3) NMR analysis

Quantitative analysis of PEG modification was performed on nuclear magnetic resonance (NMR, D2O, 500 MHz) spectroscopy by using internal standard method [28]. Potassium hydrogen phthalate (KHP) was chosen as the internal standard (δ at 7.50 ppm) and prepared in the same solution with the analytes at a constant concentration of 0.4 wt%. First, a standard curve was plotted using the integration ratio of 1H NMR peak area between 0.4 wt% KHP (δ at 7.50 ppm) and ethylene units on pristine PEG (varied from 0.05 wt% to 0.4 wt%, δ at 3.56 ppm). All analyzed samples prepared for the standard curve contained 0.8 wt% of pristine alginate. PEG-grafted alginate samples were then dissolved at 0.8 wt% in D2O solvent containing 0.4 wt % of KHP. The concentrations of PEG in those samples were quantified by correlating the integration ratio of 1H NMR peak area between KHP and PEG grafted on alginate to the standard curve. The molar ratio between PEG and alginate in these samples were calculated according to their mass fraction and molecular weight, indicating the efficiency of PEG modification.