1. food and drink
2. food
3. grains and pasta

Effect of Ultrasonic Treatment on the Nucleation and Growth of Cloxacillin
Benzathine
Jieqiong Li, Ying Bao, Jingkang Wang *
State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology,
Tianjin University, Tianjin, People’s Republic of China.
Abstract
The effect of ultrasonic irradiation on the behavior of primary nucleation of cloxacillin
benzathine in supersaturation solutions with different levels were studied in the present work. The
solubility of cloxacillin benzathine in binary solvent mixtures of ethanol and water were measured by a
dynamical method, using a medium-throughput multiple reactor (Crystal 16™, Avantium, Netherlands).
Based on the solubility data, the induction periods of this system were measured under different
experiment conditions. Compared to the induction time without ultrasonic irradiation, the induction
period for different supersaturations decreased with increasing ultrasonic irradiation energy and
subsequently increased to a certain degree. Above this level of ultrasonic irradiation, the induction
period decreased dramatically. Besides that, the composition of solution and feeding rate of the reactant
were investigated to figure out the influential factors to the primary nucleation behaviors for this system.
Measurements of crystal size and crystal habits for irradiated and silent experiments showed that the
particle size and aggregates were both affected by ultrasonic irradiation and is highly correlated with the
supersaturation. The products with desired crystal size could be obtained by optimally controlling the
conditions of supersaturation and ultrasounic irradiation.
Introduction
The application of ultrasound to the crystallization systems offers a significant potential for
modifying the processes and improving both of the product properties and yields 1. Ultrasound can
induce primary nucleation in nominally particle-free solutions and, noteworthy, it can be surmised that
the metastable zone is narrowed by ultrasound or that the sonicated reactive crystallization can nucleate
at relatively lower supersaturation levels 2, 3.
Cloxacillin benzathine ((C 19 H 18 ClN 3 O 5 S) 2 ·C 16 H 20 N 2 , CAS Registry No. 23736-58-5) is a
valuable half-synthesized antibiotic with strong antibacterial activity against Gram-positive bacteria 4-6.
As we all know that features like morphology and particle size influence the quality of the product 7. The
aggregation and agglomeration phenomenon have some bad effects on the final crystal size distribution
and morphology, as well as decrease the purity and stability of the products 8. Sonication has been used
for the breakage and dispersion of aggregations and agglomerates, where the high energy generated by
acoustic cavitation has been found to be more effective than a mechanical turbulence 9, 10. Miyasaka et al.
11
suggested the possibility that the crystal size of a final product could be controlled by controlling the
number of primary nucleation sites with an appropriate amount of ultrasonic energy. The influence of
sonication on the aggregations and agglomerates is mainly depended on the ultrasonic frequency,
*Corresponding author Tel.: 86-22-27405754. Fax: 86-22-27374971.
E-mail: [email protected]
sonication power, treatment time, and some solution characteristics.
A bath will deliver non-homogeneous acoustic fields throughout the medium with maximum
amplitude at multiples of the half-wavelength of sound 12. The non-homogeneity of the acoustic field
means that one must be careful with the positioning of the reaction/crystallizing vessel (depth,
positioning with respect to where the transducers are mounted) if direct comparison is made for a series
of experiments. According to previous literature work, the induction time and the number of nuclei were
found to be correlated with only ultrasonic energy13.
Experimental Methods
Preliminary Experiments
The solubility of cloxacillin benzathine in binary solvent mixtures of ethanol and water were
measured by a dynamical method, using a medium-throughput multiple reactor (Crystal 16™,
Avantium, Netherlands). Slurries with different concentrations were prepared in the vials and stirred at
1000 rpm. For the accuracy of the experimental data, the heating rate was set at 0.1 °C /min, while the
cooling rates employed were 0.4 °C /min. The heating and cooling cycles were repeated at least 6 times
for every sample to get a better determination of the solubility. The system temperature variation for all
the measurements was found to be within ±0.1 K. All of the masses were measured using a balance
(Model AE240, Mettler−Toledo, Switzerland) with an accuracy of ±0.00001 g.
Sonocrystallization Procedures
Cloxacillin benzathine was generated by double decomposition reaction of the raw materials of
cloxacilin sodium and N, N-dibenzylethylenediamine (DBED) (purity >0.99 in mass fraction). Ethanol
(99.7% in mass fraction) and distilled water were used as a mixtured solution for all the experiments. To
conduct the experiment, cloxacilin sodium solution (20g CS/100g water; volume 30mL) and DBED
solution (9g DBED/100g water; volume 30mL) were prepared and charged into the jacketed reactor. The
feeding rates of the two reactant solutions were controlled using two high precision pump and the
solutions were delivered to the jacketed reactor smoothly for the whole duration of the experiment. The
jacketed reactor was filled with mixture solution of ethanol and water and the filling volume in the vessel
was 80mL. Fig. 1 shows the scheme of experimental setup.
