CRYSTAL GROWTH & DESIGN 2003 VOL. 3, NO. 6 869-872
Communications Inclusion of the Stable Form of a Polymorph within Crystals of Its Metastable Form Caitriona Cashell,†,‡ David Sutton,† David Corcoran,†,§ and B. Kieran Hodnett*,†,‡ Materials and Surface Science Institute, Department of Chemical and Environmental Sciences, and Department of Physics, University of Limerick, Limerick, Ireland Received June 9, 2003;
Revised Manuscript Received July 31, 2003
ABSTRACT: We have used a variety of microscopy and spectroscopy techniques to study the crystal interior of L-glutamic acid (L-Glu) during a solvent-mediated polymorphic transformation process. Remarkably, we have found the encapsulation of the stable form of L-Glu within the metastable form. In particular, we have verified the encapsulation using the novel approach of focused ion beam microscopy, which although extensively used in the semiconductor industry, has not previously been used in the study of polymorphism. It has long been predicted that during cooling crystallization, nucleation of more than one polymorph can occur at any one time, if the solution is supersaturated with respect to more than one form. This work presents experimental evidence to support this hypothesis. On the basis of these results, we propose a new mechanism for polymorphic transformation, with implications for the crystal growth of every compound that exhibits polymorphism. Compounds that exist in more than one crystalline form are known as polymorphs.1,2 The metastable form crystallizes first, and subsequently transforms to the next most stable state, until the energy is minimized and the most stable form is achieved.1,3 Transformation can occur in the solid state, as exemplified by aspartame,4 or in solution as exemplified by taltireline,5 carbamazepine,6 and sulfamerazine.7 Crystal polymorphism occurs extensively in organic compounds,2,8 and in particular in pharmaceuticals.1 To date the mechanism of polymorphic transformation is not well understood. Secondary nucleation of the stable form on the surface of the metastable form was observed recently for 2,6-dihydroxybenzoic acid9 and L-glutamic acid.10,11 Here we show that the mechanism of polymorphic transformation involves inclusion of the stable form of a polymorph within the metastable form, after secondary nucleation of the stable form on the surface of the metastable crystals, suggesting that nucleation of the stable polymorph proceeds at a much earlier stage in the crystallization process than previously believed. In a polymorphic system, kinetics favors the formation of the metastable form, while thermodynamics favors formation of the stable form.2 Nucleation and growth of the metastable form will therefore predominate initially in accordance with Ostwald’s law of stages.3 In crystallization kinetics, the critical step for the production of different polymorphs is nucleation.1 For a dimorphic monotropic system, a solution that is supersaturated with respect to the stable polymorph only, can only result in nucleation of this form. Conversely, nucleation of both forms is predicted, * To whom correspondence should be addressed. E-mail: kieran.hodnett@ ul.ie. † Materials and Surface Science Institute. ‡ Department of Chemical and Environmental Sciences. § Department of Physics.
although it has not been verified experimentally, if the solution is supersaturated with respect to both the metastable and stable forms.9,12 L-Glutamic acid (L-Glu) is dimorphic,13 occurring in both R- and β-forms; the crystal structures of which were previously determined.14,15 The metastable and stable forms are termed R- and β-, respectively. The polymorphic L-Glu transformation is solutionmediated, and proceeds via a dynamic process of dissolution of R- and growth of β-16 until the free energy of the system is minimized.17 Nucleation provides the starting point for the transformation from R- to β-, which was found to occur by secondary nucleation of β- on the surface of R-L-Glu crystals.10 Various techniques have been used for the identification of polymorphs including crystallography, microscopy, thermal and spectroscopic methods.1,18-20 The observations detailed in this paper provide first time evidence for inclusion of β-L-Glu crystals within R-L-Glu crystals, using a combination of focused ion beam (FIB) microscopy and Raman spectroscopy, and they are supported by X-ray diffraction and scanning electron microscopy (SEM). The results not only strengthen previous findings on secondary nucleation,9,10 but advance a new theory that the transformation process begins at a much earlier stage than previously believed. The R-polymorph of L-Glu was synthesized by acidification of L-Glu monosodium salt monohydrate (Sigma-Aldrich) using 37% HCl (BDH), as previously outlined.21 Recrystallization of R-L-Glu was performed by quench cooling a 0.3 M supersaturated aqueous solution from 80 to 45 °C.21,22 The crystallizing solution was maintained at 45 °C for 6 h. Supersaturation is defined as σ ) (C-Ce)/ Ce where C and Ce are the supersaturation and equilibrium concentrations, respectively. All experiments were performed without agitation or scratching of the crystal
10.1021/cg034094a CCC: $25.00 © 2003 American Chemical Society Published on Web 09/05/2003
870
Crystal Growth & Design, Vol. 3, No. 6, 2003
Communications
Figure 1. Evidence of inclusion of β-crystals in R-L-Glu. (a) FIB image, (b) FIB cross-section, and (c) X-ray diffractogram with electron micrograph (inset): [Glu]0 ) 0.3 M; σ ) 1.12; Tsat ) 80 °C; Tcrys ) 45 °C; tcrys ) 6 h (a and b) and 1 h (c).
