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Langmuir 2007, 23, 1988-1994
Formation of Single-Crystalline Aragonite Tablets/Films via an Amorphous Precursor Fairland F. Amos,† Denise M. Sharbaugh,‡ Daniel R. Talham,‡ and Laurie B. Gower*,† Departments of Materials Science and Engineering and of Chemistry, UniVersity of Florida, P.O. Box 116400, GainesVille, Florida 32611
Marc Fricke and Dirk Volkmer Faculty of Chemistry (AC 2), UniVersity of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany ReceiVed July 6, 2006. In Final Form: October 23, 2006 Thin tablets and films of calcium carbonate have been grown at the air-water interface via an amorphous precursor route using soluble process-directing agents and a Langmuir monolayer based on resorcarene. By using appropriate concentrations of poly(acrylic acid-sodium salt) in combination with Mg2+ ion, an initially amorphous film is deposited on the monolayer template, which subsequently crystallizes into a mosaic film composed of a mixture of singlecrystalline and spherulitic patches of calcite and aragonite. Of particular importance is the synthesis of singlecrystalline “tablets” of aragonite (∼600 nm thick), because this phase generally forms needle-like polycrystalline aggregates when grown in vitro. To our knowledge, a tabular single-crystalline morphology of aragonite has only been observed in the nacreous layer of mollusk shells. Therefore, this in vitro system may serve as a useful model for examining mechanistic issues pertinent to biomineralization, such as the influence of organic templates on nucleation from an amorphous phase.
Introduction Scientists have long been fascinated with how organisms form their highly regulated biomineral morphologies in aqueous solution and under ambient conditions. Because of the unique properties these biocomposites possess, which often result from an organic-inorganic nanostructured architecture, much effort hasbeenmadetomimictheprocessesinvolvedinbiomineralization.1-3 It is generally believed that interactions with organic materials, such as proteins and polysaccharides, are used to modulate the growth of the inorganic crystals. For example, living systems use macromolecular matrices or amphiphilic assemblies (most often in the form of phospholipid membranes) to modulate the formation of the inorganic crystals, such as through compartmentalization of the growing mineral to control crystal morphology or as a nucleating template to regulate crystal location, phase, and orientation.2,4-7 These insoluble matrices, combined with soluble additives, e.g., proteins enriched with aspartate, glutamate, or phosphoserine residues, often lead to complex nonequilibrium (i.e., nonfaceted) morphologies of the biomineral crystals that distinguish them from crystals grown in the beaker. The role of the soluble proteins has been more ambiguous, but recent studies find a transient amorphous precursor in a variety of calcific * Correspondingauthor.Fax(+01)352-846-3355;
[email protected]. † Department of Materials Science and Engineering. ‡ Department of Chemistry. (1) Bauerlein, E. Angew. Chem. Int. Ed. 2003, 42 (6), 614. (2) Heuer, A. H.; Fink, D. J.; Laraia, V. J.; Arias, J. L.; Calvert, P. D.; Kendall, K.; Messing, G. L.; Blackwell, J.; Rieke, P. C.; Thompson, D. H.; Wheeler, A. P.; Veis, A.; Caplan, A. I. Science 1992, 255 (5048), 1098. (3) Berman, A.; Hanson, J.; Leiserowitz, L.; Koetzle, T. F.; Weiner, S.; Addadi, L. Science 1993, 259 (5096), 776. (4) Murphy, W. L.; Messersmith, P. B. Polyhedron 2000, 19 (3), 357. (5) Lieske, J. C.; Walshreitz, M. M.; Toback, F. G. Am. J. Physiol. 1992, 262 (4), F622. (6) Collier, J. H.; Messersmith, P. B. Annu. ReV. Mater. Res. 2001, 31, 237. (7) Scheid, C.; Koul, H.; Hill, W. A.; LuberNarod, J.; Jonassen, J.; Honeyman, T.; Kennington, L.; Kohli, R.; Hodapp, J.; Ayvazian, P.; Menon, M. J. Urol. 1996, 155 (3), 1112.