The experiments were carried out in a 400mL jacketed vessel thermostated using an external
thermostatic bath (accuracy of ±0.1°C). A four-leaf stainless steel mixing propeller was placed 20mm
above the bottom and the initial stirring rate was set at 200rpm to make sure that crystal suspension was
well mixed. Ultrasound waves were generated by a stainless steel low powered ultrasound bath
(SB-5200, DTD, SCIENTZ, Ningbo, China), with a maximum nominal power of 200W and a frequency
of 40 kHz. The ultrasonic bath was filled with water as a cavitating medium with a height of 14 cm. The
ultrasonic irradiation was applied at various levels of energy input. The energy input applied to the
system was determined by an adiabatic measurement of the temperature rise due to the ultrasonic
irradiation13-15. Ultrasonic irradiation was stopped as soon as nucleation was observed in the crystallizer.
Figure 1: Schematic diagram of the experimental setup:(A) jacketed reactor; (B) stirrer driver;
(C) four leaf propeller; (D and E) peristaltic pump; (F and G) feed tanks; (H) thermostatic bath;
(I) ultrasonic bath.
Results and Discussion
Preliminary experiments
The experimental solubility data of cloxacillin benzathine in binary solvent mixtures of ethanol
and water with different solution composition within the temperature range from 283.15K to 313.15K
were determined in our previous works 16 (see Fig. 2). The final results were used to calculate the mole
fraction solubility ( x1 ) according to eq 1.
x1 =
m1 / M 1
∑ i=1mi / M i
3
(1)
where mi represents the mass of the solute and solvents, and the M i means the molecular weight of the
solute and solvents. Among them, m1 and M 1 separately represent the mass and the molecular weight of
the solute, while m2 , m3 and M 2 , M 3 represent the mass and the molecular weight of the ethanol and
water, respectively.
Figure 2: Experiment solubility of cloxacillin benzathine vs the water mole fraction in binary
solvent mixtures at different temperatures ((○) 283.15K; (▲) 288.15K; (▽) 293.15K; (◆)
298.15K; (△) 303.15K; (▼) 308.15K; (☆) 313.15K).
The dissolving capacity of cloxacillin benzathine in the selected solvent mixtures increases with
increased temperature within the studied temperature range. The mole fraction of the solute ( x1 ) reaches
its maximum value at a specific initial composition of water ( x3 ) in the ethanol + water systems.
The effect of ultrasound on the reduction of agglomeration
Fig. 3 shows optical photographs of cloxacillin benzathine crystals just precipitate from the
solution with or without ultrasonic irradiation. In the conventional silent experiments, non-uniform
mixing of the reactant will result in very high supersaturation and dense nucleation in some locations in
solution. Small nuclei intend to cluster together and grow further to become agglomerates. With well
mixing of the reactant, when under the interruption of transient calvitation, the nuclei formed in the
system can grow separately and the aggregates can barely be found.
Figure 3: Optical photographs of cloxacillin benzathine crystals just precipitate from the
solution under different process conditions, both of them were not interrupted by seeding. (1)
uninsonated experiment; (2) insonated experiment (200W, 40kHz).
According to Mullin17, increasing agitation at first reduces the supercooling required for
nucleation in aqueous solutions of ammonium dihydrogen phosphate, magnesium sulfate, and sodium
nitrate, but further increasing in agitation actually retard nucleation. Finally, nucleation again increases
at the highest usable rates of agitation. On the basis of previous results, it was concluded that ultrasonic
irradiation inhibits and activates primary nucleation at various degrees of supersaturation. Furthermore,
the number of crystals related to final crystal size, and ultrasonic energy could yield the desired crystal
size by inducing suitable nucleation 11. The relationship between the number of crystals N and the
average crystal size L is expressed as
(2)
X = αρc NL3
Where α is the average volume shape factor and X is the total crystal weight. If X is fixed and N
increases, the amount of solute crystallized on each nucleus decreases, which decreases the size of the
final product L .
Sonication can reduce the agglomeration via two pathways. One is the control of the magnitude
of the primary nucleation number 18. The second is the enhanced distribution of nuclei and improvement
in the environment of the growing crystal. Considering the two aspects, the nuclei were growing under
control of more powerful hydrodynamics and mass transfer performance.
