Structural Characterization of Mg-Stabilized Amorphous Calcium

Mar 10, 2015 - Biogenic amorphous calcium carbonates (ACCs) play a crucial role in the mineralization process of calcareous tissue. Most biogenic ACCs...
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Structural Characterization of Mg-Stabilized Amorphous Calcium Carbonate by Mg-25 Solid-State NMR Spectroscopy Cang-Jie Lin,† Sheng-Yu Yang,† Shing-Jong Huang,*,‡ and Jerry C. C. Chan*,† †

Department of Chemistry and ‡Instrumentation Center, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan S Supporting Information *

ABSTRACT: Biogenic amorphous calcium carbonates (ACCs) play a crucial role in the mineralization process of calcareous tissue. Most biogenic ACCs contain Mg ions, but the coordination environment of Mg, which may influence the kinetics of the phase transformation of an ACC, remains poorly understood. We demonstrate that Mg-25 solid-state NMR can be used to probe the coordination shells of Mg in synthetic ACCs. The variation in Mg25 chemical shifts suggests that Mg−O bond lengths increase as Mg content increases. On the basis of the Van Vleck second moments obtained from the double-resonance NMR experiments, we infer that the average number of carbonates surrounding the central Mg ion is in the range of 4−4.5 and that there is at least one water molecule coordinated to each Mg ion for the synthetic Mg-ACC samples. We suggest that the stability of Mg-ACC is owing to the structural water bound to Mg ions, which increases considerably the activation energy associated with the dehydration of Mg-ACC.

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INTRODUCTION Calcite and aragonite are polymorphs of calcium carbonate that are widely present in biominerals.1,2 Previous studies have indicated that amorphous calcium carbonate (ACC) is a precursor of biogenic calcareous materials and a temporary storage site of calcium carbonate in certain organisms.3 A distinct short-range order observed in the biogenic ACC of different species and phyla has led to the conclusion that biogenic ACC has an intrinsic structural order that is actively controlled in these organisms.3 The term “CaCO3 polyamorphism” was coined to describe recent findings regarding the role of ACC in biomineralization.4 Because most biogenic ACCs contain Mg, and the distribution of Mg is specific in different species and tissues,5 the structural order in the vicinity of Mg ions may be associated with the stability of biogenic ACC. The most commonly used methods for identifying the cationic species in biominerals are the X-ray absorption fine structure (EXAFS) and the X-ray absorption near-edge structure (XANES) techniques.3 Although X-ray absorption spectroscopy (XAS) has been successfully applied in characterizing the short-range order of Ca in biominerals, conducting XAS measurements in the regime of Mg K-edge is technically challenging.6 In recent studies, high-energy X-ray total scattering has been used to obtain atomic pair distribution functions (PDFs) for synthetic7,8 and biogenic ACC.9 Although valuable structural information regarding the first-neighbor coordination around carbon and calcium can be obtained from the PDFs, discerning the structural order of Mg is difficult, especially when Mg is present as a minor component.9 To date, the structural difference in Mg-ACC samples containing various © 2015 American Chemical Society

amounts of Mg remains poorly understood. To clarify the functional role of Mg in biogenic ACC, it is crucial to develop a research strategy that can directly characterize the Mg coordination environment. In this study, we demonstrated that 25Mg solid-state NMR spectroscopy can be used to study the structural order of Mg ions in synthetic Mg-ACC. By conducting 25Mg{13C} and 25 Mg{1H} double-resonance experiments, we successfully obtained the 25Mg/13C and 25Mg/1H Van Vleck second moments for our Mg-ACC samples containing various amounts of Mg. From the second-moment data, which were used to quantify the strength of the magnetic dipole−dipole interaction between the central 25Mg nucleus and the surrounding nuclei, the coordination shells of Mg were elucidated. We suggest that the stability of Mg-ACC is owing to the structural water bound to Mg ions, which increases considerably the activation energy associated with the dehydration of Mg-ACC.

