Achiral Metastable Crystals of Sodium Chlorate Forming Prior toChiral Crystals in Solution GrowthHiromasa Niinomi,*,†,§ Tomoya Yamazaki,† Shunta Harada,§ Toru Ujihara,§ Hitoshi Miura,†

Yuki Kimura,*,† Takahiro Kuribayashi,† Makio Uwaha,‡ and Katsuo Tsukamoto†

†Department of Earth and Planetary Materials Science, Graduate School of Science, Tohoku University, Aramaki, Aoba, Sendai,980-8578, Japan‡Department of Physics, Graduate School of Science, Nagoya University, Japan, Furo-cho, Chikusa, Nagoya 464-860, Japan§Department of Materials Science and Engineering, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8603, Japan

ABSTRACT: Chiral symmetry breaking in NaClO3 crystallization from an aqueoussolution with perturbations has been of great interest. To understand the mechanism,several models focusing on the early stage of the crystallization have been proposed.However, they are ambiguous because the early stage has been barely explored directly.Here, we investigate the early stages of the crystallization process driven by dropletevaporation using a combination of direct in situ microscopic observations andcryogenic single-crystal XRD experiments. We demonstrate that an achiral crystalhaving P21/a symmetry, which is newly discovered for a solution growth, first appears in the droplet and then transforms into thechiral crystals. Additionally, determination of the lattice constants by XRD experiments (a = 8.42 Å, b = 5.26 Å, c = 6.70 Å, β =109.71°) revealed that the achiral phase should be identical to Phase III (a = 8.78 Å, b = 5.17 Å, c = 6.83 Å, β = 110°), which is ahigh-temperature phase from a melt growth of NaClO3. We advocate further assessment of the achiral crystal and a new pathwayfor the formation of chiral crystals via crystalline phase transition from achiral Phase III.

1. INTRODUCTION

Chiral asymmetry is ubiquitous in nature; it has been observedacross various levels from elementary particle physics to themorphology of winding plants. One example is homochiralitywhere living organisms preferentially select one type from twomirror isomers (enantiomorphs), e.g., L-type amino acids andD-type sugars, as a constituent component. However, theprocess that produces the asymmetric chiral state is not yet fullyunderstood. Therefore, the transition from the chiral symmetricstate, also known as the achiral state, to the chiral state is ofgeneral interest.Recently, reports on chiral symmetry breaking in sodium

chlorate (NaClO3) crystallization from aqueous solution arearousing great interest,1−7 because these are one of a fewexamples of the transition achieved by only abiotic physicalprocesses and offer the possibility of an analogical under-standing of homochirality. NaClO3 molecules are achiral; theyform chiral crystals having cubic symmetry with space groupP213 during crystallization.8 Because the two enantiomorphshave equal thermodynamic stability, when NaClO3 is crystal-lized from a static solution by solvent evaporation, equalproportions of both the enantiomorphs are yielded in eachcrystallization.9 However, Kondepudi and co-workers foundthat only one type from the two enantiomorphs crystallizes ifthe solution is stirred during crystallization.1 It is surprising thatthe resulting chiral asymmetry contradicts the thermodynamicequality between both the enantiomorphs. Moreover, Viedmashowed that a completely asymmetric state can be achieved bygrinding a 50:50 racemic mixture of NaClO3 crystals in asaturated solution.7 These two experiments are sometimes

distinguished by their degree of deviation from the equilibriumstate.10 Whereas the transition from the achiral state to thechiral state in Kondepudi’s experiment should occur undersupersaturation, that in Viedma’s experiment should occurunder near equilibrium because the crystals are already presentin the solution in the initial state. Thus, in this study, wedifferentiate these two processes as chiral symmetry breakingvia nucleation and chiral symmetry breaking via recrystallizationat equilibrium, respectively.Several crystallization experiments of chiral symmetry

breaking via nucleation have been carried out in addition toKondepudi’s experiment.1−5,11−16 Generally, these crystalliza-tions are interpreted using kinetic theory involving a secondarynucleation process.11−20 The single crystal that initiallyappeared because of primary nucleation, called the “Eve”crystal, produces many secondary nuclei when it collides withthe stir bar or is exposed to shear flow. Because the secondarynuclei possess the same handedness as the “Eve” crystal, thehandedness of “Eve” is amplified. In contrast, the oppositehandedness is suppressed by the reduction in the solutionconcentration. Because this secondary nucleation modelqualitatively explains chiral crystallization via nucleation events,it builds a certain level of consensus (except for the process ofchiral symmetry breaking via recrystallization at equilibriumsolution21−23).

