Chromatography Forum

Dear All,
My peptide sample is precipitating during HPLC analysis and I believe this is because of the acidic pH of my 0.1 % TFA water and MeCN mobile phase.
I was wondering if there is an alternative pairing agent to TFA which is basic but has a similar effect on peak separation and sharpness and is compatible with water and MeCN?
Cheers,
Sat

I was wondering if there is an alternative pairing agent to TFA which is basic but has a similar effect on peak separation and sharpness

Strictly speaking, "no". To the extent that TFA is acting as an ion-pairing agent, it is behaving like an anionic surfactant. Any anionic surfactant will have an acidic functional group.

That said, if all you want to do is to increase the pH, then you could simply neutralize the TFA with a base of your choice. Or, you could use a weaker acid like formic or acetic, or heptafluorbutyric or . . .

Be aware, however, that this would not be a "plug and play" change. It would almost certainly change the selectivity of your separation, and would probably also affect the peak shape. You would have to re-develop the separation conditions from the beginning.

Do you have any idea of your peptide's hydrophobicity? How did you come up with the conclusion that your peptide is precipitating in the column? What are your separation conditions (i.e. exact mobile phase composiiton)?