Detecting Crystalline Nonequilibrium Phases on the Nanometer Scale

Article pubs.acs.org/crystal

Detecting Crystalline Nonequilibrium Phases on the Nanometer Scale Tatiana E. Gorelik,† Adnan Sarfraz,‡ Ute Kolb,† Franziska Emmerling,*,‡ and Klaus Rademann*,‡,§ †

Institute of Physical Chemistry, Johannes Gutenberg-Universität Mainz, Welderweg 11, 55128 Mainz, Germany BAM Federal Institute for Materials Research and Testing, Richard-Willstätter-Strasse 11, 12489 Berlin, Germany § Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany ‡

ABSTRACT: The use of Automated electron Diffraction Tomography (ADT) is presented as a novel approach for crystallization studies at the nanometer scale for nonequilibrium phases. Here, ADT was applied to elucidate the structural identity of the recently reported hexagonal morphology of caffeine crystals, which grow only on specific surfaces. Caffeine was crystallized from solution on a specially treated TEM carbon grid. The analysis of ADT data revealed that the lattice parameters of these hexagons match those of the high temperature αform of caffeine. Furthermore, it was observed that in this hexagonal morphology, the α-form remained stable for a prolonged period of time. The stabilization of hexagons can be interpreted in terms of enhanced interactions with the supporting surfaces.

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INTRODUCTION Organic compounds are known for their polymorphism. The phenomenon characterizes the propensity of a compound to crystallize in more than one structure.1 Polymorphism is of great importance for the pharmaceutical, pigment, and fine chemical industries, since the crystal structure of a product affects directly its properties, that is, stability, dissolution rate.2,3 The deliberate and selective crystallization of a distinct polymorphic compound still remains a challenging task. Particularly the initial nucleation event is difficult to control due to the various thermodynamic and kinetic factors affecting the crystallization process. Furthermore, the crystals formed transiently are often too small to be considered for conventional single-crystal experiments. Nevertheless, these stages might have a great impact on the processes that determine the future fate of a crystal. Hence, crystal creation, stabilization, and investigation is of great interest.3−9 Consequently, it is not only a prerequisite to understand in detail the key physical processes that occur during nucleation and crystal growth but also to characterize the formed nanoscale crystals structurally. In this context, the well-known molecule caffeine (1,3,7trimethylpurine-2,6-dione, C8H10N4O2, see Figure 1) is experiencing a renaissance in scientific discussion. Caffeine is a widely used food and pharmaceutical agent. Surprisingly, the two polymorphs of caffeine (α, β) were only recently structurally characterized (α in 2005; β in 2007).10,11 The room-temperature modification (β) crystallizes in the monoclinic space group C2/c and contains five independent caffeine molecules in the asymmetric unit, whereas the high-temperature α-form (R3c̅ ) is dynamically disordered. Moreover, the α form is metastable at room temperature and is known to © 2012 American Chemical Society

Figure 1. Molecular structure of caffeine (1,3,7-trimethylpurine-2,6dione, C8H10N4O2).

transform to the β form at different rates depending on temperature and humidity. Besides their polymorphism, organic compounds are also known to exhibit different morphologies during crystallization. A large number of studies have been devoted to the appearance of various morphologies on different length scales. Usually, these investigations are based on light or electron microscopic studies. A detailed description of the morphology becomes readily available, but the structural identity of the crystals remains obscure.12−15 As for caffeine, typical morphologies are needles, hollow needles, and the recently reported hexagons. The formation of hexagons is strongly influenced by the nature of the surface. By appropriate choice of conditions (surface, solvent, temperature), they can be grown selectively and in Received: March 21, 2012 Revised: April 10, 2012 Published: April 23, 2012 3239

