pubs.acs.

org/crystal Article

Milling-Induced Phase Transformations, Underlying Mechanisms,

and Resulting Physical States in an Enantiotropic System: The Case
of Bezafibrate
Emeline Dudognon,* Florence Danède, and Mathieu Guerain
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ABSTRACT: The mechanisms driving the milling-induced transformations of an

enantiotropic polymorph system, bezafibrate, are depicted. The phase trans-
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formations promoted by milling at two different temperatures and the resulting

physical states were carefully investigated by the cross-use of structural (X-rays
powder diffraction), thermodynamic (differential scanning calorimetry), and
dynamic (dielectric relaxation spectroscopy) techniques. Our results highlight that
milling of the commercial α phase at Tg50 °C (−10 °C) leads to a complete
amorphization (Tg ∼ 40 °C), whereas at Tg15 °C (25 °C), it leads to the stable
β phase. We establish that, as for monotropic situations, the solid−solid conversion
is mediated by a transient amorphous state resulting from a milling-induced
disordering of the crystalline structure partly counterbalanced by a slower re-crystallization. However, the monitoring of the
transformation kinetics (phase ratio and crystallites’ size) reveals that at least 10% amorphous phase is required to trigger the re-
crystallization to the stable β form instead of the metastable α form. For the first time, the molecular mobility of the physical states
resulting from milling is finely investigated by dielectric relaxation spectroscopy. Strikingly, it evidenced, for the crystalline phases
produced by milling, a residual mobility (detection of localized intra-molecular motions but absence of wide-amplitude motions
characterizing the amorphous state), which originates through the mobility of part of molecules on the surface of crystallites. This
outstanding result emphasizes and unravels the highly defective nature of the crystalline phases generated by milling.

1. INTRODUCTION understood despite milling-induced phase transitions, that is,

Milling is an important stage in the processing of all types of amorphization or polymorphic transformations, have been
materials. Indeed, in addition to reducing the size of particles, extensively studied over the years.
For molecular compounds, it has been shown that a
high-energy milling is able to trigger physical transitions and
preponderant parameter is the milling temperature with regard
chemical reactions.1 As it allows to reach nonequilibrium
to the glass transition (Tg) of the compound:15 amorphization
states, called driven materials by Martin and Bellon,2 it is used
is observed when milling is performed well below Tg whereas
for the formation of alloys (from intermetallics3−5 till
solid−solid conversion between polymorphic crystalline forms
molecular crystals6), or on a more fundamental point of
is observed when milling is performed above Tg. Regarding the
view, it can be used to explore the different physical states of a
involved mechanisms, the amorphization appears to result
compound and the screening of polymorphs and their relative
from the increasing disorder generated by the mechanical
stabilities.7 Moreover, milling avoids the use of solvents or
perturbation.16 However, as far as solid−solid transformations
high-temperature processing. It is thus an interesting
are concerned, the situation is blurrier as it refers to the relative
technology that develops in many different fields from
thermodynamic stability of the different crystalline phases of
metallurgy to biology.1 In particular, in the pharmaceutical the compound. On the one hand, the two crystalline
field, originally used to decrease the size of particles in order to polymorphic phases can form a monotropic set: one of the
enhance the dissolution, the powder flowability, or the two phases is stable in the entire temperature range whereas
tableting,8 it becomes more and more a tool for mechanosyn- the other one is always metastable (the Gibbs free energy of
thesis of promising multi-components systems aiming to
overcome solubility issues of drugs such as nanocrystals,9 co-
crystals,10,11 amorphous solid dispersions in early-stage Received: August 30, 2021
research,12 co-amorphous system,13 or the loading of drugs Revised: November 12, 2021
into mesoporous matrices.14 However, in order to use Published: November 25, 2021
efficiently this technology, the mechanisms underlying these
transformations have to be controlled. However, even the
simple case of the milling of a pure compound is still not fully

© 2021 American Chemical Society https://doi.org/10.1021/acs.cgd.1c00997

363 Cryst. Growth Des. 2022, 22, 363−378
Crystal Growth & Design
the first is always lower than that of the latter).17,18 Then, it
seems from previous investigations of the mechanisms ruling
the solid−solid conversion under milling that it proceeds
through an intermediate amorphous state: milling leads to an
amorphization of the compound but the mobility enables a
rapid re-crystallization.19 On the other hand, the system can be
enantiotropic: each phase is stable in a temperature range, and
the temperature at which the inversion of stability occurs
(crossing of the two corresponding Gibbs free energy curves)
being the theoretical temperature of the solid−solid trans-
formation.17,18 Then, because this transition point exists, a
solid−solid conversion may directly occur under milling
without the requirement of a transient amorphous state.
However, unfortunately, too few cases of such enantiotropic
sets are reported to solve this issue. It has been shown that the
milling at room temperature of the stable phase (II) (at this
temperature) of sulindac leads to a complete amorphization of
this compound.20 In the case of sulfamerazine, milling of both
the stable and the metastable phases leads to the crystalline
stable phase.21 It is also the case of sulfathiazole but a complex
behavior is observed for the milling of the stable form (III) (at
the milling temperature). Indeed, at the initial stage of milling,
the metastable form (I) appears, presumably assisted by the
development of a transient amorphous fraction. This results
after 10 min milling in a mixture of stable (III) and metastable
(I) crystalline phases; however, upon further milling, the
transformation reverses back.22,23 Therefore, this kind of
milling-induced transformations and their mechanism still
remain unclear.
Bezafibrate is an organic compound used for its efficiency as
an antihyperlipoproteinemic drug. It can appear in two
crystalline forms: α and β, commercial bezafibrate being in
the α form. From a detailed investigation of both crystalline
phases, Lemmerer and co-workers evidenced that they form an
enantiotropic set: the β form is the stable form at room
temperature and solid−solid transition to the α form occurs on
heating at around 160 °C.24 The crystalline structure of the α
form, which melts at 185 °C (ΔHm = 52 kJ mol−1),24 is in the
orthorhombic system with the space group P212121,25 whereas
the β form crystallizes in the centrosymmetric space group
P21/c.24 Besides, this active pharmaceutical ingredient is poorly
water soluble (0.016 ± 0.003 mg mL−126 ). Because the use of
the amorphous state can improve the bioavailability, the glass-
forming ability of bezafibrate was investigated.27 ,28 Wytten-
bach and co-workers28 showed that a glass of bezafibrate could
be obtained by the quenching of the liquid (Tg = 37 °C) and
did not crystallize on subsequent heating. However, to the best
of our knowledge, the glassy state of bezafibrate, in particular
the dynamic, has not been further explored.
In this article, we report a detailed investigation of the
behavior of bezafibrate under high-energy milling. The aim is
to take advantage of the thermodynamic features of this
compound to enhance the knowledge of the mechanisms
ruling the milling-induced transitions. For this purpose and
given the key role of the milling temperature on triggered
phase transformations, milling is performed at two different
temperatures, −10 °C and the room temperature (∼25 °C). In
both cases, milled bezafibrate is then deeply characterized by
three complementary techniques: X-rays powder diffraction
(XRPD) to probe the structure and microstructure, differential
scanning calorimetry (DSC) to probe the thermodynamic
behavior, and the dynamic relaxation spectroscopy (DRS) that
allows to probe the global and localized molecular motions due
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to the wide accessible frequency range (mHz to MHz). The
cross-use of these techniques allows us to fully describe the
nature of the physical states of bezafibrate generated by the
mechanical perturbation.
2. EXPERIMENTAL SECTION
2.1. Material. Bezafibrate (2-[4-[2-(4-chlorobenzamide)ethyl]-
phenoxy]-2-methylpropanoic acid, C19H20ClNO4, cf. Figure 1, CAS:
41859-67-0, and Mw = 361.82 g mol−1) was furnished by Sigma-
Aldrich and was used without further purification (purity ≥ 98%).
The crystalline form is the α form.
Figure 1. Bezafibrate molecule.
2.2. Methods. 2.2.1. Milling. Milling of bezafibrate was performed
in a high-energy planetary mill (Pulverisette 7 from Fritsch GmbH). 1
g of material was placed in ZrO2 milling jars of 43 cm3 containing
seven ZrO2 balls (Ø = 15 mm), corresponding to a ball/sample
weight ratio of 75:1. The milling was performed at two different
temperatures: −10 °C and room temperature. In the former case, the
milling device was placed in a cold room with a dehumidifier so that a
temperature of −10 °C and a null relative humidity percentage could
be maintained during milling. In order to limit the overheating of the
compound, cycles of 20 min milling (rotation speed of the solar disk
set to 400 rpm) and 10 min pause periods were applied. The total
effective milling time was tmill = 14 h at −10 °C and ranged from tmill =
5 min to tmill = 14 h at room temperature.
2.2.2. XRPD. The XRPD experiments were performed with an
XPERT PRO MPD diffractometer in Debye−Scherrer geometry
(λCu Kα = 1.5406 Å) equipped with an X’celerator linear position-
sensitive detector. Samples were enclosed in Lindemann glass
capillaries (Ø = 0.7 mm) that were mounted on a rotating sample
holder in order to avoid effects of preferential orientations of
crystallites if any. X-rays diagrams were recorded from 5 to 70° (2θ)
with 50 s per step and a step size of 0.0167° (2θ), mostly at room
temperature. For X-rays diagrams recorded at higher temperatures (in
the range of 54−190 °C), an Huber furnace was used. Microstructural
analyses and phase quantification were performed thanks to Rietveld
refinements using MAUD software29 from the data obtained by
Lemmerer et al. for α and β polymorphs (CSD Reference
VAMBOA01 and VAMBOA02).24
2.2.3. Differential Scanning Calorimetry. DSC measurements
were performed in a differential scanning calorimeter Q200 from TA
Instrument equipped with a refrigerated cooling system. Samples were
encapsulated in a standard aluminum pan T0 (2 mg < m < 3 mg) and
measurements were conducted under nitrogen flow (50 mL min−1).
In order to separate when needed the events depending on Cp change
(reversing heat flow), in particular to evidence the Cp-jump
characteristic of a glass transition, from those depending on physical
transformations (non-reversing heat flow), a modulated temperature
protocol (MDSC) was followed. A ramp of 5 °C min−1 with a
temperature modulation of ±0.663 °C per 50 s or ±0.530 °C per 40 s
was applied. Temperatures and enthalpies were calibrated using
indium at the same heating rate and under the same environmental
conditions as the experiments. Specific heat capacity was measured
using sapphire as a reference.
2.2.4. Dielectric Relaxation Spectroscopy. Dielectric relaxation
spectroscopy (DRS) measurements were performed using a
Novocontrol Technologies GmbH Alpha analyzer. A small amount
of the milled sample powder was put between two gold-coated
electrodes (diameter of 10 mm), and inert quartz spacers (50 μm
thickness) were added to avoid short circuits between electrodes. The
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Cryst. Growth Des. 2022, 22, 363−378
Crystal Growth & Design pubs.acs.org/crystal Article

