When I was at Amgen we used an HP1100 almost exclusively at low pressure
with Pharmacia 10x30 FPLC size exclusion columns. The pump gave very
reproducible flows at low pressure, and the fact that you can flush behind
the pistons is helpful for long-term reliability when you use buffers with
high amounts of salts.
For our work, the other nice feature of the 1100 is the built-in vacuum
degasser. This significantly reduces baseline noise for our on-line laser
light scattering and RI detectors, and is far superior (and far cheaper over
the long run) than helium sparging.
John Philo
Alliance Protein Laboratories
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of Deb McMillen
Sent: Friday, July 24, 1998 10:53 AM
To: Recipients of ABRF List
Subject: FPLC system
Well, I feel I have to add my 5 cents worth.
First of all, I remember a study that was done by the HP HPLC R & D group
in Germany (sorry, I can't quote the reference) that looked at the
activity of proteins run on glass vs stainless steel columns. If I
remember correctly (and please correct me if I am wrong) they found that
the column was not what was killing the activity of the proteins--but
rather the pH and/or salt conditions or temperature of the separation
conditions/or how long the protein sat at room temperature and in the
elution conditions post-run. So,the
most important thing to look at during a separation was not steel vs
teflon, but just to what extent the biological material was being
tortured.
That said, PEEK tubing is a god send in terms of maintaining a
system--soooo much easier to replace.
In terms of systems that pump, for low pressure column work what seems
important is a system that can reproducibly create the
flow rate/mixing/gradients that you require. For instance, when I want to
use a low back pressure column (under 1000 psi) I don't use my Beckman
Gold system as the check valves just don't work properly at low flow
rates/back pressures. I instead walk over to the Waters 625 and use its
reciprocating pump system.
I was wondering how the new Waters system (the Alliance?) stacks up in
this and how does the HP 1100 behave under low pressure settings.
Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR