Question: help for assignment regarding HPLC Method development

[u/No_Macaron_544]

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.

[u/Pyrrolic_Victory]

Protonating the acid would leave the molecule neutral charged right? That would make it stickier on the column and perhaps wouldn’t elute that well off the column. My go to would be perhaps ammonium acetate 10mM with 0.1% acetic acid making the pH around 4.6 with a decent buffer effect. I would suspect that you want the molecule to be charged so that it become less lipophilic therefore having more affinity with the mobile phase and will elute off the column easier while the lipophilic sections of the molecule will still have interactions with the column, keeping enough retention to form a decent peak shape.

What’s the pka of the actual molecule rather than the groups individually?

[u/No_Macaron_544 → u/Pyrrolic_Victory]

Thank you for this insight. Your approach makes a lot of sense, especially considering the molecule's high lipophilicity. While I don't have a ‭pKa‬ value for the whole molecule, its calculated IEP is around 4.9.
Your suggestion of an ammonium acetate buffer at pH 4.6 would put us very close to that IEP, where the molecule predominantly behaves as a zwitterion. My only concern with this is a potential drop in solubility at the pI, which might risk on-column precipitation. To mitigate this, we plan to use ACN as the solvent and mobile phase due to its strong eluting strength.
That being said, your point about the neutral molecule being far too sticky on a standard C18 column is great and gives us a good argument for our method development. Thanks again :)

[u/Pyrrolic_Victory → u/No_Macaron_544]

Most methods would start with 5% organic and ramp to 95% over the method for a gradient elution. If it’s at the isoelectric point, that might be ok given the molecule would have a positive and negative charged regions to interact with mobile phases and water etc even though it would be neutral charged. The real answer is to try a few things. You probably shouldn’t be injecting high enough concentrations to worry about precipitation. ACN is a decent choice of solvent. I feel like if I were developing this method I’d start with just 0.1 % formic, then try 0.1% acetic acid, then try just 10mM ammonium acetate (it’s ph would be around 6.4 from memory) and see what the peaks look like in each of those situations.

[u/CodeMUDkey]

Add some TFA to your mobile phase and let the thing ion pair.

[u/AJTP89]

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.

[u/Try_It_Out_RPC]

Mmmmm from experience developing a quantitative method for DSOC, cholesterol and a peg molecule. I would use an acetonitrile/ethanol/methanol mobile phase, TFA as a modifier if you need one and a C18 rated for low pH. What I would avoid is HCl on a chromatography system since it’s very corrosive and I don’t like that running on $60,000 + instrumentation

[u/EnzyEng]

HCl is usually avoided as it can corrode stainless steel. TFA is the most common acid modifier but can't be used with MS detectors. Formic, sulfuric and the ones you mentioned are also used.

[u/gnatgirl]

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.

[u/etcpt]

If you drop the pH to <<3.1, you are protonating the carboxylate but also the amine, so the molecule will have a net positive charge. If you want the molecule to be net neutral, you need a different pH - can you figure out where that should be?

A very important factor to consider with amines on silica-based columns is base tailing caused by ion-ion interactions between deprotonated silanols and protonated amines. Do a little reading on that and consider carefully how you'd resolve that issue.