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org/JPCB Article

Impact of Low Concentration of Strongly Hydrogen-Bonded Water

Molecules on the Dynamics of Amorphous Terfenadine: Insights
from Molecular Dynamics Simulations and Dielectric Relaxation
Spectroscopy
Jeanne-Annick Bama, Emeline Dudognon, and Frédéric Affouard*
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sı Supporting Information

ABSTRACT: The impact of low water concentration of strongly hydrogen-bonded water

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molecules on the dynamical properties of amorphous terfenadine (TFD) is investigated

through complementary molecular dynamics (MD) simulations and dielectric relaxation
spectroscopy (DRS) experiments. In this article, we especially highlight the important role
played by some residual water molecules in the concentration of 1−2% (w/w) trapped in
the TFD glassy matrix, which are particularly difficult to remove experimentally without a
specific heating/drying process. From MD computations and analyses of the hydrogen
bonding (HB) interactions, different categories of water molecules are revealed and
particularly the presence of strongly HB water molecules. These latter localize themselves
in small pockets in empty spaces existing in between the TFD molecules due to the poor
packing of the glassy state and preferentially interact with the polar groups close to the
flexible central part of the TFD molecules. We present a simple model which rationalizes at
the molecular scale the effect of these strongly HB water molecules on dynamics and how
they give rise to a supplementary relaxation process (namely process S) which is detected for the first time in the glassy state of TFD
annealed at room temperature while this process is completely absent in a non-annealed glass. It also explains how this
supplementary relaxation is coupled with the intramolecular motion (namely process γ) of the very flexible central part of the TFD
molecule. The present findings help to understand more generally the microscopic origin of the secondary relaxations often detected
by DRS in the glassy states of molecular compounds for which the exact nature is still debated.

1. INTRODUCTION crystalline state due to strong inter- and intra-molecular HBs

Amorphous solids (glasses)1 composed of small and large
organic molecules are ubiquitous in natural systems, and they
are commonly formed both intentionally and unintentionally
during technological processes. These materials have partic-
ularly attracted strong interest in the development of
pharmaceutical2 and food3 products for their remarkable
advantages in terms of solubility, mechanical properties, or
their capabilities for preserving biological agents and cells.4−7
In almost all cases, molecular amorphous solids contain a
certain amount of water and may also adsorb even more water
upon storage since environmental moisture is generally
unavoidable.8 The reported values for the water content
usually range from low concentrations of less than 1% (w/w)
when specific heating and drying treatments have been
employed, to high concentrations of about 30% (w/w) as
often found in protein systems mixed with sugars.8 These
different concentrations are actually very dependent on the
hydrogen properties of the molecules that compose the
amorphous solids and particularly on the number of groups
that can donate or accept hydrogen bonds (HBs). It is
remarkable that the systems that are almost insoluble in the
can be highly hygroscopic in the amorphous solid state.9 The
amount of water content and water uptake are also highly
dependent on the processes used to generate the amorphous
solids. In practice, it can be produced by many different
routes:10 (i) the classical thermal quench from the liquid
state,11,12 (ii) the mechanical route (compression and milling)
occurring entirely in the solid state,13−17 and (iii) the solvent
removal processes (freeze-drying and spray-drying) requiring
the dissolution of the compound in a solvent.18−20 It is well
recognized that water uptake can be particularly important for
amorphous solids obtained by milling since this technique
intrinsically increases the specific surface area of the
materials.13,14 Water content might also be important in
some freeze-dried or spray-dried systems since they are first
Received: July 8, 2021
Revised: September 15, 2021
Published: September 30, 2021

