Amorphous state and delayed ice formation in sucrose solutions

Internntionnl Journal of Food Science nnd I'echnology (1991) 26, 553-566

Amorphous state and delayed ice formation in sucrose solutions

Y . ROOS" & M . K A R E L Rutgers University, New Brunswick, USA

Summary

Phase transitions of amorphous sucrose and sucrose solutions (20- 100% sucrose) were studied using differential scanning calorimetry, and related to viscosity and delayed ice formation. Glass transition temperature ( Tg) was decreased by increasing water content. Ice formation and concurrent freeze concentration of the unfrozen solution increased apparent Tg. Tg could be predicted weight fractions and Tg values of components. Williams-Landel-Ferry (WLF) relation could be used to characterize temperature dependence of viscosity above Tg. Crystallization of water above Tg was time dependent, and annealing of solutions with less than 80% sucrose at -35°C led to a maximally freeze-concentrated state with onset of glass transition at -46"C, and onset of ice melting at -34°C. The state diagram established with experimental and predicted Tg values is useful for characterization of thermal phenomena and physical state at various water contents.

Keywords

Cryostabilization, differential scanning calorimetry, freezing, phase transitions, sucrose-water-viscosity .

Introduction

The physical state and phase transitions of various carbohydrate solutions at low temperatures have been studied extensively because of their importance to freeze drying of food and pharmaceutical materials, cryoprotection of biological materials, and cryostabilization (Rey, 1960; Rasmussen & Luyet, 1969; Simatos & Turc, 1975; Franks et al . , 1977; Maltini, 1977; Roos, 1987; Levine & Slade, 1988; Williams & Carnahan, 1990). Freezing of biological materials leads to separation of pure ice crystals, simultaneous concentration of dissolved substances, and resultant melting point depression.

In carbohydrate solutions the solutes in their freeze-concentrated state have been shown to form an amorphous matrix which exhibits a glass transition at a concentration-dependent temperature ( Tg) (Luyet & Rasmussen, 1968; Rasmussen & Luyet, 1969; Bellows & King, 1973). The amount of ice formed during cooling depends on the cooling rate. Rapidly cooled solutions usually exhibit less ice and show low Tg values. However, ice formation may occur during rewarming probably

Authors' address: Rutgers University, Department of Food Science and Center for Advanced Food Technology, PO Box 231, New Brunswick, NJ 08903, USA. * Correspondent. Present address: University of Helsinki, Department of Food Chemistry and Technology, Viikki, B-talo, SF-00710 Helsinki, Finland.

553

554 Y. Roos & M . Karel

because of decreasing viscosity above the 7'g of the unfrozen matrix. As the temperature rises a sufficiently decreased viscosity allows molecular movement required for crystallization.

In differential scanning calorimetric (DSC) rewarming scans of rapidly cooled solutions Tg is followed by an exotherm which precedes the melting of ice. The typical transitions reported are glass transition ( Tg), devitrification or ice formation ( Td), ante-melting (Tam) which has been referred to as a temperature of melting of small ice crystals and represents an endothermal step change above Tg and incipient melting of ice (Ti,) (Luyet & Rasmussen, 1968; Rasmussen & Luyet, 1969; Simatos et al. 1975; Blond, 1989). In the glassy state water is not able to crystallize, and devitrification above the T, is the major cause for injury and loss of biological activity of cells (e.g. Fahy et al . , 1984; MacFarlane, 1986; Williams & Carnahan, 1990).

Levine & Slade (1986) have reported for carbohydrate solutions a minor Tg below Tam and Tim, which were combined into a single thermal transition referred to as glass transition of the maximally freeze-concentrated amorphous matrix (TL), and melting of ice. This concept has been criticized by Izzard et al. (1991) because the TL as defined by Levine & Slade (1986, 1988) was found to be an indication of ice melting rather than a glass transition. Roos & Karel(1991a) showed that annealed sucrose solutions have only one glass transition which is followed by ice melting endotherm. In that study the onset temperature of the reported concentration- independent glass transition (in the range of 20-6580 sucrose) was considered as the Tg of maximally freeze-concentrated amorphous matrix (7';). An endothermal step change close to the end-point of the TL region had also a concentration-independent temperature value. This transition was considered as onset of ice melting ( T k ) , and it was also the onset temperature for a single ice-melting endotherm in annealed solutions with high initial sucrose concentrations.