surface. Crystals of the R-polymorph of L-Glu (approximately 500 µm in size) were placed in a suitable mould, and mounted in slow-setting Araldite resin. Once set, the mounted sample was polished using successive coarse and fine silicon carbide paper (1200 and 4000 grit). Distilled water was employed during polishing as a differential etch to the individual polymorphs, while also acting as a coolant. This procedure provided a mounted cross-section through a number of R-L-Glu crystals. Whole crystals were also imaged after dispersion onto conductive adhesive tape, which was then mounted on an aluminum stub. Microscopy of the mounted samples was facilitated using an FEI FIB 200 microscope: 10-30 keV, 1 pA - 11 500 pA beam current, 9 nm - resolution, 50 × 50 mm stage. Mounted crystals were gold coated prior to imaging. Raman spectroscopy was performed on the uncoated samples using a LABRAM laser Raman spectrometer, as previously outlined.10 Excitation radiation at 632.8 nm was employed using a He-Ne red laser. Spectra of R- and β-crystals in the mounted composite R-β sample were compared to pure R- and β-spectra. Scanning electron microscopy was performed as previously described,10 and X-ray diffraction was performed using a Philips X’Pert-MPD diffractometer with nickel filtered copper KR radiation (λ ) 1.542 Å) as the X-ray source. Microscopy of R-L-Glu crystals, displayed extensive occurrence of β-crystals within R-crystals, i.e., composite R-β crystals. Figure 1a displays a FIB electron image of an individual R-L-Glu crystal. From this image, there is clear evidence for growth of β-crystals out of R-L-Glu crystals, suggesting that the β-crystals become encapsulated in the R-crystals. The surface structure of the two polymorphs is distinct, as evidenced by the dissolving R-crystals and the well-facetted β-crystals. Figure 1b displays a FIB electron image of a mounted cross-section of a composite R-β crystal. The R-crystal appears to contain several β-crystals within its bulk, and there is further evidence for the
emergence of the β-crystals out of the R-crystal. Differences in the solubility between the two polymorphs,23 lead to raised β-features and increased contrast between the two phases in composite R-β crystals. This facilitated identification of the R-β phase boundaries, for subsequent characterization by Raman spectroscopy. Encapsulation was observed on many occasions, and accounts for the extensive presence of β-form using X-ray diffraction, in what appeared from microscopy to be pure R-form crystals. One such example of this is presented in Figure 1c, in which an electron micrograph of L-Glu crystallized for 1 h displays pure R-form, but the diffractogram displays a considerable quantity of the β-polymorph, suggesting that polymorph identification cannot always be made on a visual basis. Figure 2a depicts a milled cross-section of an R-β interface, which clearly displays that the β-form extends deeply into the bulk of the R-form crystals (Figure 2b), and does not remain on the surface. All the FIB images presented here provide dramatic evidence for the nucleation of β-crystals within, and emerging from R-crystals. The β-form crystals that emerge from the R-form crystals display a preponderance of (001) and (010) facets. However, it is not possible on the basis of this investigation to associate the origin of this growth with any particular facet of the R-form because of the manner in which the R-form encapsulates the β-form. Confirmation of the observations obtained for all crosssectioned crystals examined was facilitated using Raman spectroscopy. Figure 3 displays optical micrographs and Raman spectra of the R- and β-constituents of a composite R-β crystal, showing β-crystals within a large R-L-Glu crystal (Figure 3a, ×10), with the R- and β-sections denoted c and b, respectively. Higher magnification images (×50) of the R- and β-sections display the surface structure of the two polymorphs, as shown in Figure 3, panels c and b. Raman spectra of the different phases evident in these sections were obtained (Figure 3d). The spectra of the
Communications
Crystal Growth & Design, Vol. 3, No. 6, 2003 871
Figure 2. Milled cross-section of the R-β interface of L-Glu. (a) Milled section showing R- and β-crystal, (b) phase interface of a β-crystal growing within R-L-Glu: [Glu]0 ) 0.3 M; σ ) 1.12; Tsat ) 80 °C; Tcrys ) 45 °C; tcrys ) 6 h.
