Published as part of a special issue of selected papers presented at the 8th International Workshop on the Crystal Growth of Organic Materials (CGOM8), Maastricht, Netherlands, September 15-17, 2008
CRYSTAL GROWTH & DESIGN 2009 VOL. 9, NO. 6 2719–2724
Characterization of Defects Inside Single Crystals of Ciclopirox N. Couvrat,† A. S. Blier,‡ B. Berton,*,‡ Y. Cartigny,† V. Dupray,‡ and G. Coquerel† Unite´ de Croissance Crystalline et Mode´lisation Mole´culaire, EA3233 UniVersite´ de Rouen, F-76821 Mont-Saint-Aignan Cedex, France, and La2B, EA 3233 Centre UniVersitaire d’EVreux, 1 rue du 7e`me Chasseurs - BP 281, F-27002 EVreux Cedex, France ReceiVed December 14, 2008; ReVised Manuscript ReceiVed March 30, 2009
ABSTRACT: Different types of defects observed inside single crystals of ciclopirox (obtained by recrystallization in ethyl acetate) are characterized. Confocal Raman spectroscopy and optical microscopy analyses revealed the coexistence of three types of defects: (i) liquid inclusions containing saturated solution with or without gas bubbles inside these inclusions, (ii) open cavities with the shape of channels, (iii) isolated gas bubbles inside the crystal matrix. It seems that the two first types of defects originate from the trapping of mother solution during crystal growth. The trapped supersaturated solution continues to crystallize inside the defect, until the formation of a negative crystal (liquid inclusion having the shape of a single crystal). This crystallization is associated with a decrease in volume, responsible in some cases for the formation of a gas bubble. However, if the liquid pocket is not sealed when the crystal is extracted from the mother solution, it produces open channels by evaporation of the solvent. The formation of isolated gas bubbles seems to result from another mechanism. Introduction In the pharmaceutical industry, the internal quality of organic crystals is a crucial point in the optimization of crystallization processes.1 In some cases, the appearance of large ill-defined defects in crystalline particles can result from many factors controlling the crystal growth: supersaturation, presence of impurities of the solute and/or of the solvent, colloidal particles, etc. Among these defects, liquid inclusions are rather frequent and have been defined as “small pockets of liquid impurities entrapped in crystals”.2 Only a few studies have been devoted to their characterization,2-4 usually with the aim to control and minimize the pollution of pharmaceutical powders; indeed, the final purification step in many pharmaceutical drug processes is a crystallization. The incorporation of mother liquor in bulk materials during this step can compromise the purification efficiency (i.e., solvent may contain dissolved impurities4). Furthermore, trapped solvent may be toxic or cause degradation of the drug. In the case of inorganic crystals,5 the study of liquid inclusions have sometimes revealed that this type of defect may present a particular morphology similar to that of the crystal, called “negative crystals”.6-8 The reasons for the formation of liquid inclusions remain unclear, but it may have various causes and probably does not proceed through a single mechanism. Recently, Berton et al.9 have observed another type of defect inside trehalose dihydrate single crystals, which appeared to consist of gas inclusions. The influence of gas defects on enduse properties has not been studied, but it was proved that they affect locally the dehydration mechanisms of trehalose. This phenomenon is frequently observed in geological crystals * To whom correspondence should be addressed. Phone: +33 (2) 32 39 90 87. E-mail:
[email protected]. † UC2M2, EA3233 Universite´ de Rouen. ‡ La2B, EA 3233 Centre Universitaire d’Evreux.
