Jim,
The fact that liquid propane can exist at a range of temperatures is
actually a MAJOR advantage. While you must ensure that the
temperature of the liquid propane is just above its own freezing
point, the very high boiling point of propane ensures that there is
liquid-to-solid contact with your sample for good heat transfer.
When the cryogen boils, there is a gas-to-solid contact, slower
cooling, and a greater chance of ice crystal formation.
Liquid-to-solid contact is important for getting the sample below
~180K as fast as possible to get good sample freezing (i.e. glass
formation), regardless if the sample is a crystal, an EM grid, or a
small tissue block. Thus, the jump from the temperature where a
crystal/mother liquor freezes to the boiling point of the cryogen is
a critical regime to pass through quickly. That is why I prefer
freezing in propane. But as you correctly point out, you must ensure
that the temperature of the liquid propane is just its freezing point
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: [EMAIL PROTECTED]
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On Jun 6, 2008, at 1:09 PM, Jim Pflugrath wrote:
I would like to point out that flash-cooling in liquid propane has
the added complication that the liquid propane can have a range of
temperature and still be liquid. If you use propane you may not
know which temperature you are actually using. The temperature in
the exposed layer of the propane will be warmer (could be 231 K)
than the bottom layer unless you stir the propane. Luger's group
has published their work on flash-cooling in propane. [Raji, who
posts here often must be away from the internet :) ] See
Edayathumangalam and Luger (2005) Acta Cryst D 51, 891-898.
As an alternative to propane and ethane, one might consider carbon
tetrafluoride instead. It is a liquid between about 88 K and 145
K, so not quite the range of propane, but if you see liquid, you
know you are are below 145 K. And it's non-flammable. Note that
the CRC has the wrong boiling point for this cryogenic gas which I
believe is why CF4 is not used as much.
For more tips on cryo-cooling, see also the PDF linked at http://
www.rigaku.com/cryo/ and the references therein.
Jim
On Fri, 6 Jun 2008, R.M. Garavito wrote:
Tommi,
The question has been asked and answered not by protein
crystallography, but by cyroelectron microscopy and EM freeze etch
research. Even as far back as the early 1960's, people noticed
that liq. N2 was really slow at cooling. Read the cyroEM work on
the bacteriorhodopsin photocycle and check out the wicked
guillotine device for freezing.
The slower freezing in liq. N2 is partly due to nitrogen's low
heat capacity, which can be seen in the fact that there is only
about a 13 degree difference between the freezing and boiling
points of N2 (~64K vs. ~77K). In contrast, difference between the
freezing and boiling points for propane is almost 148 degrees
(~83K vs. ~231K). Thus, it makes sense to freeze in liq. propane,
but then shift to liq. N2 for storage and shipping. Making
propane popsicles for storage, shipping, and mounting is not
necessary.
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: [EMAIL PROTECTED]
****************************************************************
On Jun 5, 2008, at 5:11 PM, Tommi Kajander wrote:
according to literature,see below and references
http://www.px.nsls.bnl.gov/courses/papers/ZD_EG_papers.html,
it is not clear that liq. propane plunged item would cool
faster. (whilst i havent tested this)...
Would anyone have actual experimental data with protein crystals
on the hyperquenching suggested by
Warkentin, V. Berejnov, and R. E. Thorne, J. Appl. Cryst. (2006) 39,
805-811. (no diffraction data in the paper). in particular with
small samples.
thanks,
Tommi
Quoting Petr Leiman <[EMAIL PROTECTED]>:
yes you are right, but I assumed if people see a cloud of
condensed
fog over their LN2 bath they should remove that by
a) filling up the bowl completely e.g. some LN2 drips out of
the bowl
b) blow the fog away before you dip
I think the original poster meant the relatively low heat
conduction of
liquid N2, which causes boiling around the crystal immediately
after
plunging.
The best way to freeze things is to put a small container of liquid
ethane
or propane into a liquid N2 bowl, and plunge into the ethane/
propane
(this
methods was suggested earlier).
Petr
--
Tommi Kajander, Ph.D.
Macromolecular X-ray Crystallography
Research Program in Structural Biology and Biophysics
Institute of Biotechnology
P.O. Box 65 (Street address: Viikinkaari 1, 4th floor)
University of Helsinki
FIN-00014 Helsinki, Finland
Tel. +358-9-191 58903
Fax +358-9-191 59940
