[clemi2310]

Hello everyone,

I am currentlky planning to do stability studies of Diketo acids coumpounds by HPLC and I am optimizing gradients and buffer to have proper peak shape and response.

My compounds are a bit tricky because they have an -COOH group, an enol group, and a protonable piperidine (see simulated Pkas below).

In a classical buffer (A= H2O + 0,1% Formic Acid, B= MeCN + 0,1% Formic Acid), the peak is very broad and comes out over 6 min

I tried then to do ion pairing buffer with Triethylammonium Acetate pH 7,5 (A= TEAA 0,05M H2O, B= TEAA 0,05M MeCN/H2O 80/20), my peak gets thiner but is tailing on the right ...

The column is a Nova-pak C18 4µm 3.9x150mm from waters and works perfectly for other compounds.

pH might be the issue for this topic I'm afraid...

Would you have any advice to get proper peaks ?

Thank you !!

Clémence

[Arkcon]

That you have 3 pKa's isn't too much of a problem, since two are close to each other.  The wide range for the other one is something of a problem.

You've gone to two extremes:  Very low pH, to somewhat higher for the ion-pair buffer.  You may have to experiment with some other points to build a conclusion as to where you need to go.

[Arkcon]

Here's an online resource to some tips you should always start with:

http://www.chromatographyonline.com/hplc-mobile-phases-10-bad-habits-avoid-0