Pardon my skepticism, but it will be hard to get adequate retention of a
30-residue peptide if the only basic group is the N-terminus (assuming it is
unblocked). At best, you'll get elution about 3-4 column volumes past the
void when eluting isocratically with a low-salt buffer (e.g., 5 mM K-PO4, pH
2.7, with 20% ACN). Jenny stands a better chance by using 50 mM K-PO4, pH
2.0, with 10% ACN, to elute her peptide from the anion-exchange column.
These conditions will uncharge the Glu- residues.
One possibility may be that her Glu- residues are in close proximity and that
they're chelating heavy metals in the frits. If this is the case, then she
can passivate her column by eluting 24 hours with 40 mM EDTA.2Na.
Best regards,
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045 USA
tel: (410) 992-5400 FAX: (410) 730-8340
*********************************************************************
<< Subj: Re[2]: IE HPLC
Date: 12/01/1999 9:14:17 AM Eastern Standard Time
From: M.Schrader@biovision.de (Schrader, Michael)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
Dear Jenny
We have very good experience with cation-exchange chromatography for
peptides and I would suggest to try that. Your peptide won't be sticky then.
Try strong exchanger pH around 3, usually you get very sharp peaks.
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
> -----Urspržngliche Nachricht-----
> Von: Jenny Shipway [SMTP:jennys@biols.susx.ac.uk]
> Gesendet am: Dienstag, 30. November 1999 18:26
> An: Recipients of ABRF List
> Betreff: IE HPLC
>
>
> Hello you ABRFers,
>
> Help!
>
> It's easy, I thought, just get an anion-exchange column (DEAE) and get a
> salt gradient going. Hah!
>
> I have a highly charged peptide (30aa, 4 Glu's, no positive charges) which
> is refusing to budge from the column. I've been running 10mM Tris (pH 8.5
> - I want to seperate this guy from another with pI about 7, and didn't
> want to do a pH gradient) and shoving loads of NaCl in, but nothing comes
> off. I just ran 2M NaCl for 1.5h (this is how Vydac say I should clean the
> column of any highly charged stuff) and still nothing.
>
> Is there anything I can do to avoid doing a pH gradient? (Kosmotropic
> salts?) I don't want to fill the entire HPLC system with salt crystals.
> This negative peptide has a pI of about 3, so what buffer systems would be
> good for going from pH8 to pH3? Is it okay doing a pH gradient with
> DEAE? (I read somewhere that it's much better to do salt gradients(?))
>
> Any thoughts would be appreciated!
>
> Jenny Shipway
> Centre for Biomolecular Design and Drug Development
> University of Sussex