Diluent pH for Peptide analysis

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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I am developing a method for the quantification of a peptide in a drug product. It is an ion-pairing chromatography method on a 150 mm Waters CSH column. MP-A is water and MP-B is ACN, both with 0.1% TFA.
My diluent is 50:50 Water/Methanol with 0.01 N HCl. When I am not using 0.01 N HCl in the diluent, I am getting two separated peaks for the API (Peptide molecule). Using an acidified diluent would fix the problem.

This might very simple, but I am new to biochemistry and large molecule analysis. I know that this has to be something with the PI number of peptides (8.2 in my case) and acid/base chemistry. Can anyone suggest any reading material explaining this phenomenon?
What salt form is your peptide in? If it's a drug product, then there's a good chance that it's in the acetate salt form (i.e., the basic residues have acetate as a counterion). Let us know and we'll proceed from there.
PolyLC Inc.
(410) 992-5400
aalpert@polylc.com
Andy Alpert wrote:
What salt form is your peptide in? If it's a drug product, then there's a good chance that it's in the acetate salt form (i.e., the basic residues have acetate as a counterion). Let us know and we'll proceed from there.



Hi Andy, Thank you for taking the time to answer my question. The DP is in form of acetate salt.
I speculate that the two peaks correspond to the peptide with acetate as a counterion and trifluoroacetate as a counterion. The resulting ion pairs differ in polarity. This can be a problem in HILIC as well, with the elution orders reversed. When HCl is present in your diluent, then that would convert much or most of the peptide to the chloride salt form, which evidently migrates similarly to the TFA salt form. Having TFA in the diluent instead of HCl should be even more effective for this; try the experiment.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Andy Alpert wrote:
I speculate that the two peaks correspond to the peptide with acetate as a counterion and trifluoroacetate as a counterion. The resulting ion pairs differ in polarity. This can be a problem in HILIC as well, with the elution orders reversed. When HCl is present in your diluent, then that would convert much or most of the peptide to the chloride salt form, which evidently migrates similarly to the TFA salt form. Having TFA in the diluent instead of HCl should be even more effective for this; try the experiment.


I appreciate your detailed answer Andy. I used 0.1% TFA solution and it turned out to be very strong as it degraded some of the excipients. I should probably shoot for a lower conc. of TFA.
I just did not understand one part of your answer. Where did trifluoroacetate come from? The formulation lab mentioned that the only counter ion in API is acetate.

Thank you!
The trifluoroacetate is in your mobile phase! It contains 0.1% TFA. Yes, use less TFA in the diluent.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Andy Alpert wrote:
The trifluoroacetate is in your mobile phase! It contains 0.1% TFA. Yes, use less TFA in the diluent.


Thank you so much Andy! It is completely tangible for me now.
Andy Alpert wrote:
The trifluoroacetate is in your mobile phase! It contains 0.1% TFA. Yes, use less TFA in the diluent.


I understand that it mostly comes from your experience. But, do you happen to have a reference for this?

Thank you again.
As it happens, I published an example in 2008: https://pubs.acs.org/doi/full/10.1021/ac070997p
Look at Fig. 14. This shows the consequences when a charged analyte's counterion doesn't match the counterion in the mobile phase. You can get two peaks (cf. your own observations), each corresponding to the analyte with one of the two counterions. The continuum connecting them consists of molecules of the analyte that started with one counterion but which exchanged it for the other counterion during the migration through the chromatography column.

I've thought of an alternative explanation for your two peaks. You say that this involves a peptide. Does this peptide have either of the following structural features?
a) N-terminal glutamine;
b) An aspartyl- residue with an unhindered residue (Gly; Ser; Ala) on its C-terminal side in the primary sequence?
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Thank you for sharing your work. My peptide is made of Ala, Lys, and Phe.
How many lysines does it have? The more it has, the longer it's going to take for all of them to exchange a counterion that isn't trifluoroacetate for one that is trifluoroacetate. That's why I'm trying to get you to complete that exchange in the sample solvent prior to its injection onto the column.

It isn't obvious why the low pH of your initial dilution with TFA resulted in degradation of the peptide. Ala, Lys and Phe aren't especially sensitive to low pH.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
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