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RESULTS Characterization of Mg-ACC. A series of Mg-ACC samples were prepared by mixing solutions of CaCl2/MgCl2 and Na2CO3 in a microreactor with a volume of 1.2 μL. The samples are henceforth referred to as MgXACC, where X is the molar Mg/Ca ratio of the mother liquor. The sample of amorphous magnesium carbonate (AMC) was prepared similarly. All the characterization data obtained using powder Received: December 29, 2014 Revised: March 10, 2015 Published: March 10, 2015 7225

DOI: 10.1021/jp512971a J. Phys. Chem. C 2015, 119, 7225−7233

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The Journal of Physical Chemistry C

Structural Characterization by NMR. The Mg content of ACC could have a substantial effect on the crystallization kinetics.3,12 Therefore, determining whether the molecular structures of high-Mg and low-Mg ACC exhibit any differences is an important issue. For the solid-sate NMR measurements, the amorphous samples were enriched in 13C and 25Mg. The 13 C NMR spectra of Mg0.6ACC and Mg5ACC contained a single symmetrical broad peak (line width at half-maximum ≈4 ppm) positioned at 168.3 and 167.7 ppm, respectively (Figure S9). As discussed by Gebauer et al.,13 the structural environment of the carbonate species resembled the local environment of carbonate in calcite, which produces a 13C NMR signal at 168.7 ppm. Figure 2 illustrates the 25Mg Bloch

X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, and transmission electron microscopy (TEM) were consistent with those reported for ACC (Figures S1−S3). The Mg content, defined as Mg/(Mg + Ca), of the samples was determined by inductively coupled plasma mass spectrometry (ICP-MS) and energy-dispersive X-ray spectroscopy (EDX). The ICP-MS and EDX results are presented in Figure 1.

Figure 1. Plot of the Mg content of the MgXACC samples determined using EDX and ICP-MS. The numbers next to the symbols indicate the X of the sample. The dashed line denotes perfect agreement.

Because the ICP-MS data reflected the average Mg amount of the entire sample batch and the EDX data revealed the Mg content of a micrometer-sized area, the favorable agreement between the ICP-MS and EDX data implies that the distribution of Mg ions did not exhibit any heterogeneity at the micrometer scale. We found no evidence for the presence of Mg(OH)2 neither in crystalline form nor in an amorphous state. Furthermore, the Mg amount incorporated into the sample was correlated to the Mg/Ca ratio of the mother liquor (Figures S4 and S5). The Mg content of the sample prepared from the solution with a similar Mg/Ca ratio to that of seawater (Mg/Ca = 5) can reach 42%. Based on the Mg/Ca ratio and the weight loss of the samples (Figures S6−S8), the molecular formulas of the Mg0.6ACC, Mg5ACC, and AMC samples were determined (Table 1). The hydration levels of the samples are Table 1. Summary of the TGA Data Obtained for the Amorphous Samples

samples

weight loss due to loosely bound watera (%)

weight loss due to tightly bound waterb (%)

Mg/Ca ratio determined by ICP-MS

Mg0.6ACC

6.0

4.0

0.20

Mg5ACC

9.0

4.6

0.78

11.3

5.0

AMC

Figure 2. 25Mg MAS spectra acquired for (a) magnesite and (b) Mg5ACC at a spin rate of 10 kHz. For the spectrum of Mg5ACC, the shoulder peaks flanking the central transition (indicated by arrows) are caused by satellite transitions.

water per formula unitc Mg0.17Ca0.83CO3· 0.60H2O Mg0.44Ca0.56CO3· 0.81H2O MgCO3·0.91H2O

decay (direct excitation) spectra acquired for Mg5ACC and magnesite under magic-angle spinning (MAS). On the basis of the distinctive spectral features of magnesite, we extracted the 25 Mg NMR parameters, the isotropic chemical shift (δcs), and second-order quadrupolar effect (χQ), by conducting line shape analysis. The obtained results were consistent with data that have been reported in relevant literature.14 The χQ is sensitive to the electric field gradient at the nuclear site, whereas the chemical shift is sensitive to the chemical environment, such as the coordination number of the probe nucleus. Although the 25 Mg signals of MgXACC and AMC are featureless, the NMR parameters had been extracted by analyzing the centers of gravities of the spectra acquired at two magnetic fields (Figure S10 and Table S1). Table 2 summarizes the 25Mg NMR data obtained for Mg0.6ACC, Mg5ACC, AMC, and magnesite. The

Weight loss from ambient temperature to 100 °C. bWeight loss from 100 to 200 °C. cCalculated based on the total water content. a

largely consistent with those reported for synthetic ACC.10 The weight loss below 100 °C is mainly due to the physisorbed water, but a recent study suggests that this low-temperature weight loss also has non-negligible contribution from structural water.11 Therefore, it is difficult to determine accurately the amount of structural water per formula unit based on the TGA data. 7226

