I appreciated your comment regarding ion-exchange for peptides. Even being
still quite young, I experienced that ion-exchange complements
reversed-phase separations of peptides very well. The nice thing is, that
you can directly load an ion-exchange eluent on a reversed-phase column and
vice versa - no desalting, lyophilisation or dialysis in between.
Regards
Michael Schrader
> ----------------------------------------
> BioVisioN GmbH & Co. KG
> Feodor-Lynen-Str. 5
> D-30625 Hannover
> GERMANY
> fon +49 (0)511-53 88 96-12
> fax +49 (0)511-53 88 96-66
> Internet www.biovision.de
> E-mail m.schrader@biovision.de
>
> -----Original message -----
> Von: Roger Murphy [SMTP:Roger.Murphy@ludwig.edu.au]
> Gesendet am: Freitag, 17. September 1999 02:31
> An: Recipients of ABRF List
> Cc: abrf@aecom.yu.edu
> Betreff: Re: base-deactivated HPLC
>
> Hi Jenny,
>
> The simplest way to separate these peptides is by ion-exchange. With
such
> vastly differing pI values you can virtually use any ion exchanger and
use
> conditions where one peptide will stcik and the other won't. Simple.
>
> Just a general comment (don't take it personally).......many younger
> researchers have only ever association HPLC with reversed phase
> separations. The selectivity, and indeed the huge loading capacity, of
ion
> exchange chromatography is often overlooked for peptides, but I've found
it
> to be an extremely useful, complementary technique, to be followed
perhaps
> by RP-HPLC to buffer exchange into a voltile solvent system.
>
> Maybe I'm showing my age, but I would recommend to all young researchers
to
> look at ion exchange very closely....
>
> Cheers,
>
> Roger
>
> At 04:59 PM 16-09-99 +0100, you wrote:
> >
> >
> >Hello all,
> >
> >I'm trying to use reversed-phase HPLC to seperate and quantify an acidic
> >(A) peptide and a basic (B) peptide. The A peptide is only soluble > pH
> >6ish, but at this pH the B peptide sticks irretrievably to the column
> >(Vydac TP218, C18).
> >
> >I presume it's getting stuck to silanol groups, as it is very basic,
> >definitely soluble, and elutes fine at pH2. I also suspect that although
> >changing my buffer system (Phosphate/MeCN) may help, it won't completely
> >negate the problem, meaning that I'm still not getting quantitative
> >results.
> >
> >>From reading the archives, I suspect a base-deactivated column is what
I
> >need. I can't find anything specifically labelled such in our catalogues
-
> >are there other terms for the same thing, or is it fairly specialised?
> >Does it cost loads more?
> >
> >Alternatively, is there a way to solve my silanol problem without buying
a
> >new column? Or is there a completely different way to seperate these
very
> >differently charged peptides (PI's about 3 & 10) quantitively?
> >
> >Thanks!
> >
> >Jenny Shipway
> >Centre for Biomolecular Drug Design and Drug Development
> >University of Sussex
>
>
> ------------------------------------------------------------
> Roger Murphy, Ph.D.
> Biological Production Facility
> Ludwig Institute for Cancer Research
> Austin & Repatriation Medical Centre
> Studley Road,
> Heidelberg, Vic. 3084
> Australia.
>
> Tel 61-3-94965463
> Fax 61-3-94965436
> Email Roger.Murphy@Ludwig.edu.au
>