Feb 06, 2026
Molecular weight determination of lignin via gel permeation chromatography
- Lisa Stanley1,
- Rui Katahira1,
- Gregg T. Beckham1
- 1Renewable Resources and Enabling Sciences Center, National Laboratory of the Rockies, Golden, CO, USA
- National Laboratory of the Rockies (NLR)Tech. support email: [email protected]
Protocol Citation: Lisa Stanley, Rui Katahira, Gregg T. Beckham 2026. Molecular weight determination of lignin via gel permeation chromatography. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj1r15vk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 29, 2025
Last Modified: February 06, 2026
Protocol Integer ID: 236015
Keywords: lignin, molecular weight, gel permeation chromatography (GPC), reductive catalytic fractionation (RCF), lignin extraction, lignin depolymerization, diode array detector (DAD), molar mass, size exclusion chromatography, lignin oxidation, molecular weight determination of lignin, gel permeation chromatography lignin, physicochemical properties of lignin, lignocellulosic biomass, major components of lignocellulosic biomass, organic gel permeation chromatography, important for lignin valorization, lignin valorization, insoluble lignin, lignin, molecular weight determination, molecular weight of water, cellulose, molecular weight, aromatic biopolymer, physicochemical property, hydrophobicity, hemicellulose
Funders Acknowledgements:
This work was authored by the National Laboratory of the Rockies, operated by Alliance for Energy Innovation, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. This work was supported by the Office of Critical Materials and Energy Innovation and Bioenergy Technologies Office (BETO). The views expressed in this protocol do not necessarily represent the views of the DOE or the U.S. Government.
Grant ID: DE-AC36-08GO28308
Disclaimer
This protocol is for research purposes only.
Abstract
Lignin is a complex, aromatic biopolymer found in the cell walls of plants, where it imparts rigidity, hydrophobicity, and pathogen resistance. It is one of the three major components of lignocellulosic biomass, along with cellulose and hemicellulose.
The physicochemical properties of lignin depend on several factors including the plant species, the method of isolation, its chemical structure, and the abundance of functional groups. Among these characteristics, the molecular weight (MW) or molar mass distribution is important for lignin valorization to fuel, chemicals, and materials.
This protocol describes the most commonly used and versatile technique for quantifying the molecular weight of water-insoluble lignin using an organic gel permeation chromatography (GPC) system.1-6
Guidelines
This protocol utilizes a high-performance liquid chromatography system with diode array detection (HPLC-DAD) manufactured by Agilent Technologies as referenced in 'Materials'. A similar chromatography and detection system can be utilized; however, some parameter nomenclature may deviate depending on the manufacturer.
Materials
Standards:
Polystyrene Calibration Kit (Mp 162-20,000 Da)Agilent TechnologiesCatalog #PL2010-0101
Polystyrene Calibration Kit (Mp 580-3,000,000 Da)Agilent TechnologiesCatalog #PL2010-0100
Toluene, HPLC plusMerck MilliporeSigma (Sigma-Aldrich)Catalog #650579
Reagents:
Acetic AnhydrideMerck MilliporeSigma (Sigma-Aldrich)Catalog #320120
Pyridine anhydrousMerck MilliporeSigma (Sigma-Aldrich)Catalog #270970
Methanol (HPLC)Fisher ScientificCatalog #A452-1
Tetrahydrofuran, suitable for HPLC, inhibitor-freeMerck MilliporeSigma (Sigma-Aldrich)Catalog #34865
Heating and stirring module for sample preparation :
Equipment
Reacti-Therm
NAME
Triple block heating and stirring module
TYPE
Thermo Scientific
BRAND
PI18823
SKU
LINK
Consumables:
Syringes and 0.2 μm syringe filters for sample filtration to remove any undissolved particles
Equipment
