Effect of Amino Acid Additives on the Crystallization of L-Glutamic Acid Caitriona
Cashell,†,‡
David
Corcoran,*,†,§
and B. Kieran
Hodnett†,‡
Materials and Surface Science Institute, University of Limerick, Limerick, Ireland, Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland, and Department of Physics, University of Limerick, Limerick, Ireland Received August 18, 2004;
CRYSTAL GROWTH & DESIGN 2005 VOL. 5, NO. 2 593-597
Revised Manuscript Received November 1, 2004
ABSTRACT: The influence of a range of amino acid additives on the crystallization of L-glutamic acid has been investigated. The presence of a bulky side chain (phenyl) in L-phenylalanine and L-tyrosine was identified as an essential feature in achieving stabilization of the R-polymorph at low additive concentrations. Minute quantities of these additives were incorporated into the R-L-glutamic acid product crystals when the additive/L-glutamic molar ratio was 1:30 in solution; in this condition no β-form inclusions were observed inside the R-form crystals. At higher additive/L-glutamic acid molar ratios in solution (1:6), a significant amount of additive uptake was observed and changes in R-form morphology were noted under these conditions. A key feature was the disappearance of the {011} and {001} facets of R-L-glutamic acid and the emergence of the {111}and {110} facets. The overall hypothesis is that additives disrupt the developing β/R interface and this stabilizes the R-form. Introduction Selective crystallization of polymorphs requires control and manipulation of the nucleation and growth processes and may be achieved using additives.1 Currently it is believed that additives operate by adsorption onto crystal facets, which consequently changes the surface free energy and may block sites which are necessary for incorporation of solute into the crystal lattice. This may ultimately result in kinetic and morphological changes.2 Additives which are used to control polymorphism can also induce morphological changes which cannot be avoided.3 In a dimorphic system, if an additive adsorbs on the fast growing face of one polymorph and hinders its growth significantly, the other polymorph may be crystallized selectively.4 Sakata et al. examined the effect of impurities such as amino acids or inorganic salts on polymorphism in L-glutamic acid (L-Glu).5 The R-form of L-Glu predominated when crystallized in the presence of L-aspartic acid (L-Asp), L-cysteine (L-Cys), L-phenylalanine (L-Phe), or L-tyrosine (L-Tyr); while β-crystals predominated when crystallized with L-methionine (L-Met), L-arginine (L-Arg), L-lysine (L-Lys), and a racemic mixture of D- and L-serine (DL-Ser). It was postulated that additives adsorb onto existing β-nuclei, and only amino acids with the same steric configuration as that of L-Glu were found to be effective in stabilizing the R-form.5 D-Phe was found to have no effect on the L-Glu transformation.6 Kitamura and co-workers examined the effect of L-Phe and D-Phe on the R to β polymorphic transformation of L-Glu at 45 °C over a 72 h interval. Low concentrations of L-Phe preferentially restricted the nucleation rate of the β-form to the advantage of the R-form. The precipi* To whom correspondence should be addressed. Mail: Material and Surface Science Institute, University of Limerick, Plassey Technological Park, Limerick, Ireland. Phone: +353 (0)61 202509. Fax: +353 (0)61 202423. E-mail:
[email protected]. † Materials and Surface Science Institute. ‡ Department of Chemical and Environmental Sciences. § Department of Physics.
