platform between peaks?

[guoyang]

Hi!
Recently I do a purity method for a compound(containing a cyclopentane, a -COOH, a -C=O and two -OH ). I met a strange peak shape. There is a platform between a impurity peak and main peak with different column and mobile phase.
I tried different column, like: C8, C18, Phenyl, PFP with different brand. And tried different pH of mobile phase, such as: CH3CN and Water, 0.05% TFA in CH3CN and 0.05% TFA in H2O, K2HPO4 buffer conditioned by H3PO4 (pH = 7.0, 5.0, 2.5).
What's wrong with it? Any suggestion?

[Bruce Hamilton]

Assuming that the sample solvent is very similar to the initial mobile phase ( a common potential cause of such platforms ), then another possible cause could be on-column degradation of your compound.

I would first look at the sample solvent, especially ensuring correct organic content and sufficient buffer capacity. If that's OK, I'd consider on-column degradation.

[Uwe Neue]

There are many different possible causes of such phenomena. After you can exclude the external events, as suggested by Bruce, a frequent cause of double peaks with intermediate steps is an equilibrium between the main peak and the "impurity" peak. If you have enough of the standard for the "impurity" peak, you can inject it and see if it converts back into the main peak.

[guoyang]

Thank you very much!
My sample concentration is about 5mg/ml (buffer capacity is enough)and I also tried sample solvent. So I guess the problem is on-column degradation.
So how coud I avoid this phenomena?

[Uwe Neue]

Why do you think that it is "on-column degradation" rather than the much more frequent phenomenon of the interconversion of the two species?

The most famous example of this event is the interconversion of alpha and beta anomers of sugars, but any molecule that contains a proline structure will do the same thing (enalapril, captopril...).

You claim that you have used all kinds of different columns, so I find "on-column degradation" rather unlikely.

[Bruce Hamilton]

Perhaps Guoyang is like me, and considered unwanted interconversion as a form of "on-column degradation". Incorrect usage, I know, but such is life .
Thanks for the correction.

If the impurity isn't available, perhaps they could individually collect the two peak fractions from a concentrated sample injection and re-inject each without further workup.

Further guidance may also be obtained by changing the mobile phase elution strength so the compound resides on the column for a different time, and see whether the peak ratios change.

[guoyang]

Thank you very much for your knowledge and suggestion!

[juddc]

I've seen this produce peaks with unusual shapes and while you can vary the pH to favor one tautomer over the other, it's tough to get completely away from. One way to tell is to collect the peaks in several fractions and reinject them. If they all come up with the same pattern regardless of the RT collected, then it's a reasonable bet.