**Purification of Erythromycin by Antisolvent Crystallization or Azeotropic Evaporative Crystallization**

Kui Chen, Li-Jun Ji and Yan-Yang Wu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52934

### **1. Introduction**

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Crystallization plays an important role in separation and purification of the antibiotics. And it is also an indispensable step in preparation of pharmaceuticals with biological activities and specific crystal form. As the last step in purification, crystallization determines the puri‐ ty, crystal habit, granularity and its distribution as well as pharmacologic effect, biologic ac‐ tivity and product stability [1], which are actually dependent on specific mechanism for its processes and operational conditions. So it's necessary to study thermodynamics, kinetics and conditions of crystallization process, which helps increase the yield and reduce cost.

As a representative of macrolide antibiotics, erythromycin has been widely used since its in‐ troduction in 1952 [2]. As erythromycin derivatives, clarithomycin and azithromycin have exhibited remarkable improvement on stability in acid solutions and metabolism dynamics [3, 4]. A lot of researches have been done on new derivatives with features of combating drug resistance [5, 6]. In the meanwhile, high-purity erythromycin, as the raw material, is fundamental to produce its pharmaceutical derivatives.

Erythromycin is obtained from microbial fermentation in industry. Subsequent separa‐ tion and purification involve multiple unit operations, such as extraction, absorption, chromatography and crystallization. Different process involves different combinations of unit operations [7].

Among them, solvent extraction accompanied with intermediate precipitation is widely used, in which butyl acetate is commonly adopted to extract erythromycin from the fermen‐ tation filtration. It is followed by reactive crystallization to form an intermediate prior to conversion into erythromycin alkaline and dissolving in acetone. Lastly, erythromycin is pu‐

© 2013 Chen et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

rified by antisolvent crystallization [8]. That is to say, both reactive crystallization and anti‐ solvent crystallization have to be employed in this separation process.

In this paper, two crystallization processes of erythromycin in different systems, which include the antisolvent crystallization for mixed solvents of acetone and water and the azeotropic evaporative crystallization for butyl acetate-water system, are described in de‐ tails. The thermodynamics and kinetics of the crystallization of erythromycin, which help to thoroughly understand the effect of a variety of factors on the nucleation, crystal growth and crystal habit, are summarized systematically. On the basis of these funda‐ mental studies, effective control techniques are proposed to improve the quality of er‐

Purification of Erythromycin by Antisolvent Crystallization or Azeotropic Evaporative Crystallization

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45

In the solvent extraction process for purification of erythromycin, erythromycin alkaline was converted from erythromycin thiocyanate by adding ammonia or NaOH solution and dis‐ solving in acetone. Then erythromycin product was prepared by antisolvent crystallization,

The traditional crystallization process, which was too simple, only involved modulating two

Water was poured into erythromycin acetone solution at room temperature, and then the product was obtained by filtration after standing for a period of time. Such operation made obvious differences of supersaturation, nucleation rate and crystal growth rate and then

As we know, the phase equilibrium between solid and its solution is fundamental to choose crystallization method and also determines the maximum yield of solution crystallization [12]. Erythromycin is soluble in acetone, but insoluble in water [13]. Thus, erythromycin can

The solubility of erythromycin in acetone increases with the increasing temperature, where‐ as it becomes less soluble with the higher temperature in water. So, the solubility of erythro‐ mycin in acetone-water binary solvent system is influenced by the solvent composition and

Some data on solubility of erythromycin in acetone-water solution was reported in litera‐ tures [14,15]. In this paper, the solubility above 303.15K has been measured. As can be seen in Figure 2, the solubility of erythromycin in the medley acetone-water solution increased with increasing acetone concentration and increasing temperature, respectively. In the same range of acetone content, the slope of the solubility curve increased with increasing temper‐

ature, which meant the rate of increase of erythromycin solubility increased.

**2. Purification of erythromycin by antisolvent crystallization**

indicators including antisolvent quality and crystallization temperature.

caused the discrepancy in product quality for different batch.

be precipitated by adding water into erythromycin acetone solution.

**2.1. Solubility and metastable limit of erythromycin**

ythromycin product.

*2.1.1. Solubility*

temperature.

in which water was served as antisolvent.

In contrast, the technological process with membrane separation and resin absorption [9] is drawing more attention compared with the traditional solvent extraction in the above [10]. This process usually goes as follows: firstly, microfiltration is used to remove solid impurities from the fermentation broth, and the filtrate is purified by macroporous ab‐ sorption resin, and then the adsorbed erythromycin is eluted with butyl acetate. Finally, either evaporative crystallization or reactive crystallization can be used to obtain the fi‐ nal product [11].

**Figure 1.** Schematic diagram for the purification erythromycin

The flowsheet of these two technological processes is demonstrated in Figure 1. It can be seen that crystallization is the final step to prepare erythromycin no matter which one is adopted. Different crystallization method has been used for different pretreatment.

Crystallization is a complex process involving mass transfer, heat transfer and surface reactions, which includes the formation of a supersaturated solution, nucleation and crystal growth. The operating parameters of crystallization process, such as temperature, agitation intensity and seed crystals, can affect the generation rate and scale of the su‐ persaturation. The structure of the crystallizers and stirrer will influence the fluid me‐ chanics properties of the crystallization system. Thus it can be seen that all these factors profoundly influence crystal nucleation and growth [12]. Over a long period of time, the crystallization processes have been carried out on according to experiences rather than theoretical researches due to the little study on thermodynamics and kinetics. Not sur‐ prisingly, it's hardly to obtain erythromycin with high purity, complete crystal form, narrow distribution of crystal size, less crystal bonding, which are very important for the stability and bioavailability of the drug.

In this paper, two crystallization processes of erythromycin in different systems, which include the antisolvent crystallization for mixed solvents of acetone and water and the azeotropic evaporative crystallization for butyl acetate-water system, are described in de‐ tails. The thermodynamics and kinetics of the crystallization of erythromycin, which help to thoroughly understand the effect of a variety of factors on the nucleation, crystal growth and crystal habit, are summarized systematically. On the basis of these funda‐ mental studies, effective control techniques are proposed to improve the quality of er‐ ythromycin product.

### **2. Purification of erythromycin by antisolvent crystallization**

In the solvent extraction process for purification of erythromycin, erythromycin alkaline was converted from erythromycin thiocyanate by adding ammonia or NaOH solution and dis‐ solving in acetone. Then erythromycin product was prepared by antisolvent crystallization, in which water was served as antisolvent.

The traditional crystallization process, which was too simple, only involved modulating two indicators including antisolvent quality and crystallization temperature.

Water was poured into erythromycin acetone solution at room temperature, and then the product was obtained by filtration after standing for a period of time. Such operation made obvious differences of supersaturation, nucleation rate and crystal growth rate and then caused the discrepancy in product quality for different batch.

### **2.1. Solubility and metastable limit of erythromycin**

As we know, the phase equilibrium between solid and its solution is fundamental to choose crystallization method and also determines the maximum yield of solution crystallization [12]. Erythromycin is soluble in acetone, but insoluble in water [13]. Thus, erythromycin can be precipitated by adding water into erythromycin acetone solution.

### *2.1.1. Solubility*

rified by antisolvent crystallization [8]. That is to say, both reactive crystallization and anti‐

In contrast, the technological process with membrane separation and resin absorption [9] is drawing more attention compared with the traditional solvent extraction in the above [10]. This process usually goes as follows: firstly, microfiltration is used to remove solid impurities from the fermentation broth, and the filtrate is purified by macroporous ab‐ sorption resin, and then the adsorbed erythromycin is eluted with butyl acetate. Finally, either evaporative crystallization or reactive crystallization can be used to obtain the fi‐

The flowsheet of these two technological processes is demonstrated in Figure 1. It can be seen that crystallization is the final step to prepare erythromycin no matter which one is

Crystallization is a complex process involving mass transfer, heat transfer and surface reactions, which includes the formation of a supersaturated solution, nucleation and crystal growth. The operating parameters of crystallization process, such as temperature, agitation intensity and seed crystals, can affect the generation rate and scale of the su‐ persaturation. The structure of the crystallizers and stirrer will influence the fluid me‐ chanics properties of the crystallization system. Thus it can be seen that all these factors profoundly influence crystal nucleation and growth [12]. Over a long period of time, the crystallization processes have been carried out on according to experiences rather than theoretical researches due to the little study on thermodynamics and kinetics. Not sur‐ prisingly, it's hardly to obtain erythromycin with high purity, complete crystal form, narrow distribution of crystal size, less crystal bonding, which are very important for

adopted. Different crystallization method has been used for different pretreatment.

solvent crystallization have to be employed in this separation process.

**Figure 1.** Schematic diagram for the purification erythromycin

the stability and bioavailability of the drug.

nal product [11].