Received: April 23, 2013Revised: October 7, 2013

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This model should be inapplicable to some chiralcrystallization experiments by homogeneous nucleation froma supersaturated solution.2−4 For instance, Viedma showed thatstirring a highly supersaturated solution, where primarynucleation should occur at a high rate (σ ∼ 54%, 60 °C ofsupercooling), results in a chiral asymmetric state.2 This resultcontradicts the secondary nucleation model, because the modelassumes the existence of a single “Eve” crystal, and yet such ahigh rate of primary nucleation should significantly decrease thepossibility that the primary nucleation functions as an “Eve”crystal. Thus, chiral crystallization from a highly supersaturatedsolution is often interpreted by deracemization during theprimary nucleation event,2 or even before it, in a period of aprecritical cluster.3,4

Here, we believe that an in situ investigation of chiralcrystallization from a highly supersaturated solution will explainchiral symmetry breaking via nucleation, in contrast to most ofthe previous studies that relied on measurements of theresulting crystals. To confirm the early stage crystallizationprocess involving nucleation, we carried out an in situ directmicroscopic observation of crystallization driven by dropletevaporation and demonstrate a new pathway to chiral crystalformation.

2. CRYSTALLIZATION AND OBSERVATION

All experiments were performed starting with a saturatedsolution prepared using the following procedure. An aqueoussolution of NaClO3 was prepared by dissolving 110 g ofNaClO3 (Analytical grade, Wako Pure Chemical Industries,Ltd., Osaka, Japan) in distilled water (100 mL) at roomtemperature (22 °C). The resulting solution was heated to 30°C under stirring with a magnetic hot plate stirrer and then leftfor a week at room temperature to precipitate the excess soluteand thus to equilibrate the solution. The crystallizationexperiments were performed by pipetting a drop (6 μL) ofthe supernatant saturated solution on a glass slide. The glassslide was placed on a Peltier stage with the temperature set at22 °C. Then, the solution was allowed to crystallize (formingNaClO3 crystals) by evaporation. After approximately 10 min,crystals appeared in the droplet because of nucleation from asupersaturated solution. We observed in situ the process of thiscrystallization with a polarized-light microscope [BX51-P(custom-made); Olympus Corp., Tokyo, Japan] equippedwith a video recording system.

3. RESULTS OF IN SITU OBSERVATION

Figure 1a−d shows time-lapse images of the in situ polarized-light microscopic observation. Figure 1a shows an image takenjust after the nucleation of NaClO3. Nucleation started fromthe droplet fringe 5−10 min after pipetting the droplet on theglass slide. The crystal that first appeared in the dropletexhibited a bright color under the crossed Nicol prism (whentwo polarizers are orthogonally oriented). The needle-shapedbright crystal grew toward the center of the droplet. After a fewseconds of growth, the bright color started to become extinctfrom the fringe (Figure 1b). Then, the extinct region graduallyspread (Figure 1c) along with the bright crystal. Eventually, theentire bright region became extinct within a few minutes.Noncrossed Nicol observation showed that the extinct regionscomprised the crystal (Figure 1d).Because all crystals except for the cubic ones exhibit bright

color under crossed Nicol owing to birefringence, the bright

crystal appearing first is noncubic. In addition, the dark crystalformed after the noncubic crystal is a cubic chiral crystal. Figure1e shows a noncubic crystal observed in a similar experiment,with the perfect shape of a parallelogram (Figure 1e) unlikethat of the chiral cubic crystal (Figure 1f). This observationdemonstrated that a noncubic crystal nucleates prior to theformation of chiral cubic crystals; it transforms into the cubiccrystal, indicating that the noncubic crystal is less stable thanthe chiral cubic crystal. Thus, the noncubic crystal should be ametastable phase.This observation raises a question: whether this trans-

formation is a transition from an achiral state to a chiral state.To answer this question, it is necessary to determine whetherthe crystal is chiral. Therefore, we performed an X-raystructural analysis on the metastable crystal.