tation ratio of R-L-Glu increased with L-Phe concentration, and almost 100% R-form was observed at concentrations of L-Phe above 2.6 × 10-3 M. At and above 5.2 × 10-3 M, L-Phe not only hindered the nucleation and growth rate of β-L-Glu but also retarded growth of the R-form. L-Phe is believed to adsorb onto the crystal surface of L-Glu via the common part of the amino acid molecules and hinders further attachment of L-Glu molecules, due to steric hindrance caused by the bulky phenyl group.7 L-Amino acids have also been observed to adsorb preferentially onto the three dominant {101}, {010}, and {001} facets of the β-form of L-Glu, resulting in selective crystallization of the R-form and hence its stabilization.8 The {010} facets of the β-form were suppressed to the greatest extent by L-Lys, while the {001} facets were especially suppressed by L-Phe. All molecules possessing the moiety of L-R-amino acid are capable of forming hydrogen bonds to the three dominant facets of the β-form, namely, {101}, {010}, and {001}, with the ultimate consequence of stabilizing the R-form.8 In the absence of any additive, the growth rate of R-{111} was already several times greater than that of β-{101}.9 In addition, the critical concentration of L-Phe at which close to 100% growth inhibition of β-{101} was observed was 7.5 × 10-4 M; the corresponding value for R-{110} was 1.7 × 10-3 M, explaining the preferential inhibition of the β-form by L-Phe.4 In addition, there have been some reports of changes in morphology in R-L-Glu in the presence of amino acid additives.10 Typically growth of R-{111} is stopped by 3 × 10-3 M L-Phe, favoring growth of the R-{001} facets.10 The uptake of impurity species into a crystal has important implications in industrial crystallization.11 Additives that are effective for control of polymorphism may be adsorbed on the surface of the crystal and hence become occluded, which gives rise to decreased purity of crystals.3 Additive molecules with similar structure or conformation to that of the bulk crystal can selectively inhibit the crystallization of the stable polymorph
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Figure 1. Additive concentration at which 10% (blue), 50% (magenta), and 90% (orange) stabilization of R-L-Glu was obtained. Graph columns are truncated at 5 × 10-2 M for stabilization concentrations of >5 × 10-2 M. The structures annotated on this figure refer to the side chain attached to [-CH2-CH(NH2)(COOH)]; [L-Glu]0 ) 0.3 M; σ ) 1.12; Tsat ) 80 °C; Tcrys ) 45 °C; tcrys ) 24 h.
by molecular mimicry.12 It is therefore possible that such molecules become incorporated into the host crystal lattice, by virtue of their structural similarities.10 This paper describes the influence of amino acid additives on the crystallization of L-Glu in the light of a recent report13 that the crystals of β-L-Glu can nucleate on the surface of and become encapsulated inside rapidly growing R-form crystals.14 Experimental Section Recrystallization of L-Glu with Additives. The R-form of L-Glu was crystallized from 98% pure L-Glu monosodium salt monohydrate (Sigma-Aldrich) using 37% HCl (BDH, Analar), as previously reported.15 Recrystallization of L-Glu was performed in the presence of D- or L-Phe, L-Tyr, L-Asp, L-Lys, L-Arg, L-Cys, L-Ser, or L-Met in the concentration range 6 × 10-4 to 5 × 10-2 M. The selected amino acids were added to solutions of L-Glu at 80 °C and brought to supersaturation by cooling to 45 °C. All amino acids were obtained from SigmaAldrich. In general, crystallizations were allowed to continue for 24 h at 45 °C, unless otherwise specified. Crystal Characterization. L-Glu crystal samples were characterized using powder X-ray diffraction (XRD), scanning electron microscopy (SEM), focused ion beam microscopy (FIB) Raman spectroscopy (RS), differential scanning calorimetry (DSC), and particle size analysis (PSA). Full details have been presented elsewhere.16 HPLC Analysis of L-Glu Crystals. HPLC was used to quantify the uptake of additives in L-Glu crystals. This technique was applied to L-Glu crystal samples that were recrystallized in the presence of 6.25 × 10-4 to 5 × 10-2 M L-Phe, D-Phe, L-Tyr, or L-Met. Standards were prepared for all additives in the concentration range 10-400 ppm from a 1000 ppm stock solution, by serial dilution. The solvent used was reverse osmosis (RO) water, and solutions were filtered using 0.22 µm Sarstedt filters. The stationary phase was a Jupiter Phenomenex C18 column, with dimensions of 250 × 4.6 mm. The mobile phases were as follows: phase A ) 0.1% trifluoroacetic acid (TFA) obtained from Aldrich Chemicals (99+% spectrophotometric grade) in RO water; phase B ) 60% acetonitrile obtained from ROMIL (HPLC grade) with 0.1% TFA in RO water. Solvents were filtered (0.22 µm) and degassed before use; the injection volume was 20 µL, and UV detection at 215 nm was employed. All calibration graphs yielded R2 values of >0.99.