dx.doi.org/10.1021/cg300377j | Cryst. Growth Des. 2012, 12, 3239−3242

Crystal Growth & Design

Article

large quantities.16 However, individual hexagons remain always small, which hinders a direct structural characterization. This contribution presents the first structural identification of caffeine hexagons. As reported previously, caffeine hexagons can be formed selectively but can also be formed simultaneously in the presence of β-phase needles. The β-structure dominates the X-ray powder patterns, which complicates structural studies. Attempts to characterize caffeine hexagons by synchrotron Xray investigations were also not successful. The only possibility to elucidate the structure in such a case, is electron crystallography. Electron crystallography applied to organic materials has a long history; the first structure reported using electrons appeared in 1936.17 Organic samples have a distinct advantage of consisting mainly of weak scatterers and therefore showing reduced dynamical effects in the electron diffraction data. This allows ab initio structure solution of organic materials once a data set with sufficient completeness is available.18,19 The major limitation of the electron diffraction studies is the radiation damage to the material during investigation.20 Better control over the electron radiation dose during data acquisition was obtained by development of automated routines for data collection (automated diffraction tomography, abbreviated as ADT). These routines are based on a sequential electron diffraction pattern collection from a single crystal while it is being tilted around a goniometer axis.21,22 The sample is imaged in scanning transmission electron microscopy (STEM) mode using a very low illumination rate. From a mixture of crystals, one specimen can be selected, and an electron diffraction tilt series can be collected from only this crystal. Importantly, the ADT approach combines the possibility to control the electron dose and collect rich electron diffraction data. This approach has already demonstrated its power for lattice parameter determination as well as ab initio structure analysis of molecular crystals20,23 and is applied successfully to study caffeine hexagons.

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measurements, a 626 GATAN cryo-transfer holder was kept at liquid nitrogen temperature. The imaging was done in STEM mode. Electron diffraction was collected using the automated acquisition module as described elsewhere.21,22 The data was collected using a dose rate of 0.3 e−/(Å2 s). The exposure time for obtaining single diffraction patterns was 3 s. The beam size for nanodiffraction was about 100 nm in diameter. For lattice parameter determination, the diffraction data was collected in the tilt range of ±30°. The lattice parameters were calculated using ADT3D software (Nanomegas, Belgium).

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RESULTS AND DISCUSSION Formation of Hexagons. In Figure 2a, ESEM images of caffeine crystals grown on carbon-coated grids without plasma

Figure 2. ESEM images of (a) caffeine crystals on a carbon-coated, untreated TEM grid and (b) caffeine crystals on a plasma-treated grid.

activation are shown. These grids mainly contain needle-like crystals. The formation of hexagonally shaped crystals was not observed under these conditions. In order to modify the properties of the carbon surface and thereby facilitate the growth of the target phase, the grids were hydrophobized in a plasma cleaner. Figure 2b shows a representative image of typical crystal morphologies (needles and hexagons) obtained under these conditions. Afterward the plasma-treated grids were used for the TEM investigations. Figure 3a shows a STEM image of hexagonally shaped crystals. It turned out that these particles were unstable in the

EXPERIMENTAL SECTION

Materials. Caffeine (anhydrous 99%) was obtained from Fluka GmbH and used without further treatment. The X-ray diffraction (Bruker AXS, D8, Cu Kα radiation) pattern of this sample consists of 100% β polymorph. Dichloromethane was purchased from Carl Roth GmbH + Co., Karlsruhe, Germany. After the deposition of a small volume (∼3 μL) of an aqueous caffeine solution, the formation of hexagons was observed on different surfaces (e.g., pure silicon, silver surfaces, soda lime silicate glasses) at room temperature. Further details of the formation mechanism have been published elsewhere.16 ESEM. Environmental scanning electron microscopy (ESEM) experiments were carried out on a tabletop microscope TM-1000 (Hitachi High-Technologies Europe GmbH) using a precentered cartridge filament electron gun and a high-sensitivity semiconductor BSE (solid state backscattered electron) detector. The accelerating voltage was 15 kV. The obtained magnification was between 20 and 10 000. The evacuation system consisted of a turbo molecular pump (30 L/s) and a diaphragm pump (1 m3/h). TEM. Samples for transmission electron microscopy (TEM) measurements were prepared on holey-carbon covered copper grids by immersing the grids into dichloromethane solution for a few seconds. In order to facilitate the growth of hexagons on the carbon film, the surface of the grids was activated using a plasma cleaner in argon flow for 30 s prior to the caffeine deposition. The grids were dried in air. After a few minutes, the grids were sufficiently dry and were then transferred into the TEM. TEM measurements were performed using a FEI TECNAI F30 transmission electron microscope with a field emission gun operating at 300 kV. For cryo-TEM

Figure 3. STEM images of caffeine crystallites on plasma-treated carbon film (a) immediately after the insertion into the TEM and (b) the same area after 30 min keeping the sample in the TEM (without illumination).

