Hakon Hope hhope at
Sat Nov 4 16:38:53 EST 1995

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 the crystal 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 volume.

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 into 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 intervals.

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 longterm storage and transport to a remote site.

Designs are available on request.

Hakon Hope

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