CRYSTAL GROWTH & DESIGN
Nucleation Behavior of D-Threonine on Different Faces of L-Threonine Crystals
2008 VOL. 8, NO. 8 2716–2720
Akihiko Ito and Masakuni Matsuoka* Department of Chemical Engineering, Tokyo UniVersity of Agriculture and Technology, 24-16 Naka-Cho 2, Koganei, Tokyo, Japan 184-8588 ReceiVed August 15, 2007; ReVised Manuscript ReceiVed March 28, 2008
ABSTRACT: Three different faces of L-threonine (L-Thr) crystals growing from supersaturated solutions enriched with D-Thr were observed under an optical microscope to make clear the mechanism of optical purity decrease during preferential crystallization operation, in particular the effect of washing the seed crystals. The solution concentration corresponded to those in the later period of optical resolution processes. On the {210} faces small platelike crystals of D-Thr were found to appear by the mechanism of surface epitaxial nucleation. For seed crystals washed with water, such D-Thr crystals were not observed. A closer observation of the surfaces with an atomic force microscope (AFM) revealed that the {210} faces washed with water had changed to the combination of the {100} and {110} faces. With a forced nucleation method on the {210}, {100}, and {110} faces, deposits of D-Thr particles showed very different appearances, indicating that surface nucleation occurred only on the {210} faces. On the basis of these observations, the purity decrease in the later period of batch preferential crystallization was explained as the surface nucleation of the undesired isomer. 1. Introduction When optically active compounds are chemically synthesized, racemates are generally produced. Racemates are mixtures of equal amounts of both isomers and can be classified into racemic mixtures and racemic solids.1 These isomers have identical physical properties except for optical rotations, and their activities as foods and medicines are different; therefore, separation of racemates into individual isomers is essential. For the separation of racemic mixtures, preferential crystallization has been applied in which desired isomer crystals are seeded, allowed to grow, and finally separated.2 However, the purity of the crystals is reported to decrease during preferential crystallization, because of spontaneous crystallization of the undesired optical isomer;3,4 therefore, it is necessary to understand the mechanism of such spontaneous crystallization, particularly in the later period of the operation. The time of starting the purity decrease is known to be influenced by various factors, e.g., agitating speed,5 suspension density,3 and supersaturation.6 In addition, in the case of threonine (Thr),3,4 washing the seed crystals delayed the start of the purity decrease by 4 h compared with the seeds without washing. As possible mechanisms of purity decrease or generation of undesired isomer, the followings have been proposed; growth of undesired isomer already present in the seed crystals,3 bulk nucleation7 and heterogeneous nucleation on the seed crystals.8,9 When lamellar racemic twin crystals were crystallized during preferential crystallization, optical purity of products was low.10,11 For the effect of washing, the growth mechanism of undesired isomers was proposed as a possible one3. However, it is not clearly understood which mechanism is dominant. The purpose of the present study is to observe phenomena occurring on the different surfaces of growing seed crystals and to make
* Corresponding author. Phone: 81-42-388-7059. Fax:81-42-387-7944. E-mail:
[email protected].
Figure 1. Typical rodlike L-Thr single crystal grown from aqueous solution: (a) orthographic projection, (b) photomicrograph images.
