CRYOBIOLOGY
12, 202-208 (1975)
The Physical State of Water at Low Temperatures in Plasma with Different Water Contents as Studied by Differential Thermal Analysis and Differential Scanning Calorimetry D. SIMATOS, M. FAURE I.B.A.N.A., UnioeTsitt? de Dijon, France AZID
E. BONJOUR
M. COUACH
AND
Centre d’Etudes Nucldaires, Grenoble, France
The physical state of biological systems at low temperatures has been the object of numerous studies, since information in that field is necessary for a better understanding of, first, the changes induced by freezing, low temperature storage, or freeze-drying, and, second, the funct:ons of cryoprotective agents. Moreover, these studies can provide insight on interactions of water with the other components of bioIogica1 sys,tems. Different ‘techniques can be used for that purpose, especially diIatometry (711 ), measurement of electrical conductivity (lo), nuclear magnetic resonance (19, 20 ). However, most studies have made use of thermal and caIorimetric methods. We have to recognize that despite the great amount of work done, some of the observed thermal phenomena are still of uncertain nature. In the present study, differential thermal analysis (DTA) and differential scanning calorimetry (DSC) have been used for the anaIysis of the physica state of blood plasma at low temperatures, which has been considered as a mode1 for bioIogica1 systems, Received December 4. 1974.
MATERIALS
Sample Preparation The product is freeze-dried plasnia,1 separated from human bl,ood diluted with the ACD Z anticoagulant solution. The plasma is rehydrated to different water contents 3 foIlowing two procedures, ( 1) Samples prepared with a view toward DTA are obtained by mixing a rather large quantity of dry plasma (1 g) with the necessary quantities of water. They are stored at 4°C for at least 24 hr before DTA. This procedure results in a SatisfactoriIy homogenous rehydration, since different samples taken from the mixture provide quite reproducible DTA diagrams. (2) Samples to be submitted to DSC are prepared directly in the DSC cell by adding the necessary quantities of water to a 1 Freeze-dried plasma obtained from the Centre Regional de Tramfusion Sanguine. 2 One volume of ACD solution (citric acid 0.8 g, sodium citrate 2.2 g, dextrose 2.45 g, water 100 g) is added to 5.5 voIumes of blood. 8 Water contents are indicated as grams of water per grams of dry material, taking into account: the residual moisture of plasma, as determined by Fischer titration.
202 Copyright 0 1975 by Academic Press, Inc. All rights of reprcdoctionin any form reserved.
AND METHODS
PHYSICAL
STATE
OF WATER
AT LOW
TEMPERATURES
203
IN PLASMA
constant weight of dry plasma (approximately 20 mg), the combined weight of water and plasma being determined with the aid of a microbalance. The system reaches an equilibrium state of hydration after 24 hr (see Results: melting section), Diflerential
Thermal Analysis (DTA)
The DTA is performed with an apparatus built in the laboratory in collaboration with Centre d’Etudes CryogAniques ( Grenoble). The sample and the refrence (powdered silver ) are in pans hermetically cIosed by crimping with a special tool. Thermocouples are made with chrome]-alumel. The sample temperature is determined by reference to calibrat:on performed with standards having known transition temperatures. The DTA cell is cooled to -150°C through the evaporation of liquid nitrogen in the double wall. The ceI1 is then evacuated to 0.1 Torr and filled with helium. Cooling
FIG. 2. Differential thermal analysis (DTA) of rehydrated plasma (water content = 0.48 g-g-‘). (a) DTA obtained in the same conditions as in Fig. 1. (b) DTA after the sample has been first rewarmed to -44”C, then cooled again,
and heating are controlled by a monitoring device and can progress at different speeds. The usual conditions are: for cooling, 22°C mn-1 and for rewarming, 2°C mn-1 (between 0 and - 100°C and conversely). Differential
Scanning Calorimetry
(DSC)
Calorimetry is done with the CPC 600 (3), an apparatus of the DSC type. Samples are in hermeticalIy closed pans. A rather sl:ow cooIing is obtained by circulating a cold gas (cooling rate: 6 to 8°C mn-1 about 0°C). Immersion of the sample holder land sample pan in liquid nitrogen results in a higher cooling rate. More rapid cooling is obtained by immersion of the sampIe pan in liquid nitrogen and then introducing it into the cold sample holder. It has been demonstrated by electrical simulation of a reaction in the apparatus and by measurement of the heat of fusion of pure materials that the energy absorbed or evolved in the sam,ple can be measured with a precision of T 1% (U). RESULTS
FIG. 1. Differential thermal analysis (DTA) of freeze-dried plasma rehydrated to different water contents. Temperature difference is recorded as a function of time. Temperature indicated on the figure as “C (cooling rate = 22°C mn?; rewarming rate = 2°C n-x-‘).
