This is a repository copy of Precipitation of Amorphous Calcium Oxalate in Aqueous Solution. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/87207/ Version: Accepted Version Article: Ihli, J, Wang, YW, Cantaert, B et al. (5 more authors) (2015) Precipitation of Amorphous Calcium Oxalate in Aqueous Solution. Chemistry of Materials, 27 (11). 3999 - 4007. ISSN 1520-5002 https://doi.org/10.1021/acs.chemmater.5b01642 Reuse Unless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version - refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher’s website. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Precipitation of Amorphous Calcium Oxalate in Aqueous Solution Johannes Ihli1*, Yun-Wei Wang1, Bram Cantaert1, Yi-Yeoun Kim1, David C. Green1, Paul H. H. Bomans2, Nico A. J. M. Sommerdijk2 and Fiona C. Meldrum1* 1 School of Chemistry, University of Leeds, Leeds, LS2 9JT, UK. 2 Department of Chemistry and Chemical Engineering, Eindhoven University of Technology, Eindhoven, 5612 AZ ,NL KEYWORDS: Calcium oxalate, amorphous, biomineralization, confinement ABSTRACT Inspired by the observation that crystalline calcium carbonate and calcium phosphate biominerals frequently form via amorphous precursors, a wide range of studies have been performed which demonstrate that many inorganic crystals can precipitate from solution via amorphous phases. This article considers the crystallization mechanism of calcium oxalate, which is a significant biomineral in many plants and the primary constituent of kidney stones in vertebrates, and shows that this can also precipitate via an amorphous precursor phase from aqueous solution. A range of approaches were employed to study calcium oxalate formation, including precipitation in bulk solution in the presence and absence of additives and in the spatially confined volumes offered by track etched membranes and a crossed cylinders apparatus. A freeze concentration method was also used to generate sufficient quantities of amorphous calcium oxalate (ACO) for analysis. The results show that amorphous calcium oxalate crystallizes rapidly in bulk solution, but can be significantly stabilized through the concerted activity of additives and confinement. We also demonstrate that ACO has a composition of CaC2O4:H2O. These data suggest that calcium oxalate biominerals, in common with their carbonate and phosphate counterparts, may also precipitate via amorphous phases. 1 INTRODUCTION It is now widely accepted that crystalline calcium carbonate1, 2, 3 and calcium phosphate4, 5 biominerals often form via amorphous precursor phases. Employed by vertebrates, invertebrates and plants alike,3, 5, 6 this strategy would appear to offer many advantages, such as access to more rapid mineralization rates and potentially greater control over the crystallization process. While it is recognized that some crystalline iron oxide biominerals, for example magnetite in magnetotactic bacteria and chiton teeth,7 can form via the poorly-ordered phase ferrihydrite, there is as yet insufficient data available to determine whether this mechanism is common across the large number of known crystalline biominerals. Indeed, this can be a difficult question to answer, given the challenges associated with identifying amorphous materials when they are present in combination with crystalline phases and in characterizing biominerals that are only generated in small quantities. Valuable insight into the possible mechanisms of formation of biominerals can therefore be gained by studying crystallization mechanisms in solution, as this gives the chance to study rapid reactions and to rigorously characterize products. While obviously not proving that the same mechanisms operate within biogenic systems, it is notable that organisms frequently retard processes as compared with the parallel reactions occurring in bulk solution, such that metastable polymorphs are commonly observed.8, 9 This can be attributed to the action of soluble organic macromolecules, association with organic matrices and localization within confined volumes.1, 10, 11, 12, 13, 14 Thus, observation of a crystallization pathway that goes through an amorphous phase provides strong evidence that this may also operate in biology. Conversely, identification of calcium sulfate hemihydrate identified as statoliths (gravity sensors) within deep-sea medusae8 where this polymorph of calcium sulfate had only