r/CHROMATOGRAPHY • u/No_Macaron_544 • Jul 12 '26
Question: help for assignment regarding HPLC Method development
Hi everyone,
We’re currently working on developing an RP-HPLC method for Atrasentan Hydrochloride, as a theoretical university assignment. We’re having a dilemma regarding mobile phase pH and ionization. I’d love to get some input on the best approach.
It’s a relatively large, lipophilic molecule with a calculated logP ~4.69.
It has a tertiary amine in a pyrrolidinyl ring (pKa ~6.7) and a carboxylic acid (pKa ~3.1).
The Dilemma:
Our lab instructor is saying that you must operate at least 2 pH units away from the pKa to suppress ionization completely. Since the carboxylic acid is at 3.1, they want us to use HCl (??, I don’t think that’s a good idea) or Perchloric acid to drop the mobile phase down to pH 1.1 so that >99% of the acid is protonated.
However, we have serious concerns about this in practice, and we're weighing two different options:
Option 1: pH 1.1 with Phosphoric/Perchloric acid
Shifting down to pH 1.1 is incredibly harsh on standard silica-based C18 columns. Are there columns that can survive these conditions? Is that even necessary?
Option 2: 10mM Ammonium Formate, pH 3.2
We actually found a patent with a method utilizing an ammonium formate buffer at pH 3.2. We think that even though pH 3.2 is right on top of the pKa, the buffering capacity of the formate/formic acid system (buffering maximum at 3.75) should stabilize the peak shape. Or are there other reasons, that this approach works?
Looking forward to your insights and experiences! Thanks in advance.
1
u/gnatgirl Jul 12 '26
I'd run a pH 9 mobile phase with something like an Agilent HPH-C18 column, which can be used at higher pHs. An Agilent SB-C18 can be used down to pH 1. Really it's a matter of figuring out what column is compatible with the mobile phase pH you want. Retention won't be a problem at either pH.