The DTA and DSC reoordings are quite similar. They also look very much like those which have been obtained with similar products: beef muscle (17: scanning calorimetry) or egg white (12: DSC). If we look, for example, at DTA scans provided by pIasma with a water c0nten.t of 0.48 g* g-l, we observe the following events (Fig. 2):
SIMATOS
204
-100
-80
-60
-40
-20
‘C
FIG. 3. Differential scanning calorimetry ( DSC ) of rehydrated plasma (water content = 0.5 g.g-‘), The figure indicates the graphical bases for the measurements.
(1) an inflexion of the curve (at G) which indicates a change in the specific heat of the sample, (2) a peak (D) corresponding to an exothermal event, and (3) a peak (M) corresponding to an endothermal event. These curves are very comparable to DTA curves obtained with aqueous solutions of glycerol (2, 9, 15, 16), sugars and ethyleneglycol (9, 15), polyvinyl pyrrohdone (8). By referring to these curves, we can ascribe the above events to the following transitions: (1) G: glass transition; (2) D: devitrification of an amorphous phase, or recrystallization; and (3) M: melting, beginning at TIM, and ending at T M* The interpretation will be confirmed by the following results, but we must emphasize that it is somewhat different from previous ones; for beef muscle with a water content of 0.34 g *g-r, Rasmussen ( 14) identifies a glass transition at -92°C but indicates the incipient melting at a point which would be equivalent to the maximum of devitrification ( D ).
ET AL.
of samples (g per g of dry weight); H, = constant; L = constant. L is equal to 77.2 calmg-l, a value quite close to 79.6 calag-I, latent heat of melting of ice. H, can be considered as the nonfreezable water. H, = 0.466 g per g of dry weight. The DSC scans considered here have been performed on samples having reached a homogenous state of hydration, as is inferred from the constant melting area obtained in successive DSC on the same sample. Samples with the lowest water content usually provide a larger melting peak immediately after preparation, which decreases and eventually vanishes in subsequent scans. We feel that the small, persistent mehing peaks which are observed for water content near 0.47 g/g are due to still imperfect equilibrium between water and dry substance. As can be seen on Fig. 5, the incipient melting temperature, Tl,v, was the same for all water content. This had also been observed with aqueous solutions of glycerol ( 2,15), sugars
150,
Malting The area of the endotherma DSC peak (Fig. 3) is a measure of the amount of heat which is absorbed by the sample whiIe mehing. Figure 4 gives the regression line for A, = L (H - H,). A, = area of endothermal peak (cal per g of dry weight); H = total water content
FIG. 4. Area of the melting peak measured on differential scanning calorimetry (DE) scans as a function of water content. Data represented by hollow circles have not been considered in the calculations.
PHYSICAL
STATE
OF WATER
AT LOW
and ethyleneglycol (9, 15) and PVP (8). It was then concluded that melting was concerned with a solution of constant concentration, whatever the dry matter content of the sample. The same conclusion can be drawn here. Whast was not observed with the above solutions and can be seen in Fig. 1, is that melting occurs in plasma in two stages, giving two fused peaks. The first of these is the only one at the lowest water content; it undergoes a Iimited development when water content increases. On the contrary, the second one is greatIy enhanced above water contents ‘of 0.50 g *g-l. Euolution
of Glass Phase
During cooling, a part of the water of the system under study is soEdified into an amorphous state. During rewarming, the glass phase undergoes a second-order transitlon (glass transition: G ), then a partial crystallization (D). The nature of these phenomena is demonstrated by the changes in the DTA or DSC scans when a thermal treatment, annealing, is d.one; after being rewarmed to temperature Tn, the sample is cooled, then rewarmed again (Fig. Zb ). During the second scan, there is no further sign of an exothermal peak, the glass transition is #of a reduced amplitude and ,occurs at a higher temperature. These changes are the consequence of the irreversible character of the crystalIization which has occurred during the first rewarming process.
TEMPERATURES
205
IN PLASMA
w.g.’
CL----J 0
20
40
60
concentration
80 wiw 9(
FIG. 6. Change in the rate of energy absorption during the G transition ( watt per g of dry weight ). l , after slow cooling (643”C*mn~‘); a, after quicker cooling; 0, after very rapid cooling (set Methods ); *, after anueolinp.