© 2021 American Chemical Society https://doi.org/10.1021/acs.jpcb.1c06087

11292 J. Phys. Chem. B 2021, 125, 11292−11307
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Figure 1. Schematic representation of a molecule of TFD (α-[4-(1,1-dimethylethyl)phenyl]-4-(hydroxydiphenylmethyl)-1-piperidinebutanol).
dissolved in water before imposing subsequent freezing and
drying stages to systems.4−7
Even in small quantities, the presence of water has a major
influence on the physicochemical properties of the amorphous
solids including their effective stability against recrystallization
or their chemical degradations.2,8,21,22 A significant piece of
work has been devoted to this topic aiming to understand the
exact mechanisms of action of water. Numerous studies have
particularly focused on the structural and dynamical properties
of water molecules themselves. Several investigations have
demonstrated the existence of different categories of water
molecules, that is, free water, loosely bound water, or tightly
bound water, depending on their positions and how they
interact with the molecules forming the amorphous solid.23,24
Evidence of the inhomogeneous organization of water and the
formation of water clustering8 of various sizes depending on
the water content has also been obtained by various methods
that include neutron and X-ray scattering, molecular dynamics
(MD) simulation, and nuclear magnetic resonance. At very low
water contents on various glassy carbohydrate matrices, based
on the positron annihilation lifetime spectroscopy, it was also
particularly proposed that water might act as a hole filler
between molecules.25
Major efforts have been particularly devoted to the
investigation of the impact of water on the dynamical
properties of the amorphous solids for which water uptake
has been shown to generate greater molecular mobility and
consequently a depression of the glass-transition temperature
Tg.21,22,26 The possibility for water molecules to diffuse in the
glassy matrices has been demonstrated by several works from
NMR spectroscopy and neutron scattering experiments.27,64
MD studies of water diffusion in a number of complex systems
have also been undertaken in which the mechanisms of
translational jumps for water diffusion were of particular
interest in this context. Based on computations, several
extremely complex processes were proposed to describe the
“tortuous” paths, followed by the water molecules.28,29 The
problem of diffusion of a small species in another frozen matrix
is not restricted to molecular systems, and it is also seen in
metallic30 and ionic conductor glasses.31
Dielectric relaxation spectroscopy (DRS) is a well-suited
technique to follow rotational dynamics since it probes both
global and localized dipolar reorientation motions over a wide
accessible frequency range (mHz to GHz).32,33 DRS signals
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can be strongly impacted by the presence of water because of
its high polarity.5,34−38 Traces of water that can be present
even in very dry products can be detected by DRS and not by
some other techniques. Above Tg, in the liquid and
supercooled regime, dynamics as seen by DRS is often
characterized by a single relaxation usually called the primary
α-relaxation.32 This relaxation is associated with the global
dynamics of the liquid or the supercooled liquid. It measures
the structural relaxation of the systems, and the characteristic
time associated with the α-relaxation approximately equals 100
s at the glass-transition temperature. Addition of water makes
the α-relaxation faster and thus lowers the glass-transition
temperature.32 This effect is well known as plasticization and
has been investigated in many works.22,26,35,39−42 It seems well
accepted that this effect in polymers can be interpreted as due
to an increased free volume and the consequent loosening of
the interchain and/or intrasegmental motions.35 A similar
interpretation has also been provided for systems made of low
molecular weight molecules. Oppositely, below Tg, dynamics as
probed by DRS, is typically characterized by a number of faster
relaxations called secondary relaxations, which are far from
being so well understood.23,32,36,37 A deeper understanding on
these local relaxations is important since they may have some
impacts of different phenomena including sub-Tg crystalliza-
tion processes or the stability of dried biological systems.5
These secondary relaxations originate from often unclear local
dynamics of molecules associated with some polar groups of
molecules (moiety and functional groups). The influence of
water on these processes has been studied, and it has been
shown, by analogy with the primary α-relaxation, that water
may have some kind of plasticization or anti-plasticization
effect on them. Some other works have particularly suggested
that some secondary relaxations could directly originate for the
orientation of water molecules themselves.34 The molecular
origin behind these relaxations and the peculiar role of water
are still under controversy.
In the present study, the model pharmaceutical terfenadine
(TFD) C32H41NO2 has been investigated. The literature on
this system is relatively scarce. Calorimetric studies of TFD
have shown the existence of two polymorphic forms I and II
with melting temperatures TmI = 424.1 K and TmII = 416.5 K,
respectively, but others forms are suspected.43,44 Details about
the crystalline structure of the two forms are lacking. The
molecular mobility of TFD has been recently investigated by
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Dudognon et al. by means of DRS experiments and MD
simulations.45 TFD was shown to be a good glass former (Tg =
330 K) with fragile nature (m = 112),45 which also has the
advantage to be easily vitrified by different amorphization
routes including melt-quenching or milling.46,47 However, it
was evidenced that these two routes of preparation weakly
influence its dynamics.45 TFD is a hydrophobic molecule with
solubility in water equal to 10.2 μg mL−1,48 and consequently,
the interactions between TFD and water are poorly
documented. Interestingly, based on scanning electron
microscopy, a peculiar interaction of amorphous TFD with
water was shown to generate a self-limiting crystallization
process.49 This work particularly demonstrated that when
amorphous TFD is exposed to water vapor, a small amount of
the surface crystallizes, which inhibits the whole crystallization
process. It was suggested that water molecules may localize in
different pores or cavities at the surface of the amorphous
material and trigger the process of crystallization of TFD in
these pores. However, these newly crystallized regions at the
surface also seal the access of further water molecules, which
stops the continuation of the crystallization process.49
In this article, based on complementary DRS experiments
and MD simulations, we performed a thorough investigation of
the structural organization of water in TFD amorphous solids
and how it impacts dynamics and in particular secondary
relaxations.
2. EXPERIMENTAL SECTION
2.1. Materials. The racemic TFD (C32H41NO2) was
furnished by Sigma-Aldrich (purity ≥ 97.5%, Lt
MKBX6318V) and was used as received. TFD is a quite
large molecule (Mw = 471.67 g mol−1) compared to the
majority of active pharmaceutical ingredients (see Figure 1). It
contains three polar sites (two hydroxyl groups and one
nitrogen) numbered 1, 2, and 3 as presented in Figure 1. At
room temperature, TFD is in the form of a white crystalline
powder. TFD melting point is found at Tm = 425 K.49 It can be
vitrified by quenching from the melt, and the glass-transition
temperature reported in the literature is Tg = 327−332 K with
a heating rate of 10 K min−1.43,45
2.2. Preparation of Amorphous TFD. The amorphous
TFD was prepared by quenching from the melt using a
differential scanning calorimetry (DSC) Q200 from TA
Instrument equipped with a Refrigerated Cooling System.
About 5 mg of crystalline TFD was heated in an aluminum pan
until 453 K with a heating rate of 5 K min−1. This latter
temperature was chosen since it is about 30 K above the
melting point and thus allows us to obtain a melt with no
crystalline germs. A cooling of 20 K min−1 to room
temperature vitrified the melt. The obtained glass was
immediately stored either at room temperature (RH ≈ 36%)
for 2−47 days or in a desiccator containing saturated sodium
chloride solution (RH ≈ 76% at RT) for 3 weeks for further
analysis. The annealing times were arbitrarily chosen.
It should be noted that the annealed glass of TFD prepared
by melt-quenching and stored either at room temperature or in
a controlled humidity environment (RH ≈ 76%) was found to
be X-ray amorphous at room temperature. Assessment of
chemical degradation by 1H NMR analysis (data not shown)
revealed that the chemical integrity of TFD, is maintained.
For dielectric measurement, about 60 mg of crystalline TFD
was placed between two gold-coated electrodes (diameter 10
or 20 mm) with quartz spacers (50 μm thickness) to avoid
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short circuit between the two electrodes. A heating to 453 K/
cooling cycle at 5 K min−1 was applied while recording the
complex permittivity at some frequencies. This ensures that the
melting of the sample is achieved and verifies that no re-
crystallization occurs on cooling. After cooling to room
temperature, the electrodes were removed from the dielectric
cell and immediately stored in the conditions previously
described.
2.3. Dielectric Relaxation Spectroscopy. Dielectric
measurements of amorphous annealed TFD were carried out
using a Novocontrol Technologies GmbH Alpha Analyzer in
the frequency range from 10−1 to 106 Hz at temperatures
ranging from 173 to 453 K. The temperature was controlled
through the Quatro Cryosystem (supplied by Novocontrol
Technologies) with a stability condition of 0.5 K. The complex
permittivity ε*(ω) = ε′(ω) − iε″(ω) was recorded while
samples were submitted to a sinusoidal electrical field of 1 V
magnitude.
Dielectric relaxation data were analyzed using the Havriliak−
Negami (HN) model function50 as expressed by
Δε
ε*(ω) = ε∞ +
[1 + (iωτHN)αHN ]βHN (1)
where Δε = εs − ε∞, defined as the difference between the
dielectric constant measured at low and high frequencies, gives
the magnitude of the relaxation process. τHN is the Havriliak−
Negami characteristic relaxation time of the process, and αHN
and βHN are the shape parameters taking into account the
broadening and asymmetry (0 < αHN ≤ 1, 0 < βHN ≤ 1). In the
case of the existence of dc-conductivity, we used an additional
term (−i σdc/(ε0 ω)), where σdc is the dc-conductivity and ε0 is
the permittivity of the vacuum. From the HN fitting parameter
values, the model-independent relaxation time τmax = 1/2πFmax
ÄÅ É Ä É
was calculated by
ÅÅ i α π yÑÑÑ−1/ αHN ÅÅÅ i α β π yÑÑÑ−1/ αHN
ÅÅ jj zzÑÑ ÅÅ jj HN HN zzÑÑ
= τHNÅÅsinjjj zzÑÑ ÅÅsinjj zÑ
ÅÅ j 2 + 2β zzÑÑÑ
ÅÅ 2 + 2βHN z{ÑÑÑ
ÅÅÇ k ÅÅÇ k HN {Ñ
ÑÖ ÑÖ
HN
τmax
(2)
2.4. MD Simulations. In the present work, MD
simulations were performed on pure TFD (see Figure 1)
and TFD/water mixtures using the all-atom OPLS and the
TIP3P water force field.51,52 Four systems have been
investigated: pure TFD and three TFD/water mixtures
corresponding roughly to 1, 10, and 99% w/w water
concentrations. To prepare the pure TFD system, a pseudo-
crystal made of 64 TFD molecules (4 × 4 × 4 simple cubic
cells) was constructed. Then, this system was rapidly melted at
a high temperature T = 500 K in order to generate an initial
disordered configuration. For TFD/water mixtures at 1 and
10% water concentrations, the same procedure was used and
either 16 or 192 water molecules were just included in the
construction of the pseudo-crystal. For the TFD/water mixture
at the 99% w/w water concentration, a single TFD molecule
was inserted into a large TIP3P water box pre-equilibrated at
300 K. Then, water molecules overlapping with the TFD
molecule were removed to generate a system made of a single
TFD molecule and 1338 water molecules. This latter system
aims at investigating TFD in a fully hydrated situation.
Computations have been performed at different temperatures
by rapidly cooling the pure TFD and the 1 and 10% TFD/
water mixtures from 500 to 300 K. The simulation times range
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from 10 ns at 500 K to 50 ns at 300 K. This procedure has

