crystals

Article
Agglomeration Control during Ultrasonic
Crystallization of an Active Pharmaceutical Ingredient
Bjorn Gielen 1,2 , Jeroen Jordens 1,2 , Leen C. J. Thomassen 1,2 , Leen Braeken 1,2 and
Tom Van Gerven 1, *
1 KU Leuven, Department of Chemical Engineering, Celestijnenlaan 200 F Box 2424, 3001 Leuven, Belgium;
bjorn.gielen@kuleuven.be (B.G.); jeroen.jordens@kuleuven.be (J.J.); leen.thomassen@kuleuven.be (L.C.J.T.);
Leen.braeken@kuleuven.be (L.B.)
2 KU Leuven, Faculty of Engineering Technology, Agoralaan building B box 8, 3590 Diepenbeek, Belgium
* Correspondence: tom.vangerven@kuleuven.be; Tel.: +32-16-32-23-42

Academic Editor: Judy Lee

Received: 16 January 2017; Accepted: 2 February 2017; Published: 8 February 2017

Abstract: Application of ultrasound during crystallization can efficiently inhibit agglomeration.

However, the mechanism is unclear and sonication is usually enabled throughout the entire
process, which increases the energy demand. Additionally, improper operation results in significant
crystal damage. Therefore, the present work addresses these issues by identifying the stage in
which sonication impacts agglomeration without eroding the crystals. This study was performed
using a commercially available API that showed a high tendency to agglomerate during seeded
crystallization. The crystallization progress was monitored using process analytical tools (PAT),
including focus beam reflectance measurements (FBRM) to track to crystal size and number and
Fourier transform infrared spectroscopy (FTIR) to quantify the supersaturation level. These tools
provided insight in the mechanism by which ultrasound inhibits agglomeration. A combination of
improved micromixing, fast crystal formation which accelerates depletion of the supersaturation
and a higher collision frequency prevent crystal cementation to occur. The use of ultrasound
as a post-treatment can break some of the agglomerates, but resulted in fractured crystals.
Alternatively, sonication during the initial seeding stage could assist in generating nuclei and prevent
agglomeration, provided that ultrasound was enabled until complete desupersaturation at the seeding
temperature. FTIR and FBRM can be used to determine this end point.

Keywords: ultrasound; crystallization; Active Pharmaceutical Ingredient (API); agglomeration;

crystal shape; Process Analytical Technology (PAT)

1. Introduction
Crystallization is one of the most important processes during the manufacturing of active
pharmaceutical ingredients (APIs). Precise process control allows tuning the final physical properties of
the crystals such as purity, size distribution and polymorphic form. On the one hand, these properties
should be controlled to facilitate processing during filtration, drying and formulation. On the other
hand, these characteristics also provide the opportunity to control the drug release profile after
intake into the human body. As a result, industrial processes exploit numerous particle engineering
techniques in combination with crystallization to produce crystals that meet both the bioavailability
and processing requirements [1–6].
Particle formation during crystallization in agitated vessels is governed by two primary processes:
crystal nucleation and growth. Furthermore, secondary mechanisms such as breakage, Ostwald ripening
and agglomeration can have a substantial influence on the obtained crystal polymorph, shape and particle
size distribution (PSD) [7,8]. Agglomeration should be clearly distinguished from aggregation since these

Crystals 2017, 7, 40; doi:10.3390/cryst7020040 www.mdpi.com/journal/crystals

Crystals 2017, 7, 40 2 of 20

phenomena describe two distinct types of particle assemblage. Aggregation denotes the association of
individual (primary) particles into larger structures which can easily disintegrate since the particles are
not cemented together. These are stabilized by attractive or adhesion forces including Van der Waals
interactions that depend on the microscopic structure of the solvent and the solute, the orientation of
the crystals faces and the presence of impurities. On the contrary, agglomerates require brute force to
break the solid bridges binding the individual particles. The origin of the agglomerative bonds is unclear
although it is believed that liquid bridges are involved in the process. The actual structure is, however,
very complex and might even involve solvent inclusions during the cementation [9,10]. In terms of
flowability, filterability and compaction properties, agglomerated particles can exhibit a clear benefit
over single crystals, depending on the final morphology. In contrast, significant disadvantages such as
reduced purity due to entrapment of mother liquor or other impurities, elevated generation of fines
during transport and lower dissolution rate have been reported [11].
The mechanism of agglomeration involves three consecutive steps. Firstly, individual crystals
collide due to fluid motion in agitated systems, followed by stabilization of these adhered or aggregated
particles due to attractive forces. Finally, molecular growth forms solid bridges which transform
the aggregates into agglomerates [7,8,10,12–14]. However, if the crystal–crystal adhesion is insufficient,
the aggregates will disintegrate before agglomeration can occur. As a result, agglomeration during
crystallization strongly depends on the collision frequency. In order for particles to combine, contact by
means of a collision is necessary. Nonetheless, aggregated particles that have not yet been cemented
together can be released by the impact of other colliding particles. The kinetics of agglomeration
can be described by means of an agglomeration rate constant or kernel β (m3 ·s−1 ) that depends
on the particle size and the mechanism for agglomeration [7,10,15]. In general, Equation (1) can be
used to determine the agglomeration rate ragg (m−3 ·s−1 ), although several adoptions were proposed
in literature to incorporate the kernels dependency on crystal size and processing conditions [16].
Despite these modifications, several authors emphasize that a size-independent kernel usually
describes the experimental data best [16–18].

r agg = η · β · N 2 (1)

where η (-) denotes the efficiency of the collisions between the particles, and N (m−3 ) represents
the number concentration of particles.
To control or prevent agglomeration, numerous factors were identified that influence this
phenomenon. The most dominant parameters are the degree of supersaturation, particle concentration,
particle size and agitation rate. An increase in supersaturation level results in a higher agglomeration
rate as evidenced by the strong presence of agglomerates in anti-solvent crystallization processes
that operate under high supersaturation. In these conditions, particles have an increased tendency
to stick together, inhibiting the disruption into separate elements [19]. The effect of the particle
concentration exhibits a maximum. At low concentrations, the collision frequency is too low for
efficient aggregation, while a high collision rate breaks up any pre-agglomerated structures at high
particle concentration. The particle size determines the mechanism for agglomeration, which is
either perikinetic or orthokinetic. The former one applies to small particles that collide due to
Brownian or laminar fluctuations in a static solution. In a stirred crystallizer orthokinetic or shear
induced agglomeration clearly dominates. This type of agglomeration requires a velocity gradient
to be present before efficient particle collisions can occur. David et al. considered that particles
above the Kolmogorov microscale (~20–40 µm) collide by the orthokinetic mechanism, while smaller
particles collide due to the laminar microshear stresses [7,20]. In general, smaller particles exhibit
a higher degree of agglomeration, with a clear abundance in the range of 1 µm to several tens of
microns. Finally, a combination of computational modeling and experimental results indicated that
agglomeration shows a maximum as a function of the agitation rate. Initially, increased agitation
and shear promotes interparticle collisions that may lead to agglomeration. However, at some point
Crystals 2017, 7, 40 3 of 20

the stress induced by the collisions overcomes the force of the crystal–crystal interactions and separates
the aggregated or partially agglomerated particles [15,16,21].
A possible technique to inhibit agglomeration is the use of ultrasound during crystallization [13,22–25].
The benefits of this technology for crystallization were demonstrated before and include reduced
metastable zone width, narrow particle size distribution and homogeneous crystal morphology [5,26–28].
Although the exact mechanism of these phenomena is not yet understood, the proposed hypotheses
attribute this to acoustic cavitation, involving the formation and implosion of micron-sized bubbles,
and its accompanying mechanical effects [29,30]. Additional work on the effect of ultrasound during
crystallization indicated that agglomeration can be suppressed, yielding individual crystals with
a smooth surface [31]. Guo et al. investigated this aspect in more detail by high speed imaging
and evidenced that both mechanical effects from imploding cavitation bubbles and an enhanced
collision rate contribute to the breakage of agglomerated particles [25]. Although numerous reports
demonstrate the beneficial effect of sonication on the final crystal morphology, industrial applications
are scarce. Most likely, this is due to the additional energy consumption, crystal breakage and
surface erosion caused by continuous or improper operation of the sonication probe throughout
the crystallization process in most cases [32–34]. However, it is not clear whether such a long treatment
time is required for the production of non-agglomerated particles since the moment of initiation
and the length of sonication have not yet been optimized. Prolonged operation of the ultrasonic
source can also result in erosion of the probe which can subsequently contaminate the crystal
suspension. In addition, most research only targets the behavior of inorganic compounds or small
organic substances [5,28,35–37]. Although some active pharmaceutical ingredients were studied, more
investigation is needed to evaluate the feasibility of sonication in pharmaceutical processes [5,27,38].
Therefore, this study aims to evaluate the potential of ultrasound for agglomeration control during
the recrystallization of a commercially available API produced by Johnson & Johnson. The selected
substance is sensitive to formation of agglomerated, star-shaped crystals during conventional seeding
crystallization with dry seed material. Therefore, the present work evaluates whether the application
of a short ultrasonic post-treatment or sonication as a seeding agent is able to reduce agglomeration
for this compound. These results are compared to a process that successfully inhibited agglomeration
by use of continuous sonication throughout the entire crystallization protocol. In the end, this study
provides more insight into the mechanism by which ultrasound prevents the formation of agglomerated
crystals. Furthermore, it shows how a targeted application of ultrasound in the correct stage of
the crystallization process can aid in the production of desired crystal morphology, ruling out excessive
energy demands during the entire process.

2. Materials and Methods

2.1. Experimental Setup

A Mettler Toledo Easymax 102 workstation was used for accurate and programmable temperature
control throughout the entire experiment. This equipment was fitted with a 100 mL glass reactor
and sealed with a Teflon lid mounted with a water cooled condenser. In both silent and sonicated
experiments, macromixing was provided by a half-moon blade stirrer operating at 250 rpm. In the latter
tests, ultrasound was supplied by a 30 kHz Hielscher UP50H sonotrode which operated at 10 W and
was introduced from the top. In addition, the setup could be equipped with process analytical
technology (PAT) such as a focus beam reflectance measurement probe (FBRM, S400A, Mettler Toledo,
Columbus, OH, USA) or a Fourier Transform Infrared probe (FTIR, ReactIR 15, Mettler Toledo,
Columbus, OH, USA) to gather real time data on the chord length distribution and solute concentration,
respectively. Figure 1 shows a schematic view of the experimental setup in which the Teflon cover and
condenser were not shown to simplify the picture.
Crystals 2017, 7, 40 4 of 20
Crystals 2017, 7, 40 4 of 20