Effect of higher amounts of ultrasonic energy on the induction period
Ultrasonic irradiations were performed at solutions of cloxacillin benzathine in solvent mixtures
of ethanol and water. The induction periods were measured using samples with different supersaturation
levels under interruption of ultrasonic irradiation. The results are shown in Figure 4. In the figure, each
point corresponds to a certain concentration of the solution and nominal power of ultrasound. Under the
same supersaturation, dramatic decreased induction periods were observed with increasing ultrasonic
energy at lower levels of ultrasonic irradiation. Additionally, the induction periods gradually became
even shorter when the measurements were performed using higher levels of ultrasonic irradiation in the
supersaturated solutions. This phenomenon implies that sonication can induce nucleation more intensely
at lower supersaturation solutions. Therefore it can be surmised that the metastable zone is narrowed by
ultrasound or that the sonicated reactive crystallization can nucleate at lower supersaturation levels.
Figure 4: Induction period vs. ultrasonic power for cloxacillin benzathine in binary solvent
mixture of ethanol and water with water mole fraction x3 = 0.754 at 288.15K: (■)S=8.28; (●)
S=10.19; (▲) S=12.735; (▼) S=14.642.
Impact of initial solvent compositions
According to the experimental solubility data of cloxacillin benzathine in binary solution mixture
of ethanol and water with different compositions, the solubility reaches its maximum value at a specific
initial composition of water in the ethanol + water systems. Therefore, the insonated experiments were
carried out between the solvent compositions range, these extra supersaturation data were added to the
data from Figure 2 and the plots are shown in Figure 5. As ultrasonication was applied to the reactive
crystallization process, the supersaturation levels of solution were relatively small when water mole
fraction of the solvent mixture was less than 0.75. Based on the optical photographs we can found that
sonication clearly prevented aggregation/agglomeration based on re-dispersing the crystal aggregates
during the crystallization in the solvent mixture with relative small water mole fraction. But this kind of
interruption was gradually weaker than the attachments between the nuclei, and the formation of giant
aggregation/agglomeration became irreversible.
Figure 5: Impact of initial solvent compositions on the supersaturation levels with interruption
of the sonication (200W, 40kHz) at 288.15K: (left) solubility x1 (●) and supersaturation S (■) of
cloxacillin benzathine in binary solvent mixtures of ethanol and water when the fines were
visible; (right) optical photographs of cloxacillin benzathine crystals just precipitate from
solution with different water mole fraction: (1) x3 = 0.288 ; (2) x3 = 0.352 ; (3) x3 = 0.447 ;
(4) x3 = 0.617 ; (5) x3 = 0.673 ; (6) x3 = 0.754 ; (7) x3 = 0.826 ; (8) x3 = 0.861 .
Effect of Different Feed Rate of the Reaction Material Solution
Considering the dosage and properties of the crystal seeds magnificently influenced the final
PSD, series of experiments were performed to generate seed crystals in situ by transient cavitation
caused by continuous ultrasonic disturbance (nominal power of 100W). At first, the feeding rates were
varied from 0.1 to 1.0 mL/min. The ultrasonic burst was stopped a few seconds after the fine can be
visibly detected. Figure 6 showed the solution concentration when fines can be detected varied with the
increased feeding rate, as well as the corresponding crystal habits.
Even under continuous ultrasonic disturbance, the supersaturation levels of the system gradually
increased with the increased feeding rate. Non-uniform mixing of the reactant will result in very high
supersaturation and dense nucleation in some locations in solution. Some nuclei and small grains in
solution will cluster together due to attractive interactions and will grow further. At the same time, the
number of the small needle crystals generated in situ become larger and the fines inclined to aggregates,
even under continuous interruption of ultrasound at the early stage of the reaction. The agglomerated
product contains occlusions of the mother liquor that will detrimentally affect the purity of the product.
Figure 6: Effect of feeding rate of the reactant solutions on the crystallization process of
cloxacillin benzathine with interruption of the sonication (200W, 40kHz) at 288.15K: (left) the
concentration of the solution when the crystals were detectable; (right) optical photographs of the
crystals after precipitation from the solution under different feeding rate: (a) 0.1mL/min; (b)
0.2mL/min; (c) 0.4mL/min; (d) 0.6mL/min; (e) 0.8mL/min; (f) 1.0mL/min.
Conclusions
In the reaction crystallization of cloxacillin benzathine, crystal agglomeration and aggregation
occurred simultaneously. The effect of ultrasonic irradiation on the induction periods and final crystal
habit in supersaturation solutions were studied. Firstly, low levels of ultrasonic irradiation slightly
inhibited primary nucleation and high levels of ultrasonication induced it. These results provide a useful
guideline for achieving the desired effect of ultrasonic irradiation on primary nucleation. Secondly,
under the continuous interruption of ultrasonic irradiation, the supersaturation levels of the system
gradually increased with the increasing feeding rate and reached the minimum when the water mole
fraction was 0.673. Besides that, ultrasonication clearly prevented aggregation/agglomeration based on
re-dispersing the crystal aggregates. But this kind of interruption was gradually weaker than the
attachments between the nuclei, and the formation of giant aggregation/agglomeration became
irreversible.