Figure 6 indicates the amplitude, related to the unit of dry weight, of the change in specific heat during the transition G as a function of concentration. The transition can be observed only when water content is higher than 0.25 g* gl, The amplitude increases when water content is raised from 0.25 to about 0.45 g-g-l and remains constant for higher water contents. As can be seen on Fig. 7, after a rather slow cooling, the temperature of transition G is approximately constant for water contents between 0.4 and 5 g*g-“. But Tn is lower when cooling has been quicker, The area of exothermal peak D (Fig, 8) is proportional
‘C
-20
i
concenfmtion
w/w
*
FIG. 5. Temperature of incipient melting (7) and of Anal melting (A) as a function of concent,ration (g of dry substance per 100 g of sample).
1 0
20
40
60 concentration
80 wviw 04
FIG. 7. Temperature ?‘a. For symbols see Fig. 6, 0 represents means of values obtained in DTA with the same conditions as in Fig. 1.
206
! .I.( I
. !
SIMATOS ET AL.
FIG. 8. Area of the exothermal peak D, measuring the amount of water crystallized during rewarming (cal per g of dry weight). Symbols as in Fig. 6.
plitude which remains proportional to the amount of dry substance in the sample, even when the water content is strongly increased (Fig. 6). After annealing, a glass transition is still observable (Fig. 2b and S), The values for To suggest that the glass phase has the same water content as after a slow cooling. One might thus think that, in all samples having a water content higher than about 0.45 gag-l, a glass phase with this water content is present, even after a slow cooling or a thermal treatment. Making use of an anaIysis method which has been developed for the study of crystallinity in polymers (13) one might state: AwG/v*Acp
to the
quantity
of ice which
is farmed
during rewarming. As has been explained in sever.al papers (1, 6, 18), the increase in concentration and the decrease in temperature during cooling are responsible for a great increase in the viscosity of the interstitial solutions and the crystal growth is inhibited; the remaining interstitial solutions are solitied into a glass. The water content of the thus formed amorphous phase is higher, the m’ore rapid the cooling rate, During rewarming, visoosity can be lowered enough to enable a more compIete crystallization of water. As can be seen on Figs. 6 and 8, the amplitude
of the G transition
and the
area of ,peak D are larger after a more rapid cooling. For a rather slow cooling, the proportion of water incorporated in the glass phase and CrystaIlizing ,during rewarming is especially importanmt in sampIes with a water content close to 0.6 gag-l. Diagrams simiIar to Fig. 7 have been published for various simple aqueous solutions undergoing slow cooling: glycerol (2, 15) ethylenegIyco1 and glucose (15). The fact that To was independent of the original concentrati,on was explained (15) by a constant water content in the glass phase. This oonclusion is directly confirmed by the observation of the G transition am-
= 1 - (AN - A,)/77.2.
AWo = change in the amount of energy absorbed by the sample during the G transition per unit of time, and by unit of weight; ACP = change in the specific heat of the glass phase; o = rewarming rate; AWo/v . ACp is thus the fraction of the
sampIe solidified into glass during coohng; (AM - A,)/77.2 is the fraction of the sampIe sohdified into ice during coobng. Figure 9 represents AWo as a function of (A,, - A,). A straight Iine is obtained with a satisfactory approximation. The w-g-’ 0.02 “I . I\
*
. l
0.01
**
0
.
r.
3 c
\\\\
o1 0
0
’ Y,, 20
40
60
80 c.I.g-’
FIG. 9, Representation of the extent of crystaIlization at low temperatures (see text).
PHYSICAL
STATE OF WATER AT LOW TEMPERATURES
extrapolation of the ,line to Aaw-AD = 0 provides the specific heat change revealing the glass transition: AC, = 0.13 cal*gml”C?.
