ORIGINAL RESEARCH

Thermal behavior of trehalose dihydrate (Th) and b-anhydroustrehalose (Tb) by in-situ laboratory parallel-beam X-ray powderdiffraction

Paolo Ballirano Æ Claudia Sadun

Received: 14 July 2008 / Accepted: 6 May 2009 / Published online: 23 May 2009

� Springer Science+Business Media, LLC 2009

Abstract Thermal behaviors of trehalose dihydrate (Th)

and b-anhydrous trehalose (Tb) have been investigated by

in-situ laboratory parallel-beam X-ray powder diffraction.

Data indicate that both phases show essentially the same

volume expansion but expansion of the anhydrous form is

markedly anisotropic due to the features of the hydrogen-

bond network. Under the present experimental conditions,

dehydration starts at 66 �C and within the 75 \ T \114 �C the presence of the Ta anhydrous polymorphic form

has been detected.

Keywords Trehalose dihydrate Th �b-Anhydrous trehalose Tb � Thermal behavior �Parallel-beam X-ray powder diffraction � Rietveld method

Introduction

It is widely known that almost all living organisms are

stressed by critical environmental conditions determining

the loss of a consistent part of their water content. The

temperature increase or decrease causes dehydration of cell

membranes and protein denaturation resulting in the death

of the organism unless the biomolecules are able to

maintain their conformation.

Several studies have been undertaken to preserve living

bodies and foods from freezing by the addition of cryo

protecting materials, including synthetic chemicals and

natural substances. Sugars are widely used as cryo- and

lyo-protectants during the freezing drying process of lipo-

somes and proteins [1].

a,a-Trehalose, a naturally occurring sugar, is used by

some vegetal and animal organisms to survive under

extreme conditions of dehydration, but the mechanism by

which this occurs is still now not at all understood [2].

Trehalose seems to play many roles in increasing tolerance

to dehydration. The researchers are discussing their various

theories as to how it works within the organisms. The

vitrification theory and the water displacement theory are

the most bear out. In the first case, trehalose, through the

formation of intracellular carbohydrate glass, maintains

limited molecular activity. Its role is to bind to the cell

membrane, with its high viscosity and hydrogen bonding

interactions, and low-melting temperature, keeping thereby

the membrane in its liquid crystalline phase. In the second

theory, trehalose molecules could directly replace the water

molecules at the membrane–fluid interface and at the

protein surface preserving the integrity of biological

structure. Therefore, it was suggested that combined fac-

tors are involved in membrane stabilization, and there are

still open questions about the protection mechanism [3].

Trehalose shows a wide range of functional properties

that permit widespread applications in different industrial

fields [4, 5]. It can be utilized in novel drying technologies,

and its relationship with water appears to be important in

terms of stabilizing effects. This feature has been largely

studied with regard to both trehalose solutions and its

crystal polymorphs. The intent was to provide an insight

into the way water mobility and sugar structure can be

related to each other.

Trehalose possesses two stable crystalline forms [6–9],

one involving two water molecules per trehalose unit while

P. Ballirano (&)

Dipartimento di Scienze della Terra, ‘‘Sapienza’’ Universita di

Roma, P. le A. Moro, 5, 00185 Rome, Italy

e-mail: paolo.ballirano@uniroma1.it

C. Sadun

Dipartimento di Chimica, ‘‘Sapienza’’ Universita di Roma,

P. le A. Moro, 5, 00185 Rome, Italy

123

Struct Chem (2009) 20:815–823

DOI 10.1007/s11224-009-9473-5

the other is an anhydrous form. These crystal structures

have been known for a long time and the transition from

one to the other has been largely studied due to the widely

justified relevance of the dehydration process mechanism.

Moreover, it is well known that the transition from one

form to the other is widely influenced by the operating

conditions of relative humidity and pressure up to obtain-

ing different polymorphous and amorphous forms [10–14].

Therefore, the authors’ choice to focus their attention on

the dehydration process of sugar trehalose is justified by the

relevance of this mechanism. In this work, an X-ray powder

diffraction study was carried out with the aim to evaluate

the thermal behavior of these two crystalline forms.

