Nicolet 5dxb spectrometer

FTIR analysis of chitosan and collagen powders was performed using a Nicolet 5DXB spectrometer (Thermo Scientific, Boston, MA, USA). The polymer powders were analyzed by adding the polymer to a KBr powder. The polymer and KBr mixture were then pressed to form a thin film. The films were analyzed by averaging 32 scans at a resolution of 2 cm⁻¹ over a range of 500–4000 cm⁻¹. ¹H NMR analysis was performed to verify the degree of deacetylation of the chitosan scaffold. Chitosan was first dissolved in 2.5% DCl D₂O at 10 mg/mL. A 300 MHz spectrometer (Bruker Avance III 300, Billerica, MA, USA) was used to acquire the spectra in triplicates at 25 °C. The degree of deacetylation was determined by calculating the integrals of the protons on the H-Acetyl group and the protons on the H2/H6 group. The following equation was then used to determine the degree of deacetylation: $$\mathrm{D}\mathrm{D}\left(\mathrm{\%}\right)=\left[1-2\left(\frac{{\mathrm{I}}_{\mathrm{H}-\mathrm{A}\mathrm{c}\mathrm{e}\mathrm{t}\mathrm{y}\mathrm{l}}}{{\mathrm{I}}_{\mathrm{H}2/\mathrm{H}6}}\right)\right]\times 100$$
where DD (%) is the degree of deacetylation in percent, I_H-Acetyl is the integral of the protons on the acetylated group, and I_H2/H6 is the integral of the protons on the monomer hexose rings.