RE: base-deactivated HPLC
Breslav, Michael [PRI] (MBreslav@prius.jnj.com)
Fri, 17 Sep 1999 15:42:30 -0400
Jenny,
I think that your problem is a general problem of solubility. I would
suggest that you use ACN/water with up to 1% TFA. If this does not help,
then use 70C for your column provided peptides are stable during HPLC.
Usually they are. I got better results with high temperature than with IPA
in mobile phases. To dissolve your peptides prior to injection you may use
trifluroethanol/water/1%TFA mixtures. Also, you may get more suggestions at
the chromatography discussion forum at www.lcresources.com or
www.lcgcmag.com.
Michael Breslav
R.W.Johnson PRI
Spring House, PA
> -----Original Message-----
> From: Jenny Shipway [SMTP:jennys@biols.susx.ac.uk]
> Sent: Thursday, September 16, 1999 12:00 PM
> To: Recipients of ABRF List
> Subject: base-deactivated HPLC
>
>
>
> 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