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Polymorphism of 3,3′-dihydroxy-β,β-carotene-4,4′-dione (Astaxanthin)
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1648–1652
Contents lists available at ScienceDirect
Chemical Engineering Research and Design journal homepage: www.elsevier.com/locate/cherd
Polymorphism of 3,3 -dihydroxy-,-carotene-4,4 -dione (Astaxanthin) Jingfei Guo, Matthew J. Jones, Joachim Ulrich ∗ Martin-Luther-Universität Halle-Wittenberg, Zentrum für Ingenieurwissenschaften, Verfahrenstechnik/TVT, D-06099 Halle (Saale), Germany
a b s t r a c t Astaxanthin (AXT) 3,3 -dihydroxy-,-carotene-4,4 -dione, from the group of carotenoid, specifically as xanthophyll, is investigated in this work on its polymorphism. The method to obtain a second polymorph of AXT is described. Polymorph I can be purchased as a commercial product or obtained via phase transformation from polymorph II. Polymorph II can be prepared from polymorph I by a thermal treatment carried out in a differential scanning calorimetry (DSC). The resulting crystals are characterized by different analytical techniques. X-ray powder diffractometer (XRPD) and resonance Raman spectroscopy prove the existence of two different crystalline polymorphs. Simultaneous thermal analysis (STA) gives additional information on their thermal behaviors. Polymorph I melts at 230 ◦ C and polymorph II melts at 216 ◦ C. Phase transition experiment carried out by slurry conversion experiment reveals two polymorphs are monotropically related. Polymorph II is the metastable form. © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Polymorphism; Characterization; XRPD; Raman spectroscopy; Phase transformation
1.
Introduction
Carotenoids are the most important group of natural pigments in the vegetable and animal kingdoms. The rate of progress in the research of carotenoids increased rapidly, in 1971, the famous monograph of Isler (1971) recorded around 230 carotenoids with well-established constitutions. In 1993, the number of basic structures exceeded 600 (Britton, 1995), which mainly attributed to the improvements in chromatographic procedures and spectroscopic methods. Here the solid-state properties of 3,3 -dihydroxy-,carotene-4,4 -dione (Astaxanthin) (see Fig. 1) are investigated. Astaxanthin (AXT) is a carotenoid responsible for the pink coloration in salmonidea, shrimp and lobster, and is of interest due to its antioxidant properties resulting from the specific molecular structure consisting of a linear chain with conjugated double bonds (polyene). Like all carotenoids, AXT is insoluble in water and has a low solubility in fats and oils, which highly restricts the application as feed additive for aquatic animals. Different methods have been described to improve the properties, e.g. decreasing the crystal size to a particle size range of smaller than 10 m
∗
(Runge et al., 2003) and increasing amorphous content up to 70–100% (Auweter et al., 2005). However, few papers described about polymorphism in carotenoids which could be an option to improve their limited properties. In 2001, Mori suggested the presence of polymorphism or dimorphism as a common characteristic of carotenoid crystals according to their absorption spectra. The results showed further controllability of the molecular arrangement indicating the existence of solid phases other than those observed (Mori, 2001). Since polymorphism is a common phenomenon of organic substances, AXT is selected as a model substance from the carotenoid family used to study the existence of polymorphs in these compounds, and thus, to select a desired polymorph with better properties, to provide insight into similar research work for other carotenoids. In 2007, Bartalucci reported the crystal structure of solvated AXT and one non-solvated AXT (Bartalucci et al., 2007). Two solvated AXT, chloroform solvate and pyridine solvate can exist at extremely low temperature (100 K) which are not to be chosen as the commercial crystalline forms for production. For non-solvated AXT, one triclinic crystal structure was reported.
Corresponding author. E-mail address: [email protected] (J. Ulrich). Received 5 December 2008; Received in revised form 15 October 2009; Accepted 8 February 2010 0263-8762/$ – see front matter © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cherd.2010.02.005
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1648–1652
1649
Fig. 1 – Chemical structure of the carotenoid AXT. However, no direct information can be found regarding the existence of polymorphism for non-solvated AXT. In this paper, the preparation of a second polymorph of AXT is described. The crystals were characterized by simultaneous thermal analysis (STA), X-ray powder diffraction (XRPD) and resonance Raman spectroscopy. Slurry conversion experiment is performed to investigate on the phase stability.
2.
Methods and materials
2.1.