NORM-JECT® Luer slip 1 mL syringe
NAME
Lab grade polypropylene/polyethylene syringe
TYPE
B. Braun
BRAND
NJ-9166017-02
SKU
LINK
Equipment
0.2 µm PTFE syringe filters
NAME
syringe filter
TYPE
Agilent Technologies
BRAND
5191-5912
SKU
LINK
13 mm
SPECIFICATIONS
GC vials and caps for sample preparation
Equipment
Crimp top amber vials - 2 mL
NAME
GC vials
TYPE
Agilent Technologies
BRAND
5183-4496
SKU
LINK
certified, deactivated (silanized), 12 x 32 mm
SPECIFICATIONS
Equipment
11 mm silver aluminum crimp caps
NAME
GC vial caps
TYPE
Agilent Technologies
BRAND
6183-4498
SKU
LINK
press-fit PTFE/natural red rubber bi-layer septum
SPECIFICATIONS
Equipment:
Pre-filter
Equipment
Inline filter assembly and frits
NAME
stainless steel inline filter
TYPE
Agilent Technologies
BRAND
5067-1551 and 280959-904
SKU
LINK
2.1 mm, 0.2 µm, max 600 bar, 3 frits, 0.12 x 70 mm connecting capillary
SPECIFICATIONS
Analytical columns
Equipment
PLgel 10^4 Å column
NAME
polystyrene-divinylbenzene copolymer filled column
TYPE
Agilent Technologies
BRAND
PL1110-6140
SKU
LINK
inner diameter 7.5 mm, length 300 mm, particle size 10 µm, MW range 10-450 kDa
SPECIFICATIONS
Equipment
PLgel 10^3 Å column
NAME
polystyrene-divinylbenzene copolymer filled column
TYPE
Agilent Technologies
BRAND
PL1110-6130
SKU
LINK
inner diameter 7.5 mm, length 300 mm, particle size 10 µm, MW range 0.2-60 kDa
SPECIFICATIONS
Equipment
PLgel 50 Å column
NAME
polystyrene-divinylbenzene copolymer filled column
TYPE
Agilent Technologies
BRAND
PL1110-6115
SKU
LINK
inner diameter 7.5 mm, length 300 mm, particle size 10 µm, MW range 0.1-1.5 kDa
SPECIFICATIONS
HPLC System
Equipment
1260 Infinity HPLC System
NAME
High performance liquid chromatography system
TYPE
Agilent Technologies
BRAND
G1311C Quat Pump, G1329B Autosampler, G1316A TCC, G4212B DAD
SPECIFICATIONS
Troubleshooting
Safety warnings
All chemicals used for this procedure are hazardous. Read the Safety Data Sheet (SDS) for each chemical listed and follow all applicable chemical handling and waste disposal procedures. Manufacturer specific SDS information can be found by following the catalog numbers of compounds in 'Materials' list.
Before start
All solvents and chemicals used are listed in the ‘Materials’ section. These are excluded from in-line references to maintain clarity and keep the steps concise.
Preparation of standards
Standards
- Prepare polystyrene (PS) stock solutions by grouping the eighteen PS standards with varying molecular weights as shown in Table 1.
- Dissolve 5 mg of each standard in the group into 50 mL of tetrahydrofuran (THF) along with 1 mL of toluene.
- Transfer 1 mL of each stock solution to a 2 mL amber GC vial. These will be used to create the calibration curve.
Note
Stock solutions should be kept in a refrigerator capable of maintaining a temperature of 4 °C.
PS standard stock solutions' molecular weights
a Retention times may varying depending on the HPLC system used.
Preparation of samples
Acetylation procedure
- Dissolve 20 mg of air-dried lignocellulosic material in a mixture of 0.5 mL pyridine and 0.5 mL acetic anhydride in 5 mL conical vials.
- Heat vials at 40 °C in a heating and stirring module, as described in 'Materials', for 24 h with stirring.
- Terminate the reaction with an addition of 0.2 mL of methanol.
- Subsequently, add 1 mL aliquots of methanol to each sample and dry under nitrogen.
- Repeat methanol additions 7-10 times.
- Further dry samples in a vacuum oven at 40 °C overnight.
- Suspend the dried, acetylated samples in THF to achieve a final dilution of 2 mg of lignin residue to 1 mL of THF.
- Filter the samples through a 0.2 µm PTFE filter into a GC vial.
Note
If insoluble material remains, collect the solids before filtering the liquids through a 0.2 μm filter. Dry the collected solids and weigh the amount to determine what ratio of sample is not represented in the GPC analysis.
