Ascorbic Acid Ion Exchange Oddity

[Chris Pohl]

DawnRazor,

You could have an highly retained component in your sample (at least relative to ascorbate and phosphate at pH 6.5) tying up your ion exchange sites until eluted with 500mM buffer. What is in the sample besides ascorbate?

[Chris Pohl]

DawnRazor,

This may possibly be avoided at higher ionic strength. What is your sample matrix, just ascorbate and buffer? Surely there is more in the sample than this.

[Mark Tracy]

Ascorbate oxidizes rapidly at neutral or alkaline pH, and is catalyzed by transition metal ions. Are you sure your sample is stable?

[Jack C]

Our laboratory does a good deal of vitamin C analysis and we use a weak anion exhange column using an amine phase column. The column is conditioned with mobile phase of 75% acetonitrile and 25% 0.15M Sodium Acetate Buffer, pH=5.0. This chromatography is similar to carbohydrate analysis (mono- and di-saccharides). We use a calibration from 2.5 ug to 50 ug with good linearity.
People who do this analysis use dithiothreitol as a reducing agent (to keep asocorbic acid from oxidizing to dehyroascorbic acid and then to di-ketogluconic acid). Once oxidized to di-ketogluconic acid, it will not be able to be reduced back to asocorbic acid. So the analysis and results you are achieving might be from oxidation of your standard. In the food industry, meta-phosphoric acid is also used in the extraction solvent (up to 5%) and this does not seem to affect the linearity of standards. Our lab also adds a small amount of EDTA to complex any free metals (mostly iron) to prevent oxidation.
On the other side of this, some labs do a total oxidation of the material and a derivatization and then HPLC (C18, methanol:water combination).
Our lab actually uses a narrow bore (2 mm) column for solvent savings. I hate wasitng all that acetonitrile. Good Luck.

[HW Mueller]

Is there a reason, here, for not using mobile phases at lower pH where ascorbic is little ionized and stable against oxidation? (No ion pairing needed)

Incidentally, if one knows somebody with an electron spin resonance machine one may check if there is the characteristic radical present in ones samples. If yes the pH is too high.