Although recent literature on carbohydrate solutions reports studies resulting in state diagrams, and quantitative data for transition temperatures and concentrations (Levine & Slade, 1988; Blond, 1989; Williams & Carnahan, 1990; Izzard et al., 1991) the high viscosity of the freeze-concentrated state and resultant time- dependent phenomena have not been widely discussed with experimental data. The purpose of this study was to determine phase transitions of amorphous sucrose at varying levels of water plasticization, to relate the transitions to those of frozen sucrose solutions, and to determine factors leading to time-dependent ice formation in frozen sucrose solutions.

Materials and methods

Preparation of samples Amorphous sucrose was prepared by freeze drying of 20% sucrose (Sigma, grade

11) solutions (Roos & Karel, 1990) which is known to produce an amorphous state (To & Flink, 1978; Roos & Karel, 1990). The freeze-dried material was further dehydrated to 'zero' moisture content (mh in a vacuum desiccator over P205 for 6 months to study the thermal behaviour of anhydrous sucrose. Rehumidification of the freeze-dried material over saturated salt solutions was used to obtain samples of low moisture contents (rn < 0.05 g H20 g-' solute) as reported by Roos (1987) and Roos & Karel (1990). Sucrose solutions having sucrose Concentrations of 20, 30, 40, 50, 65, 68, 70, 75 and 80% for the determination of thermal phenomena at low

Amorphism of freezing sucrose solutions 555

temperatures were prepared by weighing respective amounts of crystalline sucrose (Sigma, grade 11) and water (Fisher, HPLC grade) to obtain l o g of the solution in a 50-ml beaker. Sucrose was dissolved until a clear solution was obtained. Heating was used to dissolve sucrose at concentrations above 65%, and the amount of evap- orated water was added on an analytical balance (Mettler AE240) after cooling of the solutions.

Differential scanning calorimetry Phase transitions of amorphous sucrose and frozen sucrose solutions were analysed

by using differential scanning calorimetry (Mettler TA4000 thermal analysis system with a TCll TA processor and DSC30S DSC cell). The instrument was calibrated for heat flow and temperature using n-hexane (m.p. -94"C, AH,n 151.8Jg-'), water (m.p. O"C, AH,,, 334 Jg-'), gallium (m.p. 29.8"C, AH,,, 79.3 J g-') and indium (m.p. 156.6"C, A H , 28.5 Jg-'). The thermograms were analysed for onset temperature of glass transition (Tg), change of the specific heat at the glass transition (Ac,,), onset temperature of devitrification (Td), onset temperature of ice melting ( T k ) and latent heat of melting (AHm) using a Mettler TA72 PS.l software (see Figs 1, 3 and 4). Samples were prepared by weighing 10-25mg of the material in 4 0 ~ 1 aluminum DSC pans (Mettler) which were subsequently hermetically sealed. An empty aluminum pan was used as reference in all measurements. The samples were cooled to -100°C at the maximum cooling rate (30"Cmin-') in the DSC cell. After cooling the samples were either scanned at 5"Cmin-I to 0 or 20°C to detect their thermal behaviour in the non-annealed state, or heated at 10"Cmin-I to -35°C. Isothermal annealing at -35°C (see Figs 3 and 5 ) was used to observe time-dependent ice formation at a temperature slightly below the onset of melting, Tk = -34°C (Roos & Karel, 1991a). After annealing the samples were recooled to - 100°C at 10"Cmin-', and scanned from -100°C to 0 or 20°C at S'Cmin-'. The latent heat of ice melting (AH,) was obtained by integration of the melting endotherm of annealed samples. Annealing was found to be necessary for AH, determination because only then could maximum ice formation be achieved and confirmed from the Tg= Ti value of the unfrozen matrix. The integration limits were set to a temperature slightly below TL and to a temperature above end-point of the melting endotherm (see Fig. 3). Triplicate samples were used to obtain average transition temperatures, and Acp and AH, values.