4. CRYOGENIC SINGLE-CRYSTAL X-RAY STRUCTURALANALYSIS

It was difficult to conduct a typical X-ray diffraction experimentbecause the metastable crystals are extremely unstable. Toovercome this problem, we preserved the metastable crystalinside the droplet by flash-freezing with liquid nitrogen at−195.8 °C. We constructed a cryogenic single-crystal X-raystructure-analysis system for the frozen droplet. A similartechnique has been used in the analysis of protein crystals.24

Figure 2 shows a schematic overview of the experimental setupthat consists of three systems: (1) the low-temperatureinstrument; (2) the X-ray diffraction system; and (3) thepolarized-light microscopy system.A metastable single crystal was produced upon a glass sheet

mounted on a sample holder of the X-ray diffraction system by

Figure 1. Micrographs of NaClO3 crystals captured by in situobservation using polarized-light microscopy. (a−c) Time-lapsemicrographs of NaClO3 crystallization process driven by evaporationof solvent, under the crossed Nicol prism. (d) Crystallization processobserved under the noncrossed Nicol prism. (e) Micrograph showingthe perfect parallelogram shape of noncubic crystal. (f) Micrographshowing the perfect square shape of cubic crystal. Micrographs (e) and(f) were taken from separate observations.

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means of the droplet-evaporation method described above.When the single crystal had grown to 100−300 μm, thesolution surrounding the crystal was removed using a wipe, andthe crystal was covered with glycerin that acts as acryoprotectant. The glycerin droplet containing the crystalwas instantaneously frozen using liquid nitrogen. The frozensample was then installed into the cryogenic single-crystal X-raystructure-analysis system.A full X-ray diffraction analysis requires the specimen to be

kept as-is throughout the measurement, which means that thedroplet should be kept frozen for several hours. Thisrequirement was achieved by the continuous blowing of low-temperature nitrogen gas generated by a Cryostream system(Oxford Cryosystems, Oxford, U.K.). The temperaturesurrounding the droplet was maintained at −27(±1) °C,which was measured using a K-type thermocouple, throughoutthe measurement. Diffraction data were collected from thesample with an approximately 100 × 80 × 10 μm size by theoscillation method using R-AXIS IV++ (Rigaku Corp., Tokyo,Japan) with an imaging plate. All the oscillation images wereprocessed using the CrystalClear software (Rigaku) todetermine the crystallographic parameters. The experimentalconditions for the X-ray diffraction measurements are listed inTable 1.

5. RESULTS OF X-RAY DIFFRACTION EXPERIMENTThe cryogenic single-crystal X-ray diffraction experimentssuccessfully provided 4314 reflections. Through an analysis ofthe distributions of the diffraction spots, the lattice parametersof the metastable crystal were determined to be a = 8.42(2) Å,b = 5.260(7) Å, c = 6.70(1) Å, β = 109.71(1)°, and V =279.8(8) Å3, where V is the volume of the unit cell. Theseresults indicate a monoclinic symmetry. In addition, three

systematic absence rules were observed in the analytic data setof the X-ray diffraction intensities: h = 2n + 1 in the h0l series, k= 2n + 1 in the 0k0 series, and h = 2n + 1in the h00 series.These results indicate that the space group of the monoclinicphase is P21/a. Figure 3 shows a photograph of the X-ray

oscillation image with a 4° oscillation range, showing asystematic absence rule of h = 2n + 1 in h0l. One shouldnote that crystals with space group P21/a are achiral becausethis space group contains an inversion center as thesymmetrical element. Therefore, the noncubic metastablecrystal is achiral.

6. PHASE DETERMINATION OF NONCUBIC CRYSTALIt has been previously reported that a monoclinic phase namedPhase III occurs in the NaClO3 melt growth.

