You should be aware that some separation or retention might be attributed to
interactions with the residual Si groups and possible metal contaminates in
the column base material. This might be the case for peptide B. If you
eliminate or minimise these interactions, as high purity silicas usually do,
you might lose your separation or retention. So, a high purity Si base
material will not always be your answer.
Alternatively, you can add TEA to your mobile phase using your current
column. (try 0.1% first.) However, there are a number of disadvantages
using this approach including reproducibility and undesirable interactions.
High purity columns were designed to overcome these problems.
Hope this helps!!
Nicolas Angell
Zeneca / SmithKline Beecham Centre for Analytical Sciences
Department of Chemistry
Imperial College
South Kensington, London, UK
Main Lab: (44) 0171 594 5865
MS Lab : (44) 0171 594 5857
Fax: (44) 0171 594 5859
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu] On Behalf Of Jenny Shipway
Sent: 16 September 1999 17:00
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