Prediction of glass transition temperatures The glass transition temperatures of compatible polymer mixtures can be calcu-

lated using equation (1) (Gordon & Taylor, 1952). This equation has also been used to predict glass transition temperatures of polymer-water mixtures (Ellis, 1988; Orford et al . , 1989). We used equation (1) to calculate the Tg curve of sucrose to establish the state diagram using both experimental and predicted Tg values. The Tg value of water at -135°C (Johari et al., 1987) was used. The empirical value for the constant k was obtained by solving equation (1) for the experimental weight fractions and corresponding Tg values. The value k=4 .7?0 .2 was obtained for sucrose solutions (65-80% sucrose), and it was used to calculate the Tg curve shown in the state diagram (see Fig. 7).

556 Y. Roos & M. Karel

where Tg is glass transition temperature of mixture ( K ) , Tgl is glass transition temperature of component 1 (sucrose, 335K), Tg? is glass transition temperature of component 2 (water, 138K), w l is weight fraction of component 1 (sucrose), w2 is weight fraction of component 2 (water), and k is constant.

Determination of composition at maximum ,freeze concentration Various methods have been used to predict unfrozen water content (WL, g H20

g-' solute) or solute concentration (CL, weight % solute) in the maximally freeze- concentrated state. The method proposed by Levine & Slade (1986, 1988) is based on determination of the AH,,, for a 20% by weight solution, which reportedly provides a fast method to determine the unfrozen water content. The unfrozen water content is then obtained from the difference between total water content and calculated amount of ice [equation (2)]:

where W,,, is total amount of water (g), Ctot is total amount of solute (g), AH,,,, is total heat of melting of ice (J), AHrnw is latent heat of melting of ice (334Jg-'). AHmtOt/AH,, is the amount of ice (g) in the sample and W,,, - AHmtot/AHmW gives the amount of unfrozen water (g).

Simatos et al. (1975) and Roos (1987) have determined thermal behaviour of carbohydrate materials at various water contents. The DSC scans showed a decreasing Tg with increasing water content, and a certain water content at which a melting endotherm was observed. The AH, values were also determined for materials with varying water contents, and the unfrozen water content was calculated from the linear relationship between AH,,,,/Ct,,, and m by extrapolating equation (3) to A H,,,,IC,,, = 0 J g-' .

AHmtot/Ctot = am + b ( 3 )

where AH,,,,/C,,, is total latent heat of melting of ice in sample divided by total amount of solute (Jg-' solute), m is water content (g H 2 0 g-' solute), a is slope (J g H20) and b is constant (J g-' solute).

Since Ti is the glass transition temperature of the unfrozen matrix in the maximally freeze-concentrated state with a solute concentration of Ci the unfrozen water content obtained with various methods should agree with the composition shown in the state diagram at TL (see Fig. 7). In this study we compared all the above methods for the determination of the unfrozen water content at TL. The CL corresponding to the experimental TL as shown in the state diagram (see Fig. 7) was calculated using equation (1).

Prediction of viscosity above glass transition Bellows & King (1973) reported viscosity values for 70 and 75% sucrose solutions

above -30°C. The Tg values for these concentrations obtained experimentally in this study were used to calculate the viscosity above Tg using Williams-Landel-Ferry equation [equation (4)] (WLF, Williams et al., 1955). The viscosity at Tg was calculated by solving equation (4) for all experimental points at respective T - Tg values to obtain its average value (qg = 1010.66 Pas).


r -17.44 (7'- Tg) qg 51.6 + ( T - Tg)

log -= (4)

where T is temperature, Tg is glass transition temperature, q is viscosity at T , and qg is viscosity at Tg.