Crystals 2017, 7, 40 4 of 20

FigureFigure 1. Experimental
1. Experimental setup
setup used
used duringsilent
during silentand
and sonicated
sonicated experiments.
experiments. (a)(a)
A 30
A 30kHzkHzprobe is is
probe
submerged from the top to supply ultrasound to the solution. (b) Overhead stirring
submerged from the top to supply ultrasound to the solution; (b) Overhead stirring is provided is provided by a
half-moon blade impeller. (c) A thermometer allowed accurately logging and controlling the
by a half-moon blade impeller;
Figure 1. Experimental (c) Aduring
setup used thermometer
silent and allowed
sonicated accurately
experiments.logging
(a) A 30andkHz controlling
probe is
temperature within this equipment. (d) PAT tools such as FBRM and FTIR were fitted to collect data
the temperature within
submerged from thethis
top equipment; (d) PATtotools
to supply ultrasound such as (b)
the solution. FBRM and FTIR
Overhead were
stirring fitted to by
is provided collect
a
on chord length distribution and solute concentration.
data on chord length
half-moon bladedistribution
impeller. (c)and
A solute concentration.
thermometer allowed accurately logging and controlling the
temperature within
2.2. Experimental this equipment. (d) PAT tools such as FBRM and FTIR were fitted to collect data
Procedure
on chordProcedure
2.2. Experimental length distribution and solute concentration.
In this study, the effect of ultrasound on agglomeration during the crystallization process of a
In this study, the
commercially
2.2. Experimental effectactive
available
Procedure of ultrasound on agglomeration
pharmaceutical ingredient wasduring the crystallization
investigated. The selected process
API is of
crystallized available
a commercially from ethanol denatured
active with methylingredient
pharmaceutical ethyl keton was
(MEK). Figure 2 displays
investigated. Thethe solubility
selected API is
In this study, the effect of ultrasound on agglomeration during the crystallization process of a
curve for
crystallized fromthisethanol
solute-solvent system,
denatured which
with methylis obtained experimentally
ethyl keton by determination
(MEK). Figure 2 displaysofthe
thesolubility
clear
commercially available active pharmaceutical ingredient was investigated. The selected API is
point during
curve crystallized
for this slow heating (<0.1 °C·min
solute-solvent
−1) after addition of a fixed amountby of API. The temperature
of theatclear
from ethanol system,
denaturedwhich
with is obtained
methyl ethylexperimentally
keton (MEK). Figuredetermination
2 displays the solubility
which complete dissolution ◦occurred−1was assessed by a combination of visual detection and FBRM
point during
curve forslow
this heating
solute-solvent C·min which
(<0.1 system, ) after is addition of a fixed amount
obtained experimentally of API. The temperature
by determination of the clear at
measurements. This data can be reasonably fitted with an exponential expression, given in Equation (2).
whichpoint
complete dissolution occurred was assessed by a combination of visual detection
during slow heating (<0.1 °C·min ) after addition of a fixed amount of API. The temperature
−1 and FBRM
at
measurements. This data
which complete can be reasonably
dissolution C =
occurred was 0.0521
fitted ⋅ exp(0.0838
with by
assessed ⋅ T)
an aexponential
combinationexpression, given inand
of visual detection (2) (2).
Equation
FBRM
measurements. This data can be reasonably fitted with an exponential expression, given in Equation (2).
where C represents the concentration of the solute in g/100 g solvent and T is the absolute
C = 0.0521 · exp(0.0838 · T) (2)
temperature in °C. C = 0.0521 ⋅ exp(0.0838 ⋅ T) (2)

C represents
wherewhere the concentration
C represents of the solute
the concentration of the insolute
g/100ing g/100
solvent
g and T is and
solvent the absolute
T is thetemperature
absolute
in ◦ C.temperature in °C.

Figure 2. Solubility data of the API in ethanol denatured with MEK. The dashed line corresponds to
the exponentially fitted solubility curve, given in Equation (2).

FigureFigure
At Solubility
2.the start
2. of data of the
the experiment,
Solubility data APIAPI
of the in
25ethanol
g of
in thedenatured
ethanol active with
with MEK.
pharmaceutical
denatured The dashedline
ingredient
MEK. The dashed line
was corresponds
dissolved
corresponds into75tog
the exponentially
ethanol
the denatured fitted
exponentially with solubility
MEK,
fitted incurve,
order
solubility given
to given
curve, inin
prepare Equation (2).
a solution
Equation (2).with a concentration of 33 g API/100 g
solvent. Based on the exponential fit of the solubility data, this solution has a solubility temperature
of the
At Atstart
about the
77 start of experiment,
°C.the
of the experiment,
Hence, 25 25
to completely g of theactive
dissolve
g of the active pharmaceutical
the substance, ingredient
the solution
pharmaceutical was dissolved
was maintained
ingredient was in 75
in g75 g
at reflux
dissolved
ethanol denatured
temperature (~78 °C)with
for MEK,
45 mininunder
ordercontinuous
to prepare stirring.
a solution withthe
Next, a concentration
solution was of 33 gatAPI/100
cooled a linear g
ethanol denatured with MEK, in order to prepare a solution with a concentration of 33 g API/100 g
solvent. Based
cooling rate ofon0.5the exponential
°C·min −1 until fit
theofdesired
the solubility
seedingdata, this solution
temperature washas a solubility
obtained. Thetemperature
latter was
solvent. Based on the exponential fit of the solubility data, this solution has a solubility temperature
of about 77 °C. Hence, to completely dissolve the substance, the solution was maintained at reflux
of about 77 ◦ C. Hence, to completely dissolve the substance, the solution was maintained at reflux
temperature (~78 °C) for 45 min under continuous stirring. Next, the solution was cooled at a linear
temperature ◦
cooling (~78
rate ofC)0.5 for°C·min
45 min under
−1 until thecontinuous
desired seedingstirring. Next, the was
temperature solution was cooled
obtained. at awas
The latter linear
cooling rate of 0.5 ◦ C·min−1 until the desired seeding temperature was obtained. The latter was
evaluated at 57 ◦ C, 62 ◦ C and 70 ◦ C, which corresponds to a supersaturation ratio (C0 /Csat ) of 5.28,
 2017, 7, 40
Crystals 2017, 40 5 of 20

evaluated at 57 °C, 62 °C and 70 °C, which corresponds to a supersaturation ratio (C0/Csat) of 5.28,
3.47
3.47 and
and1.77,
1.77,respectively.
respectively. Seeding waswas
Seeding eithereither
performed by addition
performed of dry material
by addition of dry or by activation
material or by
of the ultrasonic field at 10 W at one of these seeding temperatures. In the
activation of the ultrasonic field at 10 W at one of these seeding temperatures. In the experiments experiments performed at
aperformed
supersaturation ratio of 3.47 and
at a supersaturation 5.28,ofthe
ratio seeding
3.47 and 5.28, temperature
the seedingwastemperature
maintained was for 80 min, after which
maintained for 80
the solution was subjected to a cooling trajectory. This includes cooling to 50 ◦ C at 0.1 ◦ C·min−1 ,
min, after which the solution was subjected to a cooling trajectory. This includes cooling to 50 °C at
followed
0.1 °C·min by−1,afollowed
temperature by areduction
temperature 15 ◦ C at 0.3
untilreduction ◦ C·min−1 . When the seeding
until 15 °C at 0.3 °C·min−1. When temperature
the seeding of
70 ◦ C was evaluated,
temperature of 70 °C thewasinitial waiting
evaluated, thetime of 80
initial min was
waiting timealso
of employed,
80 min wasbut theemployed,
also solution was butfirst
the
cooled to 62 ◦ C at 0.1 ◦ C·min−1 . This intermediate temperature was maintained for 80 min, after which
solution was first cooled to 62 °C at 0.1 °C·min . This intermediate temperature was maintained for
−1

the previously
80 min, described
after which cooling trajectory
the previously describedwascooling
started.trajectory
This adjustment in the This
was started. protocol was necessary
adjustment in the
to prevent
protocol wasbuild-up
necessaryof the
to supersaturation
prevent build-upduring of the cooling which could
supersaturation result
during in secondary
cooling which couldnucleation,
result
increasing the possibility to produce agglomerated crystals. Hence, the
in secondary nucleation, increasing the possibility to produce agglomerated crystals. Hence, the additional waiting time at
62 ◦ C provided isothermal
additional waiting time atdesupersaturation,
62 °C provided isothermal and allowed operating as close
desupersaturation, to the
and solubility
allowed curve as
operating
possible during the cooling profile after the initial desupersaturation at the seeding
close to the solubility curve as possible during the cooling profile after the initial desupersaturation temperature.
at theDuring
seeding the experimental runs, data was obtained from the FBRM and IR probes, submerged in
temperature.
the crystallization vessel. Subsequently,
During the experimental runs, datathese
was measurements
obtained from were processed
the FBRM and IR byprobes,
the IControl software
submerged in
of Mettler Toledo. The chord length distributions were averaged over
the crystallization vessel. Subsequently, these measurements were processed by the IControl a 30 s time interval and displayed
by a normalized
software of Mettlersquare weighted
Toledo. The distribution.
chord lengthThe concentration
distributions were of averaged
the solute was
overquantified
a 30 s time byinterval
tracing
aand
characteristic −1 in the IR-spectra. In the studied concentration
displayed peak area at a wavelength
by a normalized of 1122 cm
square weighted distribution. The concentration of the solute was
range, this measurement
quantified provided a linear
by tracing a characteristic peak correlation with an R2of
area at a wavelength of1122
at least
cm−10.99. Similar
in the as the FBRM
IR-spectra. In the
measurements,
studied concentration the IR-data were
range, thisaveraged
measurementover 30provided
s in which 256 spectra
a linear were collected.
correlation with an RThe 2 ofIR-plots
at least
shown in thisaspaper,
0.99. Similar the FBRMdisplay the relative supersaturation
measurements, the IR-data were level which can
averaged overbe30calculated
s in which from
256 the API
spectra
concentration
were collected.measurements
The IR-plots usingshownEquation (3). display the relative supersaturation level which
in this paper,
can be calculated from the API concentration measurements using Equation (3).
C − Csat
Relative supersaturation level = C − C (3)
Relative supersaturation level = 0 − Csat
C sat
(3)
C 0 − C sat
where C (g API/100 g solvent) represents the actual concentration, and C0 and Csat are the initial
where C (g API/100
concentration g solvent)
(33 g API/100 represents
g solvent) the actuallevel,
and solubility concentration,
respectively.and C0 and Csat are the initial
The slurry was stirred overnight at the isolation temperature of 15 ◦ C, followed by separation
concentration (33 g API/100 g solvent) and solubility level, respectively.
of the The slurry by
crystals wasvacuum
stirred overnight
filtration.atNext,
the isolation temperature
the crystals were driedof 15in°C,a followed
fume hood by separation
by exposure of
thethe
to crystals by vacuum
air. Finally, filtration.
the dried Next,are
samples theclassified
crystals were dried in
as mono- or astar-shaped
fume hood by exposure
crystals, to the air.
displayed in
Finally,3.the
Figure Thedried samples
latter refer toarestrongly
classifiedagglomerated
as mono- or star-shaped
structures, crystals,
consisting displayed in Figure
of multiple 3. The
intergrown
latter refer
crystals. Ontothestrongly
contrary,agglomerated structures,
individual crystals consisting
or very of multiple intergrown
small agglomerates consisting ofcrystals.
a maximumOn the of
contrary,
three individual
crystals cemented crystals or very
together small agglomerates
are considered consistingThe
as mono-crystals. of aanalysis
maximum of three crystals
is performed using
acemented
combination together are considered
of optical as mono-crystals.
and scanning microscopy, and The analysis
thereforeisprovides
performed using a combination
qualitative assessment.
of optical and some
Alternatively, scanning microscopy,
authors suggested and thetherefore provides
use of shape qualitative
parameters suchassessment. Alternatively,
as the aspect ratio and
some authors suggested the use of shape parameters such as the aspect
perimeter to quantify the degree of agglomeration [7]. However, application of this approach using ratio and perimeter to
quantify
image the degree
processing by a of agglomeration
Malvern G3 provided [7]. inconsistent
However, application
classificationofforthis
thisapproach
particular using
API andimage
this
processingwas
procedure by atherefore
Malverndiscarded
G3 provided inconsistent
for sample classification
allocation. Prior to SEMfor this particular
imaging with aAPI and Pro,
Phenom this
procedure was therefore discarded for sample allocation. Prior to SEM imaging
the samples were coated with gold for 2 min using a constant current of 30 mA. Optical images of with a Phenom Pro,
samples
the dry were
crystals coated
were made with
as agold for 2with
Z-stack minausingNikona constant current of without
Eclipse microscope 30 mA. Optical images ofstep.
any preparation the
dry crystals were made as a Z-stack with a Nikon Eclipse microscope without any preparation step.

