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4.2 Deep Chill: Undercooled Water
It has long been known that water, especially when purified to some
extent, does not freeze spontaneously when cooled to 01C. Instead, the
liquid state is maintained at subzero temperatures, until at some arbitrary
temperature, freezing occurs spontaneously and abruptly. Although the
undercooled state may not appear to have any relevance to freeze-drying,
because chill depends on the avoidance or prevention of freezing, it is
nevertheless important because some degree of undercooling, often down
to ca.
151C, can never be avoided during an industrial freezing process,
especially one carried out in highly purified water. That undercooling can
have useful, even beneficial results, becomes apparent from an inspection
of Figure 3, where it is shown that enzyme activity in solution can be
completely maintained for long periods in an undercooled aqueous solvent
medium, without the necessity for any cryoprotectant additive addition.
The usefulness of undercooled water as a reaction medium has also
been demonstrated in biochemical studies. Thus, the kinetics of bio-
chemical reaction sequences can be sufficiently slowed down to become
time resolved at subzero temperatures in the unfrozen state. Even more
importantly, complex reaction pathways (mechanisms) are unaffected
by undercooling, whereas the use of conventional cryoprotectants
(glycerol, ethane diol, dimethylsulfoxide, etc.) alters the pathways,
although not necessarily the nature of the end products.
15
Finally, single
cells or even cell clusters can be stored and kept intact for considerable
periods in undercooled aqueous media.
16
100
Undercooled -12 and -20
°
C
80
+ 4
60
40
20
Room temperature
Frozen -12 and -20°C
0
2
3
4
5
6
60
1
Storage period (weeks)
Figure 3 Maintenance of enzyme activity by LDH, stored under differing conditions.
LDH concentration: 10 mgml
1
in phosphate buffer (pH 7). Reproduced from
Hatley et al.,
14
with permission by Elsevier
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