biominerals,8-11 suggesting that these proteins may play a role in generating and/or stabilizing the amorphous phase. Work in our biomimetics laboratory has made use of in vitro model systems to examine crystallization reactions that proceed from an amorphous precursor, and we have found that a variety of nonequilibrium morphologies of calcium carbonate can be formed via a polymer-induced liquid-precursor (PILP) mineralization process.12 In this mineralization route, an anionic polymer (e.g., polyaspartate or polyacrylate) is used to induce the formation of an amorphous phase that is so highly hydrated it has fluidic character. When the amorphous precursor transforms (i.e., solidifies and crystallizes), a variety of nonequilibrium CaCO3 morphologies are produced, because the crystals retain the shape of the initially isotropic, amorphous phase upon crystallization. Therefore, by manipulating the deposition of the precursor phase, we have been able to produce CaCO3 crystals that mimic several biomineral structures found in marine organisms, such as CaCO3 tablets and films of the same thickness and morphology as the nacreous tablets of mollusk shells (although composed of the calcite polymorph),13-16 calcite “fibers” similar to the “rods” found in sea urchin teeth,17 and (8) Aizenberg, J.; Lambert, G.; Weiner, S.; Addadi, L. J. Am. Chem. Soc. 2002, 124 (1), 32. (9) Gotliv, B. A.; Addadi, L.; Weiner, S. Chembiochem 2003, 4 (6), 522. (10) Raz, S.; Hamilton, P. C.; Wilt, F. H.; Weiner, S.; Addadi, L. AdV. Funct. Mater. 2003, 13 (6), 480. (11) Nassif, N.; Pinna, N.; Gehrke, N.; Antonietti, M.; Jager, C.; Colfen, H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (36), 12653. (12) Gower, L. B.; Odom, D. J. J. Cryst. Growth 2000, 210 (4), 719. (13) Gower, L. A.; Tirrell, D. A. J. Cryst. Growth 1998, 191 (1-2), 153. (14) DiMasi, E.; Patel, V. M.; Sivakumar, M.; Olszta, M. J.; Yang, Y. P.; Gower, L. B. Langmuir 2002, 18 (23), 8902. (15) Kim, Y.; Gower, L. B. In Materials Research Society; Materials Research Society: San Francisco, 2003. (16) Volkmer, D.; Harms, M.; Gower, L.; Ziegler, A. Angew. Chem., Int. Ed. 2005, 44 (4), 639. (17) Olszta, M. J.; Gajjeraman, S.; Kaufman, M.; Gower, L. B. Chem. Mater. 2004, 16 (12), 2355.
10.1021/la061960n CCC: $37.00 © 2007 American Chemical Society Published on Web 01/19/2007
Formation of CaCO3 Via an Amorphous Precursor
“micromolding” of CaCO3 to mimic the bicontinuous morphology of the sea urchin spine.18 The nacreous layer of mollusk shells is composed of tablets of [001] oriented aragonite crystals, which are either stacked in columns or layered sheets, depending on the species. Although the aragonite polymorph has an orthorhombic crystal lattice, thin tablets in the [001] orientation have a roughly hexagonal appearance. These pseudohexagonal tablets are reportedly singlecrystalline aragonite, yet when aragonite is grown synthetically (such as with the use of Mg2+ additive19-21), it forms a spherulitic texture, producing dumbbell- or pincushion-shaped aggregates composed of needle-like polycrystals. Through the use of polyanionic additives, we and others have been able to mimic the thin film and tablet morphologies of CaCO3, but these have most often resulted in the formation of calcite films.13,22,23 There have been some reports of synthetic aragonite thin films; however, these mineral films are polycrystalline with the spherulitic texture that aragonite so often favors.24-27 Recently, Gehrke et al.28 have formed single-crystalline calcite plates within the confines of a demineralized insoluble organic matrix of a nacreous shell via an amorphous precursor using poly(acrylic acid). In this paper, we present the formation of not only single-crystalline calcite but also aragonite tablets and films via the PILP process. This is achieved by precipitating an amorphous precursor from a supersaturated solution of CaCO3(aq), using the synergistic effects of a amphiphilic resorcarene monolayer, rccc-5,11,17,23-tetracarboxy-4,6,10,12,18,22,24octa-O-methyl-2,8,14,20-tetra(n-undecyl)resorc[4]arene,29 spread at the air-water interface, and water-soluble additives, Mg2+ and poly(acrylic acid) (PAA), as process-directing agents to induce the amorphous precursor. The same resorcarene monolayer, compressed to low pressures, has been reported by Volkmer et al.29 to template the formation of 〈012〉 oriented calcite crystals (when grown without the soluble additives). Experimental Section A sample of rccc-5,11,17,23-tetracarboxy-4,6,10,12,18,22,24octa-O-methyl-2,8,14,20-tetra(n-undecyl)resorc[4]arene, was kindly donated by Mattay et al. (University of