Exampleofof
Figure 3. Example thethe final
final morphology
morphology of API.
of the the API. Dry samples
Dry samples are classified
are classified by SEM byand
SEM and
optical
optical microscopy: (a) mono-crystal (CE diameter: 233.2 µm, length: 329.1 µm, width:
microscopy: (a) mono-crystal (CE diameter: 233.2 µm, length: 329.1 µm, width: 207.5 µm); and (b) 207.5 µm);
and (b) star-shaped
star-shaped crystal
crystal (CE (CE diameter:
diameter: 191.5
191.5 µm, length
µm,247.5
length µm,247.5 width:
µm, 197.9
width: 197.9 µm).
µm).
Crystals 2017, 7, 40 6 of 20

Crystals 2017, 7, 40 6 of 20

3. Results and Discussion

3. Results and Discussion
3.1. Evaluation of the Conventional Crystallization Process
3.1. Evaluation of the Conventional Crystallization Process
In the conventional crystallization process, seeding by the addition of 0.2 m% dry API at 62 ◦ C
In the
initiated theconventional crystallization
nucleation process. After an process, seeding
aging time by the
of 80 min, theaddition of 0.2 m%
aforementioned dry API atprofile
temperature 62 °C
initiated the nucleation process. After an aging time of 80 min, the ◦aforementioned temperature
with two gradients was started in order to cool the solution down to 15 C. This conventional process
profile with two gradients was started in order to cool the solution down to 15 °C. This conventional
yields strongly agglomerated crystals, shown in Figure 4a.
process yields strongly agglomerated crystals, shown in Figure 4a.

Figure 4.4.SEMSEMimages
imagesof the APIAPI
of the produced by thebyconventional
produced crystallization
the conventional process in
crystallization the Easymax
process in the
102 setup with addition of 0.2 m% dry seed material at 62 ◦ C. The crystal were imaged after filtration
Easymax 102 setup with addition of 0.2 m% dry seed material at 62 °C. The crystal were imaged after
at 15 ◦ C and
filtration air°C
at 15 drying. Crystals
and air drying.were takenwere
Crystals at different
taken atmagnifications, given by thegiven
different magnifications, length
byof thelength
the scale
bar:
of the(a)scale
300 µm; (b) 300
bar: (a) 10 µm;
µm;and
(b) (c) 2 µm.
10 µm; and (c) 2 µm.

These crystals
These crystals have
have aa predominantly star-shaped form
predominantly star-shaped form inin which
which thethe faces
faces are
are growing
growing outwards
outwards
from one
from onecenter.
center. This
This effect
effect can
can be be observed
observed even more even morein clearly
clearly the earlyinstagethe ofearly stage of the
the crystallization.
crystallization.
Figure 4b,c showsFigure
a SEM4b,c capture
shows aofSEM capture
crystals of crystals
which which were
were obtained by hotobtained by hot
filtration filtration
immediately
immediately
after after visual
visual detection detection ofThe
of nucleation. nucleation.
elementary The elementary
particles haveparticles
a rod-likehave a rod-like
shape, implying shape,
that
implying that the fastest growth direction is oriented along the length axis of
the fastest growth direction is oriented along the length axis of these rods. In general, fast growingthese rods. In general,
fast growing
faces exhibit faces exhibit the
the smallest areasmallest area surface
in the total in the total surface
of the crystalof the
andcrystal
develop andstronger
developattractive
stronger
attractive
forces forces compared
compared to slow-growing
to slow-growing faces [8,19,39].
faces [8,19,39]. As a result,As atheresult, the fast-growing
fast-growing faces will faces will
contact
contact each other via liquid bridges and orient around one central core that
each other via liquid bridges and orient around one central core that cements the crystals together. cements the crystals
together.
This This is
foundation foundation is largely
largely maintained maintained
through through
further growth andfurther growthprocesses,
agglomeration and agglomeration
resulting in
processes,
the resulting in the
star morphology. star morphology.
In addition, some ofIn addition,
the some ofshown
agglomerates the agglomerates
in Figure 4ashown have in Figure
multiple
smaller fragments grown into the surface of a larger crystal. Most likely, this phenomenonthis
4a have multiple smaller fragments grown into the surface of a larger crystal. Most likely, is
phenomenon
caused is caused bycollisions
by interparticle interparticle
andcollisions
aggregation and aggregation between
between crystals of crystals
different of sizes
differentthatsizes
are
that are subsequently
subsequently cemented cemented together
together into into an agglomerative
an agglomerative bond. This
bond. This process process
is strongly is strongly
dependent on
the collision frequency between the crystals and can therefore result in a strong variety in the ashape
dependent on the collision frequency between the crystals and can therefore result in strong
of
variety
the in the shape
agglomerates of the agglomerates
[12,15,17,18,40,41]. [12,15,17,18,40,41].
In turn, this inconsistency Ininturn, this inconsistency
the final morphology might in thecause
final
morphology
strong might in
fluctuations cause strong fluctuations
post-processing operations in post-processing
such as filtrationoperations
and tableting, suchaltering
as filtration and
the drugs’
tableting, altering the drugs’ bioavailability. Therefore, the effect of seed mass and
bioavailability. Therefore, the effect of seed mass and stirring rate, two parameters that have an impact stirring rate, two
parameters
on that have
the formation an impact onwere
of agglomerates, the formation
studied inof agglomerates,
more detail [7,8].were studied in more detail [7,8].

3.1.1. Effect of Seed Mass

The effect of seed mass was evaluated at a constant stirring rate of 250 rpm and seeding
temperature of 62 ◦°C. C. Figure 5a,b shows the obtained crystals
crystals after
after seeding
seeding with
with 11 m%
m% and
and 5 m%,
respectively. InInboth
respectively. both tests,
tests, thethe presence
presence of agglomerates
of agglomerates is strongly
is strongly reducedreduced
compared compared to the
to the reference
reference
case with case
0.2 m%with 0.2 m%
seeds seeds
(Figure 4).(Figure 4). This
This effect effect
can be can be attributed
attributed to an collision
to an increased increasedfrequency
collision
frequency
at at higher
higher seed loading,seed
whichloading, which
splits the splits the
aggregates intoaggregates intobefore
single crystals singlethe
crystals before into
cementation the
cementation into agglomerates takes place [10]. In addition, the SEM images indicate that smaller
crystals are produced when a higher seed load is used. Hence, as the growth is distributed across a
Crystals 2017, 7, 40 7 of 20

agglomerates takes place [10]. In addition, the SEM images indicate that smaller crystals are produced
Crystalsa2017,
when 7, 40seed load is used. Hence, as the growth is distributed across a larger number of particles,
higher 7 of 20

the size of each individual element is reduced. Overall, it is clear that a seed loading of 1 m% and more
larger number of particles, the size of each individual element is reduced. Overall, it is clear that a
yields non-agglomerated mono-crystals.
seed loading of 1 m% and more yields non-agglomerated mono-crystals.

Figure 5. Effect
Figure 5. Effect of
of seed
seedmass
masson
ondegree
degreeofofagglomeration,
agglomeration,while
whileoperating
operating at at
a constant stirring
a constant rate
stirring of
rate
250 rpm andandseeding temperature of 62 ◦ C: (a)
of 250 rpm seeding temperature of 62 °C:1(a)
m% seedseed
1 m% mass; and (b)
mass; and5 (b)
m%5 seed
m% mass. TheseThese
seed mass. SEM
images were magnified
SEM images 265 and
were magnified 260
265 times,
and 260 respectively, and theand
times, respectively, scalethe
barscale
represents a length aoflength
bar represents 300 µm.of
300 µm.
3.1.2. Effect of Stirring Rate
3.1.2. Effect of Stirring Rate
Firstly, the crystallization was evaluated in the absence of stirring to minimize the interaction between
Firstly, the
the particles. Thiscrystallization
experiment was was evaluated
performed in ain25the
mLabsence
test tube,ofplaced
stirring to minimize
inside the interaction
a temperature-controlled
between
jacket theEasymax
of the particles.setup.This During
experiment was performed
the dissolution stage, thein asolution
25 mL was teststirred
tube, with
placed inside a
a magnetic
temperature-controlled
stirrer to provide a homogeneous jacket of thetemperature
Easymax setup. During the
distribution. dissolution
However, stage,
during thethecooling
solution was
phase,
stirred with
stirring a magnetic
was disabled stirrerany
to avoid to grinding
provide aofhomogeneous
sedimented crystals temperature
by thedistribution.
stirrer bar. To However,
initiate
during the cooling
nucleation, the tubephase, stirringwith
was seeded was 3disabled
m% milled to avoid any Figure
crystals. grinding 6aof sedimented
shows crystals
the crystals thatby the
were
stirrer bar.byTothis
obtained initiate nucleation,
procedure. the tube
Although some wassmall
seeded with 3are
particles m%still
milled crystals.
grouped intoFigure
a single 6a cluster,
shows
crystals that
the majority were
of the sampleobtained by of
consists this procedure.
individual Although some small particles are still grouped
mono-crystals.
into aSecondly,
single cluster, the majority
an experiment with ofthe
theaddition
sample consists
of 3 m%ofmilledindividual
seedsmono-crystals.
was evaluated under agitated
Secondly,
conditions (250 an
rpm)experiment
in a 100 mL with the by
vessel addition
meansof of3comparison.
m% milled This seedsseed
wasmaterial
evaluated willunder agitated
contain many
conditions
fines (250 rpm)
and damaged in a 100
crystals withmL vesselsurface.
a rough by means of comparison.
Crystals produced from Thisthese
seedmilled
material willshown
seeds, contain
in
many fines and damaged crystals with a rough surface. Crystals produced
Figure 6b, again exhibit a clear star-shaped morphology due to the strong degree of agglomeration. from these milled seeds,
shown
This in Figureclearly
observation 6b, again differsexhibit a clear star-shaped
from previous experimentsmorphology
in which addition due to them%
of >1 strong
seedsdegree of
resulted
agglomeration.
in a non-agglomeratedThis observation clearly differs
crystal morphology, from previous
as illustrated experiments
in Figure 5. On the in one
whichhand,addition
this canof be
>1
m% seeds resulted
attributed in a non-agglomerated
to an increased tendency of milled crystal morphology,
particles as illustrated
to aggregate in Figure 5.both
and agglomerate, On the one
before
hand, this
dosing in drycan be attributed
conditions and aftertoseeding
an increased tendency
in the solution of milled
[8,19,42]. Underparticles
influence to aggregate and
of supersaturation,
agglomerate, both
agglomerative bonds before
will be dosing
formedin which
dry conditions
irreversiblyandlockaftertheseeding in the solution
seed material [8,19,42].
into a clustered Under
structure.
influence
On of supersaturation,
the other hand, the milledagglomerative
particles might bonds
havewill be formed
multiple whichedges
fractured irreversibly
or faceslock
thatthecanseed
act
material
as into and
nucleation a clustered structure.
growth sites, On the
resulting other hand,
in dendritic the growth
crystal milled which
particles might
yields an have multiple
agglomerated
fractured edges
structure. or faces
Additional that can
breakage act asdendrites
of these nucleation and growth
introduces moresites,
smallresulting
particlesin in dendritic
the solution crystal
that
growth promote
further which yields an agglomerated structure. Additional breakage of these dendrites introduces
agglomeration.
moreFinally,
small theparticles in the solution
conventional process inthat further
which 0.2 m% promote agglomeration.
non-micronized seed material is added, was evaluated
at 500Finally,
rpm. Thesethe conventional
results are displayedprocess inin which
Figure 6c.0.2 m% non-micronized
In comparison seed material
to the conventional is added,
procedure was
operating
evaluated
at at 500 rpm.
250 rpm (Figure 4), it isThese results
clear that are displayed
a higher stirring rate inyields
Figure 6c.agglomerated
less In comparison to the conventional
crystals.
procedure operating at 250 rpm (Figure 4), it is clear that a higher stirring rate yields less
agglomerated crystals.
Crystals 2017, 7, 40 8 of 20
Crystals 2017, 7, 40 8 of 20

Figure 6. 6.Effect
Figure Effectofofstirring
stirring rate on
on degree
degreeofofagglomeration
agglomeration after
after seeding
seeding at a at a temperature
temperature of 62 of (a)◦ C:
°C:62
(a)33m%
m%milled
milledseeds,
seeds, nono stirring;
stirring; (b)(b) 3 m%
3 m% milled
milled seeds,
seeds, 250 rpm;
250 rpm; and
and (c) 0.2(c)m%
0.2seeds,
m% seeds, 500All
500 rpm. rpm.
AllSEM
SEMimages
imageswerewere magnified
magnified 270270 times
times andand
thethe scale
scale bar bar represents
represents a length
a length of 300ofµm.
300 µm.

