Application of preferential crystallization for racemic compound integrating thermodynamics, kinetics and optimization 6

That means only final crystal product from 96% mole fraction S-Kp initial composition was almost pure S enantiomer considering the measure error in HPLC and impurities in products.. The

Chapter 7 Application of direct crystallization for racemic

compound ketoprofen

7.1 Introduction

As mentioned before, the direct crystallization for partially resolved enantiomers can be used for the racemic compound According the phase diagram, whether we can obtain the desired pure enantiomer depends on the partially resolved mixture’s initial position and eutectic composition When the crystallization initial solution composition is located inside the existence region of pure enantiomers, this crystallization process will afford a pure enantiomer In the last chapter, a systematic preferential crystallization process was successfully applied for the favorable racemic compound mandelic acid In this chapter this strategy was extended to another racemic compound system: unfavorable racemic compound For this compound, the solubility of racemate is smaller than that of pure enantiomer and it shows the most narrow operation region to obtain pure enantiomers by crystallization Ketoprofen was chosen for this study

There were some methods already applied for the ketoprofen enantioseparation, including chiral chromatography and enzymatic separation

For a chromatography, several direct/indirect liquid chromatographic methods involving a variety of chiral phases have been reported for the ketoprofen enantioseparation and its enantiomer analysis For example, a new chiral stationary phase using flavoprotein, a glycoprotein present in chicken egg-white, was developed for high-performance liquid chromatography by Nariyasu et al in 1992 The column with this chiral stationary phase could achieve baseline separations for ketoprofen Also, Zhu (1999) reported that β-cyclodextrin (CD) and its derivatives HP- β -CD, DM- β -CD, and TM- β -CD had been employed as chiral selectors for the separation

of ketoprofen by capillary zone electrophoresis And also, Pehourcq (2001) found that

flurbiprofen and ketoprofen were resolved from their racemic forms using a vancomycin chiral stationary phase known as ChirobioticV In addition, according to Aboul-Enein (2003), ketoprofen was also resolved on Kromasil tartardiamide-DMB chiral stationary phase In this process, optimum resolution was achieved using a mobile phase consisting of hexane: tert-butyl methyl ether: acetic acid (75 :25: 0.1 v/v/v) at flow rate of 1 ml/min

An enzymatic separation can be used for ketoprofen enantioseparation too Antona et al (2002) observed that Immobilized lipase from Candida antarctica

(Novozym 435) can catalyze the enantioselective etherification of (RS)-ketoprofen

He found that the use of methanol in dichloropropane allows large scale separation for

ketoprofen This method gave the desired (S)-ketoprofen with 96% ee as unreacted enantioform The (R)-enantiomer, recovered as ester, can easily undergo chemical

racemising hydrolysis and can be reused in the process

Though several chiral separation methods were used for the enantioseparation

of ketoprofen, few studies have reported on using direct crystallization for partially

resolved enantiomers to get the pure (S)-ketoprofen And there is little information

available on whether the coupling the directly crystallization with chromatography could be used for chiral separation of ketoprofen

Therefore, this chapter presents a study to obtain a enantiomerically enriched ketoprofen by using the HPLC with a semi-preparative column for the subsequent systematical study of direct crystallization process The critical supersaturation control strategy and crystallization progression were investigated

7.2 Experiment and methods

7.2.1 HPLC collection of ketoprofen

The collection experiments were carried out with a Shimadzu chromatographic system and used a chiralpak preparative HPLC AD-H column (dimension 250mm L x 10mm I.D) to collect ketoprofen whose mole fraction should be more than 0.96 The mobile phase contained 90%hexane and 10% IPA The temperature was 25oC and flow rate was 3.5ml/min

