pubs.acs.org/crystal Published on Web 12/17/2009 r 2009 American Chemical Society DOI: 10.1021/cg901279t 2010, Vol. 10 963–969 Nucleation and Polymorphism of Calcium Carbonate by a Vapor Diffusion Sitting Drop Crystallization Technique Jaime Gomez-Morales,* Angeles Hernandez-Hernandez, Gen Sazaki, § and Juan Manuel Garcı´a-Ruiz Laboratorio de Estudios Cristalogr aficos, IACT (CSIC-UGR) Ed. Inst. Lopez Neyra. Avda. Conocimiento s/n. P.T. Ciencias de la Salud, 18100 Armilla, Granada, Spain. § Present address: The Institute of Low Temperature Science, Hokkaido University, N19-W8, Kita-ku, Sapporo 060-0819, Japan. Received October 14, 2009; Revised Manuscript Received December 2, 2009 ABSTRACT: The nucleation and polymorphism of calcium carbonate have been studied using a microdevice named the crystallization mushroom. This setup allows carrying out precipitation experiments reproducibly by the vapor diffusion sitting drop technique. Within the range of concentrations investigated (from 10 to 500 mmol/L CaCl 2 and from 1 to 25 mmol/L NH 4 HCO 3 ), the dominant polymorph to appear first in the drops was calcite, or mixtures of calcite and vaterite followed by aragonite. Additionally, amorphous calcium carbonate (ACC) was not observed. The order of appearance of the polymorphs in the droplets is explained by intrinsic features of the crystallization mushroom, that is, the slow increase in the ionic activity product caused by slow diffusion of NH 3 and CO 2 gases, which favors the least soluble phase calcite to crystallize before other more soluble polymorphs. The appearance of calcite as the first nucleating dominant polymorph in the drops allowed us to calculate its surface free energy from induction time measurements assuming the mononuclear nucleation model. The experimentally calculated result of 35 mJ/m 2 is lower than the value predicted for homogeneous nucleation. The cause is the existence of heterogeneous nucleation taking place at the air-solution and solution-support interfaces. 1. Introduction The precipitation of calcium carbonate (CaCO 3 ) plays an important role in a number of fields, such as geology, 1 environmental sciences, 2 and biomineralization, 3,4 in addition to a broad number of applications in industry, where it is used, with a controlled polymorphism, morphology, and crystal size distribution, as a filler or pigment in rubber, paper, plastics, paints, food, etc. 5,6 Moreover, CaCO 3 provides a poly- morphic model system to study nucleation and growth of minerals in classical 7 and nonclassical crystallization. 8 Hence, the precipitation processes of CaCO 3 have been so far in- vestigated by various methods, in various size scales. In the field of biomimetic mineralization of CaCO 3 , most studies employ a vapor diffusion technique based on the decomposition of (NH 4 ) 2 CO 3 or NH 4 HCO 3 into CO 2 and NH 3 gases, which in turn are used as reagents to precipitate CaCO 3 in flasks containing solutions of Ca ions. 9 The whole reaction is carried out in normal laboratory desiccators which are used as isolated environmental chambers. This method has shown some disadvantages such as low reproducibility of the precipitation process, difficulty of monitoring, depen- dency of the precipitation process upon the volumes of the solution and of the desiccator, 10 and a relatively high con- sumption of “tailored additives”. To overcome these disadvantages, our laboratory has developed a device called the “crystallization mushroom”, 11,12 by which precipitation is carried out by slow vapor diffusion using the sitting drop method. The crystallization mushroom is a multidrop set up that enables us (i) to crystallize several samples in the volume range of microliters under different conditions in the same run, (ii) to perform the precipitation with high reproducibility, (iii) to monitor the experiment in situ using an ion-selective electrode or a pH-electrode, and (iv) to observe the evolution of precipitates in situ using any type of optical microscopy and video recording. This micro- method has been successfully employed to study the effect of a number of macromolecules on the precipitation of CaCO 3 : lysozyme, 11 carbonic anhidrase, 13 ribonuclease-A, 14 mioglobin, 14 R-lactalbumin, 14 eggshell matrix proteins fractions, 15 hen uterine fluids proteins, 16 chitosan, 17 and many others. This study aims to clarify the intrinsic features in vapor- diffusion crystallization of CaCO 3 in absence of additives, using a crystallization mushroom. In particular, we have focused our study on the following aspects: (i) the relationship between the rate of increase of the ionic activity product, and also, of the supersaturation reached in droplets, with the type of dominant polymorph of CaCO 3 first observed, (ii) the efficiency of the technique for quantitative studies on nuclea- tion kinetics of this first observed CaCO 3 polymorph by applying the classical theory, and (iii) comparison with results obtained in batch and microbatch precipitation of CaCO 3 . 2. Experimental Section 2.1. Apparatus. All precipitation experiments of CaCO 3 were carried out by vapor diffusion using a crystallization mushroom (Triana Sc. & Tech, S.L.). The set up of the experiment is shown in Figure 1. A crystallization mushroom is composed of two cylind- rical glass chambers and a glass cover. The upper and lower chambers are connected to each other through a hole 6 mm diameter to allow vapor diffusion. In this mushroom, nine polystyrene microbridges (a small plastic block with a shallow well to facilitate sitting drop crystallization) are placed concentrically every 36° inside the upper chamber. Each microbridge holds 40 μL of a CaCl 2 solution. The NH 4 HCO 3 solution is placed in the lower chamber. *To whom correspondence should be addressed. E-mail: [email protected]; tel.: þ34 958 181643; fax: þ34 958 181632.
