SAXS Study of the Nucleation of Glycine Crystals from a

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SAXS Study of the Nucleation of Glycine Crystals from a Supersaturated Solution Chattopadhyay,‡

Erdemir,‡

Evans,§

Soma Deniz James M. B. Jan Heinz Amenitsch,| Carlo U. Segre,*,⊥ and Allan S. Myerson‡

Ilavsky,#

CRYSTAL GROWTH & DESIGN 2005 VOL. 5, NO. 2 523-527

Department of Chemical and Environmental Engineering, Illinois Institute of Technology, Chicago, Illinois 60616, Glaxo Wellcome Manufacturing Pte Ltd., 1 Pioneer Sector 1, Singapore, 628413, Argonne National Laboratory, Building 438 E, APS, 9700 South Cass Avenue, Argonne, Illinois 60439, Institute of Biophysics and X-ray Structure Research, Austrian Academy of Sciences, Schmiedlstrasse 6, A 8042, Graz, Austria, and Physics Division, Illinois Institute of Technology, Chicago, Illinois 60616 Received July 30, 2004;

Revised Manuscript Received December 13, 2004

ABSTRACT: Nucleation of crystalline solids, the first stage of crystallization from solution, is not yet fully understood. This is true for both small molecules of low molecular weight and more complicated large molecules. To obtain direct structural information about the process of nucleation and crystallization of small molecules, smallangle X-ray scattering (SAXS) has been used to study the crystallization of the amino acid glycine from its supersaturated aqueous solution. The scattering data was analyzed using the unified fit model, which is well-suited for studying complex systems that may contain multiple levels of related structural features. The results suggest that glycine molecules exist as dimers in supersaturated solution. The system obeys power-law behavior that indicates the presence of fractals in the solution. A transformation from mass fractal structure to surface fractal structure is observed during the crystallization process, which could be the signature of a two-step nucleation process. Introduction The nucleation of crystals from solution is of great importance since it is the primary method for the preparation and purification of industrially important chemicals such as pharmaceuticals, explosives, dyes, photographic materials, etc. The crystallization process is also used for the synthesis of single crystals of proteins, amino acids, etc., as well as very small crystals whose large surface area makes them important for applications in gas sensing and catalysis, among others. The early stages of the formation of the crystals from solution can play a decisive role in determining the properties of the solid in its final state. Hence, it is highly desirable to understand the process of crystallization so that one can predict and control the size, morphology, structure, and the quality of the crystals. Such studies will also enable scientists and engineers to design new functional solids with technological importance. Solution crystallization is considered to be a two-step process: nucleation, or the birth of crystals, and crystal growth, which involves subsequent growth of existing crystals. One of the requirements for bulk crystallization is that the solution should exceed its solubility at a given temperature, i.e., the solution should be supersaturated. Classical nucleation theory1,2 postulates that concentration fluctuations in supersaturated solutions give rise * To whom correspondence should be addressed. E-mail: [email protected]. ‡ Department of Chemical and Environmental Engineering, Illinois Institute of Technology. § Glaxo Wellcome Manufacturing Pte Ltd. # Argonne National Laboratory. | Austrian Academy of Sciences. ⊥ Physics Division, Illinois Institute of Technology.

