The question of TFA remaining after HPLC is to be expected.
TFA is a strong acid. It will protonate (create the TFA salt) any amino
group. The same goes for HCl.
Acetic acid is a weak acid. It will only protonate more-basic amino groups
such as that on the side chain of lysine and of course the guanidino group
of arginine. As such, AcOH cannot displace TFA from a salt.
The above means that a simple peptide does not form an acetate salt, but it
forms a monoacetate for each basic group on side chains (Lys, Arg).
But the simple peptide does form an HCl or TFA salt, and even more so if the
peptide has side-chains with basic groups.
The only way to remove the TFA is to displace it (evaporate in the presence
of) with a stronger acid - Hydrochloric acid will displace TFA -, which may
or may not be better for you, or remove the TFA using an anionic
ion-exchange resin in the hydroxide or acetate form. The anion of the
stringer acid displaces that of the weaker acid.
Leo B
At 05:40 PM 98/7/17 -0400, you wrote:
>Hello:
>
> I have been following this discussion, and I was thinking that the
>TFA could be protonating the side-chains of Glu and Asp, and the
>C-terminus. In the presence of acetate, Glu and Asp should deprotonate,
>based on respective pKa values. Does anyone have an opinion on whether
>this is, in fact, what is occurring?
>
>Chris Halkides
>
>
>
>
>
>>> Federico,
>>> I tried to freeze-dry TFA salts from acetic acid several times measuring
>>> the amount of TFA before and after and this did not work well. But an
>>> ion-exchange resin in an acetate form worked fine for my peptides.
>>> Trifluoroacetate was transferred to an acetate.
>>> Regards,
>>> Michael Breslav
>>> R.W.Johnson PRI
>>> Spring House, PA
>>>
>>> ----------
>>> From: Federico Brianza[SMTP:brianza@imb-jena.de]
>>> Sent: Friday, July 17, 1998 8:38 AM
>>> To: Recipients of ABRF List
>>> Subject: How to get rid of TFA?
>>>
>>> Hi everybody,
>>> after purification with RPC-FPLC, using acetonitril and TFA, my synthetic
>>> peptides
>>> have extremely low pH, I guess due to the presence of TFA. The problem
>>> persists
>>> also after freeze-drying. What can I do? Is it true that freeze-dryng
>>> after adding
>>> acetic acid can help remove the TFA?
>>> Thanks in advance
>>>
>>>
>>>
>>> --
>>> Federico Brianza
>>> Protein Laboratory
>>> Institute of Molecular Biotechnology
>>> Beutembergstr. 11, 07745 Jena / Germany
>>> Tel: +49 3641 656432
>>> Fax: + 49 3641 656431
>>>
>>>
>
>
>Christopher Halkides
>Dept. of Chemistry, UNCW
>601 S. College Road
>Wilmington, NC 28403-3297
>(910) 962-7427
>
>
>
N. Leo Benoiton
Department of Biochemistry
University of Ottawa
Ottawa, Ontario, Canada K1H 8M5
Tel: 613 562 5800, Ext. 8216
Fax: 613 562 5440
eMail: benoiton@uottawa.ca