Hi, Dick -
Elute the system at a low flow rate with 40 mM EDTA.2Na, right out of the
bottle (i.e., don't adjust pH; just filter). You can leave your HPLC columns
in line, passivating the metal parts in them as well (NOTE: Titanium frits
are "inert" in that they aren't corroded by halide salts, but they can still
interact with susceptible proteins. This varies from frit to frit, driving
HPLC column manufacturers nuts. The passivation takes care of them too). Do
this for at least 16 hours at room temp., and preferably 24 hours. You can
recycle the solution after the first hour. In a few cases with particularly
susceptible proteins, our customers had to elute all weekend before finally
getting sharp, symmetrical peaks.
Passivating the metal parts of the column improves the peak shape of
susceptible proteins. An example of the latter is basic fibroblast growth
factor. Now, with an extreme case, such as transferrin - a heavy
metal-binding protein - you'll only get reproducible results if you eliminate
all metal components from the column.
This procedure passivates the system for 3-4 weeks of sustained exposure to
NaCl; repeat it thereafter. If you don't have EDTA.2Na on the shelf, buy
some. Do NOT prepare it by adding 2 stoichiometric equivalents of NaOH to
EDTA free acid. One of our customers did that. Trouble is, this is an
unbuffered solution. He didn't bother to check his pH, which was 9.3. That
killed a silica-based column fast.
Best regards,
Andy Alpert
PolyLC Inc.
****************************************************************
<< Subj: HPLC - passivation
Date: 2/3/2000 6:46:08 PM Eastern Standard Time
From: cook@MIT.EDU (Richard F. Cook)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
Dear ABRFers,
Does any one have a good HPLC passivation SOP?
I'd like someting that I can run through the whole system from buffer
bottle to flow cell with out doing damage. I'd like to do this to a HP1100.
I've seen some talk on the ABRF List using 6N HNO3 for 30' at high flow
rate but that seems too severe. HP tech support suggests 1N HNO3 but they
do not have a time period for that step.
Thanks in advance.
Dick
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