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The Journal of Physical Chemistry C Table 2. Summary of the 25Mg NMR Parameters Obtained for the Amorphous and Crystalline Samples samples Mg0.6ACC Mg5ACC AMC MgCO3, magnesite Mg(NO3)2·6H2O

χQa (MHz) 2.8 2.7 2.5 2.25 1.496

± ± ± ± ±

0.2 0.2 0.2 0.05 0.020b

δcs (ppm) −26.9 −29.3 −32.6 −3.7 0.18

± ± ± ± ±

0.5 0.5 0.5 0.2 0.05b

MMg‑C × 104 (rad2 s−2) 2

aMg‑H × 105 (s−2)

MMg‑H × 105 (rad2 s−2) 2

± ± ± ±

15.0 ± 0.6 19.8 ± 0.7 18.6 ± 0.5 28.6c,d

2.0 ± 0.3 2.1 ± 0.3 1.65 ± 0.05

57 ± 9 60 ± 10 47.3 ± 3.1

4.97 ± 0.32

142.6d,e

1.09 1.44 1.35 2.08

0.04 0.05 0.03 0.05

χQ is defined as the product of e qQ/h and (1 + where e qQ/h and ηQ correspond to the nuclear quadrupole coupling constant and the asymmetry parameter of the electric field gradient, respectively. bTaken from ref 14. cStructure taken from ref 24. dCalculated for a distance of up to 20 Å. eStructure taken from ref 60. a

2

ηQ2/3)1/2,

aMg‑C × 104 (s−2)

2

χQ and chemical shift data of MgXACC and AMC were clearly distinct from those of magnesite. In other words, the firstcoordination shells of Mg in our amorphous samples, which is defined by the Mg−O bond length, O−Mg−O bond angles, and the number of nearest oxygen neighbors, were undoubtedly distinct from that in magnesite. According to the χQ data, we concluded that the charge distribution at the nuclear site of 25 Mg was less symmetric than that of magnesite. These results were consistent with the finding that the first-coordination shell of Mg ions in biogenic ACC is distorted compared to that in crystalline polymorphs.6 We note in passing that the attempt to acquire the 43Ca MAS spectrum of Mg0.6ACC was not successful. As demonstrated in glass research, the Van Vleck second moment (M2) is a useful parameter for characterizing structural arrangement in amorphous systems.15 For a spin system containing rare spins (S) and abundant spins (I), the heteronuclear magnetic dipole−dipole interaction between S and I can be characterized by the second moment, defined as follows:16 M 2S − I =

2 4 ⎛ μ0 ⎞ 2 2 2 −6 ⎜ ⎟ γ γ ℏ I (I + 1) ∑ r j 15 ⎝ 4π ⎠ I S j

Figure 3. 25Mg{13C} REDOR data acquired for magnesite (■) and AMC (□). The dashed line represents the parabolic fit according to eq second moment can be 2 in the main text, by which the MMg‑C 2 obtained.

parabola to the data of ΔS/S0 ≤ 0.2, we can extract the MMg‑C 2 value according to the following equations:19,20

(1)

ΔS = a Mg‐Ct 2 S0

where S is the center nucleus, I is the spin quantum number of the surrounding I nuclei, γS is the gyromagnetic ratio of spin S, and rj is the distance between S and the jth nucleus I. The other symbols retain their conventional meanings. Basically, the Van Vleck second moment represents the extent of the interaction between two spin species. Because of the inverse sixth-power distance dependence, the MS−I 2 value is primarily determined by the nearest neighbors of the probe nucleus. Experimentally, the corresponding MMg‑C data can be obtained by performing 2 rotational echo double resonance (REDOR) experiments.17 REDOR is a well-established solid-state NMR technique used to determine the heteronuclear magnetic dipole−dipole interaction under MAS conditions.18 Briefly, we first acquired a reference 25Mg spectrum of the sample by a rotorsynchronized spin-echo sequence, in which the heteronuclear dipolar coupling was averaged out using MAS. We repeated the measurement in the presence of a judiciously designed rf pulse train in the 13C channel, which reintroduced dipolar coupling and hence diminished the signal.17 Because of the signaldiminishing effect, also referred to as signal dephasing, we could quantify the heteronuclear dipolar interaction by calculating the intensity difference between the reference signal (S0) and the diminished signals (S), ΔS = S0 − S. By convention, the REDOR fractions were calculated as ΔS/S0. The REDOR data acquired for magnesite are presented in Figure 3. The corresponding raw data are shown in Figure S11. By fitting a