Experimental methods

Trehalose dihydrate (Th) from Fluka (product 90210: high

purity grade 99.5%) was used as starting material without

any further purification. A preliminary powder diffraction

pattern on a conventional Bragg-Brentano instrument

confirmed the absence of detectable impurities. The

material was gently ground, to avoid amorphization, in a

mortar and pestle. The powder was loaded in a 0.7 mm

diameter SiO2-glass capillary that was glued to a 1.2 mm

outer (1.0 mm inner) diameter Al2O3 tube by means of a

high-purity alumina ceramic (Resbond 989). The capillary/

tube assembly, opened at both ends to allow water mole-

cules evacuation, was subsequently aligned onto a standard

goniometer head. Under the present instrumental setup, it is

impossible to flow any inert gas through the capillary. This

procedure could eventually reduce the amount of unwanted

reactions, but in any case, we were interested to evaluate

the evolution of the dehydration process in ordinary con-

ditions. Nonambient data were collected on a parallel-beam

Bruker AXS D8 Advance diffractometer operating in h–hgeometry, fitted with two Soller slits (2.3� opening angle

on the incident and radial on the diffracted beam) and a

PSD VANTEC-1 detector set to a 6� 2h aperture. The

capillary heating chamber is a prototype developed with

MRI and Bruker AXS and is fitted with an MRI TCPU1

temperature control and power unit equipped with a 2404

Eurotherm controller. The capillary heater consists of a

bored corundum cylinder acting as thermal insulator.

Incident and diffracted beams pass through two 12 mm

wide windows closed by aluminum foils with a maximum

angular opening of ca. 75�h. The chamber slides along two

guiding rods allowing the insertion of the capillary via a

2 mm diameter hole. During measurements, the capillary

synchronously spins at 60 rpm. The heater element is a

Kanthal spiral. Temperature measurement is carried out by

a type K (NiCr/NiAl) thermocouple. Chamber cooling is

obtained via a water flow. The sample was heated from 30

to 201 �C with a step of 3 �C, angular range 3–70� 2h, and

using CuKa radiation. The full data set consists of 58

diffraction patterns, and its magnified view is reported in

Fig. 1 as a 3-D plot. Under such experimental conditions,

the conversion of Th to the b-anhydrous form starts at

66 �C and both phases coexist within the narrow

66 �C \ T \ 75 �C range.

A further capillary was prepared using the powder of Th

heated for 2 h at 100 �C and then cooled back to room

temperature to produce b-anhydrous trehalose. A fast

powder diffraction spectrum on a conventional Bragg–

Brentano instrument was performed to confirm the com-

pleteness of the dehydration. The capillary was then heated

Fig. 1 Magnified view of the

complete data set for the first

run shown as 3-D plot

816 Struct Chem (2009) 20:815–823

123

from 30 to 202 �C with a step of 4 �C under the same

experimental conditions of the first run.

Powder diffraction data were evaluated by the Rietveld

method using Topas [15]. This software implements the

Fundamental Parameter Approach (FPA) [16]. FPA is a

convolution approach in which the peak shape is synthe-

sized from a priori known features of the diffractometer (i.e.

the emission profile of the source, the width of the slits, the

angle of divergence of the incident beam) and the micro-

structural features of the specimen. This approach is

believed to improve the stability and the quality of the

refinement, especially with respect to the extraction of

microstructural parameters. Peak shape was modeled

through FPA imposing a simple axial model (9.5 mm).

Peak broadening was assumed to follow a Lorentzian (size)

and a Gaussian (strain) behavior [17]. Peaks position was

corrected for sample displacement from the focusing circle.

The background was fitted with a 30-term Chebyshev

polynomial of the first kind. An absorption parameter

(including aluminum contribution from the heating cham-

ber windows) was refined at 30 �C and subsequently kept

fixed for the remaining patterns. Fractional coordinates for

Th [6] and b-anhydrous (Tb) [18] were unrefined. A global

displacement parameter was refined and found to regularly

increase with temperature. The presence of texture was

checked by means of a generalized spherical-harmonic

description [19]. No significant improvement of fits was

observed as a result of texture indices J very close to one, as

expected for a capillary mount. Miscellaneous data of the

refinements are reported in Table 1. Examples of Rietveld

plots at 42 and 180 �C of the first run are shown in Fig. 2.

Results and discussion

Trehalose dihydrate Th

Thermal expansion of Th has been investigated up to 72�.

In fact, at that temperature dehydration is almost complete

as there is still 5.8(2) wt% of dihydrate remaining in

mixture with Tb. Cell parameters and volume dependence

from temperature closely follow a quadratic behavior up to

66 �C (Fig. 3). However, in correspondence with the

nucleation and growth of the anhydrous form, there is an

abrupt discontinuity consisting in a marked contraction of

the cell, mainly acting along the b axis. Moreover, it is

worth noting that in the 75 \ T \ 114 �C range weak

additional peaks (at ca. 16.1 and 17.9� 2h), attributed to the

Ta anhydrous polymorphic form [7], clearly appear. The

presence of this polymorph in mixture is not trivial to

explain. On the contrary, no amorphous phases were

observed in correspondence with the conversion Th ? Tb.

Such evolution of the process may be attributed to the

presence of a relatively high water stem pressure within the

capillary due the difficulty to evacuate water molecules via

sample porosity. In fact, as previously indicated, there is a

strong dependence of the reaction path from vapor partial

pressure [12].

