Crystal Growth & Design - ACS Publications - American Chemical

Formation of Thin Calcium Carbonate Films on Chitosan Biopolymer Substrates Simon R. Payne, Mary Heppenstall-Butler, and Michael F. Butler* Corporate Research, UnileVer R&D Colworth, Sharnbrook, Bedfordshire, MK44 1LQ, UK

CRYSTAL GROWTH & DESIGN 2007 VOL. 7, NO. 7 1262-1276

ReceiVed October 9, 2006; ReVised Manuscript ReceiVed April 27, 2007

ABSTRACT: Crystallization of two-dimensional spherulitic calcium carbonate films was performed on planar films of chitosan in the presence of poly(acrylic acid) (PAA). PAA, electrostatically bound to the chitosan substrate, increased the local concentration of calcium at the surface, thereby promoting CaCO3 crystallization there. Simultaneously, interaction between PAA and calcium ions in the surrounding solution suppressed bulk crystallization. Increasing the PAA concentration at fixed pH increased the surface supersaturation of calcium ions leading to larger spherulites. Saturation of the surface with PAA at 0.004% led to a maximum in the nucleation rate at this concentration. Above a certain PAA concentration, a regime existed at which the surface was completely covered. Above even higher PAA concentrations no crystallization occurred, since the bulk PAA sequestered all of the calcium ions in solution previously available for surface crystallization. As the pH increased, PAA became more charged and therefore interacted with more calcium ions in solution. Increasing sequestration of bulk calcium ions with increasing pH countered the increase in surface calcium supersaturation and carbonate ion formation, leading to a maximum in surface coverage at an intermediate pH value, at fixed PAA concentration. In situ crystallization experiments demonstrated that both vaterite and calcite spherulites grew simultaneously in separate parts of the film, albeit at different rates. The coexistence of polymorphs may therefore indicate the importance of local supersaturation conditions in determining the film morphology. Introduction The ability to control crystallization is a critical requirement in the synthesis of many industrially important materials. Selected examples of these are pharmaceutical molecules, triglycerides, fatty acids, and inorganic materials such as calcium carbonate. Scientifically, calcium carbonate is of enormous interest in the context of crystal control because it is produced by a wide variety of biological organisms that exhibit exquisite control over the polymorph, location of nucleation, crystal size and shape, crystallographic orientation, composition, stability, and hierarchical assembly of the crystals. Calcium carbonate can exist in six different forms, of which the three major polymorphs are in order of decreasing stability as follows: calcite, aragonite, and vaterite. Two hydrated crystalline forms and an amorphous form of calcium carbonate also exist, but they are generally unstable toward transformation into the calcite, aragonite, or vaterite polymorphs. Both calcite and aragonite are common natural polymorphs because the radius of the calcium ion is close to the limiting value for the transition from the rhombohedral calcite structure to the orthorhombic aragonite structure.1 While rare in nature, vaterite is a common synthetic product of solution precipitation, which is kinetically favored under certain conditions such as high supersaturation. Certain additives, such as biopolymers, synthetic polymers, fatty acids, and polypeptides containing acidic or basic functional groups, have been shown to influence the growth of calcium carbonate crystals.1 In their presence, different crystal forms of calcite are expressed and in some cases the metastable aragonite and vaterite polymorphs are kinetically trapped. Furthermore, many examples exist in nature where organisms grow calcium carbonate in a controlled manner under ambient conditions of temperature and pressure to form structural * To whom correspondence should be addressed. Telephone: +44 (0)1234 222958. Fax: +44 (0)1234 248010. E-mail: Michael.Butler@ Unilever.com.

components that require strength and flexibility, and shells that provide barriers.1,2 In the latter example, films of calcium carbonate (often the aragonite polymorph) are formed in the presence of biopolymer templates containing a high amount of acidic functional groups. Synthetic CaCO3 films grown according to these principles may also be practically useful because of their possible use as barrier materials resulting from the low permeability of inorganic materials. In the present study, twodimensional films of calcium carbonate grown on a biopolymer substrate were investigated with the aim of understanding the film formation process, by systematic investigation of the filmforming conditions, with particular emphasis on elucidating the conditions for the formation of complete CaCO3 films that can act as effective barriers to readily diffusing species. Biomimetic CaCO3-polymer composites can be readily produced by growing CaCO3 crystals on an organic matrix from a supersaturated aqueous solution. Numerous studies have been performed concerning CaCO3 crystallization on biopolymer substrates,3-18 largely in an attempt to mimic the growth of nacreous layers of calcium carbonate on biopolymer templates in mollusc shells. In particular, synthesis of CaCO3 films on insoluble chitosan matrices in the presence of poly(acrylic acid) (PAA) has been achieved and reported.3,4,6,7,10,13,14,17,18 The molecular structures of PAA and chitosan are illustrated in Figure 1. Chitosan is a cationic biopolymer possessing primary amine groups (NH2) that become protonated (NH3+) in acidic environments. It is obtained by partial deacetylation of chitin, a structural polysaccharide common in crustaceans and insects. PAA is a synthetic polyanion possessing carboxylate (COO-) groups. Adsorption of PAA on the insoluble chitosan matrix can be interpreted as a polyelectrolyte complex, the stoichiometry of which is determined by polymer concentration and solution pH, since the charge density on chitosan and PAA is pH-dependent. Zhang and Gonsalves3,4,6 and Kato and co-workers7-10,14,15 studied CaCO3 crystallization on chitosan matrices submerged in supersaturated solutions of calcium carbonate with PAA and succeeded in the formation of thin CaCO3 films that consisted

10.1021/cg060687k CCC: $37.00 © 2007 American Chemical Society Published on Web 06/19/2007

Calcium Carbonate Crystallization

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Figure 1. Schematic structure of (a) chitosan, (b) poly(acrylic acid).

