Understanding the Role of Citric Acid on the Crystallization Pathways

May 2, 2019 - The precipitation of calcium oxalate can be either grown into structural support in plants or precipitated as stones in human kidneys. P...
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Cite This: Cryst. Growth Des. 2019, 19, 3139−3147

Understanding the Role of Citric Acid on the Crystallization Pathways of Calcium Oxalate Hydrates Si Li,† Weiwei Tang,† Mengya Li,† Lingyu Wang,† Yang Yang,† and Junbo Gong*,† †

School of Chemical Engineering and Technology, State Key Laboratory of Chemical Engineering, The Co-Innovation Center of Chemistry and Chemical Engineering of Tianjin, Tianjin University, Tianjin 300072, People’s Republic of China

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ABSTRACT: The precipitation of calcium oxalate can be either grown into structural support in plants or precipitated as stones in human kidneys. Previously, citrate, an effective inhibitor for oxalate stone formation, was suggested to alter the crystallization pathway of calcium oxalate and enhance the formation of less stable calcium oxalate trihydrate; however, the underlying mechanism is unknown. Herein we investigated the role of citric acid on crystallization pathways of calcium oxalate hydrates and the effect of supersaturation. It was found that the presence of citric acid can modulate the water content of amorphous nucleated precipitates by TEM and XRD and hence promote the formation of different calcium oxalate hydrates. The remaining water content in amorphous precipitates depends upon the amount of citric acid employed. FTIR and XRD analyses further reveal the extreme structural similarities between amorphous precipitates and the resultant hydrates. Additionally, the role of citric acid on crystallization pathways could be completely changed by supersaturation of calcium oxalate. High supersaturation dominated by homogeneous nucleation can diminish or even invalidate citric acid roles. The findings highlight the interplay of roles between additives and supersaturations, and could improve current understandings on the formation mechanism of oxalate stone. effects of different additives,18,19 such as proteins (osteopontin,20 Tamm-Horsfall protein,21 albumin,22 and lysozyme23), small molecules (citrate and hydroxycitrate),24 and inorganic salts on the nucleation and growth of calcium oxalate. Citrate is well-known as an inhibitor for formation of calcium oxalate. It not only restrains the growth of calcium oxalate, but inhibits the nucleation process. Previously, most of the studies suggested that citrate can reduce the growth rate of crystal steps from experimental observations of atomic force microscope (AFM) and bulk crystallization.24−26 By use of titration experiments and TEM observations, Ruiz-Agudo et al. were able to examine the role of citrate on the early stages of calcium oxalate formation, and found that citrate interacts with polynuclear stable complexes and amorphous precursors and hence inhibits calcium oxalate nucleation.17 However, the mechanism of citrate acting on the crystallization pathway of calcium oxalate hydrate is still unknown; therefore, we focus on the role of citrate on calcium oxalate hydrates in this paper. In this study, we try to resolve the role of citric acid on the crystallization pathway of calcium oxalate hydrates. Calcium ion selective electrode (ISE) was applied to probe the effect of citric acid on the formation of calcium oxalate during prenucleation. The morphology and structures of nucleated

1. INTRODUCTION Biomineralization of calcium oxalate is widely observed in nature. There are different habits and sizes of calcium oxalate crystals in plants,1−3 the formation of which involves a series of processes including cell growth, crystal formation, and the production of specific organelles worked in parallel rather than a simple precipitation phenomenon.4,5 Calcium oxalate plays a beneficial role in plants because it supplies a source of calcium to the tissues and organs to achieve various activities of plants.6,7 However, it is harmful for human health, especially for the kidneys, in which calcium oxalate aggregates and becomes the primary component of kidney stones, a worldwide disease sparing no gender, race, or region.8−10 Calcium oxalate exists in the form of crystals in both plant and human body. Many studied focused on the formation process of calcium oxalate crystals. Crystallization from solution mainly involves crystal nucleation, the birth of crystals, and growth. Three crystal forms of calcium oxalate hydrates, calcium oxalate monohydrate (COM),11 calcium oxalate dihydrate (COD),11 and calcium oxalate trihydrate (COT),12 can be crystallized under different conditions.13 Both COM and COD were found existing in human urine.13 Prior to the formation of calcium oxalate crystals, amorphous calcium oxalate (ACO) is formed initially after nucleation,14,15 which are aggregated by clusters formed in prenucleation process.16,17 Because the components of urine is complex, based on kidney stones, many studied have focused on the © 2019 American Chemical Society