powder was packed as much as possible to reduce the noise due to the The thermogram of the milled compound recorded by
non-homogeneity of the sample. However, it should be noted that, modulated DSC at 5 °C min−1 ± 0.663 °C per 50 s from 0 to
due to the unavoidably entrapped air between grains, the exact 200 °C is reported in Figure 3. It exhibits four events. First, a
volume of the sample could not be measured. Nevertheless, as shown
by Kothary and co-workers,30 even if the increase in porosity leads to
a decrease in the measured dielectric constant, it has no influence on
the relaxation time. In spite of the lack of precision, we preferred this
protocol instead of the application of a pressure for the tableting of
the powder because it would not modify the physical state resulting
from milling. The complex permittivity ε*(ω) = ε′(ω) − iε″(ω) was
recorded while a sinusoidal electrical field of 1 V magnitude and of
frequency, F, varying from 106 to 10−2 Hz was applied to the samples.
Temperature was controlled through a Quatro cryosystem (supplied
by Novocontrol Technologies), and spectra were collected during
isotherms ranging from −100 to 70 °C using a nitrogen gas cryostat
with a stability condition of 0.5 °C. Data were analyzed using the
Havriliak−Negami (HN) model function31 (or a sum of HN
functions when several relaxations appeared) as expressed using the
following equation
Δε
ε*(ω) = ε∞ +
[1 + (iωτHN)αHN ]βHN (1)
where Δε = εs − ε∞ is the difference between the real permittivity
values at respectively low and high frequency limits, τHN is the
Havriliak−Negami characteristic relaxation time of the process, and Figure 3. Thermogram of bezafibrate milled for 14 h at −10 °C
αHN and βHN are shape parameters taking into account the broadening recorded using MDSC (total heat flow) at 5 °C min−1 ± 0.663 °C per
and asymmetry (0 < αHN ≤ 1 and 0 < βHN ≤ 1). From the HN fitting 50 s. The inset shows the reversing heat flow recorded between 20
parameters values, the model independent relaxation time τmax = 1/ and 50 °C for the milled sample and the glass obtained from the

ÄÅ É Ä É
2πFmax was calculated using the following equation

ÅÅ i α π yÑÑÑ−1/ αHN ÅÅÅ i α β π yÑÑÑ−1/ αHN

quenching of the liquid.

