Chromatography Forum

Hey guys. I´m having a problem with ascorbic acid. I´m trying to do linearity with an HILIC method I´m developing. Kinetex HILIC 2.6 µm, 150 x 4.6 mm, MP Acetonitrile:Ammonium acetate buffer 50 mM pH 7.0 (90:10), injection volume 15 µL. Samples are in MP.
When I´m trying to do LOD-LOQ, 5% solution of sample concentration is not a high as it should, and sometimes even peak does not appear, it varies a lot. A 50 % solution does not show a great variation like 5% solution, but the same problema, is not as high as expected.
HPLC is working well. It is not a problem with injector, precision is good. Detector Works well, lamp is under 2000 hours and when you check it passes tests. Pump does not show any problem, no leaks or pressure fluctuation.
When I changed to another HPLC, happens the same.
I´m not familiar developing HILIC methods, Little experience with them, so I do not realise which problem I´m having. But I developed one for L-Cystine and had no problems.
Any ideas? Is it a method problem or vitamin C problem?

Is it a method problem or vitamin C problem?

My guess is that it's an ascorbic acid problem. Ascorbic acid is *very* easy to oxidize. You might be able to verify that as the problem by doing a time study of one of your mid-level standards. Using degassed diluent might help.

I didn't have a problem in the past if you do not allow Ascorbic Acid to oxidize. As a mobile phase use 10% Meta-Phosphoric Acid (not ortho!) in your mobile phase 90% Water/10% Methanol (pH 2 or 3?).

Thnak you for your reply
Is that because it is an equilibrium? Because área is varying, is not decreasing constantly.
And why meta-phosphoric?

Yes! You want one form or the other in order to obtain 1 peak. Thus, I decided to have the Ascorbic Acid form solely rather than the dehydro form.

But why meta-phosphoric? I´m asking because I´ve never used it.

I have no idea. I just found an article in the 1980's.