been repeated five times to generate five independent
trajectories in order to improve the statistics and estimate
the uncertainties. It should be noted that MD simulations have
been thus performed either on equilibrated or non-equilibrated
states. At the highest temperatures, 450 and 500 K, the pure
TFD and the 1 and 10% TFD/water mixtures systems can be
considered as fully equilibrated over the duration of the MD
simulations [see some examples of time-dependent correlation
function in Figure S1 (Supporting Information)]. However, at
the lowest temperatures, only the 99% TFD/water mixtures
can be considered as fully equilibrated. The three other
systems cannot be fully equilibrated and they thus enter the
glassy state.
All calculations have been carried out using the DL_POLY
package.53 Cubic periodic boundary conditions have been
Figure 2. First recordings of tan δ = ε″/ε′ vs F from 193 to 283 K of a
applied in all simulations. To integrate the equations of
glass of TFD annealed 17 days at room temperature (full symbols)
motion, we used the Verlet algorithm, with a time step of 2 fs, where the supplementary S-relaxation appears. For the sake of
and bond lengths were constrained by means of the SHAKE comparison, the spectra of a non-annealed glass of TFD (open
algorithm. A Lennard-Jones potential has been employed to symbols) recorded at four representative temperatures (193, 213, 233,
represent van der Waals interactions. For the electrostatic and 253 K) are added. The solid lines represent the fits of the data
interactions, a pairwise damped shifted method developed by (two and one Cole−Cole functions, respectively, for annealed and
Wolf was used. The same cut-off radius of 10 Å has been used non-annealed samples).
for both van der Waals and Coulombic interactions. Thermal-
ization was carried out in the isobaric−isothermal NPT to the high-frequency shoulder of the S-relaxation found in the
ensemble at a pressure P of 1.0 Atm. The Nosé−Hoover annealed glass. This is clearly visible in Figure 3, where, for the
thermostat and barostat relaxation times have been chosen as
0.2 and 2.0 ps, respectively. The stabilized volume of the
simulation box during the NPT simulation was considered to
compute the averaged volume of the system and used to
perform the subsequent production simulation in the NVT
ensemble (constant number of particles, volume, and temper-
ature).
Dipolar properties have been obtained from the time
dependence and statistics of the individual molecular dipole
moments computed in the different systems at the different
investigated temperatures. The time-dependent dipole mo-
ment of a given molecule at time t is particularly calculated
N
from the classical expression: μ⃗ (t ) = ∑α =a 1 qα rα⃗ (t ), where qα
and r⃗α(t) are, respectively, the fixed charge localized on atom α
and its position at time t and Na is the number of atoms in the
considered molecule (Na =3 and 64 for the water and TFD
molecule, respectively). Fixed charges are directly taken from Figure 3. tan δ = ε″/ε′ recorded at 228 K for the glass annealed 17
the OPLS and TIP3P force field.51,52 days at room temperature (black symbols). The green and red lines
represent the Cole−Cole functions of the S- and γ- processes,
3. RESULTS respectively, and the black line corresponds to the sum. For the sake
of comparison, the recording at the same temperature of the non-
3.1. Dynamic Behavior. The dielectric spectra of annealed glass of TFD (blue symbols) is added, as well as the fitting
amorphous TFD annealed 17 days at room temperature have by a Cole−Cole function (blue line).
been recorded in the glassy state between 10−1 and 106 Hz
from 173 to 283 K (increment of 5 K). Figure 2 represents tan sake of comparison, the tan δ dielectric spectra recorded at 228
δ as a function of frequency (full symbols). In the temperature K for TFD non-annealed (in blue) and annealed 17 days at
range from 193 to 283 K, we can observe one relaxation room temperature (in black) are reported (the spectra showing
crossing the frequency window. It is very intense and the evolution at 228 K of the imaginary part of the permittivity
characterized by a shoulder on the high-frequency side that and of the modulus can also be seen in Supporting Information
can be seen especially at the lowest temperatures. For S2). Therefore, in order to fit the data of annealed TFD, two
comparison, some dielectric spectra of a freshly obtained Cole−Cole functions (βHN = 1)54 were used (also reported in
glass recorded in the same temperature range are also reported Figure 3). It should be noted that, since the S-relaxation is
in Figure 2 (open symbols).45 It can be seen that this about 3 times more intense than the γ-one and the γ mode is
relaxation mode does not appear on the dielectric spectra of quite broad, the two modes are difficult to deconvolute. The
non-annealed glass. It will then be called S-relaxation (for magnitude of the fitting functions can thus be biased, which
supplementary relaxation). Besides, observed in more detail, could explain the slight shift toward the low frequency of the
the γ-relaxation of the non-annealed glass seems to correspond fitted γ mode observed for the annealed glass. However, based
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on raw data which are reliable, one can note that the
magnitude of the shoulder on the spectra of annealed TFD
systematically appears slightly lower than the magnitude of the
γ-relaxation of non-annealed TFD.
The fit of these data permitted us to extract the characteristic
relaxation time τmax of the γ and S modes appearing for
annealed glass. The Arrhenius diagram can be seen in the
Supporting Information (Figure S3).
The dependence of S-relaxation time versus the reciprocal
temperature obeys an Arrhenius law
Ea
log τmax = log τ∞ +
Ln 10 R T (3)
where τ∞ would be the relaxation time at infinite temperatures,
Ea is the activation energy, and R is the perfect gas constant.
The best fit was obtained for log τ∞ = −14.48 ± 0.06 close to
the expected value (log τ∞ = −14) and an activation energy of
Ea = 50 ± 1 kJ mol−1. The shape parameter αHN slightly
increases with temperature from about 0.5 to 0.6 (βHN = 1).
This high value reflects the narrowness of this mode.
Regarding the γ-relaxation, the log τmax values extracted from
the Cole−Cole fit are close to the evolution of the relaxation
time obtained for the non-annealed glass by Dudognon and et
al.45 (log τ∞ = −14.1 ± 0.1 and Ea = 40 ± 1 kJ mol−1) (cf.
Figure S3). However, the dispersion of points prevented us to
precisely determine the behavior law of the relaxation time.
This dispersion comes from the difficulty to deconvolute the S
and γ modes, as previously mentioned. In any case, it seems
that the γ-relaxation time is not or only slightly influenced by
the appearance of the S-mode.
The results obtained at higher temperatures upon isotherms
ranging from 343 to 368 K of TFD previously annealed 45
days at room temperature are reported in Figure 4 (solid
Figure 4. Recordings between 343 and 368 K of tan δ = ε″/ε′ vs F
showing the α-relaxation of TFD annealed 45 days at room
temperature (solid lines). From 363 K, the signal collapses due to
crystallization. The increase of tan δ values at low frequencies is due
to σdc. For comparison, the recording at 348, 358, and 368 K for non-
annealed TFD is added (dashed lines).
lines). We can observe the α-relaxation which moves toward
higher frequencies with increasing temperature. Its magnitude
vanishes above 363 K. It is due to the re-crystallization of the
sample. At low frequencies, a tail of dc-conductivity also
appears. For the sake of comparison, the spectra recorded in
the same temperature range for non-annealed TFD are also
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added in Figure 4 (dashed lines). It can be seen that, contrary
to the freshly quenched sample, the annealed TFD
recrystallizes on heating, but the most striking feature is that
the α-relaxation of TFD previously annealed at room
temperature (solid lines) is enhanced compared to the one
of the freshly melted-quenched sample (dashed lines). There is
a shift of about 1 decade toward high frequencies. This
indicates that the relaxation time of the α-process which refers
to the global motions of the system decreases when this latter
is annealed a few days at room temperature.
Upon further heating, the recrystallized sample melts at 423
K. The sample of TFD obtained after the quench of this liquid
was analyzed above and below Tg and compared to the freshly
quenched one. Figure 5 is a plot of tan δ as a function of
Figure 5. Recordings at 343 K of tan δ = ε″/ε′ vs F showing the α-
relaxation of TFD freshly melted (red line), after annealing of the
glass 45 days at RT (green line), and after melting of that sample
recrystallized on previous heating (black line). The inset shows a
comparison in the glassy state at 253 K of the same samples.
frequency obtained upon isotherms at 343 and 253 K (cf. inset
Figure 5, black line). It can clearly be seen that below Tg, in
the glassy state, the S-relaxation does not appear anymore and
only the γ-process remains. The obtained spectrum is nearly
identical to the spectrum of the non-annealed glass (red line)
(cf. inset Figure 5). Additionally, the dielectric spectrum at 343
K of the obtained liquid (black line) is superimposed to that of
the freshly melt-quenched one (red line). Hence, it proves that
the phenomenon at the origin of the S-process and the shift of
the main relaxation is reversible: it is not due to a chemical
degradation of TFD glass that could have occurred during
annealing.
In order to understand the difference in dynamics that exists
between the annealed and non-annealed glasses of TFD,
progressive heatings of glass annealed 17 days at room
temperature were carried out. In the temperature range of
193−353 K, we performed cycles of successive isotherms
starting from 193 K with an increment of 5 K till an end
temperature of the “n + 1” cycle 10 K higher than that of the
“n” cycle. At the end temperature of each cycle, an annealing of
40 min was then carried out before recording the next one.
The dielectric spectra acquired at 218 K during these
successive measurements are presented in Figure 6. One can
observe that, during these cycles of heatings, the S-process
magnitude decreases and it shifts toward the lower frequencies.
In the same time, the γ-relaxation arises and its intensity
increases upon heating. This result indicates that the dynamics
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Figure 6. Recordings of tan δ = ε″/ε′ vs F, at 218 K, of a glass of TFD
stored 17 days at room temperature during cycles of successive
isotherms starting from 193 K with an increment of 5 K until an end
temperature of the “n + 1” cycle 10 K higher than the “n” cycle. An
annealing time of 40 min was carried out at the end temperature of
each cycle before recording the next one.
of these two modes are coupled. The decrease of the S-process
magnitude upon heatings added to the fact that the relaxation
time of the α-process of TFD decreases when the glass is
annealed a few days at room temperature suggests that the
absorbed water could be at the origin of these two phenomena.
Therefore, these progressive heatings could be assimilated to
drying of the sample, which would result in a loss of absorbed
water. In other words, the annealed glass of TFD could be
dehydrated during these progressive heatings, with water being
responsible of the observed changes of dynamics.
To investigate a possible effect of water, we have annealed a
glass of TFD 21 days in a desiccator containing a saturated
sodium chloride solution (RH ≈ 76% at RT) and compared it
to a glass annealed 45 days at ambient conditions (RH = 36%)
(cf. Table 1). Thermogravimetric analysis (TGA) shows that
Table 1. Annealing Conditions at Ambient Temperature of
TFD Glasses and Water Content Determined by TGA
annealing relative humidity water content
sample time (%) (%)
glass of TFD 1 21 days 76 1.73
glass of TFD 2 45 days 36 1.26
the weight loss of these two glasses occurs in two steps (cf.
Figure S4). First, there is a continuous loss from room
temperature to at least 400 K. Then, there is a second loss
starting from around 475 K, which is due to the thermal
degradation of TFD. The first loss can then be assigned to loss
of absorbed water. The measurements of the corresponding
water content for both samples were done at 448 K in order to
have results comparable to those of DRS, where the effective
heating rate is much slower (about 0.9 K min−1) and the
material quantity is 12 times greater. The sample annealed 21
days at RH ≈ 76% shows a water content of 1.73%, so about
0.5 point more than the glass annealed 45 days at RH ≈ 36%.
Thus, despite the hydrophobic characteristic of crystalline
TFD,55 the TGA experiment shows that the glass can absorb a
significant amount of water. The same behavior was observed
in the case of indomethacin (IMC).26 Figure 7A shows the
dielectric spectra obtained upon isotherm at 253 K for the
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glasses containing 1.23% water (blue line) and 1.73% water
(red line) and for a non-annealed glass (black line) (free of
water). One can observe that the intensity of the S-process
increases strongly with increasing water content. Therefore, we
can definitely relate the S-process with the presence of water,
absorbed by the sample during annealing.
Moreover, as can be seen in Figure 7B, the α-relaxation
moves toward higher frequencies with increasing relative
humidity. This enhancement of mobility of the main relaxation
above Tg can indeed be assigned to a plasticization effect
because of the well-recognized ability of water to act as a
plasticizer in lowering the glass transition and increasing the
molecular mobility.7,9,39 This remarkable increase of the
mobility at 343 K is about 1 decade for the sample annealed
at 36% RH and 2 decades for the one annealed at 76% RH,
which is a clear indication that the TFD matrix mobility is
changing due to the incorporation of water molecules. In a
detailed study of supercooled amorphous IMC, Andronis and
Zografi26 observed a similar behavior; that is, the increasing
water content significantly lowers the relaxation time of
amorphous IMC. Indeed, they provided the evidence that for
every 1% water content in amorphous IMC, the Tg is lowered
by ≈ 10 K.9 Besides, a simple rescaling of ε″/εmax″ vs F/Fmax
allows us to evaluate the effect of water on the shape
parameters of the α-relaxation peak. The spectra of TFD
previously annealed with different RH conditions superimpose
with the spectrum of the freshly melted-quenched glass (cf.
Figure S5). In other words, the shape parameters of α-
relaxation are water independent. This suggests that the
distribution of the dielectric relaxation time remains the same
as the water uptake increases. Hence, water does not generate
more heterogeneity in the main dynamics of TFD.
As a final remark of this part, it is striking that traces of the
S-relaxation which have been associated with the presence of
absorbed water are still detected after heating and annealing at
353 K (see Figure 6) and that the plasticization of the main
dynamic is still observed at 368 K (see Figure 4). Moreover, a
close inspection of the inset of Figure 5 shows that the
spectrum of the glass obtained from the melting of TFD
previously annealed 21 days at RH = 76% and recrystallized
upon subsequent heating (black line) does not perfectly
superimpose with the non-annealed glass (red line). Indeed, a
small discrepancy can be observed around 102 Hz that could
be due to the presence of a “residual” S-process. To get more
insights concerning the origin of this discrepancy, recordings in
the glassy state were carried out after successive increasing
annealing times at 453 K (Tm +30 K) of TFD previously stored
21 days at RH ≈ 76%. The spectra recorded at 253 K are
reported in Figure 8. In this figure, one can notice that, after a
simple heating until 453 K and cooling, the spectrum of the
obtained glass shows a γ-relaxation with a shoulder appearing
clearly around 102 Hz. It indicates the existence of a residual S-
process. However, one can notice that the intensity of this
residual S-process decreases for increasing annealing time at
453 K. Also, the recrystallized sample has to be heated up to
the melting temperature Tm = 425 K in order to release the
water molecules that were trapped as inclusion in the
crystalline structure. It is quite striking that the sample should
be heated up to 453 K (Tm +30 K) and annealed at this
temperature for about 1 h to find the signature of the non-
annealed glass (back line). This would suggest that some water
molecules absorbed during the storage of the glass at room
temperature really remain embedded in the sample (till 453 K)
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Figure 7. (A) Recordings at 253 K of tan δ = ε″/ε′ vs F showing the γ- and S-relaxation of TFD glasses containing 0, 1.26, and 1.73% of water. (B)
Recordings of ε″ vs F at 343 K of the same samples. The lines through the data points correspond to the HN fit of the α-relaxation after subtraction
of the σdc contribution.
Figure 8. Recordings at 253 K of tan δ = ε″/ε′ vs F showing the γ-
relaxation in the case of the non-annealed TFD (black line) and after
different annealing times (0, 5, 20, and 50 min, respectively) at 453 K
for the TFD sample previously stored at RT 21 days in a desiccator
over a saturated sodium chloride solution.
and are possibly highly hydrogen-bonded to TFD molecules.
In order to clarify this feature at the molecular scale, molecular
modeling works have been performed that will be described in
the next section.
3.2. MD Simulations. MD simulations have been
performed in order to investigate in more detail structural
and dynamical properties of TFD/water mixtures (see
simulation details in the Experimental Section) and in
particular the HB interactions between TFD and water
molecules. Our aim was to shed some light on the behavior
of TFD in the presence of water, which seems to be the
microscopic origin of the peculiar S dynamical process
detected from DRS experiments. A thorough analysis of HB
associations that can be formed in the pure TFD system and in
the different TFD/water mixtures has been thus performed.
The TFD molecule possesses two −OH hydroxyl groups and
one nitrogen atom from which it may form HBs with other
TFD molecules and/or with water molecules (see Figure 1).
The two −OH hydroxyl groups can participate to an HB either
as a donor or as an acceptor while the nitrogen atom can only
be an acceptor. It should be noted that TFD can also
potentially form an intra-molecular HB involving one of the
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two −OH hydroxyl groups and the nitrogen atom localized
close to the central part of the molecule (see Figure 9C). In
order to identify HBs in the different studied system, a
geometric criterion has been chosen. Two molecules are
considered to be H-bonded if (i) the nitrogen-oxygen or the
oxygen−oxygen distance is less than 3.4 Å and (ii) the (N−
H−O) or (O−H−O) angle is larger than 150°. This criterion
is in some way arbitrary due to the lack of information on the
electron density, but it has been successfully used in many MD
simulations and allows us to take into account a wide variety of
HBs including more deformed and weaker HBs in
statistics.56−58 Some snapshots of instantaneous configurations
of the pure TFD system and the TFD/water system at 1% w/w
water concentration at T = 300 K have been generated and are
displayed in Figure 9A,B, respectively. They illustrate the very
rich diversity of HB associations which can exist between the
TFD molecules and TFD and water molecules in a dried
sample in which some residual water is still present.
3.2.1. Statistics of Intra-molecular and Inter-molecular
HBs. Statistics on the number of intra-molecular and inter-
molecular HBs formed on average in the four investigated
systems at T = 300 K are reported in Table 2. For pure TFD
and TFD/water mixtures at low water concentrations (1 and
10% w/w), a few intra-molecular HBs are clearly detected (see
Figure 9C). Since one TFD molecule can only form one intra-
molecular HB, it corresponds to a fraction of about 12.5% of
TFD molecules in the pure TFD system. This 12.5% fraction is
simply calculated by dividing the number of intra-molecular
HBs, 8, by the total number of TFD in the system, 64. This
fraction tends to slightly decrease to about 9.8% upon
increasing the water concentration, but this trend is not
completely clear due to the large uncertainties associated with
the number of HBs. Indeed, the pure TFD and the TFD/water
mixtures at the studied low water concentrations (1 and 10%
w/w) at T = 300 K are actually in a glassy state obtained from
a rapid quench from high temperatures. Although MD
simulations were repeated five times at each concentration,
the large uncertainties associated with the number of HBs
actually reflect the possibility for the different systems to be
frozen in many different configurations with many different
kinds of HB associations between molecules. It should be thus
certainly necessary to perform a much larger number of
independent MD simulations for the different systems in order
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Figure 9. (A) Snapshots of instantaneous configurations as obtained from MD simulations at T = 300 K in the pure TFD system. Only
configurations involving the hydroxyl group and the nitrogen atom localized close to the central part of the TFD molecule are shown. (a) Simple
HB association as a dimer where two TFD molecules (#TFD1 and #TFD2) are HB-bonded by a single HB involving two hydroxyl groups. (b):
More complex dimer for which two TFD molecules (#TFD1 and #TFD2) are HB-bonded by two HBs involving two hydroxyl groups and one
nitrogen atom. Because of the formation of two HBs, this association is clearly more stable than the previous one. (c): Simple trimer where three
TFD molecules (#TFD1, #TFD2, and #TFD3) are HB-bonded through single HBs between them. A short chain-like structure is thus formed. (d)
Another example of a trimer in which the nitrogen atom is involved. HBs are indicated by dotted lines. (B) Snapshots of instantaneous
configurations as obtained from MD simulations at T = 300 K in the TFD/water mixture at 1% w/w concentration. A single water molecule is
shown to be capable to form a HB with two (a), three (b), or four (c) TFD molecules. Figures (b,c) are actually zoom-centered on the water
molecule. HBs are indicated by dotted lines. (C) TFD molecule for which the possible intra-molecular HB is shown (dashed line).

