Message-Id: <l03010d01af04ebaab2f0@[131.111.47.52]>
In-Reply-To: <9701161742.ZM12975@prion.ucsf.edu>
Date: Fri, 17 Jan 1997 08:57:57 +0000
From: Len Packman <lcp2@mole.bio.cam.ac.uk>
Subject: PepSyn: RP-HPLC at basic pH
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Haydn,
For synthetic peptides which are poorlyu soluible at pH2, we test
out solubility at pH6-7. If successful, we use 50mM ammonium acetate as
mobile phase. This is made with 50mM acetic acid which is then pH'd with
ammonia solution until the pH is 6-7 (actual value not important ususally).
If peptides are not soluble at neutral pH, we try solubilising in dilute
ammmonia solution and then adjusting the pH back to neutral for silica
columns. If that fails, we go back to acid pH and solubilise in guanidine
and inject the sample between slugs of guanidine in the injector loop so
that there is no precipitation in the lines.
One important feature of aggregation is that it is concentration
dependent and we routinely test out a small amount of material to see if it
will solubilise on dilution. At 200-300ml, a preparation can be loaded to
hplc through a pump position. Dilution tests can be done as follows. Take
several small fixed volumes (e.g. 50ul) of the insoluble peptide in
suspension (disperse by sonication and vortexing) and place in separate
tubes. To tube 1, do nothing. To each of the other tubes, try adding
increasing volumes of solution and vortex well. Then spin all the tubes in
a centrifuge and compare the sizes of the pellets; tube 1 is the control.
>From this, you can estimate the final volume that will be needed to
solubilise the whole preparation and so assess if this is reasonable. The
same approach can be used to test out other solubilisation procedures (e.g.
dissolve in conc. acid and then dilute, or, add organic modifier to
dissolve and then dilute out etc.) These small tests can avoid getting into
difficulties when treating the whole fraction and ending up with ever
increasing volumes and the likelihood of redrying everything down again and
starting over.
With these approaches, we have never found it necessary to invest
in additional high-pH-stable columns.
Len
>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.
>
******************************************************
Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW, UK
Tel: +44 (1223) 333639 (including answerphone)
FAX: +44 (1223) 333345
e-mail: lcp2@mole.bio.cam.ac.uk
Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html