Chromatography Forum

We've recently tried several chromatography methods for ascorbic acid assay:
- SAX, 0.1M AcONH4 / AcN 1/1: ideal peak and specificity
- NH2, 0.1M AcONH4 / AcN: tailing peak, no specificity
- C18, TBA HSO4 pH 5 / MeOH 95/5: tailing peak, poor specificity
Finally SAX wins
However method validation shows
+ quantification limit ~ 1 % from nominal concentration
+ recovery 100 ± 7 %
+ linearity with r2 > 0.999
- residual member ~ (minus 10 %) from nominal area
- unstable (day-to-day) slope coefficient (probably because of pH and UV spectra instability, however adsorption maximum seems to be constant)
Matrix:
- Adcorbic acid - 0.5 mg/ml
- Vinpocetine - 5 mg/ml
- Sorbitol
- Benzyl alcohol
- Metabisulfite sodium
- Tartric acid - up to pH from 3 to 4
Test solution: test sample as is.
Reference solution: ascorbic acid, sorbitol, tartric acid, water.
Any ideas are welcomed!

Any tips please?

What is your question?

One thing I can tell you is that vitamin C is a highly unstable molecule in solution. For a good analysis, you want to prepare your calibration standards daily from scratch (solid vitamin C).

It is sensitive to light, oxygen and metals, especially at higher pH. Most analytical procedures extract in an acidic environment, together with the use of reducing agents and chelating agents.

I would advice to not reinvent the wheel and use an application note or article as a fundament of your analysis.

I used ~1% meta-phosphoric acid (not ortho) in our RP mobile phase in order to prevent formation of de-hydro-AA.

Rndirk,
The cause of high negative residual member is the question.
Tartaric acid and sorbitol make solution stable for at least 24 hours at room temperature.
Please note any applications you know. For example European pharmacopoeia method (NH2) doesn't suit.

HPLC chemist,
Could you please share the method? I failed to develop RP one.