Results and discussion

Glass transition of amorphous sucrose The T, of sucrose at low-water contents is of importance to stability and control

of crystallization in materials containing amorphous sucrose (Roos & Karel, 1990). The Tg of anhydrous amorphous sucrose was found to be 62°C (onset of transition) with midpoint at 67°C. This value agreed with the midpoint value of 67°C reported by Kauzmann (1948) and Weitz & Wunderlich (1974), but was lower than 70°C reported by Orford ef al., (1990). The Tg of amorphous carbohydrates is significantly decreased by traces of water (Roos & Karel, 1990). Thus we have previously reported Tg = 56.6"C for 'anhydrous' sucrose glass dried less rigorously than in the present study (Roos & Karel, 1990), and To & Flink (1978) have reported 52°C. The Tg of amorphous sucrose was decreased with increasing water content as shown in Fig. 1. The non-annealed samples containing up to 30% water showed no ice melting endotherm during rewarming.

The change of the apparent specific heat (Ac,) for amorphous sucrose at T, was 0.6 J g-'"C-', and was lower than values reported in previous studies (0.75 J g-'"C-', Weitz & Wunderlich, 1974; 0.77 Jg-'"C-', Orford et a f . , 1990). Weitz & Wunderlich (1974) reported an endotherm for sucrose in the Tg region. This relaxation endotherm was also noticed in this study (Fig. l ) , and it may slightly affect the determination of Ac, leading to higher values. The endotherm was not apparent in a second or third scan of the samples, but the Ac, value remained the same (0.6 J g-' "C'). The Ac, value increased to a constant value of 1.0 J g-' sucrose "C-' for the sucrose solutions being slightly higher than our previous value 0.9 J g-' sucrose "C-' (Roos & Karel, 1991a). Usually Acp increases with decreasing molecular weight. Water is a low

---_ \

-- ---__ --__ -1 00 - 50 0 50 100

TEMPERATURE "Y)

Figure 1. Decrease of glass transition temperature (T,) of amorphous sucrose with increase in water content. At low-water contents thermal relaxation occurred in the glassy state at room temperature; this was observed as a small endotherm at the end of the T, rcgion (Weitz & Wunderlich, 1974). The Tg values shown are onset temperatures of the Tg determined for samples scanned at 5"Cmin-'.


molecular weight plasticizer and thus higher Ac,, values were obtained for sucrose solutions than for anhydrous sucrose, although reported values for Ac, of amorphous water vary significantly (1.94Jg-'"C-', Sugisaki el al . , 1968; 1-1.4Jg-'"C-', Angel1 & Tucker, 1980; 0.1 Jg-l"C-', Hallbrucker et al., 1989).

Thermal behaviour of non-annealed sucrose solutions The thermograms of freeze-concentrated sucrose solutions were dominated by

the ice melting endotherm. Levine & Slade (1986) have used 20% sugar solutions for the determination of the T, of the maximally freeze-concentrated state. The 20% solutions (Fig. 2) had a glass transition with onset at about -50°C followed by a small devitrification exotherm with onset at about -43°C. The exotherm was followed by a step change, and ice melting endotherm. Levine & Slade (1988) in- terpreted the midpoint value of the step change (-32°C) to be the Tg of the maximally freeze-concentrated amorphous matrix. However, it is not likely that, in a dynamic measurement, devitrification occurs before the observed Tg because that would require ice formation in the glassy state, which is known to be kinetically inhibited (Levine & Slade, 1988).

The T, values of carbohydrate solutions have been reported to vary depending on concentration and cooling rate (e.g. Luyet & Rasmussen, 1968; Rasmussen & Luyet, 1969). The apparent T, values obtained in this study initially decreased with increasing sucrose concentrations (Figs 2 and 3) and increased again at high sucrose concentrations (Figs 1 and 4). As the Tg decreased, the Td also decreased (Fig. 3), but at high sucrose concentrations devitrification occurred at a higher temperature above Tg (Fig. 5), or was not observed (Fig. 1). The decrease of the Tg value was caused by an increase of the amount of unfrozen water in the amorphous matrix as discussed by Luyet & Rasmussen (1973). This indicated delayed ice formation in concentrated sucrose solutions. Thus 65% sucrose solution in the non-annealed state showed Tg at -77°C which was followed by Td above T, at -57°C (Fig. 3).