Figure 3. Optical micrographs and Raman spectra of β-crystals growing within R-L-Glu. (a) Micrograph ×10; (b) micrograph ×50 β-L-Glu; (c) micrograph ×50 R-L-Glu; (d) Raman spectra of R- and β-L-Glu: [Glu]0 ) 0.3 M; σ ) 1.12; Tsat ) 80 °C; Tcrys ) 45 °C; tcrys ) 6 h.
morphological R- and β-features were consistent with pure R- and β-spectra, respectively.10
Since a supersaturated solution of L-Glu is saturated with respect to both polymorphs,12 and by virtue of the
872
Crystal Growth & Design, Vol. 3, No. 6, 2003
Communications (Figure 4a). The R-form therefore redissolves, as seen in Figure 1a, providing an increased local supersaturation for simultaneous continued growth of the β-crystals, formed at the initial nucleation stage. β-Growth. As the solution continues to crystallize, new β-nuclei form advantageously on the R-crystal surface, and the encapsulated β-nuclei continue to grow, once they emerge from the dissolving R-crystals and contact the supersaturated solution (schematic representation outlined in Figure 4b).
References
Figure 4. Mechanism of transformation. (a) Solubility curves (adapted from Cardew and Davey, 1985) and (b) schematic of the L-Glu transformation mechanism.
similarities in structure of R- and β-polymorphs, it is reasonable to assume that β-crystals adsorb onto, and subsequently become incorporated into, R-L-Glu crystals. The FIB electron images, combined with Raman spectroscopy provide unparalleled evidence of this, suggesting that the β-nucleation process begins at a much earlier stage than previously believed. The mechanism proposed is shown in Figure 4. R-Nucleation. In agreement with Ostwald’s law of stages, as the supersaturated L-Glu solution decreases to the solubility of the R-form, primary homogeneous nucleation of the R-form occurs. β-Nucleation. This is followed successively by secondary nucleation of β-L-Glu nuclei advantageously on the R-surface, followed by slight growth of β-nuclei. R-Growth. Growth of R-crystals occurs (kinetics favors this), until the solubility of the R-form is attained, thereby encapsulating the β-crystals in the R-crystals, by R-form overgrowth. On surface inspection, this may appear as though the β-crystals are nucleating on the R-surface, and not in the crystal interior as evidenced by FIB crosssections. R-Dissolution. Once the R-solubility is reached, the solution is only supersaturated with respect to the β-form
(1) Brittain, H. G., Ed. In Polymorphism in Pharmaceutical Solids, 95 Vols.; Marcel Dekker: New York, 1999. (2) Bernstein, J. In Polymorphism in Molecular Crystals; Oxford University Press: Oxford, 2002. (3) Ostwald, W. Z. Phys. Chem. 1897, 22, 289-330. (4) Zell, M. T.; Padden, B. E.; Grant, D. J. W.; Schroeder, S. A.; Wachholder, K. L.; Prakash, I.; Munson, E. J. Tetrahedron 2000, 56, 6603-6616. (5) Maruyama, S.; Ooshima, H. J. Cryst. Growth 2000, 212, 239-245. (6) Nair, R.; Gonen, S.; Hoag, S. W. Int. J. Pharm. 2002, 240, 11-22. (7) Gu, C.-H.; Chatterjee, K.; Young, V., Jr.; Grant, D. J. W. J. Cryst. Growth 2002, 235, 471-481. (8) Verma, A. R.; Krishna, P. In Polymorphism and Polytypism in Crystals; John Wiley & Sons: London, 1966. (9) Davey, R. J.; Blagden, N.; Righini, S.; Alison, H.; Ferrari, E. S. J. Phys. Chem. B 2002, 106, 1954-1959. (10) Cashell, C.; Corcoran, D.; Hodnett, B. K. Chem. Commun. 2003, 3, 374-375. (11) Ferrari, E. S.; Davey, R. J. Chem. Eng. Sci. 2003, in preparation. (12) Cardew, P. T.; Davey, R. J. Proc. R. Soc. London 1985, A398, 415-428. (13) Hirokawa, S., Acta Crystallogr. 1955, 8, 637-641. (14) Lehmann, M. S.; Nunes, A. C. Acta Crystallogr. 1980, B36, 1621-1625. (15) Lehmann, M. S.; Koetzle, T. F.; Hamilton, W. C. J. Cryst. Mol. Struct. 1972, 2, 225-233. (16) Kitamura, M. J. Cryst. Growth 1989, 96, 541-546. (17) Bernstein, J.; Davey, R. J.; Henck, J.-O. Angew. Chem. Int. Ed. 1999, 38, 3440-3461. (18) Vippagunta, S. R.; Brittain, H. G.; Grant, D. J. W. Adv. Drug Delivery Rev. 2001, 48, 3-26. (19) Yu, L.; Reutzel, S. M.; Stephenson, G. A. Pharm. Sci. Technol. Today 1998, 1, 118-127. (20) Threlfall, T. L. Analyst 1995, 120, 2435-2460. (21) Garti, N.; Zour, H. J. Cryst. Growth 1997, 172, 486-498. (22) Kitamura, M.; Funahara, H. J. Chem. Eng. Jpn. 1994, 27, 124-127. (23) Sakata, Y. Agric. Biol. Chem. 1961, 25, 835-837.
CG034094A
