HPLC Resolution of Analytes

[klhayes]

Hello,

I am trying to separate two analytes, 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) from its hydroxylamine metabolite. I have a method that call for Solvent A 0.1% TEA in water and Solvent B is 100% methanol. It starts out at 30%B and goes to 55% B. The system stay at 55% B for 12 minutes. The two analytes elute during the hold (15.9 for the hydroxylamine and 16.5 min for PhIP). I would like to separate them more. I know if they eluted during the gradient (30 to 55% B over 7.5 min), I could alter the gradient and the time over which the gradient changes. But they elute during the final hold at 55% B, and I cannot figure out what I can do to separate them. Any suggestions would be welcomed and much appreciated. Thank you for taking the time to read this.

[Noser222]

Maybe altering pH would probably be a good place to start.

[mohan_2008]

I do not know and did not do this before. But, I wouldn't recommend doing a gradient with TEA.

As it is strongly retained by the column and may require very long re-equilibration times. Specially if you have a non-encapped column.

Plus, it alters the pH of your column during the gradient steps.

[danko]

Hi Klhayes,

Just start with a lower percentage B – f. ex. 20 - 25% instead of 30.
Potentially, you can try a holdup for minute or so at the beginning of the separation.

Best Regards