ÅÅ jj zzÑÑ ÅÅ jj HN HN zzÑÑ
τmax = τHNÅÅÅsinjjj zzÑÑ
zÑÑ
ÅÅsinjj zÑ
ÅÅ j 2 + 2β zzÑÑÑ
ÅÅ
ÅÇ k 2 + 2βHN {ÑÑÖ ÅÅÇ k HN {ÑÑÖ
HN small and broad endothermic bump can be seen between 10
(2) and 50 °C, both on the total heat flow and the non-reversing
heat flow (not shown). Due to its location, it corresponds to
the loss of some adsorbed water. Indeed, such water sorption is
3. RESULTS usually observed for milled compounds which are more
3.1. Milling at −10 °C (Tmill = Tg50 °C). The reactive, owing to an increased specific area. However, an
commercial α form of bezafibrate was milled for 14 h at −10 inspection of the reversing heat flow in this temperature range
°C. The X-ray diffraction pattern, recorded at room temper- (cf. the inset in Figure 3) reveals that the loss of water masks
ature just after milling, can be seen in Figure 2 (bottom). The the Cp-jump characteristic of a glass transition, which
absence of Bragg peaks indicates that the compound has been corroborates that the sample has been amorphized by milling.
fully amorphized by milling. Successive recordings on It is located at Tg = 39 ± 1 °C, and we obtained the ΔCp
increasing temperature show that the sample re-crystallizes value, ΔCp = 0.41 ± 0.01 J g−1 °C−1 (cf. the inset in Figure 3).
around 50 °C in the β form, which converts into the α form At higher temperatures, an exothermic peak can be seen
between 130 and 150 °C, and then, it melts at 180 °C (cf. between 60 and 70 °C. It corresponds to the re-crystallization
Figure 2). of the sample in agreement with X-ray diffraction results.
Between 140 and 160 °C, a small endothermic peak is
observed (ΔHconv = 3.5 kJ mol−1). As reported by Lemmerer
and co-workers,24 it is due to the solid−solid conversion of the
re-crystallized β form to the α form. Finally, an endothermic
peak can be seen between 180 and 190 °C. It corresponds to
the melting of the α form (Tm = 181.5 ± 0.1 °C and ΔHm =
55.4 kJ mol−1). In addition, commercial bezafibrate has been
heated at 5 °C min−1 until 200 °C, and the melt has been
immediately quenched to −50 °C to obtain a glass. On re-
heating, a Cp jump can be seen in the same temperature range
as for the compound amorphized by milling. However, a close
inspection of the reversing heat flow (cf. the inset in Figure 3)
shows that the glass transition temperature is a little bit lower
(Tg = 37 ± 1 °C and ΔCp = 0.45 ± 0.01 J g−1 °C−1) but in
nice agreement with the value reported by Wyttenbach et al.28
(Tg = 36.9 ± 0.2 °C at 10 °C min−1). It should be added that
no re-crystallization exotherm is observed at higher temper-
ature.
Figure 2. Successive XRPD patterns recorded on increasing To check the chemical nature of the glasses obtained from
temperatures of Bezafibrate milled for 14 h at −10 °C. these two different routes, analysis by 1H NMR has been
365 https://doi.org/10.1021/acs.cgd.1c00997
Cryst. Growth Des. 2022, 22, 363−378
Crystal Growth & Design
Figure 4. Evolutions of tan δ = ε″/ε′ vs the frequency F of the applied electrical field for bezafibrate milled for 14 h at −10 °C. (a) In the glassy
state, at four representative temperatures. Solid lines: first recordings (freshly milled compound) dashed lines: recordings after dehydration at 25
°C. (b) In the supercooled liquid state, on isotherms ranging from 38 to 52 °C by steps of 2 °C (solid lines). At 52 °C, two recordings are added
after isotherms of 300 s (dashed line) and 600 s (dotted line).
performed. The spectra were compared to the spectrum of
commercial bezafibrate (see Supporting Information S1).
Spectra of the commercial and milled compounds are identical
whereas some differences appear in the spectrum of bezafibrate
amorphized by quenching of the melt (in particular between 6
and 7 ppm). It reveals first that some thermal degradation
occurs as bezafibrate melts. According to Shen and Zhou,32 it
seems to correspond to the breaking of the ether bond. Such
thermal degradation, even in small quantity, could explain why
the quenched liquid does not re-crystallize on heating. Second,
1
H NMR analysis highlights that the milling of bezafibrate at
−10 °C permits to obtain a glass of bezafibrate, which is
chemically pure and, in the authors’ knowledge, it is the only
way to obtain it.
The molecular mobility of milled bezafibrate has been
investigated by DRS. First, in order to detect motions ascribed
to local mobility, experiments have been performed in the
glassy state, below Tg. The evolution of the permittivity was
recorded between 10−1 and 106 Hz in isotherms ranging from
−100 to 25 °C, every 5 °C. As an example, some tan δ spectra
(tan δ = ε″/ε′) obtained in this temperature range are reported
in Figure 4a (solid lines). They show the shift of a peak, γw,
toward higher frequencies as the temperature increases. Its
magnitude decreases from −10 °C. Moreover, it is
dissymmetric, and a broad shoulder can be seen on its high-
frequency flank. A second recording in the same temperature
range has been performed. The spectra obtained at the
previous temperatures are added in Figure 4a (dashed lines).
The γw peak has disappeared revealing at higher frequencies a
broad peak, γ, less intense in addition to a shoulder, δ, that is
visible at high frequency for the lowest temperature. The
decrease in the magnitude of the γw mode from −10 °C, in the
first recordings to 25 °C, and its disappearance in the second
recordings indicate that the corresponding mobility is linked to
traces of adsorbed water. This kind of mobility related to
adsorbed water is usually found for compounds amorphized by
milling, owing to their enhanced reactivity.33,34 Here, this
phenomenon could also be reinforced by the fact that the
milled powder has been directly put on cold electrodes to
avoid an overheating at room temperature of the sample, which
could catch some moisture of the surrounding air. Moreover,
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the location of the maximum of the γ mode in the frequency
range where γw becomes asymmetric and the similarities of
spectra on the high-frequency side suggest that the γ and δ
modes are already present in the first recording but are masked
at low frequency by the more intense γw. As an example, a
focus on spectra recorded at −70 °C can be seen in Supporting
Information (S2). The spectra recorded in the second scan
have been analyzed using two Cole−Cole functions (HN
functions with βHN = 1, see Experimental Section) (the fit at
−70 °C of the two corresponding modes γ and δ can also be
seen in S2). In a first approximation, the spectra recorded in
the first scan have been fitted with three Cole−Cole functions,
the γ and δ modes being fixed from fit of the second scan (cf.
S2, e.g., at −70 °C). It permits to obtain an evolution versus
temperature of the frequency of the γw peak maximum similar
but more regular than by a direct reading. Experiments were
also performed at higher temperatures: the recording of the
permittivity between 10−2 and 106 Hz from 25 to 70 °C shows
a peak that enters the frequency window at 38 °C (cf. Figure
4b) and shifts toward high frequency with increasing
temperature. It corresponds to the main relaxation, α, due to
large amplitude motions that are frozen below Tg and thus
associated with the dynamic glass transition. From 50 °C, its
magnitude suddenly decreases and vanishes as the sample re-
crystallizes. One can notice in Figure 4b that, during
crystallization at 52 °C, the maximum of the relaxation slightly
shifts toward high frequency, which indicates a slight
acceleration of the dynamics of the remaining amorphous
fraction. This prevents a proper analysis of the α mode of
partly amorphous samples at higher temperature. In addition,
at low frequencies, a tail of conductivity and Maxwell−
Wagner−Sillars effects appear when the sample starts to
crystallize.
The relaxation times characteristic of the main relaxation
and the different secondary relaxations extracted from the HN
fits are reported in an Arrhenius diagram in Figure 5.
Concerning the main relaxation, α, one can see that the
evolution of the relaxation time with the reciprocal temper-
ature is not linear, even if few points are available, owing to the
re-crystallization. It is usually described using the Vogel−
Tammann−Fulcher−Hesse (VTFH) law35−37
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Cryst. Growth Des. 2022, 22, 363−378
Crystal Growth & Design
Figure 5. Relaxation map of bezafibrate showing the α, γw, γ, and δ
relaxations observed for the sample amorphized by milling at −10 °C
(full symbols). The relaxations observed for the samples milled at
room temperature are also added (open symbols): the γ mode for tmill
= 1 h (green open circles) and the γ′ mode for tmill = 4 h (red open
circles). The lines represent the corresponding fits (see text).
B Relaxation Times Associated With the Different Secondary
log τmax = log τ∞ +
ln(10)(T − T0) (3)
where τ∞ would be the relaxation time at infinite temperatures,
B is a parameter that accounts for the curvature of the
evolution, and T0 is the so-called Vogel temperature at which
the relaxation time would diverge. In a first approximation, log
τ∞ was fixed at −14 to reduce uncertainties in the fit of the
data with the VTFH law (due to the lack of points at high
temperature). It leads to B = 1656 ± 30 K, T0 = 264 ± 1 K,
and an extrapolated value of T = 36 ± 1 °C for τ = 100 s. This
latter value is in nice agreement with the glass transition found
by DSC (Tgonset = 35 ± 1 °C and Tgmid = 39 ± 1 °C). The
Vogel temperature is 45 °C below this extrapolated Tg, in
agreement with the 40−50 °C commonly observed.38 The
degree of departure from the Arrhenius law is usually
quantified through the “strength parameter”, D, related to B
ÄÅ É
by D = B/T039 or to the fragility, m, defined as40−42
ÅÅ d log τmax(T ) ÑÑÑ
Å ÑÑ
m = ÅÅÅ ÑÑ
ÅÅÇ d(Tg/T ) ÑÑÖ
BTg
= mode, the corresponding motion could involve the central part
T = Tg
ln(10)(Tg − T0)2 (4)
The VTFH fitting parameters lead to D = 6.3 ± 0.2 and m =
110 ± 12. Values of m can vary from 16, when no deviation
from the Arrhenius law is observed for the compounds
qualified as strong,39 to 17043 for the compounds qualified as
fragile for which the deviation is the most important.39
Bezafibrate thus appears as a quite fragile glass-former because
its value is comparable to that of compounds considered as
very fragile materials like flurbiprofen (m = 113),44 terfenadine
(m = 112),45 N-acetyl-α-methylbenzylamine (m = 111),46 or
celecoxib (m = 110).47
The non-exponential character of the main relaxation is
usually described in the time domain using the Kohlrausch−
ÄÅ É
Williams−Watts function48,49
ÅÅ i y βKWW ÑÑÑ
ÅÅ jj t zz ÑÑ
φ(t ) = expÅÅÅ−jj zz ÑÑ
ÅÅ jk τα z{ ÑÑ
ÑÑÑ
ÇÅÅ Ö
(5)
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where τα is the relaxation time and βKWW is a parameter varying
from 1 to 0 as the relaxation mode departs from the
exponential (Debye) decay. This parameter can be calculated
from the HN shape parameters, αHN and βHN, through the
relation50
βKWW = (αHNβHN)1/1.23 (6)
We found, in the vicinity of Tg, βKWW = 0.56 ± 0.01. This
value indicates a relatively large degree of non-exponentiality.
It is in agreement with the high fragility and the anti-