7.2.2 Direct crystallization process

The crystallization experiments were carried out in the same crystallization

set-up as described in Fig 4.2 The controlled cooling profile (convex) was used on the batch direct crystallization operation of ketoprofen The start point was the same enantiomeric composition with the partially resolved ketoprofen from HPLC

collection It was the 96% mole percent (S)-MA saturated solution at 20 oC in the mixed solvent ethanol and water with volume ration 0.9/1.0 Five batches direct crystallization experiments were carried out starting from the same solution with different modes, which are (a) Exp_01: with seeding and final temperature at 10 oC; (b) Exp_02: with seeding and final temperature at 7.3 oC; (c) Exp_03: with seeding and final temperature at 6.0 oC; (c) Exp_04: with seeding and final temperature at 0.7

oC; (f) Exp_05: without seeding until nucleation occurred

The optical purity of crystal products were also measured by using HPLC For HPLC, the analyze AD-H column was also used to analyze product ee values The

separation conditions are as followed: hexane/IPA (90/10 v/v) as mobile phase, at 25°C column temperature, flow rate of 0.8ml/min and UV-Vis detection at 254nm

7.3 Result and discussion

7.3.1 Semi-preparative HPLC separation of Ketoprofen

As discussed in chapter 6, the loading capacity of ketoprofen on Chiralcel

AD-H was determined by injecting different amount of sample onto the column It was found that ketoprofen shows partial separation when sample loading reaches to 5.0mg, shown in Fig 7.1

Fig 7.1 Partial separation of Kp on Chiralcel AD-H semi-preparative HPLC column (dimension 250mm L x 10mm I.D) at loadings 5.0mg per injection using hexane/IPA (90/10 v/v) as mobile phase, at 25°C column temperature, flow rate of 3.5ml/min and

UV-Vis detection at 254nm

Through semi-preparative chiral HPLC separation, the 96% mole percent S enantiomer and pure R enantiomer of Kp were obtained by collecting two different

fractions at two different retention time, t =17-22 mins and t =15.3-17mins, as

presented in Fig 7.2 The optical purity of 96% mole percent S enantiomer collection

was analyzed on analytical chiral column with hexane/IPA (90/10 v/v) as the mobile phase and a flow rate of 0.8ml/min, shown in Fig 7.3 Then, the volume enough 0.96

mole fraction S Kp can be obtained by using continuous HPLC separation with an

antosampler and automated fraction collector

Fig 7.2 Fraction collection under semi-preparative HPLC separation of Kp on Chiralcel AD-H column (dimension 250mm L x 10.00 mm I.D.) under separation conditions: hexane/IPA (90/10 v/v) as mobile phase, at 25°C column temperature, flow rate of 3.5ml/min and UV-Vis detection at 254nm.Fraction (a) collected at retention time 15.3-17 minutes and fraction (b) is collected at 17-22 minutes

Fig 7.3 Chromatogram of fractions (b) obtained through semi-preparative HPLC separation of ketoprofen on Chiralcel AD-H analytical column (dimension 250mm L

x 4.6 mm I.D.) under separation conditions: hexane/IPA (90/10 v/v) as mobile phase,

at 25°C column temperature, flow rate of 0.8ml/min and UV-Vis detection at 254nm

7.3.2 Preferential crystallization operation for ketoprofen

Based on the phase diagram of Kp in the chapter 4, if we want to get the pure S

enantiomer, the initial composition of the cooling crystallization should be between

pure S and eutectic composition At first, three different start compositions (96%, 94%, and 92% mole percent S ketoprofen) were tried at same cooling process in order to determine from which one pure S enantiomer products can be obtained The HPLC

analyzing results for the final crystal products of different initial composition are shown in Fig 7.4

(a)

(b)

(c) Fig 7.4 The HPLC analyzing results for the crystal products of different initial

composition, (a) 92% mole percent (S)-Kp; (b) 94% mole percent (S)-Kp; (c) 96%

mole percent (S)-Kp

The first peak in these figures presents the R enantiomer of ketoprofen, while the second one is S enantiomer We can see from these HPLC results that the first

peak area was almost negligible compared with the second peak only in Fig 7.4 c

That means only final crystal product from 96% mole fraction (S)-Kp initial composition was almost pure S enantiomer considering the measure error in HPLC

and impurities in products

It can be explained by the Fig 7.5 (Lorenz and Seidel-Morgenstern, 2002) If

we want to get the pure (S)-Kp, the system point should locate inside the pure

enantiomer existence region at ending temperature When start point P cooling to a lower temperature T2, the pure S enantiomer will come out Then, The start point at

high temperature, such as T1 should be higher than the eutectic point The bigger the cooling temperature range, the higher the start point Generally the start point always need higher than the eutectic point Therefore, in this work, the crystallization operations with start composition of 92% and 94% can not produce the optical pure ketoprofen, though their start composition was higher than eutectic point

