Chromatography Forum

Hi! I am trying to analyze ascorbic acid with HPLC. I am using a HILIC amide column 2.1 i.d. My mobile phase is acetonitrile/buffer 60/40 (10mm ammonium formate/0.15% formic acid). 0.25 ml/min. The peak elutes after 4.2 minutes. When I am analyzing soft drinks the peak areas are wery similar but when I am preparing standards the areas change very much when I am injecting from the same vial. I have tried to use some stabilizing agents such as oxalic acid an phosphoric acid. I have also changed the mobile phase composition so it contains more acetonitrile. Does anyone have an idea about what is happening? I have also tried C18 column but I could not retain AA on that one.

As I recall, when I was analysing Ascorbic Acid you have to use meta-phosphoric acid (not ortho!) in your extraction buffer and mobile phase in order to prevent the formation of the dehydro form. The final pH should be acidic.

I also used Methanol instead on Acetonitrile.