3 peaks of bromazepam?

[C.Romero]

Good afternoon,

I have a problem with Bromazepam in my HPLC. When I make the chromatograph for it I have 3 peaks. The first two peaks are together and with one line in a high level, I mean, it seems an equilibria reaction. I'm using a C18 column 12x0.46,3µm. The solvent is 50%MeOH+50%ACN and the aquous phase is formed by NaH2PO4 + H3PO4 (2.6pH).
Could you explain me the reason, please?

Thank you!

C.Romero

[MestizoJoe]

If I understand you correctly, you are using methanol and acetonitrile mixture as your sample solvent and you are using aqueous phosphate buffer as your mobile phase. Is this correct?

If so, your sample solvent is not compatible with your mobile phase. Your sample solvent should be a weaker eluter or the same as your mobile phase. When this is not the case, a pure component may look like more than one peak.

Also, at this low pH, 2.6, your api is ionized. You may want to consider a slightly higher pH and maybe also have some organic in your mobile phase. You may want to try a gradient of 2% ACN to 10% ACN and see how your API elutes. If it elutes quickly you may consider a more polar column or an ion pairing reagent.

If you chose an ion pairing reagent like octane sulfonic acid, you can work at pH 2.6 since your retention mechanism is no longer just hydrophobic interaction but also ion exchange.

Hope you find this useful.

[C.Romero]

Thank you, I've tried for a high pH and the problem persisted but with better results. I've found an article talking about a possible equilibrium and decomposition for a low pH, that's may be my case. Thank you anyway.

C.Romero