25 Phase III appearsas a high-temperature phase at 262−237 °C when the melt iscooled down; it transforms into a cubic phase when thetemperature falls below 237 °C.26 The structure of Phase III hasbeen previously characterized as follows: space group, P21/a;lattice parameters, a = 8.78(5) Å, b = 5.17(5) Å, c = 6.83(5) Å,and β = 110(1)°.27 These lattice constants are very close tothose of the metastable phase that we identified in the currentstudy (Table 2). This implies that the metastable phase shouldbe identical to Phase III. However, the differences in the valuesof the lattice constants between Phase III and the metastablephase cannot be explained exclusively in terms of statistical

Figure 2. Schematic experimental setup for cryogenic single-crystal X-ray diffraction: (1) Cryostat system using Cryostream (OxfordCryosystems Inc.), indicated by dotted−dashed blue arrow; (2) X-ray diffraction system, indicated by solid red arrow; and (3) polarized-light microscopy system, indicated by dotted green arrow.

Table 1. Experimental Conditions for X-ray DiffractionMeasurements

analytical method oscillation conditions

radiation Mo Kαwavelength (Å) 0.7107X-ray output (kV, mA) 50, 20collimator size (μm) 300crystal-to-imaging plate distance (mm) 120oscillation range (deg) 360oscillation step range per image (deg) 2exposure time (s) 300

Figure 3. X-ray diffraction image at an oscillation range of 4°. The halois caused by frozen glycerin (amorphous), and diffraction spots arisefrom the metastable phase (crystal). Arrowed spots indicate h0l series.Spots for h = 2n + 1 are absent from the series, indicating a spacegroup of P21/a.

Table 2. Comparison of Lattice Constants of MetastableCrystal Formed by Solution Growth with Those of Phase III,a High-Temperature Phase Formed by Melt Growtha

phasemetastable phase(this study)

metastable phase(calculated values) Phase III28

crystal system monoclinic monoclinic monoclinicspace group P21/a P21/a P21/aa (Å) 8.42(2) 8.54(2) 8.78(5)b (Å) 5.260(7) 5.34(1) 5.17(5)c (Å) 6.70(1) 6.80(1) 6.83(5)β (deg) 109.71(1) 110(1)temp (°C) −27 250 237−262unit cellvolume (Å3)

279.8(8) 292.2(8) 291(4)

aCalculated values are values based on α(T) and β(T).

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error; they might be caused by the difference in temperature.The crystallographic information for the metastable phase wasrecorded at −27 °C, whereas that for Phase III was obtained at237−262 °C. By taking thermal expansion into consideration,we can estimate the volume of the unit cell of the metastablephase at 237−262 °C and compare its value to that of Phase III.Here, for simplicity, we estimated the value of the metastablephase at 250 °C (V250°C) from the following equation

∫

∫

β

α

= +

= +

° − °−

− °−

⎛⎝⎜

⎞⎠⎟

⎛⎝⎜

⎞⎠⎟

V V T T

V T T

1 ( ) d

1 3 ( ) d

250 C 27 C27

250

27 C27

250

(1)

where T is the temperature (°C), β(T) is the volume thermalexpansion coefficient, α(T) is the linear thermal expansioncoefficient, and V−27°C is the volume of the unit cell of themetastable phase at −27 °C. Assuming that (1) the thermalexpansion coefficient of the cubic phase is equal to that of themetastable phase and (2) the unit cell expands isotropically,V250°C was calculated to be 292.2(8) Å3 by substituting thevalue of the linear thermal expansion coefficient of the cubicphase from the previous report.28 These calculated values areequal, within the experimental error, to the volume of the unitcell of Phase III [291(4) Å3].28 Therefore, we concluded thatthe metastable phase of crystallization from solution is identicalto Phase III. Because the noncubic metastable phase is achiral,the crystalline phase transformation observed in the currentstudy is a transition from the achiral state to the chiral state,which means that the chirality of the NaClO3 crystal emergesduring the transition (Figure 4).

