Low-Temperature ¹³C Solid-State NMR Study of Cryo-Fixed NaHCO₃ Solutions

Calcium carbonate (CaCO₃) is one of the most abundant minerals on Earth, and is mainly present in limestone and sea waters. CaCO₃ is also of major importance in Biology as it is involved in biomineralization processes at the origin of shells and skeletons of many invertebrates such as mollusks, corals, urchins…¹. CaCO₃ can be found in 3 polymorphic forms: calcite, aragonite and vaterite, from the most to the least stable form, respectively. However, amorphous calcium carbonate (ACC) is often observed as the first nucleated solid before the formation of the crystalline phases, in line with the Ostwald’s rule of stage. The nucleation mechanism has been described by Gibbs through the so-called classical nucleation theory (CNT), which involves the formation of critical size nucleus by monomer addition². Nevertheless, CaCO₃has been intensively studied by its tendency to not follow a classical pathway of nucleation. As such, it has been reported the existence of stable and soluble entities before the first nucleation of any solid phase. These so-called prenucleation clusters (PNC) are described as nanometric and highly dynamic entities in equilibrium with free ions in solution³. Since this discovery, no consensus has emerged concerning their composition and structure as well as their mechanism of formation and transformation⁴.<br/>In order to progress on the comprehension of the local environment and the dynamical behavior of these clusters, we used low-temperature (-120°C) solid-state NMR spectrometry. To allow such analysis we use a stopped flow instrument coupled to a freeze quench device. First, the solution containing PNC is prepared with the stopped-flow allowing to access very short mixing time (~10 ms). Then, the resulting out-of-equilibrium solution is vitrified by spraying it into cold isopentane (» -140°C). Thus, PNC are cryo-fixed and stay stable during the solid-state NMR analysis. We already successfully used this approach to investigate amino-acid stabilized prenucleation clusters as well as dense liquid phases of CaCO₃ but the vitrification process was not automatized.⁵<br/>As the carbonate speciation (HCO₃^- <i>vs</i> CO₃^2-) is a key parameter of PNC structure, a preliminary low-temperature ¹³C solid-state NMR study has been conducted on cryo-fixed NaHCO₃ solutions at various pH (from 7 to 11), concentrations (from 25 to 400 mM) and temperature (from -120 to -50°C). The main ¹³C NMR parameters, including ¹³C isotropic chemical shift, line width and chemical shift anisotropy (CSA), have been systematically investigated. We show that the HCO₃^-<i>/</i>CO₃^2- molar ratio is depending on the pH and that at low temperature the “apparent” HCO₃^-<i>/</i>CO₃^2- pKa is shifted to lower values compared to ambient temperature. Surprisingly, we also evidence the impact of the initial concentrations on this “apparent” HCO₃^-<i>/</i>CO₃^2- pKa, which is increasing with the molarity. Finally, analysis of ¹³C CSA parameters shows that carbonate dynamics in the vitrified solutions does not drastically change when the temperature is varied from -120 to -50°C. Hence, the present low temperature solid-state NMR study of vitrified carbonate solution is the first step toward structural investigation of CaCO₃ PNC in similar conditions.<br/><br/>References:<br/><br/>1. Lowenstam, H. A. & Weiner, S. <i>On Biomineralization</i>. (Oxford University press, New York Oxford, 1989).<br/>2. Gibbs, J. W. & Smith, A. W. On the equilibrium of heterogenous substances. <i>Trans. Conn. Acad. Arts Sci.</i> <b>Vol.III, Chapter V and IX</b>, (1874).<br/>3. Gebauer, D., Völkel, A. & Cölfen, H. Stable Prenucleation Calcium Carbonate Clusters. <i>Science</i> <b>322</b>, 1819–1822 (2008).<br/>4. Karthika, S., Radhakrishnan, T. K. & Kalaichelvi, P. A Review of Classical and Nonclassical Nucleation Theories. <i>Cryst. Growth Des.</i> <b>16</b>, 6663–6681 (2016).<br/>5. Ramnarain, V. <i>et al.</i> Monitoring of CaCO₃ Nanoscale Structuration through Real-Time Liquid Phase Transmission Electron Microscopy and Hyperpolarized NMR. <i>J. Am. Chem. Soc.</i> <b>144</b>, 15236–15251 (2022).