clear the effect of washing the seed crystals on the changes in surface appearance caused by washing. 2. Experimental Section Threonine (Thr) crystallizes as a stable conglomerate and its formation of racemic compound is not known. An illustration and a photomicrograph of the L-Thr rodlike crystal are shown in Figure 1(threonine belongs to the orthorhombic system, space group of P212121, lattice parameter: a ) 13.64, b ) 7.75, and c ) 5.16 (Å)).12 Polymorph and hydrates of D- or L-Thr have not been reported. The solubilities of each isomer and the conglomerate in water are reported.3 In addition, the purity decrease phenomena have been examined in detail. In the present study, three crystallographic faces of seed crystals were observed in supersaturated DL-Thr solutions enriched with D-Thr, under an optical microscope and an AFM, and the nucleation behaviors were compared. 2.1 Preparation of Seed Crystals of L-Thr and Washing Single Crystals of L-Thr. L-Thr crystals and the conglomerate were supplied from Ajinomoto Co., Ltd. HPLC analysis showed that the amount of impurities in L-Thr crystals was below 0.01%. The L-Thr content in
10.1021/cg700774g CCC: $40.75 2008 American Chemical Society Published on Web 06/21/2008
Nucleation Behavior of D-Threonine
Crystal Growth & Design, Vol. 8, No. 8, 2008 2717
Figure 2. Definition of saturated compositions of each isomer in the solution. crystals was measured with HPLC using a chiral Daicel Crownpak CR (+) column. To prepare seed crystals of L-Thr, saturated aqueous solutions of L-Thr at 308 K were cooled to 298 K at a cooling rate of 1 K min-1, and rod-like single crystals having representative dimensions of 1 mm × 1 mm × 10 mm were obtained after a few days. Major four side faces of obtained L-Thr crystal were identified as the {210} faces, but the {100} faces also appeared.12,13 To see the effect of washing, we washed these L-Thr crystals placed on a filter paper with about 50 mL of running ultrapure water for 30 s at 294 K. 2.2. In situ Observation of L-Thr Faces in DL-Thr Solutions. All observations were performed at 294 K. To prepare solutions that are supersaturated with respect to both isomers but with the degree of supersaturation of D-Thr being higher than that of L-Thr, a predetermined amount of D-Thr crystals were further dissolved in solutions that were saturated at 298 K; the solution was then heated to 308 K. The solutions thus prepared are referred to as enriched D-Thr solutions. The solution was then cooled to the experimental temperature. The concentration of total Threonine in solutions was determined by a gravimetric method and the ratio of D- and L-Thr in solutions was determined by HPLC analysis. The degrees of supersaturation of the enriched D-Thr solution in terms of D-Thr and L-Thr were σD ) 0.21 and σL ) 0.05, respectively, where they are defined as σD ) (wDsol - wD*)/wD* and σL ) (wLsol - wL*)/ wL* in terms of the saturated concentration (wD* or wL*) and the bulk solution concentration (wDsol or wLsol). Because the saturated concentration of each isomer varies with the bulk solution concentrations, they are defined as those shown in Figure 2. For example, for solution A (wDsol, wLsol), its corresponding saturated composition in terms of D-Thr is given as B, whereas that for L-Thr is given as C. Point B represents the ideal end point of the preferential crystallization. A single crystal (about 1 mm × 1 mm × 10 mm) of L-Thr was placed in a closed cell, as shown in Figure 3. Eighty microliters of the prepared enriched D-Thr (σD ) 0.21) was injected in the cell, and the {210} faces of the seed crystal were continuously observed for 6 h under an optical microscope and an AFM (Nanoscope III AFM of Digital Instruments) in contact mode. 2.3. Forced Nucleation of D-Thr on the Various Surface of L-Thr Crystal. To see the occurrence of nucleation on the {210}, {100}, and {110} faces of L-Thr seed crystals, we conducted the following experiments. For the observation of the {210} and {100} faces, rodlike crystals crystallized from aqueous solutions were used, whereas washed crystals with water were used for observation of the {110} faces. (The preparation method of the {110} faces will be discussed later in section 3.2.) Unwashed or water-washed L-Thr crystals were immersed in the DLThr saturated solution at 294 K for 1 min and taken out from the solution. The solution adhering to the seed crystal was then allowed to evaporate so as for D-Thr to nucleate on the crystal surface. After the completion of evaporation, the surfaces were observed with the AFM.