to clusters that coalesce under certain conditions to form nuclei. Mullin and Leci reported that isothermal columns of supersaturated citric acid solutions developed concentration gradients with higher concentrations in the bottom than the top of the column, while no gradients formed in the saturated or undersaturated columns.3 In a recent review aimed at understanding crystal nucleation, Weissbuch et al. have used the technique of grazing incidence X-ray diffraction to demonstrate that crystal nucleation can be achieved employing tailor-made auxiliaries that are either nucleation inhibitors or promoters.4 The basis of their work is the hypothesis that supersaturated solutions containing molecular clusters adopt various arrangement and shapes, some of which resemble the crystal they grow into. Myerson and Lo measured the diffusion coefficient for supersaturated aqueous solutions of glycine. The diffusion coefficient was observed to decline with age, and the effect was attributed to the evolution in time of molecular clusters in the metastable solution.5,6 They observed a large decrease in the critical cluster size with increasing supersaturation. Myerson and co-workers have used the theory of Binder7 and the concept of a cluster size distribution to analyze their diffusivity and viscosity data. These results show that after an initial period of rapid growth, the clusters grow very slowly. They predicted that glycine exists mostly as dimers in supersaturated solutions. Their data also showed that sodium chloride and potassium chloride exist mainly as monomers in solution and urea exists as oligomers.8-10 Recent studies, however, suggest that classical nucleation theory is not even qualitatively correct, and there is growing evidence that nucleation from solution is itself a two-step process. The first step involves the formation of a liquidlike cluster of solute molecules,

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while the second involves the reorganization of such a cluster into an ordered crystalline structure.11-13 ten Wolde and Frenkel considered solute-solute interactions given by a modified Lennard-Jones potential and proposed the presence of a metastable fluid-fluid critical point close to which the free energy barrier for crystal nucleation is strongly reduced and the nucleation rate increases by many orders of magnitude.11 Anwar and Boateng showed by molecular simulations that crystallization in highly supersaturated systems involves liquid-liquid-phase separation followed by nucleation of the solute phase.12 By using molecular dynamics to study the nucleation of AgBr in water, Shore et al. have provided further evidence to the conjecture that nucleation of crystals from solution may be a two-stage process.13 They have further suggested that disordered clusters are actually the stable state until the cluster size becomes rather large. However, there is little direct experimental evidence that supports this two-step nucleation model. In recent years, there has been increasing utilization of X-ray scattering techniques from ultra-small angle (USAXS), to small angle (SAXS) to wide angle (WAXS), which probe a wide range of length scales to characterize the nucleation and crystal growth of a broad range of compounds that include zeolites, polymers, colloids, and composite materials.14-17 From small angle neutron scattering (SANS) and SAXS studies of supersaturated solutions of the hen egg white lysozyme, Nimura et al. have reported that in supersaturated solutions, large aggregates of polydispersive nature and with a radius greater than 50 Å coexist with a much larger number of monodispersive smaller aggregates whose radii are between 25 and 40 Å.18 The former do not change with time, but the radius of the latter increases with time and then decreases after 14 h showing the onset of nucleation in the system. Davey et al. have performed SAXS and WAXS studies on saturated and supersaturated aqueous solutions of urea, sucrose, and citric acid.19 Their results have not revealed any evidence of long-range ordering in these systems at distance scales larger than 16 Å. To date, there have been no reports of nucleation studies on most other proteins, peptides, and amino acids and definitely no SAXS studies on crystallization of glycine. In fact, the vast majority of work has been undertaken in dilute systems with supersaturated solutions being ignored for the most part. Glycine (CH2NH2COOH) is known to exist in three polymorphic forms: R, β, and γ.20 β, the unstable form, is obtained by adding ethyl alcohol to a concentrated aqueous solution of glycine; it transforms rapidly into the R-form in air or in water.21 Single crystals of γ-glycine are grown by slow cooling of aqueous solutions in the presence of acids such as acetic acid, or bases such as ammonium hydroxide.22 Although R-glycine forms spontaneously from aqueous solution, γ-glycine is the most stable phase at room temperature, indicating that the spontaneous nucleation of glycine is kinetically rather than thermodynamically controlled.23 Grazing incidence X-ray diffraction and atomic force microscopy measurements were performed by Gidalevitz et al. on glycine, and they determined that cyclic dimers are the essential building blocks for R-glycine grown out of

Chattopadhyay et al.