Bielefeld, Germany). The raw product was further purified by recrystallization as described in ref 29. Poly(acrylic acid-sodium salt), 45 wt % solution in water (Mw ) 8000 g mol-1), CaCO3 (minimum 99.0% purity, ACS reagent), and MgCl2‚6H2O powder (minimum 99.0% purity) were purchased from Sigma-Aldrich. High-purity 18.2-MΩ deionized water was used for all solutions. Carbon dioxide utilized for bubbling was purchased from Praxair. Isotherm Measurements. Monolayers were prepared by spreading the resorcarene dissolved in chloroform (0.3-0.5 mg/mL) at the air-water interface of different subphases, i.e., (i) water, and (ii) 10 mM CaCO3(aq) (see next paragraph) and 4 µg/mL PAA, and (iii) 10 mM CaCO3(aq), 10 mM MgCl2‚6H2O, and 4 µg/mL PAA, on a Langmuir trough setup with dimensions of 45.8 cm × 15 cm. Prior (18) Cheng, X. G.; Gower, L. B. Biotechnol. Prog. 2006, 22 (1), 141. (19) FernandezDiaz, L.; Putnis, A.; Prieto, M.; Putnis, C. V. J. Sediment. Res. 1996, 66 (3), 482. (20) Morse, J. W.; Wang, Q. W.; Tsio, M. Y. Geology 1997, 25 (1), 85. (21) Meldrum, F. C.; Hyde, S. T. J. Cryst. Growth 2001, 231 (4), 544. (22) Xu, G. F.; Yao, N.; Aksay, I. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120 (46), 11977. (23) Hosoda, N.; Kato, T. Chem. Mater. 2001, 13 (2), 688. (24) Hosoda, N.; Sugawara, A.; Kato, T. Macromolecules 2003, 36 (17), 6449. (25) Sugawara, A.; Kato, T. Chem. Commun. 2000 (6), 487. (26) Belcher, A. M.; Wu, X. H.; Christensen, R. J.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. Nature (London) 1996, 381 (6577), 56. (27) Kotachi, A.; Imai, H. Chem. Lett. 2006, 35 (2), 204. (28) Gehrke, N.; Nassif, N.; Pinna, N.; Antonietti, M.; Gupta, H. S.; Colfen, H. Chem. Mater. 2005, 17 (26), 6514. (29) Volkmer, D.; Fricke, M.; Agena, C.; Mattay, J. Cryst. Eng. Commun. 2002, 288.
Langmuir, Vol. 23, No. 4, 2007 1989 to compression, the monolayers were allowed to equilibrate for 10 min. The monolayers were then compressed at a rate of 5 mm/min using a KSV 3000 Langmuir-Blodgett system. The surface pressure was monitored using a Wilhelmy balance. Phase assignments of the monolayer were confirmed using a Nanofilm Technologie GmbH (Goettingen, Germany) BAM2plus Brewster angle microscope (BAM). Crystallization Under Resorcarene Monolayer. Supersaturated CaCO3 solutions were prepared by bubbling carbon dioxide in deionized water to partially dissolve CaCO3 powder.30 The solution was bubbled overnight and filtered to remove undissolved powder. This yielded an approximate concentration of 10 mM of dissolved CaCO3(aq), as calculated from the weight of the dried filtrate powder subtracted from the initial weight of the CaCO3 mixed in the solution. Appropriate amounts of MgCl2‚6H2O powder were added into the solution to form various concentrations of Mg2+ (0-40 mM). Appropriate amounts of the polymer in water (1 mg/mL stock solution) were micropipetted into the Ca2+:Mg2+ solution to yield 0-32 µg/mL of the polymer. The resulting mixtures were placed in 6-cm glass Petri dishes. Resorcarene dissolved in chloroform was aspirated into a Hamilton syringe. A drop of the monolayer mixture was formed at the tip of the syringe and was gently tapped onto the surface of the solution. This allowed the careful spreading of the monolayer at the air-water interface. A predetermined amount of the monolayer mixture was placed at the air-water interface (based on previously measured isotherms showing the area/head group), such that the desired liquid expanded (LE) or liquid condensed (LC) phase of the resorcarene could be produced. As CO2 escapes from the solution, CaCO3 films were allowed to deposit and grow on the monolayer substrate for up to 4 days. The mineral films were examined in situ using a polarized optical microscope with first-order-red wave plate (Olympus BX-60 optical microscope with MTI 3CCD camera). Formvar-coated copper grids and glass coverslips were used to dip off the samples from the surface of the solution for transmission electron microscopy (JEOL TEM 200CX) and X-ray diffractometry (Philips APD 3720), respectively. The samples were scanned with Cu KR X-ray radiation at 40 kV and 20 mA, using a step size of 0.01° with a time of 2 s/step, over a 2θ range 20-45°. The final mineral phase was correlated with the XRD d-spacings provided by the Joint Committee on Powder Diffraction Standards (JCPDS) files. Prior to TEM analysis, the samples were carefully rinsed with ethanol (while on the grid) to help the films adhere to the sample holder and to remove extraneous salt solution. TEM and selected area electron diffraction (SAED) were performed at an operating voltage of 200 kV.
