Analysis of wall constituents by 2D spectroscopy HSQC NMR

Ball-milled de-starched, alcohol insoluble material (25 mg) was dissolved in 0.75 mL of DMSO-d6 and 10 μL of [Emim] OAc-d14 as previously described⁵⁶. The dissolved lignocellulosics were subjected to a 2D HSQC NMR experiment acquired on a Bruker AVANCE 600 MHz NMR spectrometer equipped with a 5-mm TXI ¹H/¹³C/¹⁵N cryo-probe using the pulse sequence ‘hsqcetgpsisp.2’. The experiments were carried out at 25 °C with the following parameters: spectral width 12 ppm in F2 (¹H) dimension with 4096 data points (TD1) and 160 ppm in F1 (¹³C) dimension with 256 data points (TD2); scan number (SN) of 200; inter scan delay (D1) of 1 s. The chemical shifts were referenced to the DMSO solvent peak (δ_C 39.5 ppm, δ_H 2.5 ppm). The NMR data was quantified as described previously using Bruker’s Topspin 3.1 software^56,57. The acetylation on xylan was quantified as described below. In brief, the signals in the aromatic region (H1-C1 signals of 2-O-Ac-Xyl, 3-O-Ac-Xyl, 2,3-O-Ac-Xyl, Xyl (xylan) and reducing ends of Xylan (α/β-Xyl-R)) were summed up to 100%, and the signal in the aliphatic region were integrated separately to calculate the relative content of each form of O-acetyl- xylan unit. The relative content of 2-O-Acetyl and 2,3-O-Acetyl-Xylan units were calculated from H2-C2 signal and 3-O-Acetyl-Xylan unit were calculated from H3-C3 signal. The monosaccharide composition [glucose (Glu), xylose (Xyl) and mannose (Man)] was quantified from their anomeric integrals as a fraction of 100%. The compositions of lignin; S (syringyl), G (guaiacyl), H (p-Hydroxyphenyl), FA (ferulate) and pCA (p-coumarate) lignin units were quantified from their aromatic lignin integrals as a fraction of 100%.