Results Figure 1 presents a general overview of the effectiveness of all the L-amino acids used in this study for the stabilization of the R-form of L-Glu. This figure demonstrates that the L-form of the amino acid is necessary for stabilization but is not effective for all additives investigated. Some of the L-amino acids, most notably L-Phe, L-Lys, L-Met, and L-Arg, did suppress the total crystal yield (both R- and β-forms) when employed at concentrations of 5 × 10-2 M and above. Figure 1 demonstrates the relationship between additive structure, or more specifically side chain, and the ability of the additive to stabilize the R-form of L-Glu. In this work, L-Tyr, which differs in structure from L-Phe by a single hydroxyl group, was found to be extremely effective, more so than L-Phe, at low concentrations for the selective crystallization of R-L-Glu. Both L-Phe and L-Tyr stabilized R-L-Glu at lower concentrations than the other additives, perhaps due to the bulky nature of their phenyl group, as already proposed by Kitamura and Funahara.7 L-Lys and L-Arg were extremely effective at 1 × 10-2 M and above. They behaved similarly, again due to their comparable molecular structures. L-Ser was a relatively poor additive, as even at 5 × 10-2 M it only partially stabilized the R-form of L-Glu. Its structure differs from that of L-Tyr, by a phenyl group. This strengthens the argument that the strong effect of L-Phe and L-Tyr is attributable to the phenyl group.7 While L-Cys was not as effective as L-Phe or L-Tyr, it was significantly better than L-Ser, even though the difference in structure between L-Cys and L-Ser is a thiol group and a hydroxyl group, respectively. L-Asp possesses a second carboxyl group, which renders it more effective than either L-Ser or L-Cys, as it is more comparable to the L-Glu molecular structure and can therefore readily adsorb on L-Glu crystal surfaces. L-Met was the poorest additive, as it did not stabilize R-L-Glu at any concentration. Incorporation of L-Phenylalanine in L-Glu Crystals. To compare the incorporation behavior of a pair of enantiomers, crystals of L-Glu formed in the presence
Effect of Additives on L-Glu Crystallization
Figure 2. Mole ratio (additive to L-Glu) in harvested crystals and β-L-Glu crystal yield in L-Glu crystals as a function of additive (a) L-Phe and (b) D-Phe concentration: mole ratio ((); % β-L-Glu (b). [L-Glu]0 ) 0.3 M; σ ) 1.12; Tsat ) 80 °C; Tcrys ) 45 °C; tcrys ) 24 h.
of L-Phe or D-Phe were dissolved in water and analyzed using HPLC. Figure 2a,b represents the molar ratios (Phe/Glu) of L-Phe and D-Phe, respectively, found in these crystals as a function of additive concentration. The corresponding wt % β-polymorph observed after 24 h of crystallization at 45 °C is also presented in Figure 2a,b. Below 1 × 10-2 M addition, the molar ratio of L-Phe/ L-Glu found in the final L-Glu crystals was extremely low, but it increased significantly above this concentration (Figure 2a). Indeed it would appear that very low levels of L-Phe uptake occurred at solution concentrations which resulted in almost 100% stabilization of the R-form. Little or no stabilization of the R-polymorph was observed with D-Phe, and the incorporation levels recorded for this additive were much lower than that observed using L-Phe. Note the differing Phe/Glu scales used in Figure 2a,b. Even at 5 × 10-2 M D-Phe, the Phe/ Glu ratio in the product crystals did not exceed 1:1000, whereas the molar ratio in crystal harvest from a solution containing 5 × 10-2 M L-Phe was close to 1:125. The typical morphology of R-L-Glu is displayed in Figure 3a when the L-Phe additive concentration was 2.5 × 10-3 M and below. At L-Phe concentrations of 2 × 10-2 M and above, the point at which appreciable uptake of the additive occurred, a profound effect was observed on the crystal morphology. At 1 × 10-2 M L-Phe (Figure 3b,c), elongation of crystals occurred, compared to their characteristic rhombic morphology from pure solutions.12 Increasing the L-Phe concentration induced progressive elongation of R-crystals, until long pointed crystals were formed (Figure 3d). At all concentrations of D-Phe, only the typical needlelike habit of β-L-Glu was observed. The incorporation of two other L-amino acids into L-Glu crystals was also determined by HPLC analysis.
