Apr 18, 2025

Public workspaceAromatic monomers analysis for reductive catalytic fractionation of lignin by liquid chromatography with diode array detection

DOI
dx.doi.org/10.17504/protocols.io.bp2l6x6jdlqe/v1
  • 1Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA
  • NREL
    Tech. support email: ftlb_analysis@nrel.gov
Sean P. Woodworth
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DOI: dx.doi.org/10.17504/protocols.io.bp2l6x6jdlqe/v1
Protocol Citation: Sean P. Woodworth, Kelsey J. Ramirez, Gregg T. Beckham 2025. Aromatic monomers analysis for reductive catalytic fractionation of lignin by liquid chromatography with diode array detection. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x6jdlqe/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: January 31, 2024
Last Modified: April 18, 2025
Protocol Integer ID: 94424
Keywords: lignin, c18, aromatic acids, gradient, UHPLC, DAD, HST, lignin-derived aromatics, RCF, Reductive catalytic fractionation
Funders Acknowledgements:
This work was authored by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. This work was supported by Office of Energy Efficiency and Renewable Energy (EERE) and Bioenergy Technologies Office (BETO).
Grant ID: DE-AC36-08GO28308
Disclaimer
This protocol is for research purposes only.
Abstract
An analysis method was developed to quantify the products of lignin derived reductive catalytic fractionation (RCF) by high pressure liquid chromatography paired with diode array detection (HPLC-DAD). This was achieved through chromatographic separation using a mobile phase gradient to separate aromatic analytes on a HPLC reverse phase analytical column.
Guidelines
This protocol utilizes an ultra-high pressure liquid chromatography diode array detection (UHPLC-DAD) system 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:

ReagentGuaiacolMerck MilliporeSigma (Sigma-Aldrich)Catalog #W253200
Reagent4-MethylguaiacolMerck MilliporeSigma (Sigma-Aldrich)Catalog #W267104
Reagent4-EthylguaiacolMerck MilliporeSigma (Sigma-Aldrich)Catalog #W243604
Reagent4-PropylguaiacolMerck MilliporeSigma (Sigma-Aldrich)Catalog #W359807
Reagent4-PropanolguaiacolAmBeedCatalog #A161873
ReagentSyringolMerck MilliporeSigma (Sigma-Aldrich)Catalog #D135550
Reagent4-EthylsyringolAA BlocksCatalog #AA0038V7
Reagent4-PropanolsyringolBiosynthCatalog #FD67266
Reagent4-PropylsyringolEnamineCatalog #EN300-263267
Reagent4-PropenylsyringolChemspaceCatalog #CSC102611715
ReagentPhenolMerck MilliporeSigma (Sigma-Aldrich)Catalog #PHR1047
Reagent4-EthylphenolMerck MilliporeSigma (Sigma-Aldrich)Catalog #E44205
ReagentIsoeugenolMerck MilliporeSigma (Sigma-Aldrich)Catalog #I17206
ReagentMethyl ParabenMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3647
Reagent4-Hydroxybenzoic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #H20059
Reagentp-Coumaric AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #C9008
Reagenttrans-Ferulic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #128708
ReagentMethyl trans-p-CoumarateTokyo Chemical IndustryCatalog #M2259
ReagentMethyl trans-p-FerulateAlfa AesarCatalog #B22657
ReagentMethylhydrocoumarateAlfa AesarCatalog #H64255
ReagentMethylhydroferulateTokyo Chemical IndustryCatalog #M1803



Reagents:
ReagentAcetonitrile OptimaFisher ScientificCatalog # A996SK ReagentFormic acid 98 % pureThermo ScientificCatalog # AC147932500
ReagentMethanol OptimaFisher ScientificCatalog # A454SK

Equipment:


Syringe filters:

Equipment
Syringe Filters, 13 mm PTFE
NAME
syringe filter
TYPE
Cytiva
BRAND
2400
SKU
https://www.cytivalifesciences.com/en/us/shop/lab-filtration/syringe-filters-non-sterile/wwptfe-non-sterile-syringe-filters/acrodisc-syringe-filters-with-universal-membranes-p-36635
LINK
13mm
SPECIFICATIONS


Equipment
syringe filters, 13mm nylon membrane
NAME
syringe filter
TYPE
Cytiva
BRAND
4550
SKU
https://www.cytivalifesciences.com/en/us/shop/lab-filtration/syringe-filters-non-sterile/nylon-non-sterile-syringe-filters/acrodisc-syringe-filters-with-nylon-membrane-p-36371
LINK
13mm
SPECIFICATIONS
Guard column holder:

Equipment
SecurityGuard ULTRA Holder
NAME
guard column holder
TYPE
Phenomenex
BRAND
AJ0-9000
SKU
https://www.phenomenex.com/part?partNo=AJ0-9000
LINK
2.1 to 4.6mm ID
SPECIFICATIONS
Guard column:
Equipment
UHPLC C18 guard cartridge
NAME
guard column
TYPE
Phenomenex
BRAND
AJ0-8782
SKU
https://www.phenomenex.com/part?partNo=AJ0-8782
LINK
2.1 mm ID
SPECIFICATIONS
Analytical column:
Equipment
Luna HST
NAME
reverse phase analytical column
TYPE
Phenomenex
BRAND
00D-4446-B0
SKU
https://www.phenomenex.com/products/luna-hplc-column/luna-c18-2#order
LINK
2.5 µm / 100 x 2.0 mm / 100 Å
SPECIFICATIONS
UHPLC-DAD system:
Equipment
1290 Infinity UHPLC
NAME
Ultra-high performance liquid chromatography system
TYPE
Agilent Technologies
BRAND
1290 Infinity UHPLC
SKU
https://www.agilent.com/en/product/liquid-chromatography/hplc-systems/analytical-hplc-systems
LINK


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 Mobile Phases
Preparation of Mobile Phases
Mobile Phases
  • To make the aqueous 0.2% formic acid, dilute 2.0 mL of formic acid into 1.0 L of 18.2 MΩ⋅cm ultrapure water (UPW). Volumetric preparation is optimal.
  • Acetonitrile is used as the organic mobile phase.

Note
It is advised to prepare sufficient mobile phase for the entire analysis to reduce the need to add additional mobile phase during an active sequence. Adding mobile phase during an active sequence may cause retention time shifting if the new mobile phase varies in pH or acid concentration. This method uses 4.0 mL of mobile phase per injection, calculate how much mobile phase is needed before beginning analysis.

Preparation of Standards
Preparation of Standards
Standards
Individual analytes found in 'Materials' were dissolved into methanol at a concentration of 20 g/L and combined to make mixed working stock standards which were further diluted in methanol to create the calibration using the scheme below.
Calibration Curve


Example calibration curve preparation (click to enlarge)


Note
Reporting limits and linear ranges may vary and should be determined for each instrument and analyte individually. The standard ranges in the table above are suggested starting amounts and may change depending on detector response.

Preparation of Samples
Preparation of Samples
Samples

  • Samples must be filtered through a 0.2 μm or smaller filter prior to injection on the high performance liquid chromatography system (HPLC).
  • Samples expected to be over the linear range of the instrument should be further diluted in methanol to be within the calibration range to ensure accurate analysis and avoid carryover.
UHPLC-DAD Analysis
UHPLC-DAD Analysis
Method Specifications

Analysis of reductive catalytic fractionation products is performed using an Agilent 1290 series ultra high performance liquid chromatography system with a diode array detector (UHPLC-DAD). Complete method parameters are in the tables below.


Note
This method operates at approximately 450 bar on an Agilent 1290 series ultra high performance chromatography system with a binary pump. This exceeds the maximum pressure of a traditional HPLC such as the Agilent 1100 series. Systems that can operate up to 600 bar such as the Agilent 1260 HPLC can utilize this method.


Defined HPLC parameters (click to enlarge)


Defined UHPLC-DAD parameters (click to enlarge)

Use the analytical column listed below, as well as associated guard and holder listed in 'Materials'.

Equipment
Luna HST
NAME
reverse phase analytical column
TYPE
Phenomenex
BRAND
00D-4446-B0
SKU
https://www.phenomenex.com/products/luna-hplc-column/luna-c18-2#order
LINK
2.5 µm / 100 x 2.0 mm / 100 Å
SPECIFICATIONS

Analytical Quality Control
Analytical Quality Control
Multiple strategies are utilized when performing this analysis to ensure instrument stability and
reproducibility.
Calibration Curves

  • A minimum of 5 standard levels should be used.
  • All compounds must have a correlation coefficient (r2) of 0.995 or greater using a linear calibration fit and ignoring the origin.
Calibration Verification Standards (CVS)

A calibration verification standard (CVS) is a standard from the calibration curve that is re-injected every 20 or fewer samples to ensure instrument drift remains within the determined acceptance criteria. Acceptable CVS recoveries for this analysis are within 10% of the expected amount. Acceptance criteria may differ between instruments and should be determined experimentally.
Software of Acquisition and Analysis

Agilent OpenLab CDS Chemstation Edition Rev C.01.10 [287]
Example Chromatography
Example Chromatography
The chromatogram below illustrates the elution order of analytes quantified for hardwood and softwood substrate experiments.

Example elution order chromatogram. (click to enlarge

Analysis of herbaceous substrate experiments also include the additional analytes in the chromatogram below.

Example elution order chromatogram. (click to enlarge)


Protocol references

CITATION
Hoon Choi, Manar Alherech, Jun Hee Jang, Sean P. Woodworth, Kelsey J. Ramirez, Eric M. Karp, and Gregg T. Beckham (2024). Counter-current chromatography for lignin monomer–monomer and monomer–oligomer separations from reductive catalytic fractionation oil. Green Chem.
LINK
https://doi.org/10.1039/D4GC00765D

Citations
Hoon Choi, Manar Alherech, Jun Hee Jang, Sean P. Woodworth, Kelsey J. Ramirez, Eric M. Karp, and Gregg T. Beckham. Counter-current chromatography for lignin monomer–monomer and monomer–oligomer separations from reductive catalytic fractionation oil
https://doi.org/10.1039/D4GC00765D