Mechanisms and Additive Effects in the Early Stages of Calcium Phosphate Nucleation

Calcium phosphates are present in numerous geological, industrial and biological settings, making up the majority of our bone’s inorganic content; however, their formation and transformation pathways are not fully understood. In this work, state-of-the-art potentiometric titration methods were developed, shown to be critical in understanding this system and in avoiding premature phase separation. The initial ion association, or pairing, between Ca2+ ions and the three phosphate ions (H2PO4, HPO42- and PO43-) was quantitatively evaluated; it could be shown that the Ca2+ - PO43- interaction is two orders of magnitude weaker than previously assumed, and actually becomes negligible below a pH around 9. The changed ion pair speciation means that [CaHPO4]0 is dominant between pH 6-10 and likely plays a key role in the formation of calcium phosphate clusters and early phase separation. Investigation of the following stages in the titration reveals a strong pH dependence. At low pH, nucleation directly forms a crystalline phase, while intermediate phases become more prevalent as pH increases – up to three distinct precursor phases are present. Further it could be shown that several of these intermediates likely correspond to unique amorphous calcium phosphates; at high pH it was also found that such phases could be stable in aqueous media on the timescale of days. Indeed the nucleation event itself, at least at pH 9.8, was plausibly shown to be a liquid-liquid phase separation; the binodal for this process was also determined. Remarkably the miscibility gap was seen to be based on PO43-, resulting in a tricalcium phosphate like composition. The interplay between thermodynamic and kinetic influences on the nucleation was also studied in the range containing the physiological pH 7.4. A counter-intuitive effect was observed, where slower addition produced a thermodynamically less stable product, dicalcium phosphate dihydrate, whereas fast addition produced a more stable product, poorly crystalline hydroxyapatite. Interestingly, at addition rates between the extremes, a mixed product containing both phases was recovered. Finally, multiple biologically and industrially relevant additives were screened, including poly-amino acid model systems similar to proteins involved in crystal formation. A range from no effects, to influence along all stages of the nucleation pathway was seen. The results presented in this work are of direct interest to a wide range of communities from bio-mineralisation, to geology and industrial areas, pproviding insight into the solution chemistry and nucleation behaviour of calcium phosphates.