Table 2. Number of Intra-molecular HBs Formed by TFD and Number of Inter-molecular HBs Formed between TFD
Molecules and TFD and Water Molecules at T = 300 K for the Four Investigated Systemsa
inter-molecular HB TFD−TFD inter-molecular HB TFD−water
intra- HB TFD
O2−H−N O1 O2 N total O1 O2 N total
pure TFD (0% w/w) 8.0 ± 1.0 5.7 ± 0.5 14.5 ± 1.0 2.3 ± 0.2 22.5 ± 1.6 - - - -
TFD/Water (1% w/w) 7.9 ± 1.0 5.0 ± 1.2 13.5 ± 1.5 2.1 ± 1.0 20.6 ± 1.7 5.1 ± 0.5 10.1 ± 0.5 4.0 ± 0.4 19.2 ± 1.1
TFD/Water (10% w/w) 6.3 ± 0.5 3.5 ± 0.5 10.5 ± 0.6 0.8 ± 0.2 14.8 ± 1.2 23.7 ± 0.7 41.4 ± 2.2 21.9 ± 0.8 87.0 ± 3.6
TFD/Water (99% w/w) 0 - - - - 71.7 ± 7.0 110.0 ± 5.1 53.8 ± 1.9 235.5 ± 14.1
a
For inter-molecular HBs, the number of HBs is also specifically given for the two hydroxyl groups (O1 and O2) and the nitrogen atom (N) (see
Figure 1) of the TFD molecule. For the TFD/water mixtures at 99 % w/w water concentration, the number of HBs have been multiplied by the
number of TFD molecules present in the other investigated systems, i.e., 64, to allow a direct comparison (see details in MD simulations).
Uncertainties have been calculated based on five independent MD runs and/or using block averaging.

