Spinning up the polymorphs of calcium carbonate

Controlling the growth of the polymorphs of calcium carbonate is important in understanding the changing environmental conditions in the oceans. Aragonite is the main polymorph in the inner shells of marine organisms, and can be readily converted to calcite, which is the most stable polymorph of calcium carbonate. Both of these polymorphs are significantly more stable than vaterite, which is the other naturally occurring polymorph of calcium carbonate, and this is reflected in its limited distribution in nature. We have investigated the effect of high shear forces on the phase behaviour of calcium carbonate using a vortex fluidic device (VFD), with experimental parameters varied to explore calcium carbonate mineralisation. Variation of tilt angle, rotation speed and temperature allow for control over the size, shape and phase of the resulting calcium carbonate.

C arbon dioxide fixation from the atmosphere by marine organisms as large deposits of calcium carbonate is important in the carbon cycle. The rising concentration of carbon dioxide in the atmosphere, estimated to be 40% higher than preindustrial levels and higher than at any point in the last 800,000 years 1 , has led to a shift in the equilibrium of dissolved CO 2 in seawater. This has lowered the pH of seawater and along with increasing temperatures, has greatly affected the optimum conditions for calcium carbonate (CaCO 3 ) mineralisation 2,3 . Deposition of CaCO 3 by marine organisms, in the form of aragonite in the nacreous layer or inner shell [4][5][6] , is therefore likely to be tested by the climate change 7,8 in the Anthrapocene era 9 . Controlling the formation of CaCO 3 polymorphs is important in understanding the mineralisation process with such changing environmental conditions, and for this purpose we used the recently developed, continuous flow processing, vortex fluidic device (VFD) [10][11][12][13] . While the conditions here are much more extreme and different to those likely to be encountered in the environment in the future, the results highlight the conditions required for selective preparation of different polymorphs of calcium carbonate. The vortex fluidic device (VFD), is capable of producing thin films in which high shear forces provide a mechanical energy which is effective in manipulating the growth of a range of diverse materials, including the synthesis of mesoporous silica 11 , decorating carbon nano-onions with palladium nanoparticles 12 , exfoliation of laminar material 10 , forming composite material with live algal cells and controlling organic reactions 13 .
Calcite is the most stable polymorph of CaCO 3 and is the least soluble in water as opposed to vaterite, which is the least stable polymorph and most soluble in water. These stability differences arise from the way the calcium and carbonate ions are assembled in the extended solid-state structures, Fig. 1a [14][15][16][17] . Aragonite and calcite have similar structures with the inter-planar carbonate ions in a staggered arrangement relative to each other, minimising electrostatic repulsion, while in the less dense vaterite they are almost eclipsed relative to each other. The structures have been further explored in the present study using the recently developed Hirshfeld surface analysis 18 , in gaining further insight into the interplay of the ions in the solid state. Transformation from vaterite to aragonite and calcite is possible at 0-30uC and 60-80uC respectively 19,20 . In addition, aragonite is readily converted to calcite in nature at temperatures higher than 380uC 21 . The nucleation and growth of the three different polymorphs can be manipulated through the addition of crystal modifiers, including bio-polymers, inorganic salts, and macromolecules 22,23 . Developing protocols for gaining access to the different polymorphs of calcium carbonate is central to mimicking processes in nature, for example in generating synthetic aragonite, which has only recently been developed using polymer-mediated mineral growth, in addition to layer-by-layer deposition of a porous organic material 24 . Also important is gaining control over the interconversion of the different polymorphs of calcium carbonate where particular applications demand the presence of one phase of specific particle size and morphology (e.g. paper coating) 25 while another application may require the in situ conversion of CaCO 3 from one phase to another, such as in drug delivery 26 .