Transcript
pubs.acs.org/crystalPublished on Web 12/17/2009r 2009 American Chemical Society
DOI: 10.1021/cg901279t
2010, Vol. 10963–969
Nucleation and Polymorphism of Calcium Carbonate by a Vapor
Diffusion Sitting Drop Crystallization Technique
Jaime G�omez-Morales,* �Angeles Hern�andez-Hern�andez,Gen Sazaki,§ and Juan Manuel Garcıa-Ruiz
Laboratorio de Estudios Cristalogr�aficos, IACT (CSIC-UGR) Ed. Inst. L�opez Neyra. Avda.Conocimiento s/n. P.T. Ciencias de la Salud, 18100 Armilla, Granada, Spain. §Present address: TheInstitute of Low Temperature Science, Hokkaido University, N19-W8, Kita-ku, Sapporo 060-0819,Japan.
Received October 14, 2009; Revised Manuscript Received December 2, 2009
ABSTRACT: The nucleation and polymorphism of calcium carbonate have been studied using a microdevice named thecrystallization mushroom. This setup allows carrying out precipitation experiments reproducibly by the vapor diffusion sittingdrop technique. Within the range of concentrations investigated (from 10 to 500 mmol/L CaCl2 and from 1 to 25 mmol/LNH4HCO3), the dominant polymorph to appear first in the drops was calcite, or mixtures of calcite and vaterite followed byaragonite. Additionally, amorphous calcium carbonate (ACC)was not observed. The order of appearance of the polymorphs inthe droplets is explained by intrinsic features of the crystallization mushroom, that is, the slow increase in the ionic activityproduct caused by slow diffusion of NH3 and CO2 gases, which favors the least soluble phase calcite to crystallize before othermore soluble polymorphs. The appearance of calcite as the first nucleating dominant polymorph in the drops allowed us tocalculate its surface free energy from induction time measurements assuming the mononuclear nucleation model. Theexperimentally calculated result of 35 mJ/m2 is lower than the value predicted for homogeneous nucleation. The cause is theexistence of heterogeneous nucleation taking place at the air-solution and solution-support interfaces.
1. Introduction
The precipitation of calcium carbonate (CaCO3) plays animportant role in a number of fields, such as geology,1
environmental sciences,2 and biomineralization,3,4 in additionto a broadnumber of applications in industry,where it is used,witha controlledpolymorphism,morphology, and crystal sizedistribution, as a filler or pigment in rubber, paper, plastics,paints, food, etc.5,6 Moreover, CaCO3 provides a poly-morphic model system to study nucleation and growth ofminerals in classical7 and nonclassical crystallization.8 Hence,the precipitation processes of CaCO3 have been so far in-vestigated by various methods, in various size scales.
In the field of biomimetic mineralization of CaCO3, moststudies employ a vapor diffusion technique based on thedecomposition of (NH4)2CO3 or NH4HCO3 into CO2 andNH3 gases, which in turn are used as reagents to precipitateCaCO3 in flasks containing solutions of Ca ions.9 The wholereaction is carried out in normal laboratory desiccators whichare used as isolated environmental chambers. This methodhas shown some disadvantages such as low reproducibility ofthe precipitation process, difficulty of monitoring, depen-dency of the precipitation process upon the volumes of thesolution and of the desiccator,10 and a relatively high con-sumption of “tailored additives”.