Received: August 30, 2018 Revised: March 25, 2019 Published: May 2, 2019 3139

DOI: 10.1021/acs.cgd.8b01305 Cryst. Growth Des. 2019, 19, 3139−3147

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Figure 1. (a) Titration curves: Development time of free Ca2+ concentration in the absence and presence of different amounts of citrate at pH 6.1 and their repetitions. Black line: the amount of Ca2+ added; red lines: experiments run in the absence of citric acid; blue lines: experiments run in the presence of 5 mg/mL citric acid; purple lines: experiments run in the presence of 10 mg/mL citric acid. Two replicates are presented for each run. (b) FTIR data of precipitates obtained in the titration experiments without citric acid (red line), with the amount of 5 mg/mL (blue line) and 10 mg/mL (purple line) citric acid and FTIR data of COM (black line) and COT (olive line). and TG were performed. In order to improve the reliability of the experiments, two or three replicates are presented for each run. 2.4. Monitor Ca2+ Concentration after Nucleation. The calcium ions free concentration during the reaction were monitored by calcium ion selective electrode (ISE, Thermo Scientific Orion 9720BNWP) to reflect the amount change of Ca2+ in solution at the room temperature under constant stirring, about 500 rpm. During the measurement, calcium chloride solution (0.01 and 0.025 M) at a rate of 17.90 mL/min was added into sodium oxalate solution (0.01 and 0.025 M). Before the experiment, different concentration (5−10 mg/ mL) of citric acid (pH = 6.00−6.40) were added into the sodium oxalate solution. 2.5. Characterization. X-ray diffraction (XRD, Type, R-AXISRAPID, Rigaku, Japan) was used to analyze the crystalline form of precipitates by Cu Ka radiation (1.5405 Å) over the 2θ range of 10− 45° with a scanning rate of 2°/min for ACOs improving the S/N ratios, and 8°/min for other samples. The polymorphs of precipitates were determined by Bruker ATR-FTIR (Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy), with the resolution value of 4 cm−1, scan time of 16, and wavenumber ranging from 400 to 4000 cm−1. The morphology of precipitates were determined by scanning electron microscope (SEM) and transmission electron microscope (TEM). TEM images and the selected area electron diffraction (SAED) patterns were performed by field emission transmission electron microscope (FE-TEM, Tecnai G2 F20), operated at 200 kV and room temperature. The water content of precipitates were determined by thermogravimetric analysis (TGA \DSC 1, Mettler Toledo) with the heating rate of 10 K/min under 30 mL/min N2 flow.

precipitates were analyzed by TEM, XRD, and FTIR to investigate the influence of citric acid on the nucleation process. Moreover, the role of citric acid on postnucleation process was also examined by calcium ion selective electrode. The manipulation of solution supersaturation on the performance of citric acid roles was found and the underling formation mechanism was further probed.

2. MATERIALS AND METHODS 2.1. Materials. CaCl2:2H2O (>99%), tartaric acid (99%, TA), malic acid (99.0%, MA), and glutaric acid (GA, 99%) were bought from Aladdin. Na2C2O4 (99.0%), citric acid (99.5%, CA), transaconitic acid (98.0%, TAA), and succinic acid (SA, >99.5%) were purchased from GENERAL-REAGENT, MACKLIN, TCI, and Sinopharm Chemical Reagent Co. Ltd., respectively. All materials were used without retreatment, and deionized water was used in all experiments. 2.2. Free Ca2+ Concentration Determination during Prenucleation. Titration setup (Injection Pump, LGpump, LSP04-1A) was used to determine the free Ca2+ concentration in solution before and after nucleation with a CaCl2 drop rate of 0.1 mL/min. Ca2+ potentials were monitored continuously by calcium ion selective electrode (ISE, Thermo Scientific Orion 9720BNWP) at room temperature under constant stirring, about 500 rpm. Before the experiment, different concentrations of citric acid (pH = 6.00−6.40) were added into the sodium oxalate solution. Prior to ISE measurements, the electrode was calibrated by calcium ion standard solution (0.1 M, Orion 92206). Calcium ionic strength adjuster (Orion 932011) was used to adjust the ionic strength of each solution. Between the experiments, the electrode was stored in 0.01 M solution diluted with 0.1 M of standard solution. 2.3. Precipitates after Nucleation Preparation. Precipitates of calcium oxalate were prepared by quickly adding 5 mL NaC2O4 (0.01−0.1 M) solution into 5 mL CaCl2 (0.01−0.1 M) solution in the presence of the magnetic stirring at a speed of 600 rpm, followed by stirring for about 2 s, 10 min, and 30 min. Then, the precipitates were filtered swiftly through a 0.22 μm organic filter by vacuum pump and quenched with ethanol. The purpose of quenching with ethanol is to insulate the water from the sediment obtained by the filtration, and to quicken the natural drying of the precipitates. The additives were added into CaCl2 before mixing CaCl2 and Na2C2O4. The concentration of additives range from 0.5 to 40 mg/mL with a pH of 6.00−6.40, where CA ranged from 0.5 to 10 mg/mL, TA from 5 to 10 mg/mL, MA and TAA from 5 to 40 mg/mL, and GA and SA from 10 to 40 mg/mL. After the precipitates were dried, XRD, FTIR, TEM,