(2)

and a Mg‐C = f

4 M Mg‐C 2 2 3π

(3)

where a is obtained by a parabolic fit to the initial regime of the REDOR fractions, t is the REDOR dephasing time, and f is a scaling factor accounting for the effects of various experimental artifacts, such as the rf inhomogeneity and the imperfect selective excitation of the central transitions.21 The scaling factor f, obtained by the measurement of a model compound with known geometry, can be used to obtain accurate M2 data of the amorphous systems that exhibit similar chemical composition and spin dynamics to the model compound, provided that all the NMR experiments are conducted under the same conditions.20 In this study, the 13C chemical shift anisotropies of the carbonate ions in magnesite and Mg-ACC were expected to be similar.22,23 The use of proton decoupling during the REDOR dephasing period alleviated the concerns of the variation in water content among the samples. Because the 25Mg nuclei of the model compound and the samples have comparable quadrupolar interactions, the excitation efficiency of their central transitions are about the same. Therefore, the effects of experimental imperfection should be similar for magnesite and Mg−C

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The Journal of Physical Chemistry C Mg-ACC. The scaling factor f was determined to be 0.538 ± 0.015 based on magnesite, whose structural data are known.24 The same scaling factor was used to extract the MMg‑C of the 2 amorphous samples. Table 2 summarizes the MMg‑C data of our 2 amorphous samples, which were substantially smaller than that of magnesite. Thus, the number of carbonate ions surrounding each Mg ions should be less than six (vide inf ra). Typical REDOR spectra are shown in Figures S12−S14. The MMg‑C of 2 Mg5ACC and AMC were larger than that of Mg0.6ACC. This data trend reflected a structural reorganization of the carbonate species surrounding the Mg ions upon an increase in the Mg content. Although REDOR is well suited for the characterization of MMg‑C , it is in general not applicable for the determination of 2 MMg‑H when the magnetic dipole−dipole interaction among the 2 1 H spins is significantly larger than that between 25Mg and 1H. Indeed, experimentally we found that the 25Mg{1H} REDOR fractions of AMC had significant scattering (data not shown). To alleviate this problem, we employed a variant of REDOR, viz. C-REDOR, which can suppress selectively the homonuclear dipolar interaction when the spinning frequency is sufficiently large.25,26 Figure 4 shows the 25Mg{1H} C-REDOR data

Figure 5. 25Mg{1H} C-REDOR curve acquired for AMC. Because of the spin−spin relaxation, the last data point has the least signal-tonoise ratio, resulting in having the largest error bar.

0.01. From these C-REDOR fractions, the aMg‑H and MMg‑H 2 data of Mg0.6ACC and Mg5ACC were estimated on the basis of eqs 2 and 4 (Table 2). Typical C-REDOR spectra are shown in Figures S15−S19.

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DISCUSSION Mg Content in Mg-ACC. The speciation of Mg in biogenic calcium carbonates is of paramount importance to understanding their mineralization process. The levels of Mg in numerous biogenic calcareous minerals are substantially higher than those of their counterparts prepared in vitro.3 The Mgpromoting effect of various organic additives, such as acidic polymers, has been reported in many in vitro studies of high-Mg calcite.27−29 Recently, Mg-ACC has been proposed as the key precursor of high-Mg calcite.30,31 To test the hypothesis that the Mg content in biogenic ACC is regulated by acidic proteins, the effect of simple carboxylic acids on the incorporation of Mg into ACC was systematically investigated.32 It has been found that the Mg content of the Mg-ACC samples prepared in a mother liquor of Mg/Ca ratio equal to 5.0 could be increased from 31% (without any additives) to more than 50% with the addition of aspartic or glutamic acids. Because the Mg content in biogenic calcite can reach up to 50%, researchers have suggested that biomolecules play a role in regulating the Mg composition.32 In general, the formation of Mg-ACC is a kinetically controlled process. The method of sample preparation may have affected the Mg content. Wang et al. prepared their samples by sealing the CaCl2 and MgCl2 solution in an atmosphere of ammonia and carbon dioxide.32 Although this gas-diffusion method can presumably produce samples with homogeneous compositions, its biological relevance requires further investigation. In the present study, the MgXACC samples were prepared by directly mixing the mother liquors in a microreactor. Our approach is not necessarily more effective in mimicking the mineralization process of ACC in vivo; nevertheless, the Mg content of the MgXACC samples was comparable to those observed in biogenic ACC, without using any organic additives. Additional experiments demonstrated that the amount of Mg incorporated in MgXACC can be varied at different pH conditions. This finding is consistent with what