List of Symbols and Abbreviation
L = average crystal size
N = number of crystals
mi = mass of the solute and solvents
M i = molecular weight of the solute and solvents
ρc = density of the solute
x = mole fraction in the solution
X = the total crystal weight
α = the average volume shape factor
Subscripts
1 = solute (cloxacillin benzathine)
2 = ethanol
3 = water
Acknowledgements
Funding provided by China Ministry of Science and Technology for the key technology of preparation of
edible pigment and industrialization project (no. 2011BAD23B02) is acknowledged.
References
1.
L. H. Thompson and L. K. Doraiswamy,(1999), "Sonochemistry: Science and engineering,"
Industrial & Engineering Chemistry Research, pp.1215-1249.
2.
G. Ruecroft, D. Hipkiss, T. Ly, N. Maxted and P. W. Cains,(2005), "Sonocrystallization: The use
of ultrasound for improved industrial crystallization," Organic Process Research and
Development, pp.923-932.
3.
Hossein Kiani, Zhihang Zhang, Adriana Delgado and Da-Wen Sun,(2011), "Ultrasound assisted
nucleation of some liquid and solid model foods during freezing," Food Research International,
pp.2915-2921.
4.
T. J. Keefe,(1980), "Benzathine cloxacillin as a dry-cow mastitis product," Modern Veterinary
Practice, pp.783-785.
5.
N. M. Villanada, N. P. Medina, N. S. Abes and C. N. Mingala,(2012), "Retrospective study on
the treatment of subclinical mastitis in water buffaloes," Large Animal Review, pp.201-205.
6.
Y. Gundelach, E. Kalscheuer, H. Hamann and M. Hoedemaker,(2011), "Risk factors associated
with bacteriological cure, new infection, and incidence of clinical mastitis after dry cow therapy
with three different antibiotics," Journal of Veterinary Science, pp.227-233.
7.
D. Pertig, R. Buchfink, S. Petersen, T. Stelzer and J. Ulrich,(2011), "Inline Analyzing of
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Industrial Crystallization Processes by an Innovative Ultrasonic Probe Technique," Chemical
Engineering and Technology, pp.639-646.
J. Daigneault and L. W. George,(1990), "Topically applied benzathine cloxacillin for treatment
of experimentally induced infectious bovine keratoconjunctivitis," American journal of
veterinary research, pp.376-380.
Sang-Mok Changa Jong-Min Kima, Kyo-Seon Kimb, Min-Kyu Chungc, Woo-Sik Kim,(2011),
"Acoustic influence on aggregation and agglomeration of crystals in reaction crystallization of
cerium carbonate," Colloids and Surfaces A: Physicochem. Eng. Aspects, pp.193-199.
M. Lemanowicz, A. Kus and A. T. Gierczycki,(2010), "Influence of ultrasonic conditioning of
flocculant on the aggregation process in a tank with turbine mixer," Chemical Engineering and
Processing, pp.205-211.
E. Miyasaka, S. Ebihara and I. Hirasawa,(2006), "Investigation of primary nucleation
phenomena of acetylsalicylic acid crystals induced by ultrasonic irradiation - ultrasonic energy
needed to activate primary nucleation," Journal of Crystal Growth, pp.97-101.
T. G. Leighton (1994), "The Acoustic Bubble," Academic Press, New York.
M. Kurotani, E. Miyasaka, S. Ebihara and I. Hirasawa,(2009), "Effect of ultrasonic irradiation on
the behavior of primary nucleation of amino acids in supersaturated solutions," Journal of
Crystal Growth, pp.2714-2721.
Takahide Kimura, Takashi Sakamoto, Jean-Marc Leveque, Hajime Sohmiya, Mitsue Fujita,
Shigeyoshi Ikeda and Takashi Ando,(1996), "Standardization of ultrasonic power for
sonochemical reaction," Ultrasonics Sonochemistry, pp.S157-S161.
K. Seo, S. Suzuki, T. Kinoshita and I. Hirasawa,(2012), "Effect of Ultrasonic Irradiation on the
Crystallization of Sodium Acetate Trihydrate Utilized as Heat Storage Material," Chemical
Engineering and Technology, pp.1013-1016.
J. Q. Li, Z. Wang, Y. Bao and J. K. Wang,(2013), "Solid-Liquid Phase Equilibrium and Mixing
Properties of Cloxacillin Benzathine in Pure and Mixed Solvents," Industrial & Engineering
Chemistry Research, pp.3019-3026.
W.J. Mullin (2001), "Crystallization," Butterworth-Heinemann Ltd., London.
H. Li, H. R. Li, Z. C. Guo and Y. Liu,(2006), "The application of power ultrasound to reaction
crystallization," Ultrasonics Sonochemistry, pp.359-363.