3. RESULTS 3.1. Titration Experiments. The titration experiments were performed to understand cluster formation and crystallization process of calcium oxalate. Figure 1a shows the time development of free Ca2+ concentration in a 0.01 M sodium oxalate solution in the absence and presence of different concentrations of citric acid. As can be seen, the detected free Ca2+ concentration is significantly lower than the added amounts, suggesting the formation of prenucleation clusters. The existence of calcium oxalate clusters is already proven by previously studies.16,17 The amount of free Ca2+ detected by calcium ion selective electrode increases over the titration time to a point where the free Ca2+ concentration starts to drop and calcium oxalate nucleates, then reaches a constant level that corresponds to the solubility of the 3140

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Figure 2. (a) XRD patterns of the precipitates prepared in 0.01 M (black), 0.05 M (red), and 0.01 M + 10 mg/mL citric acid (blue). (b) XRD patterns of the precipitates stirring in 0.01 M solution for 2 s (black), 10 min (red), 30 min (blue), and COM (pink). (c) XRD patterns of the precipitates stirring in 0.05 M solution for 2 s (black), 10 min (red), 30 min (blue), and COD (pink). (d) XRD patterns of the precipitates stirring in solution with 10 mg/mL citric acid and 0.01 M reactants for 2 s (black), 10 min (red), 30 min (blue), and COT (pink). The replicates are shown in Figure S2.

amorphous phase (ACO III) in 0.01 M reactants was transformed into crystalline COT (Figure 2d). These results indicate that the structure of ACO I may resemble that of COM with only short-range order, whereas the structures of ACO II and ACO III, respectively, resemble to COD and COT. The IR spectra indeed reveal the remarkable structure similarities between ACOs and crystalline hydrates (for more information, see section 3.4 below). In fact, a similar phenomenon was also reported previously in calcium carboxylate mineralization. By virtue of WAXS (wide-angle X-ray scattering) analysis, Gebauer et al.16 found two different species of amorphous calcium carbonate (ACC) of short-range order and they could transform into corresponding crystalline polymorph later (i.e., ACC I can transform into calcite, while ACC II transforms into vaterite). 3.3. TEM Data. The phases of precipitates from rapidmixing experiments were determined by TEM images and selected area electron diffraction (SAED). Figure 3 shows TEM images and SAED patterns of the precipitates obtained in the absence (a, c) and presence (b, d) of citric acid. The precipitates obtained at 0.01 M were like liquid from the TEM images (Figure 3a) which were unable to distinguish the size of the particles, while those obtained at 0.01 M with citric acid were like flowers with the size of about 1−3 μm (Figure 3b). The different morphology of precipitates may be a resulted of limited diffusion of reactants during the nucleation process in the presence of citric acid.27 The precipitates obtained at 0.025 M with and without citric acid were nanoparticles of size ranging between 100 and 500 nm (Figure 3c and d). In the absence of citric acid, the particles are not only smaller (100−