Results
The VFD used herein has a 10 mm tube with the capacity for high rotational speeds and variation in the tilt angle, h, relative to the horizontal position, Fig. 1b. The latter can dramatically affect the shear forces present within the dynamic thin films 13 , which include solutions of Milli-Q water, seawater, and a mixture of ethanol and Milli-Q water, noting that ethanol is effective in CaCO 3 polymorph control under conventional batch processing 27 . Milli-Q water spiked with NaCl matching the activity of salt in seawater and Milli-Q water spiked with 1% and 2% of Mg 21 also features in the study, noting that the presence of Mg 21 ions attenuates the growth of calcite with no effect on aragonite formation 28,29 . Aqueous solutions of NaHCO 3 as the source of carbonate ions and aqueous solutions of CaCl 2 as the source of calcium ions were fed into the VFD at a flow rate of 1 mL/ min, in parallel with classical batch processing in a round bottom flask as control experiments. The effect of varying the ratios of HCO 3 2 : Ca 21 on CaCO 3 polymorph formation was investigated with relevance to the changing atmospheric and seawater concentration of CO 2 (see experimental).
For batch processing at room temperature, 71% of the samples produced calcite quantitatively, as established using X-ray diffraction (XRD) data, Fig. 2a. A 251 ratio of HCO 3 2 and Ca 21 results in a mixture of calcite (66.8%) and vaterite (33.2%), and decreasing or increasing the ratio of HCO 3 2 : Ca 21 increased the percentage of calcite in the product at the expense of forming vaterite. For batch processing at 80uC there was little control in the formation of the CaCO 3 polymorphs, with XRD data revealing a mixture of aragonite, calcite and vaterite in all but one sample. Nevertheless, as the ratio of HCO 3 2 to Ca 21 increases, the percentage of vaterite increases and to a lesser extent the percentage of aragonite increases, whereas the percentage of calcite decreases, Fig. 2a.
At room temperature, operating the VFD at 500 rpm at a tilt angle of 45u afforded a mixture of calcite and vaterite in 57% of the samples, while at 80uC the percentage of samples with both polymorphs dropped to 29%, Fig. 2b. Operating the VFD at 4500 rpm at a tilt angle of 45u gave no difference in the outcome for the continuous flow processing at room temperature and 80uC, with calcite being exclusively formed. The choice of 45u tilt relates to the angle that provides the greatest shear below 60u tilt. This has been judged by extensive optimisation of exfoliation of graphene and simple Diels Alder reactions 13 . Vaterite formation at 80uC was favoured at 1510 and 1051 ratios of HCO 3 2 to Ca 21 . Reducing the angle of rotation of the VFD from 45u to 0u at 500 rpm also selectively afforded pure calcite for all ratios, irrespective of the temperature of the reaction.
We have shown that the VFD is effective in forming calcite or vaterite at high or low shear in aqueous systems with no evidence of the formation of aragonite despite its presence in nature in molluscan shells. Substituting Milli-Q water for seawater in the VFD and also in batch processing afforded vaterite quantitatively, Fig. 2c. Optimum conditions for obtaining vaterite in the VFD are low shear  at 45u tilt angle (500 rpm), high shear at 45u tilt angle (4500 rpm) or 0u tilt angle, all at room temperature. Aragonite was produced in 45% yield at 80uC using the VFD at a rotation speed of 4500 rpm and 0u tilt angle. These results may reflect the presence of Mg 21 ions present in seawater inducing the formation of aragonite 30 . This was corroborated by XRD data for material prepared in the presence of Mg 21 ions, with 1% and 2% Mg 21 in Milli-Q water which resulted in 1% aragonite (99% calcite) in the VFD at operating at 500 rpm and 5000 rpm, at 45u tilt at room temperature. For the same conditions in the absence of added Mg 21 , the product was exclusively calcite, and batch processing in the presence of 1% and 2% Mg 21 resulted in 1% aragonite (99% calcite) and 10% aragonite (90% calcite) respectively, in contrast to forming vaterite and calcite in the absence of Mg 21 . The presence of carbonate and calcium ions in seawater made it inherently difficult to measure the concentration of the prepared NaHCO 3 and CaCl 2 solutions. Rather saturated solutions were prepared by the addition of excess CaCl 2 and NaHCO 3 to seawater followed by filtration. ICP-AES established that the seawater had 12500 ppm Na, 1237 ppm Mg, 891 ppm S, 389 ppm K, 400 ppm Ca, 8.9 ppm Sr and 1.35 ppm Si. (The Na and Si concentrations are higher than the global mean concentration in seawater of 10561 ppm and 0.03 ppm respectively, while that of Sr is lower than the average at 13 ppm.) Matching the activity of salt in seawater had no effect on aragonite formation with both batch and VFD processing resulting in 100% calcite.