to obtain a full description of the HB associations that could
exist in the glassy state of the different TFD/water systems. It
is clear that the formation of an intra-molecular HB for TFD
tends to reduce its inter-molecular HB propensity. For
example, it might have strong consequences on the capability
of TFD to form large HB clusters as already demonstrated for
disaccharide/water mixtures from MD simulations.56 Interest-
ingly, no intra-molecular HBs are found for the fully hydrated
TFD system at 99% w/w water concentration. It shows that
the presence of a large amount of water molecules completely
inhibits the formation of this intra-molecular HB. It could be
suggested that water molecules, because of their quite small
size and high mobility, can very easily access to the central
−OH hydroxyl group (O2 in Figure 1) and the nitrogen atom
and form HB bonds with them. This suggestion is confirmed
by a closer examination on the details of the HBs formed with
the different HB groups of the TFD molecules in the 99% w/w
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TFD/water mixture. Table 2 particularly shows that 64 TFD
molecules form on average 71.7, 110.0, and 53.8 HBs with
water molecules through the −OH hydroxyl group (O1), the
−OH hydroxyl group (O2), and the nitrogen atom (N)
localized close to the central part of the molecule, respectively
(see Figure 1). It roughly corresponds to about 1.1, 1.7, and
0.9 HBs per TFD molecule. From these results, the HB
propensity of the central part of the TFD molecule approaches
an optimal situation assuming theoretically that two water
molecules can HB-bond to the −OH hydroxyl group (O2) and
one water molecule to the nitrogen atom. Oppositely, the
terminal −OH hydroxyl group (O1), localized in between
three large rings, seems much less accessible and only about
one water molecule can form a HB with this group. Upon
decreasing the water concentration, this trend is still observed.
Table 2 shows that the central −OH hydroxyl group (O2)
systematically forms from 2−3 times more HBs than the
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Figure 10. (A) Fraction of HB aggregates formed by TFD molecules at 300 K calculated from MD simulations for pure TFD and TFD/water
mixtures at 1 and 10% w/w concentrations. n represents the number of TFD molecules in the HB aggregate. For example, n = 1 corresponds to
isolated molecules, n = 2 to dimers, n = 3 to trimers, and so forth. Dotted lines are a guide for the eye. (B) Fraction of HB aggregates formed by
water molecules at 300 K calculated from MD simulations for TFD/water mixtures at 1% (inset) and 10% w/w concentrations. n represents the
number of water molecules in the HB aggregate. Dotted lines are a guide for the eye.