44 Advanced Topics on Crystal Growth

The solubility of erythromycin in acetone increases with the increasing temperature, where‐ as it becomes less soluble with the higher temperature in water. So, the solubility of erythro‐ mycin in acetone-water binary solvent system is influenced by the solvent composition and temperature.

Some data on solubility of erythromycin in acetone-water solution was reported in litera‐ tures [14,15]. In this paper, the solubility above 303.15K has been measured. As can be seen in Figure 2, the solubility of erythromycin in the medley acetone-water solution increased with increasing acetone concentration and increasing temperature, respectively. In the same range of acetone content, the slope of the solubility curve increased with increasing temper‐ ature, which meant the rate of increase of erythromycin solubility increased.

**Figure 2.** Solubility of erythromycin in acetone-water solution at different temperatures; -□-: 293.15K; -■-: 298.25K; - △-: 303.15K; -▲-: 308.15K; -○-: 310.15K; -●-: 312.15K; -◇-: 314.15K; -◆-: 323.15K

The impact of acetone on the solubility of erythromycin increased as the mass fraction of acetone increasing. It was not hard to infer that the difference of the solubility at different temperatures tended to decrease with the mass fraction of water increase.

An empirical model was proposed to relate the experimental data of the solubility of eryth‐ romycin and the parameters was obtained by fitting. The empirical equation for the solubili‐ ty of erythromycin in mixed solvents of acetone and water was expressed as below:

$$\text{C}^\* = \frac{1.02 \times e^{0.0491T}}{0.395 - 0.00383T + \chi^{(0.0138T + 1.298)}} \tag{1}$$

**Figure 3.** Comparison of simulated value and experimental data of solubility

unstable zone [16] that results in the worse product.

Metastable zone width is fundamental to choose suitable supersaturation of crystallization. It is also used as a restrictive operating condition to avoid crystallization system going to

Purification of Erythromycin by Antisolvent Crystallization or Azeotropic Evaporative Crystallization

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47

**Figure 4.** Apparatus for antisolvent crystallization of erythromycin; 1. Thermostat Bath; 2. Circulating Pump; 3. Water Storage Tank; 4. Peristaltic Pump; 5. Laser Generator; 6. Double-Wall Crystallizer; 7. Stirrer; 8. Thermometer; 9. Con‐

*2.1.2. Metastable zone*

denser; 10. Laser Power Detector

where *C\** was solubility (g Erythromycin/100g Acetone-Water Solution) and *x* was the mass proportion of water to acetone (*x*=*m*w:*m*a). Equation (1) was practicable in the range of 293.15K≤*T*≤323.15K, 1.0≤*x*≤2.0.

Equation (1) could be used to calculate erythromycin solubility *C*\* Cal under various ex‐ perimental conditions. The calculated solubility *C*\* Cal and the experimental solubility *C*\* Exp were shown in Figure 3. It was indicated that Equation (1) was appropriate to predict the solubility of erythromycin within the range of temperature and acetone concentra‐ tion for the equation.

Purification of Erythromycin by Antisolvent Crystallization or Azeotropic Evaporative Crystallization http://dx.doi.org/10.5772/52934 47

**Figure 3.** Comparison of simulated value and experimental data of solubility

#### *2.1.2. Metastable zone*

**Figure 2.** Solubility of erythromycin in acetone-water solution at different temperatures; -□-: 293.15K; -■-: 298.25K; -

The impact of acetone on the solubility of erythromycin increased as the mass fraction of acetone increasing. It was not hard to infer that the difference of the solubility at different

An empirical model was proposed to relate the experimental data of the solubility of eryth‐ romycin and the parameters was obtained by fitting. The empirical equation for the solubili‐

(0.0138 1.298)

was solubility (g Erythromycin/100g Acetone-Water Solution) and *x* was the mass

´ <sup>=</sup> - + (1)

Cal under various ex‐

Exp

Cal and the experimental solubility *C*\*

*T T*

*T x* <sup>+</sup>

proportion of water to acetone (*x*=*m*w:*m*a). Equation (1) was practicable in the range of

were shown in Figure 3. It was indicated that Equation (1) was appropriate to predict the solubility of erythromycin within the range of temperature and acetone concentra‐

ty of erythromycin in mixed solvents of acetone and water was expressed as below:

1.02 0.395 0.00383

0.0491 \*

*<sup>e</sup> <sup>C</sup>*

Equation (1) could be used to calculate erythromycin solubility *C*\*

perimental conditions. The calculated solubility *C*\*

where *C\**

293.15K≤*T*≤323.15K, 1.0≤*x*≤2.0.

46 Advanced Topics on Crystal Growth

tion for the equation.

△-: 303.15K; -▲-: 308.15K; -○-: 310.15K; -●-: 312.15K; -◇-: 314.15K; -◆-: 323.15K

temperatures tended to decrease with the mass fraction of water increase.

Metastable zone width is fundamental to choose suitable supersaturation of crystallization. It is also used as a restrictive operating condition to avoid crystallization system going to unstable zone [16] that results in the worse product.

**Figure 4.** Apparatus for antisolvent crystallization of erythromycin; 1. Thermostat Bath; 2. Circulating Pump; 3. Water Storage Tank; 4. Peristaltic Pump; 5. Laser Generator; 6. Double-Wall Crystallizer; 7. Stirrer; 8. Thermometer; 9. Con‐ denser; 10. Laser Power Detector

Supersolubility of erythromycin was measured by the method of laser scattering [15]. As shown in Figure 4, the experimental device consisted of crystallizer, mixing system, feeding system, temperature control system and detection system. Wherein, the crystallizer was a dou‐ ble-wall kettle with internal diameter 75mm and height 130mm. Stirrer with four inclined pro‐ pellers was driven by variable speed motor, the propeller diameter was 12mm, and the stirring shaft diameter was 5mm. The peristaltic pump continuously pumped antisolvent water at a fixed temperature into crystallizer. The detection system consisted of He-Ne laser generator and laser power detector. He-Ne laser generator outputted 632.8nm red line, scattering and dif‐ fraction occurred when monochrome laser beam encountered with body of similar length of wavelength, the laser intensity received by detector was drastically reduced, thus the nuclea‐ tion could be detected.

The relationship between metastable zone width ΔC of erythromycin and solvent composi‐ tion at 323.15K was shown in Figure 5. It could be seen form the figure that metastable zone width decreased gradually with the increase of the quality of water in solution. In mw:ma range of 1.0 to1.8, the supersolubility presented apparent downward trend. After mw:ma reached 1.8, the change of the metastable zone width weakened, but the metastable zone width of this region was already narrow and was not suitable for crystallization operation.

The equation was obtained by correlating the metastable zone width and solvent composi‐ tion, which was listed as follows:

$$
\Delta \mathbf{C} = \mathbf{3}.09 \times \mathbf{x}^{-2.49} \tag{2}
$$

**Figure 6.** The effect of stirring intensity on supersaturation of erythromycin at different temperatures; –▲–: 308.15K; –

Purification of Erythromycin by Antisolvent Crystallization or Azeotropic Evaporative Crystallization

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49

It could be seen form Figure 5, the calculated value was in good agreement with experimen‐

As shown in Figure 6, the metastable zone width of erythromycin decreased with the in‐ crease of temperature. The metastable zone width was similar at 308.15K and 313.15K, while it was quite different at 313.15K and 323.15K, which indicated metastable zone width was temperature sensitive in the range of 313.15K to 323.15K. The variation of metastable zone width with agitation power presented a consistency at different temperatures. The metasa‐

In this paper, the intermittent dynamic method [17] was used to study the kinetics of eryth‐ romycin antisolvent crystallization under different conditions. With the empirical models deduced from the Larson-Randolph population balance equation [18,19], the model parame‐ ters were obtained from the experimental data through the matrix convertion and the meth‐ od of linear squares regression. Thus, the equations of nucleation and crystal growth of antisolvent crystallization of erythromycin were established to help find the suitable opera‐

The experimental apparatus were shown in Figure 4. Firstly, at the start of recording the time, antisolvent water at set temperature was poured into the erythromycin-acetone solu‐ tion in the crystallizer. Once the nucleation appeared in the solution, water was stopped im‐

ble zone width was wider under the same agitation power at lower temperature.

tal data. Similar results could be obtained at other temperatures.

**2.2. Antisolvent crystallization kinetics of erythromycin**

●–: 313.15K; –■–: 323.15K

tion parameters.

where *x* was the mass ratio of water to acetone, *x=m*w:*m*a.

**Figure 5.** Effect of solvent composition on metastable zone width of erythromycin at 323.15K

**Figure 6.** The effect of stirring intensity on supersaturation of erythromycin at different temperatures; –▲–: 308.15K; – ●–: 313.15K; –■–: 323.15K

It could be seen form Figure 5, the calculated value was in good agreement with experimen‐ tal data. Similar results could be obtained at other temperatures.