water.24 Recent laser-induced nucleation studies reveal that supersaturated solutions of glycine crystallize into the polar γ-polymorph when subjected to plane polarized laser light.25 In fact, by switching between linear and circular polarization in the irradiation of supersaturated solution of glycine with intense nanosecond pulses of near-infrared laser light, one can obtain γ and R phases of glycine, respectively.26 In this paper, we report the results obtained by our group on a study of the nucleation and crystallization of the amino acid glycine from its supersaturated aqueous solution using small-angle X-ray scattering. Experimental Procedures Solution Preparation. The supersaturated solution of glycine with concentration of 3.6 M was prepared by combining solid glycine, 99.8%, purchased from Sigma, and deionized water (resistivity ) 18MΩ-cm) in a test tube with a screw-on cap. The glycine was completely dissolved by sonicating and heating the test tube in a water bath at 60 °C for several days. A total of 200 µL of solution was transferred into a glass capillary tube, using a microsyringe. The syringe and the syringe needle were placed in an oven beforehand to bring them above the solution temperature to prevent the solution from crystallizing during this process. The capillary was then immediately sealed and placed into a metal sample holder on the SAXS beamline. The sample holder was preheated to 55 °C before the capillary was inserted. Data collection was started immediately and the temperature of the sample holder was reduced at the rate of 0.5 °C/min to a final value of 10 °C (at this temperature a saturated solution of glycine has concentration csat ) 2.17 M) using a chilled water bath. The cooling of the supersaturated solution was used to mitigate the heating caused by X-ray absorption and to reduce the time of glycine crystallization to a few hours. SAXS Measurements. Small-angle X-ray measurements were performed at the Bio-CAT undulator beamline (18-ID) at the Advanced Photon Source at Argonne National Laboratory. We used a brass sample stage whose temperature can be controlled within (2 °C by a water bath. The sample holder is designed with a hole to insert a thermocouple close to the capillary. The capillary (3.5 mm in diameter) was placed vertically in the path of the beam. 12 keV X-rays (spot size ) 250 µ × 800 µ) were focused at the CCD detector located 1.8 m from the sample, and scattering data were collected until crystallization was visually observed. Exposure time was 20 s with a delay of 60 s between exposures to limit heating and radiation damage of the glycine solution. The SAXS data were corrected for the intensity of the X-ray source, and background subtraction was performed by subtracting a constant value over the entire range of q. This constant was obtained from the high q region of a water measurement taken under the same conditions. SAXS Data Analysis. SAXS data provides information about the size and shape of particles that are present in a system and also gives information about the presence of different particle populations and type of interactions. Data reduction was carried out using the Igor Pro (Wavemetrics, Inc.) based programs developed by the Intense Pulse Neutron Source (IPNS) Group at the Argonne National Laboratory. Data evaluation was done using Igor Pro package of SAS data modeling and evaluation routines called Irena.27 This package contains the implementation of the so-called unified fit model. In the unified fit method, a function is derived that models both the Guinier exponential and structurally limited power law regimes without introducing new parameters beyond those used in the local fit. The unified equation for one structural level is given as follows.28,29

I(q) ≈ G exp(-q2Rg2/3) + B{[erf(qRg/61/2)]3/q}P

(1)

SAXS Study of Nucleation of Glycine Crystals

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Results and Discussion

Figure 1. Variation of the scattered intensity I(q) versus the momentum transfer q ) 4π/λ sin(θ/2). where the Guinier prefactor G ) ne2NpIe: Np is the total number of particles in the scattering volume, Ie is the scattering factor for a single electron, and n is the number of electrons in a particle; q describes the difference between the incident and the scattered beam, and its magnitude is given by (4π/λ) sin(θ/2), where λ is the wavelength of the X-ray used and θ is the scattering angle; Rg is the radius of gyration. B is a prefactor specific to the type of power-law scattering, and it is defined according to the regime in which exponent P falls. Generally, for surface fractals 3 < P 4. For Porod’s law, P ) 4 and B ) 2πNpFe2SpIe, where Sp is the particulate surface area and Fe ) n/Vp where Vp is the average particulate volume. The unified equation27,29 describes a material with complex morphology over a wide range of q and thereby helps to obtain different sizes in terms of structural level. One structural level includes a Guinier regime describing an average structural size and a power-law regime that describes the mass or surface fractal scaling for that particular structural level.