correlation law established by Böhmer and co-workers.51
Concerning the different secondary relaxations, the Ar-
rhenius diagram in Figure 5 shows that the associated
relaxation times all obey an Arrhenius law
Ea
log τmax = log τ∞ +
ln(10)RT (7)
where Ea is the activation energy of the involved process and R
is the perfect gas constant. The fitting parameters are reported
in Table 1. The activation energy of δ relaxation is 30 ± 1 kJ
Table 1. Parameters of the Fit with the Arrhenius Law of the
Relaxations Observed for the Material Milled for 14 h at
−10 °C (Glassy: δ, γ, and γw Modes) and at Room
Temperature (β Crystalline: γ′ Mode)
mode log τ∞ Ea (kJ mol−1)
δ −14.0 ± 0.3 30 ± 1
γ −14.91 ± 0.09 48 ± 1
γw −15.6 ± 0.2 57 ± 1
γ′ −14.0 ± 0.2 50 ± 1
mol−1. If we refer to the work of Brás and co-workers on
ibuprofen,52 the corresponding motion of the δ mode could be
fluctuations of the carboxylic group as the same activation
energy was found for this motion. For γ relaxation, observed
after dehydration, the value is higher (48 ± 1 kJ mol−1). The
mode is quite broad and spreads over more than five frequency
decades. The HN shape parameter αHN slowly increases from
0.24 at −80 °C to 0.45 at 20 °C (βHN = 1). Taking into
account the higher activation energy and the broadness of the
of the molecule carrying the amide group (cf. Figure 1). For
the γw relaxation appeared in the first scan and related to a
small amount of adsorbed water, we found an activation energy
of 57 ± 1 kJ mol−1. Because it appears close from the γ mode,
it could be due to the same type of intramolecular motion but
slowed down by some hydrogen interactions between the
water molecules and the oxygen and nitrogen atoms of the
amide group.
These secondary relaxations are of intramolecular type. It
should be noted that another type of secondary relaxation can
be found: those of intermolecular origin (so-called β relaxation
of Johari-Goldstein type)53,54 that involve the motion of the
entire molecule and are regarded as a precursor of the
cooperative α relaxation according to Ngai’s coupling
model.55−57 Such relaxation appears either as a well-resolved
peak or as a change in slope (excess wing) on the high
frequency flank of the α relaxation.58 In the case of bezafibrate,
this type of relaxation could not be observed (possibly because
of the rapid re-crystallization of the sample).
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Cryst. Growth Des. 2022, 22, 363−378
Crystal Growth & Design
3.2. Milling at Room Temperature (Tmill = Tg15 °C).
3.2.1. Structural and Thermodynamic Analyses. Bezafibrate
has been milled at room temperature for a time, tmill, varying
from 5 min to 14 h. Some amounts of milled powder were
regularly sampled from the milling jars and immediately
analyzed by XRPD and MDSC.
Some of the XRPD patterns recorded at room temperature
are reported in Figure 6. Before milling, only the Bragg peaks
Figure 6. XRPD patterns recorded at room temperature for
bezafibrate milled for increasing milling times (indicated on the
right) at room temperature, showing the conversion of the
commercial α form (tmill = 0 min) to the β form (tmill = 850 min)
upon milling.
corresponding to the commercial α form are observed. From
the first minutes to 70 min milling, these Bragg peaks broaden
and their magnitude decreases. Then, from 90 to 130 min, new
Bragg peaks characteristic of the β form appear and develop
whereas those characteristic of the α form disappear. From 130
to 850 min milling, there is no noticeable evolution and only
Figure 7. Evolution vs the milling time at room temperature of the fraction of the different phases: α phase (full blue squares), β phase (full red
circles), and amorphous phase (full green triangles). The evolutions of the size of α (open blue squares) and β (open red circles) crystallites are
represented in the inset. Lines are guide for the eyes.
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peaks characteristic of the β form are observed. Therefore, the
milling of the α form at room temperature for at least 2 h
allows to obtain the β form of bezafibrate. In the authors’
knowledge, this is the only way to obtain this form directly
from the commercial product, without the use of solvents. A
deeper analysis of XRPD spectra was performed using Rietveld
refinements. It permits to determine from the width of the
Bragg peaks the evolution upon milling of the size of
crystallites and, from the peaks’ relative intensity evolution
and the background evolution, the proportion of each phase
and amorphous content. Both evolutions are reported in
Figure 7. One can see (in the inset) that, after only 5 min of
milling, the size of the α crystallites decreases from
approximately 2000 Å to less than 500 Å and it reaches a
plateau value of 250 Å after around tmill = 50 min. An
interesting point is the evidence of some amount of the
amorphous phase that appears from the start of the milling
process. The exact proportion of amorphous phase is difficult
to be precisely determined but it seems to be of the order of
10%. Then, after around 1 h of milling, the β phase appears
and develops for approximately another 1 h of milling, whereas
the amounts of the crystalline α phase and of the amorphous
phase decrease. After tmill = 2 h, the milled powder is 100% in
the β form and no change occurs until the end of the milling
(tmill = 14 h). As the β phase appears, the corresponding
crystallite size first starts to increase to around 1000 Å and then
rapidly decreases to reach a plateau value of 500 Å after tmill = 2
h and does not evolve anymore (cf. inset).
Milled powder was also analyzed using MDSC. Some
thermograms are reported in Figure 8. Three different types of
behavior are observed in the total heat flow. First, for milling
times shorter than 1 h, an exothermic peak appears between 45
and 70 °C and tends to develop with milling time. This peak is
located just above the glass transition range, and it indicates a
re-crystallization of the sample. The appearance of such an
exotherm of re-crystallization is an indirect proof of
amorphization. Nevertheless, the Cp jump characteristic of a
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Figure 8. MDSC thermograms recorded at 5 °C min−1 ± 0.530 °C
per 40 s (total heat flow) of bezafibrate milled at room temperature
for increasing milling times (indicated on the left).
glass transition cannot clearly be observed on the reversing
heat flow: the re-crystallization occurs as soon as the Cp jump
begins and the mobility is released. For most of the recordings
and despite the use of different modulation parameters, only
the start of the Cp jump is observed, but then, the signal is
disturbed by the rapid re-crystallization. The Cp jump is all the
more difficult to observe as it is quite small, which indicates
that only a small fraction of the sample has been amorphized.
In the cases where the reversing heat flow signal was the least
disturbed (tmill = 60 min), tentative analysis of the Cp jump
leads to values of amorphous content around 15%, which is of
the order of the amount extracted from XRPD analysis. At
higher temperatures, the endotherm of melting of the α form
clearly appears between 180 and 190 °C. It confirms that, after
milling, the crystalline part of the sample is still in the α form
and it indicates that a small amorphous content has re-
crystallized to this form. It should be noted that the melting
temperature onset is depressed by 3 °C from that of the
commercial product, which can be explained by the smaller
size of crystallites leading to a Gibbs−Thomson effect.
Second, for milling times longer than 120 min (cf. Figure 8),
the previous exothermic peak located in the glass transition
temperature range is no longer visible and the reversing heat
Figure 9. 3D representation of tan δ recordings: (a) for bezafibrate milled for 60 min at room temperature on isotherms between −90 and 25 °C
(every 5 °C) showing the γ relaxation; (b) for bezafibrate milled for 4 h at room temperature on isotherms between −120 and 50 °C (every 5 °C)
showing the γ′ relaxation.
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flow signal shows no event. This indicates that no amorphous
fraction is detected and that the milled compound is crystalline
(in β-form), in agreement with XRPD results. At higher
temperatures, from 145−150 to 170 °C, an endothermic peak
appears shifting toward high temperature with milling time. It
corresponds to the solid−solid conversion of the β form to the
α form, and above approximately 175 °C, the endotherm of
melting of this α form can be seen. One can notice on the
high-temperature side of the endotherm of conversion, a
shoulder that develops into a peak as the milling time increases
and an exothermic peak at the foot of the endotherm of the α
melting. The shoulder could be due to the melting of β
crystallites that are not yet converted (more numerous because
the conversion is delayed) but the corresponding endotherm of
melting could be partly masked by a rapid re-crystallization to
the α form at the origin of the exothermic event. It should be
noted that the α melting temperature onset is the same as that
of commercial bezafibrate, which indicates that α crystallites
resulting from the conversion have larger sizes than those
directly resulting from shorter milling times.
Finally, for milling times ranging from 90 min (cf. Figure 8)
up to 120 min, thermograms present both a thin exothermic
peak of re-crystallization between 40 and 60 °C, which
indicates a partial amorphization of the sample upon milling,
and an endothermic peak of conversion of the β form to the α
form, which also reveals the existence of β crystallites, in
agreement with XRPD data.
It should be pointed out that whatever be the milling time
longer than 50 min, a close inspection of thermograms reveals,
from around 60 °C until the endotherms of the α form melting
or the β → α transition, a shallow but very broad exothermic
basin. Its origin will be discussed later (see the discussion part
4.2).
3.2.2. Dynamic Analysis. 3.2.2.1. Milling Time of 1 H. The
sample milled for 1 h at room temperature was analyzed by
DRS. As for the sample milled at −10 °C, the evolutions of the
real and imaginary parts of the permittivity were first recorded
at low temperature between −90 and 25 °C upon two
successive scans. In spite of a very noisy and weak response, a
relaxation mode can be observed as illustrated in Figure 9a.
This reveals the existence of some kind of residual mobility.
It should be noted that the two successive scans are a bit
different: in the second scan, the low-frequency flank of the
mode is lower as can be seen in Figure 10 where, as an
example, the two spectra recorded at −20 °C are reported (in
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Figure 10. Recordings of tan δ (F) at −20 °C. In black (left axis), bezafibrate amorphized by milling at −10 °C: freshly milled (full symbols) and
after heating until 25 °C (open symbols). In red (right axis), bezafibrate milled for 60 min at room temperature: freshly milled (full symbols) and
after heating until 25 °C (open squares), 50 °C (open circles), and 110 °C (open triangles).