I 20% Sucrose Solution (w/w)

Figure 2. Thermal behaviour of 20% non-annealed sucrose solutions determined at S'Cmin-'. The glass transition (T,) region is shown in the inset figure. The Tg was followed by a small devitrification exotherm (Td) indicating icc formation during rewarming above Tg and subsequent melting.


c 65% SUCROSE (w/w)

1 s . -100 -80 -60 -40 -20 0

TEMPERATURE (OC)

Figure 3. Thermal behaviour of 65% sucrose solutions determined at 5"Cmin-'. (A) Non-annealed solutions showed T, at -77°C which was followed by devitrification with onset at -57°C and subsequent ice melting. (B) Annealing 30min at -35°C led to maximum freeze -concentration. Thus no devitrification during rewarming was observed, T, was increased to TA at -46"C, and the onset of the ice melting endotherm ( T h ) was at -34°C.

1 8 0 % SUCROSE (w/wl

- 60 -40 -20 TEMPE R ATU RE (O,C)

Figure 4. Thermogram of 80% sucrose solution obtained at 5"Cmin-'. The onset temperature of the glass transition (T,) region determined as the intersection of the tangents shown was at -46°C. The T, midpoint was at -40°C and the end-point of the Tg region was at -35°C. The change of specific heat (AcJ at the T, region was 1 J g-' sucrose "C-'.

These results agreed with those of Williams & Carnahan (1989, 1990), showing delayed ice formation in sucrose solutions close to T, or in solutions with high initial sucrose concentrations.

Thermal behaviour of annealed sucrose solutions In our previous study, temperature cycling below -34°C was found to lead to a


Annealing Time at

I # -100 - 80 -60 -LO -20 0

TEMPERATURE (OC)

Figure 5. Thermograms of 68% sucrose solutions annealed for varying time periods at -35°C beforc determination of the transitions during rewarming from -100 to 0°C at 5"Cmin- I . Isothermal ice formation at -35°C led to increased Tg values, decreasing size and temperature of the devitrification exotherrn, and decreasing temperature and increasing size of the ice melting cndotherm. Annealing 5 h at -35°C led to maximum freeze concentration. Thus the glass transition was at TL with onset at -46°C. The onset temperature of the ice melting endotherm ( T k ) was -34°C.

constant T, value for freeze-concentrated sucrose solutions (Roos & Karel, 1991a). In the present study isothermal annealing below the proposed TL at -34°C was used to allow time for ice formation. Annealing at -35°C resulted in disappearance of the devitrification exothem in all solutions having initial Tg below -46°C (Figs. 3 and 5 ) . Thus after annealing for 1Smin at -35"C, 20% sucrose solutions had Tg at -46°C (with midpoint of transition at -41"C), and melting with TL at -34°C. This agrees with previously reported data for materials annealed by slow cooling (Rasmussen & Luyet, 1969) or by temperature cycling (Simatos et a l . , 1975; Roos & Karel, 1991a). The thermal behaviour of the 30% and 40% solutions was similar to that of the 20% solution. Annealed samples with SO% sucrose showed a small endothermal peak between the Tg and main melting endotherm as reported by Roos & Karel (1991a). Crystallization of water in solutions with 6.5 and 68% initial sucrose concentrations was substantially delayed (Figs 3 and 5). The Tg of the 68% sucrose solution increased with increasing annealing time because of the decreased amount of unfrozen water and increased sucrose concentration in the unfrozen amorphous matrix (e.g. Luyet & Rasmussen, 1973). Ice formation was detected as increased size of the ice melting endotherm. After 5 h at -35"C, 68% sucrose solution was maximally freeze concentrated, and it had Tg at -46"C, and a single ice melting endotherm with TL at -34°C (Fig. 5) . The delayed ice formation was also observed visually as increasing opacity in solutions (6S-80% sucrose, 10ml in SO-ml beaker) held in a freezer at -35°C. Solutions with 65, 70 and 75% sucrose showed time- dependent (from a few hours to several weeks) ice formation but no sucrose crys-


tallization in the supersaturated unfrozen phase was observed after melting of the ice formed.