7. ROLE OF METASTABLE PHASE IN CHIRALSYMMETRY BREAKING

Most of the chiral symmetry breaking via nucleation can beinterpreted by the secondary nucleation model.1,5,13−15

However, some of them cannot be interpreted by thismodel.2−4 The uninterpretable crystallizations are exclusivelyfrom a highly supersaturated solution, as seen in Viedma’scrystallization experiment in 2004, where the initial super-saturation is ∼54% (ref 2). Note that this experiment isdifferent from chiral symmetry breaking via recrystallization at

equilibrium such as the so-called Viedma deracemization.7,10

The reason why the crystallization from a highly supersaturatedsolution is uninterpretable is the absence of the “Eve” crystalcaused by the high nucleation rate originating from highsupersaturation. However, this is the case when the achiralphase was not taken into consideration. Here, we discuss thepossible contribution of the achiral metastable phase on thechiral symmetry breaking from a highly supersaturated solution.Generally, the solubility of a metastable phase is higher than

that of a stable phase, meaning that the appearance of ametastable phase requires relatively high supersaturation.Therefore, the crystallizations from a highly supersaturatedsolution mentioned above are suitable for the formation of theachiral metastable phase. The nucleation and growth of themetastable crystals decrease the supersaturation of the mothersolution while consuming the solute in the solution. If the ratesof the growth and the nucleation of the metastable phase aresufficiently high relative to the induction time for the nucleationof the chiral phase, the supersaturation in which the nucleationof the chiral crystals occurs should become lower than theinitial supersaturation because the supersaturation decreasesprior to the nucleation of the chiral phase. Consequently, thenucleation rate of chiral crystals becomes lower than thatexpected from the initial supersaturation. Assuming thatformation of the achiral crystals decreases the nucleation rateof chiral crystals such that the secondary nucleation modelbecomes applicable, the model can explain even the chiralsymmetry breaking from a highly supersaturated solution.Additionally, since the metastable phase is achiral, themetastable phase does not have a chiral influence on thenucleation of chiral crystals. In conclusion, the role of theachiral metastable phase is possibly the reduction of the initialsupersaturation. In other words, the achiral metastable phaseacts as a buffer that suppresses the direct nucleation of chiralphase at extremely high supersaturation, which limits theapplication of the secondary nucleation model to the chiralsymmetry breaking from a highly supersaturated solution.Although the achiral metastable phase may not provide a newinsight for the Viedma deracemization, it demands reconsidera-tion of the process of the chiral symmetry breaking vianucleation.To discuss the contribution of the metastable phase on chiral

symmetry breaking more precisely, it would be necessary toassess the solubility of the phase and to observe the phasetransformation from the achiral crystal to chiral crystal in detail.In the future, we will provide the further assessment of theachiral metastable phase.

8. CONCLUSIONWe examined the initial stages of NaClO3 crystallization from ahighly supersaturated aqueous solution in situ using polarized-light microscopy. We for the first time observed that anunknown crystalline metastable phase appeared prior to thenucleation of chiral crystals with cubic symmetry. The crystalsystem of the metastable phase determined by cryogenic single-crystal X-ray diffraction experiments is monoclinic with latticeparameters a = 8.42(2) Å, b = 5.260(7) Å, c = 6.70(1) Å, β =109.71(1)°, and V = 279.8(8) Å3 at −27 °C. The space group isP21/a, which is chirally symmetric. This crystallographicinformation suggests that the metastable phase is identical toPhase III, which is known as a high-temperature phase observedduring the NaClO3 melt growth. When the NaClO3crystallization process follows a pathway through the metastable

Figure 4. New pathway for the formation of chiral NaClO3 crystals.Chiral cubic crystals are formed after nucleation of achiral Phase III.

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phase, the chirality of NaClO3 crystals appears during the phasetransition from the achiral metastable phase to the chiral cubicphase. Taking the achiral metastable phase into consideration,the secondary nucleation model might be applicable even tochiral symmetry breaking via nucleation from a highlysupersaturated solution, which has not been interpretable bythe secondary nucleation model.

■ AUTHOR INFORMATION

Corresponding Authors*E-mail: niinomi37@gmail.com. Tel & Fax: +81-22-795-6661.*E-mail: ykimura@m.tohoku.ac.jp. Tel & Fax: +81-22-795-5903.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We thank J. M. Garcia-Ruiz from the University of Granada,Spain, for helpful comments. We are grateful for the support byGrant-in-Aid for Challenging Exploratory Research. No.23656005 from the Scientific Research of the Ministry ofEducation, Science, and Culture of Japan.

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