31P-NMR Analysis [12 (link)]. An accurately weighed amount of lignin (approximately 30 mg) was dissolved in 425 μL of anhydrous CDCl3/pyridine solution (1:1.6 (v/v)). 100 μL of a standard solution of cholesterol (0.1 M in anhydrous CDCl3/pyridine solution) containing Cr(III) acetylacetonate as the relaxation agent was added. Finally, 75 μL of 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (Cl-TMDP, 95%, Sigma-Aldrich) was added, and the mixture was stirred at room temperature for 2 h. The mixture was then transferred into 5 mm NMR tubes, and the spectra were measured on a Bruker (Billerica, MA, USA) AVANCE 400 MHz spectrometer equipped with a 5 mm double resonance broadband BBI inverse probe (256 scans at 20 °C), operated by Topspin software (Version 3.5). All chemical shifts reported are relative to the reaction product of water with Cl-TMDP, which gives a sharp signal in pyridine/CDCl3 at 132.2 ppm. NMR data were processed with MestreNova (Version 8.1.1, Mestrelab Research, Santiago de Compostela, Spain).
HSQC Measurements. Samples of around 50 mg were dissolved in 600 μL DMSO-d6 (providing NMR sample solutions with concentrations of around 83 mg/mL); chromium acetyl acetonate was added as spin-relaxing agent at a final concentration of ca. 1.5–1.75 mg/mL. HSQC spectra were recorded at 303 K on a Bruker (Billerica, MA, USA) AVANCE 400 MHz spectrometer equipped with a 5 mm double resonance broadband BBI inverse probe operated by TopSpin software (Version 3.5). The Bruker hsqcetgp pulse program in DQD acquisition mode was used, with NS = 32; TD = 2048 (F2), 512 (F1); SW = 15.0191 ppm (F2), 149.9819 ppm (F1); O2 (F2) = 2000.65 Hz, O1 (F1) = 7545.96 Hz; D1 = 2 s; CNST2 (1J(C–H) = 145; acquisition time F2 channel = 85.1968 ms, F1 channel = 8.4818 ms; pulse length of the 90° high power pulse P1 was optimised for each sample. NMR data were processed with MestreNova (Version 8.1.1, Mestrelab Research, Santiago de Compostela, Spain).
13C-NMR Measurements. Samples of approx. 80 mg of lignin were dissolved in 550 μL DMSO-d6; 50 μL chromium acetyl acetonate in DMSO-d6 (approx. 1.5 mg mL−1) were added as a spin-relaxation agent; 50 μL of trioxane in DMSO-d6 (approx. 15 mg mL−1) was used as an internal standard. The spectra were recorded at room temperature on a Bruker (Billerica, MA, USA) AVANCE 400 MHz spectrometer equipped with a 5 mm double resonance broadband BBI inverse probe operated by TopSpin software (Version 3.5). An inverse-gated proton decoupling pulse sequence was applied with a 90° pulse width, a relaxation delay of 1.7 s and an acquisition time of 1.2 s. A total of 20000-24000 scans were acquired for each spectrum. NMR data were processed with MestreNova (Version 8.1.1, Mestrelab Research, Santiago de Compostela, Spain).
