Date: Sat, 1 Feb 1997 14:56:19 -0500 (EST)
From: Steven Birken <birks@styx.ios.com>
Subject: Re: HPLC resins for labile proteins
In-Reply-To: <v01540b01af1230b92700@[10.0.2.15]>
To: Recipients of ABRF List <abrf@aecom.yu.edu>
A Vydac C4 protein silica column was tried for this labile growth
factor at pH 6.5 NaPHOS buffer and methanol. No activity was recovered.
The protein does survive well in this buffer so it was denaturation
on the column that likely destroyed activity. I assume the deformation
of the protein on binding to the lipid surface is the problem. Ionic
interactions I assume are much less stressful. What I do not understand
in this situation is that the protein seemed to survive a C18 silica
cartridge binding and elution. Why would it not survive the column?
Is time on the column a significant factor? I do not know any ion
exchange columns with resolutions similar to reverse phase. In general,
most of my experiences with HIC columns is also much lower resolution.
When organic solvent is needed to remove proteins from HIC columns, is
not the denaturation stress similar to that of reverse phase?
On Mon, 27 Jan 1997, Amos Heckendorf wrote:
> Steven:
>
> The limitation of silica packings for proteins is more their pH stability
> than their poor interaction with the proteins. A well constructed bonded
> phase will give excellent selectivity and recovery of growth factors.
> There are a number of published and unpublished examples where silica
> phases have been used with excellent results.
>
> Each vendor has their own case to make, so it can be confusing as a user to
> determine the correct global statement to make. The generally acceptable
> benefit of silica over polymers is that there will be more surface area and
> pore volume for a silica type packing relative to a polymer, for the "same"
> general characteristics of the base material. This can be seen with the
> comparison of SW and the PW TSK SEC columns, for example. For ion exchange
> this is attenuated by varying the surface ligand density. The fact that
> one can, in ion exchange with nonporous particles, get the same capacity as
> with porous ones, bears this out. (Digressing slightly, this is an area we
> have been active in for comparisons of polymeric ion exchange and
> hydrophobic interaction NPR columns to the polymeric, perfusive, porous
> ones. The two choices give high speed separations, similar capacities, and
> both "flatten out" the Van Deempter curve at higher linear velocities, yet
> the NPR packing can do it at lower flow rates and lower pressure, with
> sharper peaks.).
>
> If one wants to use silica and one can operate at a pH of 7.0 or less, then
> there are definite benefits to doing so. The gating factor is whether the
> selectivity is there and whether one wants to disregard the advertising.
>
> Best of luck.
>
> 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
>
>
>