As shown in Figure 6, the metastable zone width of erythromycin decreased with the in‐ crease of temperature. The metastable zone width was similar at 308.15K and 313.15K, while it was quite different at 313.15K and 323.15K, which indicated metastable zone width was temperature sensitive in the range of 313.15K to 323.15K. The variation of metastable zone width with agitation power presented a consistency at different temperatures. The metasa‐ ble zone width was wider under the same agitation power at lower temperature.

### **2.2. Antisolvent crystallization kinetics of erythromycin**

Supersolubility of erythromycin was measured by the method of laser scattering [15]. As shown in Figure 4, the experimental device consisted of crystallizer, mixing system, feeding system, temperature control system and detection system. Wherein, the crystallizer was a dou‐ ble-wall kettle with internal diameter 75mm and height 130mm. Stirrer with four inclined pro‐ pellers was driven by variable speed motor, the propeller diameter was 12mm, and the stirring shaft diameter was 5mm. The peristaltic pump continuously pumped antisolvent water at a fixed temperature into crystallizer. The detection system consisted of He-Ne laser generator and laser power detector. He-Ne laser generator outputted 632.8nm red line, scattering and dif‐ fraction occurred when monochrome laser beam encountered with body of similar length of wavelength, the laser intensity received by detector was drastically reduced, thus the nuclea‐

The relationship between metastable zone width ΔC of erythromycin and solvent composi‐ tion at 323.15K was shown in Figure 5. It could be seen form the figure that metastable zone width decreased gradually with the increase of the quality of water in solution. In mw:ma range of 1.0 to1.8, the supersolubility presented apparent downward trend. After mw:ma reached 1.8, the change of the metastable zone width weakened, but the metastable zone width of this region was already narrow and was not suitable for crystallization operation.

The equation was obtained by correlating the metastable zone width and solvent composi‐

2.49 *C x* 3.09 - D= ´ (2)

tion could be detected.

48 Advanced Topics on Crystal Growth

tion, which was listed as follows:

where *x* was the mass ratio of water to acetone, *x=m*w:*m*a.

**Figure 5.** Effect of solvent composition on metastable zone width of erythromycin at 323.15K

In this paper, the intermittent dynamic method [17] was used to study the kinetics of eryth‐ romycin antisolvent crystallization under different conditions. With the empirical models deduced from the Larson-Randolph population balance equation [18,19], the model parame‐ ters were obtained from the experimental data through the matrix convertion and the meth‐ od of linear squares regression. Thus, the equations of nucleation and crystal growth of antisolvent crystallization of erythromycin were established to help find the suitable opera‐ tion parameters.

The experimental apparatus were shown in Figure 4. Firstly, at the start of recording the time, antisolvent water at set temperature was poured into the erythromycin-acetone solu‐ tion in the crystallizer. Once the nucleation appeared in the solution, water was stopped im‐ porting and the time was recorded. Then the agitation rate and temperature were maintained constant. It was sampled at different interval of time. The indexes of each sam‐ ple, such as magma density, degree of supersaturation and crystal size distribution (CSD) of production, were measured respectively.

*2.2.2. Kinetics model*

The nucleation equation

The crystal growth equation

secondary nucleation.

in this paper.

CSD.

where *M*T was magma density (kg/m3

*2.2.3. Online study of crystallization process*

decrease as temperature increased [20].

On the basis of the above, the size-independent model was adopted to describe the crystal growth rate of erythromycin. According to the study on the effects of temperature, agitation and dosing rate of antisolvent on nucleation rate and crystal growth rate, the corresponding

Purification of Erythromycin by Antisolvent Crystallization or Azeotropic Evaporative Crystallization

<sup>14</sup> <sup>2343</sup> 0.378 0 317 3 303 3.23 10 exp( ) *. .*

<sup>5</sup> <sup>4539</sup> 0.102 3 053 1.18 10 exp( ) *- . <sup>G</sup> <sup>V</sup> <sup>C</sup> PΔ*

In the antisolvent crystallization of erythromycin, slurry density had less effect on the nucle‐ ation rate than supersaturation did. The influence of stirring intensity and supersaturation on nucleation rate was greater than those on crystal growth rate. The supersaturation series 3.303 in the nucleation equation was much smaller than the primary nucleation kinetics ser‐ ies [12]. So the mechanism of nucleation of antisolvent crystallization of erythromycin was

In order to further reveal the intrinsic principles of the antisolvent crystallization process of erythromycin, the Focused Beam Reflectance Measurement (FBRM) technique was adopted to monitor in situ the variation of crystal quantity and crystal size distribution

The total number and the chord length distribution (CLD) of crystal particles were meas‐ ured by using the equipment and method shown in literature [20]. A mathematical pro‐ cedure based on Monte Carlo simulation was established to transform the CLD into

The change of the number of crystals and CSD of erythromycin antisolvent crystallization were studied under different temperature and feeding rate of antisolvent. The results indi‐ cated that the faster water was fed, the earlier new crystals came into being, the faster the crystal grew at the initial stage. The total number of crystals at the stable stage tended to

*<sup>T</sup>* = ´ (3)

*RT* = ´ (4)

), *P*V was unit volume of stirring power (W/m3

).

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51

equations for nucleation rate and crystal growth rate were shown as follows:

*V T BC P MΔ*

#### *2.2.1. Crystal size correlation of crystal growth*

The crystal nucleation and growth kinetics were solved according to the size-independent model [16], using a set of the experimental data of magma density and CSD at 323.15K. The calculated value was in good agreement with the experimental data, as shown in Figure 7. In the crystal size (*L*<sup>i</sup> ) range, erythromycin crystal population density logarithm (l*nn*<sup>i</sup> ) was basically a straight line. At the same time, the results of matrix convertion also showed that the use of size-dependent model to describe the crystal growth was of large error. Therefore, erythromycin crystal growth was size-independent.

**Figure 7.** Typical population density distribution of erythromycin

### *2.2.2. Kinetics model*

porting and the time was recorded. Then the agitation rate and temperature were maintained constant. It was sampled at different interval of time. The indexes of each sam‐ ple, such as magma density, degree of supersaturation and crystal size distribution (CSD) of

The crystal nucleation and growth kinetics were solved according to the size-independent model [16], using a set of the experimental data of magma density and CSD at 323.15K. The calculated value was in good agreement with the experimental data, as shown in Figure 7.

basically a straight line. At the same time, the results of matrix convertion also showed that the use of size-dependent model to describe the crystal growth was of large error. Therefore,

) range, erythromycin crystal population density logarithm (l*nn*<sup>i</sup>

) was

production, were measured respectively.

*2.2.1. Crystal size correlation of crystal growth*

erythromycin crystal growth was size-independent.

**Figure 7.** Typical population density distribution of erythromycin

In the crystal size (*L*<sup>i</sup>

50 Advanced Topics on Crystal Growth

On the basis of the above, the size-independent model was adopted to describe the crystal growth rate of erythromycin. According to the study on the effects of temperature, agitation and dosing rate of antisolvent on nucleation rate and crystal growth rate, the corresponding equations for nucleation rate and crystal growth rate were shown as follows:

The nucleation equation

$$\mathbf{B} \in \mathbf{3.23} \times 10^{14} \exp(\frac{\mathbf{2343}}{T}) P\_V^{0.378} M\_T^{0.317} \tag{3}$$

The crystal growth equation

$$\mathbf{G} \mathbf{G} \in 1.18 \times 10^{-5} \exp(\frac{4539}{RT}) P\_V^{0.102} \tag{4}$$

where *M*T was magma density (kg/m3 ), *P*V was unit volume of stirring power (W/m3 ).

In the antisolvent crystallization of erythromycin, slurry density had less effect on the nucle‐ ation rate than supersaturation did. The influence of stirring intensity and supersaturation on nucleation rate was greater than those on crystal growth rate. The supersaturation series 3.303 in the nucleation equation was much smaller than the primary nucleation kinetics ser‐ ies [12]. So the mechanism of nucleation of antisolvent crystallization of erythromycin was secondary nucleation.

### *2.2.3. Online study of crystallization process*

In order to further reveal the intrinsic principles of the antisolvent crystallization process of erythromycin, the Focused Beam Reflectance Measurement (FBRM) technique was adopted to monitor in situ the variation of crystal quantity and crystal size distribution in this paper.

The total number and the chord length distribution (CLD) of crystal particles were meas‐ ured by using the equipment and method shown in literature [20]. A mathematical pro‐ cedure based on Monte Carlo simulation was established to transform the CLD into CSD.

The change of the number of crystals and CSD of erythromycin antisolvent crystallization were studied under different temperature and feeding rate of antisolvent. The results indi‐ cated that the faster water was fed, the earlier new crystals came into being, the faster the crystal grew at the initial stage. The total number of crystals at the stable stage tended to decrease as temperature increased [20].

