Benning, L.G.

Bovet, Nicolas Emile

Dideriksen, Knud

Rodriguez Blanco, Juan Diego

Sand, Karina Krarup

Stipp, Susan Louise Svane

Tobler, Dominique Jeanette

English

Copenhagen University Research Information System

u n i ve r s i t y  o f  co pe n h ag e n  Københavns UniversitetThe effect of aspartic acid and glycine on amorphous calcium carbonate (ACC)structure, stability and crystallizationTobler, Dominique Jeanette; Rodriguez Blanco, Juan Diego; Dideriksen, Knud; Sand, KarinaKrarup; Bovet, Nicolas Emile; Benning, L.G.; Stipp, Susan Louise SvanePublished in:Procedia: Earth and Planetary ScienceDOI:10.1016/j.proeps.2014.08.047Publication date:2014Document versionPublisher's PDF, also known as Version of recordDocument license:CC BY-NC-NDCitation for published version (APA):Tobler, D. J., Rodriguez Blanco, J. D., Dideriksen, K., Sand, K. K., Bovet, N. E., Benning, L. G., & Stipp, S. L. S.(2014). The effect of aspartic acid and glycine on amorphous calcium carbonate (ACC) structure, stability andcrystallization. Procedia: Earth and Planetary Science, 10, 143-148.https://doi.org/10.1016/j.proeps.2014.08.047Download date: 03. Feb. 2020 Procedia Earth and Planetary Science  10 ( 2014 )  143 – 148 Available online at www.sciencedirect.com1878-5220 © 2014 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).Peer-review under responsibility of the Scientific Committee of GES-10doi: 10.1016/j.proeps.2014.08.047 ScienceDirectGeochemistry of the Earth’s Surface meeting, GES-10 The effect of aspartic acid and glycine on amorphous calcium carbonate (ACC) structure, stability and crystallization D.J. Toblera*, J.D. Rodriguez Blancoa, K. Dideriksena, K.K. Sandb, N. Boveta, L.G. Benningc and S.L.S. Stippa aNano-Science Center, Department of Chemistry, University of Copenhagen, Denmark (*Corresponding Author) bLawrence Berkeley National Laboratories, Berkeley, USA  cCohen Geochemistry Laboratory, School of Earth and Environment, University of Leeds, Leeds, LS2 9JT, UK  Abstract The effect of organic molecules on CaCO3 crystallization, in particular on the formation of the initial amorphous calcium carbonate (ACC) phase, is poorly understood despite this knowledge being crucial for designing biomimetic compounds with specific function, strength and stability. We monitored ACC crystallization in the presence of varying concentrations of aspartic acid (ASP) and glycine (GLY). We observed an increase in ACC lifetime with increasing amino acid concentrations and showed that the amino acid molecules sorbed onto the ACC particles. However, little if any difference in composition and atomic structure or the so formed ACC was observed. Similarly, the crystallization pathway of ACC via vaterite and calcite although delayed, was only slightly affected by the added amino acids. The only exemption was at the highest tested ASP concentration where ACC formation was inhibited, The calcite crystals that formed in the presence of ASP had rounded edges and rough surfaces, features that are not observed for the pure, inorganic calcite or calcite formed in the presence of GLY. Overall, the results suggest that the amino acids affected ACC lifetime through the inhibition of crystal nucleation and growth, more so in the presence of ASP than GLY.    © 2014 The Authors. Published by Elsevier B.V. Peer-review under responsibility of the Scientific Committee of GES-10. Keywords: Amorphous calcium carbonate, ACC lifetime, CaCO3 crystallization, amino acids, bimineralization  © 2014 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).Peer-review under responsibility of the Scientific Committee of GES-10144   D.J. Tobler et al. /  Procedia Earth and Planetary Science  10 ( 2014 )  143 – 148 1. Introduction The formation of calcium carbonate (CaCO3) minerals in natural environments frequently occurs in the presence of organic molecules. These are known to strongly modify CaCO3 crystal structure, morphology and properties, often leading to more resilient and stronger crystals [1,2]. Detailed understanding of how organic molecules modify CaCO3 growth kinetics and the mechanisms that control precipitation, is therefore of great interest. At high supersaturation, the initial steps of CaCO3 crystallization usually occur through the formation of poorly ordered amorphous CaCO3 (ACC). This phase is often short lived and quickly transforms to more stable CaCO3 phases such as vaterite, calcite and aragonite [3]. A great deal is known about how the stability of ACC and its crystallization kinetics are affected by temperature, pH and inorganic additives [4-6]. However, less is known about how organic molecules affect the atomic structure, composition, crystallization pathways and stability of ACC. Here, we investigated the role of two amino acids, aspartic acid (ASP) and glycine (GLY) in ACC crystallization using time resolved UV-Vis spectrophotometry combined with synchrotron radiation pair distribution function (PDF) analyses, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), thermogravimetric analyses and electron microscopy.  