Deciphering the phosphorylation of chitosan through complementary 1H and 31P{1H} DOSY NMR

Chemical modification of chitosan through phosphorylation has gained significant attention for expanding its applications. However, confirming whether phosphorylating agents form covalent bonds with the chitosan backbone or remain as non-covalently associated species has remained challenging using conventional analytical techniques. The diffusion-ordered spectroscopy (DOSY) NMR of the complementary probes 1 H and 31 P nuclei was used for distinguishing between the results of three phosphorylating agents: phosphoric acid, phosphorous acid, and dimethyl phosphite. While conventional FTIR and common 1D/2D NMR spectroscopy experiments confirmed the presence of phosphorus-containing groups in all samples, DOSY NMR analysis revealed critical differences in molecular behavior. Chitosan backbone protons exhibited consistently low self-diffusion coefficients (4-9 × 10 -12 m 2 /s) across all samples. Phosphorus species in samples treated with phosphoric acid and phosphorous acid displayed significantly higher diffusion coefficients (394-548 × 10 -12 m 2 /s), indicating non-covalent association and freely diffusing in solution. In contrast, dimethyl phosphite treatment produced a 31 P resonance at 30.3 ppm with a diffusion coefficient of 11 × 10 -12 m 2 /s, closely matching the chitosan backbone protons values and providing strong evidence for covalent phosphorylation. This work establishes DOSY NMR of complementary probes as a reliable, quick, and simple method for distinguishing between covalent or non-covalent modification of biopolymers.