3. Results and Discussion 3.1. Generation of D-Thr on L-Thr Crystal Surface from Enriched D-Thr Solution. All the {210} faces of L-Thr are identical from a viewpoint of molecular arrangements as
Figure 3. Experimental apparatus for in situ observation under optical microscope and AFM: (a) top view and (b) side view.
well the shown later, so the following observation were carried out using one of the {210} faces of each seed crystal. On the {210} faces of a L-Thr single crystal immersed in the enriched D-Thr solution (σD ) 0.21 and σL ) 0.05), platelike crystals were found to appear after 50 min. In Figure 4, growth of a platelike crystal on the {210} faces of L-Thr crystals observed is shown, of which photographs were taken after 100 and 113 min. These platelike crystals were aligned along the c-axis of the L-Thr seed crystals. To determine if these platelike crystals were either D-Thr or L-Thr, the solution in the cell surrounding the seed crystal was replaced with an L-Thr supersaturated solution for about 1 s and the changes in the shape of the crystals were observed under the microscope. Because the solution was supersaturated with respected to L-Thr, but contained no D-Thr, i.e., σD ) 0.0, σL ) 0.03, if the platelike crystals were D-Thr, they would dissolve; if they were L-Thr, slight increases in size would be observed. The observed changes in the platelike crystals are shown in Figure 5, and they began dissolving after 10 s. On the other hand, the seed crystal beneath them did not show any changes. These platelike crystals on the seed crystal were therefore identified as D-Thr. Because these D-Thr crystals were aligned along the c-axis of the seed crystal, they were concluded to have been formed by spontaneous epitaxial surface nucleation on the {210} faces of L-Thr crystals. 3.2. Observation of Washed L-Thr Single-Crystal Surface in DL-Thr Solution. To see the effect of washing of seed crystals, surfaces of the seed crystals after washing were observed with the optical microscope and AFM. As shown in Figure 6a, the crystal surface after washing with water had a large number of pits and the surface was not flat compared with the original surfaces shown as the basal planes in Figures 4 and 5. These pits were caused by the dissolution of L-Thr crystals in water. According to the AFM observation, as shown in Figure 7 as the cross-section of the surface, the {210} faces almost disappeared and new microscopic faces appeared. Figure 7 indicates the cross-section of the original {210} faces of the seed crystals and the new faces are inclined by 40 and 18° to the original {210} faces, which are indicted by the dotted lines. On the basis of the L-Thr crystal structure data,12 the {100}
2718 Crystal Growth & Design, Vol. 8, No. 8, 2008
Ito and Matsuoka
Figure 5. Observed dissolution of the platelike crystal in a L-Thr saturated solution (a) before immersion in the L-Thr saturated solution, and after immersion for (b) 20 s and (c) 1 min. Figure 4. Photomicrographs of platelike crystals on the L-Thr {210} face in the DL-Thr solution with large supersaturation (σD ) 0.21) with respect to D-Thr, after (a) 0, (b) 100, and (c) 113 min.
and {110} faces are respectively inclined theoretically by 41.8 and 19.0° to the {210} faces, therefore the new faces were identified as the {110} and {100} faces. To see the recovery process of the dissolved faces of the seed crystals, we immersed the seed crystals washed by water in the enriched D-Thr solution (σD ) 0.21, σL ) 0.05) and observed the crystal surface for 6 h. As already shown in Figure 6b, the pits had disappeared; however, the surface still showed parallel lines along the c-axis, showing that the {210} face was not completely recovered in 6 h. In addition on these surfaces, platelike D-Thr crystals were not observed in the experiments that continued for 6 h. Since the volume of the solution was so small (80 µL) and the supersaturation in terms of L-Thr was low (σL ) 0.05), the possible amount of deposition of L-Thr was not enough to recover the entire surface of the seed crystal even after the consumption of the initial supersaturation of L-Thr. Similar parallel lines can also be found in a photograph in the report3 on the seed crystals washed with water after about 3 h before the start of purity drop during preferential crystallization. They are aligned along the c-axis of the seed crystals. However, the solution supersaturation in that case was higher and the solution volume was also larger compared with the present case; the surface of the seed crystals could have
