May 08, 2026

Analysis of HDO autoxidation products by UHPLC-DAD 

 Forked from Polyethylene terephthalic acid deconstruction product analysis by UHPLC-DAD
DOI
https://dx.doi.org/10.17504/protocols.io.kqdg31ex1l25/v1
Analysis of HDO autoxidation products by UHPLC-DAD
  • 1Renewable Resources and Enabling Sciences Center, National Laboratory of the Rockies, Golden, CO, USA;
  • 2BOTTLE Consortium, Golden CO, USA
Stefan J. Haugen
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.kqdg31ex1l25/v1
Protocol Citation: Stefan Haugen, Hannah Alt, Kelsey Ramirez, William E. Michener, Gregg T. Beckham 2026. Analysis of HDO autoxidation products by UHPLC-DAD . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg31ex1l25/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: June 30, 2025
Last Modified: May 08, 2026
Protocol  Integer ID: 221344
Keywords: diode array detection, UHPLC, catalytic products, HDO, Hydrodeoxygenation, autoxidation, phthalic acids, benzoic acid, benzene tricarboxylic acids, benzene dicarboxylic acids, analysis of hdo autoxidation product, lignin oil, hdo autoxidation product, comprehensive understanding of lignin polymeric deconstruction, lignin polymeric deconstruction, lignin, terephthalic acid analysis method, referenced terephthalic acid analysis method, hydrodeoxygenation, performance liquid chromatography with diode array detection, carboxylic acid, performance liquid chromatography, hdo, additional aromatic compound
Funders Acknowledgements:
This work was performed by the National Laboratory of the Rockies, operated by Alliance for Energy Innovation. Funding was provided by the U.S. Department of Energy's (DOE) Office of Critical Minerals and Energy Innovation (CMEI), Alternative Fuels and Feedstocks Office (AFFO) under Contract No. DE-AC36-08GO28308.
Grant ID: DE-AC36-08GO28308
Disclaimer
This protocol is for research purposes only.
Abstract
This procedure is forked from an existing protocol and outlines an analytical method for the quantification of additional catalytic products from autoxidation (AO) of hydrodeoxygenation (HDO) lignin oil. Additional aromatic compounds (benzene tri/di carboxylic acids) are able to be analyzed by extending the method runtime and amending the UHPLC gradient program from the referenced terephthalic acid analysis method. This protocol similarly uses an ultra-high-performance liquid chromatography with diode array detection (UHPLC-DAD) system. The following method allows for a more comprehensive understanding of lignin polymeric deconstruction via HDO and AO.
Guidelines
This protocol utilizes an ultra-high performance liquid chromatography diode array detector (UHPLC-DAD) system manufactured by Agilent Technologies as referenced in 'Materials'. A similar UHPLC-DAD system can be utilized however, some parameter nomenclature may deviate depending on the manufacturer.
Materials
General:
UPW - ultra pure water (18.2 MΩ·cm resistivity at 25°C)

Standard stock materials:
Hemimellitic acidTCI ChemicalsCatalog #H1592
Terephthalic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #185361
Isophthalic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #I19209
Phthalic acidTCI ChemicalsCatalog #P0287
Benzoic acidThermo ScientificCatalog #149135000
1,2,4-Benzenetricarboxylic acidThermo Fisher ScientificCatalog #349032500
1,3,5-Benzenetricarboxylic acidAmBeedCatalog #Catalog #A284402
Biphenyl-3,3-dicarboxylic acidAmBeedCatalog #Catalog #A136778
Protocatechuic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #37580-25G-F

Reagents for standards and calibration preparation:
Potassium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5655
Potassium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P3786
TetrahydrofuranMerck MilliporeSigma (Sigma-Aldrich)Catalog #401757
Methanol OptimaFisher ScientificCatalog # A454SK
Dimethyl sulfoxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #34869

Mobile Phase Reagents:
Phosphoric Acid ACS 85 wt. % in WaterMerck MilliporeSigma (Sigma-Aldrich)Catalog #695017
Methanol OptimaFisher ScientificCatalog # A454SK



Guard Column
Equipment
Zorbax Eclipse Plus C18
NAME
Guard Column
TYPE
Agilent
BRAND
821725-901
SKU
https://www.agilent.com/store/productDetail.jsp?catalogId=821725-901
LINK
2.1 x 5 mm 1.8 µm
SPECIFICATIONS



Analytical Column
Equipment
Zorbax Eclipse plus C18 Rapid Resolution HD
NAME
analytical separation column
TYPE
Agilent
BRAND
959757-902
SKU
https://www.agilent.com/store/en_US/Prod-959757-902/959757-902
LINK
2.1 x 50 mm, 1.8 µm
SPECIFICATIONS