TEM (pressure in the column of approximately 10−5 Pa) and no hexagons were observed after 30 min even without being illuminated by the electron beam (Figure 3b). In contrast, the needle-shaped crystals remained stable. To preserve the material in vacuum, a freshly prepared sample was first cooled to liquid nitrogen temperature (the grid was dropped into liquid nitrogen; no vitrification procedure was used) and then inserted into the TEM using a cryo-transfer procedure. The cooling preserved hexagons under vacuum. Under these 3240



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as opposed to a nontreated surface. Therefore plasma cleaning is used as a standard technique in vitrification of aqueous suspensions; the droplets are repelled from the carbon surface and are forced to form thin membranes within the holes of the carbon film. The modified properties of the carbon surface prevent the formation of a thin homogeneous wetting layer, thus assisting the breakup of droplets to microdroplets. The concentration gradient achieved during the evaporation of the microdroplets is then responsible for the formation of hexagons on the surface. The surface interacts also with the emerging nanocrystals and stabilizes hexagons. The diameter of these hexagons stays within a limited range of less than a few micrometers. This observation suggests that with increasing size and hence a more bulk-like character of the crystals the β form is preferred. It is known that the more commonly exhibited needle-like morphology of caffeine is present in the βform.10,27,28 The surface has two functions with respect to the formation (i) and the stabilization (ii) of the hexagons. Thus further studies may show that this phenomenon of stabilizing metastable polymorphs at room temperature in novel morphologies can be triggered by an appropriate choice of surfaces. Figure 5 displays nearly perfect hexagons after a

conditions, the particles were stable in TEM for at least 3 h, which was sufficient to collect ADT data. Crystal Structure of the Hexagons. ADT data collected from hexagons delivered the following lattice parameters of a = 9.53 Å, b = 8.66 Å, c = 6.60 Å, α = 75.7°, β = 118.1°, and γ = 123.0°. These basis vectors transform into a = 14.53 Å, b = 14.55 Å, c = 6.60 Å, α = 89.89°, β = 89.17°, and γ = 119.94° of an R-centered lattice upon a transformation of (120; 11̅1; 001̅). These parameters correspond to the unit cell of α-caffeine (a = 14.831(3) Å and c = 6.7648(19) Å; R3̅c).10,24 Views of the reconstructed ADT volume along the three main directions are shown in Figure 4.

Figure 4. Views of the reconstructed ADT volume along the main directions: (a) (001), the 3-fold symmetry is not completely visible due to the missing wedge of the reciprocal space; (b) (010); (c) (100).

ADT data was also collected from a needle-shaped crystal. The lattice parameters matched those of the β-phase: ADT analysis showed a monoclinic C-centered lattice with the parameters of 42.3 Å, 14.9 Å, 6.9 Å, 91.4°, 96.2°, and 90.0°, corresponding to 42.521(9) Å, 14.948(3) Å, 6.7923(14) Å, and 97.825(4)°, and match the ones reported in literature.10 Reportedly, the high-temperature polymorph α converts to the β-phase at temperatures below the transition temperature of 153 °C. Elevated temperatures accelerate this conversion,25 but the process rate at room temperature is still significant.26 Contrary to these literature reports, the caffeine hexagons grown from dichloromethane on various surfaces were still present after a year. To verify whether a phase conversion may have affected the hexagons’ structure while the morphology was maintained, cryo-TEM ADT measurements were performed on a sample after it was kept 10 days at room temperature. Similar to the freshly prepared sample, the hexagons were observed together with the needle-like crystals. Analysis of the ADT data recorded from a hexagon gave a rhombohedral unit cell with the parameters of 14.4 Å, 14.3 Å, 6.8 Å, 88.2°, 92.5°, and 118.2°, thus confirming the room temperature stability of the α-form. This finding demonstrates the ability of the hexagons to preserve the α modification for a longer period of time. Previously, the α modification was known to convert to the β form rather rapidly (vide supra). Stability of the Hexagons. The unstable behavior of the hexagonal shape crystals in vacuum compared with the needle form also implies that needles and hexagons are different polymorphs. Here, we clearly observed polymorphs behaving differently. The two forms (α and β) obviously have different vapor pressure parameters.29 The mechanism of the formation of hexagons on specific surfaces has been described before but has never been observed on a carbon film and therefore never been studied in a TEM.16 The formation is facilitated by the creation of microdroplets of the solution on the surface. This preferred formation of hexagons is explained on the basis of increased microdroplet formation. The mechanism explains the appearance of hexagons on a plasma-cleaned carbon TEM grid