been reported to precipitate from salt-free aqueous solutions at temperatures above 97 oC15, 16 prompted us to re-examine the mechanism of precipitation of calcium sulfate in solution, and revealed the presence of a previously unidentified amorphous calcium sulfate phase.17 In this paper we explore whether calcium oxalate (CaOx) can precipitate via an amorphous precursor phase in aqueous solutions, and identify the experimental conditions that extend the lifetime of amorphous calcium oxalate (ACO). Calcium oxalate is an important biomineral and is generated within specialised cells in many plants.18 The crystals can form with a wide range of shapes and sizes and may provide structural support, or in the form of styloid and raphide crystals, as protection against predators.19, 20 Precipitation of CaOx may also serve to maintain a low concentration of 2 oxalate within the cytoplasm. The formation of CaOx within vertebrates, in contrast, is typically pathological, and in humans is most commonly seen in kidney stones.21 CaOx crystallizes in three forms, of which monohydrate (COM, CaC2O4:H2O) is the thermodynamically most stable, and the dihydrate (COD, CaC2O4:2H2O) and trihydrate (COT, CaC2O4:3H2O) are metastable. CaOx in plants commonly appears as COM or COD,22 while kidney stones are principally COM.23, 24 Interestingly, a recent report has also shown that CaOx can be precipitated in an amorphous form from ethanolic solutions.25 In our work we employ a range of methods that are known to kinetically stabilize amorphous precursor phases in solution including the freeze-concentration of saturated solutions,26 precipitation within confined volumes11, 27, 28, 29 and precipitation in the presence of polyelectrolytes30, 31, 32 to aid the identification and characterization of amorphous precursor phases of calcium oxalate in solutions. These methods demonstrate that amorphous calcium oxalate (ACO) particles with compositions of CaC2O4:H2O can form in aqueous solution, and that this amorphous phase can be significantly stabilized both in the presence of anionic polyelectrolytes and within confined volumes. That both of these conditions are characteristic or biomineralization processes suggests that calcium oxalate biominerals could well form via an amorphous phase, but that the challenges associated with characterizing calcium oxalate (it readily decomposes during analysis)33 may have precluded the identification of this phase. RESULTS Bulk Precipitation The possibility of observing amorphous calcium oxalate (ACO) in bulk solution was investigated by characterizing the precipitants formed by direct combination of equal volumes of Na2C2O4 and CaCl2 to give a final concentration of [CaOx] = 0.125-50 mM and pH of 6.8-7.05, where this procedure is consistent with those used in the preparation of ACC and ACP.34, 35 These solutions were degassed with nitrogen for 2 hours prior to use to avoid the possible formation of calcium carbonate, due to influx of CO2 into the solution from the atmosphere, and all precipitates were analyzed within 5-45 mins. In all cases the crystals were imaged using scanning electron microscopy (SEM), and the polymorph was identified by IR spectroscopy and Raman spectroscopy. The FTIR spectra of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) differ significantly in the region of OH stretching vibrations (36003000 cm1), where the sharp vibrational band at 3000 cm1 provides 3 a fingerprint for COM.36 Distinguishing features in Raman spectra are that COM possesses two defined stretching bands located at 1460 and 1490 cm-1, whereas COD displays a single sharp band -1 at 1475 cm (Supporting Figure 1). Figure 1. Electron micrographs of calcium oxalate precipitated in bulk solutions with concentrations of (a) 2.5 mM (COM), (b) 0.5 mM (COD + COM) and (c) 0.25 mM (COD). The inset in (b) shows small particles present on the surfaces of the COD crystals. While the expectation from these bulk experiments was to obtain a homogeneous population of ACO particles at early reaction times, this was not observed. Instead, COM and/ or calcium COD was identified, where their proportions depended on the initial reagent concentrations employed. COM was the principal polymorph formed at solution concentrations of 2.5 mM and above, where these crystals appear in the form of spindles (Figure 1a), while COD was observed at concentrations below 2.5 mM, appearing