The previous results indicate that agglomeration as a function of stirring rate exhibits a
The previous results indicate that agglomeration as a function of stirring rate exhibits a maximum
maximum in its profile. Hence, agglomeration can be prevented by the absence of agitation, or by
in its profile. Hence, agglomeration can be prevented by the absence of agitation, or by application
application of a high stirring rate [21]. A similar dependency was also established by computational
of a high stirring rate [21]. A similar dependency was also established by computational modeling
modeling of orthokinetic aggregation in precipitation processes [16]. This model from Mumtaz
of orthokinetic aggregation in precipitation processes [16]. This model from Mumtaz assumes that
assumes that agglomeration is initiated by collisions between particles, as already proposed by
agglomeration
Smoluchowski’s is initiated by collisions
theory, but introducesbetween particles,
an efficiency factorasthat
already
accountsproposed by Smoluchowski’s
for collisions that do not
theory, but introduces an efficiency factor that accounts for collisions that
result in agglomeration. On the one hand, this model predicts a linear dependency of do not result in the
agglomeration.
agitation
Onrate
theandonethehand, this model predicts a linear dependency of the agitation rate and
collision frequency. Since a higher stirring speed increases the velocity of the crystals the collision
in
frequency. Since a higher stirring speed increases the velocity of the crystals in solution, the
solution, the probability for a particle-particle collision is enhanced. On the other hand, the efficiency probability
forofa the
particle-particle collision
collisions to produce is enhanced.
aggregates On the other
or agglomerates hand,
reduces theincreasing
with efficiencyshear
of the collisions
rate. This can to
produce aggregates
be attributed to bothorstronger
agglomerates reducesforces
hydrodynamic with that
increasing
break up shear rate. This
aggregated can and
clusters be attributed
a reduced to
both
timestronger
frame to hydrodynamic forces that
transform aggregates intobreak up aggregated
agglomerates. clusters
In the end, theseand a reduced
opposing time
effects frame
cause an to
intermediate
transform stirring into
aggregates rate to exhibit a higher
agglomerates. In degree of these
the end, agglomeration
opposingcompared to very
effects cause an low or high
intermediate
agitation.
stirring rate to exhibit a higher degree of agglomeration compared to very low or high agitation.

3.1.3. Prompt
3.1.3. Generation
Prompt GenerationofofNuclei
Nuclei
Overall,the
Overall, theprevious
previousresults
results show
show that
thatagglomeration
agglomeration can be be
can minimized
minimized by adequate
by adequate tuning of
tuning
of the
the stirring
stirringrate
rateand/or
and/or seed loading.
seed loading.ThisThis
corresponds
correspondsto a crystallization processprocess
to a crystallization with a very
with high
a very
collision
high frequency
collision frequency in which aggregation
in which is effectively
aggregation reverted
is effectively beforebefore
reverted agglomeration can occur.
agglomeration An
can occur.
alternative approach to increase the number of collisions without addition
An alternative approach to increase the number of collisions without addition of high seed loads of high seed loads is is
prompt generation of crystals [17,43,44]. Hence, quasi-instantaneous generation
prompt generation of crystals [17,43,44]. Hence, quasi-instantaneous generation of a large number of of a large number of
crystals
crystals willwill increase
increase the the collision
collision frequency,
frequency, rapidly
rapidly deplete
deplete the supersaturation
the supersaturation and therefore
and therefore reduce
reduce agglomeration. To quickly produce crystals without addition of seeds, spontaneous
agglomeration. To quickly produce crystals without addition of seeds, spontaneous nucleation at
nucleation at high supersaturation can be used. This method facilitates nuclei generation and rapidly
high supersaturation can be used. This method facilitates nuclei generation and rapidly increases
increases the suspension density. However, it should be taken into account that this procedure will
the suspension density. However, it should be taken into account that this procedure will also
also reduce the average particle size, which could make the process more sensitive to agglomeration.
reduce the average particle size, which could make the process more sensitive to agglomeration.
Despite this possible disadvantage, spontaneous crystallization at 57 °C, corresponding to an initial
Despite this possible disadvantage, spontaneous crystallization at 57 ◦ C, corresponding to an initial
supersaturation ratio of 5.28, was performed to evaluate whether prompt generation of crystals
supersaturation
could inhibit ratio of 5.28, wasAs
agglomeration. performed
shown in to evaluate
Figure 7a, whether promptpredominantly
this sample generation of crystals
consists could
of
inhibit agglomeration. As shown in Figure 7a, this sample predominantly consists
mono-crystals, confirming the applicability of this procedure. Further analysis of the FBRM and IR of mono-crystals,
confirming the applicability
data, displayed in Figureof7b, thisallows
procedure.
gainingFurther analysis
further insightofinto
the FBRM and IR data,
the consecutive displayed
steps of the in
Figure 7b, allows gaining further insight into the consecutive steps of the
crystallization process. Both the total FBRM counts which are correlated to the number of crystals crystallization process.
Both the total FBRM counts which are correlated to the number of crystals
and the IR signal, representing the solute concentration or supersaturation level, proceed through and the IR signal,
representing the solute
three different stages concentration
after nucleation or supersaturation
[45]. Initially, theselevel,
PATproceed through
tools only three
detect different
a small stages
change,
after nucleation
attributed [45]. Initially,
to slow nucleation these
andPAT tools only detectkinetics.
desupersaturation a small Afterwards,
change, attributed to slow
the profile nucleation
of the total
andFBRM counts shows akinetics.
desupersaturation steep incline, accompanied
Afterwards, by aof
the profile fast
thedecrease in solute
total FBRM concentration.
counts shows a steep During
incline,
accompanied by a fast decrease in solute concentration. During this stage, the process is governed by
Crystals 2017, 7, 40 9 of 20

Crystals 2017, 7, 40 9 of 20
nuclei generation under influence of a high supersaturation level. In the final phase, the solute
concentration
this stage, approaches
the process isthe solubility
governed levelgeneration
by nuclei and the process evolvesofinto
under influence a growth-dominated
a high supersaturation
regime. During
level. In thethis transition,
final phase, thethe slope
solute of the FBRMapproaches
concentration and IR trends decreaselevel
the solubility as only
andsmall changes in
the process
evolves of
the amount into a growth-dominated
crystals and concentrationregime.
are During
detectedthis transition, the slope of the FBRM and IR
[19].
trends decrease as only small changes in the amount of crystals and concentration are detected [19].

(a) (b)
Figure 7. Evaluation of agglomeration during spontaneous crystallization at 57 °C (C0/Csat = 5.28),
Figure 7. Evaluation of agglomeration during spontaneous crystallization at 57 ◦ C (C0 /Csat = 5.28),
leading to fast crystal generation. (a) SEM-image (Magnification: 270×, 300 µm scale bar) showing the
leading to fast crystal generation. (a) SEM-image (Magnification: 270×, 300 µm scale bar) showing
predominant presence of mono-crystals. (b) The FBRM and IR trend allow tracking the total chord
the predominant presence
count and solute of mono-crystals;
concentration (b) The FBRM and IR trend allow tracking the total chord
during the experiment.
count and solute concentration during the experiment.
Although it is demonstrated that agglomeration can be minimized by an adequate tuning of the
stirring
Although rate,
it this parameter strongly
is demonstrated that depends on the stirrer
agglomeration can beconfiguration
minimizedand reactor
by an designtuning
adequate [46]. of
Hence, reoptimization is required if the process is performed in a different setup.
the stirring rate, this parameter strongly depends on the stirrer configuration and reactor design Similarly, it was [46].
evidenced that the occurrence of star-shaped crystals depends on the size and pretreatment of the
Hence, reoptimization is required if the process is performed in a different setup. Similarly, it was
seed crystals. Any inconsistency in the drying procedure can introduce crystal defects or size
evidenced that the occurrence of star-shaped crystals depends on the size and pretreatment of the seed
reductions which influence the final crystal morphology. This problem is further complicated if the
crystals.
seedsAny
are inconsistency
obtained after ainmilling
the drying procedure
operation, as thiscan introduce
process affectscrystal defects
the final or size reductions
seed properties in a
whichcomplex
influence the final crystal morphology. This problem is further complicated
and rather unpredictable way. Additionally, seeding by addition of dry material if cannot
the seedsbe are
obtained after a milling
implemented operation,
if a sterile processasisthis process
desired. affects the
Therefore, this final
studyseed properties
further in athe
evaluates complex
use of and
ratherultrasound
unpredictable way. Additionally,
as an alternative technology seeding by addition
since literature of dry material
has indicated cannot
that it can beas
act both implemented
a seeding if
source and agglomeration inhibitor [4,36,47].
a sterile process is desired. Therefore, this study further evaluates the use of ultrasound as an alternative
technology since literature has indicated that it can act both as a seeding source and agglomeration
3.2. Implementation of Ultrasound in the Crystallization Process
inhibitor [4,36,47].
Different ultrasound-assisted crystallization processes were evaluated based on their ability to
3.2. Implementation of Ultrasound in
reduce crystal agglomeration. the Crystallization
Firstly, it was studiedProcess
whether sonication could be implemented as a
post-treatment to destroy agglomerates. Next, it was assessed whether the use of continuous and
Different ultrasound-assisted crystallization processes were evaluated based on their ability to
pulsed sonication during the entire crystallization protocol could inhibit the formation of
reduce crystal agglomeration. Firstly, it was studied whether sonication could be implemented as
star-shaped crystals. In the end, ultrasound was implemented as a seeding agent in order to test it as
a post-treatment
an alternativetotodestroy agglomerates.
conventional Next,
seeding, while it production
the was assessed whether the use
of mono-crystals was of continuous
guaranteed by and
pulsed sonication during the
other means than ultrasound.entire crystallization protocol could inhibit the formation of star-shaped
crystals. In the end, ultrasound was implemented as a seeding agent in order to test it as an alternative
3.2.1. Ultrasound
to conventional as Post-Treatment
seeding, while the production of mono-crystals was guaranteed by other means
than ultrasound.
The crystals obtained by the conventional crystallization process were strongly agglomerated as
demonstrated previously in Figure 4a. Therefore, an ultrasonic treatment of 60 min with 15 W
3.2.1.continuous
Ultrasound as Post-Treatment
ultrasound was introduced after cooling to 15 °C. Figure 8 shows the crystal morphology
that is
The obtained
crystals by implementation
obtained of the additional
by the conventional sonication stage.
crystallization Thiswere
process procedure removes
strongly most
agglomerated
of the small particles that are attached and grown into the surface of large crystals, but does not
as demonstrated previously in Figure 4a. Therefore, an ultrasonic treatment of 60 min with 15 W
break the agglomerates of equally sized particles.
continuous ultrasound was introduced after cooling to 15 ◦ C. Figure 8 shows the crystal morphology
that is obtained by implementation of the additional sonication stage. This procedure removes most of
the small particles that are attached and grown into the surface of large crystals, but does not break
the agglomerates of equally sized particles.
Crystals 2017, 7, 40 10 of 20
Crystals 2017, 7, 40 10 of 20

Figure
Figure 8. Final crystal
8. Final crystal morphology
morphology of of the
the API
API after
after conventional
conventional seeded
seeded crystallization
crystallization protocol
protocol
followed by 60 min sonication at 15 W. SEM images are magnified 270 times and the scale
followed by 60 min sonication at 15 W. SEM images are magnified 270 times and the scale bar bar represents
arepresents
length of a300 µm. of
length The white
300 µm. arrows indicate
The white theindicate
arrows remaining
the presence
remaining ofpresence
agglomerates.
of agglomerates.