To overcome these disadvantages, our laboratory hasdeveloped a device called the “crystallization mushroom”,11,12
by which precipitation is carried out by slow vapor diffusionusing the sitting drop method. The crystallization mushroomis a multidrop set up that enables us (i) to crystallize severalsamples in the volume range of microliters under different
conditions in the same run, (ii) to perform the precipitationwith high reproducibility, (iii) to monitor the experiment insitu using an ion-selective electrode or a pH-electrode, and(iv) to observe the evolution of precipitates in situ using anytype of optical microscopy and video recording. This micro-method has been successfully employed to study the effectof a number of macromolecules on the precipitation ofCaCO3: lysozyme,11 carbonic anhidrase,13 ribonuclease-A,14
hen uterine fluids proteins,16 chitosan,17 and many others.This study aims to clarify the intrinsic features in vapor-
diffusion crystallization of CaCO3 in absence of additives,using a crystallization mushroom. In particular, we havefocused our study on the following aspects: (i) the relationshipbetween the rate of increase of the ionic activity product, andalso, of the supersaturation reached in droplets, with the typeof dominant polymorph of CaCO3 first observed, (ii) theefficiency of the technique for quantitative studies on nuclea-tion kinetics of this first observed CaCO3 polymorph byapplying the classical theory, and (iii) comparisonwith resultsobtained in batch and microbatch precipitation of CaCO3.
2. Experimental Section
2.1. Apparatus. All precipitation experiments of CaCO3 werecarried out by vapor diffusion using a crystallization mushroom(Triana Sc. & Tech, S.L.). The set up of the experiment is shown inFigure 1. A crystallization mushroom is composed of two cylind-rical glass chambers and a glass cover. The upper and lowerchambers are connected to each other through a hole 6mmdiameterto allow vapor diffusion. In this mushroom, nine polystyrenemicrobridges (a small plastic block with a shallow well to facilitatesitting drop crystallization) are placed concentrically every 36�inside the upper chamber. Each microbridge holds 40 μL of a CaCl2solution. The NH4HCO3 solution is placed in the lower chamber.
*Towhomcorrespondence should be addressed. E-mail: [email protected];tel.: þ34 958 181643; fax: þ34 958 181632.
To monitor the chemistry of the precipitation process we used a pHelectrode (Titan model, Sentron) placed on a hole situated on a sidewall of the upper chamber. This electrode allows us to measure thepH in a 40 μL drop. The glass cover and the upper chamber aresealed with silicon grease.
2.2. CaCO3 Precipitation by CO2 and NH3 Diffusion. Experi-ments were carried out at 20 ( 2 �C, by varying the concentrationsof both CaCl2 andNH4HCO3 solutions from 10 to 500mmol/L andfrom 1 to 25 mmol/L, respectively. Stock solutions were preparedunder N2 atmosphere using analytical grade reagents from Sigmaand deionized water (Elix 3, Millipore). Once the crystallizer wasclosed and sealed, the underlying NH4HCO3 solution released NH3
and CO2 gases into the free space of the crystallizer (0.158 L). Boththe NH3 and CO2 gases redissolved into the droplets of CaCl2solutions, increasing their pH and forming neutral and chargedaqueous species, such asH2CO3*(aq), HCO3
þ. The experiments were finished afterreaching the equilibrium conditions (i.e., the recorded pH reached aplateau and the number of crystals and polymorph composition, asvisualized by optical microscopy, remained unaltered).
2.3. Monitoring of the Precipitation Process and Characterization
of the Crystals Obtained. Each of the precipitation processes wereobserved in situ using an optical microscope (Leica MZ12 orOlympus SZH10) connected to a digital camera (Olympus,C3040ZOOM), or to a video camera (Sony, High ResolutionMTV-3). Observations were carried out at every 15 or 60 minintervals. The time interval was adjusted according to the inductiontime for nucleation at each experimental condition. From theseobservations, the morphology and total number Ntotal of crystalsappearing in each drop and induction time ti, at which onset ofprecipitation was first confirmed, were determined in situ.
