Evolution of calcium phosphate precipitation in hanging drop vapor diffusion by in situ Raman microspectroscopy

Abstract

The time-evolution of calcium phosphate precipitation by vapor diffusion has been studied by in situ confocal Raman microspectroscopy. A hanging drop configuration within a device known as ‘‘crystallization mushroom’’ was employed in order to improve the Raman signal coming from growing crystals. This innovative methodology allowed to identify and follow the evolution of the precipitates formed at different areas of the drops containing mixed solutions of Ca(CH3COO)2 and (NH4)2HPO4 due to the diffusion of CO2 and NH3 gases released from NH4HCO3 solutions at different concentrations (30 mM, 100 mM and 2 M). Time-dependent in situ Raman spectra indicated that amorphous calcium phosphate (ACP) was the first precipitate appearing just after mixing the Ca- and PO4-containing solutions. A few minutes later, it transformed to dicalcium phosphate dihydrate (DCPD). The lifetime of DCPD strongly depends on the concentration of the NH4HCO3 solutions and thus on the pH increase rate. The pathway for the phase transformation from ACP to DCPD and then to octacalcium phosphate (OCP) followed a dissolution– reprecipitation mechanism. Additionally, OCP acted as temporal template for the heterogeneous nucleation and crystallization of biomimetic carbonate–apatite nanocrystals (cAp). The characterization by TEM, XRPD and Raman spectroscopy of the freeze-dried powders obtained after seven days confirmed that OCP and cAp were the remaining phases when using 30 mM and 100 mM NH4HCO3 solutions. By contrast, working with the highest NH4HCO3 concentration the system evolved to the precipitation of elongated calcite crystals.