Figure 11. Evolution of tan δ vs temperature between 10 and 75 °C for different frequencies of the applied electrical field in the case of bezafibrate
freshly milled for 60 min at room temperature. It shows the collapse of the signal for the lowest frequencies (0.1, 1, and 11 Hz) between 42 and 50
°C.

red, full and open squares). For comparison, the two scans
recorded at the same temperature in the case of the sample
amorphized by milling at −10 °C are also reported (in black,
full and open squares). The vertical scale is not the same as
these are far more intense, but the position of the mode is
similar. This suggests that the detected mobility has the same
origin as for the glassy sample. Therefore, the relaxation mode
observed in the second recording would be the γ relaxation,
and the difference between the two successive scans would
result from the loss of some adsorbed water. It should be noted
that, due to the very small magnitude of the signal, it is difficult
to determine whether the observed mode is due to only one
relaxation process (γ) or two (γ and δ) like for the glass. In a
first approximation, the mode was fitted with one Cole−Cole
370
function. Because of the noisy recording, this could be carried
out only between −55 and −15 °C. The evolution of the
extracted relaxation time is reported in the Arrhenius diagram
in Figure 5 (green open circles). As expected, it appears very
close to that of the γ mode obtained for the glass of bezafibrate.
In the second step, the evolution of the permittivity was
recorded for isotherms above 25 °C, in particular, between 35
°C and 50 °C, that is, the temperature range where the α mode
could be detected at low frequencies in the case of the
amorphous sample. In the obtained scans, at high frequencies,
the γ relaxation mode can still be detected, and as the
frequency decreases, both the real and imaginary parts of the
permittivity increase. This increase is certainly due to
Maxwell−Wagner−Sillars effects resulting from the hetero-
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geneity of the sample. However, a close inspection of the scans
recorded between 42 and 48 °C reveals at low frequency some
incidents of the increase in the ε″ value. To clear things up, the
evolution of tan δ with temperature was analyzed (cf. Figure
11). One can observe that, at 0.1 Hz, tan δ increases from 30
to 42 °C and then decreases till approximately 50 °C, resulting
in a peak. At 1 and 11 Hz, the same behavior is observed but
the magnitude of the peak decreases with increasing frequency
and is slightly shifted toward higher temperature. Above 500
Hz, this phenomenon disappears. The considered temperature
range is the zone where, in the case of the amorphous sample,
the α mode appears before the re-crystallization occurs (from
50 °C). Thus, the sudden increase in tan δ observed in Figure
11 from 30 °C for the lowest frequency can reasonably be
attributed to the appearance of the α mode, leading to a peak.
The anomalous decrease in its magnitude with increasing
frequency could be explained by re-crystallization of the
sample, which agrees with the exotherm of crystallization
observed by DSC. This would result in the disappearance of
the α mode at higher temperature that should be observed for
higher frequencies. So, even if the main relaxation assigned to
large-amplitude motions cannot clearly be evidenced as a well-
resolved relaxation, its existence masked by a rapid re-
crystallization is highly suspected. It corroborates the DSC
and X-ray results indicating a partial amorphization of the
sample.
It should be added that, interestingly, after heating to 50 °C
leading to a complete re-crystallization of the sample (α-form)
and the disappearance of the α relaxation, the recordings of the
permittivity on isotherms below the room temperature
(between −90 and 25 °C) still exhibit the secondary γ
relaxation. This is illustrated in Figure 10 for the spectrum at
−20 °C (red open circles). As can be seen, the magnitude of
the mode is lower but the mode is still clearly detected. It does
not disappear even after further heating to 110 °C (cf. Figure
10, red open triangle).
3.2.2.2. Milling Time Longer than 2 H. Some samples
milled for a time longer than 2 h were analyzed by DRS. Like
before, the evolutions of the real and the imaginary parts of the
permittivity were recorded from 106 to 10−1 Hz at low
temperature for isotherms ranging from −120 to 50 °C (two
successive series of recordings). As an example, the tan δ
evolution (second series of scans) obtained for bezafibrate
milled for 4 h at room temperature can be seen in Figure 9b.
As for the sample milled for 1 h, the response is very noisy and
weak but the striking point is that a relaxation mode, γ′, is
detected in spite of the sample being fully crystalline (β form)
according to XRPD and DSC analyses. This, again, emphasizes
the existence of some kind of residual mobility. The same
response (with comparable magnitude) is obtained for other
milling times. This relaxation process, γ′, has been fitted with
the Cole−Cole function, and the evolution of the associated
relaxation time is reported in the Arrhenius diagram in Figure
5 (red open circles). It appears very close from the γw mode
observed in the case of glassy bezafibrate, only in the first
recording, and assigned to movement of a small part of the
molecule linked through hydrogen bonds to some water
molecules. However, for bezafibrate milled at room temper-
ature for tmill > 2 h, it is quite unlikely that the origin of the
relaxation is the same. Indeed, no significant difference was
observed between the first and the second recordings except
for samples that were stored in a freezer after milling: for these
samples, the low-frequency flank of the γ′ mode is more
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intense and noisy in the first recording, which is certainly due
to traces of adsorbed water. Therefore, a relaxation related to
some adsorbed water appears at lower frequency (longer
relaxation time) than the γ′ mode. The evolution of the γ′
relaxation time follows an Arrhenius behavior, and an
activation energy of 50 ± 1 kJ mol−1 is found (log τ∞ =
−14.0 ± 0.2) for tmill = 4 h. It should be underlined that this
result does not seem to depend on milling times (see
Supporting Information S3), taking into account the small
fluctuations that can result from the analysis of the noisy
recordings.
Concerning the evolution of the permittivity recorded for
isotherms above 25 °C, for all the studied milling times, in the
temperature range 35−60 °C, the γ′ relaxation is observed at
high frequency and, like in the case of the sample milled for 1
h, for decreasing frequencies, both the real and the imaginary
parts of the permittivity increase due to Maxwell−Wagner−
Sillars effects. However, this time, no accident appears at low
frequencies, in particular between 40 and 50 °C and no trace of
an α relaxation or of a sudden re-crystallization can be detected
(even in tan δ vs the temperature evolution), in agreement
with DSC results. Therefore, in the case of bezafibrate milled
for more than 2 h at room temperature, only a localized
mobility is evidenced. It should be added that, like for the
sample milled for 60 min, this mobility is still detected,
although decreasing, after further heating of the sample to 110
°C (data not shown).
4. DISCUSSION
4.1. Mechanisms of Transformations Induced by
Milling. The results presented in part 3.1. have shown that a
14 h milling performed at −10 °C leads to the amorphization
of bezafibrate. This temperature is about 50 °C below the glass
transition temperature of the compound. Therefore, the
observed behavior is in agreement with results previously
reported on other materials: when milling is performed well
below the glass transition of the compound, it amorphizes.15
Indeed, the physical state reached by milling results from a
disorganization of the crystalline network due to ballistic
shocks, the re-crystallization being blocked by the too weak
molecular mobility of the glassy state.59
In part 3.2, it was shown that bezafibrate resulting from a 14
h milling at room temperature is still crystalline but
polymorphic transformation to the β form has occurred. In
this case, although the milling is performed 15° below Tg,
there is no effective amorphization even for long milling times.
Such a behaviour has been previously reported for compounds
such as indomethacin,60 biclotymol,61 or glucose.59 However,
for glucose, it was reported that a milling at Tg13 °C leads
to non-amorphization, which is only apparent because it results
from a re-crystallization process made more efficient by the
increase in temperature that counterbalances the amorphiza-
tion due to ballistic shocks.59 Therefore, obtaining a
completely crystalline bezafibrate after a 14 h milling at
room temperature does not mean that transient amorphization
does not occur, and the analysis of the milling kinetics permits
to better understand the mechanisms of the transformation.
Indeed, the XRPD study of the beginning of the milling
process kinetics (cf. Figure 7) reveals not only a rapid decrease
in the average size of crystallites, which is due to their
fragmentation by the impacts of milling balls, but also the
appearance of some amount of the amorphous phase. The
