I would like to ask a question to the forum. We have problems with a
compound that we have to analyze by HPLC. This compound, a large molecule,
is (in part) a diketone. It is possible that we have a ketoenolic
equilibrium, and we can not obtain a single peak (apart the possible
impurities), we obtain two peaks and an ugly moor in the middle. We have
used reverse phase, and now we are trying the analysis in normal phase, but
the peaks are not very good.
Please, any suggestion about this type of equilibrium?.

Thanks in advance.

Yolanda Sainz Sánchez Ph.D.
PharmaMar
Quality Control
Control Process Group
ysainz@pharmamar.com

It appears that you are looking at a slow interconversion between two sterically different forms of your analyte. There are a few examples of this in my book on page 362 - 364.
There are a few ways in which these equilibria can be manipulated so that you get a single peak, instead of two with a step in between. First of all, you can increase the temperature. This speeds up the interconversion, and if it is fast enough the two peaks merge into one. Another possibility for many compounds is a change in pH. It may very well be that this is a possibility for your compounds as well. If a change in pH affects the way the peak looks, you can use pH to drive it into a single peak.
Alternatively, you ignore this and teach your software how to quantitate the strange beast. I have never tried this, but it should not be impossible.

If the ketone groups are close together, diketones can form complexes with metal ions. Could that be your problem?

These (probable) equilibrium problems are fascinating, too bad nobody follows up on solutions to these. Usually, enol-keto equilibria are quite fast and often all on one side, what is fascinating is whether the chromatographic equilibrium interferes. Do you have the means to check on the possibility of this equilibrium outside of the column (spectroscopically, etc.)?
Anyway, what happens if you collect different portions of the peaks and reinject them (maybe letting them stand different times, heating them....)? Such equilibria are catalyzed by acids, Uwe´s sugggestion on pH is, therefore, a "must".

Thanks a lot for the suggestions. We had tried to change different things: temperature, pH, sample preparation, normal phase (a disaster), reverse phase... Now, we are trying to get only a form (acid and basic phase: basic is better, at the moment), but it is difficult. We had collected the portions (and the moor) and the reinjection gave two peaks again. We will continue with the trials changing the pH (and the temperature).

Yolanda Sainz Ph.D.
PharmaMar

If the interconversion happens at the time scale of the chromatography, a reinjection of the collected peaks will give you two peaks again. This is actually a nice proof that your suggestion of an interconversion mechanism is correct.
My first suggestions had focussed on getting a sinlge peak. But you can - if this is acceptable to you - also try the opposite and get two clean peaks. You would try to get to this point by reducing the temperature. Or, if pH has an effect (and it is highly likely that it does), you can change the pH to get two clean peaks instead of a single somewhat broad one.
Keep us posted on the results!

Keto-enol isomerism will result in the "batman" peak you describe. We've found that high ph (like 10) on a stable column (Xterra MS, Zorbax Extend, Luna C18(2)) gave us one peak for some of these compounds. Use organic buffers like 10 mM ammonium acetate or formate (instead of borate or phosphate to avoid cleaving your C18 chains). In this case the ammonia (pKa about 9.2) is actually the buffering agent. Other organic bases (cyclic amines, DEA, TEA) work well too, but will not work for gradient (the gradient will slough off these addititves and give you a horrendous baseline, particularly at low wavelengths. As stated above, this pH equilibrium is compound dependent, but we found good luck at high pH - so don't rule that out. Try low, medium, and high pH screens and see if you can affect this equilibrium.

We have got to separate very well the equilibrium (without a horrible moor in the middle) through changes in pH (better at acid pH)and temperature (better at 0ºC). In this way, we can add both peaks (Enol and Ketone) and know the chromatographic purity. We have used a Symmetry Shield column. It has been impossible to get a single peak with a good resolution.
Thanks a lot for our suggestions.
Best regards,

Yolanda Sainz Ph.D.