Figure 4. Mg{1H} C-REDOR dephasing data acquired for Mg(NO3)2· 6H2O (■), AMC (□), and Mg5ACC (△).

acquired for Mg(NO3)2·6H2O and AMC at a MAS frequency of 40 kHz. The corresponding MMg‑H values can be extracted 2 from the parabolic fitting of the data as follows:26 a Mg‐H = f

16 M 2Mg‐H 27π 2

(4)

As described above, the scaling factor f was determined to be 0.580 ± 0.041, from which the MMg‑H of AMC was estimated 2 (Table 2). Furthermore, the full C-REDOR curve of AMC was obtained (Figure 5). Because the 25Mg{1H} C-REDOR fractions of AMC approached unity as the dephasing time increased, it can be unequivocally concluded that all Mg ions were in close proximity to water molecules. The same strategy of fitting the initial C-REDOR curve could not be applied for the samples of Mg0.6ACC and Mg5ACC because of their very limited 25Mg NMR sensitivity. Therefore, we chose to characterize a single C-REDOR fraction at the dephasing time of 1.05 ms.26 As a result, the ΔS/S0 of Mg0.6ACC and Mg5ACC were found to be 0.21 ± 0.03 and 0.22 ± 0.03, respectively. The corresponding fraction of AMC is 0.17 ± 7228

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The Journal of Physical Chemistry C has been reported for the Mg-ACC samples prepared by direct mixing in a reactor with a volume of 26 mL.33 Although the hypothesis that the Mg uptake in biogenic ACC is influenced by macromolecules is of interest, the results of our study indicated that organisms may regulate the incorporation of Mg merely through pH manipulation, provided that biogenic ACC is formed in a sufficiently confined space.34 A systematic study was recently conducted to study the energetic and structural aspects of the amorphous carbonates with a composition of MgxCa1−xCO3·nH2O.12 Samples for which x = 0.0−0.47 formed a homogeneous amorphous phase, whereas those in the region x ≥ 0.47 comprised a mixture of AMC with another amorphous phase. Consistently, the ICPMS and EDX data revealed that Mg0.6ACC (x = 0.17) and Mg5ACC (x = 0.42) were homogeneous in Mg distribution. On the basis of the 43Ca NMR chemical shifts of Mg-ACC, Singer et al. reported that the mean Ca−O bond length was not substantially influenced by the presence of Mg ions for x ≤ 0.54.35 43Ca chemical shifts exhibit a strong correlation with the mean Ca−O bond lengths of their nearest neighbors, in which the chemical shift becomes more shielded as the mean bond distance increases.35−37 A similar correlation between 25Mg chemical shifts and the mean Mg−O bond lengths was, therefore, expected. As shown in Table 2, the data trend of 25 Mg chemical shifts suggests that the average Mg−O bond length of its nearest neighbors increased as the percentage of Mg increased from Mg0.6ACC to AMC. Number of Carbonate Ions Coordinated to Mg. One of the greatest challenges in the study of biogenic ACC is the lack of a structural model describing how nucleation and phase transformation occur at the molecular level. Because of the considerable progress of the atomistic simulations of ACC,38,39,8,40−42,35 it is crucial to characterize the structural order at various length scales using experimental observables. XAS is an effective technique for characterizing the first coordination shell of Ca and Mg in biogenic ACC.6,43 The PDFs obtained from the X-ray scattering data can also provide a quantitative measure of the first coordination shell of Ca in synthetic or biogenic ACC,8,35 yet are less useful for determining the length scale of, and beyond, the second coordination shell.7 From an atomistic structural model of ACC, it is straightforward to calculate various Van Vleck second and MCa‑C . In principle, each of these moments such as MMg‑C 2 2 second moments can be individually determined using solidstate NMR. Therefore, we believe that Van Vleck second moments could serve as useful structural parameters for the study of ACC. In this study, we demonstrated that 25Mg{13C} REDOR , from which NMR can yield an experimental measure of MMg‑C 2 one may estimate the number of carbonate groups surrounding each Mg2+ ion. As an illustration, the MMg‑C of hydromagnesite 2 were calculated based on its crystal structure,44 taking into account the carbonate groups in the coordination shell of Mg only. There are three crystallographically nonequivalent Mg sites in hydromagnesite, viz., Mg1, Mg2, and Mg11. All of their coordination shells comprise four carbonates groups. Because the MMg‑C values of hydromagnesite calculated for the four 2 closest Mg−C distances are approximately 90% of the values calculated for a distance of up to 20 Å, we scaled down the experimental MMg‑C values of Mg0.6ACC, Mg5ACC, and AMC 2 by 10% accordingly. As shown in Figure 6, the MMg‑C data of 2 of Mg1 and Mg11, Mg0.6ACC are similar to the MMg‑C 2 suggesting that there are four carbonate groups surrounding