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Figure 3. Electron micrographs of L-Glu crystallized with L-Phe: (a) no additive, (b,c) ) 1 × 10-2 M; (d) ) 2 × 10-2 M. [L-Glu]0 ) 0.3 M; σ ) 1.12; Tsat ) 80 °C; Tcrys ) 45 °C; tcrys ) 24 h except for image a where tcrys ) 2 h.
Figure 4. Mole ratio and β-L-Glu crystal yield in L-Glu crystals as a function of additive concentration for (a) L-Tyr to L-Glu and (b) L-Met to L-Glu: mole ratio ((); % β-L-Glu (b). [L-Glu]0 ) 0.3 M; σ ) 1.12; Tsat ) 80 °C; Tcrys ) 45 °C; tcrys ) 24 h.
The results are summarized in Figure 4a,b for L-Tyr and L-Met, respectively, as well as the corresponding wt % of the β-L-Glu detected in the harvested crystals by quantitative X-ray diffraction. L-Tyr is an effective additive for the stabilization of R-L-Glu crystals at low concentrations. Above 10-2 M, incorporation is very high. At 5 × 10-2 M, the recorded L-Tyr/L-Glu molar ratio in the harvested crystals was 1:5 against a solution molar ratio of 1:6. At additive concentrations as low as 2.5 × 10-3 M, morphological variations were observed. At high additive concentrations, the R-L-Glu crystals
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Figure 5. Crystal morphological variations using 5 × 10-2 M L-amino acid additives.
again became more elongated and bore resemblances to the crystals modified using L-Phe. Despite the fact that L-Met did not selectively crystallize the R-polymorph, it reduced the particle size of the β-crystals that formed. An additional interesting factor is that the uptake of additive in the crystals harvested from solutions containing L-Met was larger than that observed using L-Phe, one of the most effective additives at stabilizing the R-form of L-Glu. A general overview of the morphological effects of all the L-amino acid additives used in this work is presented in Figure 5, which clearly demonstrates the loss of the {011} and {001} facets of R-L-Glu and the emergence of the {110} and {111} facets. By extension, the additives incorporated into R-L-Glu must be preferentially attached to the {110} and {111} facets, namely, the slowest growing facets. When R-L-Glu crystals harvested from additive-free solutions were examined by electron microscopy, a common occurrence was the apparent growth of β-crystals from the surface of the R-form crystals (see Figure 6a). When these crystals were cleaved, β-form inclusions that had become encapsulated inside the R-form crystals were observed. In some cases these inclusions protruded through the surface of the R-crystals (see Figure 6b,c). In this work the identities of the R- and β-form crystals and inclusions were established by laser Raman spectroscopy.14 When these techniques were applied to R-form crystals harvested from a solution with 5 × 10-2 M L-Tyr, there were no incidences of β-inclusions (see Figure 6d). Discussion A general finding of this work is that additive incorporation remains low at concentrations (generally below 1 × 10-2 M) that are effective in stabilizing the R-form of L-Glu (see for example Figure 2a, L-Phe, and Figure 4a, L-Tyr). Another general finding is that at
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Figure 6. (a) FIB images of β-crystals growing out of the surface of R-L-Glu. (b,c) FIB cross sections of β-crystals growing within R-L-Glu: [Glu]0 ) 0.3 M; σ ) 1.12; Tsat ) 80 °C; Tcrys ) 45 °C; tcrys ) 6 h. (d) FIB cross sections of R-L-Glu crystals grown with L-Tyr: [Glu]0 ) 0.3 M; σ ) 1.12; Tsat ) 80 °C; Tcrys ) 45 °C; tcrys ) 24 h; [L-Tyr] ) 5 × 10-2 M. Table 1. Additive/L-Glutamic Acid Ratio in Harvested Crystals for a Solution Ratio of 1:6 additive