It would have been expected that AC, would take different values following the water content of the glass phase. It seems however that the resulting variations for aWo are negligible beside the accuracy of the measurement. CONCLUSION
The quantitative analysis performed on DSC curves seems to provide a further confirmation of the interpretation of the observed phenomena (G = glass transition; D = ,devitrii?cation; IM = incipient melting). In simpler systems such as PVP ( 12), sucrose, or ,dextrose f S), a glass transition could be observed at very low moisture content at temperatures above 0°C. With the present material, however, the glass transition could not be observed with samples having water content below 0.25 gag-l even when the DTA or DSC scans were carried on to temperatures as high as 70°C. The low probability of occurrence of this phenomenon in very low moisture samples is confirmed by the steep increase ,of Tci when the water content is decreased. It is thus suggested that 0.25 gsg-l represents a r.ather critical level in water content for the present system. For water contents above this value, some components of the system can gain enough mobility to give rise to a detectable gIass transition. SUMMARY
Differential therma analysis ( DTA ) and differential scanning calorimetry (DSC) have been used for a study of the physical state of water at low temperatures in freeze-dried bloo,d plasma rehydrated to different water contents. Measurement ,of the amplitude of the specific heat change accompanying the glass transition, the quantity of ice formed during rewarming,
IN PLASMA
207
and the total ice content in the sampIe provided a determination of the extent of crystallization in water at low temperatures. Even after slow cooling or annealing, a fraction of water remained incorporated in an amorphous phase. It is suggested that the unfreezable water (0.47 g per g of dry substance) could be divided into two more or Iess distinct species: 0.25 g per g is the minimum water content above which the system can gain enough mobility to give rise to a detectable grass transition. ACKNOWLEDGMENT The authors thank Dr. Alan Mac Kenzie for a fruitful discussion about the present study. REFERENCES 1. Bellows, R. J. Freeze drying of liquid foods: dependence of collapse temperature on composition .and concentration. Thesis, University of California, Berkeley, 1972. 2. Bohon, R. L., and Conway, W. T. DTA studies on the glycerol-water system. T’hermochim. Acta 4, 321-341 (1972). 3. Bonjour, E., Couach, M., and Pierre, J. de la Quelques examples d’application technique de calorimktrie par compensation de puissance, Joumires de Calorimktrie, Lyon, France 1970. 4. Chevalley, J., Rostagno, W., and EgIi, R. H. A study of the physical properties of chocolate. The application of differential microcalorimetry to chocolate and confectionery. Reu. Int. Choc. 25, 3-6 ( 1970). 5. Couach, M., Bonjour, E., and Chavanel, H. Mesures de chaleur de r&action en analyse enthalpique differentielle. Int. Conf. on Thermal Analysis, Davos, France, 1971. 6. King, C. J. Apphcations of freeze-drying to food products. Vlth International Course on Freeze Drying, Lucerne, Switzerland, 1973. 7. Lusena, C. V. Ice propagation in glycero1 solutions at temperatures below -40°C. In “Freezing and Drying of Biological Materials” (H. T. Meryman, Ed.), Ann. N. Y. Acad. Sci. 85,541-548 (1960). 8. Luyet, B., and Rasmussen, D. Study by differential thermal analysis of the temperatures of instability in rapidly cooled solutions of polyvinylpyrrolidone. Biodynamics 10, 137-147 (1967). 9. Luyet B., and Rasmussen, D. Study by differential thermal analysis of the temperatures of instability of rapidly cooled solutions of
208
10.
11.
12.
13.
SIMATOS ET AL. glycerol, ethyleneglycol, sucrose and glucose. Bdodynamica 10, 167-191 ( 1968). Mac Kenzie, A. P., and Rasmussen, D. H. Interactions in the water-polyvinylpymolidone system at low temperatures. In “Water Structure at the Water-Polymer Interface” (H. H. G. Jellinek, Ed.), pp. 146-171. Plenum, New York, 1971. Moran, T. The freezing of gelatin gel. Proc. Boy. Sot. London Ser. A. 112, 30-40 (1926). Parducci, L. G., and Duckworth, R. B. Differential thermal analysis of frozen food systems. II. Micro-scale studies on egg white, cod and celery. J. Fd. TechmE. 7, 423430 ( 1972 ). Pineri, M., Bonjour, E., Gerard, P., and MartinD’Hermont, F. L’analyse enthalpique diffkrentielIe associbe a des mesures mkcaniques pour 1’8tude des prop&es physiques des polymkres. Phtiques Modernes et Elastom&es, 156-105 ( 1972 ).
14. Rasmussen, 13. A note about “phase diagrams” of frozen tissues. Biodynatmca 10, 333-339 (1969). 15. Rasmussen, D., and Luyet, B. Complementa,ry study of some nonequilibrium phase transitions in frozen solutions of glycero1, ethylene glycol, ethylene gIycc1, glucose and sucrose. Biodynamics 10, 319-331 ( 1969). 16. Rey, L. R. “Conservation de la Vie par Ie Froid,” p, 167. Hermann, Paris, 1959. 17. Riedel, L. Zum Problem des gebundenen Wassers in Fleisch. Kiiltetechnik 13, 12% 128 (1961. 18. Rollet, A. P., and Bouaziz, R. “L’Analyse Thermique. Tome I: Les Changements de Phase,” p. 357. Gauthier-Vilallars, Paris, 1972. 19. Sussman, M. V. Liquid water in hozen tissue: study by NMR. Science 151, 324-325 (1966). 20. Toledo, R., Steinberg, M. P., and Nelson, A. I. Quantitative determination of bound water by NMR. 1. Fd. Sci. 33, 315-317 (1968).
