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SEPARATIONS Pseudopolymorphic Crystallization of L-Ornithine-L-Aspartate by Drowning Out Yehoon Kim,† Seungjoo Haam,† Yong Gun Shul,† Woo-Sik Kim,† Joon Ki Jung,§ Hee-Chun Eun,‡ and Kee-Kahb Koo*,‡ Department of Chemical Engineering, Yonsei University, Seoul 120-742, Korea, Korea Research Institute of Bioscience and Biotechnology, Taejeon 305-333, Korea, and Department of Chemical Engineering, Sogang University, Seoul 121-742, Korea
Crystallization of L-ornithine-L-aspartate (LOLA) by drowning out was investigated using methanol as an antisolvent for the production of the anhydrous form of LOLA. An initial mixing method of the LOLA aqueous solution with methanol was found to be a critical factor in the pseudopolymorphic crystallization of LOLA. When methanol was added into the LOLA aqueous solution, it was found that the crystal structure of LOLA was mainly determined by the operating temperature. At a temperature higher than about 60 °C, the anhydrous form of LOLA was always formed. However, at a temperature lower than 50 °C, only LOLA hydrate was produced. On the other hand, when the LOLA aqueous solution was added into methanol, the anhydrous form was obtained by controlling the operational temperature, the feeding rate of the LOLA aqueous solution, and the methanol concentration even at a temperature lower than 50 °C. 1. Introduction It has been known that the rapid accumulation of ammonia by the brain is a main cause of the major complications such as cerebral edema and hepatic encephalopathy of acute liver failure. For patients with chronic liver failure, dosage of an ammonia-lowering agent with mild hypothermia is usually recommended. L-Ornithine (C5H12N3O2), an antidote to ammonia in the blood, is known to play an important role in the urea production of the ornithine cycle in the living body. L-Aspartic acid (C4H7NO4) also plays a role as an amine donor in the ornithine cycle and thus is used for preventing hepatic disturbance. Their simultaneous administration in a complex form of L-ornithine-Laspartate (LOLA) has been proven to be more effective in reducing the blood ammonia concentration in patients with acute liver failure.1-7 L-Ornithine has R-amino and R-carboxyl groups and one amino group in its tail, while L-aspartic acid has R-amino and R-carboxyl groups and one side-chain carboxyl group. All of the amino groups are protonated (NH3+) in water and hence are positively charged, while all of the carboxyl groups are deprotonated (COO-) and are negatively charged. Thus, molecular ions of Lornithine and L-aspartic acid carry a positive charge and a negative charge, respectively. Therefore, LOLA can be obtained by reacting L-ornithine with L-aspartic acid or by reacting an acid addition salt of L-ornithine with a metal salt of L-aspartic acid in the presence of * To whom correspondence should be addressed. Tel: (82)2705-8680. Fax: (82)2-711-0439. E-mail:
[email protected]. † Yonsei University. ‡ Sogang University. § Korea Research Institute.
water.8-10 One method of LOLA crystallization under development for commercialization involves a fermentation process using molasses and corn steep powder as carbon and organic nitrogen sources, respectively. In this process, an aqueous solution with about 33 mass % LOLA is finally obtained. LOLA has two different crystal forms: an anhydrate and a hemihydrate. The hydrate of LOLA is not desirable for commercial purposes because of coloration and swelling during storage for a long period of time, while the anhydrous form does not have such problems. Thus, it is advantageous to produce the anhydrous form of LOLA, and it is essential to understand the crystallization behavior of LOLA for the production of the desired form. However, literature on the crystallization of LOLA has been very limited to date.8-10 LOLA is highly soluble in water, but temperature dependence of solubility is reported to be relatively weak.11,12 The solubility of LOLA, on the other hand, is shown to decrease dramatically with addition of a certain solvent into the LOLA aqueous solution. For the recovery and purification of LOLA as a solid form, we have attempted a cooling crystallization with the saturated solution at 60 °C. The crystals obtained by the cooling method with various cooling rates were gellike, and they were found to be very difficult to filter and dry. Therefore, drowning out would be suitable for LOLA crystallization from an aqueous solution. In crystallization by drowning out, supersaturation is generated by the addition of an antisolvent into the solvent in which the solute is dissolved. The mixing of solvents modifies the structure, properties, and behavior of the electrolytic solution, leading to changes in the mobility and solvation of ions and thus relative permittivity of the solution.13 The dielectric property of aque-
10.1021/ie020432d CCC: $25.00 © 2003 American Chemical Society Published on Web 01/21/2003