Materials
AXT polymorph I was obtained commercially and used as it is. Solvent of ethyl acetate (EtOAc) was used from Roth (Rotisolv® ) at HPLC grade. Polymorph II can be prepared by the heat treatment or evaporation from certain solvents, such as acetone. Due to the low solubility (approximately 8 mg 100 mL−1 in acetone at 20.5 ◦ C), only a very small amount of crystals can be obtained after evaporation. Heat treatment was therefore used as the main method for crystal preparation. Polymorph II was produced by melting of polymorph I at 224 ◦ C followed by quenching process. Heating of polymorph I was carried out in a DSC (Netzsch Phoenix 204) cell. Care should be taken that AXT is decomposed after melting, considering the temperature lag between the instrument and the sample, temperature need to be set slightly below the real melting temperature of polymorph I where decomposition may occur. Approximately 6–7 mg crystals of AXT polymorph I were heated in the Al-pan at a heating rate of 5 K min−1 , from 20 ◦ C to 200 ◦ C, and then slowly heated up to 224 ◦ C at a heating rate of 2 K min−1 . Immediately after this temperature was reached, the sample was cooled down from 224 ◦ C to 20 ◦ C at a cooling rate of 40 K min−1 . The DSC system was purged with nitrogen throughout. XRPD confirms the second crystal polymorph of AXT.
polymorph I and II were heated up from 28 ◦ C to 500 ◦ C at a heating rate of 5 K min−1 while purged with helium. The mass changes during heating were recorded.
3.3. Preparation of AXT polymorph II at different maximal temperatures The best heating temperature was studied by calculating the composition of the final crystals from their melting temperatures. In order to achieve this, a calibration of melting temperatures versus compositions was built up first. The mixtures of polymorphs were prepared at different compositions from 0% to 100% at an interval of 10% and heated up at 5 K min−1 , from 20 ◦ C to 250 ◦ C. After obtaining the calibration curve, 5–6 mg AXT polymorph I was heated up by DSC with a heating rate of 5 K min−1 , from 20 ◦ C to 200 ◦ C and, heated again with a smaller heating rate of 2 K min−1 , from 200 ◦ C to 218 ◦ C, 220 ◦ C, 222 ◦ C, and 224 ◦ C respectively, cooled down at 40 K min−1 to the initial conditions. By calculation of composition of resulting crystals, the best heating temperature resulting in the highest purity of polymorph II was obtained and the heating process was optimized.
3.4.
Resonance Raman spectroscopy (FT-Raman-Specktrometer RFS 100/S, Bruker) was used to identify the polymorphs of AXT. This technique is a vibrational molecular spectroscopy which derives from an inelastic light scattering process. The measurement was performed on a FT-Raman spectrometer with Raman microscopy and temperature control unit. The laser was operated at 1064 nm with back scattering optics. A stainless sample holder is used which can hold amount of several micrograms. Crystals of polymorph I and II were measured respectively at solid state.
3.5.
3.
Methods
3.1.
X-ray powder diffraction studies
The X-ray powder diffraction patterns were obtained by collecting intensity data measured by a Bruker D4 diffractometer. The system is equipped with a Cu anode and a monochromator providing Cu K␣1 radiation ( = 1.54056 Å). Measurements were carried out using a step width of 0.005◦ and an acquisition time of 4 s per step.
3.2.
Thermal analysis
A STA (Netzsch STA-409) referring to the simultaneous measurement of caloric effects and mass changes was used here to study the thermodynamic properties of different polymorphs and their thermal stability. Approximately 5–6 mg samples of
Raman spectroscopy
Slurry conversion experiment
Slurry conversion experiment was carried out here by suspension of excess amount of polymorph II in EtOAc in double jacket beakers controlled by thermostat at a constant temperature of 50 ◦ C. The suspensions were stirred at a constant rate of 300 rpm. Samples were taken out at different retention times, filtered and dried. Finally, the resulting crystals were analyzed by XRPD.
4.
Results and analyses
4.1.
XRPD patterns of different polymorphs
Fig. 2 shows the pattern of crystals prepared by DSC compared with the original polymorph I. The pattern of crystals illustrates different characteristic peaks in the scanning range indicating a generation of second crystalline form.
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Table 1 – Mixture at different compositions.
Fig. 2 – Comparison between XRPD patterns of AXT polymorphic polymorph I and II. For example, the high intensity peak observed at 20.35◦ in polymorph I is absent in the pattern of the other crystals. Also, two peaks observed at 2 = 11.07◦ and 18.32◦ of the second crystals do not match the pattern of polymorph I. Therefore, a different crystalline form, denoted polymorph II, of AXT was successfully prepared by the thermal treatment carried out by DSC. The patterns are compared with the calculated peak positions from the reported crystal structure (Bartalucci et al., 2007), and the comparison shows good agreement which confirms that the crystal structure obtained by Bartalucci is polymorph II in our work. Concerning the generation of polymorph II by Bartalucci et al. underwent a solution crystallization process, therefore, different methods could be used for obtaining polymorph II. The XRPD pattern of polymorph I reported in our work is new.