recovered further, so that spontaneous surface nucleation would have occurred,leading to the decrease of purity after 4 h. 3.3. Forced Nucleation of D-Thr on Various Surfaces of L-Thr Crystal. As mentioned above, in the enriched D-Thr solution (σD ) 0.21, σL ) 0.05), D-Thr plate-like crystals were generated on the {210} faces by surface nucleation, while on the other hand, they did not appear on the washed crystal surfaces, which consisted of the {100} and {110} faces. To discuss the difference between these results, ease of nucleation of D-Thr on the {210} faces was compared with that on the {100} and {110} faces. Forced nucleation experiments, described in section 2.3, were performed on the {210}, {100}, and {110} faces of L-Thr seed crystals, and D-Thr crystals were observed on the {210}, {100} and {110} faces with the AFM, and Figures 8 and 9 show typical surface structures observed. The images given in the left show the height of the crystal surface and those in the right the diffraction image, which are imaged error signals during scanning the same locations. In Figure 8a, microscale crystals were observed in several areas of the {210} faces, where the droplets of the adhered mother solution might have evaporated. These microcrystals were identified as D-Thr with the same method mentioned in section 3.1. Because these D-Thr crystals on the L-Thr {210} faces (Figure 8b) had the same orientation along the c-axis, and had the same shape (thickness: between 20 and 150 nm), they
Nucleation Behavior of D-Threonine
Figure 6. (a) Surface of washed L-Thr crystal and (b) immersed in DL-Thr in the supersaturated solution (σD ) 0.21σL ) 0.05) for 6 h.
Crystal Growth & Design, Vol. 8, No. 8, 2008 2719
Figure 8. AFM images of D-Thr crystals on a L-Thr crystal after forced nucleation experiments showing many microsize crystals on the {210} face. Scan size is (a) 40 µm × 40 µm and (b) 15 µm × 15 µm.
Figure 7. Cross-section of L-Thr crystal surface washed with water before immersion in the DL-Thr saturated solution.
were concluded to have nucleated epitaxially on the {210} faces of L-Thr crystals. Images a and b in Figure 9 are AFM images of D-Thr on the {100} and {110} faces of L-Thr crystal, respectively. In this forced nucleation experiment, D-Thr crystals were also observed on the {100} and {110} faces. These crystals were different from those observed on the {210} faces in that they were substantially small, their shapes were irregular and their orientations were at random. On the basis of these observations, it can be concluded that D-Thr crystals did not nucleate with the surface exitaxial nucleation mechanism on the {100} or {110} faces, and that nucleation of these D-Thr crystals is assumed to have occurred in the solution phase during the evaporation. The crystal structure projection on the (x;y) plane is shown in Figure 10 to envisage the molecular arrangements of the {100}, {110}, and {210} faces. The {210} faces are flat in the molecular level, and both positively and negatively charged functional groups appear on {210} faces, indicating that these faces are electrically neutral. On the contrary, the {110} faces also have a negative electric charge. For the {100} faces, only negative groups appear on the surfaces; therefore, the faces are electrically charged.14 Since the crystal structure of D-Thr is a mirror image of that of L-Thr, the corresponding D-Thr structure was computed and drawn graphically and was superimposed on the L-Thr crystals
Figure 9. AFM images of D-Thr crystal on L-Thr surface after forced nucleation experiments for the (a) {100} and (b) {110} faces. Scan size is (a) 20 µm × 20 µm and (b) 2 µm × 2 µm.
to see how intermolecular bondings are changed between the {210}, {100}, and {110} planes. Figure 11 represents the interface between the {210} face of a D-Thr layer on the {210} L-Thr crystal face, showing that intermolecular bondings are well-kept so that clusters of D-Thr molecules are likely to form on the {210} faces of L-Thr crystals. Meanwhile, those on the {100} and {110} faces are unlikely because most of the bondings are broken between the molecules on those faces. Although detailed computation of bonding energies is not yet
2720 Crystal Growth & Design, Vol. 8, No. 8, 2008
Figure 10. Crystal structure of (001) faces of L-Thr. The dotted lines were indicated the (100), (110), and (210) faces, respectively.