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ous electrolyte solutions is of considerable importance for the understanding of the hydration and complexation behavior.14-16 Because the antisolvent interacts strongly with water molecules mostly by hydrogen bonding in the LOLA aqueous solution, the solubility of the LOLA is decreased and thus LOLA is forced to crystallize. In this paper, we report the experimental results undertaken to investigate pseudopolymorphic crystallization of LOLA from an aqueous solution by drowning out. The effect of the mixing methods of the LOLA solution with methanol and the operating temperature on the formation of LOLA hydrate/anhydrate is mainly discussed. 2. Experimental Section 2.1. Materials and Apparatus. LOLA (C5H13N3O2+C4H6NO4-) was supplied by Sigma Chemical Co. and was used after drying for 12 h in a vacuum oven at 50 °C. Deionized water (conductance of less than 4 µs/cm) and methanol (Merck, (99.8 mol %) were used without further purification. The experiments were conducted in a semibatch crystallizer. A jacketed cylindrical glass vessel with 100 mm inner diameter was used. Four stainless steel baffles with 2 mm thickness and 10 mm width are attached to the walls. The temperature of the crystallizer during the experiments was controlled by a heating medium supplied by a thermostat (PolyScience, model 9510). Materials were prepared by mass using a balance with an uncertainty of (0.0001 g. Weighed quantities of materials were charged to the crystallizer, and agitation was provided with a pitched-blade impeller of stainless steel. A peristaltic pump was used to transfer the feed from a stock vessel to the crystallizer at a desired feeding rate. The feeding line was a silicon tube with 3 mm inner diameter. 2.2. Characterization of Pseudopolymorphs. In the present experiments, LOLA crystallization by drowning out was conducted by two methods termed the SM (solution into methanol) and MS (methanol into solution) processes. For the SM process, the LOLA aqueous solution is added into an antisolvent, methanol. For the MS process, methanol is added into an aqueous solution of LOLA. To investigate the crystal structure and morphology, the slurry was sampled from the solution with a desired crystallization time. The crystals were filtered and dried for 24 h at 50 °C for the analysis. Each polymorph was identified using a powder X-ray diffractometer (Rigaku, D/MAX-2500H), a FTIR spectrometer (ATI-Mattson, Genesis), a CHNS elemental analyzer (Flisons, EA1108), and a solid-state 13C NMR spectrometer (JEOL, JNM ECP-300). 2.3. Measurements of Thermodynamic Properties. With a conductivity meter (Metrohm 660), the conductivity measurements of the LOLA aqueous solutions were performed as a function of the LOLA concentration in the range of temperature from 25 to 60 °C. The relative permittivity measurements of methanol/ water mixtures were made in the range of temperature from 25 to 60 °C with a relative permittivity meter (Brookhaven, BI-870). For measurement of the metastable zone width, the LOLA aqueous solution was placed into a doublejacketed equilibrium cell kept at a desired temperature. Supersaturation was achieved by adding methanol into the aqueous solution of LOLA at a constant rate. The
Figure 1. FTIR spectra of LOLA crystals: (a) hydrate; (b) anhydrate. Table 1. Molecular Formula Determined by a CHNS Elemental Analyzer (wt %) hydrate anhydrate
C
H
N
O
39.4 40.8
7.37 7.22
15.3 15.82
37.93 36.19
molecular formula C9H20N3O6.5 C9H19N3O6
metastable zone width was calculated by using the volume of methanol as soon as a turbidity caused by nucleation was noticed by the naked eye. 3. Results and Discussion 3.1. Characterization of Pseudopolymorphs. The crystal structure of LOLA hydrate can be explained by ionic interactions and hydrogen bonds. Each amino group is involved as a donor in three N-H‚‚‚O hydrogen bonds. The R-carboxylate oxygens accept three hydrogen bonds. However, side-chain carboxylate oxygens of Laspartate accept four hydrogen bonds, including one from the lone water molecule in the structure. A water molecule exists between two aspartates.17,18 Molecular formulas of two forms determined by a CHNS elemental analyzer are listed in Table 1. Clearly, one molecule of LOLA hydrate appeared to have 1/2 molecule of water as given by the report of Salunke and Vijayan.18 If a water molecule is incorporated into the crystal lattice, the molecular environment of the various nuclei in a hydrate, such as carbon nuclei, may be different from the environment in the corresponding anhydrate. This leads to a different chemical shift interaction for each nucleus and consequently leads to a different isotropic chemical shift for the same nucleus in the two different modifications.8,18 The FTIR absorption spectra (KBr disk) of LOLA crystals show that the hydrate has an absorption band near 1500 cm-1, as can be seen from Figure 1, but not in anhydrous form. Results of the solidstate 13C NMR spectroscopy also clearly exhibit characteristic peaks corresponding to the crystal structure.