2. Methods CaCO3 synthesis and crystallization: A 13 mM CaCl2 solution and several 13 mM Na2CO3 solutions with added amino acids (ranging from 2.6 to 87 mM ASP or GLY) were prepared using reagent grade chemicals and ultrapure deionized water (MilliQ). All amino acid containing carbonate solutions were pH adjusted to 11.2 (2 M NaOH) to match the pH of the pure Na2CO3 solution. To follow ACC formation and crystallization processes using UV-Vis spectrophotometry, equal volumes of a calcium and a carbonate (± amino acid) solution were mixed inside a plastic cuvette placed in the spectrophotometer. During measurement, the solutions were continuously stirred and the absorbance (at 450 nm) monitored at 1 second intervals. To obtain compositional and structural information about ACC through XPS and PDF analyses ACC was synthesized by mixing 20 ml of the calcium bearing solution into the carbonate solution (± ASP or GLY) followed by rapid separation of solid and solution using vacuum filtration. Immediately after filtration, the solid was rinsed with isopropanol to remove water and then quickly dried by blowing air onto the solid, following the same method described by Rodriguez-Blanco et al. [7].  CaCO3 characterization: The local atomic structure was determined using synchrotron radiation PDF analysis at beamline 11-ID-B (58.6 keV, λ = 0.2114 Å) at the Advanced Photon Source, Argonne, USA. Kapton capillaries were filled with dried ACC samples and measured for 5 minutes. Station set up, calibration procedures and standard data treatment with Fit2D and PDFGETX2 are detailed elsewhere [8,9]. The surface composition of ACC was quantified with XPS using a Kratos Axis UltraDLD system (AlΚα X-ray source, hv = 1486.6 eV, power = 150 W) and fast freezing techniques [10]. CaCO3 polymorphs were identified by XRD (Bruker D8, Co Κα1,2 radiation, 0.02° step-1 from 10 to 70° 2θ, 1° min−1) and imaged using scanning electron microscopy (SEM, FEI Quanta 3D, 10 kV).  To obtain solid samples, at various times during the crystallization reaction the reacting aqueous suspensions were filter-quenched, the solids rinsed with isopropanol and dried prior to analyses.  3. Results and Discussion The effect of amino acid on ACC crystallization in solution was investigated by monitoring the time dependent change in the solution absorbance using UV-Vis spectrophotometry (Fig. 1A). Previous work has shown that with this method, the formation of the initial amorphous CaCO3 precursor, as well as its subsequent transformation to crystalline phases and the aggregation and settling of the growing crystals can be followed [11]. Figure 1B&C show the results of the spectrophotometric analyses for aspartic acid (ASP) and glycine (GLY) at concentrations between 2.6 and 87 mM. Under all tested conditions (except 87 mM ASP), instantaneous ACC precipitation was observed as illustrated by an immediate increase in absorbance when the calcium and the carbonate solutions were mixed, at t = 10 s. Little to no difference was observed between the pure system and systems with added amino acids at 145 D.J. Tobler et al. /  Procedia Earth and Planetary Science  10 ( 2014 )  143 – 148 concentrations of 2.6 and 6.5 mM, where the ACC was stable for about 50 s. ACC lifetime increased considerably, however, at ASP concentrations of 13 mM (100 s) and 26 mM (175 s, Fig. 1B). Similarly for GLY, the ACC lifetime was prolonged but only at a concentration of 26 mM (75 s) and 87 mM (150 s, Fig. 1C). At 87 mM ASP concentrations, ACC formation was inhibited and direct nucleation and growth of vaterite and calcite were observed after an induction time of 360 s. XRD and SEM analyses also verified these crystallization trends and the effect of the amino acids on the morphology of the precipitates is shown in Fig. 2.   Figure 1: ACC formation and crystallization monitored by UV-Vis spectrophotometry. A) The absorbance profile for the pure system, where ACC forms first and then crystallizes to vaterite or calcite. to marks the induction time before formation of crystalline CaCO3. B & C) Normalized absorbance (450 nm) as a function of time after mixing two solutions of 13 mM CaCl2 and 13 mM Na2CO3, in a series of solutions of ASP (B) and GLY (C).                           