Figure 1. Developed formula of ciclopirox molecule.
obtained by hydrothermal synthesis5,10 but had not been reported yet in the case of organic molecules. Ciclopirox (6-cyclohexyl-1-hydroxy-4-methylpyridin-2-one) (Figure 1) is a pharmaceutical drug used as an ethanolamine salt or as a covalent compound11 in order to prevent and to heal fungal infections.12 The purification of this molecule is carried out by a recrystallization of the raw material in ethyl acetate. The observation of ciclopirox crystals by optical microscopy has revealed different types of micro- (1-5 µm) and macroscopic (10-100 µm) defects inside single crystals. This paper presents a description of these defects. Moreover, this study aims at elucidating, with the help of Raman microspectroscopy, the composition of these defects in order to better understand their formation. Experimental Section Materials. Micro and macro defects were observed inside single crystals obtained by recrystallization of ciclopirox in ethyl acetate. Ciclopirox (compliant with European and US pharmacopeias) was supplied by PCAS industry (Produits Chimiques et Auxilliaires de Synthe`se, Longjumeau, France), and HPLC grade ethyl acetate (purity >99.97%) was purchased from Acros. In order to study the influence of crystallization conditions on the formation of defects, 16 recrystallization processes were tested (Table 1). Saturated solutions of
10.1021/cg801355q CCC: $40.75 2009 American Chemical Society Published on Web 05/15/2009
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Table 1. Different Processes of Ciclopirox Recrystallization in Ethyl Acetatea process no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
T1 initial (°C) T2 final (°C) solvent cooling process CR (°C min-1) β (C/Csat)
60 20 a s ≈ 0.5 2.3
60 20 b s ≈ 0.5 1.9
60 20 a q ≈7 2.3
60 20 b q ≈7 1.9
20 4 a s ≈ 0.3 1.6
20 4 b s ≈ 0.3 1.6
20 4 a q ≈2 1.6
20 4 b q ≈2 1.6
20 7 a s ≈ 0.3 1.5
20 7 b s ≈ 0.3 1.4
20 7 a q ≈2 1.5
20 7 b q ≈2 1.4
20 13 a s ≈ 0.3 1.3
20 13 b s ≈ 0.3 1.2
20 13 a q ≈ 1.5 1.3
20 13 b q ≈ 1.5 1.2
a T1 and T2: initial and final temperature; solvent: (a) dry ethyl acetate, (b) water saturated ethyl acetate; cooling process: (s) slow cooling, (q) quenched; CR: average cooling rate and β: maximum supersaturation at T2.
Figure 2. Raman reference spectra of compounds that may be present inside ciclopirox crystals. (a) Ciclopirox powder, (b) dry ethyl acetate, (c) ethyl acetate saturated with water, (d) saturated solution of ciclopirox in ethyl acetate (mother liquor), (e) water saturated by ethyl acetate. ciclopirox in ethyl acetate were prepared at temperatures T1 in a sealed vial and then slowly or rapidly (quenching) cooled to temperature T2, without stirring. The various cooling rates are presented in Table 1. For every process, the influence of water content in solvent was also inspected by using “dry” (a) or “water saturated” (b) ethyl acetate (saturated with about 3% of water at ambient temperature). Nucleation occurs instantaneously or after a few hours depending on the protocol. Whatever the protocol that was used, most single crystals containing defects were obtained. However, these experiments failed to show any link between the crystallization conditions and the nature of defects. Microscopy Analysis of Single Crystals. Single crystals were observed by optical and scanning electron microscopies: (i) Optical microscopy observations were conducted, at ambient temperature, by Leica DMLM (LEICA Microsystems Ltd.) or Nikon SMZ-10A (Nikon imaging Ltd.) coupled to a CCD-camera connected to a computer. (ii) Scanning electron microscopy (SEM) analyses were performed by using a SEM equipped with a field emission gun (FEG) LEO type 1530 with an airlock for introducing samples. Raman Microspectroscopy. Analyses were carried out at ambient temperature by using a confocal Raman microscope (LabRam HR by Jobin-Yvon Horiba with a 600 lines/mm grating) coupled to a microscope (model BX41, Olympus) with xyz mapping stage via optical fibers.13 The excitation of Raman scattering was operated by a helium-neon laser at a wavelength of 632.8 nm. The laser beam was focused on the crystal by the microscope objective x50LWF with a 4 cm-1 spectral resolution. During the investigation of defects inside crystals, the confocal pinhole conditions determine the volume of sample analyzed. The pinhole diameter was selected s between 100 and 400 µm s to obtain volumes varying from 5 to 20 µm3 depending
on the experiment. The time of collection was then adjusted in order to obtain a good signal-to-noise ratio.