Results and Discussion The resorcarene molecule, which contains a macrocyclic tetraacid headgroup (see Figure 1a), assembles to form a highly charged, moderately rigid monolayer that serves as a template for deposition of the CaCO3 films. Studies with this amphiphilic system have shown that its configuration at the air-water interface can influence the nucleation of CaCO3 crystals grown via the traditional solution precipitation process, so it was of interest here to determine if the same templating interactions would occur for crystals grown via an amorphous precursor. Overall, little is known about the influence of substrates on nucleation of crystals from the amorphous phase, yet in light of recent evidence that many biominerals are formed via an amorphous precursor, there is reason to suspect that a high degree of crystallochemical control can be obtained by this route. Figure 1b shows the pressure vs area isotherm of the resorcarene monolayer spread over different subphases. On water, the monolayer assumes either the gaseous (G) or liquid expanded (LE) phase when in the uncompressed state as confirmed by BAM (Figure 2a). As the monolayer is compressed to a smaller area, the surface pressure increases steadily as the molecules (30) Kitano, Y.; Hood, D. W.; Park, K., J. Geophys. Res. 1962, 67 (12), 4873.
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Figure 1. (a) Planar view of the chemical structure of the rccc5,11,17,23-tetracarboxy-4,6,10,12,16,18,22,24-octa-O-methyl-2-814-20-tetra(n-undecyl)resorc[4]arene. (b) Surface pressure vs mean molecular area isotherms of the resorcarene monolayer spread over different subphases: water; 10 mM CaCO3(aq) (Solution 1); 10 mM CaCO3(aq), 4 µg/mL PAA (Solution 2); 10 mM CaCO3(aq), 10 mM Mg2+, 4 µg/mL PAA (Solution 3). Brewster angle microscopy was used to assign phase behavior (images not shown), where the phases can be distinguished from one another by a change in the differential refractive indices associated with an increase in molecular packing densities, leading to variations in brightness.
pack more closely into the LE phase (Figure 2b). The monolayer eventually collapses at ∼40 mN/m. When 10 mM CaCO3(aq) (Solution 1) is present in the subphase, the isotherms are shifted to a smaller mean molecular area with respect to that of water, indicating that the Ca2+ ions promote a more compact arrangement of the resorcarene molecules.31,32 In addition, a more ordered liquid condensed (LC) phase is stabilized at surface pressures above 12 mN/m. (It should be noted that this condensation effect on the monolayer in the presence of Ca2+ is opposite from the observation reported in the previous Volkmer paper,29 which may be attributed to variations in the pH and ionic strength associated with the different methods employed to form the supersaturated CaCO3.) The isotherm of the monolayer spread over 10 mM CaCO3 and 4 µg/mL PAA (Solution 2) is similar to that of Solution 1, showing that the polymer has little to no effect on the arrangement of the resorcarene molecules. On the other hand, the addition of Mg ions reduces the condensing effect that was seen for the same concentration of Ca ions alone, as can be seen by the monolayer spread over 10 mM CaCO3(aq), 10 mM Mg2+, and 4 (31) Evert, L. L.; Leckband, D.; Israelachvili, J. N. Langmuir 1994, 10 (1), 303. (32) Rapaport, H.; Kuzmenko, I.; Berfeld, M.; Kjaer, K.; Als-Nielsen, J.; Popovitz-Biro, R.; Weissbuch, I.; Lahav, M.; Leiserowitz, L. J. Phys. Chem. B 2000, 104 (7), 1399.
