<I know that removing TFA from peptide has been discussed before but I
cannot find my file. I need a good and convenient method for removing
TFA from peptide after c-18 column. Thanks.
Bob>
Bob:
Below you will find my digest of the extensive discussions of the
'residual TFA' - related problems.
You may also want to search ABRF archives for more relevant INFO. Why
not do the search in the first place? Of course, this is time-consuming,
but note that I have not prepared digests on every problem you may
become interested in.(:-))
Regards
Igor Rodionov
<><>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<><>
Does anyone know a way to exchange TFA salt in petides with other salts
then Cl or acetate ?
Karolina.Lawitz
================================================
In our laboratory we change the TFA salt in peptide to acetate salt by
dissolving of peptide TFA salt with Acetic acid:TDW (1:1) and liophelaze
this mixture.
Leah Efron
-----------------------------------------------------------------------
I have a problem with the below sugestion from Leah Efron. If you check
out paper by Werner WE, Demorest DM, Wiktorowicz JE: Analysis of
residual TFA in synthetic peptides. Biotechniques 1995;78 (pgs ?) you
will see that acetic acid will not replace TFA but that HCl will. Even
base solutions will not remove the TFA. I redissolve my peptides in
50-100 mM HCl, freeze and the dry in a speed vac. This replaces TFA
salt with a Cl salt.
Tom Haas
-----------------------------------------------------------------------
One way is to prepare a small column containing a strong anion exchanger
(QAE-Sephadex, QAE-cellulose, etc.). Exchange all the anionic sites on
the resin with the desired anion (e.g., sulfate; but it could be
bicarbonate, chloride, etc.) by eluting with a strong (1M) solution of
corresponding salt (sodium sulfate). Then wash out the excess salts
with water. This will give you a sulfate column. Then dissolve the
peptide in water and pass it through the column, and collect what comes
through, which should be the peptide in the desired ionic form. It is
important, though that the peptide be above its isoelectric point (i.e.,
be neutral or have a slight positive charge) or it will itself be an
anion and stick to the support. Also the column should cont at least a
10-fold (better 50- to 100-fold) excess of anion sites over peptide
sites. This procedure is a bit involved, but it can be scaled to any
amount of peptide and also used to to exchange any anion.
Richard Laursen
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
<><><><17.07.98 -22.07.98><><><>
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?
Federico Brianza
===============================================
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.
Michael Breslav
-----------------------------------------------------------------------
TFA can be quite a nuisance after chromatography. Hence there is
interest in finding solutions that either get rid of the TFA salts or
avoid them from the beginning. Let me explain.
1: Clean-up
Michael Breslav suggested ion-exchange resins which appears to me as
good solution. I know also that Axel Ducret did some work in this
direction -- perhaps he might eleborate. In that sense I would also like
to add to Jeff Hulmes' remark:
"...remember that RP-HPLC doesn't work for everything..." that is why
there are many other liquid chromatographic separation techniques....
2 Avoiding TFA in RP-HPLC
To avoid TFA alltogether there is an option in RP-HPLC. The company I'm
with developed a special reverse phase C-18 material that provides good
separations without TFA. The material is called PepMap. If you'd like
more information (technical or commercial), please contact me by e-mail.
I just don't want to reprimanded for being too commercial after a long
absence from this bulletin board. (And I'm really glad to be back...)
Jean-Pierre Salzmann
-----------------------------------------------------------------------
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
-----------------------
I'm afraid what you have suggested is NOT fact. 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 Benoiton
-----------------------------------------------------------------------
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
-----------------------------------------------------------------------
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.
-----------------------------------------------------------------------
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
-----------------------------------------------------------------------
Dear Philip Mack:
Ingenious, and the best idea I have heard of.
My compliments to you, and thanks on behalf of everyone.
Leo B
==~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~==
Leo,
Here is a copy of the discussion that I would like to share with you
since you indicated an excitement about the exchange on a column, and I
am
not so shure and would like to share with you my concerns.
The bottom line is: if TFA is responsible for the retention it can not
be exchanged. I imagine, following Philip's note, that if the peptide is
more hydrophobic, then the exchange is more probable. But again, depends
on
the environment of the TFA ion - inside C18 layer (no dissociation and
exchange with acetate) or outside (dissociation and exchange are
possible). My experimental data that does not support an ion exchange on
a C18 column. Regards,
Michael Breslav
------------------------------------------------
Philip,
Interesting, it was something that I tried. I loaded TFA salt of the
peptide on C18 column, washed with 10 column volumes with 10% acetuic
acid, then eluted with ~80% acetuic acid. And this did not work for me.
A possible explanation is that TFA is responsible for the holding of
the peptide on a C18 column. TFA counter ion helps to keep polar amine
(guanidine) groups on C18 and in this situation TFA is not accessible
for the acetate ion. Did you check the TFA content after the
experiment?
Michael Breslav
-----------------------------------------------------
It is interesting that you tell me that. I did find guanidine pair with
TFA more strongly than amines. Your theory about accessibility of TFA
could also be valid in certain cases. The effectiveness of the method
would depend on the nature of the peptide. On occasions when the peptide
is not exchanged well, I did find that TFA is only partially removed and
still show peaks on C-13 NMR. I tend to use 5% aqueous acetic acid and
acetonitrile buffers normally on a Delta Prep. For most of the peptides
that I handled, the method worked well.
Philip Mack
````````````````````````````````````````````````````````````````````````
`````
Dear Michael:
If you have read all my input, you may have realized that I was the one
who started everthing off by saying that one could NOT displace
trifluoroacetate bound to a peptide by acetate. So I get the impression
that you agree with me.
Regards. Leo.
This archive was generated by hypermail 2b29 : Fri Aug 18 2000 - 07:56:58 EDT