Results Raman Spectroscopy of Reference Samples. Reference spectra of solids and liquids encountered during this study are presented in Figure 2. The first spectrum presented is that of pure ciclopirox (a). It presents well-resolved peaks, consistent with a well-crystallized product. The identification of ciclopirox by Raman spectroscopy does not require a precise assignment of the bands present in the spectrum. Thereafter, peaks at 1571 cm-1 and at 3073 cm-1 will be used to identify the presence of crystallized ciclopirox. As mentioned in the literature,2-4 macroscopic defects inside crystals may contain saturated solutions. Reference spectra were therefore acquired for (b) dry ethyl acetate, (c) ethyl acetate solution saturated at 20 °C with ca. 3% w/w of water, (d) ethyl acetate solution saturated at 20 °C with 7.6% w/w of ciclopirox, (e) water saturated by ca. 8% w/w ethyl acetate, that is, the composition of the aqueous solution in equilibrium with ethyl acetate saturated with water at 20 °C, in the case of a miscibility gap. The Raman spectrum of dry ethyl acetate (b) is in good agreement with that reported in the literature.14 In particular, we can note the presence of the peak at 1736 cm-1 correlated to the CdO stretching vibration.
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Figure 3. Optical microscopy performed on ciclopirox single crystals: presence of three types of defects: type I, prismatic cavities; II, bubbles inside cavities; and III, isolated bubbles.
Figure 4. Evolution of internal defects vs time in ciclopirox single crystal observed by optical microscopy.
The presence of ethyl acetate in water is clearly visible by Raman spectroscopy in spectrum (e). Indeed, this spectrum presents typical broad bands related to the OH stretching modes of water in the region between 3000 and 3600 cm-1 and typical bands of ethyl acetate. However, the Raman spectra of ethyl acetate saturated with water (c) or with ciclopirox (d) are similar to the one obtained from dry ethyl acetate (b). Consequently, if Raman microspectroscopy allows the identification of ethyl acetate it cannot show the presence of dissolved species in this solvent. This is probably due to the low solubility of water or ciclopirox in ethyl acetate (lower than 10% (w/w)). Finally, Raman spectroscopy seems to be a pertinent technique to identify the presence of water, water saturated in ethyl acetate, or ethyl acetate (pure or with dissolved species in low quantities) in the inclusions observed inside the ciclopirox crystals. Identification of Micro-Macro Defects Inside Single Crystals. Most crystals contain internal defects, whatever the crystallization process used. Typically, each crystal contains one or two defects; however, some crystals can contain up to a dozen defects (results not shown). The size of defects varies from a few microns (microscopic defects) to several hundred microns (macroscopic defects). Three types of defects were identified by optical microscopy: Type I are prismatic cavities systematically oriented in the same direction as single crystal (Figure 3a-c), Type II are spherical bubbles inside cavities (Figure 3a,b),
Type III are isolated spherical bubbles (Figure 3d). No correlation was found between the type of defects obtained and the crystallization process used. Nature of the Defects. The different types of defects were observed and characterized separately in order to determine the nature of these inclusions. A Raman analysis in each defect was conducted by the same procedure as that mentioned by Berton et al.,8 which demonstrates that this method is a suitable technique for gaining local information inside single crystals without destroying the sample. Type I: Prismatic Cavities. Closed Prismatic Cavities. Figures 3a and 4 show the existence of closed cavities inside the crystals. As it can be seen in Figure 4, the shape of these cavities evolves over time, at ambient temperature, after extraction of the crystal from the solution. Starting from an irregular shape, defects reorganized themselves to adopt a final shape that is similar to the crystal. This evolution, which can last up to several days, is accompanied by a decrease in volume. The final shape of the defects is often called “negative crystal”.5 It indicates then that the crystallization of ciclopirox continues in the vicinity of the internal defects of single crystals during a long period of time after the formation of the crystal. In order to characterize precisely the nature of the fluids trapped inside closed cavities, Raman analysis was carried out.
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Figure 5. Raman spectra obtained in different regions inside the crystal for a depth of 80 µm (a) in the cavities, (b) in the crystal.