4.2.
Thermal analysis
Thermal analyses, in particular DSC and TG, have been the main methods to study polymorphism. Fig. 3 shows the results of different melting points and the heat of fusion of AXT of polymorph I and II. The thermal stability is also investigated by the TG technique. From Fig. 3, the DSC curves show one endothermic event between 220 ◦ C and 232 ◦ C for polymorph I and the other in
Sample
Composition
a. b. c. d. e. f. g. h. i. j. k.
I 100.0%, II 0.0% I 87.3%, II 12.7% I 80.8%, II 19.2% I 70.2%, II 29.8% I 60.4%, II 39.6% I 50.2%, II 49.8% I 40.3%, II 59.7% I 31.7%, II 68.3% I 20.4%, II 79.6% I 10.5%, II 89.5% I 0.0%, II 100.0%
MassI (%) 100.00 87.26 80.78 70.21 60.43 50.24 40.32 31.65 20.35 10.46 0.00
Melting point (◦ C) 230.80 226.80 225.20 224.30 223.40 222.00 221.70 219.30 220.40 217.10 216.60
the range between 200 ◦ C and 220 ◦ C for polymorph II, recognized as melting of the samples. In addition, another small endothermic peak at 215 ◦ C is also observed on the heating trace of polymorph I. The mass losses before melting are 3.57% for polymorph I and 5.50% for polymorph II which is probably due to heat instability. Since polymorph II is generated by melting and quenching process without any additional usage of solvents, no solvate exists in this work. Thermoanalytical data indicate that a decomposition process occurs immediately after melting for both polymorphs corresponding to the sudden mass loss. This result ascertains that heating up of polymorph I to 224 ◦ C is sensible as it avoids the decomposition and loss of the substance. Due to the heat instability, temperature cycle profile is not suitable for further investigation on the thermal behavior of AXT. Temperature controlled XRPD and hot-stage microscopy were also used to determine the possible events around the small peak observed in the STA at approximately 215 ◦ C. No solid–solid phase transformation is found. It is assumed that this small peak is due to a small amount of polymorph II presenting in the original crystals.
4.3. Preparation of AXT polymorph II at different maximal temperatures The crystal samples of polymorph I and II at different compositions were heated up by DSC at 5 K min−1 , from 20 ◦ C to 250 ◦ C. A standard curve of melting points versus composition was built up (Table 1). The melting point decreases with the increase of the content of polymorph II in the mixture. The points were plotted in Fig. 4 to obtain a polynomial fit between the melting points and the compositions. The equation is expressed as: C = −0.1836x2 + 89.433 x − 10757
Fig. 3 – STA analysis of AXT polymorph I and II; sample subjected to a heating rate of 5 K min−1 in a perforated Al-pan (1 hole) under helium flow.
which can be used to calculate the purity of polymorph II that exists in mixture as the final product, where “x” is the melting temperature of the mixture of polymorphs I and II. The best heating temperature is studied by calculating the composition of the final crystals from the melting points (Tables 2 and 3). Therefore, the best heating temperature is 224 ◦ C. The crystals obtained at this temperature give highest purity as calculated.
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Table 4 – Results of resonance Raman spectroscopy. CH3 (cm−1 )
Name Polymorph I Polymorph II J-aggregate (Auweter et al., 1999) H-aggregate (Auweter et al., 1999) Literature (Weesie et al., 1995)
Fig. 4 – Plots of melting points versus composition of polymorph I in the mixture.
4.4.
Raman spectroscopy
The resonance Raman spectra of AXT polymorph I and II are shown in Fig. 5 (Table 4). The most intense band at 1510 cm−1 is assigned to the C C stretch vibrations of the polyene chain. The second most intense band at 1155 cm−1 , which represents the superposition of two modes that can be ascribed to C–H in-plane bending vibrations mixed with C–C stretching and C C–C bending
1005.0 1006.0 –
C H (cm−1 ) 1155.4 1157.3 –
–
1008.0
–
1159.0
C C (cm−1 ) 1510.4 1513.4 1516.0
1522.0
1523.0
vibrations, respectively. The third intense line at 1004 cm−1 can be assigned to CH3 in-plane rocking vibrations. Although Raman spectra of polymorph I and II are similar, still differences can be found in the region from 500 cm−1 to 1000 cm−1 , for instance, polymorph II has only one sharp band at 966 cm−1 whereas polymorph I shows two bands. These differences are useful information for identification of polymorph I and II in the mixture and formulation.