12, 202-208 (1975)
The Physical State of Water at Low Temperatures in Plasma with Different Water Contents as Studied by Differential Thermal Analysis and Differential Scanning Calorimetry D. SIMATOS, M. FAURE I.B.A.N.A., UnioeTsitt? de Dijon, France AZID
E. BONJOUR
M. COUACH
AND
Centre d’Etudes Nucldaires, Grenoble, France
The physical state of biological systems at low temperatures has been the object of numerous studies, since information in that field is necessary for a better understanding of, first, the changes induced by freezing, low temperature storage, or freeze-drying, and, second, the funct:ons of cryoprotective agents. Moreover, these studies can provide insight on interactions of water with the other components of bioIogica1 sys,tems. Different ‘techniques can be used for that purpose, especially diIatometry (711 ), measurement of electrical conductivity (lo), nuclear magnetic resonance (19, 20 ). However, most studies have made use of thermal and caIorimetric methods. We have to recognize that despite the great amount of work done, some of the observed thermal phenomena are still of uncertain nature. In the present study, differential thermal analysis (DTA) and differential scanning calorimetry (DSC) have been used for the anaIysis of the physica state of blood plasma at low temperatures, which has been considered as a mode1 for bioIogica1 systems, Received December 4. 1974.
MATERIALS
Sample Preparation The product is freeze-dried plasnia,1 separated from human bl,ood diluted with the ACD Z anticoagulant solution. The plasma is rehydrated to different water contents 3 foIlowing two procedures, ( 1) Samples prepared with a view toward DTA are obtained by mixing a rather large quantity of dry plasma (1 g) with the necessary quantities of water. They are stored at 4°C for at least 24 hr before DTA. This procedure results in a SatisfactoriIy homogenous rehydration, since different samples taken from the mixture provide quite reproducible DTA diagrams. (2) Samples to be submitted to DSC are prepared directly in the DSC cell by adding the necessary quantities of water to a 1 Freeze-dried plasma obtained from the Centre Regional de Tramfusion Sanguine. 2 One volume of ACD solution (citric acid 0.8 g, sodium citrate 2.2 g, dextrose 2.45 g, water 100 g) is added to 5.5 voIumes of blood. 8 Water contents are indicated as grams of water per grams of dry material, taking into account: the residual moisture of plasma, as determined by Fischer titration.
202 Copyright 0 1975 by Academic Press, Inc. All rights of reprcdoctionin any form reserved.
AND METHODS
PHYSICAL
STATE
OF WATER
AT LOW
TEMPERATURES
203
IN PLASMA
constant weight of dry plasma (approximately 20 mg), the combined weight of water and plasma being determined with the aid of a microbalance. The system reaches an equilibrium state of hydration after 24 hr (see Results: melting section), Diflerential
Thermal Analysis (DTA)
The DTA is performed with an apparatus built in the laboratory in collaboration with Centre d’Etudes CryogAniques ( Grenoble). The sample and the refrence (powdered silver ) are in pans hermetically cIosed by crimping with a special tool. Thermocouples are made with chrome]-alumel. The sample temperature is determined by reference to calibrat:on performed with standards having known transition temperatures. The DTA cell is cooled to -150°C through the evaporation of liquid nitrogen in the double wall. The ceI1 is then evacuated to 0.1 Torr and filled with helium. Cooling
FIG. 2. Differential thermal analysis (DTA) of rehydrated plasma (water content = 0.48 g-g-‘). (a) DTA obtained in the same conditions as in Fig. 1. (b) DTA after the sample has been first rewarmed to -44”C, then cooled again,
and heating are controlled by a monitoring device and can progress at different speeds. The usual conditions are: for cooling, 22°C mn-1 and for rewarming, 2°C mn-1 (between 0 and - 100°C and conversely). Differential
Scanning Calorimetry
(DSC)
Calorimetry is done with the CPC 600 (3), an apparatus of the DSC type. Samples are in hermeticalIy closed pans. A rather sl:ow cooIing is obtained by circulating a cold gas (cooling rate: 6 to 8°C mn-1 about 0°C). Immersion of the sample holder land sample pan in liquid nitrogen results in a higher cooling rate. More rapid cooling is obtained by immersion of the sampIe pan in liquid nitrogen and then introducing it into the cold sample holder. It has been demonstrated by electrical simulation of a reaction in the apparatus and by measurement of the heat of fusion of pure materials that the energy absorbed or evolved in the sam,ple can be measured with a precision of T 1% (U). RESULTS
FIG. 1. Differential thermal analysis (DTA) of freeze-dried plasma rehydrated to different water contents. Temperature difference is recorded as a function of time. Temperature indicated on the figure as “C (cooling rate = 22°C mn?; rewarming rate = 2°C n-x-‘).