of a mixture of the calcite and vaterite polymorphs. Both groups showed that the interaction between the anionic carboxylate groups of PAA and the cationic groups on the chitosan surface produced sites on which crystals would preferentially nucleate. At the same time, mobile acid chains in solution suppressed bulk nucleation and growth, confining crystallization to the CaCO3 film forming on the chitosan matrix. Therefore, PAA fulfills opposite roles in promoting CaCO3 crystallization when it is adsorbed on the chitosan surface and inhibiting it when it is free in the bulk solution. The conformation of the PAA chains at the surface also determined the crystallographic orientation of the CaCO3 crystals grown on the films. Whereas the (104) plane of calcite is most stable and generally dominates the morphology of crystals grown from bulk solution, Zhang and Gonsalves3,4,6 found that the (110) plane was dominant in the crystals grown on chitosan films in the presence of PAA. The results indicated that calcite crystals were preferentially oriented with the (110) plane parallel to the chitosan film. This observation was explained by the coincidence of the spacing between calcium ions in the (110) plane with the distance between adjacent COO- groups of PAA (approximately 0.5 nm). Wada and co-workers17 recently studied crystallization on chitosan by the method of diffusion of ammonium carbonate vapor into a calcium chloride solution containing PAA and determined an optimum concentration of PAA for synthesis of a continuous CaCO3 film consisting purely of aragonite, in contrast to the system employed by previous studies3,4,6,8,9,13,14 that resulted in mixtures of calcite and metastable vaterite. Accordingly, Kato and co-workers proposed that the local high concentration of Ca2+ ions produced by adsorption of PAA on the chitosan film caused the kinetically controlled condition leading to preferred nucleation of metastable polymorphs.13 That is, the interaction between chitosan and PAA lowers the interface energy of a less stable polymorph, and thus it nucleates in accordance with Ostwald’s rule. This rule states broadly that less stable crystalline forms may nucleate in advance of more thermodynamically stable forms because kinetics are often more important than thermodynamics in nonequilibrium precipitating systems.19 In theory, subcritical nuclei of all potential polymorphs form and dissolve; only those exceeding a critical size (defined by crystal enthalpy and interface energy) continue to grow. In the current study, optical and electron microscopy were used to investigate the final structures and the growth kinetics of calcium carbonate films on the well-known chitosan-PAA model system, with the aim of establishing the conditions and rationale for the formation of continuous inorganic films. The structures, and the crystalline film coverage ability, were related to the underlying substrate using infrared and Raman spectroscopy that yielded information on both the crystal unit cell and the surface chemistry of the chitosan-PAA films. Experimental Section Materials and Sample Preparation. “Chitoclear” chitosan, with a 95% degree of deacetylation, was supplied by Primex, Norway. Acetic acid, used to dissolve chitosan in solution, was supplied by BDH, UK.

Ammonium hydroxide, used to neutralize acetic acid in dried chitosan films, was obtained from Sigma-Aldrich, UK. Poly(acrylic acid) with a mean molecular weight of 5100 was supplied by Fluka, UK. Calcium chloride and sodium bicarbonate, both in anhydrous form, and sodium hydroxide, used to raise the pH of the supersaturated CaCO3 solution, were supplied by Sigma-Aldrich, UK. Solutions were diluted with deionized water. Solutions of 1% (w/w) chitosan were obtained by dissolving the polymer in 1% (w/w) acetic acid aqueous solution. Films were cast by pouring 5 mL of the solution into 50 mm diameter polystyrene Petri dishes and leaving them to dry in a fume cupboard for 3 days. Chemically, the resultant film is a partial acetate of chitosan. The acetic acid was neutralized with dilute ammonium hydroxide to regenerate the chitosan film as the free base. The films were then washed extensively with deionized water, dried in air and then under vacuum at room temperature, following which they peeled readily from the dish. The thicknesses of 10 randomly selected films were measured with a micrometer, and the mean value was 17 ( 0.5 µm. The films were then soaked in aqueous solutions of PAA, raised to neutral pH and pH 10.5, to alter the surface charge density. Neutral pH reflects the conditions of the solution containing CaCl2, NaHCO3, and PAA prior to addition of sodium hydroxide, while 10.5 is the value to which the solution pH was subsequently adjusted for calcium carbonate growth. In this system, calcium carbonate is produced from the following reaction:

{

H2CO3

+ CaCl2 + 2NaHCO3 w CaCO3 + 2NaCl + HCO3 + H CO32- + 2H+

where the pH of the solution determines the configuration of the carbonate ions in solution. Above pH 10.5, carbonate ions become the dominant species. A 250 mL borosilicate glass beaker, placed on a magnetic stirrer, was filled with 40 mL of 0.025 M CaCl2, 40 mL of 0.050M NaHCO3, and 20 mL of PAA (at concentrations of 0.001, 0.004, 0.01, 0.04, 0.1, and 0.4 wt %) using graduated pipettes. A Teflon-coated stirrer bar and the electrode connected to a digital pH meter (Jenway 3150, calibrated with pH 4 and pH 7 buffer solutions) were both inserted into the solution. They were immediately followed by the chitosan film, which was submerged just below the surface. The solution was stirred continuously at a constant rate, while 1 M sodium hydroxide was added in 0.1 mL increments using a syringe, allowing the pH reading to settle between successive volume additions. Once the pH of the solution had been adjusted to the required value, the stirrer bar and pH meter electrode was removed, and a piece of filter paper was placed on top of the beaker to prevent contaminants from falling into the solution during crystallization. The pH behavior of selected solutions was monitored as they aged; in all cases, the crystallized chitosan film was removed after 7 days in solution. Experiments were set up with the concentration of PAA and the pH of the solution as variables to alter the solution chemistry and determine conditions to optimize the coverage of the chitosan film. For the variation in PAA concentration experiments, the solution pH was set to 10.5 since the concentration of carbonate ions becomes dominant at this value, and thus precipitation of CaCO3 is initiated uninhibited. For the variation in pH experiment, the PAA concentration was fixed at 0.004 wt %. Three control solutions were prepared and allowed to age for 7 days: one omitting the chitosan film template, another omitting the PAA additive, and a third solution omitting both polymers. Optical Microscopy. A Leitz DMRB transmission optical microscope was used to obtain images of CaCO3 crystal growth on the chitosan films following their 7 days’ submergence in solution. Each