Figure 6. SEM photograph of a surface in ciclopirox single crystal, containing an open cavity. Figure 8. Raman microspectroscopy in Z-mapping condition performed on ciclopirox crystal nearly along prismatic cavities region (point A).
Figure 7. Photograph of ciclopirox crystals by Raman CCD camera.
Figure 5 presents Raman spectra obtained, in the cavities and in the crystal at 80 µm depth. In the cavities, the Raman spectrum is the sum of the signals of crystallized ciclopirox and liquid ethyl acetate (peak at 1736 cm-1). We can suppose that the volume of sample analysis (about 6 µm3) does not correspond only to the inclusion but also includes a part of the crystal. Then, close cavities in the shape of negative crystals contain ethyl acetate solution saturated in ciclopirox. Opened Prismatic Cavities. Another type of prismatic cavities can be observed inside crystals of ciclopirox. They differ from the latter ones mainly by their length. Indeed, these cavities look like channels with different lengths ranging from 100 µm to almost the whole length of the crystal (ca. 1 mm) (Figure 3c). They have a diameter of ca. 10 µm, according to optical microscopy measurements. Further inspections carried out by
Figure 9. Photograph of mobile bubbles inside the cavity (optical microscopy).
scanning electron microscopy revealed the existence of open channels with a diameter consistent with the optical microscopy determination, that is, 10 µm (Figure 6). In order to obtain local information and especially the composition of these channels, the Z mapping mode of the confocal Raman spectrometer was used. Figure 7 exhibits a photograph of the channel chosen for this analysis, which was representative of different channels observed in the population
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Figure 10. Raman spectra obtained at 80 µm depth in the ciclopirox crystal: (a) in the bulk of the single crystal, (b) in the bubble, (c) in the cavities.
Figure 11. Schematic representation of mechanisms proposed for the formation of macroscopic defects inside ciclopirox single crystals, during growth in supersaturated ethyl acetate solution.
of crystals. Raman spectra were recorded from 0 to -210 µm beneath the surface, by means of successive 30 µm steps (Z mapping) along two different vertical segments represented by point A: in the channel, and point B: in the bulk of the crystal. Figure 8 shows the combined Raman spectra obtained as a function of the depth, in point A, during this experiment. A characteristic wavenumber region (1300-1600 cm-1) is presented, but the same evolution holds for the whole range of the spectra. The depth profile measured along A (long prismatic cavities) shows the characteristic signal of ciclopirox from the surface down to 90 µm depth. Between 120 and 150 µm, the signal decreased progressively. At 180 µm beneath the surface, the recorded signal was that of the background (i.e., noise). Along B, characteristic signals of the crystalline phase were obtained associated with a small decrease (about 20%) of the intensity versus depths (results not shown). So the loss of intensity
observed along A (intensity of the signal divided by 40) cannot be attributed to the natural attenuation of the Raman signal due to absorption or diffusion inside the sample. This experiment has been repeated by using the same protocol in several long channels in various crystals, and all analyzed areas have revealed the same spectra. These results, similar to those reported for gaseous inclusions in trehalose crystals,9 give evidence of gas channels inside the crystals. Nevertheless, in the present case it corresponds to open channels. As crystals were analyzed outside their mother liquors, they were probably filled by saturated solution before extraction. Type II: Bubbles Inside Prismatic Cavities. In some crystals, moving bubbles were observed inside closed prismatic cavities (Figure 9). Indirectly, it confirms that the close cavities contain a liquid.
