Advance 600

The hydroxyl group content of
the EOL was determined by ³¹P NMR spectroscopy, as described
in previous studies. Pyridine and deuterated chloroform were mixed
to prepare a solvent solution [1.6:1(v/v)]. A mixture solution was
prepared by adding 100 mg cyclohexanol (internal standard) and 90
mg chromium acetylacetonate (relaxation reagent) to 25 mL of the solvent
solution. Approximately 20 mg lignin was accurately weighed and placed
in a 4 mL vial. A total of 400 μL of the solvent solution and
150 μL of the mixture solution were used to dissolve the EOL.
The mixture was stirred for 5 min. After mixing, 70 μL of 2-chloro-4,4,5,5-tetramethyl-1,2,3-dioxaphospholane
was introduced into the mixture as a phosphorylating reagent. The
mixture was blended with a vortex mixer for a few seconds. The completely
prepared samples were transferred to a 5-mm NMR tube for analysis
by ³¹P NMR spectroscopy. The ³¹P NMR spectra
of the EOL from 17 runs were obtained using a 600 MHz NMR spectrometer
(ADVANCE 600, Bruker, Germany), equipped with a 14.095 T superconducting
51 mm bore magnet and 5 mm BBO BB-H&F-D CryoProbe Prodigy.

The intramolecular
coupling structure of EOL was investigated using quantitative 2D-HSQC
NMR spectroscopy. Quantitative 2D-HSQC NMR spectroscopy was performed
using a 600 MHz NMR spectrometer (ADVANCE 600, Bruker, Germany). Fifty
milligrams EOL was prepared by dissolving it in DMSO-d₆ for analysis. Each HSQC experiment was preformed using
Bruker’s “hsqcetgpsisp2.2” pulse program with
the following parameters: a 90° pulse, 0.08 s acquisition time,
2.0 s pulse delay, 1JC-H at 150 Hz, 48 scans, and acquisition of 1024
data points (for ¹H) over 512 increments (for ¹³C). Data processing and analysis were performed using MestReNova
v6.0 software. The coupling structure of the EOL sample according
to HSQC spectroscopy was determined by correlating the data from the
databases cited in the literature.^{44 (link),45 (link)} The C9 unit
(S unit and G unit) in aromatic/unsaturated (δC/δH 100–125/6.5–7.5)
regions and the coupling structure (β-O-4, β–β,
and β-5) in the oxygenated aliphatic side chain (δC/δH
50–90/2.5–6.0) regions were determined by a quantitative
method based on the 2D-HSQC spectra, using aromatic units as internal
standards.^{44 (link)} The internal standard (C9)
and coupling structures (I_x %) are calculated
as shown in eqs 1 and 2 I_x is obtained
as the integral value of the α-position of β-O-4, β–β,
and β-5.