Addition of ethanol induces aragonite formation 27 and in the present study the effect of the presence of ethanol was studied for a 20% volume mixture of ethanol in Milli-Q water, using the VFD and also in batch mode as a control. For a stoichiometric ratio of HCO 3 2 to Ca 21 at 251, at room temperature, the VFD resulted in the formation of calcite regardless of the speed and tilt angle, Fig. 2d. In contrast, using the batch mode at room temperature, pure vaterite was obtained, while at 80uC a mixture of 64% aragonite and 34% calcite resulted. At higher temperatures and low shear stress using the VFD, vaterite was the exclusive product, while at a higher shear stress a mixture of aragonite and vaterite resulted. This is consistent with ethanol stabilising vaterite at higher temperatures by inhibiting its dissolution. Interestingly placing vaterite powder, which was prepared using the VFD operating in continuous flow mode, in water devoid of ethanol and now operating the VFD in the confined mode at a tilt angle of 45u and room temperature resulted in vaterite to calcite conversion. (The confined mode is for a finite volume of liquid processed in the VFD, where there can be intense shear at high speed in the absence of the viscous drag shear associated with continuous flow operation of the device 13 .) Replacing the water with hexane in the VFD operating under confined mode gave no such phase change and this is consistent with the phase change in Milli-Q water arising from the dissolution of vaterite and the precipitation of the calcium carbonate as calcite.
The effect of tilt angle and speed on calcium carbonate synthesis in the VFD was studied for the ethanol/water system where reactant concentrations can be predetermined unlike in seawater, and where all three polymorphs can be produced, Fig. 2d. The effect of tilt angles at 4500 rpm and 80uC on the percentage of polymorph shows that for tilt angles below 45u, vaterite and aragonite dominate while at . 45u, calcite and aragonite dominate, Fig. 3b. Optimal conditions for aragonite synthesis occur at 45u with a yield of 80%. A plot of percentage of polymorph versus rotational speed at a tilt angle of 45u established that at 500 rpm vaterite is exclusively formed, and as the speed increases, the percentage of vaterite decreases to 0% at just under 4000 rpm, Fig. 3c. At 6000 rpm, aragonite and calcite are present in 80% and 20% respectively, a polymorph mixture that is not uncommon in molluscan shells 31 . Above 6000 rpm the percentage of aragonite decreases and that of calcite increases.
Hirshfeld surface analysis, as a recently developed tool for understanding the interplay of molecules and ions in the solid state 18 Fig. 1a 18 . C???C interactions are absent in the Hirshfeld surfaces of vaterite and calcite, whereas there is a small component in that of aragonite (5.4%). More significantly, vaterite is distinctly different in having a significant component of C???O interactions between carbonate ions, making up 9.1% of the Hirshfeld surface for the polymorph, which appears as a spike in the bottom right hand side of the fingerprint plot 18 , Fig. 1a.
As well as controlling the polymorphs of CaCO 3 , the use of the VFD is also effective in controlling the size and morphology of the particles. A comparison between the morphology using TEM and SEM for samples processed at 45u tilt angle and room temperature using the VFD establishes the ability to also control the shape and size of the calcite crystals, with an average size of approximately 1 mm, Fig. 4a-h. This is in contrast to little control in shape and size for calcite crystals produced using batch processing, Fig. 4i-p.