The proportion of particles of different size was the grain size frequency distribution. Figure 8 and Figure 9 showed the size frequency distribution curve after nucleation at 308.15K and 314.15K respectively. As could be seen from those, the curves were similar at different tem‐ peratures, which were sharp and steep. Particle size which was less than 20μm accounted

Purification of Erythromycin by Antisolvent Crystallization or Azeotropic Evaporative Crystallization

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53

It could be found from Figure 8 and Figure 9 that the number of both small size crystal and large size crystal hardly change with time. It meant that particles with small size were con‐ stantly dissolving, while saturated solute of erythromycin was precipitated to form new crystal, or the existing crystal grew larger in volume. The dissolution and precipitation of

In order to properly characterize the crystal growth, volume mean diameter *D*V (also known as *D*43) which was the equivalent diameter of the particles with same volume (or mass), was used to investigate the changes of crystal size with time at different temperatures. As shown from Figure 10, erythromycin *D*V monotonically decreased with the increasing temperature

**Figure 10.** The effect of temperature on *D*<sup>v</sup> of erythromycin crystal; -■-: 0min; -□-: 30min; -●-: 60min; -○-: 90min; -▲-:

for the vast majority and the peak of the curves was close to 20%.

erythromycin reached equilibrium.

at the same crystallization time.

120min; -△-: 150min; -◆-: 180min

**Figure 8.** CSD after peak value of overall crystal number count at 308.15K; –■–: 0min; –●–: 30min; –▲–: 90min; –◆–: 180min

**Figure 9.** CSD after peak value of overall crystal number count at 314.15K; –■–: 0min; –●–: 30min; –▲–: 90min; –◆–: 180min

The proportion of particles of different size was the grain size frequency distribution. Figure 8 and Figure 9 showed the size frequency distribution curve after nucleation at 308.15K and 314.15K respectively. As could be seen from those, the curves were similar at different tem‐ peratures, which were sharp and steep. Particle size which was less than 20μm accounted for the vast majority and the peak of the curves was close to 20%.

It could be found from Figure 8 and Figure 9 that the number of both small size crystal and large size crystal hardly change with time. It meant that particles with small size were con‐ stantly dissolving, while saturated solute of erythromycin was precipitated to form new crystal, or the existing crystal grew larger in volume. The dissolution and precipitation of erythromycin reached equilibrium.

In order to properly characterize the crystal growth, volume mean diameter *D*V (also known as *D*43) which was the equivalent diameter of the particles with same volume (or mass), was used to investigate the changes of crystal size with time at different temperatures. As shown from Figure 10, erythromycin *D*V monotonically decreased with the increasing temperature at the same crystallization time.

**Figure 8.** CSD after peak value of overall crystal number count at 308.15K; –■–: 0min; –●–: 30min; –▲–: 90min; –◆–:

**Figure 9.** CSD after peak value of overall crystal number count at 314.15K; –■–: 0min; –●–: 30min; –▲–: 90min; –◆–:

180min

52 Advanced Topics on Crystal Growth

180min

**Figure 10.** The effect of temperature on *D*<sup>v</sup> of erythromycin crystal; -■-: 0min; -□-: 30min; -●-: 60min; -○-: 90min; -▲-: 120min; -△-: 150min; -◆-: 180min

### **2.3. The antisolvent crystallization technique of erythromycin**

The thermodynamics and kinetics of the antisolvent crystallization of erythromycin were summarized systematically to understand thoroughly the effect of a variety of factors on the nucleation, crystal growth and crystal habit. On the basis of these fundamental studies, ap‐ propriate technological parameters were explored to develop the efficient industrialized crystallization process of erythromycin.

### *2.3.1. Technological parameters*

Crystal quality, such as crystal purity, crystal habit, crystal size, and CSD, was related close‐ ly to the crystallization conditions. Accordingly, the effect of the dosing rate of antisolvent, crystallization time, stirring intensity and crystallization temperature on CSD of erythromy‐ cin was studied in details in this paper.

**Dosing rate of antisolvent** For antisolvent crystallization of erythromycin, the dosing rate of antisolvent determined the generation rate of supersaturation, and also affected the rate of nucleation and crystal growth.

The definition of dosing rate of antisolvent was the importing water volume of per unit time and per unit volume of erythromycin-acetone solution.

$$
\sigma\_d = \frac{V\_w}{V} \tag{5}
$$

**Figure 11.** CSD based on cumulative volume of erythromycin at different water-pumping velocities; -▲-: 0.0138min-1; -

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**Figure 12.** CSD of erythromycin based on cumulative volume at different crystallization time; -▲-: 40min; -●-: 50min; -

●-: 0.188 min-1; -■-: 0.024 min-1; -◆-: 0.0389 min-1

■-: 70min: -◆-: 100min

where *v*d was dosing rate of antisolvent (min-1), *V*w was the volume rate of importing water (mL/min), *V* was the erythromycin-acetone volume (mL).

Figure 11 showed the relationship between the dosing rate of antisolvent *v*d and erythromy‐ cin CSD, where *d*p was the crystal diameter and *R*v was the cumulative volume fraction. It could be seen from the figure that the proportion of crystals with large size increased with the increasing dosing rate of water, but the CSD tended to disperse. While the CSD of crys‐ tals obtained in lower water dosing rate was more concentrated.

Therefore, in process of the crystallization, an appropriate increase in generation rate of su‐ persaturation could speed up the crystallization rate and improve the capability of the crys‐ tallizer. However, the rapid generation of crystals will increase the chance of crystal breakage and secondary nucleation and make the CSD disperse.

**Crystallization time** The cumulative volume distribution at different crystallization time was shown in Figure 12, where *d*p was the crystal diameter and *R*<sup>v</sup> was the cumulative vol‐ ume fraction. As could be seen from the figure, the increase of the crystallization time was conducive to crystal growth, while the CSD did not tend to concentrate. The crystal growth needed some time, however, long time crystallization couldn't promise CSD being more consistent.

Purification of Erythromycin by Antisolvent Crystallization or Azeotropic Evaporative Crystallization http://dx.doi.org/10.5772/52934 55

**2.3. The antisolvent crystallization technique of erythromycin**

crystallization process of erythromycin.

cin was studied in details in this paper.

and per unit volume of erythromycin-acetone solution.

(mL/min), *V* was the erythromycin-acetone volume (mL).

tals obtained in lower water dosing rate was more concentrated.

breakage and secondary nucleation and make the CSD disperse.

*2.3.1. Technological parameters*

54 Advanced Topics on Crystal Growth

nucleation and crystal growth.

consistent.

The thermodynamics and kinetics of the antisolvent crystallization of erythromycin were summarized systematically to understand thoroughly the effect of a variety of factors on the nucleation, crystal growth and crystal habit. On the basis of these fundamental studies, ap‐ propriate technological parameters were explored to develop the efficient industrialized

Crystal quality, such as crystal purity, crystal habit, crystal size, and CSD, was related close‐ ly to the crystallization conditions. Accordingly, the effect of the dosing rate of antisolvent, crystallization time, stirring intensity and crystallization temperature on CSD of erythromy‐

**Dosing rate of antisolvent** For antisolvent crystallization of erythromycin, the dosing rate of antisolvent determined the generation rate of supersaturation, and also affected the rate of

The definition of dosing rate of antisolvent was the importing water volume of per unit time

*w d*

where *v*d was dosing rate of antisolvent (min-1), *V*w was the volume rate of importing water

Figure 11 showed the relationship between the dosing rate of antisolvent *v*d and erythromy‐ cin CSD, where *d*p was the crystal diameter and *R*v was the cumulative volume fraction. It could be seen from the figure that the proportion of crystals with large size increased with the increasing dosing rate of water, but the CSD tended to disperse. While the CSD of crys‐

Therefore, in process of the crystallization, an appropriate increase in generation rate of su‐ persaturation could speed up the crystallization rate and improve the capability of the crys‐ tallizer. However, the rapid generation of crystals will increase the chance of crystal

**Crystallization time** The cumulative volume distribution at different crystallization time was shown in Figure 12, where *d*p was the crystal diameter and *R*<sup>v</sup> was the cumulative vol‐ ume fraction. As could be seen from the figure, the increase of the crystallization time was conducive to crystal growth, while the CSD did not tend to concentrate. The crystal growth needed some time, however, long time crystallization couldn't promise CSD being more

*<sup>V</sup> <sup>v</sup> <sup>V</sup>* <sup>=</sup> (5)

**Figure 11.** CSD based on cumulative volume of erythromycin at different water-pumping velocities; -▲-: 0.0138min-1; - ●-: 0.188 min-1; -■-: 0.024 min-1; -◆-: 0.0389 min-1

**Figure 12.** CSD of erythromycin based on cumulative volume at different crystallization time; -▲-: 40min; -●-: 50min; - ■-: 70min: -◆-: 100min

**Agitation power** The CSD was the result of the interaction of primary nucleation, secondary nucleation, and crystal growth. Meanwhile, agitation power had a significant impact on all the above. Figure 13 showed the particle volume distribution of erythromycin at different stirring intensity, where *x*v was the particle volume distribution.

decreased with the narrowing metastable zone width, so did the rate of crystal growth. Therefore the volume mean diameter of the crystals decreased as the temperature increased.