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Figure 2. anhydrate.
13C
NMR spectra of LOLA crystals: (a) hydrate; (b)
Figure 3. Powder X-ray diffraction patterns of LOLA crystals: (a) hydrate; (b) anhydrate.
Figure 2 shows the values of vCO chemical shifts for two crystal forms. In LOLA hydrate, a new chemical shift of vCO appeared at 175 ppm. Also two crystal forms of LOLA are clearly differentiated by the powder X-ray diffraction patterns, as can be seen from Figure 3. As can be seen from Figure 4, the morphology of LOLA crystals produced by drowning out was the spherical shape of crystal agglomerates. The overall appearances of the two forms look alike, but the surface features are found to be very different. The hydrate of LOLA is shown to be agglomerates of flakelike crystals (Figure 4c) and the anhydrate appears to be agglomerates of needlelike crystals, as shown in Figure 4d. Recently, the production of spherical agglomerates of pharmaceutical compound crystals has gained great attention, because the modification in the crystal habit can change the bulk density, flowability, compactibility, stability, etc.19-23 Moreover, the formation of the spheri-
cal crystal agglomerates is very important for preparing the solid dosage forms by capsule filling and tablet making. In this sense, crystallization by drowning out seems to be adequate for the separation of LOLA from the aqueous solution. 3.2. Thermodynamic Properties. Solubility is a key factor in the determination of the recovery of materials achievable by crystallization, and it is required in order to quantify supersaturation, an important parameter in nucleation and growth kinetics of crystals. In our previous study, the solubility of anhydrous LOLA was investigated both theoretically and experimentally.12 The solubility of L-aspartic acid was as low as 0.5 g/100 g of H2O at 25 °C, but a salt form with L-ornithine was found to enhance dramatically the solubility. For example, the solubility of anhydrous LOLA in pure water was 95-129 g/100 g of H2O in the range of temperature from 25 to 60 °C. The reason for the solubility enhancement of LOLA would be that the solution of LOLA maintains at neutral pH by the generation of zwitterions. Solubility measurements of anhydrous LOLA in the methanol/water mixture were also carried out as a function of the methanol content up to 90 mass %, as given in Figure 5. In this figure, one can notice that methanol reduces rapidly the LOLA solubility in water over the whole range of the methanol content studied in the present experiments, and the effect of temperature on the solubility of LOLA is relatively smaller than that by the methanol addition. Solubility data were used to determine the required concentration of methanol needed to prepare a desired saturated solution. Figure 6 is the molal conductivity of the LOLA aqueous solution measured as a function of the square root of the LOLA concentration in the range of temperature from 25 to 60 °C. This figure shows that the molal conductivity of the LOLA aqueous solution decreases as the concentration of LOLA increases. In particular, it is shown that a decrease in the molal conductivity at lower concentration can be represented by a linear function of the square root of concentration.24 The gradual decrease in the molal conductivity at low concentrations is known to be a general characteristic of a strong electrolyte.25 Interaction of ions is dependent on the relative permittivity of a solvent when the concentration of the electrolyte is constant. That is, the decrease of the relative permittivity correlates with the increase of the electrostatic interaction between ions of opposite charge and the formation of insoluble ionic species. Figure 7 shows the relative permittivity of the methanol/water mixture in the range of temperature from 20 to 60 °C. The value of the relative permittivity for pure water is about 80, while for methanol, it is about 32.6 at 20 °C. Therefore, the progressive addition of methanol to an aqueous solution of an ionic salt, LOLA, leads to a decrease of the relative permittivity and an increase of ionic cohesion forces, which implies the formation of LOLA crystals. To obtain a better understanding of the hydrate formation, it is essential to evaluate how the supersaturation is created in the system, as well as to determine the metastable zone width. In these experiments, the degree of the metastable zone width given by ∆C was calculated from the solubility data of LOLA in pure water and the volume of methanol added in each experiment until nucleation occurs. When the feeding rate of methanol was increased from 1 to 10 mL/min, the variation of the metastable zone width was found
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Figure 4. SEM photographs of LOLA crystals: (a) hydrate; (b) anhydrate; (c) surface of the hydrate; (d) surface of the anhydrate.