Fig 7.5 Cooling process to obtain pure enantiomer

The start composition for the preferential crystallization of ketoprofen was the saturated solution at 20oC with 96% mole percent S enantiomer This saturated

solution was saturated in the eutectic composition of Kp at 7.3 oC and saturated in the

(RS)-Kp at 0.7 oC The strategy used for the progression of preferential crystallization for mandelic acid was extended to unfavorable racemic compound system ketoprofen When the controlled cooling profile was used, five batch crystallizations described in section 7.2.2 were carried out The optical purities of each experiment final product were analyzed by HPLC and the results are shown in Fig 7.6 The optical purities of the final crystals with different cooling degree analyzed by HPLC are listed in Table 7.1

(a)

(b)

(c)

(d)

(e) Fig 7.6 HPLC results of the final crystal products with different cooling degree for ketoprofen: (a) Exp 01; (b) Exp 02; (c) Exp 03; (d) Exp 04; (e) Exp 05

Table 7.1 The optical purity of the final crystal products with different cooling degree

Primary nucleation occurred

From these results, the same situation found for mandelic acid in section 6.3.2.1

was observed for ketoprofen The product crystals were almost pure (S)-enantiomer

when the end temperature was higher than 0.7 oC (Exp_01-03), which was saturated

temperature for the (RS)-Kp In this region, only (S)-Kp is supersaturated When the crystallization final temperature was lower than this (RS)-Kp saturated temperature, (RS)-Kp began to supersaturate which resulted in the product crystals in the form of mixture of (RS)-Kp and (S)-Kp, for example Exp 04 It may further prove that there is

no selectivity of crystal growth of the pure enantiomer and racemate for a racemic

compound when both (S) and (RS) reach supersaturation The primary nucleation

occurred in Exp 05 when the solution was cooled to around 0.2oC without seeding The products of Exp 05 were not optical pure enantiomer because of the racemic compound property: no selectivity of nucleation between pure enantiomer and racemate All these results prove that the key factor to obtain pure enantiomer

products in direct crystallization of racemic compound is its solubility characteristic Only within the safe supersaturation critical limit, can pure enantiomer crystal products be obtained from the preferential crystallization with seeding

For the optimal cooling profile consideration, based on the ketoprofen MSZW data in chapter 4, the supersaturation should be kept within circa 2oC to avoid spontaneous nucleation of both its enantiomer and racemate However, as discussed before, the supersaturation should be kept lower than it measured under homogenous condition So, a suitable critical supercooling chosen for ketoprofen was controlled at around 0.5-1 oC The corresponding ∆c should be ca 0.0015-0.0025g/ml It is very

narrow feasible supersaturation control range compared with 0.027g/ml for mandelic acid It suggests that it is more difficult to control the supercooling for the preferential crystallization for ketoprofen and the nucleation of enantiomer and racemate of ketoprofen may be easy to occur On the other hand, considering the crystal growth kinetics of the ketoprofen (Eq 5-23), the crystal growth rate should be very small in order to control the supersaturation level within the chosen narrow range It means the batch operation time should be very long which can result in the low efficient for the whole crystallization operation

In addition, some researchers (Ströhlein et al., 2003) proposed a general design method for the hybrid process of a chromatographic and a crystallization unit They point that coupled chromatography and crystallization processes in which both units contribute to purification are useful and efficient, only if the considered crystallization system possesses a low eutectic point But, for the ketoprofen, an unfavorable racemic

compound system, the eutectic point is high about 92% more percent (S)-Kp, which

leads to our crystallization unit should start at very high composition, even as 96%

more percent (S)-Kp in order to obtain the pure desired enantiomer That situation

may make the whole coupling process less effective and less economical for the ketoprofen separation