1264 Crystal Growth & Design, Vol. 7, No. 7, 2007 film sample was washed with deionized water to remove loose sediment and a rectangular piece cut from the central region of the film. This sample was placed on a microscope slide beneath a standard glass cover slip and observed under bright-field conditions with crossed polarizers. Images were captured with a JVC TK-C1481BEG CCD digital camera and recorded using software (PicPort) supplied by Leutron Vision. Droplets of solution were examined from control samples omitting the chitosan substrate. Image analysis was conducted on the cross-polarized optical micrographs to derive data for the crystallite size distribution, nucleation density, film coverage, and birefringence using image analysis software (KS400, supplied by Carl Zeiss Vision). The diameters of at least 500 crystallites were obtained from an average of eight sample areas for each experiment. The birefringence of the CaCO3 crystallites, defined as nr - nt, where nr and nt are the radial and tangential refractive indices, respectively, was analyzed at different pH values for a PAA concentration of 0.004 wt % by use of a quarter wave plate placed between the specimen and the analyzer to impart color to the polarization patterns observed inside the crystallites.20 A separate, real-time, in-situ study of CaCO3 growth kinetics was performed using optical microscopy to follow the growth of calcium carbonate crystals on small portions of films. A chitosan film was cast from 0.1 mL of a 1% (w/w) solution onto a 13 mm diameter glass cover slip. This cover slip was placed on top of a glass microscope slide with a well made from a spacer filled with approximately 0.25 mL of a solution containing 0.1 M CaCl2, 0.2 M NaHCO3, 0.1 wt % PAA, and 0.04 m NaOH, to give a solution pH of 9.5, such that the chitosan film was presented to the solution for crystallization. Image capture was initiated 24 min after the chitosan film was brought into contact with the CaCO3 solution, as soon as crystallites became observable with the highest magnification objective lens (×63). It was terminated after approximately 2-1/2 h of crystallization time. Ten frames were sampled at regular time intervals for image analysis, and the growth rates of eight crystals among the primary nucleation set were measured. Electron Microscopy. High magnification images of the film morphology were obtained for selected film samples from the experiments where the solution pH was varied. Samples were prepared by placing them on a carbon-coated copper grid which was then sputtercoated with a 10 nm layer of gold/palladium metal. They were examined with a JEOL1200EX scanning transmission electron microscope equipped with an ASID10 scanning attachment, operating at an accelerating voltage of 20 kV. X-ray maps of each sample were obtained, enabling identification of the chemical composition of the crystallites. Images were recorded using software (INCA) supplied by Oxford Instruments. One further crystal sample was selected from the experiments where the PAA concentration was varied. Tilt and slice views of the CaCO3chitosan film composite were imaged as described above but with a JEOL6060 scanning electron microscope. Infrared Spectrosopy. The chitosan film surface structure, in the presence of PAA but before CaCO3 crystallization occurred, was characterized with a Fourier transform infrared (FT-IR) spectrometer (Bio-Rad FTS6000, equipped with a diamond attenuated total reflectance microsampler). Spectra were captured using software (Win-IR Pro) supplied by Bio-Rad Laboratories. A preliminary analysis was conducted on films cast from 2% chitosan solution, to determine the presence of the -COO- groups of PAA and the -NH3+ groups of chitosan. A series of 2% chitosan films were soaked overnight in aqueous solutions of various concentrations of neutralized PAA and then dried. Subsequent experiments were performed using the 1% chitosan films on which CaCO3 was grown. A range of pH and PAA concentrations were used. Raman Spectroscopy. Crystallization was repeated using the realtime experiment setup described in the optical microscopy section for the sample at pH 9.0 and at a PAA concentration of 0.1 wt % and was monitored in-situ with a Kaiser Optical Systems HoloLab Series 5000 Raman microscope, which facilitated optical targeting of specific crystals for Raman spectral analysis. A drop of oil was placed on the coverslip capping the O-ring spacer and a 100× magnification oil objective used to view crystal growth. Spectra were recorded by software (GRAMS AI) supplied by Thermo Galactic.

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Figure 2. IR spectra for a 2% chitosan film soaked in various concentrations of neutralized PAA.

Figure 3. IR spectra for 1% chitosan film surface modified by aqueous solutions of various PAA concentrations at (a) neutral pH and (b) pH 10.5.

Results Chitosan Film Surface Structure. Figure 2 shows the IR spectra for 2% chitosan films in the presence of different concentrations of PAA. Significant bands were observed at wavenumbers of 1553, 1405, and 1637 cm-1. The former two bands correspond to the asymmetrical and symmetrical stretching, respectively, of COO- groups from PAA, whereas the latter band corresponds to the symmetric deformation of NH 3+ groups from chitosan.21 In agreement with the results obtained by Wada and co-workers17 and Zhang and Gonsalves,3 the three strongest peaks in the IR spectrum for the film soaked in the 0.01 wt %



Figure 5. Spherulitic films grown on chitosan films made with a range of concentrations of chitosan, in the presence of 0.01% (w/w) PAA and at pH 9.0. (a) 2% chitosan, (b) 3% chitosan, (c) 4% chitosan. Figure 4. Crossed-polarized optical micrographs of control samples. (a) Small rhombohedra on chitosan film in the absence of PAA, (b) rhombohedra in solution in the absence of a chitosan substrate, (c) large aggregates of rhombohedra in solution in the absence of both PAA and chitosan.

PAA solution confirmed that NH3+ and COO- groups coexisted on the film surface. The spectra obtained for the 1% PAA-modified chitosan films acting as substrates for CaCO3 crystallization are presented in Figure 3. At both neutral and basic pH values, the PAA concentration of 0.004 wt % produced the most intense bands for both COO- and NH3+ groups present on the chitosan surface. The lower concentration of 0.001 wt % PAA also produced strong signals. Bands were weaker at pH 10.5 than at neutral pH: the asymmetrical stretching mode of the COOgroups was manifested as shoulders at 1553 cm-1 rather than independent peaks, while the band for the symmetrical stretching mode at 1405 cm-1 was very weak. The band for the symmetric deformation of NH3+ groups at 1637 cm-1 was slightly

diminished by the rise in pH. These observations were attributed to the lower degree of ionization of chitosan at the more basic pH leading to less adsorption of PAA in this case. Optical Microscopy, Control Samples. Figure 4 shows optical micrographs, taken between crossed polarizers, of the control samples. In all cases, the control samples yielded rhombohedral crystals. Figure 4a shows that sporadic nucleation and poor crystal growth occurred on the chitosan film in the absence of PAA (complete extinction of the chitosan film occurs with crossed polarizers due to the amorphous structure of the chitosan film itself). Figure 4b shows that, in a solution omitting the film template, but containing 0.004 wt % PAA, small aggregates of rhombohedra were present in the bulk. When both chitosan and PAA were absent these aggregates were much larger, as shown in Figure 4c, where individual crystals approached sizes of 20 µm. Optical Microscopy, CaCO3 on Chitosan Films: The Effect of Chitosan Concentration. Figure 5 shows optical micrographs, taken between crossed polarizers, of the calcium

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Figure 6. Crossed polarizer optical micrographs of spherulitic films grown on 1% (w/w) chitosan films made with a range of concentrations of PAA at pH 10.5. (a) 0.00001%, (b) 0.00004%, (c) 0.0001%, (d) 0.0004%, (e) 0.001%, (f) 0.004%, (g) 0.01%, (h) 0.04%, (i) 0.1%, (j) 0.4%.

carbonate crystals grown on chitosan films made at different concentrations of chitosan between 2% (w/w) and 4% (w/w), in the presence of 0.01% (w/w) PAA and at pH 9.0. In all experiments calcium carbonate crystallization occurred only on the underside of the chitosan film, which was exposed to the

bulk solution. In contrast to the control samples, the crystals grown on chitosan in the presence of 0.01% PAA at pH 10.5 did not form rhombohedral shapes but instead appeared disklike. Furthermore, they exhibited a Maltese cross pattern when observed between crossed polarizers, which was indicative of


