a robust way?