Chromatography Forum

We are separating an acid compound from other metabolites with an ion-pair (Pic A 5mM:MeOH 70:30) method (Luna Column). The problem is that this compound, the later eluted, most of the times splits in a peak with two peak tops, or looks like a wide peak.

This problem only appears for this compound (and not for the others) and is not constant over the time (some samples splits and not others in the same sample set), and if the same sample is reinjected, sometimes splits and not others.

Please, any idea where the problem comes from?

Can you share a chromatogram on this message board perhaps?

It could be either 1) a strong diluent effect - therefore try to get more retention, or 2) secondary equilibria effect - therefore try to separate the component as one particular form.

One way to achieve number 2 of Rob Burgess' suggestions is to make the mobile phase more concentrated. I had to scale up by a factor of 10 the recommended buffer concentration for one reagent I used. I've ditched that system in favor of a more appropriate mobile phase for the compound, but I think it's a good place to start.

Find out where you'd have issues with solubility, and back off a bit from there.

thank you for your suggestions
Finally we solve the problem by working with a PIC A more concentrated as mobile phase.