Spliting peak, but only one!

[saioa]

Hello!
I have an LC-(UV)-MS method for 4 acidic pharmaceuticals. The problem is that the first eluting peak (aspirin) is splited while the rest are ok. Then I guess I have no problem with chromatography part. I thought I could have an isomer but the purity of the substance before dilution is more than 99%, then I should exclude this since the peaks are quite similar in area. Has anyone any idea of what could it be? The standard solutions I am injecting are not more than 1 month old...could it be degradation?
Thanks for your help!
Saioa

[tom jupille]

What are you using as your diluant? If it's a stronger solvent than your initial mobile phase, you can see all sorts of funny peak shapes, which are more likely to affect the early (weakly retained) peaks.

[gbalock]

Saioa
If it is not a solvent strength issue as Tom pointed out, it could be degradation. Aspirin readily forms salicylic acid and this could be eluting just before or with your aspirin peak.

[Mark Tracy]

salicylic acid elutes significantly later than aspirin in most separations. If it is degradation, the ratio will change throughout the day. Since there is an MS involved, the nature of the two peaks of the split will be easily determined.

[saioa]

The mobile phase we have it is a gradient of acetonitrile 5mM ammonium formate pH 3.75. The solvent we are using when injecting aspirin is a buffer of ammonium carbonate 0.5M (i can not change that since it is required from my extraction method). However we have diluted (1:1) with water or mixtures of water acetonitrile, but it did not really help.
The 2 peaks elute quite close to each other and have the same ions masses (137 and 93 if I am not wrong). So if you think that it might be degradation, do you have any knowledge/recommendation of what I should do to avoid it? Should I prepare the standard solutions every month? Apart from sunlight should I take into account something else?
I will try to inject Aspirin different times/days and see if the ratio of the peaks change.
Thanks for your comments

[DR]

I have not looked, but I'd guess that the pK of aspirin is not too far off 3.75. If this is correct, you have a mixture of ionized and un-ionized being split by the column. To test, alter the pH of your MP and inject a sample containing just the aspirin. If you move the MP's pH at least 1 full unit from the pK, you should be down to a single peak.

[Uwe Neue]

You are dealing with a mismatch of the buffer pH (3.75) and concentration (5 mM) and the sample buffer pH (around 10) and concentration (500 mM). DR is probably right that it may affect the more acidic aspirin more than the other peaks. The solution is to neutralize your sample, probably best with a 1:1 dilution with a roughly 500 mM formic acid solution. You will get some bubbles, but the double peak should go away.

[HW Mueller]

DR, it´s not completely clear what you mean with "ionized, unionized". If you are talking about ionization in one spezies than you must have missed a recent discussion on this? The gist of that discussion: The ionization equilibrium of a carboxylic acid is way to fast for chromatography to separate the acid from its conjugate base (the anion).
Tom´s and Uwe´s explanations seem adequate.

[saioa]

Hello!
Well, actually I have tried different mobile phase pH, even at pH 3, I do not want to go lower because of the column stability. The two peaks did not disapear by decreasing the pH. I have also checked to add formic acid to my injection solution, but the truth is that aspirin had 2 peaks in any case and the sensitivity in the MS was worst when I added formic acid. This is why I did not think it could be a pH problem.
Maybe degradation is the problem?
Thanks for your answers

[DR]

DR, it´s not completely clear what you mean with "ionized, unionized". If you are talking about ionization in one spezies than you must have missed a recent discussion on this? The gist of that discussion: The ionization equilibrium of a carboxylic acid is way to fast for chromatography to separate the acid from its conjugate base (the anion).
Tom´s and Uwe´s explanations seem adequate.

That said, I think you & Uwe are probably a lot closer to"right" than I am... The speed of conversion should be too fast to split a peak (I hadn't thought of that, and probably did miss that other thread).

[tom jupille]

I would second Uwe's suggestion. A good rule of thumb is "to do the best chromatography, upset the equilibrium of the system as little as possible".