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**Figure 14.** Volume mean diameter of erythromycin at different temperatures

kaline product was obtained by filtration.

*2.3.2. The novel technique of antisolvent crystallization of erythromycin*

For the traditional antisolvent crystallization, water was poured into erythromycin acetone solution at room temperature. Then after standing for a period of time, the erythromycin al‐

It was not difficult to find the shortages of this crystallization method. Firstly, the dosing rate of antisolvent was too fast. When the antisolvent water was fed rapidly, the supersatu‐ ration formed suddenly and leaded to the outbreak of the nucleation. Nucleation was active and occupied the dominant position of the crystallization process. Meanwhile, the impuri‐ ties easily accompanied with crystals by precipitation in the fast crystallization process. Sec‐ ondly, stirrer and stirring intensity were inappropriate. Poor mixing effect made uneven distribution of supersaturation, so it was hard to obtain erythromycin with complete crystal form and narrow distribution of crystal size [12,21]. Thirdly, crystallization temperature was uncontrolled. Then the differences of solubility between erythromycin and impurities in ace‐

tone-water solution could not be fully explored to improve the separation efficiency.

The operation of the crystallization mentioned above lacked of crystallization process con‐ trol and could not play a good role in purification of erythromycin by crystallization. Then the erythromycin product would be highly influenced by fermentation broth and pre-purifi‐

It could be seen from Figure 13 that the erythromycin product had the widest CSD and the highest proportion of small size crystals when the stirring power was 13.99 W/m3 , and the distribution curve had smearing phenomenon in the range of large particle size. While the crystal had the narrowest CSD and the lowest proportion of small size crystals when the stirring power was 1.749 W/m3 , and the distribution curve had no smearing. The energy im‐ ported by stirring was conducive to nucleation and crystal growth. In the meanwhile, crys‐ tal breakage could easily occur with too strong stirring, while the obvious differences of supersaturation would occur with too weak stirring and then caused variation of rate of nu‐ cleation and crystal growth.

**Figure 13.** CSD based on volume of erythromycin at different agitation power; -■-: 0.02179W/m<sup>3</sup>; -●-: 1.749 W/m3; - ▲-: 13.99 W/m<sup>3</sup>

**Crystallization temperature**Figure 14 showed the variation of volume mean diameter (*D*V) of erythromycin at different crystallization temperature, *D*<sup>V</sup> decreased with the increasing of temperature. The previous thermodynamic study showed that the metastable zone width of erythromycin reduced with the increase of temperature. The intensified thermal motion of molecule caused by the increasing temperature accelerated the frequency of contact and col‐ lision of crystals, and then promoted the formation of tiny crystals, and decreased the super‐ saturation required for nucleation. On the other hand, the driving force of crystallization decreased with the narrowing metastable zone width, so did the rate of crystal growth. Therefore the volume mean diameter of the crystals decreased as the temperature increased.

**Figure 14.** Volume mean diameter of erythromycin at different temperatures

**Agitation power** The CSD was the result of the interaction of primary nucleation, secondary nucleation, and crystal growth. Meanwhile, agitation power had a significant impact on all the above. Figure 13 showed the particle volume distribution of erythromycin at different

It could be seen from Figure 13 that the erythromycin product had the widest CSD and the

distribution curve had smearing phenomenon in the range of large particle size. While the crystal had the narrowest CSD and the lowest proportion of small size crystals when the

ported by stirring was conducive to nucleation and crystal growth. In the meanwhile, crys‐ tal breakage could easily occur with too strong stirring, while the obvious differences of supersaturation would occur with too weak stirring and then caused variation of rate of nu‐

**Figure 13.** CSD based on volume of erythromycin at different agitation power; -■-: 0.02179W/m<sup>3</sup>; -●-: 1.749 W/m3; -

**Crystallization temperature**Figure 14 showed the variation of volume mean diameter (*D*V) of erythromycin at different crystallization temperature, *D*<sup>V</sup> decreased with the increasing of temperature. The previous thermodynamic study showed that the metastable zone width of erythromycin reduced with the increase of temperature. The intensified thermal motion of molecule caused by the increasing temperature accelerated the frequency of contact and col‐ lision of crystals, and then promoted the formation of tiny crystals, and decreased the super‐ saturation required for nucleation. On the other hand, the driving force of crystallization

, and the distribution curve had no smearing. The energy im‐

, and the

highest proportion of small size crystals when the stirring power was 13.99 W/m3

stirring intensity, where *x*v was the particle volume distribution.

stirring power was 1.749 W/m3

56 Advanced Topics on Crystal Growth

cleation and crystal growth.

▲-: 13.99 W/m<sup>3</sup>

### *2.3.2. The novel technique of antisolvent crystallization of erythromycin*

For the traditional antisolvent crystallization, water was poured into erythromycin acetone solution at room temperature. Then after standing for a period of time, the erythromycin al‐ kaline product was obtained by filtration.

It was not difficult to find the shortages of this crystallization method. Firstly, the dosing rate of antisolvent was too fast. When the antisolvent water was fed rapidly, the supersatu‐ ration formed suddenly and leaded to the outbreak of the nucleation. Nucleation was active and occupied the dominant position of the crystallization process. Meanwhile, the impuri‐ ties easily accompanied with crystals by precipitation in the fast crystallization process. Sec‐ ondly, stirrer and stirring intensity were inappropriate. Poor mixing effect made uneven distribution of supersaturation, so it was hard to obtain erythromycin with complete crystal form and narrow distribution of crystal size [12,21]. Thirdly, crystallization temperature was uncontrolled. Then the differences of solubility between erythromycin and impurities in ace‐ tone-water solution could not be fully explored to improve the separation efficiency.

The operation of the crystallization mentioned above lacked of crystallization process con‐ trol and could not play a good role in purification of erythromycin by crystallization. Then the erythromycin product would be highly influenced by fermentation broth and pre-purifi‐ cation. That was to say, the quality of erythromycin was restricted by erythromycin thiocya‐ nate. So it was hard to obtain the erythromycin product with stable and high quality and yield.

There were some other studies [8,22] on the improvement of erythromycin crystallization method by adding seed crystals.

On the basis of thorough research on the antisolvent crystallization process of erythromycin, a novel technique for antisolvent crystallization of erythromycin by dynamic control of tem‐ perature and stirring power was proposed in this paper, which was listed as follows.

**1.** Dosing the antisolvent. The polarity of mixed solvents was changed gradually when the antisolvent was imported into erythromycin acetone solution slowly. In the meantime, the solubility of erythromycin decreased gradually until crystal nucleus formed. The su‐ persaturation could be controlled within the thermodynamic metastable zone by dosing antisolvent continuously, the crystal growth was moderated and in order, and the CSD of erythromycin tended to be narrow.

**Figure 15.** Crystal shape of erythromycin from different antisolvent crystallization processes [23]; (a) traditional proc‐

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**Figure 16.** CSD of erythromycin from two antisolvent crystallization processes [23]; (a) traditional process; (b) novel

In the commercial use of the antisolvent crystallization process, erythromycin with high specific activity was obtained at high yield. Over 90% of the products met the de‐ mands per year, which was much higher than the 53% with the traditional crystalliza‐

ess; (b) novel process

process

tion process.


On the basis of the above, the key operation parameters which affect the quality of crystal, such as temperature, dosing rate of antisolvent and stirring intensity, were determined by measuring the crystal shape, titer and yield [23]. Then the novel technique of erythromycin antisolvent crystallization was established in this paper, which was characteristic of dynam‐ ic control of temperature and stirring intensity [24].

Figure 15 and Figure 16 showed the crystal shape and CSD of industrial erythromycin prod‐ ucts obtained by the traditional method (a) and novel technique (b), respectively. For the crystal shape, product (b) had a more regular and bigger size than product (a) did. For the CSD, product (b) was narrower. For titer, product (b) was 935.6 U/mg, while product (a) was 920 U/mg. Those meant that the quality of erythromycin had been improved by the novel technique of antisolvent crystallization [23].