Figure 6. Experimental MMg‑C determined for the amorphous samples 2 (multiplied by a factor of 0.90). The three solid lines indicate the values calculated based on the crystal structure of hydroMMg‑C 2 magnesite, taking into account the four shortest Mg−C distances only. Mg1, Mg2, and Mg11 denote the three nonequivalent sites of Mg. The values based on the three dashed lines indicate the projected MMg‑C 2 second coordination shell of Ca2+ ions in lobster carapace (LC), plant cystoliths (PC), and ascidian spicule (AS). The number after the dash represents the number of carbonate group coordinated to the Mg ion.

each Mg2+ ion. Nonetheless, it is more desirable to compare our experimental M2Mg‑C values with amorphous systems. EXAFS studies have demonstrated that the short-range orders of Ca2+ ions, defined by the atoms in the first and second coordination shells, are distinct for various biogenic ACC, viz., lobster carapace, plant cystoliths, and ascidian spicule.3 For the second coordination shell, if we replaced all of the Ca−C distances with the corresponding Mg−C distances, the calculated MMg‑C could be used as a reference for the structural 2 interpretation of the experimental M2Mg‑C values of the MgXACC samples. To this end, we scaled down the Ca−C distances in biogenic ACC by multiplying them by a factor of 0.917, which is the ratio of the Mg−C (2.953 Å) and Ca−C (3.220 Å) distances of magnesite and calcite, respectively. This procedure was not unreasonable because both the space groups of magnesite and calcite belong to R3c̅ ; that is, each cation was surrounded by six equidistant carbonate ions.45,46 Consequently, three reference values of MMg‑C were calculated based 2 on the second coordination shell of Ca2+ ions in biogenic ACC. As illustrated in Figure 6, the second coordination shell of Mg was similar to that of Ca in lobster carapace for the Mg0.6ACC sample and ascidian spicule for Mg5ACC. The MMg‑C data of 2 Mg5ACC and AMC were comparable. The slightly smaller MMg‑C for AMC was consistent with the longer mean Mg−O 2 bond length in AMC. Therefore, we surmised that the number of nearest carbonate ions surrounding each Mg ion was slightly higher than four in Mg5ACC and AMC. Note that our second moment data cannot determine whether the coordination of the carbonate ions to Mg is bidentate or monodentate. Water Molecules in Proximity of Mg Ions. Figure 7 compares the experimental MMg‑H data and those calculated for 2 hydromagnesite. The coordination shells of Mg1 and Mg11 comprise one water molecule (Mg−H: 2.6450 and 2.4586 Å) and one hydroxyl group (2.3898 Å), while that of Mg2 contains two equivalent hydroxyl groups (2.3118 Å). The calculated MMg‑H of Mg2 is relatively high because of the short Mg−H 2 distance. Interestingly, we find that the MMg‑H data of MgXACC 2 are comparable to the calculated MMg‑H of Mg1 and Mg11, 2 although in this study we did not find any experimental 7229

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The Journal of Physical Chemistry C

Comparison of Ca-43 and Mg-25 NMR of ACC. 43Ca SSNMR spectroscopy is, in principle, an ideal method for the study of hydroxyapatite,48−52 bones,53,54 crystalline polymorphs,36 and the amorphous form of calcium carbonate.35 These pioneering studies have demonstrated the potential of 43 Ca NMR in the study of calcified tissue.55 Recently, 43Ca NMR has been used to trace the phase transformation of calcite to aragonite in the presence of Mg ions.56 Both 25Mg and 43Ca belong to the category of low-gamma nuclei and exhibit a similar resonance frequency. The quadrupole moment of 25Mg is 5 times larger than that of 43Ca, suggesting that 25Mg NMR is more sensitive to the change in the electric field gradient at the nuclear site. The spin quantum number of 25Mg (S = 5/2) is smaller than 43Ca (S = 7/2), indicating that the intrinsic signal intensity of the central transition of 25Mg is 1.8 times higher under the condition of selective excitation. The natural abundance of 25Mg (10.13%) is substantially higher than that of 43Ca (0.145%). Consequently, 25Mg{1H} C-REDOR measurements should be applicable to the study of biogenic samples in natural abundance, provided that the sample contains sufficient amount of Mg ions. To the best of our knowledge, the use of 25 Mg NMR in the study of biomineralization has not yet been discussed in the literature, although the applications of 25Mg NMR in material research have been recognized.57 In this pilot study, we reported that 25 Mg SSNMR spectroscopy is a powerful method for probing the environment of Mg in biogenic ACC and in other Mgcontaining amorphous systems.

