May 31, 2024

Public workspaceAnalysis of monomeric aromatic compounds in alkaline lignin-rich liquors via UHPLC-DAD

DOI
dx.doi.org/10.17504/protocols.io.14egn3q2ql5d/v1
Analysis of monomeric aromatic compounds in alkaline lignin-rich liquors via UHPLC-DAD
  • 1Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA
Morgan A. Ingraham
Open access
DOI: dx.doi.org/10.17504/protocols.io.14egn3q2ql5d/v1
Protocol Citation: Sean P. Woodworth, Morgan A. Ingraham, Stefan J. Haugen, Kelsey J. Ramirez, Gregg T. Beckham, Davinia Salvachua 2024. Analysis of monomeric aromatic compounds in alkaline lignin-rich liquors via UHPLC-DAD. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn3q2ql5d/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 23, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94027
Keywords: Lignin-derived aromatics, aromatic acids, c18, UHPLC, Lignin, DAD
Funders Acknowledgement:
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. Funding was provided by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy (EERE) Bioenergy Technologies Office (BETO) for the Feedstock-Conversion Interface Consortium (FCIC).
Grant ID: DE-AC36-08GO28308
Disclaimer
This protocol is for research purposes only.
Abstract
An analysis method was developed to quantify aromatic compounds present in lignin-rich, alkaline pre-treated liquors produced from corn stover. This method employs ultra-high pressure liquid chromatography paired with diode array detection (UHPLC-DAD). A reverse phase Luna Omega PS C18 column from Phenomenex and a gradient were used to separate various aromatic compounds.
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
Reagents:
ReagentProtocatechuic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #37580-25G-F
Reagent4-Hydroxybenzoic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #240141 ReagentCatecholMerck MilliporeSigma (Sigma-Aldrich)Catalog #PHL82372-100MG
ReagentFerulic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #128708-5G Reagentp-Coumaric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #55823 ReagentVanillinMerck MilliporeSigma (Sigma-Aldrich)Catalog #V1104-2G
ReagentVanillic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #94770-10G ReagentVanillyl alcoholTCI ChemicalsCatalog #V0018
Reagent4-hydroxybenzaldehydeMerck MilliporeSigma (Sigma-Aldrich)Catalog #144088-50G

ReagentAcetonitrile OptimaFisher ScientificCatalog # A996SK
ReagentPhosphoric Acid ACS 85 wt. % in WaterMerck MilliporeSigma (Sigma-Aldrich)Catalog #695017
ReagentEthanol 99.5% ACSThermo ScientificCatalog # AC615090010



Instrumentation
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

Column
Equipment
Luna Omega PS C18
NAME
UHPLC column
TYPE
Phenomenex
BRAND
00D-4752-AN
SKU
https://www.phenomenex.com/products/luna-omega-hplc-column/luna-omega-ps-c18
LINK
2.1 mm x 100 mm, 1.6 µm
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
Guard holder

Equipment
SecurityGuard ULTRA Holder
NAME
guard column holder
TYPE
Phenomenex
BRAND
AJ0-9000
SKU
https://www.phenomenex.com/part?partNo=AJ0-9000
LINK
2.1-4.6 mm ID
SPECIFICATIONS






Safety warnings
Attention
All chemicals used for this procedure are hazardous. Read the Safety Data Sheet (SDS) for all chemicals and follow all applicable chemical handling and waste disposal procedures. Manufacturer specific SDS information can be found by following the CAS 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
Preparation of standards
By weight, create individual 10 g/L stock standards of the following analytes using ethanol (EtOH) as a diluent:
  • Protocatechuic acid
  • Catechol
  • 4-Hydroxybenzoic acid
  • p-Coumaric acid
  • Ferulic acid
  • Vanillyl alcohol
  • Vanillic acid
  • 4-Hydroxybenzaldehyde
  • Vanillin
Combine stock standards to make a 1 g/L aromatics mixed standard using EtOH as a diluent. A potential preparation can be made by adding 1 mL of each of the standards and 1 mL EtOH for an overall 10-fold dilution.

Create a calibration curve with a minimum of 5 points using ultrapure water (18.2MΩ⋅cm)(UPW) as a diluent.


ps cal w units.png
example calibration curve preparation (click to enlarge)



Preparation of mobile phases
Preparation of mobile phases
Mobile phase A consists of 10 mM phosphoric acid in UPW. This can be prepared by adding 0.67 mL of 85% (w/w) phosphoric acid per liter of UPW.

Mobile phase B was acetonitrile.
Sample preparation
Sample preparation
Confirm that the sample matrix is compatible with instrumentation. The calibration range for this method is between 1.0 µg/mL and 500 µg/mL. Any samples with analyte concentrations expected to be greater than 500 µg/mL should be diluted appropriately to minimize the potential for carryover. Filter all samples through 0.2 µm filtered prior to injection.
UHPLC analysis
UHPLC analysis
Analysis is completed on an Agilent 1290 UHPLC system utilizing the following parameters.




ps pump correct.png
UHPLC parameters (click to enlarge)


DAD configuration ps.jpg
Diode array configuration (click to enlarge)



Analysis was completed utilizing wavelengths 265 nm and 280 nm. Quantitation of these aromatic compounds can be accomplished on multiple wavelengths. Discretion should be used in choosing the quantitative wavelength that produces the optimal response.
Data analysis and quality control
Data analysis and quality control
Data analysis was completed using Agilent OpenLab CDS ChemStation Edition Rev. C.01.10

The following criteria are employed to ensure reproducibility and stability throughout the analysis:
  • All analyte calibration curves must have a correlation coefficient (r2) of greater than or equal to 0.995. A quadratic or linear fit may be applied with the origin ignored.
  • A calibration verification standard (CVS) should be analyzed every 20 or fewer samples. This is a point from the calibration curve re-analyzed throughout the instrument run to check for drift. An acceptable recovery range for this method is +/- 10% of the expected concentration.
Example chromatogram
Example chromatogram

image.png
Example chromatogram (click to enlarge)