Figure 2: SEM images of precipitates from the various ACC crystallization experiments. A) Initial ACC phase in the pure system, showing ~ 300 nm diameter particles; B) Precipitate collected at peak absorbance in the 13 mM GLY system (100 seconds, see Fig. 1C), where most of the ACC has transformed to vaterite and some to calcite; C) calcite from the end of a GLY amended experiment (13 mM GLY) showing identical morphologies to the pure system with sharp crystal edges; and (D) calcite crystals from the end of an ASP amended experiment (13 mM) showing rounded edges.  146   D.J. Tobler et al. /  Procedia Earth and Planetary Science  10 ( 2014 )  143 – 148 In the pure system, ACC crystallized rapidly (< 2 min at 25 °C) to vaterite or calcite (Fig. 2A, B). It then took between 1 and 4 hours before all vaterite transformed to calcite through dissolution / reprecipitation [3,12]. In solutions with the two amino acids, ACC crystallization proceeded as in the pure system and after 4 hours, all crystals in solution were calcite (Fig. 2 C, D). Interestingly, the calcite crystals in ASP solutions did not have as sharp edges as observed for calcite formed in GLY solutions or the pure system (Fig. 2C, D), indicating that the presence of ASP affected calcite crystal growth.  SEM analyses of ACC precipitates from all experiments showed that ACC particles consisted of smooth spheres with diameter ~300 nm (Fig. 2A). The presence, type or concentration of the amino acid did not significantly affect the resulting ACC size or morphology. Similarly, pair distribution function analyses of these ACC precipitates showed no difference in the local atomic structure of the various ASP or GLY ACC. The PDFs for all tested ACC were identical (Fig. 3A) and displayed a structural coherence of less than 15 Å. Furthermore, no detectable change in water content or incorporation of amino acids was observed with thermogravimetric measurements. Consequently, structural and compositional variation is unlikely to be the reason for the longer ACC lifetime observed in the experiments with higher amino acid concentrations (Fig. 1B-D).                      Figure 3: A) PDF of ACC synthesized by mixing 13 mM CaCl2 with 13 mM Na2CO3 solutions, two of which were amended with 13 mM ASP or GLY. The PDF data of the ACC that formed in each of these solutions were identical. B) XPS spectrum showing the nitrogen peak for ASP sorbed onto the surfaces of ACC particles.    It is important to note that both ASP and GLY sorbed to the surface of ACC particles, as demonstrated by a nitrogen peak in the XPS spectra (Fig. 3B). Somewhat higher adsorption was measured for ASP (atomic % ratio N/Ca = 0.8) compared with GLY (N/Ca = 0.3) on ACC formed from solutions with 13 mM amino acid. It is however questionable if this can explain the prolonged ACC lifetime, in particular because previous studies have shown that ACC dissolution is not strongly affected by sorbed organic molecules [13]. Indeed, Ihli et al. [13] did not detect any significant change in ACC composition or water content in the presence of aspartic acid but they observed a longer ACC lifetime, which our results confirm. They argued that the mechanism by which additives stabilize ACC is mainly by inhibiting crystal nucleation and growth. The high amino acid concentrations used in our study and the minimal sorption of amino acids that we observed show that a large fraction of the added amino acids remain in solution and that these likely inhibit nucleation and crystal growth. Further support for this comes from calcite growth studies, where the effects of amino acids on bulk precipitation rates and calcite step velocities were quantified [14,15]. Specifically, a higher growth inhibition was observed for aspartic acid than for glycine because of 147 D.J. Tobler et al. /  Procedia Earth and Planetary Science  10 ( 2014 )  143 – 148 the extra carboxylic group and the particular conformation of ASP, which provides a better fit to the calcite surface sites [15] and thus likely blocks growth sites. Our current study supports and further confirms this interpretation. In solutions with 13 and 26 mM ASP, we see longer ACC lifetime (2 – 4 times longer than equivalent pure system) and at the highest ASP concentration (87 mM) we see a 6 minute induction time for crystal nucleation. Furthermore, calcite crystals with rounded edges formed in the presence of ASP, confirming its effect on growth. In contrast, in solutions with glycine, where the amino acid also sorbed to the ACC surface, the compound does not retard ACC crystallization as much (Fig. 1C) and ACC still forms at GLY concentrations of 87 mM. Finally, calcite formed in GLY had the same morphology as crystals grown in the pure system, indicating little if any effect of GLY on calcite growth (Fig 2C).    