Model6600

For further characterization of the early stages
of calcium phosphate
precipitation in the presence and absence of citrate, samples were
collected from the reaction media at different reaction times, quenched
in ethanol, transferred onto carbon-coated copper grids, and observed
using a 30 μm objective aperture in a FEI TEM Titan working
at 300 kV. SAED patterns were collected using a 10 μm aperture,
which allowed the collection of diffraction data from a circular area
∼0.2 μm in diameter. Elemental compositional maps were
obtained in the STEM mode using a Super X EDS detector (FEI), formed
by four SSD detectors with no window surrounding the sample. STEM
images in the FEI Titan TEM of the areas analyzed by EDS were collected
with a high-angle annular dark field detector. At the end of each
titration experiment, the solution was separated from the reaction
media by filtration (Nucleopore, Ø = 200 nm),
and the solids collected were studied by X-ray diffraction (PANalytical
X’Pert Pro X-ray, Cu Kα radiation, λ = 1.5405 A°,
3°–50° 2θ range, scanning rate of 0.11°
2θ s^–1), FTIR (ATRproONE-FTIR, Jasco Model
6600, frequency range of 400–4000 cm^–1, resolution
of 2 cm^–1, and 100 accumulations), FESEM (Zeiss
SUPRA40VP), and TEM (FEI Titan, 300 kV).

The morphology and size of the synthesized nanoparticles were analyzed by transmission electron microscopy (TEM Philips Model CM20, Eindhoven, The Netherlands) equipped with an energy dispersive X-ray spectrometer (EDAX). The size of the particles was analyzed by using ImageJ 1.47 software. The ζ potential, hydrodynamic radius and hysteresis cycles were measured as already described by García-Rubia et al. [20 (link)] using Malvern Zetasizer software (Malvern Instruments, Malvern, Worcestershire, UK) and a superconducting quantum interference device (SQUID) 5 T magnetometer (Quantum Design MPMS XL, San Diego, CA, USA), respectively. Powder X-ray diffraction (XRD) analysis was performed with an Xpert Pro X-ray diffractometer (PANalytical, Almelo, The Netherlands) by using the Cu Kα radiation, 20°–60° in 2θ (0.01°; 3 s). Fourier-transform infrared (FT-IR) analysis was carried out using a FTIR spectrometer (model 6600, Jasco, Tokio, Japan) equipped with an attenuated total reflection (ATR) diamond crystal window (ATR ProOne). The surface of the sample was pressed against the ATR window and infrared spectra were acquired. A total of 100 scans were collected in the wavenumber range from 4000 to 400 cm⁻¹, at 2 cm⁻¹ of resolution.