radial growth and symmetry. The crystallites were twodimensional spherulites and will henceforth be termed simply as spherulites, for brevity. The spherulite size decreased with increasing chitosan concentration, for fixed PAA concentration and pH. Optical Microscopy, CaCO3 on Chitosan Films: The Effect of PAA Concentration. In all experiments, CaCO3 crystallization occurred only on the underside of the chitosan film, which was exposed to the bulk solution. Figure 6 shows images of crystal growth on the chitosan film at different concentrations of PAA. Above a PAA concentration of 0.001%, the films were spherulitic and exhibited the Maltese cross pattern when observed between crossed polarizers. Below 0.001% PAA, single rhombohedra and clusters of rhombohedra formed. For the spherulitic films, the crystal size increased markedly with the PAA concentration, and continuous film coverage of the chitosan substrate was achieved in 0.1 wt % PAA. Here the impingement of spherulites created a tiling effect with straightline boundaries. The morphology control exerted by the PAA additive was greatest at higher concentrations. Although Maltese crosses were evident at 0.04% PAA, they had the best definition in the primary continuous layers crystallized in 0.1% and 0.4% PAA. Visually, the nucleation density appeared to peak at 0.004 wt % PAA. At and above 4% PAA, no crystallization occurred whatsoever. The statistics from image analysis of the PAA concentration series are presented in Figure 7. The vertical error bars present in Figure 7a,b are the standard deviations of the distribution of spherulite diameters and sample area populations, respectively. Chitosan substrate coverage, expressed as a percentage, was determined by the nucleation density and the mean spherulite diameter and provided an estimate of the amount of calcium carbonate that had precipitated onto the film (see Figure 7c). The uncertainty in the surface coverage value was a statistical combination of the errors in the previous two quantities. Although CaCO3 film thickness was not taken into account by the planar analysis, the proximity of the data points for 0.1% and 0.4% PAA to 100% substrate coverage accorded with visual observation of a continuous film in each case. Comparison of the nucleation densities with the relative strengths of the IR absorption bands (see Figure 3) for each PAA concentration revealed that both were greatest at 0.004 wt % PAA, followed by 0.001 wt %, then 0.01 wt % and so on, with increasing PAA concentration. Thus CaCO3 nucleation density was largely determined by the charge density of the chitosan film surface. Optical Microscopy, Chitosan Films: The Effect of Solution pH. Figure 8 shows optical micrographs of the calcium carbonate crystals grown on chitosan films at a range of pH values. The pH to which the supersaturated CaCO3 solution was raised had a pronounced effect on crystal morphology as well as size. Large spherulites, giving particularly well-defined Maltese crosses when observed between crossed polarizers, were produced in solutions raised to pH 8.5 and 9.0. The morphology became increasingly irregular and disordered as the pH became more basic and the supersaturation increased. Figure 9 shows optical micrographs of calcium carbonate films grown at pH 9.0 and 10.5, in 0.004 wt % PAA, observed between crossed polarizers in the absence and in the presence of a quarter wave plate. In the presence of the quarter wave plate, the quadrants of the spherulites became colored blue and red. However, in both cases two populations of spherulites were observed that differed both in size and in appearance. There was a population for which quadrants 1 and 3 (the top right


Figure 7. The effect of PAA concentration on calcium carbonate crystallized onto the chitosan films, at pH 10.5. (a) Crystal size, (b) nucleation density, (c) film coverage.

and bottom left, respectively) were red and quadrants 2 and 4 were blue, indicating that the refractive index in the tangential direction was greater than that in the radial direction (negative spherulites).20 These were the larger spherulites. The smaller, brighter spherulites had the opposite distribution of colors and were therefore positive spherulites for which the tangential refractive index was less than the radial one.20 The results arising from image analysis of the optical micrographs for calcium carbonate crystallization at a range of pH values are shown in Figure 10. The error on the pH values consists of a combination of random statistical error on repeated readings from titration data and an estimated error in calibration of the pH meter. The sizes of the vertical error bars were calculated as outlined previously. The high level of aggregation and indistinct crystal morphology at pH 11.0 meant that distinguishing individual crystals for analysis was impossible at this value, however. The analysis shown in Figure 10 revealed that crystal growth was favored over nucleation at lower pH values, since the solution raised to pH 9.0 produced large spherulites ranging between 50 and 90 µm in diameter. However, as the level of supersaturation rose with increasing pH, nucleation density increased at the expense of crystal size and reached a maximum at pH 10.5. Nucleation was reduced as the pH rose to 11.5; here the film surface charge density decreased since chitosan is largely a neutral polymer at strongly


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Figure 8. Crossed polarizer optical micrographs of CaCO3 crystal growth on chitosan film for different pH values of solution, for a PAA concentration of 0.004 wt %. (a) pH 8.5, (b) pH 9.0, (c) pH 9.5, (d) pH 10.0, (e) pH 10.5, (f) pH 11.0, (g) pH 11.5, (h), pH 12.0.

basic pH. The mean spherulite diameter, however, increased slightly at pH 11.5 and pH 12.0. Electron Microscopy, CaCO3 on Chitosan Films. Figure 11 shows a scanning electron micrograph, with an optical micrograph from the same sample, for calcium carbonate

crystallized onto a chitosan substrate at a PAA concentration of 0.004 wt % and solution pH of 9.0 as a general example of the disklike morphology of the spherulites. The micrograph in Figure 11 shows that the highly ordered spherulites grown at pH 9.0 were flat disks of near-uniform thickness (approximately



Figure 9. Crossed polarizer optical micrographs of CaCO3 growth on a 1% chitosan film in the presence of 0.004% PAA. (a) pH 9.5 without quarter wave plate, (b) pH 9.5 corresponding image with quarter wave plate, (c) pH 10.5 without quarter wave plate, (d) corresponding image with quarter wave plate.

1 µm). Figure 12 shows a map of the calcium in a disk grown at pH 9.0, measured by the back-scattered X-rays in the TEM, confirming that the spherulites were indeed calcium carbonate. On closer inspection of these micrographs, and particularly apparent in the tilt view shown in Figure 11, a bulge was observed in the central region of each spherulite that corresponded with a dark region in areas at 45° to the polarizer/ analyzer axes in the optical micrograph. In this region, the magnitude of brightness suddenly increased, and the intensity of the transmitted light was greater at the crystal edge. Figure 13 shows a series of scanning electron micrographs for calcium carbonate spherulites on 1% chitosan films grown at a range of solution pH values, at a PAA concentration of 0.004 wt %. At pH 8.5, the profile of the spherulites was more convex than at higher pH values, and individual spheres were located at the center of some of the spherulites. In addition, there were some clusters of spheres present in some regions of the film that were much smaller than the spherulites. The spherulites grown at pH 9.0 were slightly convex, although much less so than those at pH 8.5. At higher pH values, the spherulites possessed very little curvature, and no individual spheres were observed in the spherulite centers. At pH 11.5, some small spheres were observed on the spherulites. In this case, however, the spheres were present at random positions on the spherulites, not exclusively at the centers. Secondary crystallization of a conical nature (see Figure 13f) was also observed at pH 11.5 that was not seen at lower pH values. In all cases, some radial texture was apparent, which was most noticeable at pH 9.5 and 10.5. Texture was also noticed at the edges of the spherulites, which was most obvious in the tilt views of the spherulites. In some cases, there was evidence for different morphologies. The SEM image at pH 9.5 possessed a central depression in the spherulites at this pH value. In addition, at pH 9.5, the depressed

