Chromatography Forum

Dear all,

The crude peptide we have is in TFA salt form. If I want to purify these peptide on a polymer RP column with Water/ACN ( without any acid or base additives). What kind of peptide I will get? Still a TFA salt form?

Without pH control, the peptide will elute unpredictably because it is a zwitterion. You probably will get a mix of TFA and trace anions from the mobile phase. If your peptide is basic, at physiological pH it will have to be a salt of some anion, so why not decide which one it should be and put that in the mobile phase? If your peptide is acidic, convert the TFA to a volatile anion like acetate that will vaporize when you lyophilize the peptide fraction.

Hi, Mark

Thank you so much for your reply. I am still a novice in HPLC (and Mass).If oyu don't mind, can I ask several more question?

1. Why TFA is so commonly used in HPLC? Why not other acids? Is this because TFA pKa is lower than other common acids?
From what I know, TFA in HPLC have several functions, like control pH, denature protein, suppress silanol interaction, but TFA is not good for Mass detection. In LC-MS, what will be the best alternative for TFA?

2. Is it true that: acidic condition is preferred for ESI-MS detection? If so, why? Can't we use negative mode under basic condition?

You are correct: one commonly gets better sensitivity under acidic conditions with formic acid. The selectivity is different, and some columns don't work as well with formic acid as with TFA, but if you have the right column, there is no disadvantage to formic acid.

We have occasionally done peptide separations with ammonia or ammoniumbicarbonate. My understanding is that sensitivity is not as good in this mode as in acidic mode, but I have no data to prove this.

Thank you very much.

I found the following article:

https://www.chem.agilent.com/Scripts/Ge ... &prodcol=Y

in this article they are using 5% acetic acid instead of TFA (5% really seems a lot to me, but maybe this is because the pKa of acetic acid is lower than that of TFA, to get to the desired pH, the concentration has to be increased). They also used basic condition and get the pretty good MS signal.

My confusion about this article is from its figure 14: how could they get the same MS at both negative and positive mode detection? I don't know exactly the structure of their peptide. I am just wondering if this is common in LC-MS.

In fact, I had posted a thread asking this question in the LC-MS forum. But no one answered me. Can you clarify it for me?

Here is the link I posted the question:

http://www.sepsci.com/chromforum/viewtopic.php?t=6578

There have been a couple discussions on this forum about TFA, what's good and bad about it, and the alternatives. You can search the forum for "TFA AND peptide" One is http://www.sepsci.com/chromforum/viewtopic.php?t=2437

In a nutshell, TFA is a strong acid, is volatile, is not highly corrosive to HPLC parts, is a mild ion-pairing agent, improves peak shapes for bases, has moderate UV absorbtion, is expensive in HPLC grade, suppresses electrospray ionization, and causes noisy baselines especially with gradients.

Peptides are zwitterionic, and both positive and negative ions can be produced. In general, negative ion mode gives less signal than positive ion mode.

Here is a poster that compares several different mobile phases for peptides:

TFA, formic acid, and ammonium bicarbonate

You may need to sign in to get access to the poster.

http://www.waters.com/watersdivision/pd ... 2091EN.pdf

Dear Uwe and Mark,

Thank you so much for your kindly help!

wen