Earlier work
workby byZeiger
Zeiger and Suslick
and studied
Suslick the effect
studied of sonication
the effect on crystal
of sonication slurries slurries
on crystal in order into
identify the mechanism of crystal fragmentation by ultrasound [48,49].
order to identify the mechanism of crystal fragmentation by ultrasound [48,49]. They concluded They concluded that the
interaction
that between particles
the interaction betweenand cavitation
particles and shockwaves is the primary
cavitation shockwaves cause
is the for this cause
primary phenomenon,
for this
while other potential sources such as interparticle, particle-horn and particle-wall
phenomenon, while other potential sources such as interparticle, particle-horn and particle-wall collisions only
marginallyonly
collisions contribute
marginallyin the fragmentation
contribute process. However,
in the fragmentation some
process. authorssome
However, also authors
believe also
that
microjets
believe directed
that towards
microjets directedthetowards
crystal surface can surface
the crystal contribute canto the disruption
contribute to the of weakly assembled
disruption of weakly
particles and
assembled to their and
particles surface erosion.
to their This erosion.
surface jet formation
This isjetcaused by anisasymmetric
formation caused by bubble collapse
an asymmetric
and cancollapse
bubble reach aandspeedcanofreach
overa100speedm·sof [50].
−1
over Wagterveld − 1
100 m·s [50]. et al. were evenetable
Wagterveld to visualize
al. were even able these
to
events by the use of high speed imaging during sonication of calcite crystals
visualize these events by the use of high speed imaging during sonication of calcite crystals [51]. [51]. In the end, the
ultrasonic
In the end, post-treatment yielded large particles
the ultrasonic post-treatment that particles
yielded large exhibit rough crystalrough
that exhibit surfaces andsurfaces
crystal edges,
besides
and edges,a significant amount of
besides a significant fines. of
amount This change
fines. in morphology
This change in morphology can strongly
can stronglyinfluence
influencethe
dissolution kinetics of the API in an irreproducible way. Although it is possible
the dissolution kinetics of the API in an irreproducible way. Although it is possible to remove these to remove these fines
by additional
fines temperature
by additional temperature cycling methods,
cycling methods, thetheultrasonic
ultrasonicpost-treatment
post-treatmentdid did not completely
not completely
remove the
remove the agglomerates.
agglomerates. Therefore,
Therefore, this
this procedure
procedure was was considered
considered unsuccessful
unsuccessful in in the
the production
production
of mono-crystals.
of mono-crystals.

3.2.2. Ultrasonic Treatment

Treatment during
during the
the Entire
Entire Cooling
Cooling Profile
Profile
In these set of experiments, ultrasound was enabled during the entire cooling profile. To reduce
energy consumption and heat generation, sonication sonication waswas operated
operated in in pulsed
pulsed mode
mode (10(10 W,
W, 20%)
20%) [52].
[52].
Firstly, pulsed
pulsed ultrasound
ultrasound waswasinitiated
initiatedimmediately
immediatelyafter afterthethe conventional
conventional seeding
seeding stepstep
withwith
0.2
m%m%
0.2 drydrymaterial
materialand was
and wasdisabled
disabledwhen whenthe thesolution
solutionreached
reached 15 ◦
15 °C,C, yielding predominantly
mono-crystals as as shown
shownininFigure
Figure9a. 9a.This
Thisresult indicates
result indicatesthat pulsed
that pulsed ultrasound
ultrasound cancanprevent the
prevent
formation
the formation of agglomerates
of agglomerates without
without anyanyfurther
furtheradjustments
adjustmentsinin the the conventional crystallization
crystallization
process. However,
process. However,ititshould
shouldbe betaken
takeninto
intoaccount
account that
that anan additional
additional energy
energy requirement
requirement is needed
is needed for
for ultrasonic
the the ultrasonic actuator
actuator during
during the entire
the entire cooling cooling trajectory.
trajectory. In addition,
In addition, the cooling
the cooling power will power will
increase
increase
as heat isas heat is introduced
introduced into the [53].
into the solution solution [53].
Secondly,pulsed
Secondly, pulsedsonication
sonication waswas enabled
enabled as soonas assoon as the reached
the solution solutionthereached
seeding the seeding
temperature
(62 ◦ C) to initiate
temperature (62 °C)
the to initiate the
nucleation nucleation
without additionwithout
of dryaddition of drySimilarly,
seed material. seed material. Similarly,
ultrasound was
ultrasoundduring
provided was provided
the entireduring
cooling thetrajectory
entire cooling
until trajectory untiltemperature.
the isolation the isolation The temperature. The
final crystals
final crystals
shown shown
in Figure 9b doinnot
Figure 9b agglomeration
exhibit do not exhibit and agglomeration
can therefore andbe can therefore
classified be classified
as similarly as
shaped
similarly shaped
mono-crystals. mono-crystals.
This This result
result demonstrates that demonstrates
sonication canthat sonication can
be implemented as be
an implemented as an
alternative seeding
alternativesince
technique seeding technique
no seed material since no seed
is needed material
to induce is neededFurther
nucleation. to induce nucleation.
application Further
of ultrasonic
application of ultrasonic energy prevents aggregation and agglomeration as observed previously in
the ultrasound-assisted tests with addition of seed material.
Crystals 2017, 7, 40 11 of 20

energy prevents aggregation and agglomeration as observed previously in the ultrasound-assisted

tests with
Crystals 2017,addition
7, 40 of seed material. 11 of 20

Crystals 2017, 7, 40 11 of 20

(a) (b)
Figure 9.
Figure 9. Pulsed
Pulsed USUS applied (a) the
applied during
during the cooling
cooling crystallization (b)of
crystallization process
process of the
the API:
API: (a)
(a) application
application after
after
addition
addition of 0.2 m% dry seed material; and (b) application to seed the solution and during cooling.
Figure of 0.2 m%USdry
9. Pulsed seed during
applied material;
theand (b) crystallization
cooling application toprocess
seed the solution
of the and
API: (a) during cooling.
application after
The scale
The scale bar represents
represents a length
length of
of 300
300 µm.
µm.
additionbarof 0.2 m% drya seed material; and (b) application to seed the solution and during cooling.
The scale bar represents a length of 300 µm.
The exact mechanism by which ultrasound inhibits agglomeration for this particular API
The exact mechanism by which ultrasound inhibits agglomeration for this particular API remains
remains unclear,exact although it by
is likely
whichthat three effects contribute in this for process as schematically
unclear,The
although itmechanism
is likely that three ultrasound
effects contribute inhibits
in thisagglomeration this particular
process as schematically summarized API
in
summarized in Figure 10. Firstly, application of ultrasound will improve
remains unclear, although it is likely that three effects contribute in this process as schematically mixing on a molecular
Figure 10. Firstly, application of ultrasound will improve mixing on a molecular scale induced by shear
scale induced by
summarized shear 10.
in Figure forces of imploding
Firstly, application cavitation
of ultrasound bubbles and acoustic
will improve mixingstreaming
on a molecular [54–56].
forces of imploding cavitation bubbles and acoustic streaming [54–56]. During cooling crystallization,
During cooling crystallization,
scale induced by shear forces this will enhance
of imploding the mass
cavitation bubblestransfer, resulting
and acoustic in homogeneous
streaming [54–56].
this will enhance the mass transfer, resulting in homogeneous particle growth and accelerated
During
particle cooling
growth and crystallization, this will enhance In
accelerated desupersaturation. theaddition,
mass transfer,
sonicationresulting in homogeneous
will rapidly increase the
desupersaturation. In addition, sonication will rapidly increase the amount of crystals in the solution.
particle growth and accelerated desupersaturation. In addition, sonication
amount of crystals in the solution. On the one hand, individual crystals arise due to fragmentation will rapidly increase the
Onamount
the oneofhand, individual
crystalsof in larger crystals
the solution. arise
Onorthe due to fragmentation
one hand,[57–59].
individual and disaggregation of larger crystals
and disaggregation crystals aggregates Oncrystals
the other arise due to
hand, fragmentation
cavitation bubbles
or and
aggregates [57–59]. of Onlarger
the other hand, aggregates
cavitation bubbles canthe
alsoother
act as direct nucleation sites to
can alsodisaggregation
act as direct nucleation crystals
sites toorenhance [57–59].number
the nuclei On [30,60].hand, cavitation
Although bubbles
this will result
enhance
can alsothe
actnuclei
as number
direct [30,60].
nucleation Although
sites to enhance this will
the result
nuclei in
numbersmaller particles
[30,60]. Althoughthat are
this more
will prone
result
in smaller particles that are more prone to aggregation, the high particle loading will also effectively
to aggregation,
in smaller the high
particles particle
thattoare more loading
prone to will also effectively
aggregation, disrupt
the high aggregates
particle loading duealso
to the increased
disrupt aggregates due the increased number of collisions. In addition, thewill
massiveeffectively
increase in
number
disruptof aggregates
collisions. In due addition,
to the the massive
increased numberincrease
of in surfaceInarea
collisions. will deplete
addition, the the supersaturation
massive increase in of
surface area will deplete the supersaturation within a shorter time frame, similar as the effect
within a shorter time frame, similar as the effect of micromixing. This inhibits
surface area will deplete the supersaturation within a shorter time frame, similar as the effect of the cementation of these
micromixing. This inhibits the cementation of these aggregates and moves the process quickly into a
aggregates
micromixing.and This
moves the process
inhibits quickly into
the cementation a growth-dominated
of these aggregates and moves regime
the [17,43,44].
process quicklyIn this stage,
into a
growth-dominated regime [17,43,44]. In this stage, new crystalline material will be deposited on the
newgrowth-dominated
crystalline material regime
will be[17,43,44].
deposited In on
thisthe
stage, new crystalline
surfaces of the priorymaterial
formed will be deposited
crystals. on the
This deposition
surfaces of the priory formed crystals. This deposition preferentially takes place on damaged edges
surfaces of the
preferentially takespriory
place formed crystals. edges
on damaged This deposition
and surfaces, preferentially
masking takes place
possible on damaged
erosion effectsedges
caused
and surfaces, masking possible erosion effects caused by sonofragmentation [5,19,61]. Finally,
and surfaces, masking possible erosion effects caused by sonofragmentation
by sonofragmentation [5,19,61]. Finally, ultrasound also increases the collision frequency by a direct [5,19,61]. Finally,
ultrasound
ultrasound
also increases the
also increases
collision frequency
the collision
by a direct interactionbetweenbetween shockwaves or
interaction between shockwaves or liquidfrequency
jets and the by aggregates
a direct interaction
which will disruptshockwaves or
the latter before
liquid jetsjets
and the aggregates which will
willdisrupt
disrupt the the latter beforeananagglomerative
agglomerativebond bond is is formed [25].
an liquid and
agglomerative the aggregates
bond is formedwhich [25]. latter before formed [25].

