Styragel hr4e column

Gel permeation chromatography was used to determine the molecular weight of lignin. First, the sample was derivatized by adding 0.9 mL glacial acetic acid and 0.1 mL acetyl bromide to 5 mg of lignin powder. The sample was stirred for 2 h at room temperature in closed vials. The solution was transferred to a round flask and evaporated in a rotary evaporator (Heidolph) at 50 °C and 50 mBar. Subsequently, the sample was washed twice with 1 mL tetrahydrofuran (THF) followed by solvent evaporation, dissolved in 1 mL THF, and filtered through 0.22-μm hydrophobic filters (Sartorius, Göttingen, Germany). Finally, the samples were analyzed by HPLC using a UV detector (set at 280 nm) and a Styragel^® HR 4E column (Waters, Milford, MA, USA), operated at 40 °C, with THF as mobile phase, and a flow rate of 0.6 mL/min. The calibration was done by using polystyrene (Sigma-Aldrich, St. Louis, MO, USA). The numbers were rounded up at 100 s due to the resolution of the method.

Molecular
weights were determined by a gel permeation chromatography (GPC) system,
Waters 1515. Isocratic high performance liquid chromatography (HPLC)
pump with a Waters 2410 refractive index detector, a Waters 717 plus
autosampler, and a Rheodyne (Cotati, CA) injection valve with a 20
μL loop. The samples were eluted with CHCl₃ (HPLC
grade) through a linear Styragel HR4E column (Waters) with a molecular-weight
range of 50–100K Da at a flowrate of 1 mL/min. The molecular
weights were determined relative to a polystyrene standards (Polyscience,
Warrington, PA) calibration curve having weight-average molecular
weight (M_w) from 600 to 50 000.

First, the samples were derivatized by adding 0.9
mL of glacial acetic acid and 0.1 mL of acetyl bromide to 5 mg of
powdered lignin. The mixture was stirred for 2 h at room temperature
in closed vials. The solution was then transferred to a round bottom
flask, and the solvents were evaporated in a rotary evaporator (Heidolph,
Schwabach, Germany) at 50 °C and 50 mbar. Subsequently, the sample
was washed twice with 1 mL of tetrahydrofuran (THF, HPLC grade without
stabilizer) followed by solvent evaporation. The sample was then dissolved
in 1 mL of THF (HPLC grade without stabilizer) and filtered through
0.22 μm hydrophobic syringe filters (PTFE; Sartorius, Göttingen,
Germany). Finally, the samples were analyzed by HPLC using a UV detector
set at 280 nm and a StyragelHR 4E column (Waters, Milford, MA), operated
at 40 °C, with THF as the mobile phase, and a flow rate of 0.6
mL/min. The calibration was done using polystyrene (Sigma-Aldrich,
St. Louis, MO). The numbers were rounded up at 100 s due to the resolution
of the method. The calibration curve was prepared with polystyrene
standard in a molecular weight (MW) range of 0.5–90 kDa.