Prediction of glass transition temperatures Couchman (1978) and Couchman & Karasz (1978) have reported that the k

value in equation (1) is equivalent to k = Acp2/Acpl. The Acp value of water has been reported to be 1.94Jg-'"C-' (Sugisaki et al., 1968), 1-1.45g-'"C-' (Angel1 & Tucker, 1980) and 0.1 Jg-'"C-' (Hallbrucker et al., 1989). These gave k values varying from 0.17 to 3.2 (Acpl = 0.65 g-'"C-') which did not correlate with the experimental data. Orford et a f . (1990) made a similar effort to predict Tg values of carbohydrate and water mixtures using the k value obtained from the changes in the specific heats, and observed poor correlation. However, the empirical value k = 4 . 7 gave a good correlation with the experimental T, values (see Fig. 7). We have previously reported a good correlation for sucrose -maltodextrin mixtures using equation (1) with empirical k values (Roos & Karel, 1991b).

Unfrozen water content at the maximum freeze Concentration The unfrozen water content calculated from the difference of total water con-

tent and that of frozen water calculated from AH,,, of ice melting assuming AHrnw = 334 J g-' for varying sucrose concentrations are given in Table 1. The Ti was found to have a constant, initial concentration independent value, and thus the unfrozen water content should be constant and also independent of the initial sucrose concentration. However, when the unfrozen water content was calculated using equation (2) varying results were obtained depending on the initial concentration. The extra- polation method [equation (3)] gave a = 304Jgp' H 2 0 and b = -92.45 g-' solute (Table 1) according to which the Wk was 0.30g H20 g-' solute or Ck = 76.7%. Equation (1) predicted the Tg of 76.7% solution to be at -54"C, and a solution with T, at -46°C to have a sucrose concentration of 80% (equivalent to Wg = 0.25 g H20

Table 1. Effect of initial sucrose concentration on glass transition temperature of thc maximally freeze- concentrated state ( T i ) , onset temperature of ice melting ( T h ) , energy of ice melting (AH,,,), and unfrozen water contcnt ( W i ) or sucrose concentration (CA) of the maximally freeze-concentrated amorphous matrix estimatcd with equation (2)

TL W) m* Wit

Tk AHm AHmtot/Ctot* (gH2O ci Sucrose (gF120 g- solute) 1 2 3+ ("C) (Jg-') (Jg-' sucrose) g-' solute) (YO) ("/I 20 4.00 -46 -41 -36 -34 225 1124 0.64 61.2 30 2.33 -46 -40 -3.5 -34 18.5 618 0.48 67.5 40 1.50 -46 -41 -36 -34 145 363 0.41 70.8 so 1 .00 - 47 -37q 104 208 0.38 72.6 65 0.54 -46 -42 -36 -34 46 71 0.31 76.2 68 0.47 -46 -41 -36 -34 37 54 0.31 76.3

* Allows calculation of WL using cquation (3).

respectively. * Calculated with equation (2). * Affected by a small endotherm observed abovc Ti (Roos & Karel. 1991a).

Numbers 1, 2 and 3 refer to the onset, midpoint and end-point value of the glass transition region,


8-l sucrose and 4.75 mol H20 mol-’ sucrose. The sucrose concentration in a maximally freeze-concentrated solution has been reported to be 80% by Williams & Carnahan (1990) and Izzard et nl. (1991). Other studies have reported values of 64% (Levine & Slade, 1988) and 77% (Rasmussen & Luyet, 1969). Using the value AH,,, = 304 J g-’ H20 for water as obtained from equation (3) instead of 334 J g-’ H 2 0 gave about constant value of C;! = 77% as estimated using equation (2). Thus the use of the latent heat of pure water for calculation of the unfrozen water content from latent heat of melting significantly below the melting point of pure water seems to lead to highly erroneous WL vaiues. It should also be noted that thermo- dynamically the AH,,, of ice decreases with decreasing temperature.