The DTA and DSC reoordings are quite similar. They also look very much like those which have been obtained with similar products: beef muscle (17: scanning calorimetry) or egg white (12: DSC). If we look, for example, at DTA scans provided by pIasma with a water c0nten.t of 0.48 g* g-l, we observe the following events (Fig. 2):
SIMATOS
204
-100
-80
-60
-40
-20
‘C
FIG. 3. Differential scanning calorimetry ( DSC ) of rehydrated plasma (water content = 0.5 g.g-‘), The figure indicates the graphical bases for the measurements.
(1) an inflexion of the curve (at G) which indicates a change in the specific heat of the sample, (2) a peak (D) corresponding to an exothermal event, and (3) a peak (M) corresponding to an endothermal event. These curves are very comparable to DTA curves obtained with aqueous solutions of glycerol (2, 9, 15, 16), sugars and ethyleneglycol (9, 15), polyvinyl pyrrohdone (8). By referring to these curves, we can ascribe the above events to the following transitions: (1) G: glass transition; (2) D: devitrification of an amorphous phase, or recrystallization; and (3) M: melting, beginning at TIM, and ending at T M* The interpretation will be confirmed by the following results, but we must emphasize that it is somewhat different from previous ones; for beef muscle with a water content of 0.34 g *g-r, Rasmussen ( 14) identifies a glass transition at -92°C but indicates the incipient melting at a point which would be equivalent to the maximum of devitrification ( D ).
ET AL.
of samples (g per g of dry weight); H, = constant; L = constant. L is equal to 77.2 calmg-l, a value quite close to 79.6 calag-I, latent heat of melting of ice. H, can be considered as the nonfreezable water. H, = 0.466 g per g of dry weight. The DSC scans considered here have been performed on samples having reached a homogenous state of hydration, as is inferred from the constant melting area obtained in successive DSC on the same sample. Samples with the lowest water content usually provide a larger melting peak immediately after preparation, which decreases and eventually vanishes in subsequent scans. We feel that the small, persistent mehing peaks which are observed for water content near 0.47 g/g are due to still imperfect equilibrium between water and dry substance. As can be seen on Fig. 5, the incipient melting temperature, Tl,v, was the same for all water content. This had also been observed with aqueous solutions of glycerol ( 2,15), sugars
150,
Malting The area of the endotherma DSC peak (Fig. 3) is a measure of the amount of heat which is absorbed by the sample whiIe mehing. Figure 4 gives the regression line for A, = L (H - H,). A, = area of endothermal peak (cal per g of dry weight); H = total water content
FIG. 4. Area of the melting peak measured on differential scanning calorimetry (DE) scans as a function of water content. Data represented by hollow circles have not been considered in the calculations.
PHYSICAL
STATE
OF WATER
AT LOW
and ethyleneglycol (9, 15) and PVP (8). It was then concluded that melting was concerned with a solution of constant concentration, whatever the dry matter content of the sample. The same conclusion can be drawn here. Whast was not observed with the above solutions and can be seen in Fig. 1, is that melting occurs in plasma in two stages, giving two fused peaks. The first of these is the only one at the lowest water content; it undergoes a Iimited development when water content increases. On the contrary, the second one is greatIy enhanced above water contents ‘of 0.50 g *g-l. Euolution
of Glass Phase
During cooling, a part of the water of the system under study is soEdified into an amorphous state. During rewarming, the glass phase undergoes a second-order transitlon (glass transition: G ), then a partial crystallization (D). The nature of these phenomena is demonstrated by the changes in the DTA or DSC scans when a thermal treatment, annealing, is d.one; after being rewarmed to temperature Tn, the sample is cooled, then rewarmed again (Fig. Zb ). During the second scan, there is no further sign of an exothermal peak, the glass transition is #of a reduced amplitude and ,occurs at a higher temperature. These changes are the consequence of the irreversible character of the crystalIization which has occurred during the first rewarming process.
TEMPERATURES
205
IN PLASMA
w.g.’
CL----J 0
20
40
60
concentration
80 wiw 9(
FIG. 6. Change in the rate of energy absorption during the G transition ( watt per g of dry weight ). l , after slow cooling (643”C*mn~‘); a, after quicker cooling; 0, after very rapid cooling (set Methods ); *, after anueolinp.