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µg/mL PAA (Solution 3), which shifts the LE-LC phase transition to 9 mN/m. Figure 2c,d depicts the phases of the resorcarene monolayer spread over Solution 3 at different surface pressures. Similar to that in water, a coexistence of the G and LE phases occurs at the uncompressed state (Figure 2c). This arrangement of the monolayer changes into LE at pressures below 9 mN/m. At the phase transition, the LC phase, in the form of dendritic domains, starts to form on the monolayer (Figure 2d). With further compression, these dendritic domains grow until the LC phase completely fills the entire surface. The bright spots observed on the monolayer that is spread over the cationcontaining subphase at any surface pressures indicate that these regions are thicker and more condensed than the other regions of the monolayer. These spots are clearly absent in the BAM images of the resorcarene spread over the water subphase, implying that the monolayer interacts with the ions (particularly Ca2+) in the subphase, since such spots are also observed on resorcarene monolayers spread over Solutions 1 or 2. To verify whether the bright spots are Ca2+-resorcarene complexes and not calcium carbonate precipitates, the bright spots were monitored through BAM during monolayer compression and decompression. As expected, the bright spots increase in size and intensity with compression of the monolayer (Figure 2c,d); with decompression, these bright spots reduce in number and brightness (Figure 3). If the bright spots were calcium carbonate precipitates, decompressing the monolayer should not decrease the number and intensity of these spots. In addition, when resorcarene was spread on a similar composition of subphase, but without the reactive counterions (10 mM CaCl2 was utilized in place of the 10 mM CaCO3(aq)), bright spots as well as LC domains are also observed (see Supporting Information), further verifying that the spots are cation-resorcarene complexes. Polarized light microscopy was used to examine CaCO3 film formation in situ, as shown in Figure 4; and the first-order-red wave plate was used to distinguish between the amorphous and crystalline regions of the CaCO3 film. For example, isotropic materials (such as amorphous mineral) will appear as the same magenta color as the background, while anisotropic materials (such as calcite, which has high birefringence) will exhibit higherorder retardation colors (except when oriented in the extinct positions). The formation of continuous mineral films was carried out on the LC phase of the monolayer in the presence of 10 mM CaCO3(aq), with varying concentrations of PAA and Mg2+. In the absence of PAA (Solution 1), mineral films were not observed, as expected. Instead, calcite crystals of conventional rhombohedral morphology and preferred 〈104〉 orientation were formed (Figure 4a). Note that this is different from the 〈012〉 oriented calcite crystals grown on the same monolayer (in the absence of any additives) reported by Volkmer et al.,29 because their experiment was done in the uncompressed state, while the LC state of the resorcarene monolayer was used here. The reason we chose to use the compressed state was because it was found to lead to more continuous films. Apparently, the precursor phase does not “wet” the entire interface if the LE phase of the monolayer is used, and only noncontiguous patches of CaCO3 films are produced, as shown in Figure 4b. It is likely that the monolayer, which is initially in the LE phase prior to the initial CaCO3 formation (based on isotherms of Solution 3), condenses into the LC phase due to interactions with the CaCO3 precursor phase and thus covers less surface area compared to the monolayer prepared as a LC phase. This incomplete template could explain the formation of these thin streaky patches of CaCO3 film, which do have a filmlike morphology similar to the large continuous
Formation of CaCO3 Via an Amorphous Precursor
Langmuir, Vol. 23, No. 4, 2007 1991
Figure 2. Brewster angle micrographs of the resorcarene monolayer on (a,b) water subphase and (c,d) 10 mM CaCO3(aq), 10 mM MgCl2, and 4 µg/mL PAA subphase (Solution 3). On water subphase (a), the gaseous (dark regions, G) and liquid expanded (bright regions, LE) phases of the monolayer coexist in the uncompressed state. (b) The liquid expanded phase is observed throughout the isotherm at pressures below 40 mN/m. On Solution 3, (c) the G and LE phases coexist in the uncompressed state. (c) During the phase transition, the LC phase, in the form of dendritic domains, coexists with the LE phase. Bright spots (arrows in c,d) increase in number and intensity as the surface pressure on the monolayer increases.
Figure 3. (a) Intense bright spots (arrows) are seen in the background of a dendritic LC phase during compression. (b) Upon decompressing the monolayer to 0 mN/m, both the G and LE phases are observed. The spots in (b) are much fewer in number and less intense than the spots in (a).
films deposited on the completely filled LC monolayer, yet are unable to completely cover the surface. Interestingly, these streaks of film transform into single-crystalline strands while maintaining their curved and convoluted morphologies.