At the end of the experiments, themushroomwas opened and theprecipitates were recovered from themicrobridges, rinsed twice withdeionized water, and dried at room temperature. The pH of themother liquor was decreased to 7.0 to prevent further precipitationof carbonate, and residual Ca concentration was measuredby atomic absorption spectrophotometry (AAS, Perkin-Elmer510 model). The crystals obtained were observed by field emissionscanning electron microscopy (FESEM, Gemini-1530). Polymorphof crystals with a rhombohedral habit was determined by X-raydiffraction (XRD, Siemens D5000 Kristalloflex diffractometer).Polymorph of crystals with a rounded shape (a relatively lowfraction) was determined by both X-ray diffraction (XRD) in agrazing incidence mode (1�, 24 h) and Fourier transform infraredspectroscopy (FT-IR, Nicolet 510). Finally, the rest of the crystals(a very small fraction) with a sheaf of wheat morphology wasdispersed in KBr, and polymorph was determined by FT-IR.
2.4. Calculation of Supersaturation. Supersaturation β expressedas the ratio of ionic activity product (IAP) to solubility product Ksp
was calculated by speciation software Visual MINTEQ18 using thefollowing equations:
β ¼ IAP=Ksp ð1Þ
IAP ¼ aðCa2þÞaðCO32-Þ ð2Þ
Ksp ¼ aðCa2þÞeq 3 aðCO32-Þeq ð3Þ
Here, a(Ca2þ) and a(CO32-) show activities of these ions, and
a(Ca2þ)eq and a(CO32-) eq for activities at equilibrium. This software
uses as inputs the temperature, the initial CaCl2 concentration, theinitial pH, the partial pressures of NH3 and CO2 (pNH3, pCO2), andeither the Debye-H€uckel or Davies equations to determine theactivity coefficients, depending on the ionic strength I (I < 0.1 Mor I < 0.5 M, respectively). As outputs, this software provides theactivities of the aqueous species Ca2þ, Ca(NH3)2
2þ, Ca(NH3)2þ,
CaClþ, CaCO30, CaHCO3
þ, CaOHþ, Cl-, Hþ,OH-, CO32-,
HCO3-, H2CO3*(aq), NH3(aq), and NH4
þ, as well as the values ofβ with respect to polymorphs calcite, aragonite, and vaterite.
The calculations of partial pressures pCO2 and pNH3, generatedby gases released from the NH4HCO3 solution to the free-space ofthe crystallizationmushroomaccording to eqs 4-7, were performedby software PHREEQC Interactive 2.8.19
NHþ4ðaqÞ þOH- T NH3ðaqÞ þH2O ð4Þ
NH3ðaqÞ T NH3ðgÞ ð5Þ
HCO-3ðaqÞ þHþ T CO2ðaqÞ þH2O ð6Þ
CO2ðaqÞ T CO2ðgÞ ð7Þ
Therefore, the values of β estimated with this procedure are thenominal supersaturations that could be reached in a droplet bycompletely disregarding the precipitation. During the course ofmost of the experiments, the droplets evolved from undersaturatedto supersaturated, with precipitation starting once the systemexceeded a certain critical supersaturation value.
3. Results and Discussion
3.1. Monitoring of the Precipitation and Determination of
Induction Times. Once the mushroom was closed, the pHstarted to increase as a consequence of the diffusion anddissolution of NH3. As examples, Figure 2, panels a and bshow, respectively, the time evolution of pH in experimentswith CaCl2 concentrations of 10 mM and 200 mM undervarious NH4HCO3 concentrations (2.5-25 mM). As shownin Figure 2a, the rate of increase in pH strongly depends onthe concentration of NH4HCO3 contained in the reservoir.In the case of 2.5 mM NH4HCO3, the increase in pH wasslow enough that the solution remained undersaturatedthroughout the run and no crystal was obtained. However,when the concentration of NH4HCO3 was doubled to 5 mMNH4HCO3, the pH increased up to a plateau of 7.4, and threecrystals were obtained (Table 1). Further increase in theconcentration of NH4HCO3 to 15 and 25 mM resulted in arapid rise in pH and formation of 70 and 80 crystals,respectively. Figure 2b demonstrates that the increase inthe concentration of CaCl2 also quickens the precipitationprocess significantly.
We also monitored the precipitation process of CaCO3 byoptical microscopy. Figure 3 shows a typical time course ofthe in situ observation of a sitting drop. In this experiment, werecorded a frame every 5 min. In the early stage of theprecipitation, the drop was clear (a). With increasing time,precipitates appeared and their size became larger. As indi-cated by a white circle in Figure 3b, we succeeded in identify-ing the precipitate to appear first in the drop. The earliest timeatwhich the first precipitate could be visualizedwas defined asthe induction time ti of nucleation. As shown in Figure 3e,after the precipitates were grown to certain sizes, we coulddifferentiate the morphologies of the precipitates. We couldalso count the total number Ntotal of crystals, after theprecipitation process had finished (Figure 3f).