Purification of Erythromycin by Antisolvent Crystallization or Azeotropic Evaporative Crystallization http://dx.doi.org/10.5772/52934 59

cation. That was to say, the quality of erythromycin was restricted by erythromycin thiocya‐ nate. So it was hard to obtain the erythromycin product with stable and high quality and

There were some other studies [8,22] on the improvement of erythromycin crystallization

On the basis of thorough research on the antisolvent crystallization process of erythromycin, a novel technique for antisolvent crystallization of erythromycin by dynamic control of tem‐

**1.** Dosing the antisolvent. The polarity of mixed solvents was changed gradually when the antisolvent was imported into erythromycin acetone solution slowly. In the meantime, the solubility of erythromycin decreased gradually until crystal nucleus formed. The su‐ persaturation could be controlled within the thermodynamic metastable zone by dosing antisolvent continuously, the crystal growth was moderated and in order, and the CSD

**2.** Appropriate stirring intensity. The suitable stirring power could be conducive to main‐ taining uniform supersaturation and crystallization rate. Meanwhile, stirring could pro‐ mote dynamic balance of crystallization and dissolution, and reduce the crystal

**3.** Increasing nucleation temperature. Substance usually had higher solubility at a higher temperature, so did the impurities. Increasing nucleation temperature could reduce the

**4.** Cooling crystallization and aging with lower stirring intensity. After nucleation at high temperature, the stirring power should be reduced to avoid excessive shear force on the crystal collision and maintain a uniform concentration distribution in the slurry at the same time. Then, the supersaturation produced by cooling maintained crystal growth at a steady rate after dosing all antisolvent. Lastly, aging with lower stirring power at low‐

On the basis of the above, the key operation parameters which affect the quality of crystal, such as temperature, dosing rate of antisolvent and stirring intensity, were determined by measuring the crystal shape, titer and yield [23]. Then the novel technique of erythromycin antisolvent crystallization was established in this paper, which was characteristic of dynam‐

Figure 15 and Figure 16 showed the crystal shape and CSD of industrial erythromycin prod‐ ucts obtained by the traditional method (a) and novel technique (b), respectively. For the crystal shape, product (b) had a more regular and bigger size than product (a) did. For the CSD, product (b) was narrower. For titer, product (b) was 935.6 U/mg, while product (a) was 920 U/mg. Those meant that the quality of erythromycin had been improved by the novel

chance of impurities precipitation and improve the purity of erythromycin.

er terminal temperature could improve the quality and yield of product.

perature and stirring power was proposed in this paper, which was listed as follows.

yield.

method by adding seed crystals.

58 Advanced Topics on Crystal Growth

of erythromycin tended to be narrow.

bonding, and then improve the purity of the crystal.

ic control of temperature and stirring intensity [24].

technique of antisolvent crystallization [23].

**Figure 15.** Crystal shape of erythromycin from different antisolvent crystallization processes [23]; (a) traditional proc‐ ess; (b) novel process

**Figure 16.** CSD of erythromycin from two antisolvent crystallization processes [23]; (a) traditional process; (b) novel process

In the commercial use of the antisolvent crystallization process, erythromycin with high specific activity was obtained at high yield. Over 90% of the products met the de‐ mands per year, which was much higher than the 53% with the traditional crystalliza‐ tion process.

### **3. Purification erythromycin by azeotropic evaporative crystallizaion**

The development of the crystallization technique of erythromycin is limited to some extent by the extraction and purification prior to the crystallization. Taking the production of er‐ ythromycin as an example, the widely used process is frame filtration of fermentation broth - solvent extraction - salting-out crystallization – alkalization - antisolvent crystallization. Due to the limited interception capability for fine particles and macromolecules impurities such as proteins by frame filtration, and the low selectivity of the object over pigment and the small un-ionized organic molecules by solvent extraction, the impurity content is high in the organic phase. Therefore, the object should be further purified by coupling two crystalli‐ zation methods in the subsequent refining process.

**Figure 17.** Principle illustration for azeotropic evaporative crystallization process of erythromycin

butyl acetate and the terminal crystallization temperature.

erythromycin could be avoided by adjusting the pressure.

volume of butyl acetate, which affected CSD of the product.

above 0.084MPa, the crystallization temperature was below 323.15K.

Once the azeotropic evaporative crystallization of erythromycin was established, the optimi‐ zation of parameters was directed by the quality and yield of crystal. The process parame‐ ters related to the crystal shape, crystal size and CSD were shown as follow: firstly, the supersaturation, which was related to the quantity of butyl acetate removed by azeotropic evaporation; secondly, the generation rate of supersaturation, which was dependent on the azeotropic evaporation rate and the cooling rate; thirdly, the crystallization temperature, which was bound up with vacuum of system and the cooling rate; fourthly, the stirring in‐ tensity, and etc,. The yield of erythromycin was determined by the evaporation quantity of

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**System vacuum and operation temperature** The butyl acetate-water azeotrope was re‐ moved from the crystallization system by vacuum evaporation, and the azeotropic tempera‐ ture varied with the pressure. Then the high temperature leading to the damage of

According to the phase equilibrium data reported in the literatures [26,27], the azeotropic temperature and composition under different vacuum was calculated by using Pro II simu‐ lation software and NRTL thermodynamic model. When the system vacuum was controlled

**Supersaturation** The supersaturation of erythromycin increased with the increasing volume of butyl acetate evaporated. The supersaturation varied with temperature and evaporation

Figure 18 showed the relationship between the cumulative volume distribution and supersatu‐ ration at 316.15K, where *d*p was the crystal diameter and *R*v was the cumulative volume frac‐ tion. As can be seen from the figure, the crystal size of erythromycin increased with the increase of supersaturation. However, it was necessary to choose the supersaturation range carefully

**Cooling rate** The cooling crystallization started after evaporating some amount of butyl ace‐ tate. The supersaturation caused by cooling made the crystallization process proceed contin‐ uously. It could improve the quality yield of the product by reducing the terminal

due to the variation of solution viscosity and the difficulties of the crystal dispersion.

**3.1. Technological parameters**

crystallization temperature.

In recent years, a different technological process by membrane separation and resin absorp‐ tion is gradually introduced into industrial application [25,11]. The process consists of sever‐ al steps including membrane separation, resin absorption, elution and crystallization. Firstly, microfiltration is used to remove mycelium, a variety of fine suspension particles and some protein from fermentation broth, then pigment and small un-ionized organics are removed by resin absorption and the elution with butyl acetate. An improvement of the pu‐ rity of erythromycin butyl acetate solution is obtained by using this pretreatment. And it makes crystallization preparation of erythromycin alkaline from butyl acetate elution be‐ come possible.

For the preparation of erythromycin alkaline from erythromycin butyl acetate solution, the product yield is low due to the high solubility of erythromycin. So the urgent task is to in‐ crease the yield. To remove butyl acetate is feasible, while high temperature for solvent evaporation may cause the destruction of erythromycin. Although erythromycin has better thermal stability than some other sorts of antibiotics, there is no precedent on the separation and purification of erythromycin with temperature being above 323.15K in industrial appli‐ cation till now. Thus, azeotropic evaporative crystallization of erythromycin is proposed in this paper. The method takes erythromycin, butyl acetate and water as crystallization sys‐ tem. Then butyl acetate-water azeotrope is removed by vacuum azeotropic evaporation to make erythromycin precipitate and disperse into water. Excessive water is added to the er‐ ythromycin butyl acetate solution for azeotropic evaporation, which can also play a role of washing crystals. The solubility of butyl acetate in water is quite small, so the azeotrope is easy to split into two phases at room temperature. The schematic diagram of azeotropic evaporative crystallization of erythromycin is demonstrated in Figure 17.

The solubility of erythromycin in butyl acetate-water saturated solution (solution A) and in water-butyl acetate saturated solution (solution B) was detected, respectively. The result in‐ dicated that the solubility of erythromycin in solution A was quite low and had little change with temperature. So for the azeotropic evaporative crystallization of erythromycin, the pro‐ portion of water was based on its effect on operation, such as the viscosity of the solution and crystal dispersion, as well as the utilization of equipment and the efficiency of produc‐ tion, rather than on the yield of crystallization.

**Figure 17.** Principle illustration for azeotropic evaporative crystallization process of erythromycin

### **3.1. Technological parameters**

**3. Purification erythromycin by azeotropic evaporative crystallizaion**

zation methods in the subsequent refining process.

come possible.