The occurrence of this contracted cell could be

explained as arising from the attempt to preserve at the

boundary between at least two of the three coexisting

phases some structural coherency. In principle, it seems

more reasonable that coherence could be more easily pre-

served between Th and the Ta anhydrous polymorphic form

for which closely related structures have been postulated in

the past. As a support of this hypothesis, re-hydration of Ta

to Th has been reported to start after 3 min and to complete

after 12 min at 25 �C, and 43% relative humidity [7].

Therefore, Ta could be considered, under the present

experimental conditions, an intermediate metastable prod-

uct of the Th ? Tb conversion.

The unit cell parameters and volume dependence from T

were fitted with a second-order polynomial [20] of type

P = a0 ? a1T ? a2T2, where a0 is the value of the corre-

sponding parameter at 0 �C, a1 is the first-order coefficient

of expansion, a2 is the second-order coefficient, and T is

the temperature in �C. The calculated parameters in the

25 \ T \ 66 �C range are shown in Table 2. The derived

expansion coefficients were found to follow the

ac [ aa [ ab trend as can be appreciated from the relative

expansion of cell parameters shown in Fig. 3e. However,

anisotropy is extremely limited.

The small anisotropic behavior of the thermal expansion

of Th is expected to be related to a small directional

weakening of the hydrogen-bond network. Because very

marginal differences on bond distances and angles of the

anhydrous molecules have been reported at temperatures of

-150 and 20 �C [18], it is reasonable to hypothesize that

even the molecular conformation of Th is substantially

unchanged within the thermal range analyzed in the present

work.

Table 1 Experimental details and miscellaneous data of the Rietveld

refinements

First run Second run

Angular range (�2h) 3–70

Step size (�2h) 0.021963

Counting time (s) 1.5

Rp 3.33–5.28 6.80–8.28

Rwp 4.38–6.91 9.37–11.04

GoF 1.04–1.77 1.02–1.11

DWd 0.74–1.96 1.73–2.00

Agreement indices as defined in [23]

Struct Chem (2009) 20:815–823 817

123

Fig. 2 Fitted X-ray diffraction pattern obtained at: a 42 �C (Th: first run) and b 180 �C (Tb: second run). The lower curve represents the

difference between observed and calculated profiles. Vertical marks refer to the position of calculated Bragg reflections

818 Struct Chem (2009) 20:815–823

123

b-Anhydrous trehalose Tb

Thermal expansion data for Tb were obtained from two

runs. The first one, referring to the dehydration process of

Th provides data limited to temperatures exceeding 66 �C

i.e. the temperature of formation of the anhydrous form. On

the contrary, the second run measured on the sample of Tb

obtained from dehydration of Th, provides data ranging

from RT to the loss of crystallinity. Not being interested in

reaching the melting point temperature, we operatively

define the process as an amorphization/degradation

process. During the first cycle, coupled amorphization and

degradation starts at 177 �C and becomes complete at

189 �C, whereas it begins at 182 �C and ends at 202 �C

during the second cycle. The occurrence of this process

may be easily detected from the color change of the sample

from white to caramel. The melting temperature of the

anhydrous b-trehalose, prepared at a temperature over

130 �C, is reported to have been determined at 216–218 �C

[21]; different melting points have also been determined

and explained as a consequence of the modulation of the

two streaming variables [22]: temperature scanning and

Fig. 3 Cell parameters and

volume dependence from

temperature of Th: a a cell

parameter; b b cell parameter;

c c cell parameter; d volume;

e relative expansion of cell

parameters and volume. Empty

circles refer to cell parameters

of Th after nucleation of Tb

Table 2 Calculated coefficients

of linear and volumetric

expansion of trehalose dihydrate

a b c V

R2 0.995 0.988 0.998 0.998

a0 12.2185(16) 17.875(2) 7.5882(3) 1657.0(4)

a1 6.7(7) 9 10-4 6.7(9) 9 10-4 4.27(5) 9 10-4 2.6(2) 9 10-1

a2 -2.8(8) 9 10-6 -4(1) 9 10-6 – -9(2) 9 10-4

Struct Chem (2009) 20:815–823 819

123

water flowing. Moreover, the X-ray patterns exclude the

possibility that the sample could be characterized as the

mixture of two crystalline forms.