Figure 1 shows the normalized scattering data for the glycine supersaturated solution as it crystallizes. We observe that initially there is an increase in the intensity with time and as the temperature decreases. However, after 3 h, the intensity decreases both in the low and the high q regions, indicating that the glycine content in the probed part of the solution is decreasing. The unified model27-29 was used to fit all the curves, and four selected fits are presented in Figure 2 to indicate the quality of the fits obtained. Radius of gyration (Rg) and power law value (P) were calculated for all data and are plotted in Figure 3 panels a and b, respectively. From Figure 3a, we see that Rg is initially 3.38 Å. For a glycine monomer, Rg is expected to lie between 2.9 and 3.0 Å, while for a dimer it is expected to be in the range of 3.7-4.0 Å. Since our initial Rg value is between the monomer and the dimer size, we presume that some of the glycine molecules exist as dimers even before superaturation is achieved. Rg then increases from 3.38 to 3.8 Å in 70 min and stays in the range of 3.8-3.9 Å for about 2 h. Hence, we infer that glycine exists as dimers in the supersaturated solution, in accordance with the results of indirect measurements reported earlier by Myerson and Gidalevitz.5,6,24 Rg then increases steadily to a maximum value of 4.24 Å followed by a decrease to 4.02 Å. We believe that the maximum in Rg corresponds to the time when nucleation occurred and the decrease implies that the dimers organize themselves into larger clusters whose scattering intensity falls outside the q range that was probed in this experiment. To interpret the change in the power-law behavior of the glycine solution, we have used the fractal interpre-

Figure 2. Power law fitting done with the unified model for four of the different frames taken at various times during the experiment. The times, t, and power law values, P, are as follows: (a) t ) 1 min, P ) 2.67; (b) t ) 105 min, P ) 2.96; (c) t ) 245 min, P ) 3.21; and (d) t ) 275 min, P ) 3.13.

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Figure 3. (a) Variation of the radius of gyration (Rg) with time. (b) Variation of the power law value (P) with time. The standard deviation of the fit values has been included in the symbol size.

tation of the small angle scattering data. A fractal structure is one that displays features such as selfsimilarity, scaling and universality;30 and an aggregation, which is formed by complicated random processes, is a typical example of a system which exhibits such features. SAXS has been used by various groups to determine the fractal structure of rough colloids such as silicate particles in solution, zeolites, porous scatterers such as lignite coal, colloidal aggregates of silica particles, etc.31-34 The intensity of small angle scattering I(q) is given by the general equation:

I(q) ) φP(q)S(q)

(2)

where φ is the number density of particles in solution, q is the momentum transfer, P(q) is the form factor that gives the distribution of material in the scattering particle. S(q) is the structure factor and is related to the spatial distribution of the scattering particles in the solution. For mass fractal aggregates, I(q) is dominated by S(q), which provides information about the various primary building units present in the solution35,36 and is given by:

I(q) ∼ S(q) ∼ q-Dm, 3 > Dm g 1

(3)

where Dm is the mass fractal dimension. On the scale of the primary particles, the form factor P(q) part of the intensity becomes important and provides information regarding the surface structure of these particles:

I(q) ∼ P(q) ∼ qDs-6, 3 g Ds g 2

(4)

where Ds is the surface fractal dimension. For uniform objects with smooth nonfractal surfaces, Ds ) 2 and eq 4 reduces to the familiar Porod law (I ∼ q-4). Therefore, this equation can be treated as a generalization of Porod’s law for fractal surfaces. Ds increases as the roughness of the surface increases. With the help of scattering measurements, one can distinguish between mass fractals (power law values between 1 and 3) and