7.4 Conclusion

In this chapter, a system preferential crystallization was applied for the unfavorable racemic compound ketoprofen coupling with the HPLC The partially

resolved 96% mole percent (S)-Kp was obtained by HPLC collection with

semi-preparative column Then the subsequent direct crystallization started from this initial

composition, which located inside the existence region of pure S enantiomers in the

phase diagram Based on the solubilities and MSZWs of ketoprofen, the direct crystallization progression was clearly investigated The optical purities of the final crystals product were analyzed by HPLC It was found that the optical pure product could be obtained by direct crystallization with seeding within certain safe supersaturation limit It may be further proved that there was no selectivity of crystal growth and nucleation of the pure enantiomer and racemate for a racemic compound

On the other hand, the supersaturation control is especially critical for the unfavorable ketoprofen system due to its high eutectic composition and narrow metastable zone widths, which cause narrow feasible region and more difficulty to control for direct crystallization Direct crystallization could be less effective and less economical as an enantioseparation process for the ketoprofen system

Chapter 8 Conclusions and Future work

8.1 Conclusions

In this present work, the preferential crystallization process itself was studied for the two racemic compound systems, namely more favorable racemic compound mandelic acid and unfavorable racemic compound ketoprofen, combining the aspects

of thermodynamics, kinetics, and optimal operation A systematic preferential crystallization was studied on solubility, metastable zone, kinetics and supersaturation control profile to obtain crystal product with good quality

In Chapter 3, two kind of racemic species, namely mandelic acid and ketoprofen, were characterized by the various spectroscopic techniques, thermal analysis, thermodynamic calculation and binary phase diagram The spectra of FTIR, Raman and PXRD were different between the pure enantiomer and racemate for the mandelic acid and ketoprofen, which indicates that the mandelic acid and ketoprofen both belong to the racemic compound Through the thermal analysis and calculation,

it was found that the ∆G 0

was negative and the enthalpy of fusion difference between

(RS) and (S) were positive for both of mandelic acid and ketoprofen Their ∆Tf were both far away from -30oC which implies that the racemic species is likely to be a conglomerate All these results suggest that the mandelic acid and ketoprofen are in form of racemic compound The binary melting phase diagrams were constructed for the mandelic acid and ketoprofen based on Schröder-Van Laar equation, the Prigogine-Defay equation and DSC measurements The calculated results were in good agreement with the DSC experiment data The shape of binary phase diagrams

of mandelic acid and ketoprofen both show the typical shape of racemic compound system, just the more favorable racemic compound system for mandelic acid and unfavorable racemic compound system for ketoprofen From the binary phase

diagram, the eutectic point was determined as 70% of (S)-MA for mandelic acid and 91.6% of (S)-Kp for ketoprofen

In chapter 4, the thermodynamic properties in solution were studied for the mandelic acid and ketoprofen using the Lasentec FERM The solubilities, ternary

phase diagram and metastable zone width at different mole percent of S enantiomer

were obtained for mandelic acid in water and ketoprofen in mixed solvent of ethanol and water with volume ration 0.9:1.0 For the case of mandelic acid, the solubility

ratio of (RS) to (S)-MA decreases with the temperature in the range 35-5oC which suggests that the preferential crystallization of mandelic acid in the chosen solvent is more favorable as temperature decreases All the MSZW results of mandelic Acid were higher than 5 oC which are favorable for preferential crystallization process Its ternary phase diagram showed a typical shape of more favorable racemic compound, which further proves that the mandelic acid is a kind of more favorable racemic compound On the other hand, the ternary phase diagram of ketoprofen shows the typical properties of an unfavorable racemic compound system The MSZWs of high

mole percent (S)-Kp were all narrow around 2oC It is more difficult to control the supsaturation level of pure enantiomer to inhibit its spontaneous nucleation during the crystallization process Through the study of the main four effects of MSZW by fractional experiment design for ketoprofen, it is obvious that the water/ethanol volume ratio in the solvent should have the most significant effect on the MSZW of