In situ examination reveals that, in the early stages of the reaction, the films formed initially via the PILP process are amorphous (Figure 4c), as is evident by the uniform magenta color of the isotropic sample, which becomes birefringent (yellow/ orange and blue) with time as regions within the precursor phase start to crystallize (Figure 4d-f). Previous work has shown that these amorphous films consist of a highly hydrated phase of CaCO3.14 The amorphous films are continuous and cover most of the air-water interface in the early stages but gradually disappear with time. In our prior work, using synchrotron X-ray reflectivity studies, we were able to measure the film thickness as it grew with time and found that the polymer concentration influences the lifetime of the amorphous phase.33 In the latter case, it was observed that the films that formed more slowly were more stable, while films that formed rapidly grew to a certain thickness, which subsequently diminished as the metastable amorphous film dissolved. Rieger et al.34 have also observed a dissolutionrecrystallization process of amorphous CaCO3 that was rapidly precipitated in the presence of polycarboxylates. The distinguishing feature of the PILP process is that, if the film is stabilized for a sufficient time, it can transform via a pseudomorphic transformation, generating crystals that retain the shape of the precursor.12 This is thought to occur through reorganization of the atoms within the phase boundaries of the precursor phase, analogous to a solid-state transformation, but aided by the presence of water. In the present report, crystallization of the amorphous precursor films deposited on the resorcarene template can apparently proceed by either of these two different pathways, which leads to two types of morphologies. Aggregates are formed when the (33) DiMasi, E.; Kwak, S. Y.; Amos, F. F.; Olszta, M. J.; Lush, D.; Gower, L. B. Phys. ReV. Lett. 2006, 97 (4). (34) Rieger, J.; Thieme, J.; Schmidt, C. Langmuir 2000, 16 (22), 8300.
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Figure 4. Polarized light micrographs (with a first-order-red wave plate) of CaCO3 grown under the resorcarene monolayer with different subphase additives. The CaCO3 films were grown for 4 days under a liquid condensed monolayer with a mean molecular area of 150 Å2/molecule, unless otherwise noted. (a) Rhombs of calcite nucleated on the monolayer template in the control solution (Solution 1). (b) Strands of CaCO3 films grown via the PILP process in Solution 3 using a liquid expanded monolayer (MMA ) 225 Å2/molecule), which does not provide full “wetting” of the mineral precursor phase. (c) Example of a continuous amorphous CaCO3 film formed in the early stages of the PILP process when grown from Solutions 2-4. (d,e) Single crystalline patches along with 3-D crystal aggregates were formed from both Solution 2 (10 mM CaCO3(aq), 4 µg/mL PAA) and Solution 3 (10 mM CaCO3(aq), 10 mM Mg2+, 4 µg/mL PAA). (f) Spherulitic crystalline film patches, which often contained a centralized 3-D spherulite, were observed in Solution 4 (10 mM CaCO3(aq), 35 mM Mg2+, 4 µg/mL PAA).
amorphous film dissolves and recrystallizes into the more conventional, three-dimensional CaCO3 structures expressing near-equilibrium morphologies (i.e., faceted calcite or spherulitic vaterite). On the other hand, crystalline films and tablets retain their shape when the amorphous film crystallizes via a pseudomorphic transformation. Due to these competing pathways, less film is observed over time as some of the metastable amorphous film dissolves. The patches of film that do crystallize become stabilized against dissolution; and as the surrounding amorphous phase dissolves, the single-crystalline patches become isolated and resemble “tablets”, being similar in thickness (∼600 nm thick) to the nacreous tablets of mollusk shells. The focus of this report is on the amorphous films that undergo pseudomorphic transformation, because this leads to either singlecrystalline (Figure 4d,e) or polycrystalline (Figure 4f) patches of CaCO3 film. The single-crystalline texture is apparent from the uniform extinction pattern observed in cross-polarized light upon sample rotation but can also be verified by electron diffraction (as discussed below). The spherulitic patches of film (Figure 4f), which are more prevalent at higher concentrations
of Mg2+ (e.g., 10 mM CaCO3(aq), 35 mM Mg2+, and 4 µg/mL PAA: Solution 4), exhibit a Maltese cross extinction pattern. Typical X-ray diffraction patterns of the crystal aggregates and films collected from the air-water interface are shown in Figure 5. A dominant peak is observed at 29.5°, corresponding to the (104) plane of calcite. Thus, the X-ray diffraction patterns suggest that only one CaCO3 polymorph was formed. However, discrete analysis using selected area electron diffraction (SAED) of individual film patches reveals otherwise. Single-crystalline film patches grown from Solution 2 were composed of the calcite polymorph (Figure 6a), while films formed from Solution 3 were composed of a mixture of calcite and aragonite (Figure 6b). To our knowledge, this is the first report of synthetic single-crystalline aragonite tablets, where prior to now, such structures have only been found in the nacreous layer of mollusk shells. The SAED patterns in Figure 6 are unambiguously calcite and aragonite, respectively, as determined from both the experimental d-spacings of and angles between the diffraction spots, which match the theoretical values well (Tables 1 and 2). Eleven of twenty singlecrystalline tablets analyzed were unambiguously aragonite, while
