Quantitative Measurement of the Polymorphic Transformation of L-Glutamic Acid Using In-Situ Raman Spectroscopy T. Ono,‡ J. H. ter Horst,* and P. J. Jansens
CRYSTAL GROWTH & DESIGN 2004 VOL. 4, NO. 3 465-469
Laboratory for Process Equipment, Delft University of Technology, Leeghwaterstraat 44, 2628 CA Delft, The Netherlands Received December 10, 2003;
Revised Manuscript Received March 15, 2004
ABSTRACT: Control and optimization of the polymorph formation in batch crystallization processes require insitu quantitative measurement of the concentration of different polymorphs. In this study, Raman spectroscopy was chosen to measure the polymorphic fraction of L-glutamic acid (L-Glu) in suspension. A sufficiently accurate calibration line was constructed using mechanical mixtures of the two forms, R and β. The calibration line was also valid for the suspension system. The polymorphic behavior in batch cooling crystallizations of L-Glu was monitored using the calibration correlation. It was clearly observed by Raman spectroscopy that at 25 °C the nucleation of the R-form from a clear aqueous solution occurred dominantly and nearly 100% R-form crystals were obtained. During the subsequent solvent-mediated transformation of the R-form crystals to the stable β-form, the R-form fraction gradually decreased. The transformation rate has a strong temperature dependency so that the temperature increase promotes the transformation. Raman spectroscopy proved to be a powerful tool for measuring the polymorphic fraction in suspension. Introduction Polymorphism is the phenomenon that a compound has more than one crystalline arrangement.1 Polymorphism plays a significant role in the fine chemicals industry, especially in the pharmaceutical industry. Different polymorphic structures exhibit different physical and chemical properties such as crystal morphology, solubility, and color, which affect the performance of the products1,2 (for example, the bioavailability of a drug). From a regulatory standpoint, the manufacturer is required to produce one of the forms or a defined mixture of forms in a reproducible way.3 In such productions, a measurement technique of polymorphic fractions during the crystallization operation would enable the confirmation of the reproducible productions and, if necessary, to adjust the operating conditions to obtain the desired modification. It is furthermore important to investigate the polymorph formation kinetics, which includes nucleation, crystal growth, dissolution of each modification, and transformation of one to another, and to determine appropriate operating conditions based on these kinetics.4-6 The polymorphic fraction in the crystalline phase can be measured with X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), solid-state NMR, infrared spectroscopy (IR), or Raman spectroscopy.7-14 In this study, we chose to use Raman spectroscopy. Raman spectroscopy can measure inelastic scatterings of light which have different wavelengths from the incident light.15,16 The differences in the wavelength are related to the vibrational modes of the molecules. If different polymorphs have different vibra‡ Current address: Process and Production Technology Center, Sumitomo Chemical Co., Ltd., 1-98, Kasugade-naka 3-chome, Konohana-ku, Osaka 554-8558, Japan. * Corresponding author. E-mail:
[email protected]. Internet: http://www.api.tudelft.nl/. Tel: +31 15 278 6678. Fax: +31 15 278 6975.
Figure 1. L-Glutamic acid morphology: (a) prismatic R-form and (b) needlelike β-form crystals.
tional modes, the Raman spectra are the fingerprints for the polymorphs. Raman spectroscopy coupled with an immersion probe, optical fibers, and CCD camera can be used in-situ in slurry systems without any sample preparation;14,15 therefore, it has great advantages over the other mentioned analytical techniques. L-Glutamic acid (L-Glu) was chosen as the model compound. It is known to have two polymorphs, the R-form and the β-form. The morphology of the R-form crystals is prismatic, and the β-form is needlelike (Figure 1). The R-form is generally preferred for industrial purposes because of its good separability from the mother liquor and ease of handling due to its morphology.17 The polymorphic behavior of L-Glu in crystallization has been investigated in previous studies.18-22 At a temperature lower than 25°C, the primary nucleation of the unstable R-form is predominant with following Ostwald’s Rule of Stages, which claims that metastable form crystals nucleate faster than stable ones from the supersaturated solution. As the temperature rises, the nucleation rate of the stable form β increases relative to the R-form, and the nucleation rates become competitive. If the R-form crystals are put in a saturated aqueous solution, a solvent-mediated transformation of the R-form to the β-form will take place. The transformation includes a dissolution step of the R-form crystals and nucleation and growth steps of the β-form crystals.