Kp MSZW would increase with decreasing ethanol ratio The temperature, stirring rate and cooling rate may affect the process slightly MSZW would also be inversely proportional to the temperature, but would be directly proportional to the cooling rate However, the MSZW of high mole percent ketoprofen are still not satisfied even in the optimal condition derived by fractional experiment design

In chapter 5, the classical Laplace transform analysis was used for deriving the crystal growth rate and nucleation rate in the batching crystallization process for both

(S) and (RS) mandelic acid and ketoprofen Also the kinetics of (S) and (RS)-Kp were

determined by the moment analysis method and the kinetic results of moments analysis are comparable with those of Laplace transform analysis for ketoprofen The enantiomer and racemate show different characteristics in crystal nucleation and growth for racemic compound mandelic acid and ketoprofen

In chapter 6, a systematic preferential crystallization combining the solubility, metastable zone, kinetics and supersaturation control profile to obtain crystal product

with good quality was proposed for mandelic acid At first, the 80% mole percent S

enantiomer enantiomerically enriched mandelic acid was obtained from a racemic composition by using a HPLC with a semi-preparative chiral column Then, through the study on the direct crystallization progression for the mandelic acid system, it was found that the optical pure product could be obtained by direct crystallization with seeding within certain safe supersaturation limit and the relative solubility and critical supersaturation control of the pure enantiomer and racemate were essential to obtain the pure enantiomer Subsequently, based on the thermodynamic and kinetic consideration, an optimal temperature control profile was derived to control the critical supersaturation in order to inhibit the induced nucleation of the racemate Compared with the forced and linear cooling profile, the final crystal products of our proposed control cooling profile were almost optical pure with high yield and good crystal size distribution The optical purity of product crystal was measured by HPLC, Polarimeter, and DSC Finally, Seed size effect on crystal size distribution was studied It was found that the weight mean size of final crystal product increased with the seed size increasing because the amount of solid deposited on crystal surfaces is

constant at constant yield However, the final product mean crystal size did not increased significantly at high seed size

In this chapter 7, a system preferential crystallization was also applied for the unfavorable racemic compound ketoprofen The partially resolved 96% mole percent

(S)-Kp was obtained by HPLC collection with semi-preparative column Based on the

solubilities and MSZWs of ketoprofen, the direct crystallization progression was clearly investigated It was found that the optical pure product could be obtained by direct crystallization with seeding within certain safe supersaturation limit There was

no selectivity of crystal growth and nucleation of the pure enantiomer and racemate

for a racemic compound Because of the narrow metastable zone width of S

enantiomer, the supersaturation control is especially critical for the unfavorable ketoprofen system The narrow metastable zone widths can cause narrow feasible region and more difficulty to control for direct crystallization Direct crystallization could be less effective and less economical as an enantioseparation process for the ketoprofen system because of its narrow MSZW and high eutectic composition The

ketoprofen eutectic point is high around 92% more percent (S)-Kp, which means that

the crystallization unit should start at very high composition, even as 96% more

percent (S)-Kp, in order to obtain the pure desired enantiomer and the existence region of pure S enantiomers in phase diagram should be very limited

8.2 Future work

The HPLC can be used to obtain an enantiomeric enrichment exceeding the eutectic composition in the racemic compound system, which sets the threshold for a subsequent enantioselective crystallization process However, the trend in preparative

scale chromatography is rapidly moving the emphasis to supercritical fluid chromatography (SFC).

SFC has two main advantages for preparative separations One is speed: a higher production rate can be obtained from a given column since the mobile phase viscosity is very low and fast, efficient separations can be achieved The other advantage is the small quantity of organic solvent used: between 10% and 20% of that needed for a HPLC separation This not only decreases the total quantity of solvent used for the separation, but also makes it easier and faster to recover the products from the small modifier volumes remaining after condensation from the CO2 Therefore, Coupling the SFC and preferential crystallization will be suggested for the racemic compound system in the future

In addition, the different seeding preparation methods were suggested to be studied in order to obtain the optimal operation strategy and get the good quality of final products

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