Figure 7. Experimental MMg‑H determined for the amorphous samples. 2 values of hydromagnesite, The three solid lines indicate the MMg‑H 2 calculated for a distance of up to 20 Å. The dashed lines represent the values after removing all contributions from the resultant MMg‑H 2 hydroxyl groups. The nearest hydrogen-containing neighbors are given after the dash.

evidence for the presence of OH− ions. On the other hand, the of AMC is significantly smaller than MgXACC. As a MMg‑H 2 crude approximation to the scenario that each Mg2+ is coordinated by one water molecule only, we recalculated the MMg‑H of Mg1 and Mg11 without considering the contribution 2 from the OH groups. The resultant MMg‑H are somewhat 2 smaller than the experimental MMg‑H of AMC, which implies 2 that the coordination shell of Mg in AMC contains at least one water molecule. In any case, the second moment data are simply a sum of the contribution from each interacting pair of nuclei (eq 1). Therefore, it is impossible to determine unequivocally the distance information and the number of contacts in a coordination shell based on the second moments alone. Although different structural interpretations of our second moment data remain possible, it is very likely that the coordination shells of the Mg2+ ions in MgXACC and AMC contain water molecules. Recently, an interesting structural model has been developed for hydrated ACC, based on the reverse Monte Carlo refinement of X-ray total scattering data.8 It has been argued that the structure of synthetic ACC comprises a porous calcium-rich framework and interconnected channels formed by carbonate ions and water molecules. In the calcium-rich region, water molecules bridging two neighboring Ca2+ ions were also found. Another model, where domains of structural water are embedded in the ACC framework, has been used to describe the dehydration process of ACC and its subsequent phase transformation.47 Both models allow local movement of water molecules to certain extent, providing a rationalization of the transient nature of ACC. By contrast, the 25Mg NMR data of Mg0.6ACC and Mg5ACC suggest that the coordination shell of Mg contains at least one water molecule. Consequently, the longer stability of MgXACC than ACC is most likely owing to the structural water bound to Mg ions, which increases considerably the activation energy associated with the dehydration of ACC. Meldrum and co-workers suggested that the majority of structural water of synthetic ACC does not play any important role in its stability.47 For biogenic ACC, on the other hand, we believe that those structural water molecules coordinated to Mg ions could indeed stabilize the structure by interfering the process of dehydration.

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CONCLUSION Mg-ACC samples of homogeneous distribution in Mg were prepared using the method of micromixing. The Mg content reached up to 42 mol % when the Mg/Ca ratio of the mother liquors was comparable to that of seawater. Solid-state 25Mg NMR was applied to obtain valuable structural information regarding the Mg environment of our Mg-ACC samples with varying Mg content. We found that the first and second coordination shells of the Mg ions were substantially different from those of magnesite, thereby ruling out the scenario that the molecular structure of Mg-ACC constitutes a random arrangement of magnesite-like nanodomains. On the basis of the systematic variation in the 25Mg chemical shifts, we suggest that the Mg−O bond lengths increased as the Mg content increased. The average number of carbonates surrounding each Mg2+ ion ranged from 4 to 4.5 in the amorphous carbonates samples, with the ratio Mg/(Mg + Ca) between 17% and 100%. There is at least one water molecule coordinated to the Mg2+ ion. Altogether, the NMR data acquired in this work provide a detailed characterization of the coordination shell of Mg for our MgXACC and AMC samples, which may serve as a benchmark for any atomistic models obtained using theoretical calculation. We believe that solid-state 25Mg NMR has great potential in the study of Mg-containing biominerals.