existence of this small amorphous content was confirmed by
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the occurrence of a re-crystallization exotherm in DSC
thermograms on consecutive heating (cf. Figure 8). For a
sample milled for 60 min, a small Cp jump can even be
observed, and from a close inspection of DRS spectra, the
existence of the α relaxation associated to the dynamic glass
transition is highly suspected (cf. Figure 11). Therefore, it
appears that, like at −10 °C (Tg50 °C), milling at 25 °C
(Tg15 °C) induces a real amorphization of bezafibrate.
However, the fact that after 50 min of milling, the crystallite
size reaches a plateau value of 250 Å (cf. Figure 7), a value
close to 100−200 Å corresponding to the critical size for
spontaneous amorphization,62 indicates the simultaneous re-
crystallization in the initial α form, which is induced by an
enhanced mobility. It leads to a stationary state composed of
approximately 10% amorphous phase and 90% crystalline α
form. The type of mobility involved in the re-crystallization of
glassy materials (below Tg) is still an issue under debate in
spite of the extensive studies devoted to the subject. The
structural relaxation (α relaxation) appears to be the main
factor because, in the last years, studies on different
compounds47,63−65 have highlighted, based on predictions of
τα in the glassy state, correlations between this relaxation and
the kinetics of re-crystallization. Other studies indicate that the
implication of more localized motions of intermolecular origin
(β relaxation of Johari-Goldstein type) cannot be excluded.66,67
Moreover, Descamps et al. have shown that milling produces
glasses of high energy with enhanced sub-Tg mobility.68
Unfortunately, in our case, the evolution of τα and τβJG below
Tg could not be extracted from the DRS experiments, which
prevents us to conclude which of these mobilities allows,
during milling at room temperature, the re-crystallization of
bezafibrate counterbalancing the amorphization due to
mechanical shocks.
Concerning the re-crystallization, it should be underlined
that it occurs to the initial metastable α phase. The existence of
a transient amorphous state and the re-crystallization to the
metastable crystalline state have been highlighted in several
studies of polymorphic transformation under milling at a
temperature close to Tg (in monotropic situations).69−72 It is
usually explained by Ostwald’s rule of stages:73,74 re-
crystallization proceeds through the fastest mechanism
(which does not lead to the most stable form). It could be
underlined for the following discussion that exception seems to
be the case of D-mannitol, for which no intermediate
amorphous phase was detected during milling at Tg + 7 °C
(in spite of in situ XRPD measurements).75
However, in the case of bezafibrate, what is puzzling is that
the XRPD analyses of the milling process kinetics reveal a
second stage in the behavior under milling at room
temperature: after more than 1 h of milling, polymorphic
transformation to the stable β form occurs because for sample
milled for 90 min, the β form is detected for the first time (cf.
Figure 7). Then, for an additional short milling time (∼20
min), the two crystalline forms and the glassy form co-exist.
However, during this time, the content of the β form increases
at the expense of the contents of the α and glassy forms, with
the size of β-crystallites being around 1000 Å: with milling, the
amorphous content and the remaining α-crystallites, which
keep on amorphizing, re-crystallize to the β form, which
explains the quite high value of β-crystallite size. After 130 min
of milling, when the sample is completely transformed to the β
form, the size of β-crystallites falls to a value of 500 Å that does
not evolve for the following 12 h milling: the decrease in size
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between 110 and 130 min of milling and the plateau value
obtained for longer milling times suggest that, like for the α-
crystallites, the milling tends to amorphize the β phase but the
mobility allows the rapid re-crystallization (faster than the
amorphization), and the β-crystallite size decreases to a steady-
state plateau value, resulting from a balance between
fragmentation by mechanical shocks and a cold welding
process.76
It is stunning that the milling process has to last more than 1
h to induce the polymorphic transformation of the α form to
the β form. Indeed, the α and β phases form an enantiotropic
system, and the β phase is the stable one below the transition
temperature (∼150 °C) and thus at the milling temperature.
Moreover, with the temperature of the transition and the
associated enthalpy change, it is possible to calculate the
difference in Gibbs free energy of the two phases at room
temperature assuming that the entropy change, ΔSconv,
associated with the transition is equal to the ratio of the
transition enthalpy, ΔHconv, and the temperature, Tconv. With
the values found by Lemmerer and co-workers (Tconv = 157 °C
= 430 K and ΔHconv = 4.3 kJ mol−124 ), it leads to ΔSconv = 10 J
mol−1. Therefore, at room temperature (T = 25 °C = 298 K),
the difference in Gibbs free energy is Gα − Gβ = ΔHconv −
TΔSconv = 1.32 kJ mol−1. This value is very small, so one could
think that the α phase would easily transform to the more
stable β phase as soon as the milling process begins, but it
needs more than 1 h. One should note however that the fact
that commercial bezafibrate is in the metastable α form
indicates that the transformation to the β phase does not occur
so easily one could expect. This means that, although the
difference in Gibbs free energy between the two forms is small,
the energy barrier that has to be overcome so that the
transformation occurs is high.
As shown by Lemmerer and co-workers,24 the propanoic
acid groups of bezafibrate molecules of the α phase are in syn-
conformation whereas they are in anti-conformation for the β
phase. This different conformations of the propanoic acid
groups induce different H bonding of the carboxylic acid
groups, which results in different conformations (different
torsion angles) of the propanoic acid group bonds. A change in
each of these torsion angles requires a large amount of energy
because of repulsive interactions with the rest of the molecules.
Therefore, it implies, according to Lemmerer et al.,24
correlated rotations of the different bonds of the propanoic
acid group. Therefore, due to the different and complex H
bonding networks of the α and β crystalline phases, the
polymorphic transformation appears to be of the reconstruc-
tive type. Under milling at 25 °C, the existence of a transient
amorphous phase highlighted in the present work could allow
this transformation to occur with the release of some
cooperative large amplitude motions (main relaxation,
evidenced by DRS for tmill = 60 min). Besides, one can note
that when the α, β, and amorphous phases co-exist (after a
milling of 90 or 110 min, e.g.), a spontaneous transformation
of the remaining α fraction to the β form is not observed. For
example, after a 2 day storage at room temperature of the
sample milled for 110 min, a XRPD recording shows that the
amorphous fraction has re-crystallized but without a noticeable
increase in the β fraction at the expense of the α fraction.
Another example is given by the analysis of the endothermic
peak of the β → α conversion observed in DSC around 140 °C
when the sample milled for 90 min is heated (cf. Figure 8). On
comparing the area of this peak with the one observed in the
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Figure 12. Milling of bezafibrate at room temperature for 70, 110, and 290 min: evolution vs the temperature of the size of crystallites for the α
form (blue squares) and the β forms (red circles). Lines are guide for the eyes. The dashed areas indicate the β → α conversion temperature range.
case of bezafibrate re-crystallized in the β form after complete
amorphization at −10 °C (assumed to represent the
transformation of a 100% β crystalline bezafibrate), we found
an amount of β phase corresponding to the sum of the β and
amorphous fractions determined at the end of the milling
process from the XRPD analysis (ΔHconv/ΔHconv 100% = 16%).
This suggests that after milling and upon heating till 140 °C,
the amorphous fraction has re-crystallized in the β form but the
α phase did not transform further to the β form. This
corroborates that the conversion under milling of the α form to
the β form is not direct and that a transient amorphous phase
(reached by further milling) is required to continue the
transformation of the α form to the β form due to the required
energy. It could be remarked, by the way, that in the case
previously mentioned of D-mannitol, presenting no transient
amorphous state, the polymorphic transformation is of
displacive type, which certainly requires less energy to occur.75
Then, one can wonder why, although an amorphous fraction
is detected in the sample already after 10 min milling, this
fraction does not re-crystallize in the β form but in the α form.
When we consider the amorphous content of milled samples
deduced from XRPD (cf. Figure 7) or DSC (cf. Figure 8,
development of the exotherm of re-crystallization around 50