4.5.
Slurry conversion experiment
Solvent-mediated polymorphic transformation is an efficient technique to study the phase stability of different polymorphs
Table 2 – Preparation of the AXT polymorph II by DSC. Name a b c d
Segment 1 (◦ C) 20–200 20–200 20–200 20–200
Heating rate (K min−1 )
Segment 2 (◦ C)
5 5 5 5
200–218 200–220 200–222 200–224
Heating rate (K min−1 ) 2 2 2 2
Purged gas Nitrogen Nitrogen Nitrogen Nitrogen
Table 3 – Calculated composition of polymorph II obtained at different heating condition. Name 1 2 3 4
Heated temperature (◦ C) 218 220 222 224
Melting point (◦ C) 226.4 223.9 218.4 216.6
Calculated massI of polymorph I (%) 80.91 64.50 17.84 0.00
Fig. 5 – Comparison of Raman spectra of AXT polymorph I and II in the different regions, (a) Raman spectra of AXT polymorph I and II in the range from 500 cm−1 to 1800 cm−1 ; (b) Raman spectra of AXT polymorph I and II in the range from 500 cm−1 to 1000 cm−1 .
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chemical engineering research and design 8 8 ( 2 0 1 0 ) 1648–1652
mic peak observed at 215 ◦ C on the heating trace of polymorph I is likely to be the presence of a small amount of polymorph II in the original crystals. Decomposition up to 95% of two polymorphs indicates their thermal instability. The best heating temperature for obtaining pure polymorph II is at 224 ◦ C. Raman spectra provide sufficient information to identify the polymorphs. Differences in peak position and shifts of Raman bands between polymorph I and II make it possible to distinguish the mixture of both polymorphs at ambient condition. Finally, slurry conversion experiments reveal that two polymorph are monotropically related. Polymorph I is the thermodynamically stable form.
Fig. 6 – XRPD patterns of crystals obtained from suspensions in EtOAc at the temperature of 50 ◦ C for different retention time, (a) 60 min; (b) 120 min; (c) 180 min; (d) 1 day; (e) 3 days; (f) 5 days; (g) 9 days. and to obtain the most stable polymorph. In the case of EtOAC the phase transformation is observed at a temperatures of 50 ◦ C after 3 days. Fig. 6 shows the results of XRPD measurements at different retention time. In the XRPD patterns, characteristic peaks of polymorph II disappear with the increase of the retention time. Some new peaks belonging to polymorph I appear after 3 days. The color change of the suspension from red to violet is observed when the transformation takes place simultaneously. After 5 days, this transformation is completed. Further experiments at the temperatures above 50 ◦ C show the same tendency of the phase transformation from polymorph II to polymorph I. However, the experiments carried out below this temperature demonstrate a relatively longer transformation period. Therefore, conclusion can be draw that the polymorph I and II are monotropically related. Polymorph I is the thermodynamically stable form.
5.
Discussions and conclusions
AXT, a super antioxidant from the carotenoid family, is found to be polymorphic substance with two non-solvated polymorphs. A second crystal polymorph generated by heat treatment is carried out in a DSC cell. DSC can be used as a reproducible and easy method for the preparation of the second form. Different analytical techniques are used to investigate the polymorphs. XRPD proved the existence of different crystal polymorphs. Thermal analysis shows polymorph I melting at 230 ◦ C while polymorph II melts at 216 ◦ C. A small endother-
Acknowledgements The authors appreciate and acknowledge DSM nutritional products (Kaiseraugst, Switzerland) for the donation of crystalline materials and financial support; the authors also would like to thank Prof. Dr. H.C. Reinhard Neubert and his group member, Ms. Heike Rudolf at Pharmaceutical Analytic–Central Laboratory, Martin-Luther Universität Halle-Wittenberg for their valuable measurements of Raman spectroscopy.