Figure
Figure 10.
Figure Schematic
10.10.
Schematicoverview
Schematic of theof
overview
overview proposed
of mechanism
the proposed
the proposed by which sonication
mechanism
mechanism by whichinhibits
by which agglomeration
sonication
sonicationinhibits
inhibits
during crystallization.
agglomeration during crystallization.
agglomeration during crystallization.

3.2.3. Targeted Ultrasonic Treatment at Seeding Temperature

3.2.3. Targeted Ultrasonic Treatment at Seeding Temperature
As demonstrated before, agglomeration can be effectively inhibited by the application of
As demonstrated before, agglomeration can be effectively inhibited by the application of
ultrasound throughout the entire cooling trajectory. However, as the formation of agglomerates will
ultrasound throughout the entire cooling trajectory. However, as the formation of agglomerates will
Crystals 2017, 7, 40 12 of 20

3.2.3. Targeted Ultrasonic Treatment at Seeding Temperature

As demonstrated before, agglomeration can be effectively inhibited by the application of ultrasound
Crystals 2017, 7, 40 12 of 20
throughout the entire cooling trajectory. However, as the formation of agglomerates will predominantly occur
Crystals 2017, 7, 40 12 of 20
in the initial stage of theoccur
predominantly crystallization process
in the initial stage under
of theinfluence of a high
crystallization supersaturation,
process under influenceprolonged of a exposure
high
to ultrasound
predominantlyis not required.
supersaturation, occur Hence,
prolonged
in the in stage
exposure
initial a later toofstage the process
ultrasound is not operates
the crystallization processatHence,
required. a lower
under insupersaturation
a laterofstage
influence a high thelevel,
reducing the cementation
process
supersaturation,operates of
at aggregates
a lower
prolonged into agglomerates
supersaturation
exposure to ultrasound level,[7,8,14].
not Therefore,
isreducing additional
the cementation
required. Hence, atests
in of were
aggregates
later stage performed
into
the
agglomerates
process
to determine operates
the minimum [7,8,14].
at a lower Therefore,
sonication additional
supersaturation
period for tests
level, were
thereducing performed
production to determine
the cementation
of single theInminimum
of aggregates
crystals. these into
sets of
sonication
agglomerates period
[7,8,14].for the production
Therefore, of
additional single crystals.
tests were In these
performed
experiments, ultrasound was used to start the nucleation while the total FBRM counts were monitored sets of
toexperiments,
determine ultrasound
the minimum was
used to period
sonication start the for nucleation
the field
productionwhile of the
single total FBRM counts wereof monitored in ultrasound
time. Next,was the
in time. Next, the ultrasonic was disabled atcrystals.
variousInFBRM these sets
counts experiments,
and the crystallization protocol
ultrasonic field was disabled at various FBRM counts and the
used to start the nucleation while the total FBRM counts were monitored in time. Next, the crystallization protocol was completed
was completed
as described
as described
earlier.
earlier.
In theat end,
In the end,
the crystal
the crystal morphology was analyzed to determine
ultrasonic field was disabled various FBRMmorphology
counts and the wascrystallization
analyzed to determine
protocol was the completed
presence of
the presence of
agglomerates. agglomerates. This
ThisInassessment assessment
was performed was performed
for three for
supersaturationthree supersaturation
ratios: 3.47, ratios: 3.47,
as described earlier. the end, the crystal morphology was analyzed to determine the1.77 and
presence 5.28.
of
1.77 and 5.28. 11
Figure
agglomerates. Figure This11the
shows shows thewas
total FBRM
assessment total
countsFBRM
performed counts
that were that
measured
for three were measured
at the
supersaturation moment at the
sonication
ratios: moment
3.47, wasand
1.77 sonication
disabled.
5.28.
was disabled.
Ultrasound Ultrasound
was started was
as started
soon as the as soon
solution as the
reached solution
the seeding
Figure 11 shows the total FBRM counts that were measured at the moment sonication was disabled. reached
temperature.the seeding
After a temperature.
certain time
nucleation
AfterUltrasound
a certain time was induced
nucleation by
wasultrasound,
induced which
by was
ultrasound,detected by
which a rise
was in
was started as soon as the solution reached the seeding temperature. After a certain time the
detectedFBRM by signal.
a rise The
in onset
the FBRM
signal. of nucleation
The onset
nucleation wasof is considered
nucleation
induced as t = 0, and
is considered
by ultrasound, the
whichas additional
was ultrasonic
t =detected
0, and by exposure
thea additional time
rise in the FBRM afterwards
ultrasonic is
signal.exposure varied
The onset time
to yield a certain
of nucleation numberasoft =FBRM
is considered 0, and counts. This exposure
the additional ultrasonicperiod to ultrasound
exposure after nucleation
time afterwards variedis
afterwards is varied to yield a certain number of FBRM counts. This exposure period tois ultrasound
plotted on the x-axis of the FBRM and IR plots.
after to yield a certain
nucleation number
is plotted on of theFBRM
x-axiscounts.
of the This
FBRM exposure
and IRperiodplots. to ultrasound after nucleation is
plotted on the x-axis of the FBRM and IR plots.

Figure 11. Measured total FBRM counts at the moment that sonication is disabled as a function of the
Figure 11. Measured total FBRM counts at the moment that sonication is disabled as a function of
ultrasonic exposure time after the detection of nucleation. The final morphology after completion of
the ultrasonic
Figure 11.exposure
Measuredtimetotalafter
FBRM the detection
counts atand of nucleation.
theexhibits
moment The finalismorphology
thatdistinct
sonication a after
disabled asexposure completion
function the I of
the crystallization protocol is indicated two areas. Insufficient inof
zone
the crystallization
ultrasonic protocol
exposure
results in
is indicated
timecrystals,
star-shaped after thewhileand
detectionexhibits two distinct
of nucleation.
mono-crystals The final
are obtained
areas. Insufficient
inmorphology
zone II.
exposure in
after completion of zone I
results
theincrystallization
star-shaped protocol
crystals,iswhile mono-crystals
indicated and exhibits are
twoobtained in zone
distinct areas. II.
Insufficient exposure in zone I
results
Theincrystals
star-shaped crystals, while
produced undermono-crystals are obtained
these conditions in zone
were II.
analyzed to determine whether
agglomerates (star-shaped crystals) or mono-crystals were present,
The crystals produced under these conditions were analyzed to determine as shown in Figure
whether12. agglomerates
The crystals produced under these conditions were analyzed to determine whether
(star-shaped crystals) or mono-crystals were present, as shown in Figure 12.
agglomerates (star-shaped crystals) or mono-crystals were present, as shown in Figure 12.

Figure 12. Cont.

Crystals 2017, 7, 40 13 of 20
Crystals 2017, 7, 40 13 of 20

Figure 12. SEM

Figure 12. SEMimages
imagesofofcrystals
crystalsresulting
resulting from selected
selectedexperiments
experiments displayed
displayed in Figure
in Figure 11 which
11 which
are used
are used to classify
to classify crystals
crystals into into mono-
mono- or star-shaped
or star-shaped morphology.The
morphology. Theultrasonic
ultrasonicexposure
exposure time
time after
nucleation for these images are: (a) 0 min; (b) 3.25 min; (c) 5.75 min; (d) 37 min; (e) 63.8 min; and (f)and
after nucleation for these images are: (a) 0 min; (b) 3.25 min; (c) 5.75 min; (d) 37 min; (e) 63.8 min; 317 min.
(f) 317
All SEM min. All
pictures SEMmagnified
were pictures were
270 magnified
times and270 thetimes
scaleand
barthe scale bar arepresents
represents length ofa300
length
µm.of 300 µm.

In all experiments, the ultrasonic energy was supplied at a constant temperature of 62 °C,
In all experiments,
corresponding the supersaturation
to an initial ultrasonic energy ratio was supplied
of 3.47. at a constant
Since a waiting time of 80temperature
min is maintainedof 62 ◦ C,
corresponding to an
after addition of initial supersaturation
seed crystals ratio of 3.47.
in the conventional Sincea asimilar
process, waitingtimetime
spanof for
80 min is maintained
application of
after ultrasound
addition of wasseed crystalsWithin
evaluated. in thethis
conventional
window, theprocess,
FBRM shows a similar timeresponse
a similar span for as application
discussed of
before during
ultrasound spontaneous
was evaluated. nucleation
Within this at 57 °C (Figure
window, 7b). After
the FBRM showsthe initial low-response
a similar responsephase, the
as discussed
total counts sharply rise with increasing ◦
before during spontaneous nucleation at 57 C (Figure 7b). After the initial low-response at
exposure time to the ultrasonic field and start to level off phase,
around
the total 25,000
counts counts.
sharply Furthermore,
rise this plot
with increasing indicates
exposure time that a sufficient
to the ultrasonic exposure
field and timestart
to the
to level
ultrasonic field, yielding a total FBRM count close to the plateau value, will predominantly result in
off at around 25,000 counts. Furthermore, this plot indicates that a sufficient exposure time to
mono-crystals (zone II), while agglomeration occurs by application of shorter sonication periods
the ultrasonic field, yielding a total FBRM count close to the plateau value, will predominantly
(zone I). Although some degree of agglomeration still occurs, especially for the smallest crystals, the
resultreduced
in mono-crystals
presence of (zone II), while morphology
the star-shaped agglomeration for occurs by application
large particles is clearlyof shorter sonication
noticeable when
periods (zone I). Although some degree of agglomeration
sonication is maintained until 25,000 FBRM counts are reached. still occurs, especially for the smallest
crystals, the reduced
Finally, the presence of the star-shaped
use of continuous ultrasound morphology
throughoutfor thelarge particles
entire is clearlyprocess
crystallization noticeable
when(indicated
sonicationbyisanmaintained
exposure time until
of 25,000
317 min) FBRM
is alsocounts are as
evaluated reached.
a mean of comparison. Similarly as
the teststhe
Finally, with
usethe use of pulsed
of continuous ultrasound
ultrasound during thethe
throughout entire process
entire (Figure 9),process
crystallization this method
(indicated
by an exposure time of 317 min) is also evaluated as a mean of comparison. Similarlyprocess.
produces mostly mono-crystals with a strongly reduced size compared to the conventional as the tests
with the use of pulsed ultrasound during the entire process (Figure 9), this method produces mostly
mono-crystals with a strongly reduced size compared to the conventional process. This prolonged
sonication period will further increase the total FBRM counts to values close to 40,000 as more chord
Crystals 2017, 7, 40 14 of 20
Crystals 2017, 7, 40 14 of 20

This prolonged sonication period will further increase the total FBRM counts to values close to
lengths are detected due to the higher amount of crystals in the solution. In general, these results show
40,000 as more chord lengths are detected due to the higher amount of crystals in the solution. In
that ageneral,
minimal ultrasonic exposure at the seeding temperature is necessary to avoid agglomeration
these results show that a minimal ultrasonic exposure at the seeding temperature is
of thenecessary
crystals.toTo gainagglomeration
avoid further understanding of To
of the crystals. thegain
mechanism, the FBRM and
further understanding of theIRmechanism,
trends during
sonication were analyzed for tests yielding either star-shaped or mono-crystals. This comparison
the FBRM and IR trends during sonication were analyzed for tests yielding either star-shaped or is
shown in Figure 13a,b, respectively.
mono-crystals. This comparison is shown in Figure 13a,b, respectively.