To make the movie sufficiently clear for the demonstra-tion in this paper (Figure 3), we specially illuminated thisdrop continuously with a halogen lamp throughout the
Figure 1. Set up of a “crystallization mushroom” used for the pre-cipitation of CaCO3 by vapor diffusion using the sitting dropmethod.
precipitation. Hence the temperature of the drop (23 �C)became higher than the other at the room temperature of20 �C (observed by discontinuous illumination), resulting ina shorter induction time (225 min) than that listed in Table 1(400 min) under the same conditions. The discontinuouslighting gave pictures of worse quality, but did not give anydifficulty in identifying the induction time and the phases ofthe precipitates. Therefore, the data summarized in Table 1does not reflect any effect of the rise of temperature shown inFigure 3.
In previous studies in batch experiments,20,21 changes inpH or conductivity of the supersaturated solutions weremainly used to determine the induction times ti in experi-ments initiated by rapid mixing of Ca2þ and CO3
2-/HCO3-
solutions in larger volumes. As summarized in Table 1, tidetermined in this study by visual examination roughlycorresponds to the shoulder of the pH evolution curve.
3.2. Growth Morphology and Polymorph. We basicallyobserved three types of morphologies: precipitates withrhombohedral, sheaf of wheat, and rounded shapes. Wecharacterized these precipitates using either powder XRDor FTIR. The XRD diffractogram of the rhombohedral
crystals clearly exhibited diffraction peaks belonging tocalcite (JCPDS 5-586): the reflections at 2θ = 29.45 (d =3.035 A), 39.45 (d = 2.285 A), 36.00 (2.495 A), and 39.45(d = 2.285 A). The XRD patterns of rounded precipitateswere more noisy than calcite because of smaller amounts ofprecipitates available; however, the diffraction peaks at 2θ=20.85 (d=4.26 A), 2θ=24.85 (d=3.58 A), 2θ=27.00 (d=3.30 A), and 2θ=32.80 (d=2.73 A) coincided with those ofvaterite (JCPDS 13-192). Finally, precipitates with a sheafof wheat morphology presented adsorption bands at1470 cm-1, 1087 cm-1, 856 cm-1, and 712 cm-1 in the FTIRspectrum. These bands likely corresponded to vibrationalmodes of the aragonite mineral,10,22 that is, 1466 cm-1 (υ3),1087 cm-1 (υ1), 866 cm-1 (υ2), and 712 cm
_
1 (υ4).As shown in Figure 4, the FESEMmicrographs show that
calcite crystals appear as rhombohedra bounded by the{104} faces, whereas vaterite is formed as agglomerates oftiny crystals displaying a hexagonal (hexalobulated) rosette-shape morphology. Such agglomerates of vaterite wereprobably formed by the aggregation of nanocrystals via anoriented attachment mechanism.10 Aragonite exhibitsmainly a sheaflike morphology. The formation mechanism
Figure 2. Evolution of the pH in the droplets containing 10mMCaCl2 (a) and 200mMCaCl2 (b), under various concentrations ofNH4HCO3.
Table 1. Summary of Crystallization Conditions, Supersaturation (β), Induction Time (ti) Number, and Percentage (%) of CaCO3 Crystals in Three
Polymorphs: Calcite (C), Vaterite (V), and Aragonite (A)
β no. (% polymorphs)
CaCl2 (mM) NH4HCO3 (mM) pH0 (CaCl2) C V A ti (min) Ntotal C V A
of this type of morphology in CaCO3 and in other systemssuch as Bi2S3 and β-FeO(OH) has been explained by a crystalsplitting growth mechanism.23-25
3.3. Number of Crystals and Polymorph Distribution As a
Function of Supersaturation. In this work, by selecting anappropriate range of crystallization conditions, we obtainedrelatively low numbers of crystals with well-definedmorphologies in a very reproducible way, either among thedifferent drops in the same mushroom or among duplicatedruns of the same conditions (two duplicated runs wereperformed in some cases). The total numberNtotal of crystals
obtained under the same conditions exhibited a maximumdeviation of( 5%. The results of the experiments performedunder a range of CaCl2 and NH4HCO3 concentrations aresummarized in Table. 1. As shown in this table, as thenominal supersaturation βcalcite increases from 10 to 292,so does the Ntotal from 0 to 440 (0-11 crystals/μL).