References Auweter, H., Benade, J. and Bettermann, H., 1999, Association and self-assembly of carotenoids: structure and stability of carotenoid aggregates, In Proceed. Pigments in Food Technology Sevilla, , pp. 197–201. Auweter, H., Bohn, H. and Lüddecke, E., 2005, Stable, aqueous dispersions and stable, water dispersible dry powders of xanthophylls, and production and use of the same, Patent No. 6.863.914 B1. Bartalucci, G., Coppin, J. and Fisher, S., 2007, Unravelling the chemical basis of the bathochromic shift in the lobster carapace; new crystal structures of unbound astaxanthin, canthaxanthin and zeaxanthin. Acta Crystallogr B, 63: 328–337. Britton, G., (1995). Carotenoids. (Birkhäuser, Basel). Isler, O., (1971). Carotenoids. (Birkhäuser, Basel). Mori, Y., 2001, Introductory studies on the growth and characterization of carotenoid solids: an approach to carotenoid solid engineering. J Raman Spectrosc, 32: 543–550. Runge, F., Lüddecke, E. and Auweter, H., 2003, Production of dry powders of one or more oxygenated carotenoids, Patent No. 6.639.113. Weesie, R.J., Merlin, J.R., Groot and De, H.J.M., 1995, Resonance Raman spectroscopy and quantum chemical modeling studies of protein-astaxanthin interactions in ␣-Crustacyanin (major blue carotenoprotein complex in carapace of lobster, homarus gammarus). Biospectroscopy, 5: 358–370.
Contents lists available at ScienceDirect
Chemical Engineering Research and Design journal homepage: www.elsevier.com/locate/cherd
Polymorphism of 3,3 -dihydroxy-,-carotene-4,4 -dione (Astaxanthin) Jingfei Guo, Matthew J. Jones, Joachim Ulrich ∗ Martin-Luther-Universität Halle-Wittenberg, Zentrum für Ingenieurwissenschaften, Verfahrenstechnik/TVT, D-06099 Halle (Saale), Germany
a b s t r a c t Astaxanthin (AXT) 3,3 -dihydroxy-,-carotene-4,4 -dione, from the group of carotenoid, specifically as xanthophyll, is investigated in this work on its polymorphism. The method to obtain a second polymorph of AXT is described. Polymorph I can be purchased as a commercial product or obtained via phase transformation from polymorph II. Polymorph II can be prepared from polymorph I by a thermal treatment carried out in a differential scanning calorimetry (DSC). The resulting crystals are characterized by different analytical techniques. X-ray powder diffractometer (XRPD) and resonance Raman spectroscopy prove the existence of two different crystalline polymorphs. Simultaneous thermal analysis (STA) gives additional information on their thermal behaviors. Polymorph I melts at 230 ◦ C and polymorph II melts at 216 ◦ C. Phase transition experiment carried out by slurry conversion experiment reveals two polymorphs are monotropically related. Polymorph II is the metastable form. © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Polymorphism; Characterization; XRPD; Raman spectroscopy; Phase transformation
1.
Introduction
Carotenoids are the most important group of natural pigments in the vegetable and animal kingdoms. The rate of progress in the research of carotenoids increased rapidly, in 1971, the famous monograph of Isler (1971) recorded around 230 carotenoids with well-established constitutions. In 1993, the number of basic structures exceeded 600 (Britton, 1995), which mainly attributed to the improvements in chromatographic procedures and spectroscopic methods. Here the solid-state properties of 3,3 -dihydroxy-,carotene-4,4 -dione (Astaxanthin) (see Fig. 1) are investigated. Astaxanthin (AXT) is a carotenoid responsible for the pink coloration in salmonidea, shrimp and lobster, and is of interest due to its antioxidant properties resulting from the specific molecular structure consisting of a linear chain with conjugated double bonds (polyene). Like all carotenoids, AXT is insoluble in water and has a low solubility in fats and oils, which highly restricts the application as feed additive for aquatic animals. Different methods have been described to improve the properties, e.g. decreasing the crystal size to a particle size range of smaller than 10 m
∗
(Runge et al., 2003) and increasing amorphous content up to 70–100% (Auweter et al., 2005). However, few papers described about polymorphism in carotenoids which could be an option to improve their limited properties. In 2001, Mori suggested the presence of polymorphism or dimorphism as a common characteristic of carotenoid crystals according to their absorption spectra. The results showed further controllability of the molecular arrangement indicating the existence of solid phases other than those observed (Mori, 2001). Since polymorphism is a common phenomenon of organic substances, AXT is selected as a model substance from the carotenoid family used to study the existence of polymorphs in these compounds, and thus, to select a desired polymorph with better properties, to provide insight into similar research work for other carotenoids. In 2007, Bartalucci reported the crystal structure of solvated AXT and one non-solvated AXT (Bartalucci et al., 2007). Two solvated AXT, chloroform solvate and pyridine solvate can exist at extremely low temperature (100 K) which are not to be chosen as the commercial crystalline forms for production. For non-solvated AXT, one triclinic crystal structure was reported.