region at the center of the spherulite was surrounded by a concentric ring that suggested a change in radial growth between the inner and the outer regions of the spherulite. An SEM image of the continuous CaCO3 film crystallized in the presence of 0.1 wt % PAA is presented in Figure 14, alongside an optical micrograph of the same sample. The spherulites in this case appeared bulbous in the plan view, shown in Figure 15, with a plethora of smaller spheres located mostly along the lines of impingement. A slice through the composite revealed that the film thickness was approximately 8 µm. In-Situ Monitoring of Crystallization, Optical Microscopy. Figure 16 shows the evolution in spherulite size with time for a series of spherulites grown in the presence of 0.1% PAA, observed crystallizing in-situ, accompanied by three video frames where individual crystals that contribute to the data are labeled. Two separate growth rates were measured, corresponding to two distinct groups of crystals with noticeably different birefringent properties. Both growth rates were approximately linear during the first 100 min of crystallization time, after which crystal growth was retarded by a reduction in solution supersaturation of the crystallizing species (which was a consequence of the removal of a certain amount of calcium carbonate from solution). The spherulites that were brighter (labeled 6-8) in Figure 16 grew at about half the rate of the other spherulites (examples of which are labeled 1-5 in Figure 23). At the later stages of crystallization, both populations of crystals exhibited Maltese cross patterns when observed between crossed polarizers, although the more rapidly growing spherulites also possessed a more disordered outer region, with a speckled appearance between crossed polarizers. At early stages of crystallization, however, shown in Figure 17, the extinction patterns of the different spherulite populations were different.


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Figure 11. Scanning electron micrograph (top) and optical micrograph (bottom) from the same sample, for calcium carbonate crystallized onto chitosan at pH 9.0 in the presence of 0.004 wt % PAA. Figure 10. The effect of initial solution pH on calcium carbonate crystallized onto the chitosan films, at a PAA concentration of 0.004 wt %. (a) Crystal size, (b) nucleation density, (c) film coverage.

While the faster growing spherulites contained a Maltese cross at all times, the slower growing, brighter crystals did not. These spherulites possessed a diagonal extinction pattern at the early stages of crystallization. In Situ Monitoring of Crystallization, Raman Spectroscopy. Crystallization was repeated and monitored in-situ with the Raman microscope, with the aim of observing the distinct types of spherulites with different growth rates and subsequently obtaining a Raman spectrum from which the constituent polymorph could be identified. Figure 18 shows optical micrographs of each type of spherulite, with the corresponding Raman spectrum obtained from a point within each spherulite. The spectra showed that, as well as appearing different between crossed polarizers when viewed in the optical microscope, the two types of spherulite were formed from different crystal polymorphs of calcium carbonate. The faster growing spherulites consisted of calcite, whereas the slower growing spherulites consisted of vaterite. After crystal growth had arrested and a complete film of calcium carbonate was obtained, the film was washed and dried and further Raman spectra were taken, from which the relative proportions of calcite and vaterite present across the entire sample were obtained. From these spectra, the ratio of calcite to vaterite in the calcium carbonate films grown in 0.1 wt %

PAA at pH 10.5 was estimated to be 3:1. This estimate was in general agreement with the visually observed proportion of the calcite spherulites to the vaterite ones in the micrographs obtained from the video capture images. Discussion Film Morphology. The spherulitic morphology of the calcium carbonate thin films formed in the present study, characterized by the Maltese Cross observed in the micrographs obtained between crossed polarizers, was very similar to that observed by previous workers on a variety of substrates, including chitosan3,4,13,17,18 and poly(vinyl alcohol).15,18 The spherulitic nature of the calcium carbonate formed on the films in the present cases indicates that it was in the form of polycrystalline aggregates that nucleated at single points and grew radially outward. The impinging spherulites with straight boundaries are expected for the case of simultaneous nucleation followed by linear radial growth. The disklike nature of the spherulites shown in the presence of SEM was also shown in previous studies.9,10,13,15,17 In those cases, as in the present one, the thickness of the spherulitic disks was about 1-2 µm. Studies of calcium carbonate film formation in synthetic systems broadly divide into two classes: first, those in which an insoluble substrate (often in combination with a soluble polymer) is believed to template crystallization and, second, those in which calcium carbonate is initially deposited in an amorphous state and subsequently undergoes transformation to



Figure 12. (a) Scanning electron micrograph and (b) calcium map, of a calcium carbonate spherulite grown on a chitosan substrate at pH 9.0 in the presence of 0.004 wt % PAA.

a crystalline state. Biomineralization studies have demonstrated that amorphous calcium carbonate (ACC) exists as a precursor to growth of a crystalline aragonite form in natural systems,22 and studies of calcium carbonate film formation on glass and silica substrates in the presence of PAA have also shown the existence of an ACC phase that subsequently crystallizes into a multicrystalline morphology.23-25 In the case of calcium carbonate growth on polymeric substrates, such as chitosan, cellulose, and poly(vinyl alcohol) (PVA) in the presence of PAA, no ACC formed and calcium carbonate crystallized directly on the polymer substrate. Although the existence of an ACC phase was not investigated in the present study, the use of a chitosan substrate made it likely that, in accordance with previous work, crystallization occurred directly onto the film. The essential feature of all of these studies is the interaction of PAA with the substrate film, which results in charged PAA molecules present at the interface causing nucleation of calcium carbonate.3,4,6 In the present study, IR spectroscopy confirmed the interaction between the cationic protonated amine groups of chitosan and anionic carboxylate groups of PAA. In previous studies where modified chitosan and cellulose substrates that were unable to bind to PAA were used, no calcium carbonate films formed.13 Simultaneously, PAA not adsorbed onto chitosan inhibits crystal growth both on the surface of the calcium carbonate film and in the bulk since mobile anionic groups in the bulk adsorb onto growing crystal faces. It is repeatedly reported in literature that above a certain concentration of PAA this effect surpasses the promotion of crystallization on the chitosan film surface, effecting sporadic nucleation on the film surface.3-10,13-18 This result was reproduced in the present study at very high concentrations of PAA (4%) bringing about the same paucity of crystal growth that was observed in its absence. No precipitates were observed in the bulk, indicating formation of a PAA-Ca2+ polyelectrolyte species, which stayed below a critical molar ratio and remained soluble.26 Some authors have suggested that the coexistence of vaterite, calcite, and, in some cases, aragonite, in the films is due to local differences in the supersaturation of calcium ions at the interface, controlled by the balance between adsorbed PAA at the interface and mobile PAA in the bulk. Higher PAA concentrations at the interface lead to a higher local supersaturation of calcium ions, thus favoring the formation of the less stable polymorphs, vaterite and aragonite.17 It has been speculated that when the value of the ionic product (Ca2+)(CO32-) is greater than 2.4 × 10-6 M2 then the less stable polymorphs

