From: Olwyn Byron <o.byron@bio.gla.ac.uk>
  To  : rasmb@bbri.harvard.edu
  Date: Mon, 11 May 1998 12:21:13 +0100

organic solvents summary

Dear RASMBers

Thanks to the following for their helpful replies during the past week to
the questions I asked on organic solvents:

Emory Brasswell
Helmut Coelfen
Neil Errington
Steve Harding
Walter Maechtle
Jeff Messer
Michael Morris
Paul Voelker

Here is a summary of the responses:


Qu 1.
Is it OK to do runs in either chloroform or dichloromethane in the XL-A?

An 1.
Both solvents are OK. Dichlormethane appears to be preferable in certain
cases because chloroform has a very high density and most polymers will
float in it. Chloroform is useful as a co-solvent to built up density
gradients.

The specific gravity of chloroform is 1.49. If proteins are being
centrifuged, they will float (this probably goes for dichloromethane too).

Dichloromethane absorbs below 230nm and chloroform below 240 nm.

Both solvents are compressible to a much greater extent than water this
means that at finite rotor speeds a density gradient will be established.
This needs to be corrected for.


Qu 2.
If so which is preferable?

An 2.
See An 1!


Qu 3.
Are there any specific precautions I should take (eg should I only use
Aluminium centrepieces or perhaps Kel-F ones)?

An 3.
There are certain necessary precautions: since both solvents have
relatively high vapour pressure, it is wise to start the rotor spinning as
soon as the vacuum is applied. At speed there shouldn't be any leakage and
leaks are definitely to be avoided as organic solvents may give problems
with vacuum seals and may get into the diffusion pump oil, although this is
probably not disastrous with the amounts of solvent in a typical
equilibrium run. One respondent used a full column height of heptane in a
sedimentation equilibrium run for 2 weeks at 37C with out any problems.
Another reason leaks are bad is because the solvent vapours can be drawn
out by the pump into the lab: this clearly is both a health risk and a fire
risk.


I received slightly contradictory advice from people about which
centrepieces were best:

Some recommend the carbon (charcoal-filled) Epon centrepieces for either
methylene chloride or chloroform and advise that aluminium on the other
hand can react explosively with either solvent. Others were aware of this
hazard but remarked that this concerns very fine powdered metal dust which
can only occur in a rotor accident. And in any case, the rotor and housings
are made of titanium or aluminium! Even in the event of a rotor accident
this explosion hazard is likely to be negligible again due to the small
volumes of solvent used. These warnings are more likely to concern
preparative centrifuges where such solvents definitely cannot be run.

Others maintain that Epon centrepieces should not be used but rather
titanium (or possibly aluminium) or Kel-F centrepieces although the same
respondents note that Beckman solvent compatibility sheets say that the
resistance of Epon is satisfactory with chloroform and dichloromethane.
However there is a risk that organic solvents may swell Epon resulting in a
loss of mechanical strength. Yet another respondent knows of an instance in
which Kel-F centrepieces swelled and were destroyed by this type of organic
solvents.

Polyethylene gaskets are recommended.


The following references were recommended

1. Maechtle, W., Analysis of polymer dispersions with an
eight-cell-AUC-multiplexer: high resolution particle size distribution and
density gradient techniques, in Analytical Ultracentrifugation in
Biochemistry and Polymer Science, S.E. Harding, A.J. Rowe, and J.C. Horton,
Editor. 1992, Royal Society of Chemistry: Cambridge. p. 147-175.

2. Professor Schubert's new paper (which was given at the 1997 Regensburg
Meeting).

3. Morgan, P.J., Harding, S.E. and Petrak, K., Hydrodynamic properties of a
polyisoprene/poly(oxyethylene) block copolymer. Macromolecules, 1990. 23:
p. 4461-4464.



So the centrepiece controversy sort of remains, although my belief is that
aluminium or titanium centrepieces will be best for this work.
Unfortunately I don't have any of these so if anyone has any that they
could lend me for a couple of runs I'd be extremely grateful!

In the meantime best wishes,
Olwyn Byron.


_____________________________________________________________________
Olwyn Byron
Division of Infection and Immunity
Institute of Biomedical and Life Sciences
Joseph Black Building, University of Glasgow
Glasgow, G12 8QQ, Scotland.

tel: +44 (0)141 330 3752  email: o.byron@bio.gla.ac.uk
fax: +44 (0)141 330 4600  web:   www.gla.ac.uk/Acad/IBLS/II/ob/ob.htm
_____________________________________________________________________