nucleated phase in all cases. In the absence of citric acid, the detected amounts of free Ca2+ increase over titration time to a maximum where the free Ca2+ concentration starts to drop and calcium oxalate nucleates, then reaches a plateau corresponding to the solubility of nucleated phase. FTIR data (Figure 1b) shows that the precipitate phase should be COM. The presence of citric acid displays similar curves but different depletion rates of Ca2+ and plateau concentrations. Increasing amounts of citric acid flatten the increase of Ca 2+ concentration and increase the time of nucleation onset, suggesting more Ca2+ bound into prenucleation clusters and more stable clusters.17 The data further indicates the complex interactions of citric acid with clusters and the formation of different types of clusters during prenucleation stage. Moreover, the higher plateau concentration in the presence of citric acid suggests the formation of a new precipitate phase, which is COT as revealed by FTIR. 3.2. XRD Analysis. From the powder XRD patterns (Figure 2), the absence of any Bragg reflections demonstrates the initially formed solid phases are amorphous after rapidly mixing at different concentrations (Figure 2a). The transformation process from amorphous to crystalline hydrates of calcium oxalate was detected by XRD and illustrated in Figure 2. As can be seen, the metastable amorphous phase (ACO I) generated from 0.01 M reactants in solution gradually evolved into crystalline COM after stirring about 30 min (Figure 2b). At high initial reactant concentration (0.05 M), the produced amorphous phase (ACO II) was found transformed only into crystalline COD after stirring about 30 min (Figure 2c). By contrast, in the presence of 10 mg/mL citric acid, the obtained 3141

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evidence has demonstrated that amorphous calcium oxalate was produced prior to crystalline calcium oxalate.14,15 3.4. FTIR Spectra. FTIR spectra were employed to identify the crystal forms of calcium oxalate hydrates and probe ionicbonding and hydrogen-bonding interactions (for more information, see SI). Figure 4 displays the detailed comparisons of IR spectra between hydrates and ACOs produced in the absence and presence of citric acid. The remarkable resemblance can be found between ACOs and three hydrates, suggesting the existence of three types of ACOs serving as crystallization precursors of hydrates, which is consistent with the results from XRD patterns. The IR spectrum (Figure 4a) of amorphous precipitates produced at 0.01 M reactants (ACO I) resembles that of COM apart from several weak bands at 3000−3500, 1500, and 596 cm−1, which are related to the bending and wagging vibrations of water molecules in precipitates.28−30 The identical calcium and oxalate peaks at 1313, 779, and 511 cm−1 in both amorphous precipitate and COM indicate the similar ionic interactions of Ca2+ and C2O42−.31−33 The different water peaks in 3000− 3500 cm−1, 1500 cm−1, and 660−590 cm−1 in amorphous precipitates and COM suggest that water molecules exist in a more free state in ACO than that in crystal, and the hydrogen bonding between water molecules and oxalate ions is weaker.29,32,33 Based on these similarities, the structures of amorphous precipitates is thus envisaged as shown in Figure 4d. In the structure of ACO I, a calcium ion is surrounded by three oxalate ions, which is similar to COM (for more information see SI), but the surrounding water molecules are in a free state compared to the fixed state in the crystal. The same case also exists in ACO II and COD (Figure 4b). The identical calcium and oxalate peaks (1613, 1320, 778, and 513−490 cm−1) and slightly different water peaks (3000− 3500, 1473, and 913 cm−1)28−33 in ACO II and COD demonstrate the similar ionic bonds between Ca2+ and C2O42−,

Figure 3. Characterization of calcium oxalate precipitates formed at 0.01 M (a) and 0.01 M with 10 mg/mL citric acid (b), 0.025 M (c), and 0.025 M with 5 mg/mL citric acid (d).

300 nm) than that obtained in the presence of citric acid (250−500 nm), but varying in size. From the SAED patterns inserted in Figure 3 and Figure S3 and the high-resolution patterns (Figures S3 and S5), all the tested precipitates were amorphous. That is to say, amorphous calcium oxalate can be obtained after nucleation with the operation of rapid mixing in the absence and presence of citric acid, which is consistent with the results from XRD patterns. Moreover, recently

Figure 4. FTIR data of COM (red line) and precipitates (black line) formed at the concentration of CaOx 0.01 M (a), COD (red line), and precipitates (black line) produced at the concentration of CaOx 0.025 M with 5 mg/mL citric acid (b) and COT (red line) and precipitates (black line) with the concentration of citric acid 10 mg/mL at 0.01 M reactants (c). (d, e, f) show the possible structures of ACO I, ACO II, and ACO III, respectively. 3142