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MATERIALS AND METHODS Sample Preparation and Characterization. 25Mgenriched MgO (99%) was obtained from CortecNet (Tilleuls, France). 13C-enriched anhydrous Na2CO3 was obtained from Cambridge Isotope Laboratories (Andover, MA). All other chemicals were obtained from Acros and used as received, without further purification. Deionized (DI) water was purged with N2 gas before use. The sample of Mg5ACC was prepared 7230

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The Journal of Physical Chemistry C as follows. The solution of CaCl2 (100 mM, 6% 43Ca enriched) and MgCl2 (500 mM, 55% 25Mg enriched) was adjusted to pH 8.0. A volume of 5 mL of the solution was then mixed with 5 mL of 13C-enriched Na2CO3 solution (100 mM, pH 11.2) at an ice-cooled temperature in a microreactor with a volume of 1.2 μL. The flow rate of the reactor was adjusted to 1 mL/min. After discarding the first 0.3 mL of the mixture, the remaining mixture was collected in 30 mL of acetone that had been precooled in an ice bath. Precipitates were collected by repeatedly conducting centrifugation (15000g for 10 min) and washing with precooled ethanol and acetone. The samples were lyophilized and stored in a desiccator. The same procedure was repeated for the preparation of the Mg0.6ACC, except that the 25 Mg enrichment level is 100%. Other MgXACC samples were prepared without isotopic enrichment. The AMC sample was similarly prepared at pH 8.75 and was 100% 25Mg and 13C enriched. The sample of magnesite was only 13C enriched. It was prepared by adding 0.304 95 g of MgCl2·6H2O, 0.2058 g of 13 C-enriched Na2CO3, and 0.080 25 g of NH4Cl to 15 mL of DI water. After being stirred for 5 min, the solution mixture was sealed in a Teflon-lined autoclave and aged at 160 °C for 72 h. The sample was collected and stored as previously described. The samples with isotope enrichment were prepared for the NMR measurements only. All other characterizations were conducted for the natural-abundance samples. XRD analysis was performed on a Philips X’pert diffractometer using Cu Kα radiation (λ = 1.5418 Å). FT-IR spectra were collected using a Varian 640-IR spectrometer in the range of 400−4000 cm−1. The TEM images were taken using a Hitachi H-7100 at 75 kV. The EDX analysis was performed on a LEO 1530 field emission scanning electron microscope equipped with EDX accessories. The ICP-MS measurements were performed on an Agilent 7500ce system. The standard solutions of 1000 mg/L Ca2+ and Mg (Merck) were diluted into ppb levels for the calibration measurements. Solid-State NMR. All 25Mg NMR experiments were conducted on Bruker Avance III spectrometers at 36.7 MHz (14.1 T) and 24.5 MHz (9.4 T). 25Mg chemical shifts were externally referenced to a freshly prepared MgCl2 solution of 1.0 M in natural abundance. Double-frequency sweep (DFS) pulses with a frequency sweep from 100 to 800 kHz, a length of 500 μs, and an rf field of 20 kHz were applied to enhance the initial 25Mg central transition for all measurements.58 The selective 25Mg π/2 pulse was set to 10 μs for MgXACC and MgCO3, whereas the pulse was adjusted to 40 μs for Mg(NO 3) 2·6H 2O samples. All the measurements were conducted at a temperature between 248 and 270 K. The recycle delay was set to 0.5, 3, and 20 s for the MgXACC, Mg(NO3)2·6H2O, and MgCO3 samples, respectively. The 25 Mg{13C} REDOR experiments were conducted at 14.1 T using a 3.2 mm probe with a sample mass of ∼20 mg. The spinning frequency was set to 10 kHz. The trains of 13C π pulses (10 μs) were phase cycled according to the XY-8 scheme.59 Proton decoupling during the REDOR dephasing period was applied at 75 kHz. To enhance data reproducibility, the memory buffer for acquisition was manipulated in such a way that the odd- and even-numbered scans were accumulated with and without recoupling pulses, respectively. The 25Mg{1H} C-REDOR experiments were conducted at 14.1 T using a 1.9 mm probe with a sample mass of ∼6 mg. The spinning frequency was 40 kHz. The recoupling pulse fulfilling the

symmetry of POST-C313 was applied in the 1H channel with the rf field set to 80 kHz.26

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S19 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.C.C.C.). *E-mail: [email protected] (S.J.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Technology (100-2628-M-002-009-MY3 and 102-2731-M002-002-MY2). The NMR measurements were carried out at the Instrumentation Center of National Taiwan University. We thank the reviewers for their insightful comments.

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REFERENCES

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