Corresponding author. E-mail address: [email protected] (J. Ulrich). Received 5 December 2008; Received in revised form 15 October 2009; Accepted 8 February 2010 0263-8762/$ – see front matter © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cherd.2010.02.005
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1648–1652
1649
Fig. 1 – Chemical structure of the carotenoid AXT. However, no direct information can be found regarding the existence of polymorphism for non-solvated AXT. In this paper, the preparation of a second polymorph of AXT is described. The crystals were characterized by simultaneous thermal analysis (STA), X-ray powder diffraction (XRPD) and resonance Raman spectroscopy. Slurry conversion experiment is performed to investigate on the phase stability.
2.
Methods and materials
2.1.
Materials
AXT polymorph I was obtained commercially and used as it is. Solvent of ethyl acetate (EtOAc) was used from Roth (Rotisolv® ) at HPLC grade. Polymorph II can be prepared by the heat treatment or evaporation from certain solvents, such as acetone. Due to the low solubility (approximately 8 mg 100 mL−1 in acetone at 20.5 ◦ C), only a very small amount of crystals can be obtained after evaporation. Heat treatment was therefore used as the main method for crystal preparation. Polymorph II was produced by melting of polymorph I at 224 ◦ C followed by quenching process. Heating of polymorph I was carried out in a DSC (Netzsch Phoenix 204) cell. Care should be taken that AXT is decomposed after melting, considering the temperature lag between the instrument and the sample, temperature need to be set slightly below the real melting temperature of polymorph I where decomposition may occur. Approximately 6–7 mg crystals of AXT polymorph I were heated in the Al-pan at a heating rate of 5 K min−1 , from 20 ◦ C to 200 ◦ C, and then slowly heated up to 224 ◦ C at a heating rate of 2 K min−1 . Immediately after this temperature was reached, the sample was cooled down from 224 ◦ C to 20 ◦ C at a cooling rate of 40 K min−1 . The DSC system was purged with nitrogen throughout. XRPD confirms the second crystal polymorph of AXT.
polymorph I and II were heated up from 28 ◦ C to 500 ◦ C at a heating rate of 5 K min−1 while purged with helium. The mass changes during heating were recorded.
3.3. Preparation of AXT polymorph II at different maximal temperatures The best heating temperature was studied by calculating the composition of the final crystals from their melting temperatures. In order to achieve this, a calibration of melting temperatures versus compositions was built up first. The mixtures of polymorphs were prepared at different compositions from 0% to 100% at an interval of 10% and heated up at 5 K min−1 , from 20 ◦ C to 250 ◦ C. After obtaining the calibration curve, 5–6 mg AXT polymorph I was heated up by DSC with a heating rate of 5 K min−1 , from 20 ◦ C to 200 ◦ C and, heated again with a smaller heating rate of 2 K min−1 , from 200 ◦ C to 218 ◦ C, 220 ◦ C, 222 ◦ C, and 224 ◦ C respectively, cooled down at 40 K min−1 to the initial conditions. By calculation of composition of resulting crystals, the best heating temperature resulting in the highest purity of polymorph II was obtained and the heating process was optimized.
3.4.
Resonance Raman spectroscopy (FT-Raman-Specktrometer RFS 100/S, Bruker) was used to identify the polymorphs of AXT. This technique is a vibrational molecular spectroscopy which derives from an inelastic light scattering process. The measurement was performed on a FT-Raman spectrometer with Raman microscopy and temperature control unit. The laser was operated at 1064 nm with back scattering optics. A stainless sample holder is used which can hold amount of several micrograms. Crystals of polymorph I and II were measured respectively at solid state.
3.5.
3.
Methods
3.1.
X-ray powder diffraction studies
The X-ray powder diffraction patterns were obtained by collecting intensity data measured by a Bruker D4 diffractometer. The system is equipped with a Cu anode and a monochromator providing Cu K␣1 radiation ( = 1.54056 Å). Measurements were carried out using a step width of 0.005◦ and an acquisition time of 4 s per step.
3.2.
Thermal analysis
A STA (Netzsch STA-409) referring to the simultaneous measurement of caloric effects and mass changes was used here to study the thermodynamic properties of different polymorphs and their thermal stability. Approximately 5–6 mg samples of
Raman spectroscopy
Slurry conversion experiment
Slurry conversion experiment was carried out here by suspension of excess amount of polymorph II in EtOAc in double jacket beakers controlled by thermostat at a constant temperature of 50 ◦ C. The suspensions were stirred at a constant rate of 300 rpm. Samples were taken out at different retention times, filtered and dried. Finally, the resulting crystals were analyzed by XRPD.
4.
Results and analyses
4.1.