4. Summary Results from this study show that the sorption of ASP and GLY both affect the crystallization kinetics of CaCO3 biominerals that form in their presence, to a greater extent in solutions with ASP than GLY. This occurs through enhanced stabilization of the precursor ACC and thus an amino acid inhibition effect during the formation of the crystalline calcium carbonates. However, the ACC composition is little affected and thus any ASP or GLY associated with the ACC plays a minor role in the crystallization process. The stronger impact observed for ASP is explained by ASP binding more strongly to calcite surfaces than GLY, thereby blocking calcite nucleation more effectively. This resulted in calcite crystals with rounded edges whereas in the pure system or in the presence of GLY, crystals had smooth terraces and sharp edges.   Acknowledgements This work was funded by the Engineering and Physical Sciences Research Council (EPSRC) program grant (EP/I001514/1) for the Materials Interface with Biology (MIB) Consortium and by the European Commission Framework 7, through the CarbFix Project, Grant Agreement No: FP7 283148. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. We are grateful for the help provided by Karina Chapman, Peter Chupas, Rick Spence and Kevin A. Beyer, APS on beamline 11 ID-B. References 1. Addadi L, Raz S, Weiner S. Taking advantage of disorder: amorphous calcium carbonate and its roles in biomineralization. Adv Mater 2003; 15: 959–970.  2. Sand KK, Rodriguez-Blanco JD, Makovicky E, Benning LG, Stipp SLS. Crystallization of CaCO3 in water-ethanol mixtures: spherulitic growth, polymorph stabilization and morphology change. Cryst Growth & Des 2012; 12: 842-853.  3. Rodriguez-Blanco JD, Shaw S, Benning LG. The kinetics and mechanisms of Amorphous Calcium Carbonate (ACC) crystallization to calcite, via vaterite. Nanoscale 2011; 3: 265-271.  4. Sawada K. The mechanisms of crystallization and transformation of calcium carbonates. Pure & Appl Chem 1997; 69: 921-928.  5. Loste E, Wilson RM, Seshadri R, Meldrum FC. The role of magnesium in stabilising amorphous calcium carbonate and controlling calcite morphologies. J Crystal Growth 2003; 254: 206–218. 6. Morse JW, Arvidson RS, Lüttge A. Calcium Carbonate Formation and Dissolution. Chem Rev 2007; 107: 342−381 7. Rodriguez-Blanco JD, Shaw S, Benning LG. How to make ‘stable’ ACC: protocol and preliminary structural characterization. Mineral Mag 2008; 72: 283-286.  8. Hammersley AP. FIT2D: An Introduction and Overview. ESRF Internal Report, ESRF97HA02T. Avaliable at http://www.esrf.eu/computing/scientific/FIT2D/FIT2D_INTRO/fit2d.html; 1997 9. Qiu X, Thompson JW. PDFgetX2: a GUI-driven program to obtain the pair distribution function from X-ray powder diffraction data. J Appl Crystallogr 2004; 37: 110-116. 10. Shchukarev A. Electrical double layer at the mineral-aqueous solution interface as probed by XPS with fast-frozen samples. J Electron Spectrosc Relat Phenom 2010; 176: 13–17. 11. Rodriguez-Blanco JD, Vallina B, Blanco JA, Benning LG. The role of REE3+ in the crystallisation of lanthanites. Mineral Mag 2014; (accepted). 148   D.J. Tobler et al. /  Procedia Earth and Planetary Science  10 ( 2014 )  143 – 148 12. Bots P, Rodriguez-Blanco JD, Roncal-Herrero T, Shaw S, Benning LG. Mechanistic insights into the crystallization of amorphous calcium carbonate to vaterite. Cryst Growth Des 2012; 12: 3806-3814. 13. Ihli J, Kim YY, Noel EH, Meldrum FC. The effect of additives on amorphous calcium carbonate (acc): janus behavior in solution and the solid state. Adv Funct Mater 2013; 23: 1575–1585. 14. Orme CA, Noy A, Wierzbicki A, McBride MT, Grantham M, Teng HH, Dove PM, DeJoreo JJ. Formation of chiral morphologies through selective binding of amino acids to calcite surface steps. Nature 2001; 411: 775-779. 15. Montanari G, Bovet N, Stipp SLS. Do amino acids inhibit calcite growth? Mineral Mag 2013; 77: 1781.   

The effect of aspartic acid and glycine on amorphous calcium carbonate (ACC) structure, stability and crystallization

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The effect of aspartic acid and glycine on amorphous calcium carbonate (ACC) structure, stability and crystallization

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