10.1021/cg0342516 CCC: $27.50 © 2004 American Chemical Society Published on Web 04/10/2004
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In this paper, the performance of Raman spectroscopy measuring polymorphic fractions of L-Glu in solid phase was evaluated. The performance of the monitoring the polymorphic fraction change in-situ in suspension was also evaluated using batch cooling crystallizations from an aqueous solution. The in-situ monitoring technique using Raman spectroscopy will make it possible to control polymorph formation on-line during the process. The kinetic data obtained by in-situ Raman measurements will help to construct kinetic models, which can be used for the optimization studies of the batch crystallization condition to achieve the desired polymorph formation, cycle time, quality, and high yield. Experimental Section Raman Spectroscopy. A HoloLab Series 5000 Raman spectroscopy (Kaiser Optical Systems, Inc.) was used for the measurements. Raman spectra were recorded using NIR excitation radiation at 785 nm and multichannel CCD camera. This instrument has an immersion probe (15 mm φ × 250 mm L) sealed with a sapphire glass window which facilitates insitu measurements in the slurry system. To measure the polymorphic fraction of L-Glu quantitatively, a calibration line was constructed using dry binary mixtures of the R-form and the β-form. The mixtures were created by mixing nearly pure R- and β-form samples, which were prepared by batch crystallization (see below). The probe was used to measure the polymorphic fraction of the created dry samples as well as insitu measurements in the slurry system. Preparation of Polymorphs r and β. The nearly pure R- and β-form samples were prepared by batch cooling crystallization using the method of Kitamura.19 Purchased L-Glu (chemical purity > 99%, Sigma chemicals) was recrystallized from an aqueous solution at the isoelectric point (pH ) 3.2). To obtain the pure R-form, a clear solution (4.8 g/100 g of solution) was prepared at 80 °C using L-Glu and ultrapure water. The solution was cooled to 25 °C rapidly under constant stirring. At 25 °C nucleation and growth of the R-form occurred dominantly. The R-form crystals were filtered and rinsed by ultrapure water, and the wet cake obtained was dried immediately at room temperature. To obtain the pure β-form the clear solution (5.4 g/100 g of solution) was prepared at 83 °C. The solution was cooled to 55 °C and held at the temperature for 5 h. Competitive nucleation of the R-form and the β-form was observed followed by a solvent-mediated transformation of the R-form to the β-form. The transformation was completed in 5 h, and the β-form crystals were treated similarly to the R-form. The purity of the R-form (97 wt %) and the β-form (>99 wt %) samples was measured by XRPD (R-form: d ) 4.87 Å and β-form d ) 4.17 Å17). A Calibration Line for Quantitative Analysis. A total of eight binary mixtures was created by mixing the nearly pure dry samples. The mixing was done by hand for over 10 min. The polymorphic fractions of the samples were calculated from the purity and the amounts of the samples used for the mixing. In this way, samples with R-form fraction x(R) ) 0.3, 9.8, 28.4, 31.0, 44.5, 67.1, 87.0, and 96.5 wt % were obtained, which in the following will be referred to as “the calculated R-form fraction x(R)”. At first, we attempted to construct the calibration line using Raman spectra from slurries in which the created samples were dispersed under stirring in the saturated aqueous solution. However, gradual evolutions in Raman spectra were recognized during the measurements. The evolutions were caused by the transformation. Although the evolutions were slight, to avoid the disturbance, a calibration line was determined using Raman spectra of the dry mixtures. The validity of the calibration line for in-situ measurements was confirmed by the comparison between the results of in-situ and off-line Raman measurements. In-Situ Measurements. In-situ Raman measurements were performed during batch cooling crystallizations. A 1-L
Ono et al.
Figure 2. Temperature profile employed in the crystallization experiment of L-Glu: I, nucleation region; II, Trans-formation region. jacketed glass vessel with an impeller was used for the crystallizations. Agitation rate was 440 rpm and kept constant during the crystallizations. The temperature was controlled from the jacket using a thermostat bath. The Raman probe was inserted into the crystallizer. Raman measurements were performed in-situ intermittently during the crystallization experiment. Total exposure time was 2 min for one Raman spectrum. Figure 2 shows the temperature profiles employed in the experiments. At first, a clear solution with a saturation temperature at 72 °C for the β-form was prepared at 80 °C using 39 g of L-Glu and 800 g of ultrapure water. Then the solution was cooled to 25 °C within 20 min and maintained at the temperature for 1 h to obtain pure R-form crystals (I: nucleation region). Subsequently, the temperature of the slurry was raised to a certain temperature within 30 min (except in the case of the 25 °C experiment) and maintained constant at the temperature for several hours. During this time, the solvent-mediated transformation of the R-form to the β-form was expected to occur (II: transformation region). Off-Line Measurements. In addition to the in-situ measurements, off-line Raman measurements were performed using dried samples obtained from the slurry during the crystallization experiments as well. The dried samples were obtained by sampling, filtering, and drying it. The result of the off-line measurement at a certain sampling time was compared with the result of the in-situ measurement performed at the same time. Raman spectra obtained by in-situ and off-line measurements were analyzed using HoloReact, which is the software for HoloLab Series from Kaiser Optical Systems, Inc.
Results and Discussions Polymorphic Quantitative Analysis. Raman spectra of the nearly pure samples of the R-form and the β-form in the range of 200 to 1400 cm-1 are presented in Figure 3. Some specific differences between the R-form and the β-form can clearly be observed. For instance, the peaks at 623, 665, 1003, and 1179 cm-1 are characteristic for the R-form23 and the peaks at 575, 705, 800, 1145, and 1214 cm-1 are characteristic for the β-form. The large differences in Raman spectra of the two forms are caused by the conformational differences of the molecules in the crystal lattice of the R and the β-form. The molecular conformations are significantly different through a torsion angle in the main carbon chain.24 The polymorphs of L-Glu, therefore, can be discriminated by Raman spectroscopy. Raman spectra in the range of 600 to 850 cm-1 obtained from the dry mixtures are shown in Figure 4. It is observed that as the fraction of the R-form changes the height and the area of the characteristic peaks
Polymorphic Transformation of L-Glutamic Acid
Crystal Growth & Design, Vol. 4, No. 3, 2004 467
Figure 3. Raman spectra of L-Glu in the range of 200 to 1400 cm-1: (a) R-form and (b) β-form. Round and squared dots indicate main characteristic peaks for the R-form and the β-form, respectively.
Figure 5. The calibration line constructed from the value for the relative peak height HR/(HR + Hβ) and the measured R-form fraction: Raman measurements (squares) and the fit (solid line). Table 1. Standard Deviations in the Errors between x(r) and the Predicted r-Form Fraction by Correlation Equations Raman shift [cm-1]
Figure 4. Raman spectra obtained from the created dry mixtures. The R-form fraction is (a)
