Single reflection atr accessory

The isolated HA stretching frequencies were investigated using a Shimadzu Fourier transform infrared spectrophotometer (FT-IR) with a single reflection ATR accessory (Shimadzu, Kyoto, Japan) in a spectral range of 4000–650 cm⁻¹. Thermogravimetric analysis (TGA) was performed (STA 600 Perkin Elmer, Waltham, MA, USA). The morphology of the isolated HA was examined using field emission scanning electron microscopy (FE-SEM) (Carl Zeiss, Oberkochen, Germany). Energy-dispersive X-ray spectroscopy (EDS; Oxford instrumentation, ZEISS, Begbroke Science Park, UK) was used to confirm the presence of the elements calcium (Ca) and phosphorus (P) in the material. Powder X-ray diffraction (P-XRD) patterns confirmed the isolated HA by matching the crystallographic phases. P-XRD was documented with a Bruker D8 (Ettlingen, Germany) diffractometer using the Cu K = 1.5406 radiation range of 20°–80° with a step size of 0.01. Transmission electron microscopy analyses were performed (JEOL, JEM 2100, Dearborn Road Peabody, MA, USA). SAED analysis was also performed to evaluate the crystallinity of the isolated nano HA (JEOL, 6390LA/OXFORD XMX N, Dearborn Road Peabody, MA, USA).

Optical rotations were determined using a JASCO P-2200 digital polarimeter (JASCO, Tokyo, Japan). The UV spectra were measured with a Thermo Scientific Genesys 50 spectrometer (Thermo Scientific, Waltham, MA, USA). ECD spectra were measured with a JASCO J-820 spectrometer. Infrared spectra (IR) were recorded with a Shimadzu IRSpirit FT-IR spectrometer (Shimadzu, Kyoto, Japan) with a QATR-S single reflection ATR accessory, using neat samples. The NMR spectra were obtained via the JEOL JMN-ECA600 and JMN-ECS400 NMR spectrometers (JEOL, Akishima, Japan), with tetramethylsilane as an internal standard; chemical shifts are stated as δ values. HRMS data were recorded on a JMS-700 MStation (FAB) mass spectrometer. MPLC analysis was performed with C₁₈ cartridges (Sfär C18 D, Biotage, Charlotte, NC, USA). Preparative HPLC analysis was conducted on a GL Science recycling system using an InertSustain C₁₈ column (5 μm; 20 × 250 mm) (GL Sciences, Tokyo, Japan). LC/MS analysis was recorded with a Shimadzu LCMS-9030 connected to an Imtakt Cadenza CX-C18 (2 × 50 mm; 3 μm) (Imtakt, Kyoto, Japan).