Re: How to get rid of TFA?

Sillydog (semabok@ozemail.com.au)
Tue, 21 Jul 1998 19:49:55 +1000

A technique which I have developed in my lab (and which I am particularly
fond of) is to do an anion exchange on the same reversed phase HPLC on
which the peptide was purified. I will load the peptide (dissolved in an
aqueous acetic acid buffer with minimal acetonitrile if necessary) on the
column, hold it on the top while washing it with copious amount of acetic
acid buffer, and then elute it out with an aqueous acetic acid/acetonitrile
gradient. After freeze drying, the counterion TFA will be completely
exchanged. The technique of course relies on the hydrophobicity of the
peptide. An very hydrophilic peptide will require a proper anion exchange
resin.

Philip Mack
Peptide Production
Chiron Technologies
11 Duerdin St
Clayton Vic 3168
Australia
----------
> From: Leo Benoiton <benoiton@uottawa.ca>
> To: Recipients of ABRF List <abrf@aecom.yu.edu>
> Cc: abrf@aecom.yu.edu
> Subject: Re: How to get rid of TFA?
> Date: Tuesday, July 21, 1998 4:43 AM
>
> I would like to assure you and everyone that I do not want to get myself
> into any controversy or argument. I just thought I might be able to help
> someone.
>
> I do not at all disagree that a few equivalents of trifluoroacetic acid
> might get displaced by lots of equivalents of acetic acid. But, from my
> 'knowledge' and recollection of views on this network, swamping a peptide
> with acetic acid and evaporating it does not displace/force out all of
the
> trifluoracetic acid.
>
> Leo B.
>
> At 02:22 PM 98/7/20 -0400, you wrote:
> >At 09:52 AM 7/20/1998 -0400, Leo Benoiton wrote:
> >>
> >>
> >>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 only way to remove the TFA is to displace it (evaporate in the
presence
> >>of) with a stronger acid - Hydrochloric acid will displace TFA ...
> >
> >On an equimolar basis I'm sure you're right. But I wonder if things are
a
> >bit different if one has first dried the peptide to remove excess TFA,
then
> >redisolved/resuspended in acetic acid?
> >
> >This principle (mass action) is the basis of the "TFA fix" introduced by
> >Alex Apffel and his colleagues for dealing with the suppresion caused by
> >TFA when introducing an LC eluate into an electrospray mass spec. The
> >eluant (with the usual 0.05-0.10% TFA) is mixed with half its volume of
a
> >mixture consisting of 1:1 acetic acid:acetonitrile. It is impressively
> >good in displacing TFA and improving the signal in the mass spec.
> >
> >See: Apffel A, Fischer S, Goldberg G, et al: Enhanced sensitivity for
> >peptide mapping with electrospray liquid chromatography-mass
spectrometry
> >in the presence of signal suppression due to trifluoroacetic
> >acid-containing mobile phases. J.Chromatogr.A. 1995; 712:177-190.
> >
> >Rod Levine
> >
> >NIH
> >Bldg 3, Room 106 MSC 0320
> >Bethesda, MD 20892-0320
> >
> >email: rlevine@nih.gov
> >voice: 1 (301) 496-2310
> >fax: 1 (301) 496-0599
> >
> 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
>