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.
At 09:16 AM 98/7/22 -0400, you wrote:
>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 amino
>(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?
>Regards,
>Michael Breslav
>R.W.Johnson PRI
>Spring House, PA
>-----------------------------------------------------
>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
>
>> ----------
>> From: Sillydog[SMTP:semabok@ozemail.com.au]
>> Sent: Tuesday, July 21, 1998 5:49 AM
>> To: Recipients of ABRF List
>> Cc: abrf@aecom.yu.edu
>> Subject: Re: How to get rid of TFA?
>>
>> 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
>> ----------
>>
>
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