terminal −OH hydroxyl group (O1) either considering the
inter-molecular HBs between the TFD molecules or the inter-
molecular HBs between the TFD and water molecules. It
should be noted that the same trends are also observed at
higher temperatures (data not shown). Overall, at all
investigated concentrations and temperatures, about 75% of
all inter-molecular HBs originate from the HB groups localized
close to the central part of the TFD molecule, that is, the −OH
hydroxyl group (O2) and the nitrogen atom. These results thus
clearly provide evidence of the importance of these HB groups
through which TFD preferentially interacts.
For pure TFD, the total number of HBs formed is about
22.5. This number seems relatively weak compared to the
potential capability of the TFD molecule to form HBs. Indeed,
theoretically, one could expect that five HBs could be formed
for one TFD molecule: two for each −OH hydroxyl group and
one for the nitrogen atom. In the present simulations, the pure
TFD system is composed of 64 molecules. It thus indicates
that each TFD molecule does form less than one HB on
average and potentially many TFD does not form HBs at all.
This result will be confirmed in the following by analyzing the
TFD HB cluster distribution. Upon increasing water
concentration, Table 2 reveals that the number of HBs formed
between TFD molecules decreases, while at the same time, the
number of HBs formed between TFD and water molecules
significantly increases. This result is obviously expected
because of the change in the concentration of water in the
mixtures. However, overall, the total number of HBs increases
upon increasing water concentration because of the higher
capability of water to access HB sites of TFD. Interestingly, for
the 1% w/w water concentration, one may particularly notice
in Table 2 that the number of HBs formed between TFD
molecules remains almost the same within uncertainties
compared to the pure TFD systems while TFD also forms
HBs with water. This result suggests that, at this very low water
concentration, the few water molecules in the system actually
fill the pre-existing empty spaces between the TFD molecules.
This feature has been already nicely shown from MD
simulations in lysozyme/trehalose/glycerol and trehalose/
glycerol glasses at low hydration.7
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Interestingly, the obtained results suggest that the small
amount of water molecules that mainly localized close to the
central part of the TFD molecules through HB may potentially
have a stronger impact on its peculiar dynamics, which was
associated with the γ-mode as suggested in ref 45. More
discussions on the link between MD simulations results and
DRS experiments will be provided in the following.
3.2.2. TFD and Water HB Clusters. Based on the HB
geometric criterion used in the present study (see above), the
distribution of clusters composed of n TFD molecules or n
water molecules have been calculated from MD simulations at
T = 300 K. In the following, a cluster of size n is defined as a
set of n molecules connected by at least one HB between them.
The TFD cluster distribution is shown in Figure 10A,B.
Figure 10A shows that a large variety of HB aggregates
composed of TFD molecules are theoretically possible (see
Figure 9A,B for snapshots of HB associations). The fraction of
HB aggregates mostly ranges from isolated molecule (n = 1) to
large aggregates made of n = 8 molecules. Additional even
larger (n > 8) associated structures could also be transiently
observed over a very short time, but their fraction remains
really very small at all investigated temperatures. It should be
noted that the extension of the TFD HB clusters does not
seem very developed compared to similar studies performed by
some of the authors on other small organic molecules for
which clusters involving more than n = 10 molecules can be
found.57,58 Figure 10A particularly shows that the maximum of
the cluster size distribution corresponds to n = 1, that is,
isolated molecules. Then, the probability of forming a cluster
of size n monotonically decreases upon increasing n. It thus
again indicates that a large fraction of TFD molecules (45−
62% depending on the water concentration) actually does not
form HBs with other TFD molecules and about 20% of them
may just form HB dimers (see Figure 9A). Large HB
associations seem rare or very rare (10% or below).
Nevertheless, snapshots displayed in Figure 9A(c,d) reveals
the possibility for TFD molecules to form short HB chains,
which remind the presence of HB linear chains in HB liquids
such as monohydroxy alcohols and amides.59 The cluster size
distribution shows the inability for TFD to form very large
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clusters. It certainly stems from the TFD-specific molecular
architecture (see Figure 1) as already mentioned for which the
three HB groups of the TFD molecule unfavor the HB inter-
molecular associations: (i) the −OH hydroxyl group (O1) is
strongly sterically screened because of its position surrounded
by three large rings, (ii) the central −OH hydroxyl group (O2)
is more accessible, but because of its “not terminal” position
(as in monohydroxy alcohols), it is not possible to build very
large supramolecular associations through it, and (iii) the
nitrogen atom can only be the HB acceptor. In addition, the
fact that an intra-molecular HB (see Table 2) may also form
between the central −OH hydroxyl group (O2) and the
nitrogen atom (see Figure 9C) is another strong factor which
contributes to the limited extend of the TFD HB clusters.
For the pure TFD system, on average, isolated molecules
represent about 45% of all TFD molecules. This fraction
increases to 50 and 62% upon increasing the water
concentration because water molecules progressively inter-
calate between TFD molecules, and HBs between TFD
molecules are progressively replaced by HBs between TFD and
water molecules (see Table 2 and Figure 9B). At the first
glance, the increase of the fraction of isolated TFD molecules
upon increasing water content may seem surprising. In this
calculation, it should be reminded that only HB interactions
between TFD molecules were actually taken into consideration
excluding HB interactions between TFD and water molecules.
Obviously, the fraction of TFD molecules forming no HB
either with other TFD molecules or with water molecules
decreases upon increasing water concentration. It roughly
corresponds to 35 and 14% at 1 and 10% w/w water
concentration, respectively. Interestingly, one may note that
the TFD cluster size distribution for the pure TFD system and
the 1% w/w TFD/water mixtures are actually very close.
Therefore, a very small amount of water does not seem to
strongly impact on the HB network existing between TFD
molecules. As already indicated, it shows that, at this low water
concentration, the few water molecules present in the system
preferentially localize in the empty holes in between TFD
molecules (see Figure 9B(b,c)). Upon a further increase of the
water concentration to 10% w/w, once most of the empty
spaces between TFD molecules are actually filled by water
molecules, the action of water molecules actually seems to
separate TFD molecules as proven by the sudden jump of the
fraction of isolated TFD molecules in Figure 10A from 50 to
65% and a decrease of the probability to form large associated
HB structures such as trimers, tetramers, and so forth.
Figure 10B shows the fraction of HB aggregates formed by
water molecules in the TFD/water mixtures at 1 and 10% w/w
concentrations. A snapshot of an instantaneous configuration
of the simulation box at 1 and 10% w/w concentrations is also
displayed in Figure S6.
The distribution of HB water cluster size at 1 and 10% w/w
water concentration is shown in Figure 10B. At 1% w/w water
concentration, the distribution of the cluster size does not
indicate the presence of large water aggregates. It is confirmed
from the snapshot displayed in Figure S6a which clearly shows
that water molecules are quite dispersed in the 1% w/w TFD/
water mixtures. From the distribution shown in Figure 10B,
about 70% of water molecules do not form HBs with other
water molecules and about 25% may only form dimers. The
remaining 5% of the water molecules may form trimers or even
more rarely tetramers.
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Oppositely, calculations performed at the 10% w/w water
concentration show the formation of one large water aggregate
(see Figure S6b). It could be hypothesized that water
molecules present in such a large water cluster (see Figure
S6b) would be much easy to remove by standard heating
processes than the small fraction of quite isolated water
molecules embedded in the TFD matrix at the 1% w/w water
concentration (see Figure S6a). The presence of large
percolating water volume which may facilitate dehydration
was particularly suggested in freeze-drying processes.60 It
should also be mentioned that both situations encountered at
1% w/w and 10% w/w are perfect illustrations of “unclustered
water” and “clustered water” found in pharmaceutical systems,
which have been extensively reviewed in ref 8. In order to
confirm this hypothesis, some snapshots of instantaneous
configurations of the 1% w/w water concentration system at T
= 300 K have been generated and are displayed in Figure 9B.
They illustrate the type of interactions which can exist between
TFD and water in a dried sample in which some residual water
is still present. Interestingly, a single water molecule is
particularly shown to be capable of forming at least transiently
HBs with two (Figure 9B(a)), three (Figure 9B(b)), or even
four (Figure 9B(c)) TFD molecules. Obviously, situations for
which water molecules form only one HB or even no HB are
also frequently detected. It is particularly remarkable that a
single water molecule may form simultaneously four HBs with
its surrounding made of non-water molecules. Excluding the
deformed HBs, it is actually the maximum number of HBs that
a single water molecule can form. It should be noted that
several occurrences of this type of HB associations have been
detected in MD runs. The snapshots displayed in Figure 9B
suggest that the presence of water may contribute to reinforce
the affinity between TFD molecules by creating some kind of
bridges through HBs. This type of effects have been reported
in another context related to the water anchorage hypothesis,
which assumes that residual water in freeze-dried biomaterial
products anchors the protein dynamics to its surroundings.61,62
From MD simulations, it was also particularly demonstrated
that the presence of water molecules reinforce molecular
coupling for some bioprotectants such as trehalose dis-
accharide and proteins.7
In order to complete this analysis and to be more
quantitative, the fraction of water molecules forming either 0,
1, 2, 3, or 4 HBs with TFD molecules at different temperatures
has been calculated and is shown in Figure 11. This figure
clearly demonstrates that at the highest investigated temper-
atures, water molecules are weakly HB with TFD. At T = 500
K, about 68, 23, and 4% of water molecules form no HB, one
HB, and 2 HBs with TFD, respectively. Upon decreasing
temperature, at about 400 K, the number of water molecules
forming no HB with TFD significantly drops below 50%, while
the number of water molecules forming 1 or more HB
progressively increases. One may particularly notice the
continuous increase of the small fraction of water molecules
forming three or four HBs with TFD molecules. It should be
noted that these features corroborate particularly well in line
with the present reported experimental results, which show
that water molecules only leave the samples in reasonable
conditions above 453 K. At T = 300 K, it remains only about
32% of water molecules forming no HB with TFD. This
decrease seems to continue even at lower temperatures. In
other words, at least at low temperatures and at low water
contents, that is, 1% w/w in the present case, it seems that
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molecules forming none, one, or two HBs with TFD (see

Figure 11. Fraction of water molecules forming either 0, 1, 2, 3, or 4
HBs with TFD molecules at different temperatures in the TFD/water
mixture at 1% w/w water concentration. Uncertainties have been
statistically estimated by performing five independent MD simulations
at the different investigated temperatures.
water molecules because of their smaller size and higher
mobility tend to interact through HB with TFD more
preferentially than with other water molecules well in line
with the data reported in Table 2. From the calculations
performed at T = 300 K, water molecules could be thus
classified into different categories depending on the average
number of HBs they may form with TFD: weakly HB water
molecules forming either 0 or 1 HB with TFD, moderately HB
water molecules forming two HBs with TFD, and strongly HB
water molecules forming three or four HBs with TFD. It is
thus tempting to suggest that different categories of residual
water molecules could coexist in dried samples of TFD: (i) a
majority of weakly or moderately hydrogen-bonded water
particularly Figure 9B(a)), which could be easy to remove
from the sample by a simple heating/drying process and (ii) a
minority of much more highly HB water molecules embedded
in the dried TFD matrix (see particularly Figure 9B(b,c)) that
could be much more difficult to remove without a specific
heating/drying treatment.
3.2.3. Link between Inter-molecular HBs and Molecular
Mobility. A detailed investigation of the link between inter-
molecular HBs formed by water molecules with TFD
molecules and water-specific translational and rotational
molecular mobility has been performed. As already mentioned,
water is recognized as an efficient plasticizer.22,26,35,39−41 The
increase of molecular mobility upon increasing the water
content has thus been checked from the present MD
simulations by calculating the time-dependent dipolar
correlation of the TFD molecules ϕT(t) as well as the time-
dependent correlation function of the TFD−TFD HBs ϕHB(t).
Both functions are shown in Figure S1 (Supporting
Information) at the temperature T = 450 K for pure TFD
and TFD/water mixtures at 1% and 10% w/w concentrations.
A faster decay is found for both ϕT(t) and ϕHB(t) upon
increasing the water content, which clearly confirms the
plasticization effect of water on the TFD matrix.
In the following, we will mostly focus on the 1% w/w TFD/
water mixture which is more relevant for comparison with the
experimental works performed in the present paper in which
water removal was found to be problematic. Figure 12b
represents the number of HB bond formed by some targeted
water molecules (among the 16 water molecules included in
the simulation box at this concentration) with TFD as a
function of temperature. Two targeted water molecules have