60 Advanced Topics on Crystal Growth

The development of the crystallization technique of erythromycin is limited to some extent by the extraction and purification prior to the crystallization. Taking the production of er‐ ythromycin as an example, the widely used process is frame filtration of fermentation broth - solvent extraction - salting-out crystallization – alkalization - antisolvent crystallization. Due to the limited interception capability for fine particles and macromolecules impurities such as proteins by frame filtration, and the low selectivity of the object over pigment and the small un-ionized organic molecules by solvent extraction, the impurity content is high in the organic phase. Therefore, the object should be further purified by coupling two crystalli‐

In recent years, a different technological process by membrane separation and resin absorp‐ tion is gradually introduced into industrial application [25,11]. The process consists of sever‐ al steps including membrane separation, resin absorption, elution and crystallization. Firstly, microfiltration is used to remove mycelium, a variety of fine suspension particles and some protein from fermentation broth, then pigment and small un-ionized organics are removed by resin absorption and the elution with butyl acetate. An improvement of the pu‐ rity of erythromycin butyl acetate solution is obtained by using this pretreatment. And it makes crystallization preparation of erythromycin alkaline from butyl acetate elution be‐

For the preparation of erythromycin alkaline from erythromycin butyl acetate solution, the product yield is low due to the high solubility of erythromycin. So the urgent task is to in‐ crease the yield. To remove butyl acetate is feasible, while high temperature for solvent evaporation may cause the destruction of erythromycin. Although erythromycin has better thermal stability than some other sorts of antibiotics, there is no precedent on the separation and purification of erythromycin with temperature being above 323.15K in industrial appli‐ cation till now. Thus, azeotropic evaporative crystallization of erythromycin is proposed in this paper. The method takes erythromycin, butyl acetate and water as crystallization sys‐ tem. Then butyl acetate-water azeotrope is removed by vacuum azeotropic evaporation to make erythromycin precipitate and disperse into water. Excessive water is added to the er‐ ythromycin butyl acetate solution for azeotropic evaporation, which can also play a role of washing crystals. The solubility of butyl acetate in water is quite small, so the azeotrope is easy to split into two phases at room temperature. The schematic diagram of azeotropic

The solubility of erythromycin in butyl acetate-water saturated solution (solution A) and in water-butyl acetate saturated solution (solution B) was detected, respectively. The result in‐ dicated that the solubility of erythromycin in solution A was quite low and had little change with temperature. So for the azeotropic evaporative crystallization of erythromycin, the pro‐ portion of water was based on its effect on operation, such as the viscosity of the solution and crystal dispersion, as well as the utilization of equipment and the efficiency of produc‐

evaporative crystallization of erythromycin is demonstrated in Figure 17.

tion, rather than on the yield of crystallization.

Once the azeotropic evaporative crystallization of erythromycin was established, the optimi‐ zation of parameters was directed by the quality and yield of crystal. The process parame‐ ters related to the crystal shape, crystal size and CSD were shown as follow: firstly, the supersaturation, which was related to the quantity of butyl acetate removed by azeotropic evaporation; secondly, the generation rate of supersaturation, which was dependent on the azeotropic evaporation rate and the cooling rate; thirdly, the crystallization temperature, which was bound up with vacuum of system and the cooling rate; fourthly, the stirring in‐ tensity, and etc,. The yield of erythromycin was determined by the evaporation quantity of butyl acetate and the terminal crystallization temperature.

**System vacuum and operation temperature** The butyl acetate-water azeotrope was re‐ moved from the crystallization system by vacuum evaporation, and the azeotropic tempera‐ ture varied with the pressure. Then the high temperature leading to the damage of erythromycin could be avoided by adjusting the pressure.

According to the phase equilibrium data reported in the literatures [26,27], the azeotropic temperature and composition under different vacuum was calculated by using Pro II simu‐ lation software and NRTL thermodynamic model. When the system vacuum was controlled above 0.084MPa, the crystallization temperature was below 323.15K.

**Supersaturation** The supersaturation of erythromycin increased with the increasing volume of butyl acetate evaporated. The supersaturation varied with temperature and evaporation volume of butyl acetate, which affected CSD of the product.

Figure 18 showed the relationship between the cumulative volume distribution and supersatu‐ ration at 316.15K, where *d*p was the crystal diameter and *R*v was the cumulative volume frac‐ tion. As can be seen from the figure, the crystal size of erythromycin increased with the increase of supersaturation. However, it was necessary to choose the supersaturation range carefully due to the variation of solution viscosity and the difficulties of the crystal dispersion.

**Cooling rate** The cooling crystallization started after evaporating some amount of butyl ace‐ tate. The supersaturation caused by cooling made the crystallization process proceed contin‐ uously. It could improve the quality yield of the product by reducing the terminal crystallization temperature.

**3.2. The technique of erythromycin azeotropic evaporative crystallization**

lization stages.

**4. Conclusion**

On the basis of the studies above, the crystallization technique combining the azeotropic evaporation with cooling crystallization was established to prepare the erythromycin from erythromycin butyl acetate solution directly. This process included mainly the following steps: firstly, introduction of entrainer. Adding entrainer (water) to erythromycin butyl ace‐ tate solution could form azeotropic crystallization system and decrease the evaporation tem‐ perature; secondly, vacuum evaporation. Adjusting the vacuum could promise the azeotropic evaporation temperature of butyl acetate and water was low enough to avoid the destruction of erythromycin; thirdly, appropriate evaporation quantity of butyl acetate. The supersaturation could be maintained within the thermodynamic metastable zone by adjust‐ ing the evaporation quantity of butyl acetate; fourthly, modulating cooling rate. The rate of crystallization could be regulated by adjusting cooling rate, so the supersaturation produced by cooling also could be maintained within the thermodynamic metastable zone to promise crystal growth; finally, the agitation power should be adjusted with the variation of crystal‐

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There was an application for erythromycin purification by azeotropic evaporative crystalli‐ zation. The technological conditions were listed as follows, the raw material of erythromycin was provided by a pharmaceutical company, the volume of water in the crystallization sys‐ tem was three times the volume of butyl acetate, the supersaturation was about 45g erythro‐ mycin/100g butyl acetate, cooling rate was 273.22K/min, the terminal crystallization temperature was 303.15K. With the conditions above and the technology in this paper, the purity of erythromycin A in the product was 95.87% and the yield in mass was 75.7%, which was higher than the yield 64.6% of erythromycin product by traditional antisolvent crystalli‐

In this paper, the thermodynamics, crystallization kinetics and operating conditions were studied systematically for the antisolvent crystallization of erythromycin. A brand-new tech‐ nique with dynamic control of temperature and agitation intensity was henceforth present‐ ed. This process included nucleation at high temperature (313.15K~323.15K), regulation of temperature and agitation power according to the different stage of nucleation, crystal growth and crystal aging. It made the operation parameters of crystallization process more reasonable, and the erythromycin with high specific activity had high yield. The commercial

Meanwhile, a novel purification method of erythromycin by azeotropic evaporative crystal‐ lization was also put forward. With this method, erythromycin could be produced from er‐ ythromycin butyl acetate solution directly. By the introduction of water, the evaporation temperature of azeotrope of butyl acetate and water was decreased and the supersaturation was induced. Then, crystallization nucleation and crystal growth were controlled by the reg‐ ulation of cooling rate. With the azeotropic evaporative crystallization, qualified erythromy‐

zation process using the same batch of raw materials.

use of the antisolvent crystallization technique had been successful.

**Figure 18.** CSD based on cumulative volume of erythromycin at different supersaturation -■-: 39.91 g/100g; -▲-: 40.76 g/100g; -●-: 41.26 g/100g; -◆-: 41.60 g/100g

Figure 19 showed the relationship between the cumulative volume distribution of erythro‐ mycin and cooling rate, where *d*p was the crystal diameter and *R*<sup>v</sup> was the cumulative vol‐ ume fraction of the crystal. It could be seen form the figure that speeding up the cooling rate was not conducive to the growth of crystal and made the crystal size decrease.

**Figure 19.** CSD based on cumulative volume of erythromycin at different cooling rate -■-: 273.17K/min; -●-: 273.20K/min; -▲-: 273.28K/min

### **3.2. The technique of erythromycin azeotropic evaporative crystallization**

On the basis of the studies above, the crystallization technique combining the azeotropic evaporation with cooling crystallization was established to prepare the erythromycin from erythromycin butyl acetate solution directly. This process included mainly the following steps: firstly, introduction of entrainer. Adding entrainer (water) to erythromycin butyl ace‐ tate solution could form azeotropic crystallization system and decrease the evaporation tem‐ perature; secondly, vacuum evaporation. Adjusting the vacuum could promise the azeotropic evaporation temperature of butyl acetate and water was low enough to avoid the destruction of erythromycin; thirdly, appropriate evaporation quantity of butyl acetate. The supersaturation could be maintained within the thermodynamic metastable zone by adjust‐ ing the evaporation quantity of butyl acetate; fourthly, modulating cooling rate. The rate of crystallization could be regulated by adjusting cooling rate, so the supersaturation produced by cooling also could be maintained within the thermodynamic metastable zone to promise crystal growth; finally, the agitation power should be adjusted with the variation of crystal‐ lization stages.

There was an application for erythromycin purification by azeotropic evaporative crystalli‐ zation. The technological conditions were listed as follows, the raw material of erythromycin was provided by a pharmaceutical company, the volume of water in the crystallization sys‐ tem was three times the volume of butyl acetate, the supersaturation was about 45g erythro‐ mycin/100g butyl acetate, cooling rate was 273.22K/min, the terminal crystallization temperature was 303.15K. With the conditions above and the technology in this paper, the purity of erythromycin A in the product was 95.87% and the yield in mass was 75.7%, which was higher than the yield 64.6% of erythromycin product by traditional antisolvent crystalli‐ zation process using the same batch of raw materials.