Figure 13. Comparison of the FBRM and IR trend of an experiment from: zone I (a); and zone II (b),
Figure 13. Comparison of the FBRM and IR trend of an experiment from: zone I (a); and zone II (b),
yielding star-shaped and mono-crystals, respectively. The solid lines represent the FBRM data and the
yielding star-shaped and mono-crystals, respectively. The solid lines represent the FBRM data and
dashed lines show the relative supersaturation level.
the dashed lines show the relative supersaturation level.
In both experiments, the FBRM and the IR data again proceed through three different stages,
dominated by either nucleation
In both experiments, the FBRM or growth.
and the WhenIR continuous
data againsonication
proceed isthrough
applied threewithindifferent
the
seeding stage (Figure 13b), the supersaturation is already completely depleted within ca. 30 min,
stages, dominated by either nucleation or growth. When continuous sonication is applied within
while the saturation level is not reached after 70 min if sonication is disabled in an earlier stage
the seeding stage (Figure 13b), the supersaturation is already completely depleted within ca. 30 min,
(Figure 13a). Although the IR signals initially show an identical progress in both cases, the rate of
whiledesupersaturation
the saturation level is not reached after 70 min if sonication is disabled in an earlier stage
decreases as soon as sonication is disabled. Most likely, this is caused by two
(Figure 13a). Although
phenomena. On the one thehand,
IR signals initially
the disruption show
of the an identical
ultrasonic progress
field will in botheffect
have a negative cases,on the
the rate
of desupersaturation
overall mass transfer and degree of micromixing [54–56]. In turn, this reduces the nucleation and by
decreases as soon as sonication is disabled. Most likely, this is caused
two phenomena.
growth kinetics, On andthe one
hence hand, the disruption
the depletion of the of the ultrasonic
solute concentration field willOn
[57]. havethe aother
negative
hand,effect
on theultrasound increases
overall mass the available
transfer and degree surface area in the solution
of micromixing [54–56].byInformation
turn, this of reduces
additional thecrystals,
nucleation
disruption
and growth of aggregates
kinetics, and sonofragmentation
and hence the depletion of phenomena [48,49,57–59]. Disabling
the solute concentration [57]. Onthethe ultrasonic
other hand,
field will
ultrasound thus reduce
increases these effects,
the available surface affecting
area in thethe
desupersaturation
solution by formation profile. This hypothesiscrystals,
of additional is
supported by comparison of the FBRM data. Under continuous sonication, the total counts keep
disruption of aggregates and sonofragmentation phenomena [48,49,57–59]. Disabling the ultrasonic
increasing although the most significant effect is obtained as long as the solution is supersaturated.
field will thus reduce these effects, affecting the desupersaturation profile. This hypothesis is supported
The slow increase once the solubility level is reached can be mainly attributed to the disruption and
by comparison of the FBRM data. Under continuous sonication, the total counts keep increasing
breakage phenomena. In contrast, a much lower FBRM count is observed when sonication is
although the most
disabled in an earlysignificant
stage, aseffect
shown is obtained
in Figure 13b.as long
Onlyas a the solution
limited rise inisFBRM
supersaturated.
counts is detectedThe slow
increase once the solubility level is reached can be mainly attributed
after the stop of the ultrasonic field due to some additional nucleation under the remaining to the disruption and breakage
phenomena. In contrast,
supersaturation a much lower
level. Although the FBRM FBRM count
signal is observed
stabilizes, when sonication
the concentration keeps is disabled in
declining,
indicating
an early a growth-dominated
stage, as shown in Figure 13b. process.
Only Asa alimited
result, rise
the final crystal
in FBRM size will
counts be higherafter
is detected in case
the stop
of the ultrasonic field due to some additional nucleation under the remaining supersaturationalevel.
sonication is interrupted in an earlier stage of the process. The latter statement is supported by
comparison
Although the FBRM of the SEMstabilizes,
signal images andthe of concentration
the final chord length distributions,
keeps declining, displayed
indicating in Figure 14.
a growth-dominated
These results indicate that a longer exposure to ultrasound, corresponding to a higher FBRM
process. As a result, the final crystal size will be higher in case sonication is interrupted in an earlier
count, reduces the particle size and the span. This reduced span is mainly caused by the reduction of
stage of the process. The latter statement is supported by a comparison of the SEM images and of
agglomerates in the crystallization processes under continuous sonication. Since the agglomerates
the final
varychord length
in size, distributions,
configuration and numberdisplayed in Figure
of crystals, 14.
the FBRM will pick up multiple chord lengths,
These results indicate that a longer exposure
resulting in a wide distribution. The experiment in which ultrasoundto ultrasound, corresponding
was enabled until to a2140
higher
countsFBRM
count,even
reduces
showsthe particle size
a multimodal and the span.
distribution. It is, This reduced
however, spanwhether
not clear is mainly thecaused
two peaks by the reduction
on the right of
agglomerates
shoulder in canthe
be crystallization
correlated to theprocesses
presence under continuous
of agglomerates sonication.
or eroded Since the agglomerates
and suspended coating of the vary
FBRM
in size, probe. In the
configuration andend, continuous
number sonication
of crystals, exhibits
the FBRM thepick
will narrowest distribution,
up multiple chordalthough
lengths, similar
resulting in
results can be obtained by a sufficient exposure time at the seeding temperature.
a wide distribution. The experiment in which ultrasound was enabled until 2140 counts even shows
a multimodal distribution. It is, however, not clear whether the two peaks on the right shoulder can
be correlated to the presence of agglomerates or eroded and suspended coating of the FBRM probe.
In the end, continuous sonication exhibits the narrowest distribution, although similar results can be
obtained by a sufficient exposure time at the seeding temperature.
Crystals 2017, 7, 40 15 of 20
Crystals 2017, 7, 40 15 of 20
Crystals 2017, 7, 40 15 of 20

Figure 14. Normalized square-weighted chord length distribution of final samples obtained after
Figure 14. Normalized square-weighted chord length distribution of final samples obtained after
various sonication
Figure 14. periods
Normalized and corresponding
square-weighted FBRM
chord counts
length at a seeding
distribution temperature
of final samples of 62 °C. after
obtained
various sonication periods and corresponding FBRM counts at a seeding temperature of 62 ◦ C.
various sonication periods and corresponding FBRM counts at a seeding temperature of 62 °C.
Finally, the IR and FBRM data provide further evidence to explain why a minimum ultrasonic
Finally,
exposure the
time
Finally, IR
theand FBRM
isIRneeded
and FBRM data
to provide
inhibit
data the further
provide furtherevidence
formation to
to explain
of agglomerates.
evidence explain why why aaminimum
Sonicationminimum enhances ultrasonic
the
ultrasonic
micromixing
exposure time is during
needed the
to initial
inhibit stage,
the resulting
formation in
of a homogeneous
agglomerates.
exposure time is needed to inhibit the formation of agglomerates. Sonication enhances the distribution
Sonication of
enhancesthe supersaturation
the micromixing
and
during equal
micromixing
the growth
initial during
stage, of theinitial
the
resulting particles,
instage, further
resulting
a homogeneous aided abyhomogeneous
an enhanced
indistribution mass transfer.
of thedistribution
supersaturation of theConsequently,
supersaturation
and the
equal growth
desupersaturation
of theand equal growth
particles, furtherrate
ofaided
theaccelerates
particles, and
further
by an enhanced theaided
crystals
mass willenhanced
by transfer.
an spendConsequently,
less
masstime at high
transfer. supersaturation
theConsequently,
desupersaturation the
where agglomeration
desupersaturation rate phenomena
accelerates are
and more
the likely
crystals to
will
rate accelerates and the crystals will spend less time at high supersaturation where agglomeration occur.
spend After
less time thisat initial
high phase, a
supersaturation low
supersaturation
phenomena are morelevel
where agglomeration likelyisphenomena
tomaintained
occur. After arethroughout
more
this initial the
likely
phase,entire crystallization
to aoccur.
low After this since
supersaturation initial the
level issolution
phase, a low
maintained is
completely
supersaturation desupersaturated
level is before the
maintained start of the
throughout the cooling
entire profile. This minimizes
crystallization since the secondary
solution is
throughout the entire crystallization since the solution is completely desupersaturated before the start
nucleation
completelywhich could lead to before
desupersaturated a broadthe sizestart
distribution and reduces
of the cooling profile. theThiscementation
minimizes of aggregates
secondary
of the cooling profile. This minimizes secondary nucleation which could lead to a broad size
into agglomerates.
nucleation which could Secondly,lead tothe FBRMsize
a broad trend shows that
distribution andthe use ofthe
reduces ultrasound
cementation afterof nucleation
aggregates
distribution and reduces the cementation of aggregates into agglomerates. Secondly, the FBRM trend
rapidly boosts the amount of nuclei in the solution
into agglomerates. Secondly, the FBRM trend shows that the use of ultrasound after nucleation as a result of disaggregation and
shows that the use of ultrasound
sonofragmentation. In turn, after
this nucleation
fast generation rapidly
of boosts
crystals thepromptly
will amount of nuclei the
increase in the solution as
suspension
rapidly boosts the amount of nuclei in the solution as a result of disaggregation and
a result of disaggregation
density and collision
sonofragmentation. Inand sonofragmentation.
frequency.
turn, this fastAlthough
generation Inofturn,
aggregates this
crystals andfast generation
agglomerates
will promptly increaseoforiginate
crystalsthewill
from promptly
these
suspension
increase the
collisions,
density andsuspension
disintegration density and
will dominate
collision frequency. collision frequency.
beyondaggregates
Although Although
a certain suspension aggregates
density. Finally,
and agglomerates and
originate agglomerates
ultrasound will
from these
originate
also fromdisintegration
directly
collisions, these
enhance collisions,
the will disintegration
collision frequency
dominate beyond bywill dominate
aformation
certain beyond
of shockwaves
suspension a certain
density. andFinally,
liquidsuspension
jet density.
that interact
ultrasound will
Finally,
alsoultrasound
with the aggregates,
directly will also
enhance the directly
separating enhance
these
collision thebycollision
into individual
frequency frequency
crystals.
formation of shockwavesby formation
and liquid of shockwaves
jet that interact and
liquid jetTo
with thevalidate
that interact
aggregates, thewithmechanism of agglomeration
the aggregates,
separating these intoseparating
individual inhibition
these by application
into
crystals. individual of a threshold ultrasonic
crystals.
treatment,
To validate the
To validate thesame procedure
the mechanism
mechanism ofwas evaluated forinhibition
of agglomeration
agglomeration two additional
inhibition supersaturation
by application
by application of levels. Figure
ofaathreshold
threshold ultrasonic15
ultrasonic
shows
treatment, that
treatment, for both
the same
the same supersaturation
procedure
procedure was was levels,
evaluatedfor
evaluated a certain
fortwo treatment
twoadditional time is indeed
additional supersaturation needed
supersaturation levels. to produce
levels.Figure
Figure 1515
showsmono-crystals,
shows
that that
for bothforsimilarly
both as evidenced
supersaturation
supersaturation before
levels,a for
levels, the conventional
acertain
certain treatment time
treatment protocol.
time is
is indeed
indeedneeded neededtotoproduce produce
mono-crystals,
mono-crystals, similarly
similarly as evidenced
as evidenced before
before forforthe theconventional
conventionalprotocol.
protocol.