Under the conditions used in Table 1, calcite was the mostabundant polymorph. Vaterite and aragonite were obtainedin smaller numbers. The number of calcite crystals obtainedfollowed a tendency similar to Ntotal. It varied from 0 to 396with increasing CaCl2 and NH4HCO3 concentrations. How-ever, its percentage became smaller under the middle con-centration range ofNH4HCO3 (5-15mM). The number andpercentage of both aragonite and vaterite crystals did notshow any tendency.
An interesting feature found in the series of experimentswas the “first nucleating polymorph”, which was determinedby in situ optical observation during the experiments, asdemonstrated in Figure 3. In the experiments at 2.5 mMNH4HCO3, calcite was the first and only precipitated poly-morph. In the experiments at 10 mMNH4HCO3 and 20 mMCaCl2 as well as at 25mMNH4HCO3 and 10mMCaCl2 andat 25 mM NH4HCO3 and 200 mM CaCl2, the precipitationof calcite was followed by vaterite and aragonite. In the restof the experiments we could not determine the first nucleat-ing phase with enough accuracy, either because calcite andvaterite appeared almost simultaneously, or because thenumber of crystals that appeared in the drops was too largeto identify individual phases by optical microscopy. In theseexperiments, there was no evidence of the appearance ofamorphous calcium carbonate (ACC).
Furthermore, in order to study the evolution of the poly-morph composition with time, we monitored an experiment(20 mM CaCl2, 10 mM NH4HCO3) for 5 days by opticalmicroscopy. The observations showed that after precipita-tion, the polymorph composition remained constant. How-ever, after 4 days we observed the dissolution of vateritecrystals, which was followed by the dissolution of aragoniteand simultaneous growth of existing calcite crystals.
For the findings of this work, a reasonable thermody-namic explanation can be given considering the intrinsicfeatures of this vapor diffusion method. In experiments witha low NH4HCO3 concentration (2.5 and 5 mM), the partialpressures of released gases NH3 (pNH3) and CO2 ( pCO2)were different but very low, and the diffusion rates of thesegases into the aqueous droplets were slow, particularly thatof NH3, which is reflected by the smooth increase in pH(Figure 2). Because of the slow increase of pH, and thus, ofthe ionic activity product of the solution, the droplets firstreached the critical supersaturation of the least soluble
Figure 4. Field emission scanning electron (FESEM) micrographs of precipitates obtained by the sitting drop method using crystallizationmushroom: (a) calcite, (b) aragonite, (c) vaterite.
Figure 3. A sequence of photomicrographs of the time course ofprecipitation: (a) 70 min, (b) 225 min, (c) 500 min, (d) 1000 min, (e)2000 min, and (f) 2990 min. A white circle in (b) shows the positionwhere a first appeared precipitate could be observed with a veryfaint contrast level. A dotted square in (f ) corresponds to the areashown in (a-e). Scale bars in (a-e) and (f ) show 0.5 and 1 mm,respectively. Initial concentrations of CaCl2 and NH4HCO3 are 20and 10 mM, respectively.
polymorph calcite (log Kspcalcite = -8.453),26 and then
calcite had enough time to nucleate and grow before nuclea-tion and growth of vaterite and aragonite took place. Asthe NH3 and CO2 gases further diffused into the dropscontinuously, the ionic activity product increased and sur-passed the solubility products of aragonite and vaterite(log Ksp
aragonite =-8.306, log Kspvaterite =-7.873),26 which
resulted in the nucleation and growth of these polymorphs.At higher NH4HCO3 concentrations, the diffusions of CO2
and NH3 gases in the drops were faster, the rates of devel-opment of the ionic activity product were higher and thesupersaturation within the drops increased. In these condi-tions, calcite and vaterite appeared sequentially or simulta-neously. Therefore, the crystallization mushroom worksfrom lower to progressively higher supersaturations(bottom up approach). Nevertheless, the critical supersa-turation for the nucleation of ACC was never reached in theseries of experiments.
Kawano et al.27 used a microbatch method by rapidmixing of CaCl2 and Na2CO3 solutions and monitored theprecipitation by opticalmicroscopyThe concentration of themixed solutions was adjusted to be higher than the solubilityof the ACC phase (20 mM/L). They showed that ACCappeared first, and then vaterite and calcite nucleated andgrew by consuming the amorphous phase precipitated pre-viously and creating around the crystals a precipitation freezone. They also reported that in some cases calcite appearedbefore vaterite. In order to observe the precipitation of ACCparticles as the primary precipitated phase by a vapordiffusion technique, it has been necessary to use a rectangu-lar quartz cell in which we have placed two drops containingthe reagents, and to increase largely the concentrations to1 M CaCl2 and 1 M NH4HCO3. This experiment behavedlike the microbatch experiment of Kawano et al.27 Figure 5shows pictures of ACC particles obtained by optical micro-scopy, as well as vaterite and calcite crystals creating adissolution zone around them. It is unclear whether calcitenuclei are heterogeneous nuclei forming on existing ACCparticles or form spontaneously, but what is clearly shown isthat growth of calcite or vaterite takes place at the expense ofACC dissolution.