Ito and Matsuoka
the mechanisms involved. During the growth of L-Thr seed crystals from supersaturated solutions enriched with D-Thr, platelike crystals were observed on the {210} faces of the seed crystals. These crystals were identified as D-Thr, and because they were aligned in the same orientation, surface epitaxial nucleation mechanism was concluded. On the contrary, these platelike crystals did not appear when the seed crystals were washed with water. The AFM observation revealed that the {210} faces disappeared and the new surface composed of the {100} and {110} faces appeared. The forced nucleation experiments showed that surface epitaxial nucleation occurred only on the {210} faces, and it was not expected on the {100} and {110} faces. In addition, the recovery of the dissolved {210} faces needed longer periods of time; therefore, the nucleation of D-Thr on such surface would be delayed. Acknowledgment. This study was supported by the Japan Society for the Promotion of Science (JSPS), Grant-in Aid for the Scientific Research (B)(2), 16360380, 2004-2006.
Figure 11. Slice of the {210} face of L-Thr crystals is bound to that of D-Thr crystals.
made, nucleation of D-Thr on the {210} faces of L-Thr can be much more likely compared with that on other faces. 3.4. Mechanism of Delay of Purity Decrease during Preferential Crystallization. The delay of starting purity decrease caused by washing the seed crystals with water in the preferential crystallization experiments as reported in the literature3,4 may therefore be attributed to the changes occurring in the surface structures by the dissolution of seed crystals, which causes disappearance of the {210} faces and forms new stepwise structures composed of the {110} and {100} faces on which spontaneous surface nucleation of D-Thr is difficult to occur. The observed delay of purity decrease is therefore considered to occur because of the structure changes in the surface from the {210} face, on which surface epitaxial nucleation is possible, to the combination of the {100} and {110} faces, for which surface nucleation is not expected. 4. Conclusions With threonine, phenomena of purity decrease in the later period of preferential crystallization and the effects of washing of seed crystals were experimentally examined to make clear
References (1) Jacques J. Collet, A.; Wilen, S. H. Enantiomers, Racemates and Resolutions; John-Wiley & Sons: New York, 1991. (2) Coquerel, G. Top. Curr. Chem. 2007, 269, 1–53. (3) Profir, V. M.; Matsuoka, M. Colloids Surf., A 2000, 164, 315–324. (4) Matsuoka, M.; Hasegawa, H.; Ohori, K. ACS Symp. Ser. 1990, 438, 251–260. (5) Yokota, M.; Oguchi, T.; Arai, K.; Toyokura, K.; Inoue, C.; Naijo, H. ACS Symp. Ser. 1990, 438, 898–907. (6) Hongo, C.; Yamada, S.; Chibata, I. Bull. Chem. Soc., Jpn. 1981, 54, 1905–1910. (7) Matsuoka, M. ACS Symp. Ser. 1997, 667, 59–72. (8) Yokota, M.; Toyokura, K. Kagaku Kogaku Ronbunshu 1993, 19, 265– 270. (9) Yokota, M.; Toyokura, K. Kagaku Kogaku Ronbunshu 1992, 18, 832– 838. (10) Gervais, C. P.; Beilles, S.; Cardina, P.; Petit, S.; Coquerel, G. J. Phys. Chem. B 2002, 106, 646–652. (11) Torbeev, Y. V.; Lyssenko, A. K.; Kharybin, N. O.; Antipin, Y. M.; Kostyanovsky, G. R. J. Phys. Chem. B 2003, 107, 13523–13531. (12) Clark, R. L.; Krc, J., Jr. Anal. Chem. 1952, 24, 1378–1379. (13) Janczak, J.; Zobel, D.; Luger, P. Acta Crystallogr., Sect.C 1997, 53, 1901–1904. (14) Shoemarker, D. P.; Donohue, J.; Shoemarker, V.; Corey, R. B. J. Am. Chem. Soc. 1950, 72, 2328–2349.
CG700774G