°C), we observe that it reaches its maximum when the β phase
appears, just after in XRPD or just before in DSC, depending if
the technique probes the structural disorder (XRPD) or the
dynamic disorder (DSC). This points out that the trans-
formation under milling of the α form to the β form requires
not only a transient amorphous phase but also in sufficient
quantity for the remaining α phase to be defective enough
(coherent domains small enough, network of H bonding
enough disorganized) so that the transformation from the
metastable α phase to the stable β form occurs and the new H
bonding network rebuilds.
4.2. Origin of the Remaining Mobility in the
Crystalline Milled Samples. In the case of bezafibrate
milled at room temperature, the results of the DRS
experiments suggested the existence of the α relaxation
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associated with large-amplitude motions that are frozen
below Tg, only for the sample milled for 60 min, in spectra
recorded just after milling (before it disappears when the
sample re-crystallizes on heating to 50 °C). These results are in
agreement with the DSC analysis showing a re-crystallization
exotherm around 60 °C that partly masks a Cp jump and the
XRPD analysis reporting an amorphous content of around
10%. For longer milling times (4−11 h), the α mode cannot be
detected, which emphasizes the absence of any amorphous
fraction, in agreement with DSC and XRPD results. Indeed,
the sensitivity of DRS is such that it can reveal very low
content of the amorphous phase as in the case of a recent
dielectric study, where an α relaxation due to 1 to 2% of the
remaining amorphous phase confined by surrounding crystal-
lites could be detected, whereas no sign was visible by DSC or
XRPD.45
Concerning the more localized intramolecular mobility, the
DRS study reveals interesting original results for bezafibrate
milled at room temperature. For the sample milled for 60 min,
a weak relaxation, the γ mode, is detected even after the re-
crystallization of the sample (heating at 50 °C resulting in the
disappearance of any trace of an α mode). The activation
energy and the temperature dependence of the associated
relaxation time are in agreement with those of the γ relaxation
observed in the case of completely amorphous bezafibrate
(resulting from milling at Tg50 °C). For samples milled for
4 h or 11 h (completely crystalline), a weak γ′ relaxation is also
detected. These remaining localized mobilities cannot thus be
assigned to the remaining of the, strictly speaking, amorphous
phase. Moreover, such localized residual mobilities are
detected neither for commercial α crystalline bezafibrate nor
for β crystalline bezafibrate obtained from re-crystallization on
heating of the amorphous state or for α crystalline bezafibrate
obtained on heating from the conversion of the β form
resulting from long milling times. Thus the localized mobilities,
γ or γ′, detected in crystalline milled samples are not due to an
intrinsic mobility of the crystal but seem to be rather generated
by the milling. Besides, when the samples milled at room
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temperature are heated, these residual mobilities decrease
because the magnitude of the γ and γ′ modes clearly decreases
(cf. Figure 10, heating up to 110 °C for the γ mode).
To better understand the origin of the residual localized
mobility, samples of bezafibrate milled at room temperature
have been analyzed by XRPD when heated. The evolution with
temperature of the size of crystallites extracted from Rietveld
refinements is reported in Figure 12 for milling times of 70,
110, and 290 min. It shows a continuous increase in the
crystallites’ size until temperatures closed to the melting (not
shown) of the α phase (tmill = 70 min) or until the conversion
of the β phase to the α phase (tmill = 110 and 290 min) occurs.
Moreover, as mentioned in part 3.2.1, a close inspection of
DSC thermograms reveals a broad exotherm in this temper-
ature range. As shown in Figure 13, this exotherm starts
Figure 13. MDSC thermograms recorded at 5 °C min−1 ± 0.530 °C
per 40 s (total heat flow) of bezafibrate milled at room temperature
for 1 h (top) and 4 h (bottom). Top: scans recorded at the very end
of milling (in blue), after a few minute storage at room temperature
(in green), and after heating until 110 °C (in red). Bottom: scans
recorded at the end of milling (in green) and after heating until 110
°C (in red). In both figures, the thermogram of the crystalline
unmilled sample is added for comparison (in black).
around 60−70 °C and extends till the melting of the α phase
(case of tmill = 60 min) or until the β → α transformation (case
of tmill = 4 h). It is interesting to note that, when the heating of
the sample is stopped at a temperature in this temperature
range (e.g., 110 °C in Figure 13), the exotherm starts from this
temperature on the subsequent heating. This exotherm is
characteristic of the coarsening of crystallites. The heat given
off corresponds to a decrease in the interfacial enthalpy that is
induced, upon heating when the mobility is high enough, by
the cure of crystalline defects and a coalescence of crystallites.
This leads to an increase in the average crystallite size observed
by XRPD (cf. Figure 12). These results show that the
crystalline phases generated by milling (α or β forms) are
highly defective. Besides, when heated, the highlighting of the
coarsening of crystallites (which induces a decrease in the total
surface area) and a coupled decrease in the magnitude of the γ
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and γ′ relaxations strongly suggest that the residual localized
mobilities are due to motions of part of molecules located at
the surface of crystallites.
DRS results help us to be more precise concerning the
involved motions associated with the γ and γ′ relaxations. We
have seen in the dielectric study of bezafibrate amorphized by
milling (part 3.1) that, taking into account the activation
energy and the broadness of the γ mode, this relaxation could
result from the motion of the central part of the molecule
linked to the amide group. The development on the low-
frequency side of the γw mode, only visible during first heating
to 50 °C, was assigned to the same motion slowed down by
some hydrogen bonds between the amide group and a small
amount of adsorbed water molecules. Concerning the γ′ mode
observed for bezafibrate samples converted by milling to the β
form, the fit with the Arrhenius law of the associated relaxation
times gives an activation energy of the corresponding
movement, which is roughly the same as that of the γ
relaxation (Eaγ′ = 50 ± 1 kJ mol−1 and Eaγ = 48 ± 1 kJ mol−1).
Besides the γ′ relaxation times are of the order of those found
for the γw mode. However, in this case, water molecules cannot
be involved as a slight adsorption of water has an effect at even
lower frequencies. It is therefore tempting to assign the γ′
mode to the motion of the central part of bezafibrate
molecules, as for the γ mode, but slowed down by the
development of hydrogen bonds linking the amide group, not
with water molecules, but with other bezafibrate molecules. To
go further, it is necessary to consider the possible hydrogen
bonds between bezafibrate molecules that could involve the
amide group. Lemmerer et al.24 have shown that, in the
crystalline state, the differences in conformations of the
carboxylic hydrogen atoms (syn-conformation in the α form
and anti-conformation in the β form) result for the two
polymorphs in different and complex H bonding networks. For
the α form, bezafibrate molecules form cyclic dimers through
two OH···O hydrogen bonds between the carboxylic acid
group of one molecule and the oxygen atom of the amide
group of the other molecule. These dimers are inter-connected
to two adjacent dimers by NH···O hydrogen bond between the
amide group and the carboxylic O atom, forming tetrameric
enclosed rings. This leads to a packing structure with one-
dimensional tube-shaped chains.24 For the β form, bezafibrate
molecules are linked through NH···O hydrogen bonds
between the amide group and the carboxylic O atom, forming
chains connected to adjacent ones by OH···O hydrogen bonds
between the carboxylic acid group and the oxygen atom of the
amide group. This results in tetramers forming a two-
dimensional hydrogen-bonded sheet network.24 The oxygen
atom and the NH function of the amide group are both
involved in these hydrogen bonds, irrespective of the
crystalline polymorphs. One can therefore suspect that it
blocks the mobility of the amide group and the linked −CH2−,
which is consistent with the fact that no localized mobility is
detected by DRS in the nondefective crystalline forms. When
the crystalline structure is made highly defective by milling (α
or β forms), molecules on the surface of crystallites are
certainly only partially involved in the network of hydrogen
bonds, which would partially release the local mobility,
resulting in the detection of the γ and γ′ modes. The
difference between these two modes could be due to a
difference in the hydrogen bonding in which the oxygen atom
and the NH function of the amide group are involved, its
motion coupled to that of the linked −CH2− being more or