The two different temperature ranges, registered for the

two runs carried out on samples differently dehydrated, can

then be attributed to the possible different quantities of

residual water. In the first run, the dehydration, leading to

the b-phase, was carried out in the capillary during a

continuous heating; the second run was carried out on a

b-phase obtained by dehydration of the sample at 100 �C

for 2 h. No extra reflections attributable to the Ta anhy-

drous polymorphic form were observed during the second

run. As in the case of Th, cell parameters and volume

dependence from temperature closely follow a quadratic

behavior (Fig. 4), the only exception being represented by

the a cell parameter whose behavior is linear (Fig. 4a). The

two heating cycles lead to the same cell parameters within

experimental error. Extrapolation to 20 �C gives cell

Fig. 4 Cell parameters and

volume dependence from

temperature of Tb (filleddiamonds: first run; emptytriangles: second run; for

comparison purposes reference

data [18] are also reported as

circles): a a cell parameter;

b b cell parameter; c c cell

parameter; d b angle; e volume;

and f relative expansion of cell

parameters and volume (data

refer to the second run)

Fig. 5 Variation of e0 strain, defined as bi = 4e0 tan h, with

temperature within the two runs of Tb

820 Struct Chem (2009) 20:815–823

123

parameter and volume in good agreement with those of

reference data [18], differently from the extrapolated

parameters at -150 �C that are significantly smaller indi-

cating that the chosen quadratic model for approximating

the thermal dependence of cell parameters and volume

apply only for the investigated range and that an Ein-

steinian approach could be more appropriate in evaluating

low-temperature data.

From evaluation of the integral breadths bi of the indi-

vidual reflections, microstructural parameters like e0 mi-

crostrain (lattice strain), defined as bi = 4e0 tan h, and

volume-weighted mean column height Lvol, defined as

bi = k/Lvol cos h [17], were extracted.

During the first heating cycle, e0 is progressively

released, from 0.085 to 0.045, starting at 159 �C (Fig. 5),

but such relevant reduction does not significantly affect the

Table 3 Calculated coefficients of linear and volumetric expansion of b-anhydrous trehalose

a b c b V

R2 0.999 0.998 0.999 0.995 0.999

a0 12.9887(3) 8.2512(8) 6.7906(2) 98.374(3) 720.01(9)

a1 3.74(2) 9 10-4 2.3(2) 9 10-4 1.66(5) 9 10-4 8.9(7) 9 10-4 5.7(2) 9 10-2

a2 – 9.3(8) 9 10-7 1.9(2) 9 10-7 1.3(3) 9 10-6 1.02(9) 9 10-4

Coefficients have been calculated from data of the second heating cycle as they are extended to RT

Fig. 6 Structural comparison

between dihydrate and

anhydrous forms. Superposition

may be obtained by a 22�rotation of one pyranosidic ring

around its glycosidic bond

Fig. 7 Possible cooperative

rotation of trehalose molecules

resulting from dehydration. It is

worth noting that a further

rotation of one pyranosidic ring

around its glycosidic bond is

also required

Struct Chem (2009) 20:815–823 821

123

values of the cell parameters obtained from the second run.

During the second run, e0 is constant until the beginning of

the amorphization/degradation process in correspondence

with which we observe a minimal reduction of strain. It is

possible to hypothesize that the extra strain present at the

first run was responsible for the lowering of the amorphi-

zation/degradation temperature with respect to the second

cycle.

As in the case of Th, the unit cell parameters and volume

dependence from T were fitted with the second-order

polynomial P = a0 ? a1T ? a2T2. The calculated linear

and volumetric expansion parameters are shown in

Table 3. The derived expansion coefficients are markedly

anisotropic and follow the aa � ab & ac trend (Fig. 4f).

Explanation of the anisotropic behavior of the thermal

expansion of Tb is related to the geometry of the hydrogen-

bond network whose weakening preferentially acts along

the a axis. In fact, it consists of a long infinite chain run-

ning along the a axis only weakly cross-linked along the

two remaining directions. Further weakening of the cross-

linking is expected to result in a destabilization of the

structure.

Concluding remarks

The trehalose molecule shows very modest conformational

differences in the two investigated structures. Overlapping

the two a-glycosidic bonds of the molecules of the two

crystalline forms, one of the pyranosidic rings overlaps

with good approximation, while the other needs to be

rotated of ca. 20� around its glycosidic bond to obtain a

good superposition (Fig. 6). This rotation moves away the

OH groups involved in hydrogen bonds with water mole-

cules and with other trehalose molecules. The Th ? Tb

conversion is probably governed by a more complex

cooperative rearrangement of the molecules (Fig. 7) not

easily justifiable by small configurational changes that

could perhaps explain the raise of the Ta anhydrous poly-

morphic form.

Comparison of non ambient data indicates that, within

the common 25 \ T \ 66 �C thermal range, the relative

volume expansion of both phases is substantially identical

being 0.45% for Th and 0.39% for Tb.

Moreover, dependence of Lvol from temperature has

been also investigated (Fig. 8). In the case of Th, the value

is constant [ca. 127(5) nm] up to the conversion tempera-

ture, whereas Tb is characterized by a regular increase from

69(7) to 112(6) nm from 66 to 72 �C. The value remains

remarkably constant to ca. 110 nm until amorphization/

degradation. Hypothesizing a spherical morphology of the

particles and applying the well-known relation L0 = 4/3

Lvol, the L0 diameter of the Tb particles may be estimated as

145(8) nm.

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