preferentially nucleate and grow,17,27 with the precise value determining whether vaterite or aragonite forms. The coexistence of the calcite and vaterite polymorphs was shown in the present study by the in-situ experiments. It is possible that local differences in calcium supersaturation caused the coexistence of the different polymorphs in the present study because the value of the ionic product was greater than 2.4 × 10-6 M2. Other authors have suggested that the conformation and local orientation of the PAA chains at the interface influences the crystal orientation and polymorph of the calcium carbonate crystals. It has been proposed that matching of the carboxylate groups with particular calcium carbonate crystallographic planes leads to those planes being preferentially oriented with respect to the substrate film. Analysis of the crystal orientation in calcium carbonate films crystallized on glass substrates with adsorbed PAA indicated that calcite crystals eventually grew with the calcite (001) plane parallel to the substrate,28 while the calcite (104) plane dominated the observed morphology of rhombohedra precipitating from the bulk. In this case, the separation of adjacent calcium ions in the (001) plane (0.499 nm) and different directions in the (104) plane (0.499 and 0.405 nm, respectively) matched the distances between carboxylate groups in two conformations of the PAA chains (0.410 and 0.502 nm). A similar analysis by Gonsalves et al.3,4 led to the conclusion that the (110) plane of calcite, with a spacing between calcium ions of 0.500 nm, was preferentially nucleated at the interface of a chitosan film coated with PAA. Wada et al.17 found that films comprised of aragonite and vaterite were formed on chitosan films in the presence of PAA, with the aragonite (110) planes being partially oriented parallel to the chitosan surface and the vaterite (100) and (110) planes parallel to the chitosan surface. In this case, no lattice matching calculations were performed to explain these findings, although they were consistent with growth of the dominant crystallographic c-axis parallel to the chitosan surface, as expected for a locally supersaturated layer at the film surface.17,29,30 Aragonite films were also grown on PVA substrates,15 where it was argued that the PAA was induced to orient by its interaction with the underlying crystalline PVA. The separation of the hydroxyl groups on the PVA chains was 0.504 nm, and the separation of the PVA chains in the underlying substrate was 0.781 nm, which matches the calcium ion spacing in the a and b axes in the (001) plane of aragonite, respectively. It was believed that the high level of orientation of the underlying film combined with the cooperative effects of PVA and PAA were necessary for nucleation of the aragonite polymorph in the PVA case, since


Payne et al.

Figure 13. Scanning electron micrographs of CaCO3 spherulites grown on 1% chitosan films at a range of pH values, in the presence of 0.004 wt % PAA. (a) pH 8.5, (b) pH 9.0, (c) pH 9.5, (d) pH 10.0, (e) pH 10.5, (f) pH 11.5. Left, low magnification plan view; middle (a-e) high magnification plan view; middle (f) high magnification tilt view showing precipitates; right, tilt view, showing spherulite flatness.

the use of poly(glutamic acid) (PGA) instead of PAA yielded mainly (>90%) vaterite films. In studies of calcium carbonate growth on glass,28 an effect of PAA molecular weight was found, with higher molecular weight PAA promoting the orientation of crystals grown on the substrate. The explanation for this effect was that the higher molecular weight PAA chains (250 kDa) were more effectively bound to the glass substrate compared to the more highly mobile small molecular weight PAA chains (2 kDa). In the studies of

PAA bound to chitosan films,3,4,6 even the low molecular weight PAA (2 kDa) was able to direct the growth of calcium carbonate because they were bound to the chitosan substrate. In the present case, the PAA chains were of low molecular weight (5.1 kDa) but, as for the system studied by Gonsalves et al.3,4,6 and shown by the IR spectra, were still immobilized at the interface and were therefore likely to cause oriented growth of calcium carbonate. This speculation is supported by evidence from Wada et al., who showed that there was no difference between low



Figure 14. (a) Scanning electron micrograph and (b) optical micrograph, of the continuous film of calcium carbonate grown on a chitosan film in the presence of 0.1 wt % PAA at pH 10.5.

Figure 15. Tilt view of the continuous calcium carbonate film grown in the presence of 0.1 wt % PAA at pH 10.5, showing the film thickness to be approximately 8 µm.

molecular weight PAA (2 kDa) and medium molecular weight PAA (45 kDa) in the ability of calcium carbonate to form films on chitosan substrates. It should be noted that comparison of the results of the present study with previous ones should be performed with caution, however. The method of calcium carbonate formation, and hence the rate of change of supersaturation and subsequent driving force for nucleation and growth of the calcium carbonate polymorphs, varies between studies, with the majority of studies using the bubbling of carbon dioxide through a saturated calcium carbonate solution or the decomposition of ammonium carbonate above a calcium chloride solution to cause crystallization. Where calcium carbonate is formed by bubbling carbon dioxide into a saturated calcium carbonate solution, the calcium carbonate films

grown on chitosan substrates in the presence of PAA are mainly comprised of calcite and vaterite,3,13,18 with aragonite being formed in the presence of magnesium ions and poly(aspartic acid).10 Calcium carbonate films grown on chitosan substrates via the decomposition of ammonium carbonate contained the aragonite and vaterite polymorphs.17 Films formed by a diffusion method, in which a chitosan or PVA covered membrane was placed between two glass containers separated by the membrane, with one container being filled with a solution containing a source of calcium ions and the other being filled with a solution containing a source of carbonate ions, contained aragonite.18 Films formed by an immersion method, in which both glass containers contained a mixture of sources of calcium and carbonate ions and the membrane was either PVA or chitosan coated, formed aragonite in the presence of PAA. For this method, the concentration of the calcium and carbonate ions was similar to that used in the current case (0.010 M Ca2+ and CO32- compared to 0.025 M Ca2+ and CO32- in the present case), and the range of PAA concentrations used were also similar. However, the precise value of the supersaturation, which will be influenced by the presence of other ions in solution, will be important in determining the polymorph. The current experiment differed in detail from the previously reported immersion experiment; therefore, the presence of calcite and vaterite in the present case instead of aragonite is not necessarily surprising. It should be noted that the molecular weight of the PAA used in the previous immersion study was lower than that used in the present study (2 and 5.1 kDa, respectively), and therefore the formation of aragonite in the previous study was not a PAA molecular weight effect.6 Effect of PAA Concentration. The occurrence of a maximum nucleation density at an intermediate PAA concentration in the present study is in agreement with previous results on the influence of PAA concentration on calcium carbonate film formation on chitosan, chitin, and cellulose substrates.3,4,6,13,15 In those studies, the crucial factor was shown to be the balance between the number of carboxylate groups available at the film surface that bind calcium and increase the surface supersaturation and the number of ionized carboxylate groups in solution that function as growth inhibitors. In previous studies on chitosan films, it was found that the amount of PAA adsorbed at the chitosan surface reached a maximum amount at 0.06 wt %.3,4,10,15 At low PAA concentrations, the amount of adsorbed PAA at the chitosan surface that was able to bind to calcium ions from solution and hence influence the local supersaturation at the film surface was very


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Figure 16. The growth of calcium carbonate spherulites on chitosan films in the presence of 0.1% PAA at pH 10.5, dependent on time exposed to solution, with video frames displaying the crystals that constitute the data.

