THERMAL ANALYSIS
13
«
Uncertainty‘ as to the precise ?mechetismof‘actiobf such protective additives has in
most cases necessitatedtheir
to be 'of an empirical nature. Damage on
considered to be caused either by intracellular' ice formation or salt concentration upon the
dehydration ofthe cell during the'formation of extracellular iCe. Lovelôclc (1953) sh0we'd
that glyc'erol effectively reduced the concentration of salt in equilibrium with ice at any
temperature below the freezing point—of the solution. This has been claimed to be ”imerely
an example of the colligativepropcärües' of solütiOns that the freezing point
dépressed
.
and that the
reduced before the conæntra‘ti0n of *the electr‘olyte
reaches any particular level—.— Lôveloc‘:k—Ÿtherefore supported the theory that glycerol acted
by preventing salt concentration. » More recent support’for this theory of
zing has come from Farrant (1965) working With dimèthyl sulph‘0xide.
found that the toxicity of the‘ compound was temperature dependent he
whereby dimethyl sulphoxide Was added in suitable increasing concentrations during
cooling and was therefore able to increase the elÏectivecÔncentration at lower temperatures
and so prevent a build up in salt concentration. From
diagram
of water, dimethyl sulphoxide andsodium chloride he was able to show that at ——4 79°C
the cells Were in fact being stored in the liqu‘id‘state
of 5
V/V dimèthyl .
sulphoxide. He was also able to deduce
temperature-for the triple phase
system should be lower than —“ 136 °C Which was—the feutectic temperature
.
sulphoxide and water alone.
-
'
—
…
,
.
,
,
__
The alternative theory that protectives acted by prevention Of
formations
was put forward by Mazur (1960). “Workingw1th the yeast Saccharomyces cèfèvz‘sia‘e he
found that the most lethal combination of events to the Cell
extracéular freezing,
a critical low temperature and a slow rate of warming. From his results he concluded
that at the temperatures involVed the size of the extracellular iceorystals were such that
they could pass through water lled pores Within the cell membrane and so induce crystal—
lis’ation in the supercooled intracellular water. Slow warming then permitted growth of
such crystals Within the cell. Sucrose was found to prevent ice crystals penetrating holes
in a polystyrene sheet (Lusena & Cook, 1953) and it was suggested that such additives
a"orded
protection by preventing seeding across membranes land the resulting
intracellular ice formation.
,
‘
Proposals as to the damage which occurs on freeze7drying depend to alarge extent on
hypotheses as to the mechanism of protection by various additives‘. The success achieved .
with sugars has been attributed
to buffer the residi1al moisture‘to a parti—
;
cular level, which is generally accepted to be appr0XimatÏely * 1 %However, sugars have
'
also been found to protect againstÿfr‘eezing injury and! the ‘lethàl en‘ecrs
of
durmg
_
'
'
storage. Serum and other pÿotætiŸe Colloids provide asœfom
to forma
The protective action of such colloids may depend upon
their
to make the
Withdrawal of water less “Violent, Whilst not inuendng”th6hàlwater content.
It
'
been suggested that they may comiaress
cbnträded plasma membrane in plasmolysed cells-“and débæ'aSe-the_volumëËîôf
the intenspace
between the two-=
_
membranes and
associated—osmotic;
;
e‘eCts. Sodium glutamate
reaction betWeen“
_
groups andanino
At this
1107,
iclëâr' distinction betWeen any
a great—deal ef oveap ?
.
"betweeneach additive and thevarmuspr0perüesdiscussed(aVIeS1963)
At the
”
13
«
Uncertainty‘ as to the precise ?mechetismof‘actiobf such protective additives has in
most cases necessitatedtheir
to be 'of an empirical nature. Damage on
considered to be caused either by intracellular' ice formation or salt concentration upon the
dehydration ofthe cell during the'formation of extracellular iCe. Lovelôclc (1953) sh0we'd
that glyc'erol effectively reduced the concentration of salt in equilibrium with ice at any
temperature below the freezing point—of the solution. This has been claimed to be ”imerely
an example of the colligativepropcärües' of solütiOns that the freezing point
dépressed
.
and that the
reduced before the conæntra‘ti0n of *the electr‘olyte
reaches any particular level—.— Lôveloc‘:k—Ÿtherefore supported the theory that glycerol acted
by preventing salt concentration. » More recent support’for this theory of
zing has come from Farrant (1965) working With dimèthyl sulph‘0xide.
found that the toxicity of the‘ compound was temperature dependent he
whereby dimethyl sulphoxide Was added in suitable increasing concentrations during
cooling and was therefore able to increase the elÏectivecÔncentration at lower temperatures
and so prevent a build up in salt concentration. From
diagram
of water, dimethyl sulphoxide andsodium chloride he was able to show that at ——4 79°C
the cells Were in fact being stored in the liqu‘id‘state
of 5
V/V dimèthyl .
sulphoxide. He was also able to deduce
temperature-for the triple phase
system should be lower than —“ 136 °C Which was—the feutectic temperature
.
sulphoxide and water alone.
-
'
—
…
,
.
,
,
__
The alternative theory that protectives acted by prevention Of
formations
was put forward by Mazur (1960). “Workingw1th the yeast Saccharomyces cèfèvz‘sia‘e he
found that the most lethal combination of events to the Cell
extracéular freezing,
a critical low temperature and a slow rate of warming. From his results he concluded
that at the temperatures involVed the size of the extracellular iceorystals were such that
they could pass through water lled pores Within the cell membrane and so induce crystal—
lis’ation in the supercooled intracellular water. Slow warming then permitted growth of
such crystals Within the cell. Sucrose was found to prevent ice crystals penetrating holes
in a polystyrene sheet (Lusena & Cook, 1953) and it was suggested that such additives
a"orded
protection by preventing seeding across membranes land the resulting
intracellular ice formation.
,
‘
Proposals as to the damage which occurs on freeze7drying depend to alarge extent on
hypotheses as to the mechanism of protection by various additives‘. The success achieved .
with sugars has been attributed
to buffer the residi1al moisture‘to a parti—
;
cular level, which is generally accepted to be appr0XimatÏely * 1 %However, sugars have
'
also been found to protect againstÿfr‘eezing injury and! the ‘lethàl en‘ecrs
of
durmg
_
'
'
storage. Serum and other pÿotætiŸe Colloids provide asœfom
to forma
The protective action of such colloids may depend upon
their
to make the
Withdrawal of water less “Violent, Whilst not inuendng”th6hàlwater content.
It
'
been suggested that they may comiaress
cbnträded plasma membrane in plasmolysed cells-“and débæ'aSe-the_volumëËîôf
the intenspace
between the two-=
_
membranes and
associated—osmotic;
;
e‘eCts. Sodium glutamate
reaction betWeen“
_
groups andanino
At this
1107,
iclëâr' distinction betWeen any
a great—deal ef oveap ?
.
"betweeneach additive and thevarmuspr0perüesdiscussed(aVIeS1963)
At the
”