Figure 15. Measured total FBRM counts at the moment that sonication is disabled as a function of the
ultrasonic
Figure 15. exposure
Measured time after
total FBRM thecounts
detection ofmoment
at the
the nucleation, performed at disabled
two seeding temperatures.
Figure 15. Measured total FBRM counts at momentthat thatsonication
sonicationis is disabledas a as
function of theof
a function
Solid lines represent
ultrasonic exposure the
timedata obtained
after the at 57 °C
detection of(C0/Csat = 5.28),
nucleation, while theatdashed
performed two lines indicate
seeding results
temperatures.
the ultrasonic exposure time after the detection of nucleation, performed at two seeding temperatures.
at 70 °C
Solid lines 0/Csat = 1.77).
(Crepresent The obtained
the data final morphology
at 57 °C (Cafter
0/Csat completion
= 5.28), whileofthe
thedashed
crystallization protocol
lines indicate is
results
Solid lines represent the data obtained at 57 ◦ C (C0 /Csat = 5.28), while the dashed lines indicate
indicated.
at 70 °C◦ (C0/Csat = 1.77). The final morphology after completion of the crystallization protocol is
results at 70 C (C0 /Csat = 1.77). The final morphology after completion of the crystallization protocol
indicated.
is indicated.
Crystals 2017, 7, 40 16 of 20
Crystals 2017, 7, 40 16 of 20

Analysis
Analysis of of the
the FBRM
FBRM and and IRIR signals
signals again
again supports
supports the the previously introduced mechanism.
previously introduced mechanism.
These plots are shown in Figures 16 and 17 for an initial supersaturation
These plots are shown in Figures 16 and 17 for an initial supersaturation ratio of 1.77 and ratio of 1.77 and 5.28,
5.28,
respectively.
respectively. ItIt isisimportant
importanttotonoticenoticethat thatthethe transition
transition intointo mono-crystals
mono-crystals is less
is less pronounced
pronounced in
in the
the latter case. Hence, the combination of a high supersaturation and sonication
latter case. Hence, the combination of a high supersaturation and sonication promotes the formation promotes the formation
of
of fines. These will
fines. These will aggregate
aggregate more more easily
easily andand can can therefore
therefore transform
transform intointo agglomerates
agglomerates before
before
collisions or interaction with cavitation bubbles disrupt these clusters.
collisions or interaction with cavitation bubbles disrupt these clusters. As a result, some As a result, some agglomerates
of small crystals
agglomerates of are
small produced
crystals together
are producedwith the mono-crystals.
together with the mono-crystals.
In
In both
both cases,
cases, the
the threshold
threshold exposure
exposure time time is is obtained
obtained whenwhen thethe total
total FBRM
FBRM counts
counts tend
tend to
to level
level
off. Since the counts increase more rapidly at high supersaturation, corresponding
off. Since the counts increase more rapidly at high supersaturation, corresponding to a faster to a faster depletion
in supersaturation,
depletion a shorter treatment
in supersaturation, a shorter time to obtaintime
treatment mono-crystals was needed when
to obtain mono-crystals wassonication
needed when was
started ◦
at 57wasC (C ◦
sonication 0 /Csat at
started = 5.28),
57 °Ccompared
(C0/Csat = to 70 C
5.28), (C0 /Csat =to1.77).
compared 70 °CFurthermore, a different
(C0/Csat = 1.77). responsea
Furthermore,
by the FBRM is obtained after the ultrasound is switched off. At a low
different response by the FBRM is obtained after the ultrasound is switched off. At a low seeding seeding temperature and high
initial
temperature and high initial supersaturation, the signal continues to increase after sonication in
supersaturation, the signal continues to increase after sonication is disabled, while it stagnates is
the experiments performed at 70 ◦ C with a low supersaturation level at t = 0. This effect is attributed
disabled, while it stagnates in the experiments performed at 70 °C with a low supersaturation level
to
at the remaining
t = 0. This effectabsolute supersaturation
is attributed ratio afterabsolute
to the remaining ultrasound is stopped, which
supersaturation ratiodetermines whether
after ultrasound is
growth
stopped,orwhich
nucleation
determinesdominates
whetherthe growth
crystallization process.
or nucleation The latter
dominates thewill be more likely
crystallization at higher
process. The
supersaturation
latter will be more levels,
likelyresulting
at higher in additional
supersaturation nucleilevels,
formation and ainrise
resulting in FBRM
additional counts
nuclei instead
formation
of deposition onto the available surface area. For example, in the experiments at 57 ◦ C the absolute
and a rise in FBRM counts instead of deposition onto the available surface area. For example, in the
supersaturation
experiments at 57ratio °C the equals almost
absolute 5.28 if the FBRM
supersaturation is disabled
ratio equals almost at 10,000
5.28 if the counts.
FBRM isAs a result,
disabled at
more secondary crystals will be formed in the solution if sonication is
10,000 counts. As a result, more secondary crystals will be formed in the solution if sonication is disabled and the FBRM will
only leveland
disabled off after a certain
the FBRM willdelay period
only level off(Figure
after a 17a).
certain delay period (Figure 17a).

Figure 16. FBRM, IR and SEM analysis of the crystal morphology obtained after a targeted
Figure 16. FBRM, IR and SEM analysis of the crystal morphology obtained after a targeted application
application of ultrasound during the seeding stage at 70 °C, corresponding to C0/Csat = 1.77. The solid
of ultrasound during the seeding stage at 70 ◦ C, corresponding to C0 /Csat = 1.77. The solid lines
lines represent the FBRM data and the dashed lines show the relative supersaturation level. A
represent the FBRM data and the dashed lines show the relative supersaturation level. A comparison is
comparison is made between: (a,c) limited exposure time which yields star-shaped crystals; and (b,d)
made between: (a,c) limited exposure time which yields star-shaped crystals; and (b,d) the production
the production of mono-crystals after sonication until complete desupersaturation.
of mono-crystals after sonication until complete desupersaturation.
Crystals 2017, 7, 40 17 of 20
Crystals 2017, 7, 40 17 of 20

Figure 17. FBRM, IR and SEM analysis of the crystal morphology obtained after a targeted application of
Figure 17. FBRM, IR and SEM analysis of the crystal morphology obtained after a targeted application
ultrasound during the seeding stage at 57 °C, corresponding to C0/Csat = 5.28. The solid lines represent the
of ultrasound during the seeding stage at 57 ◦ C, corresponding to C0 /Csat = 5.28. The solid lines
FBRM data and the dashed lines show the relative supersaturation level. A comparison is made between:
represent the FBRM data and the dashed lines show the relative supersaturation level. A comparison is
(a,c) limited exposure time which yields star-shaped crystals; and (b,d) the production of mono-crystals
made between: (a,c) limited exposure time which yields star-shaped crystals; and (b,d) the production
after sonication until complete desupersaturation.
of mono-crystals after sonication until complete desupersaturation.

4. Conclusions
4. Conclusions
Agglomeration is an often encountered problem during crystallization and precipitation
Agglomeration is an often encountered problem during crystallization and precipitation processes.
processes. In a conventionally seeded crystallization process, adequate tuning of the shear rate, seed
In a conventionally seeded crystallization process, adequate tuning of the shear rate, seed loading and
loading and seeding temperature can avoid this phenomenon, but this method is highly sensitive to
seeding temperature can avoid this phenomenon, but this method is highly sensitive to the properties
the properties of the seed material and therefore not robust. Although ultrasound was proven to be a
of the seed material and therefore not robust. Although ultrasound was proven to be a successful
successful alternative in the literature, the exact mechanism is unknown, leading to improper
alternative in the literature, the exact mechanism is unknown, leading to improper operation.
operation. In most cases, it is enabled throughout the entire process which results in a high energy
In most cases, it is enabled throughout the entire process which results in a high energy demand,
demand, crystal breakage and surface erosion. This study identified the mechanism and can
crystal breakage and surface erosion. This study identified the mechanism and can therefore reduce
therefore reduce the operating time of ultrasound, while maintaining the production of
the operating time of ultrasound, while maintaining the production of non-agglomerated crystals.
non-agglomerated crystals. This research was performed on a commercially available API in order
This research was performed on a commercially available API in order to evaluate the potential
to evaluate the potential of the technology in pharmaceutical industry. Ultrasound was assessed
of the technology in pharmaceutical industry. Ultrasound was assessed both as a post-treatment
both as a post-treatment technique to break agglomerates and as an alternative seeding agent. The
technique to break agglomerates and as an alternative seeding agent. The former method proved to
former method proved to be unsuccessful since all crystals exhibited strong surface erosion and only
be unsuccessful since all crystals exhibited strong surface erosion and only part of the agglomerates
part of the agglomerates could be destroyed. Sonication during the initial stage of crystallization is
could be destroyed. Sonication during the initial stage of crystallization is able to inhibit the formation
able to inhibit the formation of agglomerates as long as a sufficient exposure time is provided.
of agglomerates as long as a sufficient exposure time is provided. Analysis with PAT tools identified
Analysis with PAT tools identified that ultrasound should be enabled until the FBRM and IR signal
that ultrasound should be enabled until the FBRM and IR signal counts reach a steady state,
counts reach a steady state, corresponding to a complete desupersaturation of the solute at the
corresponding to a complete desupersaturation of the solute at the seeding temperature. The ultrasonic
seeding temperature. The ultrasonic treatment enhances the degree of micromixing, boosts the
treatment enhances the degree of micromixing, boosts the amount of particles and increases
amount of particles and increases the collision frequency. As a result, aggregates are efficiently
the collision frequency. As a result, aggregates are efficiently disrupted before they are cemented into
disrupted before they are cemented into agglomerates. In the end, this strategy of a targeted
agglomerates. In the end, this strategy of a targeted application of ultrasound during the seeding stage
application of ultrasound during the seeding stage enables the user to tune the properties of in-situ
enables the user to tune the properties of in-situ generated nuclei and prevent their agglomeration.
generated nuclei and prevent their agglomeration. Therefore, this method provides an alternative
Therefore, this method provides an alternative for conventionally seeded crystallization processes and
for conventionally seeded crystallization processes and thus increases the potential of ultrasound in
thus increases the potential of ultrasound in pharmaceutical industry.
pharmaceutical industry.
Crystals 2017, 7, 40 18 of 20

Acknowledgments: The research leading to these results has received funding from the European Community’s
Seventh Framework Programme (FP7/2007-2013) under grant agreement No. NMP2-SL-2012-309874 (ALTEREGO).
J. Jordens acknowledges funding of a grant by the Agency for Innovation by Science and Technology (IWT).
The CTU-group of Johnson & Johnson in Beerse is acknowledged for their collaboration in this project,
providing the API and allowing access to their experimental facilities.
Author Contributions: B. Gielen conceived the research, designed and performed the experiments and wrote
the paper. J. Jordens, L.C.J. Thomassen, L. Braeken and T. Van Gerven contributed in the analysis and discussion
of the data.
Conflicts of Interest: The authors declare no conflict of interest.

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