Figure 17. Different extinction patterns observed in the first frame of the images for spherulites that grew at different rates.

low.3,4,15 The low surface supersaturation led to low surface nucleation rates. Increasing the PAA concentration beyond 0.06% simply increased the amount of PAA in the bulk solution, and no further surface adsorption occurred,3,4,6,15 leading to increased sequestration of calcium in the bulk, and therefore low surface nucleation rates. The PAA concentration of 0.06 wt % therefore represented the maximum concentration of free carboxylate groups at the surface, leading to high values of calcium surface supersaturation and therefore high nucleation rates. In the present study, the IR spectra of the chitosan/PAA films showed that the maximum surface charge, hence the ability to bind calcium ions, coincided with the PAA concentration of 0.004 wt % at which the maximum nucleation rate was observed. The same mechanism, of maximum surface adsorption of PAA (at 0.004% in the present case rather than 0.06% measured in the previous study), was therefore responsible for the maximum in nucleation rate, and the film morphology at and below this concentration was therefore dominated by the nucleation kinet-

ics. The maximum nucleation density at 0.004 wt % in the present study compared to 0.06% in the previous study may be explained by the difference in pH conditions of the different experiments. The present study was performed at a higher pH (pH 10.5 compared to pH 6 used previously), where the amount of charged carboxylate groups, and hence propensity for calcium binding, will be higher for a given concentration of PAA. It should be noted from previous work, however, that the local nature of the interface is highly important since it was shown that the PAA concentration ranges in which thin films of calcium carbonate formed depended on the nature of the underlying substrate and were different for chitosan, chitin, and cellulose.13 In the PAA concentration range up to 0.4%, the reduction in nucleation density combined with minimal growth inhibition of calcium carbonate by PAA in solution led to a continual increase in spherulite size as growth became the dominant factor influencing the film morphology. At higher PAA concentrations, the growth inhibition effects became dominant, since no calcium carbonate crystallization was observed at 4% PAA. The dominance of calcium carbonate growth over nucleation at PAA concentrations of 0.1 and 0.4% led to the formation of continuous films of calcium carbonate at these concentrations. It may be speculated that between 0.4 and 4% PAA the film coverage will decrease again, as both nucleation and growth become inhibited. It should also be noted that previous studies also showed that increasing the concentration of PAA (with a molecular weight of 2 kDa) from 0.0024 to 0.01 wt % led to a dramatic increase in the calcite/vaterite ratio from approximately 0:100 to 90:10. Since the less thermodynamically stable polymorphs form in conditions of higher supersaturation and the maximum in nucleation density and hence surface supersaturation occurred toward the lower end of the concentration range used in the



Figure 18. Raman point spectra obtained during crystallization. The calcite and vaterite crystals were characterized after 57 and 148 min, respectively. The Raman-active vibration modes of calcite give bands located at 280, 712, 1085, and 1450 cm-1. Vaterite is distinguished by two degenerate modes, each manifested by a doublet: the first occurs at 1074 and 1090 cm-1, and the second occurs at 740 and 750 cm-1. A further vibration mode gives a band at 300 cm-1.

present study, the predominance of calcite in the films is therefore expected and in agreement with previous work.13 Effect of Solution pH. The other variable investigated in the present study was the solution pH, adjusted with sodium hydroxide. There have been few systematic studies of solution pH on calcium carbonate film formation on insoluble substrates, and those that have investigated the influence of pH studied values between 4.5 and 8.5, below the range in the present study. The pH of the supersaturated solution determined the film morphology via its influence on the ionization of the chitosan substrate and PAA at the surface, the ionization of PAA in bulk, the calcium surface supersaturation, and the bulk supersaturation of calcium carbonate. First, the charge density of the chitosan and PAA at the film surface, and PAA in bulk solution, change with pH. The dissociation constant, pKa, is 4.5 and 6.3 for PAA and chitosan, respectively.31 Therefore, at high pH (pH > 6.3), corresponding to the conditions used in the present experiment, PAA is charged, while chitosan is close to neutral. The increase in PAA surface charge with increasing pH will therefore lead to increased surface supersaturation of calcium. Conversion of hydrogen carbonate ions to carbonate ions also rises with pH, with carbonate ions becoming the dominant species above pH 10.5. The increase in calcium carbonate supersaturation with increasing pH therefore favors nucleation and growth with increasing pH. However, the increase in calcium supersaturation will be countered by an increase in the sequestration of calcium in the bulk solution with increasing pH as the bulk PAA also becomes increasingly charged. The nucleation density and spherulite growth rate (indicated by spherulite size) therefore pass through maximum values with increasing pH that are determined by the competition between the increase in surface PAA charge and conversion of carbonate ions, which promote nucleation and growth, and the increase in bulk PAA charge, which inhibits nucleation and growth. The maximum surface

coverage at pH 9.5 was therefore a result of the maximum spherulite size at pH 9 and the maximum nucleation rate at pH 10.5. While studies on CaCO3-chitosan film composites do not examine the effect of pH in the range presently studied, Cheng and co-workers32,33 recently reported data on the precipitation of monodisperse calcite rhombohedra from solutions containing PAA and observed a maximum crystal size at pH 9.0, which is consistent with the maximum spherulite size in the present study being measured at pH 9.0. They also attributed this observation to greater morphology control afforded by low supersaturation. The maximum value of the film coverage at a pH value between 9.0 and 10.5 is a consequence of the combination of the maximum spherulite size at pH 9.0 and the maximum nucleation density at pH 10.5. The change in appearance of the spherulites observed in the electron micrographs can also be explained by the crystallization inhibition effect of PAA and the control of calcium carbonate supersaturation at the surface. As the pH decreases from pH 12.5 to pH 8.0, the PAA becomes more protonated and therefore less charged and therefore interacts to a lesser extent with the calcium carbonate crystal surface. The growth inhibition effect of PAA on the calcium carbonate spherulites therefore decreases with decreasing pH, allowing the calcium carbonate to grow to a greater extent perpendicular to the surface. The low supersaturation at the surface at low pH values also limits the lateral spread of the spherulites. Less inhibition perpendicular to the surface and slower growth rates parallel to the surface therefore result in the more convex spherulite profiles observed at the lower pH values studied. The presence of the spherical crystals at the centers of some of the spherulites at pH 8.5 is less easy to explain, however. A similar observation on the growth of vaterite and aragonite spherulitic films showed the presence of spherical particles at the centers of the spherulites that were