Discussion
The results establish that at high temperature and high shear (4500 rpm) in Milli-Q water in the VFD results in the selective formation of the most stable polymorph, namely calcite. In contrast, operating the VFD at room temperature and low shear (500 rpm) favours exclusively vaterite, and this is consistent with previous experiments where CaCO 3 produced in a spinning disc processor (SDP) at 500 rpm consists of the vaterite polymorph 32 . (The SDP relates to the VFD in providing a dynamic thin film, but on a disc rather than in a tube.) The high shear forces in the VFD resulting in the formation of calcite are akin to the mechanical energy associated with milling which is capable of inducing a vaterite to calcite transformation 33 .
Replacing the Milli-Q water with seawater in the VFD, however, results in the quantitative formation of vaterite at high shear (4500 rpm), at room temperature and 80uC. Presumably this arises from the high ionic activity of metal ions and carbonate ions in seawater preferentially precipitating CaCO 3 and circumventing the dissolution of vaterite, Eq. 1.
The ability to generate a mixture of aragonite and calcite at high shear (4500 rpm) and in 20% ethanol in the VFD, highlights the delicate balance between the these polymorphs, which is consistent with a relatively small energy difference between their formation energies 34,35 . This relates to similar crystal structures, which are distinctly different to that of vaterite, and this is further highlighted in the Hirshfeld surface analysis.
We have demonstrated the differential formation of crystalline CaCO 3 polymorphs under shear using a vortex fluidic device (VFD), unlike in batch processing. The ratio of Ca 21 to HCO 3 2 is important in controlling the polymorph(s) using batch processing while using the VFD, calcite is obtained selectively at high shear, regardless of the tilt angle and temperature. Selective formation of vaterite using seawater under batch conditions occurs at room temperature and 80uC, and in the VFD at 45u and high shear. In the VFD up to 45% aragonite is accessible in seawater and up to 80% (and 20% calcite) in a mixture of ethanol and water, which is similar to the ratio of the polymorph in molluscan shells. Overall, the variation in shear and temperature in controlling the polymorphs of calcium carbonate has implications in understanding how marine organisms create their shells as conditions in the oceans change. The ability to control the size, shape and morphology of polymorphs of calcium carbonate is also significant, for a range of applications, from paper coatings to catalysis, to drug delivery, to templates for carbonaceous materials. Moreover, such control establishes a precedent for using the device to control the crystallisation outcome of a raft of materials.

Methods
The VFD consists of a 10 mm NMR tube as the reaction chamber with reactants fed into the chamber with controlled flow rates using a peristaltic pump, Fig. 1b. The tilt angle of the VFD can be varied from 0u to 90u and the rotational speed can be varied from 500 rpm to 7000 rpm. A heat jacket equipped with a heat gun can be used to operate reactions above room temperature.
The CaCO 3 was prepared by the reaction of sodium hydrogen carbonate (NaHCO 3 ) and calcium chloride (CaCl 2 ) to give calcium hydrogen carbonate (Eq. 2) which then decomposes to CaCO 3 , H 2 O and CO 2 (Eq. 3). NaHCO 3 was purchased from Optigen Scientific Pty. Ltd. and was of Optigrade TM , and CaCl 2 was purchased from Chem-Supply Pty. Ltd. and was of laboratory reagent quality.  Stock solutions of NaHCO 3 and CaCl 2 were made in Milli-Q water to give a final concentration of 1 M. The reactants were prepared in 151, 152, 251, 153, 351, 1510 and 1051 ratios of HCO 3 2 to Ca 21 . In the batch process, 15 mL of CaCl 2 solution with the target concentration was added drop wise to 15 mL of NaHCO 3 solution with the target concentration in a 50 mL conical flask. For the VFD under continuous flow, Fig. 1b, solutions of NaHCO 3 and CaCl 2 with the target concentrations were fed into the VFD at a flow rate of 1 mL/min using a peristaltic pump with the VFD set to the desired angle and at the desired temperature. The CaCO 3 obtained was filtered, washed with Milli-Q water and left to dry at room temperature.
Full methods and any associated references are available in the online version of the paper at www.nature.com/nature.