Figure 5. Solubility of LOLA measured with the methanol concentration in a methanol/water mixture (b, 25 °C; 9, 40 °C; 2, 50 °C; [, 60 °C).
Figure 6. Molal conductivity of a LOLA aqueous solution measured with the LOLA concentration (b, 25 °C; 9, 40 °C; 2, 50 °C; [, 60 °C).
to be negligible. Therefore, the data obtained at the feeding rate of methanol of 1 mL/min were selected as a representative and were illustrated in Figure 8. This figure indicates that the metastable zone width is significantly affected by temperature. For instance, ∆C ) 18.6 (LOLA g/100 g of solution) decreases linearly to ∆C ) 9 as the temperature increases from 25 to 60 °C. In principle, the system which has a larger metastable zone width should be correlated with the higher dehydration energy.13 Inversely, a small degree of the metastable zone width indicates that the solution is in a state with the lower dehydration energy. Therefore, the present experimental results are readily explained by the fact that the increase of temperature correlates to the increase of the electrostatic interaction between ions of opposite charge and results in the decrease of the metastable zone width.
3.3. Crystallization of LOLA Hydrate/Anhydrate in the MS Process. Figure 9 shows typical crystal structures obtained from the MS process, i.e., the crystallization by the addition of methanol into the LOLA aqueous solution (feeding rate of methanol: 1 mL/min). Solid lines in this figure represent the relative permittivity taken from Figure 7. All data in Figure 9 were obtained by the experiments given below. In general, at any given temperature, nucleation of LOLA was observed to occur at a methanol content of about 60 mass %. The first sample of LOLA crystal was taken when the methanol content was 62 mass %, and the crystal structure was determined by the powder X-ray diffraction pattern. The same experiment was restarted, and the second sample was taken when the methanol was 65 mass %, and so on.
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Figure 7. Relative permittivity of the methanol/water mixture (1, 20 °C; b, 30 °C; 9, 40 °C; 2, 50 °C; [, 60 °C).
Figure 8. Metastable zone width of LOLA measured with temperature.
Figure 9. Effects of temperature and methanol concentration on the crystal structure of LOLA for the MS process (2, hydrate; 4, anhydrate).
From the experiments conducted at 60 °C, we found that the anhydrous form of LOLA was always produced and the hydrate did not appear even though methanol was added up to 80 mass %. However, it is interesting to note that only the hydrous form of LOLA was generated below 50 °C, as can be seen from Figure 9. The present results agree qualitatively with those reported previously,8 and it seems that the anhydrous form of LOLA is a stable structure at a temperature of above 60 °C. This implies that formation of the LOLA
Figure 10. Effects of temperature and methanol concentration on the crystal structure of LOLA in the SM process (b, hydrate; O, anhydrate). The feeding rate of LOLA solution into methanol: (a) 1 mL/min; (b) 4 mL/min.