formed from vaterite on the vaterite spherulites and aragonite on the aragonite spherulites.27 No mechanism was postulated for their formation, although it was noted that the presence of vaterite and aragonite in close proximity on the film surface demonstrated the importance of local film conditions for polymorph determination. Possibly, the spheres form first from the initial nuclei on the film and act as nuclei themselves for the spherulite growth. The depressions in the centers of the some of the spherulites may represent regions where the initial spherical nucleus had dissolved. Without further evidence, the reason for the presence of spheres in the center of some of the spherulites remains uncertain. It is likely, however, that the spheres and conical structures observed at off-center positions on some of the spherulites grown at higher pH values are the results of secondary crystallization (possibly an artifact of sample preparation for the electron microscopy measurements) leading to the formation of crystallites on the surface of existing spherulites. Conclusions Calcium carbonate films, with a thickness on the order of a few microns, were grown on chitosan films in the presence of PAA. Some of the PAA bound to the oppositely charged chitosan film, where via interaction with calcium ions from solution it promoted crystallization of calcium carbonate at the film surface by increasing the local concentration of calcium at the surface. At the same time, interaction between PAA and calcium ions in solution inhibited bulk crystallization, thereby confining calcium carbonate crystallization to the film surface. The crystals that formed at the surface were in the form of disklike entities with radial symmetry parallel to the film surface, i.e., two-dimensional spherulites. Without PAA only sporadic nucleation was observed. The effect of the growth conditions on the calcium carbonate morphology was explained by the competing influences of nucleation and growth at the chitosan surface, which were determined by the local supersaturation of calcium ions at the biopolymer surface. The surface nucleation rate passed through a maximum with increasing PAA concentration, at fixed pH. Below this concentration, the film morphology was determined by the nucleation kinetics. Above this concentration, up to 0.4% PAA, nucleation rates decreased but growth was relatively unaffected, and the film morphology was determined by the growth kinetics. Therefore, the spherulite size, and hence film coverage, increased. At 0.1 and 0.4% PAA, 100% film coverage was achieved. It was therefore possible to obtain and explain a regime at which a continuous film of calcium carbonate, which may be an effective barrier, could be produced. Increasing the pH at fixed PAA concentration caused the nucleation rate and spherulite size to pass through maximum values. These were determined by the competition between nucleation and growth promotion due to the increase in surface PAA charge, hence surface supersaturation of calcium ions, and nucleation and growth inhibition due to the increase in bulk PAA charge and hence sequestration of bulk calcium ions. At low pH values, nucleation kinetics were low owing to the low calcium carbonate supersaturation. At higher pH values, nucleation kinetics were promoted owing to higher local supersaturation of calcium at the interface. Growth was, however, inhibited by the PAA that was more charged at higher pH and therefore interacted to a greater extent with the calcium ions.

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The maximum surface coverage was determined by the combination of nucleation and growth and occurred at pH 9.5. In-situ monitoring of the crystallization process, combined with polymorph identification using crossed-polarizers and Raman microscopy, revealed two coexisting populations of spherulites that were formed from calcite and vaterite, respectively. The vaterite spherulites were optically positive (as measured by the change in birefringence on insertion of a quarter wave plate between crossed polarizers) and grew at a slower rate than the optically negative calcite spherulites. Acknowledgment. The authors thank Unilever for permission to publish this paper. References (1) Meldrum, F. C. Int. Mater. ReV. 2003, 48, 187-224. (2) Mann, S. Biomineralization: Principles and Concepts in Bio-Organic Materials Chemistry; Oxford University Press: New York, 2001. (3) Zhang, S.; Gonsalves, K. E. Mater. Sci. Eng. C 1995, 3, 117-124. (4) Zhang, S.; Gonsalves, K. E. J. Appl. Polym. Sci. 1995, 56, 687695. (5) Manoli, F.; Koutsopoulos, S.; Dalas, E. J. Cryst. Growth 1997, 182, 116-124. (6) Zhang, S.; Gonsalves, K. E. Langmuir 1998, 14, 6761-6766. (7) Kato, T.; Suzuki, T.; Amamiya, T.; Irie, T.; Komiyama, M.; Yui, H. Supramol. Sci. 1998, 5, 411-415. (8) Kato, T.; Amamiya, T. Chem. Lett. 1999, 3, 199-200. (9) Kato, T.; Suzuki, T.; Irie, T. Chem. Lett. 2000, 2, 186-187. (10) Sugawara, A.; Kato, T. Chem. Commun. 2000, 6, 487-488. (11) Falini, G. Int. J. Inorg. Mater. 2000, 2, 455-461. (12) Dala, E.; Klepetsanis, P. G.; Koutsoukos, P. G. J. Coll. Int. Sci. 2000, 224, 56-62. (13) Hosoda, N.; Kato, T. Chem. Mater. 2001, 13, 688-693. (14) Sugawara, A.; Ishii, T.; Kato, T. Angew. Chem., Int. Ed. 2003, 42, 5299-5303. (15) Hosoda, N.; Sugawara, A.; Kato, T. Macromolecules 2003, 36, 64496452. (16) Ajikumar, P. K.; Lakshminarayanan, R.; Valiyaveettil, S. Cryst. Growth Des. 2004, 4, 331-335. (17) Wada, N.; Suda, S.; Kanamura, K.; Umegaki, T. J. Coll. Int. Sci. 2004, 279, 167-174. (18) Iwatsubo, T.; Sumaru, K.; Kanamori, T.; Yamaguchi, T.; Sinbo, T. J. Appl. Polym. Sci. 2004, 91, 3627-3634. (19) Mullin, J. W. In Crystallization; Butterworth-Heinemann: Woburn, MA, 2001. (20) Meeten, G. H.; Haudin, J. M. In Optical Properties of Polymers; Elsevier Applied Science: London, 1986. (21) Peniche, C.; Argu¨elles-Monal, W.; Davidenko, N.; Sastre, R.; Gallardo, A.; San Roma´n, J. Biomaterials 1999, 20, 1869-1878. (22) Weiner, S.; Addadi, L. J. Mater. Chem. 1997, 7, 689-702. (23) Gower, L. B.; Odom, D. J. J. Cryst. Growth 2000, 210, 719-734. (24) Xu, X.; Han, J. T.; Cho, K. Chem. Mater. 2004, 16, 1740-1746. (25) Volkmer, D.; Harms, M.; Gower, L. B.; Ziegler, A. Angew. Chem., Int. Ed. 2005, 44, 639-644. (26) Fantinel, F.; Rieger, J; Molnar, F.; Hu¨bler, P. Langmuir 2004, 20, 2539-2542. (27) Wada, N.; Yamashita, K.; Umegaki, T. J. Cryst. Growth 1995, 148, 297-304. (28) Kotachi, A.; Miura, T.; Imai, H. Chem. Mater. 2004, 16, 31913196. (29) Schwartz, A.; Eckart, D.; Connell, J. O.; Francis, K. Mater. Res. Bull. 1971, 6, 1341. (30) Ota, Y.; Inui, S.; Iwashita, T.; Kasuga, T.; Abe, Y. J. Am. Ceram. Soc. 1995, 78, 1983-1984. (31) Chibowski, S. J. Coll. Int. Sci. 1990, 140, 444-449. (32) Cheng, B.; Lei, M.; Yu, J.; Zhao, X. Mater. Lett. 2004, 58, 15651570. (33) Yu, J.; Lei, M.; Cheng, B.; Zhao, X. J. Solid State Chem. 2004, 177, 681-689.

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