Actually, I did mean anion-exchange at pH 2.0, not cation-exchange. The
gatekeeper(s) of this bulletin board did not see fit to post my message to
Jenny, which explained my rationale. Nothing daunted, I'm game to try again:
Jenny's problem is inordinate retention of a peptide with 4 Glu's. The usual
way of eluting acidic peptides from anion-exchangers - displacing them with a
lot of salt - wasn't working. If she elutes the column with a sufficiently
acidic mobile phase, then she will uncharge the carboxyl- groups in the Glu-
residues. From personal experience, 50 mM K-PO4, pH 2.0, should do the
trick. There would then be no mechanism whereby the peptide's retained, and
it should elute. Needless to say, you would want to adsorb the peptide to
the column at a higher pH initially. You also might not want to try this
trick with a silica-based anion-exchanger with which the coating is attached
via silane bonds; they'll start to hydrolyze at this low pH (ours doesn't
have any).
Michael Schrader (BioVision) and I discussed alternative approaches to this
problem. She probably doesn't need an HPLC column to separate her two
peptides. All she needs to do is to pass the mixture through a Solid Phase
Extraction (SPE) cartridge with a cation-exchange material. The peptide with
4 Glu's will pass right through, while the other peptide will stick and can
subsequently be eluted with salt (SCX) or dilute acetic acid (WCX).
Best regards,
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD
tel: (410) 992-5400 FAX: (410) 730-8340 e-mail: PolyLC@aol.com
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<< Subj: Re: IE HPLC
Date: 12/02/1999 5:24:33 PM Eastern Standard Time
From: nestgrp@world.std.com (Amos Heckendorf)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
Jenny
Alpert was probably suggesting pH 2 for strong cation exchange, not anion
exchange. At that pH your carboxyls will be protonated and thus not
exchangeable. Lower the pH to 5.5 and try it with the 10% MeCN . Wait for
10 column volumes of solvent to equilibrate it. You might consider a
wavelength of 214 instead.
Please let us know how you are doing. This is very instructive
Amos
Amos Heckendorf (nestgrp@world.std.com)