GPC analysis
Method specifications
Analysis is performed using an Agilent 1260 series HPLC system with a DAD. Complete method parameters are in the tables below. Use the analytical columns listed in 'Materials'. Total run time for each sample is 40 minutes.
Note
To eliminate air in the HPLC system, purge the solvent lines by bypassing the columns and increasing the flow rate of THF from 1.0 mL/min to 2.5 mL/min in 0.5 mL increments. Once this has been completed, reduce the flow rate to 0.2 mL/min, return the columns to the flow path and increase the flow rate by 0.2 mL/min increments to a final flow rate of 1.0 mL/min.
Note
Lignin components are analyzed using the 260 nm wavelength (bandwidth 80 nm).
Vials and experimental run set up
- Fill the first and second vials in the autosampler with THF (the first vial will be used to clean the injection needle after each injection and the second will be used to run 3-5 blanks at the beginning of the run).
- Place the five PS standard solutions (as prepared in Step 1) after the THF vials, going from PS1 to PS5.
- Run them in order with two runs of PS1 at the beginning.
- Place samples after the standards.
- After every ten samples, run a PS5 and again at the end of the run. This ensures instrument drift remains within the determined acceptance criteria.
- Finish with one THF blank to clear any residues from the system, then shutdown the system.
Note
If at the end of the run the peak retention times of the five standards from PS5 are +/-0.1 minute out of range of each other, then re-run the samples that occurred after the deviation.
Data analysis
Data analysis:
Data analysis is completed using Agilent Cirrus GPC Offline GPC/SEC software version 3.4.1 and Agilent OpenLab CDS, ChemStation Edition version 1.167.13.0. Other GPC analysis software can be utilized; however the depiction of the data and results provided may vary from the examples presented.
Example chromatograms
- Figure 1. Example of a calibration curve with PS5 standard in the background as viewed in Agilent Cirrus GPC Offline GPC/SEC software version 3.4.1.
- Figure 2. Example of a sample chromatogram as viewed in Agilent Cirrus GPC Offline GPC/SEC software version 3.4.1.
Figure 3. Example of a final molecular weight plot a viewed in Microsoft Excel software version 2025.
Protocol references
(1) Palumbo, C. T.; Gu, N. X.; Bleem, A. C.; Sullivan, K. P.; Katahira, R.; Stanley, L. M.; Kenny, J. K.; Ingraham, M. A.; Ramirez, K. J.; Haugen, S. J. Catalytic carbon–carbon bond cleavage in lignin via manganese–zirconium-mediated autoxidation. Nat. Commun. 2024. 15(1), 862.
(2) Gu, N.; Palumbo, C.; Bleem, A.; Sullivan, K.; Haugen, S.; Woodworth, S.; Ramirez, K.; Kenny, J.; Stanley, L.; Katahira, R. Autoxidation catalysis for carbon-carbon bond cleavage in lignin. ACS Cent. Sci. 2023. 9, 2277–2285.
(3) Stone, M. L.; Anderson, E. M.; Meek, K. M.; Reed, M.; Katahira, R.; Chen, F.; Dixon, R. A.; Beckham, G. T.; Román-Leshkov, Y. Reductive catalytic fractionation of C-lignin. ACS Sustain. Chem. Eng.. 2018. 6(9), 11211-11218.
(4) Anderson, E. M.; Stone, M. L.; Katahira, R.; Reed, M.; Beckham, G. T.; Román-Leshkov, Y. Flowthrough reductive catalytic fractionation of biomass. Joule. 2017. 1(3), 613-622.
(5) Salvachúa, D.; Katahira, R.; Cleveland, N. S.; Khanna, P.; Resch, M. G.; Black, B. A.; Purvine, S. O.; Zink, E. M.; Prieto, A.; Martínez, M. J. Lignin depolymerization by fungal secretomes and a microbial sink. Green Chem. 2016. 18(22), 6046-6062.
(6) Anderson, E. M.; Katahira, R.; Reed, M.; Resch, M. G.; Karp, E. M.; Beckham, G. T.; Román-Leshkov, Y. Reductive catalytic fractionation of corn stover lignin. ACS Sustain. Chem. Eng. 2016. 4(12), 6940-6950.