Figure 5. ESEM images (a,b) of caffeine hexagons formed on a soda lime silicate glass surface after evaporation of droplet of an aqueous caffeine solution. The hexagons were transferred from a glass surface on adhesive tape. The backside of the magnified hexagons (yellow frame in b) shows a brain coral like structure, which is indicative for spinodal decomposition and crystallization.

transfer process from glass surface to an adhesive tape, similar to the famous exfoliation process of graphene. It requires a certain force to overcome adhesion and to lift the hexagons from the surface. The same adhesion might also be responsible for stabilizing the hexagons. Once transferred, the structure of the interface between hexagons and surface becomes visible. The backside of the hexagons appears brain-coral-like (see Figure 5a). The distinct structure is a characteristic indication of the spinodal decomposition process, which precedes the formation of the hexagons and is still active during the formation of the crystals. This observation suggests that the surface provides a sufficient stabilization for the α modification in the hexagonal morphology. Such a supporting effect has also been observed for structurally related molecules (adenine, 3241



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(17) Rigamonti, R. Gazz. Chim. Ital. 1936, 66, 174−182. (18) Vainshtein, B. K. Structure Analysis by Electron Diffraction, 1st ed.; Pergamon Press: New York, 1964. (19) Dorset, D. L. Structural Electron Crystallography; Springer: New York, 1995. (20) Kolb, U.; Gorelik, T. E.; Mugnaioli, E.; Stewart, A. Polym. Rev. 2010, 50, 385−409. (21) Kolb, U.; Gorelik, T.; Kübel, C.; Otten, M. T.; Hubert, D. Ultramicroscopy 2007, 107, 507−513. (22) Kolb, U.; Gorelik, T.; Otten, M. T. Ultramicroscopy 2008, 108, 763−772. (23) Gorelik, T. E.; van de Streek, J.; Kilbinger, A. F. M.; Brunklaus, G.; Kolb, U. Acta Crystallogr. 2012, B68, 171−181. (24) Derollez, P.; Correia, N. T.; Danède, F.; Capet, F.; Affouard, F.; Lefebvre, J.; Descamps, M. Acta Crystallogr. 2005, B61, 329−334. (25) Kishi, Y.; Matsuoka, M. Cryst. Growth Des. 2010, 10, 2916− 2920. (26) Pirttimäki, J.; Laine, E. Eur. J. Pharm. Sci. 1994, 1, 203−208. (27) Habgood, M. Cryst. Growth Des. 2011, 11, 3600−3608. (28) Eddleston, M. D.; Jones, W. Cryst. Growth Des. 2010, 10, 365− 370. (29) Emel’yanenko, V. N.; Verevkin, S. P. J. Chem. Thermodyn. 2008, 40, 1661−1665.

guanine). Thus further studies may show that this phenomenon of stabilizing metastable polymorphs at room temperature in novel morphologies can be triggered by an appropriate choice of surfaces.

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CONCLUSION The most salient features of this study can be highlighted as follows: (i) For the first time, the electron diffraction tomography technique allowed the phase identification of the unique hexagonal morphology exhibited by caffeine. Other methods failed due to the small sample size. (ii) The evaluated lattice parameters showed that the hexagonal crystals are present in the α-form. (iii) For the first time, hexagonally shaped, metastable αcaffeine crystals were grown on a carbon surface facilitated by a plasma activation process. (iv) The influence of the surface on the formation mechanism was investigated, and the stabilization was explained in terms of surface interaction energies. These findings and observations may also be applicable to other organic polymorphic metastable compounds that could be stabilized in certain morphologies induced by surfaces.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We gratefully acknowledge financial support by the Deutsche Forschungsgemeinschaft through SPP 1415 (Crystalline nonequilibrium phases): Ra494/15-1 Em198/4-1, and SFB 625 (From single molecules to nanoscopically structured materials).

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Detecting Crystalline Nonequilibrium Phases on the Nanometer Scale

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