Chromatography Forum

Dear all,

Your help on the following will be highly appreciated.

We are trying to analyze an oral solution of ascorbic acid in terms of related substances. The method is as follows:
Mobile phase : 2.2g of sodium hydrogen phosphate +2.0 ml of tetrabutylammonium hydroxide are dissolved to 800 ml of water. pH is adjusted to 6.5 with o-phosphoric acid and then diluted to 1000ml with water. 970 ml of the above buffer was mixed with 30ml ACN. Column used BDS Hypersil C18 5μm 250x4.6 mm. Column temperature: 45 oC. Detection 265 and 220 nm.
The problem we are experiencing is that we cannot get stable peak area of the standard solution (50 mg of Ascorbic Acid into 100ml of mobile phase). Analysis time is 10min.

Thank you

How unstable (%RSD, number of injections)?
Is there a trend?
Is the retention time varying? Peak width varying? Peak height varying?

Hi Kliaros, I am quite interested in knowing the impurities in ascorbic acid. Is it ok for you to disclose what they are? Thanks

I have not analyzed ascorbic acid but I have its cousin sodium iso ascorbate. My recollection is that it is not stable chemical. I could literally see the peak area decrease with its succesive injection. I used citric acid as my mobile phase and if I use the moblie phase as the sample solvent the deterioration rate is decreased. I also tried ribose as antioxidant and it helps somewhat.

Jimmy

At pH 6.5, I might expect to see a more rapid degradation of ascorbate than at lower a pH. I would suggest lowering the pH of your sample preparation solvent to about 2.5 with phosphoric acid, though be careful with your injection volume because you don't want to disrupt the pH on column. Depending on the size of your column and system, this may or may not be a significant worry. If your injection volume is small in comparison already, then don't sweat it. If you're injecting 50-ul on a 2.1mm diameter 5 cm length column in a low disersion system, then worry.

It has been documented before that ascorbic acid is degraded in autosampler vials and maybe that is your problem. Have a look at the paper below which has some suggestions on how to minimize/eliminate the problem...

Stability of Ascorbic Acid in Solutions Stored in Autosampler Vials Clinical Chemistry 47: 1463-1464, 2001;

This forum is filled with info on the stability of Ascorbic acid.
At a pH near 7 one immediatly gets radicals (one can see them in an ESR instrument) and also dehydroascorbic acid. The latter converts quite well to gulonic acid, etc., etc., etc.
So Liu, you can spend a long lifetime on figuring out the possible impurities in ascorbic.

It seems that I have seen this ref. given by Kostas before. If I am not halucinating it was not clear whether the authors could have been tricked by some strange surface phenomena which might be imaginable if traces of ascorbic acid or some oxidating catalysts/agents are present. It is not easy to oxidize ascorbic in fairly strong acid. Also, one should not forget that the oxygen oxydation to the radical is an electronically forbidden reaction, one needs metal ions . . .

This method can be used for dehydroascorbic acid and related substances:
http://www.imtakt.com/TecInfo/TI341E.pdf

This new method requires Unison UK-Amino.

Bryan, does my memory fail me in thinking that there still is the unanswered question about what this peak #1 actually is?

Hans -

Yes, I'm pretty sure I now know what the first peak is.

But I'm sorry, I prefer to have that discussion in private with customers
(not on the forum). I understand using ODS with ion pairing, but I believe we need to
switch people to Unison UK-Amino for this assay. It's important.

Anyone outside North America who has questions concerning
this method should contact Imtakt:

info@imtakt.com

I'm going to have to try this next week just for giggles. I don't use ion pairing, but have used 100% Aq RP on this one for years and haven't ever been particularly fond of it. It has worked well enough for what we're playing with, but I don't like 100% Aq RP in general...Also, I might call wrt to peak #1 - I'm curious.

My guess is that first peak is sodium, it elutes before void on amino column, void is about 1.5 minutes on column in this dimension. Any amino column will work in similar way (or mixed-mode anion-exchange)..and it is understandable that it is important (for Silverstone) to switch to this method

Thanks for the thoughts on peak 1, Vlad...

While I thought the "important" comment was a bit amusing, I do have one of these columns in my stockpile and it seems to be a pretty good one. That and it won't take much time at all to check the application, so why not?

Vlad, I see your point. I'll try to keep it more objective on the forum.

I am unable to edit my comment - so perhaps this is more appropriate:
"Phases other than ODS should be evaluted for this assay"

Thank you for your remarks.
Unfortunately the method is the registered one and you cannot easily make such drastic changes.
In terms of the questions raised, the peak area is varying day to day or even within the same day.
My injection volume is 5μl.
To avoid breakdown of the ascorbic acid the solution is injected immediately after preparation. Neverthless, the problems still exist.
We have trying using new column, with no results.