ARTICLE IN PRESS

Journal of Crystal Growth 293 (2006) 110–112

www.elsevier.com/locate/jcrysgro

Melting point of ice in aqueous saccharide solutions

Tadanori Sei, Takehiko Gonda
Division of General Education, Aichi Gakuin University, Araike 12, Iwasaki, Nisshin, Aichi 470-0195, Japan
Received 14 March 2006; received in revised form 24 April 2006; accepted 26 April 2006
Communicated by M. Uwaha
Available online 27 June 2006

Abstract

The melting point of ice in trehalose and sucrose solutions was measured by in situ observations of a minute ice crystal. It was found
that the concentration dependence of the melting point of ice in both trehalose and sucrose solutions was identical. Such a concentration
dependence of the melting point was in agreement with the equation of freezing point depression for dilute aqueous solutions up to about
1 molal. In addition, the measured values of the melting point decreased rapidly with an increase in the concentration. These
experimental results for an equilibrium state were discussed by comparing them with the results for a nonequilibrium state, i.e., the
results obtained for growing ice crystals in saccharide solutions.
r 2006 Elsevier B.V. All rights reserved.

PACS: 81.10.Dn

Keywords: A1. In situ observation; A1. Melting point; A2. Growth from solutions; B1. Ice; B1. Sucrose; B1. Trehalose

1. Introduction We precisely measured the melting point of ice in trehalose

It is known that trehalose and sucrose—disaccharides
with the same molecular weight—show different abilities
with regard to the protection of plant and animal cells
against both cold and drought stress [1]. We have studied
why trehalose is more protective than sucrose; we studied
this from the viewpoint of the suppression of the growth
rate of ice crystals in saccharide solutions [2,3]. It is
important to precisely measure the melting point of ice in
saccharide solutions of various concentrations in order to
specify the degree of supercooling, which is the driving
force that causes ice crystal growth. However, such a
research to precisely measure the melting point of ice in
saccharide solutions has hardly been performed. DTA or
DSC is usually used to measure the melting point of ice in
aqueous solutions [4]. However, it is impossible to precisely
measure the melting point of ice in saccharide solutions
using these apparatuses because the morphology and
growth rate of ice crystals depend on the degree of
supercooling and the concentration of saccharide [2,3,5].
Corresponding author. Tel.: +81 561 73 1111; fax: +81 56 173 1860.
E-mail address: tadanori@dpc.aichi-gakuin.ac.jp (T. Sei).
and sucrose solutions by in situ observations of a minute
ice crystal. In this paper, we discuss the reason why
trehalose is more protective than sucrose against cold
stress. For this, we compare the measured results of the
melting point of ice, which reveal the physical properties in
the equilibrium state, with those of the growth rate and
morphology of ice crystals [2,3], which reveal the physical
properties in the nonequilibrium state.
2. Experiment
Trehalose dihydrate (Hayashibara Biochemical Labora-
tory Inc., Lot no. 002041) or sucrose (Yoneyama Reagent,
Lot no. MRB0640) was dissolved in purified water
(Yoneyama Reagent, Lot no. MXA0115) and saccharide
solutions of various concentrations (approximately 10 g of
each solution) were prepared. Trehalose solutions of more
than 2 molal were prepared using a water bath of
approximately 90 1C.
Fig. 1 shows the growth cell used for the melting point
measurements. This growth cell is identical to that used for
the growth of ice crystals in saccharide solutions [3]. After