anhydrate depends on the diffusivity of water molecules into the methanol phase, which depends on the temperature. 3.4. Crystallization of LOLA Hydrate/Ahhydrate in the SM Process. In the SM process, the starting point of every experimental run is the pure methanol side and thus the fraction of methanol in the solution decreases as the LOLA aqueous solution is added. Figure 10 shows typical results of the SM process represented on a plot of the relative permittivity with the methanol content. In this figure, all of the data also were taken from the separate experimental runs as the data shown in Figure 9. First of all, it is interesting to note that anhydrous forms of LOLA crystals were obtained at the initial stage of the SM process with a small flow rate, for example, 1 mL/min in Figure 10a, in the entire range of temperature studied here. These results contrast with those obtained from the MS process in which hydrates are formed at a temperature lower than 50 °C (see Figure 9). This may be explained by the behavior of intermolecular interaction between water and methanol in their mixture. As soon as the LOLA aqueous solution with a small flow rate is added into the bulk methanol phase, the bulk methanol phase immediately takes away all water molecules from the LOLA complex by the intermolecular force between them. Therefore, a high supersaturated state of the LOLA complex will be immediately created and the anhydrous form of LOLA crystals tends to be nucleated. However, as the water
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Table 2. Effects of Temperature and Methanol Feeding Rate on the Formation of LOLA Hydrate in the SM Process (A, Anhydrate; H, Hydrate) SM process temp (°C)
MS process
instantaneous mixing
20 30 40 50 60
H H H H A
H H H A A
4 mL/min
1 mL/min
H H A A A
A A A A A
content in the bulk methanol phase increases, i.e., as the amount of the LOLA aqueous solution added into the methanol phase increases, attractive force of methanol with water molecules freshly added is weakened and thus the LOLA hydrate is crystallized, as can be seen from the results obtained at the operating temperature of 20 or 40 °C (solid circles in Figure 10a). Here, it should be noted that all hydrates were generated in the solution with a relative permittivity higher than about 42. On the other hand, when the flow rate of the LOLA solution was 4 mL/min (Figure 10b), for the formation of the LOLA anhydrate the operating temperature was shown to be higher than 40 °C, but the anhydrous form was found to be produced only in the solution with the relative permittivity lower than 42. The extended results of the LOLA crystal structure initially formed with the feeding rate of the LOLA solution in the SM process are summarized in Table 2. As can be seen from Table 2, the anhydrous form was obtained at 50 °C even when the LOLA aqueous solution was instantaneously mixed with methanol. All of those results indicate that the nucleation of the anhydrous form is possible if the temperature is high enough to diffuse water molecules into the bulk methanol phase even at a higher feeding rate. In summary, two factors seem to be responsible for the pseudopolymorphic crystallization of LOLA in the SM process: the diffusion of water molecules from the LOLA complex to the bulk methanol phase and the relative permittivity. When a feeding rate of LOLA SM is relatively larger, for example, 4 mL/min, it seems that migration of water molecules coordinated with the LOLA complex to the bulk methanol solution is the controlling step at a temperature lower than 30 °C. As the feeding rate of the LOLA solution decreases to 1 mL/ min, the diffusion rate of water molecules is faster, the anhydrous form of LOLA is generated, and subsequently the temperature for the formation of the LOLA anhydrate can be lowered, as can be seen from Figure 10a. However, when the feeding rate of the LOLA solution is small enough, the value of the relative permittivity of the solution may be a criterion for the anhydrate formation of LOLA. Therefore, we conclude that the LOLA anhydrate that is a desired crystal structure is obtained by controlling the feeding rate and the operational temperature in the SM process. 4. Conclusions Pseudopolymorphic crystallization of LOLA was investigated by drown out using methanol as an antisolvent. In general, the initial mixing method of the LOLA aqueous solution with methanol was found to influence the structure of LOLA crystals initially formed. When methanol was added into the LOLA aqueous
