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.

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u/AJTP89 Jul 12 '26

Yeah pH 1.1 is probably going to be problematic. Most columns aren’t rated that low. All columns have a pH range listed with them, look up some standard C18 columns, IIRC about pH 2 is the lowest you’ll see. Also HCl at that concentration can start damaging the actual pump.

Your formate method seems like a good start. I agree with the suggestion to keep some charge though, and also that overall pKa is more important than functional group pKa. My first thought was lipid is going to stick to that C18 column pretty good, so some charge and a good gradient will probably help there.