0022-0248/$ - see front matter r 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jcrysgro.2006.04.113
 ARTICLE IN PRESS
T. Sei, T. Gonda / Journal of Crystal Growth 293 (2006) 110–112
50 ml saccharide solution was poured on a glass stage at
room temperature, the solution was cooled at a cooling
rate of 0.58 1C/min until the solution was completely
frozen. The frozen specimen was then heated slowly, and
only a single disc-like ice crystal with a diameter of several
ten microns remained in the solution. Subsequently, the
temperature of the glass stage was increased and decreased
in steps of 0.1 1C; the temperature at which the minute ice
crystal neither grew nor melted was identified as the
melting point of ice. The growth and melting of an ice
crystal were repeated several times in order to determine
the melting point of ice at a certain concentration.
Fig. 1. Growth cell.
Fig. 2. In situ observation of an ice crystal in 52.0 wt% sucrose solution: ((a)–(c)
111
3. Experimental results
Fig. 2 shows an ice crystal changing from an equilibrium
state (a–c: 8.6 1C) to a growth state (d–f: 8.7 1C) in
52.0 wt% sucrose solution. In this case, 8.6 1C was
determined as the melting point of ice.
Fig. 3 shows the saccharide concentration dependence of
the melting point of ice in trehalose and sucrose solutions.
The melting point of ice in these solutions showed the same
concentration dependence for the measured saccharide
concentration. The dotted line is a plot of the equation of
freezing point depression, which holds for dilute aqueous
solutions. This equation usually holds for concentrations
below the order of millimolal. However, the measured
melting points agreed with the equation up to a concentra-
tion of approximately 1 molal. In the figure, the arrows on
the left and right show the concentrations above which the
free water disappears. These concentrations—1.1 and
1.5 molal—were calculated from the dynamic hydration
numbers of trehalose and sucrose, respectively [6]. It was
found that the melting point of ice in these saccharide
solutions rapidly decreased above the concentrations at
which the free water disappears.
4. Discussion
As shown in Fig. 3, the melting point of ice in these two
saccharides was identical. This result implies that in the
equilibrium state, the structures of water in the vicinity of
the ice crystals in both saccharide solutions are identical;
further, the saccharide molecules do not adhere to the ice
crystals.
On the other hand, the growth rate (Fig. 4) and
morphological instability of ice crystals were more
8.6 1C; and (d)–(f) 8.7 1C).
 ARTICLE IN PRESS
112 T. Sei, T. Gonda / Journal of Crystal Growth 293 (2006) 110–112
Fig. 3. Saccharide concentration dependence of the melting point of ice.
Fig. 4. Growth rate along the a-axis of ice crystal versus supercooling [3]:
J 41.5 wt% sucrose solution, & 20.8 wt% trehalose solution; ’ 41.7 wt%
trehalose solution; and B 52.1 wt% trehalose solution.
restrained by trehalose than by sucrose [2,3]. When an ice
crystal grows in the trehalose solution, the volume
diffusion of water molecules toward the ice crystal in
trehalose solution encounters greater obstruction than that
in sucrose solution; this is because trehalose has a larger
hydration shell than sucrose. Therefore, it is expected that
trehalose reduces the growth rate more than sucrose.
Moreover, it is expected that the concentration gradient of
free water in the vicinity of the ice crystals growing in the
trehalose solution becomes smaller than that in the sucrose
solution. Therefore, trehalose suppresses the morphologi-
cal instability of the ice crystal more significantly than
sucrose.
Above the concentration at which the free water
disappears, it is expected that the viscosities of these
saccharide solutions increase discretely because the self-
diffusion coefficient of the water molecules within the
hydration shell is smaller than that of the free water.
Therefore, above the concentration at which the free water
disappears, the growth rate of an ice crystal is discretely
restrained, as shown in Fig. 4; further, the melting point of
ice decreases below the points obtained by the equation of
freezing point depression.
In order to discuss the growth and melting of ice crystals
in saccharide solutions in detail, it is necessary to measure
the saccharide concentration and temperature dependences
of the self-diffusion coefficient of water molecules within
the hydration shell and the viscosities of the saccharide
solutions.
5. Conclusions
The saccharide concentration dependence of the melting
point of ice in both trehalose and sucrose solutions was
found to be identical. However, in a nonequilibrium state,
i.e., in a growth state, trehalose causes a greater suppres-
sion of the growth rate and morphological instability than
sucrose by changing the structure of the saccharide
solution and consequently obstructing the volume diffusion
of water molecules toward the ice crystal.
Acknowledgements
Trehalose dihydrate used in our experiments was
obtained from the Hayashibara Biochemical Lab. Inc.
The authors wish to express their gratitude toward the
hearty cooperation that went into this study.
References
[1] T. Hirata, H. Yokomise, T. Fukuse, K. Muro, K. Inui, K. Yagi,
S. Hitomi, H. Wada, Thorac. Cardiovasc. Surg. 41 (1993) 59.
[2] T. Gonda, T. Sei, Y. Arima, Bull. Glaciol. Res. 19 (2002) 13.
[3] T. Sei, T. Gonda, Y. Arima, J. Crystal Growth 240 (2002) 218.
[4] M.J. Burke, L.V. Gusta, H.A. Quamme, C.J. Weiser, P.H. Li, A. Rev.
Pl. Physiol. 27 (1976) 507.
[5] T. Sei, T. Gonda, Y. Arima, Cryobiol. Cryotechnol. 47 (2) (2001) 13
(in Japanese).
[6] H. Kawai, M. Sakurai, Y. Inoue, R. Chuujoo, S. Kobayashi,
Cryobiology 29 (1992) 599.