XRPD patterns of different polymorphs
Fig. 2 shows the pattern of crystals prepared by DSC compared with the original polymorph I. The pattern of crystals illustrates different characteristic peaks in the scanning range indicating a generation of second crystalline form.
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Table 1 – Mixture at different compositions.
Fig. 2 – Comparison between XRPD patterns of AXT polymorphic polymorph I and II. For example, the high intensity peak observed at 20.35◦ in polymorph I is absent in the pattern of the other crystals. Also, two peaks observed at 2 = 11.07◦ and 18.32◦ of the second crystals do not match the pattern of polymorph I. Therefore, a different crystalline form, denoted polymorph II, of AXT was successfully prepared by the thermal treatment carried out by DSC. The patterns are compared with the calculated peak positions from the reported crystal structure (Bartalucci et al., 2007), and the comparison shows good agreement which confirms that the crystal structure obtained by Bartalucci is polymorph II in our work. Concerning the generation of polymorph II by Bartalucci et al. underwent a solution crystallization process, therefore, different methods could be used for obtaining polymorph II. The XRPD pattern of polymorph I reported in our work is new.
4.2.
Thermal analysis
Thermal analyses, in particular DSC and TG, have been the main methods to study polymorphism. Fig. 3 shows the results of different melting points and the heat of fusion of AXT of polymorph I and II. The thermal stability is also investigated by the TG technique. From Fig. 3, the DSC curves show one endothermic event between 220 ◦ C and 232 ◦ C for polymorph I and the other in
Sample
Composition
a. b. c. d. e. f. g. h. i. j. k.
I 100.0%, II 0.0% I 87.3%, II 12.7% I 80.8%, II 19.2% I 70.2%, II 29.8% I 60.4%, II 39.6% I 50.2%, II 49.8% I 40.3%, II 59.7% I 31.7%, II 68.3% I 20.4%, II 79.6% I 10.5%, II 89.5% I 0.0%, II 100.0%
MassI (%) 100.00 87.26 80.78 70.21 60.43 50.24 40.32 31.65 20.35 10.46 0.00
Melting point (◦ C) 230.80 226.80 225.20 224.30 223.40 222.00 221.70 219.30 220.40 217.10 216.60
the range between 200 ◦ C and 220 ◦ C for polymorph II, recognized as melting of the samples. In addition, another small endothermic peak at 215 ◦ C is also observed on the heating trace of polymorph I. The mass losses before melting are 3.57% for polymorph I and 5.50% for polymorph II which is probably due to heat instability. Since polymorph II is generated by melting and quenching process without any additional usage of solvents, no solvate exists in this work. Thermoanalytical data indicate that a decomposition process occurs immediately after melting for both polymorphs corresponding to the sudden mass loss. This result ascertains that heating up of polymorph I to 224 ◦ C is sensible as it avoids the decomposition and loss of the substance. Due to the heat instability, temperature cycle profile is not suitable for further investigation on the thermal behavior of AXT. Temperature controlled XRPD and hot-stage microscopy were also used to determine the possible events around the small peak observed in the STA at approximately 215 ◦ C. No solid–solid phase transformation is found. It is assumed that this small peak is due to a small amount of polymorph II presenting in the original crystals.
4.3. Preparation of AXT polymorph II at different maximal temperatures The crystal samples of polymorph I and II at different compositions were heated up by DSC at 5 K min−1 , from 20 ◦ C to 250 ◦ C. A standard curve of melting points versus composition was built up (Table 1). The melting point decreases with the increase of the content of polymorph II in the mixture. The points were plotted in Fig. 4 to obtain a polynomial fit between the melting points and the compositions. The equation is expressed as: C = −0.1836x2 + 89.433 x − 10757
Fig. 3 – STA analysis of AXT polymorph I and II; sample subjected to a heating rate of 5 K min−1 in a perforated Al-pan (1 hole) under helium flow.
which can be used to calculate the purity of polymorph II that exists in mixture as the final product, where “x” is the melting temperature of the mixture of polymorphs I and II. The best heating temperature is studied by calculating the composition of the final crystals from the melting points (Tables 2 and 3). Therefore, the best heating temperature is 224 ◦ C. The crystals obtained at this temperature give highest purity as calculated.
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Table 4 – Results of resonance Raman spectroscopy. CH3 (cm−1 )
Name Polymorph I Polymorph II J-aggregate (Auweter et al., 1999) H-aggregate (Auweter et al., 1999) Literature (Weesie et al., 1995)
Fig. 4 – Plots of melting points versus composition of polymorph I in the mixture.
4.4.
