2.8. Characterization Analysis

The quinone compounds content was measured using a Cary 8454 UV-Vis (Agilent Technologies, Santa Clara, CA, USA) analysis, with the configured sample solution of 3.5 g/L and a measuring absorbance at 450 nm.

The morphology of pulp fiber was analyzed using a Regulus 8220 FE-SEM (Hitachi High-Technologies Corporation, Tokyo, Japan) operated at 5 kV accelerating voltage, with 2000 times magnification.

The surface lignin content of pulp was determined using an X-ray photoelectron spectrometer (XPS), according to the literature approach [35,36], where a linear relationship between the O/C ratio and surface lignin content was applied. The calibration was done using an O/C ratio of 0.74 for pure cellulose (Whatman filter paper), and 0.33 for pure lignin. Before XPS analysis, the pulp sample of the hand-sheets was pumped in a drying oven at 60 °C, for 24 h, to eliminate residual water. The XPS analysis was carried out with Kratos Axis Ultra spectrometer, using a monochromatic AI k (alpha) source (10 Ma,15 V), probing the surface of the sample to a depth of 5–7 nm, ranging from 0.1 to 0.5 atomic percent, depending on the element. The Kratos charge neutralizer system was used on all specimens. Survey scan analysis was taken with an analysis area of 300 × 700 μm and a pass energy of 160 eV, while the high-resolution scan was also recorded with an analysis area of 300 × 700 μm and a pass energy of 20 eV. Measurements were taken at three different spots on each sample to attain an average over the heterogeneity of the samples.

The Fourier transformed infrared (FT-IR) spectra of the hand-sheets and lignin were recorded using a spectrophotometer (IR Irdison-21, Shimadzu, Japan), with a resolution ratio of 4 cm⁻¹, scanning speed of 32 s⁻¹, and scanning range of 4000–500 cm⁻¹.

The crystallinity of the pulp fiber was measured by X-ray diffraction (XRD D8-DVANCE Bruker, Germany). The measurements were conducted under the conditions of X-ray 40 kW and 35 mA, with an angle from 5 to 60°. Crystallinity was determined according to Equation (1) [31,37]:

where, CrI is the crystallinity of cellulose, I₀₀₂ is the scattering intensity of the diffraction of 002 plane, and I_am is the diffraction intensity at 2θ = 15.6°.

NMR (models for Bruker AVANCE III 500 MHz, Bruker, Karlsruhe, Germany) were recorded, according to the literature method [34], by using a spectrometer equipped with a DCH cryoprobe. HSQC spectra were recorded at 25 °C, using the Q-CAHSQC pulse program. Matrices of 2048 data points for the ¹H-dimension (13 to −1 ppm) and 1024 data for the ¹³C-dimension (160 to 0 ppm) were collected, with the relaxation delay set at 6 s. The lignin samples were dissolved in 0.5 mL of dimethylsulfoxide-d6 (DMSO-d₆), and chemical shifts were referenced to the solvent signal (2.50/40.21 ppm).