Another very interesting result is the appearance of ara-gonite (a high temperature polymorph) at 20 �C, in theabsence of any additive such asMg2þ, SO4
2-, or any externalfactor such as a magnetic field. This would require a furtherstudy in itself. In fact, to the best of our knowledge, thepresence of aragonite has not been reported in pure CaCO3
systems where the supersaturation is obtained by directmixing of calcium and carbonate/bicarbonate solutions at20 �C. The minimum reported temperature for aragonite
formation was 24 �C (pH > 9)28 or 26 �C,29 with optimaltemperature for maximum relative abundance above 60 �C.The authors suspect that very small fractions of aragonitecrystals in mixtures of CaCO3 polymorphs obtained in largesolution volumes could not be identified because of thedetection limit of powder XRD. Therefore, in these casesthe use of a micromethod can help us to better define theprecipitation range of a polymorphwhen studying the effectsof certain operating variables.
3.4. Nucleation Kinetics. We have succeeded in observingthe nucleation of calcite as the first nucleating phase byoptical microscopy. Hence we analyzed the induction time tias a function of supersaturation β of calcite, and attemptedto evaluate surface free energy of calcite. The induction timeti includes three time periods: relaxation, nucleation, andgrowth time. The relaxation time is the time the clusterdistribution needs to respond to the imposed supersatura-tion, whereas the nucleation and growth times are the timeneeded to the formation of stable nuclei and the time for theirsubsequent growth to observable size, respectively.
Kashchiev et al. reported a general equation for thecalculation of the induction time that takes into accountboth the mononuclear (MN) and polynuclear (PN) mechan-isms.30 When nucleation is dominated by the MN or PNmechanism, the relationship between ti and β can be respec-tively expressed as follows:
ln ti � B=ln2 β ðthe case of MNÞ ð8Þ
lnðtiβ1=8ðβ1=2 -1Þ6=4Þ�B=ð4 3 ln2 βÞ ðthe case of PN :
spiral growth is assumedÞ ð9Þ
with B ¼ 16πσ3Ω2
ðkTÞ3 ð10Þ
Here, σ is the surface free energy of the nuclei, Ω is themolecular volume (6.13 � 10-29 m3 for calcite,31 k is theBoltzmann constant, and T is the absolute temperature.
Figure 6a shows the plot of ti vs β obtained experimentallyin this study. To evaluate the MN and PN mechanisms, thedata were replotted according to eqs 8 and 9, as shown inFigure 6, panels b and c, respectively. In the case of the PNmechanism, we assumed the parabolic rate law of calcitecrystals according to several previous reports.32-35 Theexperiments carried out showed that calcite was the firstpolymorph to appear except in the experiments wherecalcite and vaterite appeared at the same time, or its deter-mination was ambiguous. Taking these results into account,to obtain these plots we have employed the supersaturationβ expressed with respect to the polymorph calcite.
The correlation coefficients corresponding to Figure 6,panels b and c are 0.92 and 0.47, respectively. The better fit ofFigure 6b indicates that in our nucleation experiments usingthe micromethod (drops of 40 μL) the MN mechanismprevail, although the correlation coefficient obtained (0.92)was not very good. This result roughly agrees with thehypothesis of Verdoes et al.,36 who assumed that the MNmechanism operates only when volume of a solution phase issmaller than 10 μL. This is the first experimental evidencethat confirm this hypothesis. In theMNmechanism, the timeof nucleation dominates ti, and thus, the use of eq 8 islegitimate. From the slope of the straight line of Figure 6b,we obtained the surface free energy of calcite σ=35 mJ/m2.
Figure 5. Optical microscopy pictures of a droplet containing(a) ACC, and (b) ACC transforming in vaterite and calcite crystals.The experiment was carried out by vapor diffusion using a closedrectangular quartz cell with two drops containing 1 mol/L CaCl2and 1 mol/L NH4HCO3.