https://doi.org/10.1021/acs.cgd.1c00997
Cryst. Growth Des. 2022, 22, 363−378
Crystal Growth & Design
less hindered. When the material is amorphized by milling, the
crystalline network and thus the hydrogen bonding network
that stabilizes it are destroyed and the mobility of the central
part of the molecule is fully released. It is therefore intriguing
that the same relaxation mode is found for the sample milled
for 60 min (defective α crystalline form) and the glassy sample.
An explanation could arise from the calculation of the
hydrogen bond energies for the different conformations
made by Lemmerer and co-workers24 showing that, for the α
form, the largest stabilization effect comes from the two OH···
O hydrogen bonds between the carboxylic acid group and the
oxygen atom of the amide group, whereas the NH···O
interaction contribution is small. It is thus likely that, when
milled, the NH···O hydrogen bonds are first destroyed, while
the cyclic dimers resulting from the OH···O interactions
persist. This could then indicate that these dimers also remain
in the glassy sample. For the β form, the energy difference
between the two types of hydrogen bonds is not so
important.24 Consequently, for the defective β crystalline
sample, the probability that, on the surface of crystallites, the
NH group of the bezafibrate molecule remains H-bonded
could be more important than for the α polymorph, leading to
a more hindered local motion of the central part of the
molecule. It should nevertheless be noted that although the
relaxation times extracted from the maxima of the γ modes for
the glassy bezafibrate and the defective α form correspond, the
relaxation found in the amorphous sample is broader. It could
be due to its much higher intensity but one cannot exclude that
it could result from the convolution of the preponderant γ
relaxation and also to a small extent of the γ′ relaxation. In any
case, these results raise the question of the remaining of some
peculiar arrangements of bezafibrate molecules (cyclic dimers
or linear dimers, trimers...) in the glassy state. One can wonder
whether these hydrogen-bonded arrangements, if any, would
be the same depending that the glassy state is obtained from
the milling of the crystalline material, undercooling of the
liquid, or another way. It would be interesting to determine by
experimental techniques such as solid-state NMR or FTIR or
by molecular dynamics simulation (as degradation exper-
imentally occurs on melting) the different types of bezafibrate
molecule associations that could remain in the glassy state
obtained through different routes. Besides, the link between
the milled polymorphic form and the conformational and
molecular packing of the amorphous form obtained by milling
is still an open question. For example, in the case of piroxicam
that can appear in two polymorphic phases that form
hydrogen-bonded dimers (form I) or continuous hydrogen
bond chains (form II),77 it has been shown by pair distribution
function analysis of XRPD78 that, under milling, the molecular
packing of form I evolves continuously to the amorphous one,
whereas in the case of the milling of form II, a transient
disordered state that maintained the local packing of the
crystalline polymorph was observed. Such analysis of the
evolution of the local packing of bezafibrate molecules under
milling of each polymorph could help to better understand the
origin of the difference in localized mobilities evidenced in this
work by DRS.
5. CONCLUSIONS
In this article, the complementary use of XRPD, DSC, and
DRS allowed us to carefully investigate the behavior upon
milling of bezafibrate (α form). It was shown that a 14 h
milling at −10 °C (Tg50 °C) enables to obtain the glassy
375
pubs.acs.org/crystal Article
form (Tg = 39 ± 1 °C and ΔCp = 0.41 ± 0.01 J g−1 °C−1),
which is chemically pure unlike the glass obtained from the
quenching of the melt, whereas the same milling performed at
25 °C (Tg15 °C) enables to obtain the β polymorph (stable
form at room temperature). This nicely illustrates how varying
the milling temperature permits to play with the physical state
of compounds. Moreover, the monitoring under milling of the
kinetics of this latter transformation allowed us to shed some
light on the mechanisms of the induced conversion of the
metastable to the stable form of this enantiotropic system. It
was evidenced that at first, two antagonist processes occur: an
amorphization (due to mechanical shocks) partly counter-
balanced by a slower re-crystallization (enabled by the
mobility) to the starting α form (metastable at room
temperature). This prevails until the crystalline structure is
disorganized enough (mean α crystallite size of 250 Å) and the
amorphous content is high enough (around 10%) after
approximately 1 h of milling to trigger the transformation to
the stable (at room temperature) β form. Once the conversion
is achieved (after 2 h of milling), a further milling leads to β
crystallites with a mean size of 500 Å. It thus appears that the
polymorphic transformation induced by milling in the case of
bezafibrate requires going through an intermediate amorphous
state, certainly because the conversion is of the reconstructive
type. Such transformation to the stable form triggered after 1 h
of milling has already been observed for sulfamerazine,21
another compound for which the conversion between the two
polymorphic phases that form an enantiotropic set is also of
reconstructive type.79 Milling-induced transformation in this
kind of systems thus seems to be ruled, as for monotropic
situations, by a transient amorphous state. However, due to the
very short lifetime of this state, only the use of complementary
techniques probing the structure, the thermodynamic and the
dynamic, as in this work, allows to reveal its existence. These
outcomes also underline the complexity of such trans-
formations and the need to follow the kinetics to fully
understand which crystalline form can be reached and the
underlying mechanisms.
Moreover, the detailed characterization by DRS of the
molecular mobility of the amorphized bezafibrate has
permitted us to establish that it belongs to the class of fragile
liquids (m = 110 ± 12), and to highlight, in the glassy state,
two secondary relaxations of intramolecular origin, in addition
to a relaxation, γw, originating through a small amount of water
in the first scans: the faster δ mode probably due to small
fluctuations of the carboxylic group and the γ mode (Ea = 48 kJ
mol−1) that could be assigned to movements of the central part
of the molecule carrying the amide group. Furthermore, a
striking result arose from the investigation by DRS of the
mobility of the milled crystalline forms (α or β): a residual
localized mobility was evidenced that is neither due to an
intrinsic mobility of the crystal nor to a strictly speaking
amorphous phase but to a residual mobility at the surface of
crystallites, which diminishes as the coarsening of crystallites
occurs with heating and defects are cured. A difference in this
dynamics for the two crystalline forms could arise from a
difference in the hydrogen bonding network between
bezafibrate molecules. Owing to its high sensitivity, the
dielectric relaxation spectroscopy thus appears as an interesting
tool for distinguishing the threshold between a defective
crystalline state and an amorphous one. The more frequent use
of this technique to characterize milled compounds could allow
https://doi.org/10.1021/acs.cgd.1c00997
Cryst. Growth Des. 2022, 22, 363−378
Crystal Growth & Design
a better understanding of the process-induced disorder in
crystalline structures of material.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.cgd.1c00997.
1 Composition. J. Pharm. Sci. 2002, 91, 1573−1580.
H NMR spectra of commercial Bezafibrate, sample
milled for 14 h at −10 °C and sample obtained from the
quenching of the liquid, DRS recording of ε″(F) at −70
°C for bezafibrate milled for 14 h at −10 °C, and
Arrhenius diagram showing the γ′ mode observed for
bezafibrate milled at room temperature for 4 and 11 h
(PDF)
■
AUTHOR INFORMATION
Corresponding Author
Emeline Dudognon − Univ. Lille, CNRS, INRAE, Centrale
Lille, UMR 8207UMETUnité Matériaux et
Transformations, Lille F-59000, France; orcid.org/0000-
0003-4176-1925; Phone: +33 (0)3 20 43 69 90;
Email: emeline.dudognon@univ-lille.fr; Fax: +33 (0)3 20
43 40 84
Authors
Florence Danède − Univ. Lille, CNRS, INRAE, Centrale Lille,
UMR 8207UMETUnité Matériaux et
Transformations, Lille F-59000, France; orcid.org/0000-
0001-5139-9187
Mathieu Guerain − Univ. Lille, CNRS, INRAE, Centrale Lille,
UMR 8207UMETUnité Matériaux et
Transformations, Lille F-59000, France; orcid.org/0000-
0002-7254-9014
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.cgd.1c00997
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This project has received funding from the Interreg 2 Seas
programme 2014−2020 co-funded by the European Regional
Development Fund (FEDER) under the subsidy contract
2S01-059_IMODE. The FEDER, the French National Centre
for Scientific Research (CNRS), the Nord-Pas de Calais
region, and the French Ministry of National Education, Higher
Education and Research, are also acknowledged for funding of
the X-ray diffractometer.
■
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