Viscosity of concentrated sucrose solutions The glass transition temperature of 70% sucrose solution was at -68°C and that

of 75% sucrose solution at -59°C. The viscosity data of these solutions were reported by Bellows & King (1973) and is shown as a function of T-Tg with the WLF prediction of viscosity in Fig. 6. The viscosity of amorphous mixture of sucrose and fructose (Soesanto & Williams, 1981) and amorphous fructose and glucose (Ollett & Parker, 1990) have been shown to have WLF-type temperature dependence above Tg, and the viscosity data of 70 and 75% sucrose solutions also showed good correlation with the WLF prediction. The WLF prediction for viscosity at the Tg was 1010.7Pas which agreed well with the viscosity of the glassy stage being lo1’ to 10l’Pas (White & Cakebread, 1966; Bellows & King, 1973). The WLF prediction for viscosity of 80% sucrose solution at Tk = 34°C was 107.4Pas. It is evident that crystallization of water became time dependent at lower viscosities, and extremely slow above 10’Pas.

12 -

70% Tg - -68°C 75%. Tg = -59°C 80%, Tg = -46°C

TEMPERATURE (“C)

- WLF Prediction of Viscosity 0 - 0 Viscosity of 70% Sucrose Solution

, 0 Viscosity of 75% Sucrose Solution

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0

(Bellows and King, 1973) -2 . ’ “ ” . ‘ . ’ . ” . ’ ” ~ ~ ’ ’ . .

T-Tg (“C)

Figure 6. Viscosity of 70 and 75% sucrose solutions reported by Bellows & King (1973), and the WLF (Williams et al . , 1955) prediction of viscosity above the glass transition temperature ( T g ) . The predicted viscosities of 64, 70, 75 and 80% sucrose solutions are shown in the inset figure with the Tg values obtained from equation (1). Increasing viscosity caused by freeze concentration of sucrose solutions led to time-dependent ice formation. Maximum ice formation is probably always time dependent because of the increasing concentration, increasing T,, and increasing viscosity. The viscosity at the end-point of the Tg region of 80% sucrose solution was about 10’Pas.


The WLF prediction of viscosity for a solution with Tg at -77°C at the onset temperature of devitrification at -57°C (65% sucrose solution, Fig. 3) was 1O5.'Pas. Once ice began forming above Td, concentration of the solute probably increased viscosity, while the increasing temperature decreased viscosity. Thus devitrification proceeded until melting of ice occurred above TL. It is likely that nucleation may occur during fast initial cooling, but crystallization in solutions with more than 62% sucrose is delayed, and usually occurs during slower rewarming (e.g. Fahy et al. 1984; MacFarlane, 1986; Williams & Carnahan, 1989).

We assume that the viscosity at the end point of the Tg region was close to the value of 1O7Pas. Roos & Karel (1991~) reported that stickiness of dehydrated carbohydrates occurs at the end-point of the Tg region. Stickiness, as defined by Lazar et al. (1956), results from decreasing viscosity above Tg and the critical viscosity for stickiness has been shown to be 107Pas (Downtown et al., 1982). The melting point was also decreased to the vicinity of the end-point of the Tg region, and theoretically, it is expected that the TL and TL have the same value in the maximally freeze- concentrated state (Levine & Slade, 1986).

Physical state of amorphous sucrose and sucrose solutions The physical state of amorphous materials can be visualized by the use of state

diagrams in which the Tg and T,,, of the material are shown as a function of water content. The state diagram obtained for sucrose is shown in Fig. 7. Our results showed that the Tg at varying water contents could be predicted using equation (1), which allowed a basis for establishing the state diagram with reliable concentration and respective Tg values. The results indicated isothermal crystallization of water at -35"C, which led to increased Tg values. The Tg value of the unfrozen maximally freeze-concentrated solution was constant corresponding to an initial concentration invariant composition of 80% sucrose. The concentration could also be obtained from the concentration axis of the state diagram (Fig. 7). The temperature region between TL and TL was the glass transition region where the viscosity changed by several decades. Maximum ice formation was found to occur only in this temperature range, where it also became kinetically controlled. (Thus in rapid cooling through this range crystallization may not occur.) Partially freeze-concentrated solutions had lower Tg values, showed Td above the observed Tg, but probably they could not devitrify to the maximally freeze-concentrated state until the temperature was slightly below T:, because freeze concentration simultaneously increased Tg and decreased the rate of crystallization until ice formation became inhibited.