Raman spectroscopy
The resonance Raman spectra of AXT polymorph I and II are shown in Fig. 5 (Table 4). The most intense band at 1510 cm−1 is assigned to the C C stretch vibrations of the polyene chain. The second most intense band at 1155 cm−1 , which represents the superposition of two modes that can be ascribed to C–H in-plane bending vibrations mixed with C–C stretching and C C–C bending
1005.0 1006.0 –
C H (cm−1 ) 1155.4 1157.3 –
–
1008.0
–
1159.0
C C (cm−1 ) 1510.4 1513.4 1516.0
1522.0
1523.0
vibrations, respectively. The third intense line at 1004 cm−1 can be assigned to CH3 in-plane rocking vibrations. Although Raman spectra of polymorph I and II are similar, still differences can be found in the region from 500 cm−1 to 1000 cm−1 , for instance, polymorph II has only one sharp band at 966 cm−1 whereas polymorph I shows two bands. These differences are useful information for identification of polymorph I and II in the mixture and formulation.
4.5.
Slurry conversion experiment
Solvent-mediated polymorphic transformation is an efficient technique to study the phase stability of different polymorphs
Table 2 – Preparation of the AXT polymorph II by DSC. Name a b c d
Segment 1 (◦ C) 20–200 20–200 20–200 20–200
Heating rate (K min−1 )
Segment 2 (◦ C)
5 5 5 5
200–218 200–220 200–222 200–224
Heating rate (K min−1 ) 2 2 2 2
Purged gas Nitrogen Nitrogen Nitrogen Nitrogen
Table 3 – Calculated composition of polymorph II obtained at different heating condition. Name 1 2 3 4
Heated temperature (◦ C) 218 220 222 224
Melting point (◦ C) 226.4 223.9 218.4 216.6
Calculated massI of polymorph I (%) 80.91 64.50 17.84 0.00
Fig. 5 – Comparison of Raman spectra of AXT polymorph I and II in the different regions, (a) Raman spectra of AXT polymorph I and II in the range from 500 cm−1 to 1800 cm−1 ; (b) Raman spectra of AXT polymorph I and II in the range from 500 cm−1 to 1000 cm−1 .
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mic peak observed at 215 ◦ C on the heating trace of polymorph I is likely to be the presence of a small amount of polymorph II in the original crystals. Decomposition up to 95% of two polymorphs indicates their thermal instability. The best heating temperature for obtaining pure polymorph II is at 224 ◦ C. Raman spectra provide sufficient information to identify the polymorphs. Differences in peak position and shifts of Raman bands between polymorph I and II make it possible to distinguish the mixture of both polymorphs at ambient condition. Finally, slurry conversion experiments reveal that two polymorph are monotropically related. Polymorph I is the thermodynamically stable form.
Fig. 6 – XRPD patterns of crystals obtained from suspensions in EtOAc at the temperature of 50 ◦ C for different retention time, (a) 60 min; (b) 120 min; (c) 180 min; (d) 1 day; (e) 3 days; (f) 5 days; (g) 9 days. and to obtain the most stable polymorph. In the case of EtOAC the phase transformation is observed at a temperatures of 50 ◦ C after 3 days. Fig. 6 shows the results of XRPD measurements at different retention time. In the XRPD patterns, characteristic peaks of polymorph II disappear with the increase of the retention time. Some new peaks belonging to polymorph I appear after 3 days. The color change of the suspension from red to violet is observed when the transformation takes place simultaneously. After 5 days, this transformation is completed. Further experiments at the temperatures above 50 ◦ C show the same tendency of the phase transformation from polymorph II to polymorph I. However, the experiments carried out below this temperature demonstrate a relatively longer transformation period. Therefore, conclusion can be draw that the polymorph I and II are monotropically related. Polymorph I is the thermodynamically stable form.
5.
Discussions and conclusions
AXT, a super antioxidant from the carotenoid family, is found to be polymorphic substance with two non-solvated polymorphs. A second crystal polymorph generated by heat treatment is carried out in a DSC cell. DSC can be used as a reproducible and easy method for the preparation of the second form. Different analytical techniques are used to investigate the polymorphs. XRPD proved the existence of different crystal polymorphs. Thermal analysis shows polymorph I melting at 230 ◦ C while polymorph II melts at 216 ◦ C. A small endother-
Acknowledgements The authors appreciate and acknowledge DSM nutritional products (Kaiseraugst, Switzerland) for the donation of crystalline materials and financial support; the authors also would like to thank Prof. Dr. H.C. Reinhard Neubert and his group member, Ms. Heike Rudolf at Pharmaceutical Analytic–Central Laboratory, Martin-Luther Universität Halle-Wittenberg for their valuable measurements of Raman spectroscopy.
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