Chromatography Forum

Dear Chemists,

I'm experiencing a problem involving Lactic, Malic, fumaric, and Succinic acid using LC/MS ESI with a Phenomenex Hydro RP C18 column.
the retention times for lactic, malic do not change and are rather stable. Succinic acid changes a little (seen as quick as 7.1 min and as long as 9.3 min) and fumaric acid changes drastically from injection to injection (rt 6.7 to 12.5min); sometimes eluting before succinic acid, sometimes after.

mobile phase is 100% 0.1% aq formic acid

samples are in culture broth with the following matrix recipe (1L broth):
2 g of asparagine,
1 g of KH2PO4,
2.5 g of Na2HPO4,
10 mg of MgSO4 · 7H2O,
50 mg of ferric ammonium citrate,
0.5 mg of CaCl2,
0.1 mg of ZnSO4,
0.1 mg of CuSO4, and
0.05% Tween 80
0.5 g of Casitone (Difco) The pH of the medium was 6.8. Carbon source is10mM Glucose.

the rt problem did not exist at first when just using standards and water, but has persisted since first exposure to the above broth. I have tried EDTA sol'n and column wash with 100% acetonitrile and problem continues (even if injecting standards with water).

My questions are this:

#1 what would be a good clean up method to use on the sample prior to injection? ie keep the organic acids, lose the inorganic salts, sugar, and that tween 80 polymer/lipid etc.

#2 does anyone know if the retention time variability is a column contamination issue, or ion-complexing in broth issue? Fumaric acid has a double bond in it. Other acids do not.

#3 are there any ESI compatible methods for organic acid analysis with an Aminex HPX-87H column?

any suggestions or assistance shall be greatly appreciated.

thank you.

I'd start by making up a mixture of your analytes in mobile phase (0.1% formic acid?) -forget the broth for the while-and seeing if the problem goes away.

Do you know that an 87H with sulfuric acid and UV detection will not work for some reason? People underestimate the sensitivity of UV for these acids unless you actually try it. We can easily detect very low ppm quantities of acetic acid for example, at 205 nm and the acids you list will be way more sensitive.

PS I don't know the answer to your question. I have never seen that problem before.

I think your problem is poor pH capacity of your mobile phase in acidic direction. While 0.1% formic is good for basic analytes or weak acids strong acid such as fumaric pKa 3.1 (formic pKa 3.7) can be just within own dissociation equilibrium point. Small changes in temperature, contaminations on a column or sample can significantly change retention.
I would change mobile phase to ammonium acetate or ammonium formate with 5-20 mM of ammonium. I don’t know though if this MP is a problem for ESI.
Ammonia will also increase ion-strength of the MP and suppress ion-exchange if you have any.

Hi everyone,

thanks for the feedback.

Bill,
yes, 13.5mN Sulfuric acid with the Aminex column is working rather well with uv 214nm especially with fumaric acid. The double bond in that acid lights up like a christmas tree in that particular uv range and provides sub ppm LOD. I'm making progress on the project with this suggestion!

When there is a peak in question, I collect it and run on the MSD (and hope it isn't too dilute).

Although it has been good for detecting and quantifying the acids, sadly, it will not suffice for part II of the project which involves carbon 13 doping the bacteria. We would like to learn about the metabolism of this organism and therefore a method using LC/MS is necessary to distinguish the acids from there 13C counter parts.


Victor,
I gave it a shot. Several trial samples later, still a problem.

Yury,

I'll try this next. yes ammonium formate and acetate are compatible with ESI and your explanation sounds rather plausible. I'm first going to try those buffers with the aminex column (which seems to be the main column of choice in the literature) and if that falls through, either try it with the C18 or HILIC column.

thank you all.