Figure 12. (a) Mean-squared displacement of water ⟨u2w⟩ and TFD ⟨u2T⟩ molecules as a function of temperature in the TFD/water mixture at 1%
w/w water concentration. The mean-squared displacements for water molecules are calculated at time t = 10 ns. The mean-squared displacement at
time t = 10 ns of bulk TIP3P water at room temperature is also indicated for comparison (dashed line) and (b) number of HBs formed between
water and TFD molecules. The curves labeled “max HB” or “min HB” represent the results obtained for the water molecule (among the 16 water
molecules included in the simulation box at this concentration) forming the maximum or the minimum number of HBs with TFD, respectively.
The curves labeled “avg HB” represent the average values obtained considering the 16 water molecules. Uncertainties have been statistically
estimated by performing five independent MD simulations at the different investigated temperatures. (c) Time-dependent dipolar correlation
function ϕw(t) at the temperature T = 300 K (full symbol) and T = 450 K (open symbol) for three targeted water molecules labeled #1(circle), #2
(square), and #3 (triangle up), which are strongly, moderately, or weakly HB to TFD molecules. Water molecules have been selected to match with
the definition max/min/avg defined in Figure 12a,b. On average, water molecules #1 and #2 form the highest and the lowest number of HBs with
TFD, while water molecule #3 forms an intermediate number of HBs with TFD. The function ϕw(t) is calculated from the normalized
autocorrelation function ⟨μ⃗ (t)·μ⃗ (0)⟩/⟨μ⃗ 2⟩, where μ⃗ (t) is the dipole moment of a given water molecule (either #1, #2, or #3) at time t. The brackets
mean averaging over the different time origins. Dashed lines (T = 300 K) and dotted lines (T = 450 K) are a guide for the eye.

