hhope at ucdavis.edu
Sat Nov 4 22:04:15 EST 1995
This version may be easier to read on some newsreaders
Joachim (Jo) Seemann wrote
>who know's useful cryo protectants other than
>PEG, glycol, glycerol, sucrose, MPD ?
>My xtals tolerate glycerol but get destroyed on moving the loop to>the cold stream or freezing.
The best way to proceed is not to search for another cryoprotectant,
but rather to learn a simple method that we have found to be very
effective. The following paragraphs are excerpts from a document
that I prepared for a cryoworkshop held recently at SSRL:
It has become apparent that in virtually all cases of ice formation on
cooling to cryogenic temperature the process starts in a layer of liquid
on the surface of the crystal. It is therefore necessary to remove or
modify this layer.
In the simplest cases one can begin by covering the crystal and some
of the mother liquor with oil. One can then blot off any visible liquid, or
else strip it off by moving thecrystal through the oil.
More difficult cases require that the surface layer be modified with a
cryo-protectant. Cryoprotectants are compounds that prevent ice formation
on cooling when added to aqueous solutions. Typically the cryoprotectant
is added to a portion of mother liquor, usually in the range 10-25% by
Because the action is at the surface of the crystal, it is normally not
necessary to soak the crystal for any length of time. A few seconds
(<10 s) of rinsing will often suffice. There may be crystals that require
more time to disrupt the water layer on the surface. Some crystal do
not tolerate the modified mother liquor well, so they should be cooled
as soon as possible after rinsing.
An effective way of cooling is to plunge the crystal on its mounting pin i
nto liquid nitrogen. Cool-down to 140 K can be accomplished in 0.6 sec
for a crystal of 0.75 mm cross section. (Further cooling is of course also
very quick. 140 K was used as a reference because at 140 K crystal are
generally safe, and because it is a temperature often seen in gas stream cooling; it was not used because that temperature is desirable.)
***The use of special coolants (e.g. liquid propane) is not recommended.
Such coolants complicate the operation significantly, and add non-trivial
hazards, with no cooling advantage (see below). ***
When we use an open-flow gas-stream apparatus there are in practice two
ways of cooling the crystal: either directly in the cold stream, or by immersion
in a cold liquid before the crystal is moved to the gas stream. Cooling in the
gas stream is a simple procedure, and should be used whenever practical.
However, some crystals do not tolerate the delay caused by carrying the
crystal from the mounting station to the diffraction apparatus, so that
immediate cooling is needed. Other crystals, especially larger ones, may
fare better with liquid cooling. This is best done by immersing the crystal
in a cryogenic liquid.
When a larger object at room temperature is immersed in liquid nitrogen
at its boiling point, large amounts of gas form. This gas creates an insulating
layer around the object, so that cooling to liq N2 temperature is relatively slow.
A similar experiment, using liquid propane near its freezing point as the coolant,
results in less bubble formation, and a more rapid cooling. This observation
on the behavior of large objects has led to the belief that liquid propane is a
better coolant than liq N2 also for crystals intended for x-ray diffraction
experiments. This belief has no foundation in reality. I have measured the
cooling rates of crystal-sized objects in three different media:
(1) Cold nitrogen gas (100 K)
(2) Liquid nitrogen (77 K)
(3) Liquid propane (100 K)
Probes were 0.25 mm thermocouples coated with glue to a total
diameter of 0.75 mm. Temperature related EMFs vs time were
measured with a 12-bit, multichannel A/D converter board
mounted in an Intel based PC. Readings were taken at 0.01 sec
N2 gas: Target temperature (140 K) reached in about 2 s.
Liquid N2: 140 K reached in about 0.6 s from the time of immersion .
Liquid propane: 140 K reached in about 1.2 sec.
The unambiguous conclusion to be drawn from these measurements is
that liquid N2 yields the highest cool-down rate among the three media tested. Because the rate of heat transfer is proportional to the temperature difference
there is an a priori advantage to liquid N2. For the small samples used, heat
transfer is not impeded by bubble formation.
One disadvantage of liquid propane is the tendency for the upper layer
of liquid to warm up toward the boiling point, unless it is stirred. I have
measured more than 100 K temperature difference between top and
bottom of a small container. (Propane has fp 83 K and bp 228 K.)
The simplicity of the liquid N2 method should make it the method of choice.
The use of liquid propane adds both hazards and operational complications,
without any apparent cooling advantage. A large number of protein crystals
have been successfully cooled in liquid nitrogen.
The liq N2 technique requires some special tools. Such tools have been
designed and thoroughly tested. They include devices needed for long-term
storage and transport to a remote site.Designs are available on request.
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