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. 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