Figure 4. A model showing the nucleation of glycine crystals from its aqueous supersaturated solution: (a) Existence of monomers (dark colored circles) and dimers (ellipsoids) in the solution; (b) formation of liquid like clusters in the supersaturated solution; (c) reorganization of the liquid-like clusters.

surface fractals (power law values between 3 and 4) from the slope of log(I(q)) versus log(q) plot. Figure 3b shows the variation of the power law value (P ) Dm or P = 6 - Ds) with time. We observe that P is initially 2.68, increases to around 2.95 in 35 min, and remains there for about 3 h. On the basis of these data, the sample evolution is shown schematically in Figure 4. Since a power law value between 2 and 3 indicates the existence of mass fractals in the solution, we conclude that our solution is in a mass fractal state consisting of monomers and dimers (Figure 4a) even before supersaturation is achieved. We attribute the increase of the P values to 2.95 to the formation of liquid like clusters with primary building blocks of dimers in the solution (Figure 4b). This is the stable state until the power law exponent value changes to 3.14 and reaches a maximum of 3.21, which indicates the transformation of mass fractals to surface fractals (Figure 4c). This means that the liquid like clusters are reorganizing themselves, leading to nucleation of glycine crystals. This transformation may be the signature of a two step nucleation process which has been proposed in the literature.11-13 The observed change in the power law value occurs at the same time as Rg values reach a maximum and start decreasing. After reaching a maximum, the power law value starts decreasing, which can be explained by the fact that after nucleation is achieved, the surface fractal structures grow rapidly resulting in a shift of their X-ray scattering to smaller values of q not probed in this experiment. On the basis of these

SAXS Study of Nucleation of Glycine Crystals

findings, we propose that the maxima in Rg and P likely correspond to the time where nucleation occurred. However, the scattering intensity starts decreasing 50 min earlier than the observed maxima in Rg and P. This could be attributed to some of the clusters growing to larger size and sinking out of the portion of the solution being probed by the X-rays. Conclusions Using small-angle X-ray scattering, we have directly studied the nucleation of the amino acid glycine from its aqueous supersaturated solution. Our results indicate that glycine molecules exist as dimers in the supersaturated solution. The system follows a power law behavior, which is a signature of fractal structures, and the increase of the power law value indicates a transformation of the system from mass fractals to surface fractals. This is consistent with a two-step nucleation process. Further studies are in progress on supersaturated solutions of glycine and urea to investigate if the cluster size is dependent on the concentration of the supersaturated solution and if the size is altered by the presence of additives. Acknowledgment. The authors would like to thank Dr. P. Thiyagarajan for fruitful discussions and Dr. Elena Kondrashkina for her help in setting up and support of the experiment at the beamline. S.C. would also like to thank Dr. L. Fan, Dr. J. Jacob, and Dr. S. Seifert for help with the software programs from IPNS, Argonne National Laboratory. Bio-CAT is a National Institutes of Health-supported Research Center RR08630. The APS is funded by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. W-31-109-Eng-38. References (1) Lewis, B. Nucleation and Growth Theory: Crystal Growth; Pamplin, B. R., Ed.; Pergamon: Oxford, 1980. (2) Zettlemoyer, A. C. Nucleation; Dekker: New York, 1969. (3) Mullin, J. W.; Leci, C. Philos. Mag. 1969, 19, 1075. (4) Weissbuch, I.; Meir, L.; Leiserowitz, L. Cryst. Growth Des. 2003, 3, 125. (5) Myerson, A. S.; Lo, P. Y. J. Cryst. Growth 1991, 110, 26. (6) Myerson, A. S.; Lo, P. Y. J. Cryst. Growth 1990, 99, 1048. (7) Binder, K. Phys. Rev. B 1977, 15, 4425. (8) Ginde, R. M.; Myerson, A. S. J. Cryst. Growth 1992, 116, 41.