additive/L-glutamic acid ratio in crystals
L-Phe
1:125 1:1000 1:5 1:33
D-Phe L-Tyr L-Met
high concentrations of additives (typically 5 × 10-2 M) significant amounts of L-amino acid additives are incorporated into the product. A summary of additive/ L-Glu molar ratios observed at 5 × 10-2 M additive is presented in Table 1. The amounts of additives incorporated in molar ratio terms are, in general, much less than the molar ratio of additive to L-Glu present in solution. L-Tyr is an exception for which a very high ratio was observed. In a previous observation, crystals were harvested at short to intermediate crystallization times and β-form crystals appeared to grow from the surface of R-form crystals.13 In conjunction with a FIB-RS study, β-form crystals of L-Glu were demonstrated to occur inside R-form crystals,14 suggesting a mechanism in which the nucleation point for the β-polymorph is the surface of the R-polymorph. The very small amounts of additives, particularly L-Phe and L-Tyr, required to suppress β-polymorph formation and the small amounts incorporated into the harvested crystals further support this hypothesis and point to a mechanism whereby additives attach to the developing β/R interface. This disrupts β-form growth prior to its development beyond the critical size of nucleus which defines the end of the nucleation phase. At this point the subcritical nuclei can redissolve, also leading to the redissolution of any attached additive resulting in its nonincorporation into the harvested crystals. In the absence of additives, however, β-form crystals can grow from the R-form surface. The faster growth of the latter leads to the
Effect of Additives on L-Glu Crystallization
inclusions displayed in Figure 6b,c. These findings, taken in conjunction with FIB images confirming the absence of β-inclusions inside R-form crystals when an effective additive (L-Tyr) is present during crystallization (see Figure 6d), point to a role for additives in suppressing the nucleation of the β-polymorph. A further general finding, summarized in Figure 6, is that each additive that stabilized the R-form of L-Glu generated modifications in morphology at high concentrations. The morphology changes favored the {110} and {111} facets of R-L-Glu at the expense of the {011} and {001} facets. For the work presented here, these findings are indicative of the {011} or {001} facets being the favored location from which the β-polymorph may develop. References (1) Blagden, N. Powder Technol. 2001, 121, 46-52. (2) Davey, R. J. J. Cryst. Growth 1976, 34, 109-119. (3) Kitamura, M.; Nakamura, T. Powder Technol. 2001, 121, 39-45.
Crystal Growth & Design, Vol. 5, No. 2, 2005 597 (4) Kitamura, M.; Ishizu, T. J. Cryst. Growth 1998, 192, 225235. (5) Sakata, Y. Agric. Biol. Chem. 1961, 25, 829-834. (6) Black, S. N.; Davey, R. J. J. Cryst. Growth 1988, 90, 136144. (7) Kitamura, M.; Funahara, H. J. Chem. Eng. Jpn. 1994, 27, 124-127. (8) Sano, C.; Kashiwagi, T.; Nagashima, N.; Kawakita, T. J. Cryst. Growth 1997, 178, 568-574. (9) Kitamura, M.; Ishizu, T. J. Cryst. Growth 2000, 209, 138145. (10) Sano, C.; Nagashima, N. J. Cryst. Growth 1996, 166, 129135. (11) Kubota, N.; Yokota, M.; Mullin, J. W. J. Cryst. Growth 2000, 212, 480-488. (12) Davey, R. J.; Blagden, N.; Potts, G. D.; Docherty, R. J. Am. Chem. Soc. 1997, 119, 1767-1772. (13) Cashell, C.; Corcoran, D.; Hodnett, B. K. Chem. Commun. 2003, 3, 374-375. (14) Cashell, C.; Sutton, D.; Corcoran, D.; Hodnett, B. K. Cryst. Growth Des. 2003, 3, 869-872. (15) Garti, N.; Zour, H. J. Cryst. Growth 1997, 172, 486-498. (16) Cashell, C.; Corcoran, D.; Hodnett, B. K. J. Cryst. Growth 2004, 273, 258-265.
CG049712H
