3.5. Physico-Chemical Inclusion Compounds’ Characterization

UV-Vis spectra of the compounds in buffers were recorded in the λ interval 200–900 nm using a Hewlett-Packard 8453 spectrophotometer running 845× UV-Visible ChemStation Software (Agilent, Mulgrave, VIC, Australia). Solutions were inserted in a quartz cell with a path length of 1 cm.

Fourier transform infrared spectroscopy (FTIR) spectra of hesperetin and its derivatives, HP-β-CD and their inclusion compounds were obtained using and analyzed using an FT-IR spectrometer Nicolet 6700 (Thermo Scientific, Waltham, MA, USA) in the range of 350–4000 cm⁻¹. The FT-IR spectra of the inclusion complexes were compared with those of pure hesperetin, HP-β-CD and physical mixture.

NMR spectra (¹H) were obtained at Avance Neo 400 MHz spectrometer (Bruker, Germany) in D₂O (Deutero Gmbh, Kastellaun, Denmark). Chemical shifts were referenced to the solvent (¹H: 4.79 ppm for D₂O) [66] and were expressed in parts per million (ppm, δ).

Thermal analysis was performed using a TA Discovery DSC 2500 (New Castle, DE, USA). Samples weighing about 1.2–2.5 mg were contained in corrugated aluminum pans with a small hole in the lid. Two heating cycles were used. First cycle: heating to 100 °C and holding this temperature for 5 min to remove water; second cycle: samples were cooled to 25 °C and reheated to 300 °C. The constant heating rate of the measurement was 10° K·min⁻¹ in a nitrogen atmosphere with a flow rate of 10 mL·min⁻¹.

The detailed methodology of powder X-ray diffraction (PXRD) was described in the previously paper by Sykuła et al. [19].

The structure and morphology of the samples were examined using JEOL JSM-6610LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) working in low vacuum mode.