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water content (∼26%) with COD (21.95%). The slightly higher water content in ACOs than in crystalline hydrates suggests the existence of loosely bound water molecules in amorphous state, and the crystalline hydrates formed need to be restructured and the loosely bound water molecules removed. Furthermore, the law of water content in precipitates formed in the presence of different amounts of citric acid was found by the operation of rapidly mixing 0.05 M. The loosely bound water in ACOs was further confirmed by the analyses of FTIR and TGA data (Figure 5) in which the significantly increased water content does not remarkably affect the interactions and IR spectra. In the presence of citric acid, water content in ACOs was found to increase with the addition of citric acid initially and then level off at 25−26%. FTIR data shows that all these ACOs should be of the similar structures. 3.6. Effect of Citric Acid on Nucleation. The effect of citric acid on nucleation was examined at different amounts of additive and various supersaturations. Figure 6 shows IR spectra of nucleated precipitates at different reactants and additive concentrations. As can be seen in Figure 6a, in the absence of citric acid, the ACO I was obtained below 0.01 M reactant concentration, while the ACO II was precipitated above 0.05 M. The mixture of ACO I and ACO II was produced at 0.025 M. When the additive concentration reaches 5 mg/mL, the IR spectra (Figure 6b) do not show significant change except for 0.025 M reactant concentration where the mixtures of ACO I and ACO II turn to be pure ACO II. Thus, the role of citric acid was explored at two reactant concentrations 0.01 and 0.025 M. The structure of amorphous precipitates gradually changes from ACO I to ACO III with the increasing amounts of citric acid (Figure 6c). The high concentration of citric acid favorable for the formation of COT was also reported in the literature.17 At higher reactant concentration, the amorphous structure varies from ACO I and ACO II mixtures to pure ACO II (Figure 6d). A citric acid molecule contains three carboxyl groups and one hydroxyl group. At the pH of 6.2, two or three carboxyl groups in citric acid molecules were ionized,24 then they can combine with water molecule by forming hydrogen bonds and coordinate to Ca2+, which are similar to oxalate ions. Compared with oxalate ions, there are an extra carboxylate and an extra hydroxyl group in citric acid molecule apart from the presence of carboxylates at both ends of the carbon chain.

and a free state of water in ACO II. Therefore, in the shortrange structure of ACO II (Figure 4e), a calcium ion connects with two oxalate ions, and the water molecules are randomly distributed around calcium ions and oxalate ions. Again, the similar bands (1606, 1321, 781, 638, and 498 cm−1) and different peaks (3130 cm−1, 548 cm−1)32,33 appear in both ACO III and COT (Figure 4c). In the structure of ACO III (Figure 4f), a calcium ion links with two oxalate ions, and the free water molecules surround calcium ions and oxalate ions at random.34 Different from the indistinguishable XRD patterns of the amorphous phase, there are distinct, identifiable differences of these amorphous phases in IR patterns. Therefore, the IR spectra were used to identify these three species of ACO. 3.5. Water Content in Precipitates. To explore the structure of ACOs, the water content in amorphous precipitates was examined by TGA/DSC. Table 1 shows the Table 1. Water Content of Precipitates Obtained under Different Conditions concentration (mol/L) 0.01 0.01 0.01 0.025−5a 0.025−5a 0.025−5a 0.01−10a 0.01−10a 0.01−10a

water content

water molecule number

kinds

16.89% 15.45% 15.92% 12.33% 25.61% 25.88% 25.74% 21.95% 28.26% 28.96% 28.33% 29.67%

1.45 1.30 1.35 1.00 2.45 2.48 2.47 2.00 2.80 2.90 2.81 3.00

ACO I ACO I ACO I COM ACO II ACO II ACO II COD ACO III ACO III ACO III COT

Concentration of reactants (mol/L) − the concentration of citric acid (mg/mL).

a

water content in both amorphous precipitates and hydrates. It was found the water content in the absence of citric acid is around 15−17% matching well with 12.33% in COM. The presence of citric acid increases the water content to 28−29%, which is comparable though slightly less than 29.67% in COT. At a concentration of 0.025 M reactant and 5 mg/mL citric acid, the amorphous precipitates were found with a similar

Figure 5. FTIR data (a) and the water content (b) of precipitates formed at CaOx 0.05 M with different added amounts of citric acid. 3143

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Figure 6. FTIR spectra of precipitates obtained in absence of citric acid (a) and in the presence of 5 mg/mL citric acid (b), and obtained in 0.01 M (c) or 0.025 M (d) of reactants with different amounts of citric acid.

