2.2.4. Preparation of chitosan nanofibers and nanoparticles
Chitosan nanofibers and nanoparticles were prepared using direct ultrasonication of d-chitin NFs dispersion. Before sonication, concentration of the dispersion was adjusted to 0.1% (w/v) in a 100 ml glass beaker. Ultrasonication was carried out using ultrasound tip operating at 24 kHz frequency and 360 W power (UP400S, Hielscher Ultrasound Technology, Germany) for 15 min. Centrifugation technique was utilized for separation of chitosan nanoparticles (chitosan NPs) and chitosan nanofibers (chitosan NFs) from water soluble chitosan oligomers. Before centrifugation, the solution was diluted in 9 fold in distilled water. Then the solution was centrifuged at 9000 rpm to separate water soluble chitosan oligomers. Separation of chitosan NPs and chitosan NFs was difficult with centrifugation technique as resulting dispersion is not stable. However, chitosan NPs could be separated using simple gravity filtration using ash-less cellulose filter papers (Whatman 42). Filtrate contained chitosan NPs which are almost free of chitosan NFs. Residue consisted of mostly chitosan NFs and some chitosan NPs.