### **4. Conclusion**

**Figure 18.** CSD based on cumulative volume of erythromycin at different supersaturation -■-: 39.91 g/100g; -▲-:

Figure 19 showed the relationship between the cumulative volume distribution of erythro‐ mycin and cooling rate, where *d*p was the crystal diameter and *R*<sup>v</sup> was the cumulative vol‐ ume fraction of the crystal. It could be seen form the figure that speeding up the cooling rate

**Figure 19.** CSD based on cumulative volume of erythromycin at different cooling rate -■-: 273.17K/min; -●-:

was not conducive to the growth of crystal and made the crystal size decrease.

40.76 g/100g; -●-: 41.26 g/100g; -◆-: 41.60 g/100g

62 Advanced Topics on Crystal Growth

273.20K/min; -▲-: 273.28K/min

In this paper, the thermodynamics, crystallization kinetics and operating conditions were studied systematically for the antisolvent crystallization of erythromycin. A brand-new tech‐ nique with dynamic control of temperature and agitation intensity was henceforth present‐ ed. This process included nucleation at high temperature (313.15K~323.15K), regulation of temperature and agitation power according to the different stage of nucleation, crystal growth and crystal aging. It made the operation parameters of crystallization process more reasonable, and the erythromycin with high specific activity had high yield. The commercial use of the antisolvent crystallization technique had been successful.

Meanwhile, a novel purification method of erythromycin by azeotropic evaporative crystal‐ lization was also put forward. With this method, erythromycin could be produced from er‐ ythromycin butyl acetate solution directly. By the introduction of water, the evaporation temperature of azeotrope of butyl acetate and water was decreased and the supersaturation was induced. Then, crystallization nucleation and crystal growth were controlled by the reg‐ ulation of cooling rate. With the azeotropic evaporative crystallization, qualified erythromy‐ cin product could be obtained without recrystallization, which leaded to less solvent consumption, simplified purification process and crystal product with narrow size distribu‐ tion and perfect crystal shape.

[7] Fujita S., Takatsu A., Shibuya K. Process for purifying erythromycin 1971; US Patent

Purification of Erythromycin by Antisolvent Crystallization or Azeotropic Evaporative Crystallization

http://dx.doi.org/10.5772/52934

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[8] Zhao, Q,; Gao, D.W.; Yu, S.J.; Li, G.J. Study on improvement of erythromycin lactate

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[11] Zhu, J.W.; Sun, Y.; Chen, K.; Zhu, S.; Xu, J.; et al. A method of purification of erythro‐

[13] Fukumori, Y.; Fukuda, T.; Yamamoto, Y.; Shigitani, Y.; Hanuy, Y.; et al. Physical characterization of erythromycin dihydrate, anhydrate and amorphous solid and their dissolution properties. Chemical and Pharmaceutical Bulletin 1983; 31(11)

[14] Hang, A.G; Wu, Y.Y. Solubility of erythromycin in acetone-water solution. Chinese

[15] Chen, K.; Zhu, J.W.; Ji L.J.; Wu, B. The Metastable Characteristic of Erythromycin Slovent-out Crystallization Process. Journal of Chemical Engineering of Chinese Uni‐

[16] Allan S. Myerson. Handbook of Industrial Crystallization. Oxford: Butterworth;

[17] Bennema, P. Crystal growth from solution-Theory and experiment. Journal of Crystal

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[19] Randolph, A.D.; Larson, M.A. Transient and Steady state size distributions in Contin‐

[20] Chen, K.; Cao, B.; Zhu, J.W.; Wu, B.; Ji, L.J. In-situ measurement of erythromycin crystal size distribution by focused beam reflective measurement technology. Asia-

[21] Kim, K.J.; Ryu, S.K. Nucleation of thiourea adduct crystals with cyclohexane-methyl‐ cyclopentane system. Chemical Engineering Communications 1997; 159(1) 51-66. [22] Gao, D.W.; Zhao, Q.; Li, G.J. The mechanism and enhancing methods of solventingout crystallization. Journal of South China University of Technology 1998; 26(11)

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### **Acknowledgments**

The authors are grateful to the Jiawen Zhu and Bin Wu for helpful discussions. Thanks are extended to Qing Zhang, Bin Cao, Wenjian Zheng and Peixue Mao for technical assistance.

### **Author details**

Kui Chen\* , Li-Jun Ji and Yan-Yang Wu

\*Address all correspondence to: chenkui@ecust.edu.cn

Chemical Engineering Research Center, East China University of Science and Technology, Shanghai, China

### **References**


cin product could be obtained without recrystallization, which leaded to less solvent consumption, simplified purification process and crystal product with narrow size distribu‐

The authors are grateful to the Jiawen Zhu and Bin Wu for helpful discussions. Thanks are extended to Qing Zhang, Bin Cao, Wenjian Zheng and Peixue Mao for technical assistance.

Chemical Engineering Research Center, East China University of Science and Technology,

[1] Han, J.Y.; Yan, Y.C.; Chang, H.Y.; Wang, H. Progress in drug crystallization technolo‐

[2] McGuire, J.M.; Bunch, R.L.; Andersen, R.C. et al. Erythromycin a new antibiotic. An‐

[3] Morimoto, S; Takahashi, Y.; Watanabe, Y.; Omura, S. Chemical modification of eryth‐ romycin. I. Synthesis and antibacterial activity of 6-O-methylerythromycin A. Journal

[4] Retsema, J.; Girard, A.; Schelkly, W.; Manousos, M.; Anderson, M.; Bright, G. et al. Spectrum and mode of action of azithromycin (CP62,993), a new 15-membered-ring macrolide with improved potency against gram-negative organisms. Antimicrobial

[5] Chu, D.T.; Plattner, J.J.; Katz, L. New direction in antibacterial research. Journal of

[6] Strigl S, Roblin P M, Reznik T, Hammerschlag M R. In vitro activity of ABT 773, a new ketolide antibiotic, against Chlamydia pneumoniae[J]. Antimicrobial Agents

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tion and perfect crystal shape.

**Acknowledgments**

64 Advanced Topics on Crystal Growth

**Author details**

Shanghai, China

**References**

, Li-Jun Ji and Yan-Yang Wu

\*Address all correspondence to: chenkui@ecust.edu.cn

tibiotics and Chemotherapy 1952; 2(2) 281-283.

Agents and Chemotherapy 1987; (31) 1939-1947.

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of Antibiotics 1984; 37(2) 187-189.

Kui Chen\*


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**Chapter 3**

**Crystal Growth of Inorganic and Biomediated**

Precipitation of carbonate minerals is tightly linked to water chemistry. After hydration of dissolved carbon dioxide, two pH-dependent partitioning-reactions govern the abundance of

where the O-H covalent bond in the oxyacid makes carbonate salts moderately soluble. The most common metal cations forming carbonate minerals are Ca2+, Mg2+, Mn2+, Fe2+, Pb2+, Sr2+, Co2+, Ni2+, Zn2+, Cd2+ and Cu2+. Continental and marine waters are enriched in Ca and Mg and are known to be saturated with respect diverse Ca-Mg carbonates such as calcite (CaCO3), aragonite (CaCO3) and dolomite (MgCa(CO3)2).[3] The concentration of

their respective oxyacids are stronger than those of carbonic acids.[2] Because of this, phosphates are more stable than carbonates at low pH (<5). Chemical composition of phosphate minerals is more variable than that of carbonate minerals, and crystalchemical

Limestones and dolostones constitute the most important carbonate reservoirs on the Earth, but phosphates are much more diluted within the Earth crust, as phosphate rocks are much

and reproduction in any medium, provided the original work is properly cited.

, F– , Cl–

2–, OH–

2–, and PO4

© 2013 Sánchez-Navas et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

2–) formed in aqueous solution:[1,2]

3–) is also a function of pH, and

, etc, is rather common. In addition,

, Sr2+, Ce3+, La3+, Ba2+, and Pb2+ can be incorporat‐

**Carbonates and Phosphates**

Mónica Sánchez-Román,

http://dx.doi.org/10.5772/52062

chemical species (H2CO3, HCO3

–+ H+↔CO3

the phosphate species (H3PO4, H2PO4

numerous metals as Ca2+, Mg2+, Fe2+, Na+

ed into the structure of the phosphate minerals.

Antonio Ruiz-Bustos

**1. Introduction**

H2CO3↔HCO3

substitution of the PO4

Antonio Sánchez-Navas, Agustín Martín-Algarra,

Concepción Jiménez-López, Fernando Nieto and

–

2–+ 2H<sup>+</sup>

3– group by CO3

and CO3

– , HPO4

Additional information is available at the end of the chapter

