Chromatography Forum

I developed a method which can be used as an assay if I can stop the decomposition of my compound on the column.

I am using 70% [14mM octane sulphate, 20mM phosphate at pH 4] and 30% methanol on a c18 column

I know decomposition is taking place because before my peak of interest elutes there is what I will refer to as a foot. What appears to be a small peak at first, then plateaus until it merges with my peak of interest and then after the peak of interest elutes the baseline returns to its original height before the foot appears. Here is the general idea of what it looks like (I'm don't have access to my chromatogram at the moment).



I also collected the fractions of this peak in an HPLC vial and tested it again. The idea was that if I saw something like the following picture, then it means I definitely have decomposition on the column.



What I observed was only the first peak and the peak of interest had disappeared.

So finally, my question is... Can I use an antioxidant like sodium metabisulfite in my mobile phase to prevent oxidation of my peak of interest? I am fairly certain what I am seeing is oxidation.

Thanks

What makes you believe that the chemical modification takes place on (in) the column?
And why exactly oxidation?

Best Regards

I know decomposition is taking place because before my peak of interest elutes there is what I will refer to as a foot. What appears to be a small peak at first, then plateaus until it merges with my peak of interest and then after the peak of interest elutes the baseline returns to its original height before the foot appears.

Your description, to me, seems like you're describing your observations over time in regard to the foot-plateau-peak development - e.g., over several injections. I would then ask, how do you 'know' you're not getting degradation in the injection vial, and that you're seeing the degradation product increase over time as you inject your sample?

In response to Danko,

Well I may be wrong, but I believe it is ON the column because the 'foot', as I am referring to it, plateaus and merges with the peak of interest as I have explained before.

If it were to form a peak and then tail off into the baseline I may feel differently. However, I see a hump merge with my peak of interest.

In TLC if you see a spot is streaking (analogous to tailing or fronting in HPLC) it may be an alcohol or carboxylic acid, which is due to hydrogen bonding, or a molecule which is decomposing into a more or less polar molecule (depending on which direction the streak ventures off to).

I injected my sample several times and saw the same chromatogram. So, what I observe, I believe, is not from decomposing in the HPLC vial.

Only when I collect the fractions from the detector-waste line and re-inject it do I see that the peak of interest has a much smaller response and there is a new peak near where the 'foot' begins.

Also, one of the known degs is an oxidized form of my peak of interest. there is an alcohol which becomes a carbonyl. The 'foot' begins where this known deg elutes at.

I probably could have explained that before. I just wonder if antioxidants have been used in mobile phases before to prevent degradation.

You are using an ion-pair system, adding further molecules is likely to affect your chromatography in unpredictable ways, meaning more method development.

In my opinion, you should fix the method the simplest way possible, and adding molecules to inhibit specific reactions isn't simple, especially in an ion-pair situation.

I'd develop another chromatography system where your analyte is stable.

Please keep having fun,

Bruce Hamilton

Assuming the above are highly stylized chromatograms , my guess would be that this is not decomposition on the column. But I didn´t do the development of the method so I didn´t see how flow rate effets this, how pH effects this (why a pH 4 with phosphate??), etc.
Also we don´t know what fractions you took, and why you only tell of the outcome of one fraction, and why you say one time that there was only the rt of the "foot", another time a peak at the "foot" and a little "peak of inerest".
We also don´t know under what conditions the "carbonyl" forms from the alcohol.
Also, is this another one of those methods which uses ion pairing agents without analyte ions?

Decomposition on column is a reasonable explanation. It could also be an equilibrium between isomers.

While there have been some rare cases where the column is catalyzing such decomposition, it is more likely the mobile phase. Dissolve your sample in mobile phase and see what you get. If you get now only the first peak, or only the first peak after a short period of time, you have a mobile-phase triggered degradation. If you continue to observe the strange peak shape, it could either be a specific on-column degradation, or - more likely - the separation of interconverting stereoisomers. There have been previous threads on such things.

This is an ion that I am working with. I'm not sure if pH is 4, now that you mention it, I haven't been in the lab and don't have access to my exact conditions. I'm sure that my molecule is ionized, however.

I definitely will keep it simple from this point on and definitely see if it is my mobile phase that is decomposing my molecule. The diluent I have been using is just water.

Thanks a lot for your help guys.

Since your compound is dissociable and your mobile phase is essentially not pH buffered it could be a pH incompatibility. In what is your sample, which you inject, dissolved?
We still don´t know what the fractions are and why you only tell us what one of them looked like.

Hans is right, and I changed my mind. Phosphate is not a buffer at pH 4, and to inject something dissolved in something else is prone to give all kinds of issues. Lousy buffer with undefined sample is the most likely problem.

Sorry for the confusion. The pH buffer is not 4, but 7.

pH 7 phosphate(20mM) and 14mM octane sulfate.

Now I am in lab and have access to the chromatogram shown below.

What is you sample diluent?

water

I'd dissolve your sample in 70:30 20 mM Phosphate pH=7 : MeOH, and see what that looks like, and also try dissolving the sample in the mobile phase.
If peak shape significantly improves, you could double the phosphate buffer concentration in the sample solution.

I'm assuming your sample is soluble, but it might be worth quickly investigating the effect of reduced sample concentration.

If the peak still looks bad after those runs, I'd re-evaluate the suitability of the method and look at alternatives.

Bruce Hamilton