Crystal Growth & Design, Vol. 5, No. 2, 2005 527 (9) Ginde, R. M.; Myerson, A. S. J. Cryst. Growth 1993, 126, 216. (10) Myerson, A. S.; Ginde, R. M. Handbook of Industrial Crystallization; Myerson, A. S., Ed.; Butterworth-Heinemann: Stoneham, MA, 1992; Chapter 2. (11) ten Wolde, P. R.; Frenkel, D. Science 1997, 277, 1975. (12) Anwar, J.; Boatang, P. K. J. Am. Chem. Soc. 1998, 120, 9600. (13) Shore, J. D.; Perchak, D.; Shnidman, Y. J. J. Chem. Phys. 2000, 113, 6276. (14) de Moor, P.-P. E. A.; Beelen, T. P. M.; van Santen, R. A. J. Phys. Chem. B, 1999, 103, 1639. (15) de Moor, P.-P. E. A.; Beelen, T. P. M.; van Santen, R. A.; Beck, L. W.; Davis, M. E. J. Phys. Chem. B 2000, 104, 7600. (16) Thiyagarajan, P. J. Appl. Crystallogr. 2003, 36, 373. (17) Heeley, E. L.; Maidens, A. V.; Olmsted, P. D.; Bras, W.; Dolbnya, I. P.; Fairclough, J. P. A.; terril, N. J.; Ryan, A. J. Macromolecules 2003, 36, 3656. (18) Niimura, N.; Minezaki, Y.; Ataka, M.; Katsura, T. J. Cryst. Growth 1995, 154, 136. (19) Davey, R. J.; Lui, W.; Quayle, M. J.; Tiddy, G. J. Cryst. Growth Des. 2002, 2, 4, 269. (20) Iitaka, Y. Acta Crystallogr. 1961, 14, 1. (21) Iitaka, Y. Acta Crystallogr. 1960, 13, 35. (22) Weissbuch, I.; Leiserowitz, L.; Lahav, M. Adv. Mater. 1994, 6, 952. (23) Sakai, H.; Hosogai, H.; Kawakita, T. J. Cryst. Growth 1992, 116, 421. (24) Gidalevitz, D.; Feidenhans’l, R.; Matlis, S.; Leiserowitz, L. Angew. Chem. Int. Ed. Engl. 1997, 36, 955. (25) Zaccaro, J.; Matic, J.; Myerson, A. S.; Garetz, B. A. Cryst. Growth Des. 2001, 1, 5. (26) Garetz, B.; Matic, J.; Myerson, A. S. Phys. Rev. Lett. 2002, 89, 175501. (27) Jan Ilavsky: http://www.uni.aps.anl.gov/∼ilavsky/irena_1.htm. (28) Beaucage, G. J. Appl. Crystallogr. 1995, 28, 717. (29) Beaucage, G. J. J. Appl. Crystallogr. 1996, 29, 134. (30) Mandelbrot, B. B. Fractals, Form, Chance and Dimensions; Freeman: San Francisco, 1977. (31) Keefer, K. D.; Schaefer, D. W. Phys. Rev. Lett. 1986, 56, 2376. (32) de moor, P. P. E. A.; Beelen, T. P. M.; van Santen, R. A. Microporous Mater. 1997, 9, 117. (33) Bale, H. D.; Schmidt, P. W. Phys. Rev. Lett. 1984, 53, 596. (34) Schafer, D. W.; Martin, J. E.; Wiltzius, P.; Cannell, D. S. Phys. Rev. Lett. 1984, 52, 2371. (35) Martin, J. E.; Hurd, A. J. J. Appl. Crystallogr. 1987, 20, 61. (36) Teixeira, J. In On Growth and Form: Fractal and Nonfractal Patterns in Physics; NATO-ASI Series E, Vol. 100; Stanley, H. E., Ostrowsky, N., Eds.; Martinus Nijhoff Publishers: Dordrecht, 1986; pp 145-162.

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