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been specifically followed: the water molecule forming the
maximum (max HB) and minimum number (min HB) of HBs
with TFD, respectively. The average number of HBs (avg HB)
formed by water molecules with TFD molecules is also shown.
It is actually the average value at each temperature of the data
represented in Figure 11. Figure 12a shows the mean-squared
displacement ⟨u2w⟩ of the two targeted water molecules as well
as the average value calculated including all water molecules in
the simulated system. Figure 12a also shows for comparison
the average mean-squared displacement ⟨u2T⟩ of the TFD
molecules. The mean-squared displacement ⟨u2w⟩ and ⟨u2T⟩ are
actually shown instead of the usual diffusion coefficients
because the diffusive regime cannot be actually reached over
the duration of the present MD simulations at the lowest
investigated temperatures since the investigated systems
progressively enter into a glassy state. The arbitrary time t =
10 ns is chosen in order to ensure that the system has left at
least the short-time dynamics regime associated with very fast
motions such as vibrations and librations and enters either
some cage dynamics or some sub-diffusive or possibly diffusive
regime depending on temperature. Indeed, previous inves-
tigations of the water mobility in glassy matrices have shown
that the paths of water molecules followed during their
somehow “tortuous” trajectories could be extremely com-
plex29,63 including several diffusive processes. A full description
of diffusion of water molecules in the TFD matrices thus goes
beyond the present investigation.
At the highest temperatures T = 450 and 500 K, Figure 12b
shows that the two selected water molecules (min HB and max
HB) form a similar number of HB with TFD, and values are
actually quite close to the average value obtained considering
all water molecules. Some small differences ranging from about
0.2 to 0.4 HBs actually exist between the two selected water
molecules. Oppositely, Figure 12a reveals that, already at those
high temperatures, some significant differences exist on
translational molecular mobility between water molecules. A
difference of about 1−2 orders of magnitude is found on the
mean-squared displacement ⟨u2w⟩ between the fastest water
molecule, which is forming the minimum number (min HB) of
HBs with TFD and the slowest water molecule, which is
forming the maximum number (max HB) of HBs with TFD.
Therefore, although the number of inter-molecular HBs
formed by those two water molecules with TFD only slightly
differs by 0.2−0.4 HBs, a significant difference on mobility is
found. Water molecules thus seem to behave quite differently
in the system. It is interesting to note that the ⟨u2w⟩ calculated
for the fastest water molecule and the average value are close to
the one obtained for bulk water at room temperature. It
suggests that most water molecules are diffusing very fast in the
high-temperature range except possibly the few water
molecules which are slightly more HBs to TFD. Upon
decreasing the temperature, from about T = 400 K, a clear
separation emerges between the water molecules (see Figure
12b) demonstrated by a difference of more than two HBs
between the water molecule forming the maximum (max HB)
and the minimum number (min HB) of HBs with TFD. The
behavior of both molecules clearly separates from the average
value, which increases monotonically upon decreasing the
temperature well in line with the densification of the system.
For the water molecule forming the minimum number (min
HB) of HBs with TFD, a quite constant value of about 0.5 HBs
is found, which seems completely independent of the
temperature. For the water molecule forming the maximum
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number (max HB) of HBs with TFD (see illustrations
displayed in Figure 9B(b,c)), a marked increase of the number
of HBs with TFD upon decreasing temperature is seen. It may
be speculated that the trend found in the investigated
temperature range will continue somehow at lower temper-
atures at which the separation between min HB and max HB
will become even more important. This difference on the
number of HBs is also clearly reflected on the mean-squared
displacement ⟨u2w⟩ with a sudden drop of more than 2 decades
between 400 and 350 K of ⟨u2w⟩ for the water molecule forming
the maximum (max HB) with TFD. Owing to the very low
value of ⟨u2w⟩ ≈ 1 Å2 at 300 and 350 K, the max HB water
molecule could be considered as almost immobilized over the
duration of the MD simulations. This sudden drop of
molecular mobility for some water molecules actually coincides
with the strong decrease of molecular mobility of TFD
molecules as measured by the average mean-squared displace-
ment ⟨u2T⟩. At the lowest investigated temperatures T = 300
and 350 K, ⟨u2T⟩ also decreases to values of about 1 Å2 or
below. It reinforces the idea of the presence of water molecules
completely slaved of the TFD matrix. Interestingly, one may
remark that the ⟨u2w⟩ calculated for the fastest water molecule
and the average value continuously decreases upon decreasing
temperature without significantly decreasing unlike the ⟨u2w⟩
for the water molecule forming the maximum (max HB) with
TFD. It suggests that the dynamical properties of some water
moleculesand possibly majority of themare decoupled
from that of the TFD matrix. These findings are in good
agreement with previous experimental studies on decoupling of
the mobility of water molecules from that of the glassy matrix
in which they are embedded. For maltose,64 the authors have
shown that water diffusion could be remarkably fast and
decouples from the matrix diffusion, and it is not strongly
affected by the glass transition. The same results were also
proposed for water mobility in glassy sucrose.60 This marked
difference in dynamical behavior between selected water
molecules is particularly well in line with the ideas previously
suggested in the present study of the two different categories of
water molecules, that is, weakly and strongly HB to TFD, that
may coexist in the 1% w/w water system (see above and also
Figure 9B).
In addition to translational dynamics of water molecules
probed by mean-squared displacements ⟨uw2 ⟩, rotational
dynamics have also been analyzed using time-dependent
dipolar correlation functions ⟨ϕw(t)⟩ based on autocorrelation
functions of an individual water molecule dipole moment.
Results are shown in Figure 12c. Functions ϕw(t) have been
computed at two temperatures, 300 and 450 K, for three
targeted molecules that are weakly, moderately, or strongly HB
to TFD molecules in the same manner as reported in Figure
12a,b. At 450 K, the time-dependent dipolar correlation
functions ϕw(t) of the three targeted molecules decay very
similarly. This behavior actually contrasts with the results
obtained at the same temperature for the mean-squared
displacements ⟨u2w⟩ (see Figure 12a) for which clear differences
were noticed between water molecules weakly and strongly HB
to TFD molecules. This feature can be understood from the
fact that a given water molecule may rotate without to
necessarily break the inter-molecular HBs, while it is required
for the water molecule to break those HBs to diffuse. In other
words, translational degrees of freedom may be more
influenced by small changes of the HB network than rotational
degrees of freedom. It might have an impact on how water
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J. Phys. Chem. B 2021, 125, 11292−11307
The Journal of Physical Chemistry B
molecules are actually removed in drying/heating processes.
Indeed, small differences on HB inter-molecular interactions
between water molecules and TFD could explain why some
water molecules may easily leave from the sample while some
others may remain. Oppositely, at T = 300 K, major differences
between the three types of water molecules are observed well
in line with the results previously reported in Figure 12a,b.
While the ϕw(t) function of the less HB water molecule to
TFD may still fully relax and decay to zero, the relaxation is
only partial for the moderately HB water molecule and ϕw(t)
does not decay to zero but to a plateau value. For the strongly
HB water molecule to TFD, rotational dynamics is almost
arrested and no long-time decay is seen over the duration of
the MD simulation. It could be hypothesized that the different
behaviors seen for water molecules embedded in the TFD
matrix, either weakly, moderately, or strongly HB to TFD
molecules, may give rise to some additional dynamical
processes, which could be observed in DRS experiments
because of the very high polarity of the water molecule.
It particularly reinforces the previously suggested ideas that
the origin of the supplementary S-process would originate from
peculiar dynamics of residual water molecules. Since these
water molecules seem to preferentially localize through HB
close to the central part of the TFD molecule, one could thus
speculate on the existence of some kind of coupling between
dynamics of these water molecules and dynamics of the very
flexible central part of the TFD molecule, which is also
suspected to give rise to the very broad γ-relaxation.45 It would
thus explain why the S-process is exactly detected in the same
frequency range as the γ-relaxation and could be then
described as a component of the γ-relaxation of slower
mobility.
4. CONCLUSIONS
In this article, the liquid, supercooled liquid, and glassy states
of TFD have been investigated by means of complementary
DRS experiments and MD simulations.
A new secondary relaxations process (namely process S) is
detected for the first time in the glassy state of TFD annealed
at room temperature (Tg30 °C). This process is completely
absent in a non-annealed glass of TFD. The molecular origins
behind secondary relaxations are often under controversy, and
therefore, thorough investigations to understand the micro-
scopic origin of the S-process have been performed. We have
shown that this mode is clearly linked to the presence of
residual water in the concentration of 1−2% (w/w). Several
results supporting this attribution were particularly obtained.
During the cycles of heating of the annealed glass, it was
shown that the S-process magnitude decreases and shifts
toward lower frequencies. These progressive heatings could be
assimilated to a form of drying of the sample, which would
result in a loss of absorbed water.
From annealing studies in controlled humidity, based on
TGA experiments, it was actually confirmed that the TFD glass
can absorb a significant amount of water (about 2%) in
contrast to the known hydrophobic characteristic of crystalline
TFD.
The temperature dependence of the S-process was shown to
possess an Arrhenian behavior with an activation energy of
about 50 kJ·mol−1, which is the value reported for processes
usually seen in DRS and associated with the influence of
water.34
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In the supercooled domain, the main α-relaxation of TFD
was found to move toward higher frequencies with increasing
relative humidity. This feature was usually assigned to a
plasticization effect because of the well-recognized ability of
water to increase the molecular mobility.
The S-process is detected in the same frequency range as a
previously detected process (namely, processes γ) observed in
the non-annealed TFD glass.45 Taking into account the
important broadness of this mode that spreads over 7 decades
and its quite high activation energy, the origin of the γ-process
was attributed to the intramolecular motion of the very flexible
central part of the TFD molecule well in line with MD
computations. During the cycles of heating of the annealed
glass, the intensity of the S-process and γ-process was shown to
behave in an opposite trend, suggesting a peculiar coupling
between these two sub-Tg relaxations. Moreover, it was also
observed that the sample had to be heated up to 453 K (Tm
+30 K) and annealed at this temperature for about 1 h to
completely remove the S-process and then find the signature of
the non-annealed glass. It was interpreted as an indication of
strongly HB-bonded water molecules trapped in the TFD
matrix.
MD simulations performed on TFD samples at different
water concentrations allowed us to clarify these two unconven-
tional features (coupling between γ- and S-processes and
strongly HB water molecules) which allowed us to progress on
the exact nature of secondary relaxations. From MD
computations and analyses of the HB interactions, two
categories of water molecules were revealed: i) a majority of
weakly or moderately HB water molecules to TFD and ii) a
minority of much more highly HB water molecules strongly
interacting with the OH hydroxyl group and the nitrogen atom
localized close to the flexible central part of the TFD molecule.
Weakly or moderately HB water molecules to TFD seem easier
to remove from the system by usual heating/drying processes.
Such water molecules particularly tend to form very large water
clusters at the highest water concentration 10% w/w (see
Figure 10A). Dynamics of this type of water molecules enter in
the pico-nanosecond time domain (GHz to THz frequency
range) accessible to the MD simulations. However, these
dynamics are too fast to be observed since they are outside of
the present DRS investigated frequency range. Oppositely,
highly HB water molecules to TFD are more dispersed in the
TFD matrix and do not form large water clusters between
them. Actually, very small water clusters made of few water
molecules may still exist in the very dried sample at 1% w/w
(see the inset in Figure 10B). These water molecules localize
themselves in small pockets in the pre-existing voids spaces
existing in between the TFD molecules due to the poor
packing. Because of their strong HB interactions with TFD and
weak mobility, they seem much more difficult to remove
without a specific treatment. These highly HB water molecules
might particularly explain why it is necessary to heat the
sample at so high temperatures to completely remove the S-
process. Molecular mobilities of these water molecules which
are typically in the [102 to 105] Hz frequency domain are not
accessible to MD simulations.
During the cycles of heating of the annealed glass or from
annealing studies in controlled humidity, it was shown that the
S-process magnitude decreases and shifts toward lower
frequencies upon decreasing the water content. This can be
understood assuming that the S-process originates from the
reorientation of the remaining water molecules that are still
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J. Phys. Chem. B 2021, 125, 11292−11307
The Journal of Physical Chemistry B
present even in the dried samples and which are bound to TFD
molecules. At 1% w/w water concentration, a given water
molecule belonging to these small clusters may interact both
with a TFD molecule, which tends to reduce its mobility and
to other water molecules of the same cluster, which tend to
accelerate its mobility. It seems obvious that water molecules
are more mobile than the TFD molecule. Upon a further
decrease of the water content, the number of molecules in the
water clusters will be decreased. Accordingly, the given water
molecule will interact less with its surrounding neighboring
water molecules and more with TFD. Its mobility will be thus
decreased well in line with the frequency of the S-process,
which decreases upon decreasing water content. Furthermore,
taking into account that the origin of the γ-process is
associated with molecular mobility of the central part, and
since the remaining water molecules tend to localize
preferentially close to this zone, it may explain the peculiar
coupling found between the S- and the γ-processes.
The present study is an attempt to clarify the microscopic
origin of secondary relaxations detected in TFD and the role of
water. It clearly motivates to perform investigation on other
systems to test the universality of our findings.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jpcb.1c06087.
Time-dependent dipolar autocorrelation function ϕT(t)
of TFD molecules and time-dependent autocorrelation
function ϕHB(t) of inter-molecular HBs formed between
the TFD molecule; comparison of tan δ, ε″, and M″
versus F at 228 K for the annealed (17 days) and non-
annealed glasses of TFD; Arrhenius diagram of annealed
and non-annealed glass of TFD; TGA description and
weight losses of crystalline TFD and TFD glasses
annealed 45 days at RH ≈ 36% and 21 days at RH ≈
76% as a function of temperature; invariance of the
shape of the α-relaxation of TFD samples containing 0,
1.26, and 1.76% of water; snapshots of instantaneous
configurations as obtained from MD simulations at T =
300 K in the TFD/water mixture at 1% w/w and 10%
water concentration; and TGA description (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Frédéric Affouard − University Lille, CNRS, INRAE, Centrale
Lille, UMR 8207UMETUnité Matériaux et
Transformations, Lille F-59000, France; orcid.org/0000-
0001-8429-6416; Phone: +33 (0)3 20 43 68 15;
Email: Frederic.Affouard@univ-lille.fr; Fax: +33 (0)3 20
43 40 84
Authors
Jeanne-Annick Bama − University Lille, CNRS, INRAE,
Centrale Lille, UMR 8207UMETUnité Matériaux et
Transformations, Lille F-59000, France
Emeline Dudognon − University Lille, CNRS, INRAE,
Centrale Lille, UMR 8207UMETUnité Matériaux et
Transformations, Lille F-59000, France; orcid.org/0000-
0003-4176-1925
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jpcb.1c06087
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pubs.acs.org/JPCB Article
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This project has received funding from the Interreg 2 Seas
program 2014−2020 co-funded by the European Regional
Development Fund (FEDER) under subsidy contract 2S01-
059_IMODE.
■
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