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Figure 10 presents Raman spectra obtained in the bubble, in the cavities, and in the crystal at 80 µm beneath the surface. In the vacuole, a very weak Raman signal is observed (20 times weaker than in the rest of the crystal), which proves the absence of condensed matter. The presence of a liquid aqueous phase has not been revealed, neither in the bubble nor in the surrounding liquid phase. There is then no liquid miscibility gap but simply a gas bubble in the saturated ethyl acetate solution. Type III: Isolated Bubbles. In Figure 3d, the arrow points at a vacuole trapped inside the crystal. A close inspection of other particles revealed that these spherical cavities were systematically located near the crystal surface. Raman spectroscopy analyses (data not shown) led to the same results as that with bubbles of gas trapped in solution (Type II). No trace of solvent was detected during these analyses. Discussion Raman microspectroscopy provides evidence that both liquid and gas fill various types of macroscopic defects present in ciclopirox single crystals. In defects of type I (closed prismatic cavities), mother liquor is trapped inside the crystals during crystal growth. For several days, the trapped solution and the surrounding solid continue to evolve until the thermodynamic equilibrium is reached. In order to minimize the surface energy of these defects, their ultimate shapes are similar to that of the host particles, that is, “negative crystals” (Figure 11c). It can be noted that ethyl acetate is a toxic solvent that should be removed by an additional step. This volatile solvent (vapor pressure: 9.7 kPa at 20 °C) may be removed by grinding. However, impurities initially dissolved in the mother solution may remain trapped in the crystalline powder. Concerning the presence of open channels, one can imagine that as soon as the crystals have been extracted from the solution before closure of the elongated cavities, ethyl acetate evaporates and the dissolved ciclopirox crystallizes on the internal walls of the defects, leaving then open channels (Figure 11b). The explanation of the presence of gas bubbles inside liquid inclusions (Type II) can be based on the work of Hu et al.6 who dealt with negative crystals in LiB3O5. Starting from a defect filled with mother liquor (Type I), the subsequent crystallization inside the defect was associated with a decrease of volume (the density of the saturated solution is lower than that of the solid) resulting in a gas bubble (Figure 11d). A decrease of the solubility of dissolved gases in supersaturated solution during the negative crystal reorganization could also explain this phenomenon. Thus, in the present case, it seems that the mechanism of formation of these gas inclusions is different from that observed by Berton et al.9 Indeed, for trehalose single crystals, gaseous inclusions are trapped inside the crystal matrix without a detectable quantity of solution at any moment of the evolution of the cavity. The isolated bubbles (Type III) are systematically located near the corners and the edges of the crystal. The question which remains is: are these bubbles open or not? Depending on the answer two mechanisms can be proposed. The formation of open isolated bubbles is likely to be similar to that of open channels (Figure 11b). Nevertheless, the reason for a sharp difference in the shape of these open cavities remains unclear. For closed isolated bubbles, it could be postulated that the gas comes from dissolved quantities in the liquid phase which nucleates and docks on rough surfaces of the growing crystal and subsequently are totally engulfed. The nucleation of the bubble of gas might
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result from a gas solubility drop as the supersaturation of ciclopirox decreases (Figure 11a). Conclusions and Perspectives Whatever the process used to recrystallize ciclopirox in ethyl acetate (solvent water content, initial and final temperature, cooling rate, supersaturation), some internal micro-macro defects do appear during the crystal growth. The content of these defects could be determined by Raman microspectroscopy which is a noninvasive suitable technique to characterize heterogeneities inside particles. By using this technique, various types of defects filled by gas and/or liquid have been detected. If the liquid pocket is not totally closed, the evaporation of the solvent after extraction of the crystal leads to the formation of open cavities containing air. If the liquid inclusion is closed, the trapped supersaturated solution continues to crystallize and the interface of mother liquor-crystal relaxes until the formation of a negative crystal associated in some cases with the apparition of gas bubbles. Moreover, this study highlighted the possible existence of isolated gas bubbles trapped inside crystals, whose formation mechanisms have to be clarified. In particular, a careful study of the evolution of gases solubility in ethyl acetate according to ciclopirox concentration and temperature could be helpful, in order to verify the assumption of direct nucleation of gas bubbles on rough surfaces of the crystal and their subsequent engulfment. Continuing efforts will be made to understand (and to avoid) the formation of these defects which have a profound impact on physical properties of the organic crystals, especially the processability for downstream operations such as milling, storage, as well as the chemical stability of the product. Acknowledgment. PCAS industry is gratefully acknowledged for their support during this work. Dr. Jean Jacques Malandain (Groupe de Physique des Mate´riaux, University of Rouen) is also acknowledged for the SEM analyses.
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CG801355Q
