Optimization of chitosan nanoparticles (ChNs) production process employing a 2(5–2) fractional factorial design was performed to analyze the influence of viscosity average molecular weight (40–120 kDa), the initial concentration of chitosan (2–5 g/L), the initial tripolyphosphate (TPP) concentration (0.8–1.2 g/L), the ratio chitosan/TPP (4/1–10/1) (V/V), and the stirring speed (300–700 rpm), on final nanoparticles size and zeta potential. The measured responses of average particle size and surface charge were determined on Zetasizer Nano ZS. ChNs were prepared using ionotropic cross‐linking of chitosan and TPP and were characterized by Fourier‐transform infrared spectroscopy (FTIR), X‐ray powder diffraction (XRD), and differential scanning calorimetry (DSC). The experiments showed that the size of synthesized nanoparticles depended on initial concentration and molecular weight of chitosan, TPP concentration and stirring speed within the chosen levels. However, the zeta potential was significantly influenced by chitosan molecular weight, chitosan concentration and stirring speed. The FTIR analysis confirmed the interaction between negative charge of TPP with positive charge of chitosan through the appearance of new peaks at 1222 and 895 cm−1 in produced ChNs. XRD and DSC analysis were used to evaluate the effect of crosslinking of chitosan on crystal structure of ChNs.

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