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

Safety warnings
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
Terephthalic acid (TPA) preparation

Prepare a buffer solution for the TPA standard
  1. Prepare a 1M potassium phosphate dibasic (K2HPO4) buffer solution by weighing 87 g of K2HPO4 into 500 mL of 18.2MΩ⋅cm ultrapure water (UPW).
  2. Prepare a 1M potassium phosphate monobasic (KH2PO4) buffer solution by weighing 68 g of KH2PO4 into 500 mL of UPW.
  3. Prepare a 1M phosphate buffer solution by combining 94 mL of K2HPO4 (dibasic) buffer and 6 mL of KH2PO4 (monobasic) buffer.
  4. Check that the pH of the buffer solution is 8.0 ± 0.1 pH units (7.9-8.1). Adjust pH with 10N NaOH or concentrated phosphoric acid if necessary.

Prepare the TPA standard
  1. Weigh out dry TPA standard and create 1000 µg/mL TPA stock standard using the phosphate buffer solution created in the previous step as the stock standard diluent. Example: Weighed 9,800 µg (9.8 mg) of powdered TPA and add 9.8 mL of phosphate buffer solution.
  2. Create a 250 µg/mL TPA working standard using the 1000 µg/mL TPA stock standard and UPW as the diluent. Example: Pipetted 2.5 mL of 1000 µg/mL TPA stock standard and add 7.5 mL of UPW.
  3. Create the following calibration standards using the 250 µg/mL TPA standard working solution and UPW as the diluent.

Example calibration curve preparation for TPA (Click to enlarge)

Mixed Standard Working Solution (mSWS) Preparations

1. Two mixes are prepared, "Aro" and "HDO", that each have their own sets of analytes for creation of two sets of mixed calibration curves, in addition to the separate set of TPA calibration curve levels.

2. Prepare all compounds for analysis at 2.0 g/L in the diluent listed in the table. The diluents indicated here are all miscible with one another. The 'Materials' section has reference to each of the analytes and diluents listed. See next step for mixing of stocks into these two standard mixes.

Calibration curve compound mix scheme. Eight total analytes into two separate mixes. (click to enlarge)


3. With all compounds at 2.0 g/L (2000 ppm each) in their respective diluents, mix stocks as outlined below.
Combination scheme for stocks into two mSWS, Aro and HDO. (click to enlarge)

4. Dilution of these mSWS will create the levels required for robust calibration curves, seen in the table below. The dilutions outlined occur once for a full set of HDO standards, and again for a full set of Aro standards. Preparation should occur from highest level (8) to lowest level (1).

Note that the mixed standard working solution (mSWS) is prepared from either the 'HDO' mSWS, or the 'Aro' mSWS from level 8, level 7, and level 5, while levels 6, 4, 3 and 2 are prepared from level 7, level 5 and level 1 respectively.

A mSWS dilution scheme into calibration levels
5. In combination with the original TPA preparation, the two mSWS (HDO + Aro) standard preparations expand the capability of the method to analyze more compounds with the same stringent analytical quality requirements.
Sample Preparation
  • Samples must be filtered through a 0.2 µm or smaller filter prior to injection on the UHPLC
  • Sample concentrations expected to be over the calibration range of the instrument should be diluted to ensure accurate analysis and avoid carryover.
Mobile Phase Preparation
Mobile Phase A:
  • a solution of 20 mM phosphoric acid in UPW using the ratio of 1.34 mL of 85% phosphoric acid per 1 L of UPW. Ensure sufficient volume for analysis of all samples and standards.
Mobile Phase B:
  • Methanol
UHPLC-DAD Parameters

Defined UHPLC parameters (click to enlarge)

Defined UHPLC-DAD parameters (click to enlarge)
All analytes in this protocol are quantified using a wavelength of 240 nm from the UHPLC-coupled diode array detector.
Analytical Quality Control
Calibrations
  • All compounds must have a correlation coefficient (r2) of greater than or equal to 0.995 using a linear fit. It is recommended to weigh all calibration curves with a 1/x weighting.

Calibration verification standards
  • A calibration verification standard (CVS) is a standard from the calibration levels that is re-analyzed every 20 or fewer samples to ensure instrument drift remains within the determined acceptable criteria. Acceptable recoveries for this analysis are ±10% of the expected value. All reported data was bracketed by acceptable CVS recoveries. Acceptance criteria may differ between instruments and should be determined experimentally.
Example Chromatogram



Example chromatogram (click to enlarge)



Acknowledgements
We would like to acknowledge and thank Kathryn M. Mains, Chad T. Palumbo, and Davide Rigo, for their collaboration and for producing samples for this analysis.