The induction time determined in this study is longer thanthe time of nucleation, because of the importance that therelaxation time have, in particular under low supersatura-tion. Therefore, the value of σ=35mJ/m2 shows the largestlimit of the surface free energy of calcite; that is, real valuewould be smaller than this value.
Concerning the surface free energy of calcite, S€ohnel andMullin reported σ=83 and 98mJ/m2 using batch reactors,37
although these values were considered intermediate betweenthose of calcite and vaterite. They also reported a theoreticalvalue of 120 mJ/m2.38 Other reported values using differentexperimental conditions in batch reactors show a big scatter(7,39 19.5,40 205,41 28042 mJ/m2). The value of σ obtained inthis study is comparable to that of Westin and Rasmuson43
(37.8 mJ/m2), which is lower than that predicted for homo-geneous nucleation37 and indicates that some degree ofheterogeneous nucleation occurred in the drops consideringthat the use of eq 8 is legitimate. After analyzing themovie ofFigure 3, we have observed the first precipitated crystals atthe outer surface or border of the drops, followed by crystalsin the center, indicating a supersaturation gradient. Aftercareful analysis, we have identified the air-solution interfaceof the drops and the surface of themicrobridges as the sourceof heterogeneous nucleation. Only the use of levitated dro-plets44 would permit a contact free crystallization. However,even using levitated droplets the solution/air interface wouldcontinue to be a source of heterogeneous nucleation.
At present, accurate evaluation of the surface free energyof calcite from induction time measurements remains adifficult subject. Potential sources of errors in the calculationof σ include those associated to the specific techniqueemployed to measure the induction times, the impossibilityto eliminate completely the heterogeneous nucleation, andperhaps the most important, in batch and microbatch pre-cipitation of CaCO3 the nucleation of ACC and/or vateritenormally precedes the nucleation of calcite.
Thus, we can highlight that the crystallization mushroomoffer several advantages to carry out kinetic studies ofcalcite: (i) it favors the nucleation of calcite as the primaryphase, (ii) it requires very small volumes thereby ensuring amononuclear mechanism, (iii) it allows easy in situ observa-tion and identification of the first nucleating phase, and (iv) itaffords high reproducibility of the precipitation processes.Therefore, we believe that this micromethod should becomea useful tool for the study of the crystallization of a widevariety of minerals in the near future.
4. Conclusions
The precipitation of CaCO3 by the vapor diffusion sittingdrop technique has been studied at 20 ( 2 �C using thecrystallization mushroom. The process has been monitoredby optical microscopy, video microscopy, and by recordingthe pH versus time. Within the range of concentrationsinvestigated (from 10 to 500 mmol/L CaCl2 and from 1 to25 mmol/L NH4HCO3), the dominant polymorph to appearfirst in the dropswas calcite, ormixtures of calcite and vateritefollowed by aragonite. ACCwas not observed. These findingsare explained by the slow diffusion of NH3 and CO2 gasesfrom the chamber containing NH4HCO3 to the chambercontaining droplets of CaCl2 solution. The gas diffusionwithin the crystallizationmushroom cell produces an increasein the ionic activity product that is slow enough to allow thenucleation of the least soluble polymorph calcite, as the firstnucleating polymorph. It was necessary to use another set up,a closed rectangular quartz cell composed of only one cham-ber, and to largely increase the concentrations of bothreagents to 1 mol/L in the droplets to observe the appearanceof ACC. The appearance of calcite as the first nucleatingdominant polymorph in the drops allowed us to calculate thesurface free energy of calcite from induction time measure-ments assuming themononuclearmodel. The calculated value
Figure 6. (a) Plot ti vs β, (b) test of the MN mechanism according to eq 8, (c) test of the PN mechanism according to eq 9.
of the surface free energy of calcite (35 mJ/m2) is lower thanthat predicted for homogeneous nucleation due to the exist-ence of heterogeneous nucleation taking place at the air-solution and solution-support interfaces.
Acknowledgment. This work has been supported byMAT2006/11701 of the Spanish Ministry of Science andInnovation, by PIE200630l133 of the Spanish CSIC and bythe Excellence Project RNM5384 of the Junta de Andalucia.A.H.H., J.G.M., and J.M.G.R. belong to the research team“Factorıa de Cristalizaci�on” (Consolider Ingenio 2010). Wealso thank Angel Justo for FTIR andXRDanalysis, Dr. LuisDavid Pati~no Lopez for image treatment, and Dr. AlfonsoGarcıa Caballero for English revision.
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