Crosslinking and periodate oxidation of extracted cellulose
Cellulose was extracted from the powdered oven-dried pine needles of P. wallichiana by alkali treatment as per our previous work [10]. Briefly, pine needles were refluxed with 4% (w/v) NaOH for 4 h followed by bleaching with 1.7% NaClO2 solution (w/v, pH 4), to remove lignin and hemicelluloses. Cellulose was then filtered, subsequently washed with distilled water until neutral pH of the washing solution was attained and cellulose was dried at room temperature. 5 g of extracted cellulose was mixed with 50 g of 17.5% NaOH and stirred at 50 °C for 20 min. Then, epichlorohydrin (50 g) and ammonium hydroxide (50 g, 28% NH4OH solution) were added and the reaction mixture was refluxed at 40 °C for 4 h [29]. The insoluble crosslinked cellulose (CC) was filtered and washed with ethanol and water and dried at room temperature. The CC (1.0 g) was oxidized with NaIO4 (1.65 g) at room temperature with constant stirring for 72 h in the dark [10]. The product, crosslinked cellulose dialdehyde (CCDA), was filtered, washed with ethanol and water, dried at room temperature and the degree of oxidation of cellulose was calculated by the determination of the aldehyde content by Schiff’s base reaction with hydroxylamine hydrochloride [10].