Ingenious, and the best idea I have heard of. My compliments to you, and
thanks on behalf of everyone.
Leo B (now I am going to do some work!)
At 07:49 PM 98/7/21 +1000, you wrote:
>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
>>
>
>
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