Figure 7. Molecule structures of oxalic acid (a), citric acid (b), trans-aconitic acid (c), glutaric acid (d), tartaric acid (e), malic acid (f), and succinic acid (g).

Figure 8. FTIR data of precipitates obtained in different amount of additives at 0.01 M (a) and 0.025 M reactants (b).

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Figure 9. Development of Ca2+ free concentration in solution at reactant concentrations of 0.01 M (a) and 0.025 M (b) in the absence of citric acid (black line) and the presence of 5 mg/mL (red lines), 6 mg/mL (blue line), and 10 mg/mL (purple lines) citric acid.

hence high supersaturation follows different rules (Figure 9b). The Ca2+ concentration after nucleation displays a decreasing first plateau, then decrease abruptly, and finally stable plateau corresponding to COM. The data suggests a different crystallization pathway at high supersaturations. The addition of citric acid does not show significant change in the depletion curve of free Ca2+ concentration, and merely sustains the time of the first plateau, indicating the stabilization role of citric acid on the first precipitates. The above data suggests that citric acid can promote both formation and stabilization of unstable forms.

Therefore, we speculate that these redundant carboxylate and hydroxyl groups located at middle of the molecule are the crucial factor determining the nucleation process of calcium oxalate, especially the formation and aggregation of clusters. Several structurally similar compounds of the carbon chain length (4−5 carbons) close to citric acid were selected to examine the speculation based on the carboxylic acid and hydroxyl groups, including trans-aconitic acid, glutaric acid, tartaric acid, malic acid, and succinic acid (Figure 7). These compounds can be classified into two categories: one contains carboxylates at both ends of the carbon chain (i.e., glutaric acid, and succinic acid); the other also includes the extra hydroxyl and/or carboxylic acid groups in the middle of the straight carbon chain of the molecule (i.e., trans-aconitic acid, tartaric acid, and malic acid). It was found that the tartaric acid, malic acid, and trans-aconitic acid indeed affect structures of amorphous precipitates, whereas succinic acid and glutaric acid have no effect on the amorphous structures (Figure 8). These data thus suggest the extra hydroxyl and/or carboxylic acid groups in the middle of straight carbon chain of the additive molecule play an important role in the structure of amorphous precipitates upon nucleation possibly by modifying the structure of clusters through the interaction with water molecules, calcium ions, and oxalate ions. 3.7. Effect of Citric Acid on Postnucleation and Hydrate Formation. Free Ca2+ concentration in postnucleation and hydrate formation was in situ monitored to examine citric acid roles. Two different concentrations of reactants 0.01 and 0.025 M were selected, and the results are shown in Figure 9. In the absence of citric acid, the depletion of Ca2+ concentration dramatically increases with the addition of Ca2+ and then diminishes rapidly after nucleation, then levels off (Figure 9a black line). The presence of citric acid leads to the free Ca2+ concentration undergoing two plateaus after nucleation, which corresponds to two types of precipitates of different solubility. Further, the time and number of plateaus can be modified by the amounts of citric acid. The larger amount of citric acid results in the longer plateau time retained, indicating more significant interactions of citric acid with calcium oxalate and water in precipitates. Three plateaus appear at even high concentration of citric acid, demonstrating the formation of three types of precipitates. The concentration change of free Ca2+ in the absence of citric acid at higher reactant concentration (0.025 M) and

4. DISCUSSION 4.1. Structural Correlation between ACO and Crystalline Hydrates. Three types of solid phases were precipitated initially and then transformed to crystalline hydrates under different crystallization conditions in the absence and presence of citric acid. TEM data and XRD patterns show that all these initially formed precipitates are amorphous. Thermal analysis data show that the water content in the three amorphous precipitates can correlate well with their crystallized hydrates. By the removal of loosely bound water molecules, each type of ACO (I, II, or III) will transform into its crystalline counterpart (COM, COD, or COT). FTIR was further applied to explore the possible structure correlation, Ca2+−C2O42− ionic interactions, and hydrogen bonding interactions with water. The remarkable similarities between ACOs and hydrates were found and the similar ionic interactions can be identified. The main difference lies in the hydrogen bonding interactions between C2O42− or citrate and water, which lead to the existence of loosely bound water molecules. The structural correlation between ACOs and crystalline hydrates suggests that ACOs are the crystallization precursor and drives the formation of resultant hydrate, which was illustrated in the following schemes. Pathway I: Ca 2 + + nC2O4 2 − + (1 − 2)H 2O → clusters I → ACOI (1)