Figure 6 indicates the amplitude, related to the unit of dry weight, of the change in specific heat during the transition G as a function of concentration. The transition can be observed only when water content is higher than 0.25 g* gl, The amplitude increases when water content is raised from 0.25 to about 0.45 g-g-l and remains constant for higher water contents. As can be seen on Fig. 7, after a rather slow cooling, the temperature of transition G is approximately constant for water contents between 0.4 and 5 g*g-“. But Tn is lower when cooling has been quicker, The area of exothermal peak D (Fig, 8) is proportional
‘C
-20
i
concenfmtion
w/w
*
FIG. 5. Temperature of incipient melting (7) and of Anal melting (A) as a function of concent,ration (g of dry substance per 100 g of sample).
1 0
20
40
60 concentration
80 wviw 04
FIG. 7. Temperature ?‘a. For symbols see Fig. 6, 0 represents means of values obtained in DTA with the same conditions as in Fig. 1.
206
! .I.( I
. !
SIMATOS ET AL.
FIG. 8. Area of the exothermal peak D, measuring the amount of water crystallized during rewarming (cal per g of dry weight). Symbols as in Fig. 6.
plitude which remains proportional to the amount of dry substance in the sample, even when the water content is strongly increased (Fig. 6). After annealing, a glass transition is still observable (Fig. 2b and S), The values for To suggest that the glass phase has the same water content as after a slow cooling. One might thus think that, in all samples having a water content higher than about 0.45 gag-l, a glass phase with this water content is present, even after a slow cooling or a thermal treatment. Making use of an anaIysis method which has been developed for the study of crystallinity in polymers (13) one might state: AwG/v*Acp
to the
quantity
of ice which
is farmed
during rewarming. As has been explained in sever.al papers (1, 6, 18), the increase in concentration and the decrease in temperature during cooling are responsible for a great increase in the viscosity of the interstitial solutions and the crystal growth is inhibited; the remaining interstitial solutions are solitied into a glass. The water content of the thus formed amorphous phase is higher, the m’ore rapid the cooling rate, During rewarming, visoosity can be lowered enough to enable a more compIete crystallization of water. As can be seen on Figs. 6 and 8, the amplitude
of the G transition
and the
area of ,peak D are larger after a more rapid cooling. For a rather slow cooling, the proportion of water incorporated in the glass phase and CrystaIlizing ,during rewarming is especially importanmt in sampIes with a water content close to 0.6 gag-l. Diagrams simiIar to Fig. 7 have been published for various simple aqueous solutions undergoing slow cooling: glycerol (2, 15) ethylenegIyco1 and glucose (15). The fact that To was independent of the original concentrati,on was explained (15) by a constant water content in the glass phase. This oonclusion is directly confirmed by the observation of the G transition am-
= 1 - (AN - A,)/77.2.
AWo = change in the amount of energy absorbed by the sample during the G transition per unit of time, and by unit of weight; ACP = change in the specific heat of the glass phase; o = rewarming rate; AWo/v . ACp is thus the fraction of the
sampIe solidified into glass during coohng; (AM - A,)/77.2 is the fraction of the sampIe sohdified into ice during coobng. Figure 9 represents AWo as a function of (A,, - A,). A straight Iine is obtained with a satisfactory approximation. The w-g-’ 0.02 “I . I\
*
. l
0.01
**
0
.
r.
3 c
\\\\
o1 0
0
’ Y,, 20
40
60
80 c.I.g-’
FIG. 9, Representation of the extent of crystaIlization at low temperatures (see text).
PHYSICAL
STATE OF WATER AT LOW TEMPERATURES
extrapolation of the ,line to Aaw-AD = 0 provides the specific heat change revealing the glass transition: AC, = 0.13 cal*gml”C?.
