From: GSCAVEY@am.pnu.com
Date: Fri, 17 Jan 1997 14:36:09 -0500
Message-Id: <00006ABC.1444@am.pnu.com>
Subject: Re: RP-HPLC at basic pH
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Haydn,
Below are previous e-mail notes on basic pH RP-HPLC separation. There are
also some interesting near neutral pH separation info. on the ABRF WWW home
page at http://www.medstv.unimelb.edu.au/ABRF.html
Hope this fulfills my lurker "dues"!
Greg Cavey
Pharmacia & Upjohn
==================
>I am looking for an alkaline HPLC gradient that can be used on a Shodex
>RS reverse phase column. Any suggestions will be greatly appreciated.
Carolyn:
In general, when using _silica_ based columns at neutral to alkaline pH,
avoid inorganic salts. Use GOOD buffers like tris, or organic amines.
Kirkland has showed that phosphate, borate and inorganic salts promote
degredation of silica columns much faster than these other buffers.
However, for your Shodex RPC column, which I assume is polymeric not
silica, you could use 10 mM ammonium phosphate with 0.1M sodium perchlorate
at pH 7.0 or higher as "A", and 60% MeCN + "A" as your "B" buffer.
(Perchlorate, which is a choatrope won't promote adsorption from HIC
mechanisms like other salts would, and it is soluble in higher amounts of
MeCN.) Perchlorate disrupts H-bonds preventing aggregation and promotes
interaction with the bonded phase in a more monomolecular fashion, to
enhace selectivity as Mant and Hodges showed in a a comparison of
conditions with and without the perchlorate buffer in their J Chromatogr,
359, 507 (1986) paper, figure 2. You could of course use a simple
phosphate (TEAP or ammonium) without perchorate as a starting point.
I hope this helps.
Amos
Amos Heckendorf (nestgrp@world.std.com) 800-347-6378 ; 508-481-6223
The Nest Group , Value Added Resellers
HPLC Operating Instructions and Molbiol. Protocols on WWW URL=
HTTP://world.std.com/~nestgrp
==============================
We have had very good luck using 10 mM ammonium bicarbonate as eluent A,
and
acetonitrile as eluent B, with a "pH stable" C8 column from Vydac. The pH
of the ammonium bicarbonate is about 7.8 at 10 mM, just slightly basic. It
is UV transparent, and you can detect your peptides at 215 nm. I haven't
tried this system with other silica-based columns - it might not dissolve
the silica, but why take chances? The "pH stable" Vydac column has a
polymeric coating protecting the silica. A polystyrene-divinylbenzene
column should be quite happy with this system also.
Hope this helps.
Angela C. Murphy
On Wed, 17 Jan 1996, Christoph Turck wrote:
> I am working with a protein that has an acid-labile posttranslational
> modification and want to analyze it. What solvent systems are out there
to
> separate peptides on a reversed phase column using a basic buffer solvent
> system? Also, for sensitivity reasons I would like to detect the peptides
> at 215 nm.
>
> Thanks,
>
> Chris Turck (turck@itsa.ucsf.edu)
>
============================
I'd suggest to call JM Science, the American Distributor for Shodex
columns:
JM Science Inc.
355 Lang Blvd
POB 250
Grand Island NY 14072-0250
phone 716-774-8706
fax 716-774-8708
One has to be familiar with the characteristics of the polymeric material
to give accurate recommendations.
Best regards: Jean-Pierre Salzmann
********************************************
Jean-Pierre Salzmann
LC Packings (USA) Inc.
80 Carolina Street
San Francisco CA 94103
Phone (415) 552-1855
Fax (415) 552-1859
E-mail schampe@aol.com
URL http://www.lcpackings.com
********************************************
______________________________ Reply Separator _________________________________
Subject: RP-HPLC at basic pH
Author: "Haydn Ball" <hlb@prion.ucsf.edu> at INTERNET
Date: 1/16/97 5:42 PM
Hello everyone,
I have a question concerning the reversed-phase purification of synthetic
peptides that have a strong tendancy to aggregate at the buffer pH most
commonly used for HPLC ie TFA/pH ~2. Does anyone have experience of separation
at higher pH's and can advise me on the types of commercially available
columns, given that silica based media are not stable at pH greater than about
8-9. Also any suggestions as to the buffer systems that are effective would be
greatly appreciated.
Many thanks in advance.
Haydn Ball
hlb@prion.ucsf.edu
Dept. of Neurology,
University of San Francisco
San Francisco
CA 94143-0518