→ COM

Pathway II: Ca 2 + + nC2O4 2 − + (2 − 2.5)H 2O → clusters II → ACOII → COD 3145

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Figure 10. Schematic summarizing the roles that citric acid plays on the crystallization pathway of calcium oxalate. The red lines represent the pathway in absence of citric acid, and the blue lines show the pathway in the presence of citric acid. Note that the energetics of calcium oxalate crystallization pathways from supersaturated solutions refer to ref 17.

5. CONCLUSION The mechanism of calcium oxalate crystallization and the role of citric acid were investigated in the stages of prenucleation, nucleation, and postnucleation using calcium ion selective electrode, XRD, TEM, and FTIR. It was found the initial precipitated solid phase of calcium oxalate is amorphous and transformed into the COD probably by structure relaxation and desolvation, which finally results in the formation of COM by polymorphic transition. The presence of citric acid additive can alter this crystallization pathway by modulating the water content and the intermolecular interactions. Large amounts of additive lead to the precipitates with more water content produced and amorphous precipitates corresponding to less stable crystal hydrate formed. Moreover, the roles of citric acid could not persist under high supersaturations, and the solute’s supersaturation was found to diminish or even invalidate the additive effects. Our findings could shed light on the roles of citric acid additive and the effect of supersaturation on crystallization pathways of calcium oxalate hydrates, which may promote the effective selection of appropriate additives for better treatment of kidney stones.

Pathway III: Ca 2 + + nC2O4 2 − + (2.5 − 3)H 2O → clusters III → ACOIII → COT

(3)

4.2. Role of Citric Acid on Crystallization Pathways. The above data suggests the resultant formation of crystalline hydrates depends upon the initially formed amorphous structures, and citric acid can affect these structures, hence altering crystallization pathways. Citric acid can not only promote the formation of pure ACO II and ACO III, but also stabilize the corresponding hydrates. Thus, at low supersaturation in the presence of citric acid, the water content of the precipitates produced first increases with the amount of citric acid, and then the structures of precipitates change from ACO I to ACO III. Therefore, the subsequent transformation process of crystal produced COT, COD, and finally COM. Moreover, the presence of citric acid results in pure ACO II obtained at moderate supersaturation. However, citric acid has no effect at high supersaturation. Therefore, we infer that the formation of ACO II and ACO III is kinetically driven in the presence of citric acid. The citric acid can stabilize the prenucleation clusters of high energy which was then crystallized to be amorphous precipitate due to the lower free energy barrier. The formation of ACOs bears resemblance to the particular structure of a hydrate, leading to the crystallization of structurally similar hydrate due to again lower free energy barrier (Figure 10). Nevertheless, the role of citric acid on crystallization pathways is also affected by reactant concentration and thus solute supersaturation. High supersaturation leads to the formation of ACO II and subsequent transformation to COD, and the additive role is no longer validated. One possible reason for this observation could be that homogeneous nucleation surpassed heterogeneous nucleation. However, the detailed mechanism at the molecular level is under investigation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.8b01305. Crystal structure and FTIR analysis of calcium oxalate hydrates; unit cells and FTIR spectra of calcium oxalate hydrates; a table of information on interaction in the unit cell of calcium oxalate hydrates; XRD and FTIR patterns of calcium oxalate hydrates and their corresponding amorphous; TEM patterns and XRD and FTIR patterns of calcium oxalate monohydrate and dihydrate; high-resolution patterns of samples in Figure 3 (PDF) 3146

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 86-22-27405754. Fax: 86-22-27314971. ORCID

Weiwei Tang: 0000-0002-7998-4350 Junbo Gong: 0000-0002-3376-3296 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the financial support of National Natural Science Foundation of China (NNSFC 21808159 and NNSFC 21676179).

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REFERENCES

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DOI: 10.1021/acs.cgd.8b01305 Cryst. Growth Des. 2019, 19, 3139−3147