It would have been expected that AC, would take different values following the water content of the glass phase. It seems however that the resulting variations for aWo are negligible beside the accuracy of the measurement. CONCLUSION
The quantitative analysis performed on DSC curves seems to provide a further confirmation of the interpretation of the observed phenomena (G = glass transition; D = ,devitrii?cation; IM = incipient melting). In simpler systems such as PVP ( 12), sucrose, or ,dextrose f S), a glass transition could be observed at very low moisture content at temperatures above 0°C. With the present material, however, the glass transition could not be observed with samples having water content below 0.25 gag-l even when the DTA or DSC scans were carried on to temperatures as high as 70°C. The low probability of occurrence of this phenomenon in very low moisture samples is confirmed by the steep increase ,of Tci when the water content is decreased. It is thus suggested that 0.25 gsg-l represents a r.ather critical level in water content for the present system. For water contents above this value, some components of the system can gain enough mobility to give rise to a detectable gIass transition. SUMMARY
Differential therma analysis ( DTA ) and differential scanning calorimetry (DSC) have been used for a study of the physical state of water at low temperatures in freeze-dried bloo,d plasma rehydrated to different water contents. Measurement ,of the amplitude of the specific heat change accompanying the glass transition, the quantity of ice formed during rewarming,
IN PLASMA
207
and the total ice content in the sampIe provided a determination of the extent of crystallization in water at low temperatures. Even after slow cooling or annealing, a fraction of water remained incorporated in an amorphous phase. It is suggested that the unfreezable water (0.47 g per g of dry substance) could be divided into two more or Iess distinct species: 0.25 g per g is the minimum water content above which the system can gain enough mobility to give rise to a detectable grass transition. ACKNOWLEDGMENT The authors thank Dr. Alan Mac Kenzie for a fruitful discussion about the present study. REFERENCES 1. Bellows, R. J. Freeze drying of liquid foods: dependence of collapse temperature on composition .and concentration. Thesis, University of California, Berkeley, 1972. 2. Bohon, R. L., and Conway, W. T. DTA studies on the glycerol-water system. T’hermochim. Acta 4, 321-341 (1972). 3. Bonjour, E., Couach, M., and Pierre, J. de la Quelques examples d’application technique de calorimktrie par compensation de puissance, Joumires de Calorimktrie, Lyon, France 1970. 4. Chevalley, J., Rostagno, W., and EgIi, R. H. A study of the physical properties of chocolate. The application of differential microcalorimetry to chocolate and confectionery. Reu. Int. Choc. 25, 3-6 ( 1970). 5. Couach, M., Bonjour, E., and Chavanel, H. Mesures de chaleur de r&action en analyse enthalpique differentielle. Int. Conf. on Thermal Analysis, Davos, France, 1971. 6. King, C. J. Apphcations of freeze-drying to food products. Vlth International Course on Freeze Drying, Lucerne, Switzerland, 1973. 7. Lusena, C. V. Ice propagation in glycero1 solutions at temperatures below -40°C. In “Freezing and Drying of Biological Materials” (H. T. Meryman, Ed.), Ann. N. Y. Acad. Sci. 85,541-548 (1960). 8. Luyet, B., and Rasmussen, D. Study by differential thermal analysis of the temperatures of instability in rapidly cooled solutions of polyvinylpyrrolidone. Biodynamics 10, 137-147 (1967). 9. Luyet B., and Rasmussen, D. Study by differential thermal analysis of the temperatures of instability of rapidly cooled solutions of
208
10.
11.
12.
13.
SIMATOS ET AL. glycerol, ethyleneglycol, sucrose and glucose. Bdodynamica 10, 167-191 ( 1968). Mac Kenzie, A. P., and Rasmussen, D. H. Interactions in the water-polyvinylpymolidone system at low temperatures. In “Water Structure at the Water-Polymer Interface” (H. H. G. Jellinek, Ed.), pp. 146-171. Plenum, New York, 1971. Moran, T. The freezing of gelatin gel. Proc. Boy. Sot. London Ser. A. 112, 30-40 (1926). Parducci, L. G., and Duckworth, R. B. Differential thermal analysis of frozen food systems. II. Micro-scale studies on egg white, cod and celery. J. Fd. TechmE. 7, 423430 ( 1972 ). Pineri, M., Bonjour, E., Gerard, P., and MartinD’Hermont, F. L’analyse enthalpique diffkrentielIe associbe a des mesures mkcaniques pour 1’8tude des prop&es physiques des polymkres. Phtiques Modernes et Elastom&es, 156-105 ( 1972 ).
14. Rasmussen, 13. A note about “phase diagrams” of frozen tissues. Biodynatmca 10, 333-339 (1969). 15. Rasmussen, D., and Luyet, B. Complementa,ry study of some nonequilibrium phase transitions in frozen solutions of glycero1, ethylene glycol, ethylene gIycc1, glucose and sucrose. Biodynamics 10, 319-331 ( 1969). 16. Rey, L. R. “Conservation de la Vie par Ie Froid,” p, 167. Hermann, Paris, 1959. 17. Riedel, L. Zum Problem des gebundenen Wassers in Fleisch. Kiiltetechnik 13, 12% 128 (1961. 18. Rollet, A. P., and Bouaziz, R. “L’Analyse Thermique. Tome I: Les Changements de Phase,” p. 357. Gauthier-Vilallars, Paris, 1972. 19. Sussman, M. V. Liquid water in hozen tissue: study by NMR. Science 151, 324-325 (1966). 20. Toledo, R., Steinberg, M. P., and Nelson, A. I. Quantitative determination of bound water by NMR. 1. Fd. Sci. 33, 315-317 (1968).