Maximum ice formation may be either advantageous or detrimental. Devitrification during rewarming can damage frozen materials, and an extremely high heating rate is required to prevent crystallization in those cases (e.g. Williams & Carnahan, 1990). Freeze drying requires a fully solid state, and thus during pre-freezing the materials should be allowed to form a maximum amount of ice. Above T;, sucrose solutions can be only in the rubbery state above Tg because ice melting dilutes the solution and simultaneously decreases the Tg of the unfrozen matrix.

Conclusions

The Tg of sucrose decreased with increasing water content until ice formation concentrated the solution, and thus increased the apparent Tg. Maximally freeze- concentrated solutions had 80% sucrose (0.25g HzO g-' solute) in the unfrozen


80 I , 60 - ,' "Rubber" ' r n Solution

I

0 2 0 4 0 6 0 8 0 1 0 0 CONCENTRATION ("A Sucrose)

Figure 7. State diagram of amorphous sucrose. The Tg line shows the dramatic effect of water on T, at high sucrose concentrations. At temperatures below Tg the material is in its glassy statc. Above T, solutions with less than 80% sucrose may exist as a freeze-concentrated glass a rubber or a solution depending on tempcrature, and abovc Tk only the rubbery state or solution may exist. The apparent Tg of partially freeze-concentrated statcs may have any tempcraturc value between Tg and TA depending on thc sucrose concentration in thc unfrozen matrix, and thus devitrification is possible below TL. The maximum amount of ice can be formed only at the temperature rangc between TL and Tk where crystallization is time depcndent because of the high viscosity of thc unfrozen matrix. Above Tk sucrose solutions are in their partially freeze-concentrated state, and T,,, is thc final melting point of icc. The T,,, values are from Bellows & King (1973). Predicted valucs arc shown with lines and the experimental values with symbols.

amorphous matrix which exhibited TL at -46°C and TL at -34°C independently of the initial sucrose concentration. Maximum ice formation in solutions containing less than 80% sucrose in the unfrozen matrix was achieved only by annealing between T i and TL. The viscosity of sucrose solutions followed Williams-Landel-Ferry (WLF)-type temperature dependence above Tgr which allows prediction of viscosities above Tgr and in the partially freeze-concentrated state if the amount of unfrozen water is known. The Tg for solutions with varying water contents can be calculated with the Gordon and Taylor equation [equation (l)]. Devitrification of rapidly cooled solutions was observed at a calculated viscosity of 105.8 Pas. Solutions with more than 80% sucrose were not expected to form ice because crystallization of water would occur at a predicted viscosity higher than lO'Pas which apparently was the limit for ice formation. Ice formation ceased in the glassy state and was inhibited above the equilibrium melting point of ice (conditions with viscosities lower than 107Pas would result in melting of ice above TL). Whether the maximum freeze concentration is entirely due to kinetic inhibition of crystallization, or whether the equilibrium hydration of sucrose in presence of ice at this temperature is being approached, is not known. The state diagram based on the experimental data and predicted Tg values gives highly valuable information for studies of physical behaviour of sucrose-containing materials.

565 Amorphism of freezing sucrose solutions

Acknowledgments

This is publication no. D-10535-18-90 of the New Jersey Agricultural Experiment Station supported by the Academy of Finland, by the state of New Jersey funds and the Center for Advanced Food Technology. The Center for Advanced Food Technology is a New Jersey Commission on Science and Technology Center.

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Received 10 December 1990, revised and accepted 17 June. 1991

Documents

Amorphous state and delayed ice formation in sucrose solutions