Lignin Characterization via SEC-RID

To analyze the lignin via size-exclusion chromatography, the lyophilized lignin samples were dissolved in a 80:20 (v/v) solution of 0.1 M NaNO₃:0.005 M NaOH/CH₃CN, syringe-filtered (0.22 μm), and loaded onto an Agilent 1260 series HPLC equipped with a Waters Ultrahydrogel ™ 250 (Milford, MA, USA) column. The mobile phase was also an 80:20 (v/v) mixture of 0.1 M NaNO₃:0.005 M NaOH/CH₃CN at a flow rate of 0.6 mL min⁻¹ at 45 °C. The lignin was detected using an Agilent 1260 infinity refractive index detector (RID) detector. Polyethylene glycol (PEG) standards (0.4, 0.6, 1.0, 1.4, 4.3, 6.7, 12.6, and 20.6 kDa; Waters™) were run on the system to generate a standard curve. Based on the reference elution volumes, the number average (M_n) and weight average (M_w) molecular weights were calculated. The number of monomeric units was calculated by utilizing an approximate molecular weight of a lignin monomer as ~187 g/mol. 2D HSQC NMR was performed on the solubilized lignin according to the procedure outlined in Li et al. [58 (link)].