Formation of CaCO3 Via an Amorphous Precursor
Langmuir, Vol. 23, No. 4, 2007 1993 Table 1. Comparison of the Experimental and Theoretical d-Spacings of, and Angles Between, the Diffraction Maxima Spots for the Calcitic Diffraction Pattern in Figure 4a solution 2
experimental values
theoretical values for calcite
assignment of peaks
d(A) d(B) d(C) θ(A-B) θ(A-C) θ(B-C)
2.456 Å 1.875 Å 1.882 Å 66.9° 112.0° 45.1°
2.495 Å 1.913 Å 1.913 Å 67.5° 112.5° 45.1°
(1h1h0) (01h8h) (108h)
Table 2. Comparison of the Experimental and Theoretical d-Spacings of, and Angles Between, the Diffraction Maxima Spots for the Aragonitic Diffraction Pattern in Figure 4b
Figure 5. X-ray diffraction of bulk CaCO3 films and aggregates grown on the monolayer with the various solution additives (as described in Figure 2) only shows the presence of calcite.
Figure 6. Selected area electron diffraction patterns of the singlecrystalline film patches grown on the resorcarene monolayer at different subphase compositions. (a) Calcite single-crystalline film grown from Solution 2. Zone axis at [88h1]. Indices are based on JCPDS 05-0586 for calcite with theoretical lattice parameters: a ) b ) 0.49896 nm, c ) 1.70610 nm; R ) β ) 90°, χ ) 120°. (b) Aragonite single-crystalline film grown from Solution 3. Zone axis at [1h14]. Indices are based on JCPDS 41-1475 for aragonite with theorectical lattice parameters: a ) 0.49611 nm, b ) 0.79672 nm, c ) 0.57404 nm; R ) β ) χ ) 90°. Legends in the pattern (A-C) are markers for comparison with the theoretical d-spacings and angles presented in Tables 1 and 2.
the rest were calcite. The diffraction patterns were taken at different zone axes, and another aragonite pattern is presented in the Supporting Information. There is a small shift in the lattice spacings from theoretical values (for both the calcite and aragonite patterns), which might have arisen from lattice distortions associated with the incor-
solution 3
experimental values
theoretical values for aragonite
assignment of peaks
d(A) d(B) d(C) θ(A-B) θ(A-C) θ(B-C)
4.134 Å 1.835 Å 2.070 Å 61.6° 88.2° 26.6°
4.212 Å 1.882 Å 2.168 Å 60.0° 86.5° 26.5°
(1h1h0) (04h1) (13h1)
poration of the polymer and/or Mg2+ during the solidification and crystallization of these films. Changes in lattice spacings have also been observed with biogenic aragonite minerals and have been attributed to incorporated molecules.35 The absence of aragonite peaks in the XRD pattern for samples grown in Solution 3 (Figure 5) means that the bulk XRD analysis is not sensitive enough to detect the thin and sparse aragonite films (average thickness of 600 nm after 2 days of mineralization) when three-dimensional calcite crystals or aggregates of much larger volumes are present, highlighting the need for selected area analysis when dealing with mixed products such as this. From our volume estimation of the CaCO3 species in each Petri dish, the calcite and aragonite films only occupy 0.2 mm3, while the volume occupied by the aggregates is 17.4 mm3, which is 100-fold larger than that of the crystalline films (Supporting Information). A phase diagram depicting the regions where single-crystalline calcite and aragonite tablets are formed is plotted on a map of initial magnesium vs PAA concentration (Figure 7). The formation of such nonequilibrium single-crystalline structures can be easily overlooked, since there is only a small window where these structures are formed (with this particular polymer and template), and particularly if only examined by bulk XRD. Mosaic thin films composed of single-crystalline calcite tablets were observed with 4-16 µg/mL PAA (without Mg2+), while 3.5-10 mM Mg2+ (at the same concentration of PAA) yielded mosaic films composed of single-crystalline calcite and aragonite tablets. The Mg ion appears to play a role in inducing the aragonite polymorph, but the significance of this specialty monolayer cannot be deduced from this one study. It is possible that the aragonite polymorph has been overlooked in other studies with simpler monolayers for the reasons mentioned above. In fact, studies done in our laboratory using arachidic acid as the monolayer yielded aragonite patches that were also only identified using SAED and not XRD (Supporting Information). A complete study on this simple monolayer was not reported, because the deposited mineral films were easily dissolved and could not be as readily traced over time. The resorcarene monolayer added rigidity and stability for the deposited CaCO3 films. Phospholipids were also explored as (35) Pokroy, B.; Quintana, J. P.; Caspi, E. N.; Berner, A.; Zolotoyabko, E. Nat. Mater. 2004, 3 (12), 900.