solution, it was found that only the temperature affected the crystal structure of LOLA produced; i.e., the hydrate of LOLA was always produced when the temperature was lower than 50 °C, and the anhydrous form was only formed at a temperature of 60 °C. This result indicates that the transition temperature of two LOLA modifications is between 50 and 60 °C. On the other hand, when the LOLA aqueous solution was added into methanol, the initial crystal form was dependent not only on the operating temperature but also on the feeding rate of the LOLA aqueous solution and the methanol concentration. When a feeding rate of the LOLA solution is small enough, it is found that the anhydrous form could be produced at a temperature as low as 20 °C. However, as the feeding rate is increased, a higher operating temperature is required for the formation of anhydrate. It was also found that the solution property given by the relative permittivity played an important role in the pseudopolymorphic crystallization of LOLA. A study of the relationship between the relative permittivity, the formation of two modifications of LOLA, and solventmediated transformation experiments is currently underway for the quantitative explanation of pseudopolymorphic crystallization behavior of LOLA by drowning out. Acknowledgment This work was supported by Korea Research Foundation Grant KRF-2001-042-E00058. Literature Cited (1) Desjardins, P.; Be´langer, M.; Butterworth, R. F. Alterations in expression of genes coding for key astrocytic proteins in acute liver failure. J. Neurosci. Res. 2001, 66, 967. (2) Rees, C. J.; Oppong, K.; Al Mardini, H.; Hudson, M.; Record, C. O. Effect of L-ornithine-L-aspartate on patients with and without TIPS undergoing glutamine challenge: a double blind, placebo controlled trial. Gut 2000, 47, 571. (3) Rose, C.; Michalak, A.; Rao, K. V.; Quack, G.; Kircheis, G.; Butterworth, R. F. L-Ornithine-L-aspartate lowers plasma and cerebrospinal fluid ammonia and prevents brain edema in rats with acute liver failure. Hepatology (Philadelphia) 1999, 30, 636. (4) Rose, C.; Michalak, A.; Pannunzio, P.; Therrien, G.; Quack, G.; Kircheis, G.; Butterworth, R. F. L-Ornithine-L-aspartate in experimental portal-systemic encephalopathy: therapeutic efficacy and mechanism of action. Metab. Brain Dis. 1998, 13, 147. (5) Stauch, S.; Kircheis, G.; Adler, G.; Beckh, K.; Ditschuneit, H.; Go¨rtelmeyer, R.; Hendricks, R.; Heuser, A.; Karoff, C. Oral L-ornithine-L-aspartate therapy of chronic hepatic encephalopathy: results of a placebo-controlled double-blind study. J. Hepatol. 1998, 28, 856. (6) Kircheis, G.; Nilius, R.; Held, C.; Berndt, H.; Buchner, M.; Go¨rtelmeyer, R.; Hendricks, R.; Kru¨ger, B.; Kuklinski, B. Therapeutic efficacy of L-ornithine-L-aspartate infusions in patients with cirrhosis and hepatic encephalopathy: results of a placebocontrolled, double-blind study. Hepatology (Philadelphia) 1997, 25, 1351. (7) Vogels, B. A.; Karlsen, O. T.; Mass, M. A.; Bovee´, W. M.; Chamuleau, R. A. L-Ornithine vs L-ornithine-L-aspartate as a treatment for hyperammonemia-induced encephalopathy in rats. J. Hepatol. 1997, 26, 174. (8) Chugai Pharmaceutical Co. Improvements in and relating to L-ornithine-L-aspartate. U.K. Patent 1,080,599, 1967. (9) Kunio, S.; Hirofuto, M. Process for the preparation of L-ornithine L-aspartate. U.K. Patent 1,067,742, 1967. (10) Tanabe Seiyaku Co. L-Ornithine-L-aspartate. U.K. Patent 965,637, 1964. (11) Greenstein, J. P.; Winitz, M. Chemistry of the Amino Acid; Krieger Publishing Company: FL, 1984. (12) Kim, Y.; Haam, S.; Koo, K.-K.; Shul, Y.; Jeong, J. Representation of Solid-Liquid equilibrium of L-ornithine-L-aspartate
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(20) Brittain, H. G., Ed. Polymorphism in Pharmaceutical Solids; Marcel Dekker: New York, 1999. (21) Kawashima, Y.; Okumura, M.; Takenaka, H. Spherical Crystallization: Direct Spherical Agglomeration of Salicylic Acid Crystals During Crystallization. Science 1982, 216, 1127. (22) Mohan, R.; Koo, K.-K.; Strege, C.; Myerson, A. S. Effect of Additives on the Transformation Behavior of L-Phenylalanine in Aqueous Solution. Ind. Eng. Chem. Res. 2001, 40, 6111. (23) Chulia, D.; Deleuil, M.; Pourcelot, Y. Powder Technology and Pharmaceutical Processes; Elsevier: Amsterdam, The Netherlands, 1994. (24) Fuoss, R. M.; Accascina, F. Electrolyte Solutions; Academic Press: New York, 1959. (25) Prausnitz, J. M.; Lichtenthaler, R. N.; de Azevedo, E. G. Molecular Thermodynamics of Fluid-Phase Equilibria; Prentice Hall: Englewood Cliffs, NJ, 1999.
Received for review June 13, 2002 Revised manuscript received December 17, 2002 Accepted December 20, 2002 IE020432D