1994 Langmuir, Vol. 23, No. 4, 2007
Figure 7. Phase diagram of the calcium carbonate crystals, either stabilized as films or recrystallized into 3-D crystals, using different concentrations of Mg2+ and PAA.
mineralization substrates, but these templates did not result in the formation of single-crystalline patches. The formation of these aragonite tablets/films from an amorphous precursor appears to be similar to the mechanism observed for the formation of the aragonite structures in mollusk shells presented by Weiss et al. and Marxen et al.36,37 In the larval stages of shell formation, a large amount of amorphous calcium carbonate (ACC), with some aragonite crystals, is observed. The relative composition of the polymorphs changes at the later stage of shell development, where the aragonite crystals become more prevalent than the ACC phase, suggesting that the amorphous phase transforms into aragonite crystals. Although there was no suggestion of the formation of a liquid-phase (36) Weiss, I. M.; Tuross, N.; Addadi, L.; Weiner, S. J. Exp. Zool. 2002, 293 (5), 478. (37) Marxen, J. C.; Becker, W.; Finke, D.; Hasse, B.; Epple, M. J. Moll. Stud. 2003, 69, 113. (38) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science 1996, 271 (5245), 67. (39) Levi, Y.; Albeck, S.; Brack, A.; Weiner, S.; Addadi, L. Chem.sEur. J. 1998, 4 (3), 389.
Amos et al.
precursor in their studies (which is difficult to detect in vivo since it rapidly solidifies upon contact with substrates), the fact that such biological structures were emulated with an in vitro mineralization reaction using anionic polymers mimicking the acidic proteins associated with biominerals (which are highly enriched with aspartate residues) lends further support to our hypothesis that the PILP process may play a fundamental role in biomineralization. At this time, we do not know how to regulate the transformation pathway to form only tablets, which in biomineralization is likely achieved through more stabilizing protein interactions. Likewise, while we have demonstrated the formation of aragonite tablets that mimic the morphology and phase found in mollusk nacre, our sample contains a mixture of calcite tablets and aggregates, lacking the high degree of polymorph selectivity found in biominerals. The distribution of phases in our system may indicate that the transformation reaction proceeds under kinetic control. On the other hand, work by Falini et al.38 and Levi et al.,39 using proteins extracted from nacre, or mimetic peptides, suggest that there are structural interactions between the protein and forming mineral that can influence which polymorph is formed; yet the single-crystalline aragonite tablet morphology was not achieved with these proteins alone. Therefore, we believe it may require a combination of polymeric process-directing agent (for morphological control) with structure-directing agent (for crystal registry) to fully regulate all properties of the crystals, as is accomplished in biomineralization. Acknowledgment. This research was supported by National Institute of Health grant no. RO1 DK59765-01 and in part by the National Aeronautics and Space Administration grant no. NRA 00-OSS-01-043 issued through the Office of Space Science. We thank the Major Analytic Instrumentation Center (MAIC) of the University of Florida for maintaining and providing exceptional analytical equipment and guidance for the SEM, TEM, and diffraction analysis. Supporting Information Available: Additional experimental data and graphics. This material is available free of charge via the Internet at http://pubs.acs.org. LA061960N
