Jun 30, 2025

Public workspacePolyethylene terephthalic acid deconstruction product analysis by UHPLC-DAD

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg3xeb7g25/v1
Polyethylene terephthalic acid deconstruction product analysis by UHPLC-DAD
  • Hannah Alt1,
  • Kelsey Ramirez1,
  • Stefan Haugen1,
  • William E. Michener2,3,
  • Gregg T. Beckham2,3
  • 1Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA;
  • 2Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory;
  • 3BOTTLE Consortium, Golden CO, USA
Hannah M. Alt
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg3xeb7g25/v1
Protocol Citation: Hannah Alt, Kelsey Ramirez, Stefan Haugen, William E. Michener, Gregg T. Beckham 2025. Polyethylene terephthalic acid deconstruction product analysis by UHPLC-DAD. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xeb7g25/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 12, 2023
Last Modified: June 30, 2025
Protocol Integer ID: 83268
Keywords: polyethylene, BOTTLE, diode array detection, TPA, MHET, BHET, UHPLC, polyethylene terephthalic acid deconstruction product analysis by uhplc, polyethylene terephthalic acid deconstruction product analysis, terephthalic acid, hydroxyethyl terephthalic acid, key pet deconstruction product, ultra high performance liquid chromatography, acid, hydroxyethyl, dad this analytical procedure
Funders Acknowledgements:
This work was authored in part by the National Renewable Energy Laboratory for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. This work was performed as part of the BOTTLE™ Consortium and was supported by Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (BETO), and Advanced Materials and Manufacturing Technologies Office (AMMTO).
Grant ID: DE-AC36-08GO28308
Disclaimer
This protocol is for research purposes only.
Abstract
This analytical procedure outlines a rapid and accurate method for the quantification of three key PET deconstruction products, namely terephthalic acid (TPA), 2-hydroxyethyl terephthalic acid (MHET), and bis(2-hydroxyethyl) terephthalic acid (BHET), using an ultra high performance liquid chromatography with diode array detection (UHPLC-DAD) system. This method combines the advantages of UHPLC for improved resolution and separation efficiency with the sensitivity of DAD for accurate and reliable quantification.
Guidelines
This protocol utilizes an ultra high performance liquid chromatography diode array detector (HPLC-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
Standard Materials
ReagentTerephthalic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #185361
Reagent2-Hydroxyethyl Terephthalic AcidToronto Research Chemicals IncCatalog #H942275
ReagentBis(2-Hydroxyethyl) Terephthalic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #465151

Standard Reagents
ReagentPotassium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5655
ReagentPotassium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P3786
ReagentTetrahydrofuranMerck MilliporeSigma (Sigma-Aldrich)Catalog #401757


Mobile Phase Reagents
ReagentPhosphoric Acid ACS 85 wt. % in WaterMerck MilliporeSigma (Sigma-Aldrich)Catalog #695017
ReagentMethanol 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

Troubleshooting
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)

2-Hydroxyethyl terephthalic acid (MHET) preparation

  1. Prepare a 1:1 solution of UPW and tetrahydrofuran (THF)
  2. Weigh out neat MHET standard and create a 1000 µg/mL MHET stock standard using the 1:1 solution of ultrapure water (18.2MΩ⋅cm) and THF created in the step 2.1. Example: weigh 8,700 µg (8.7 mg) of powdered MHET and added 8.7 mL of 1:1 UPW and THF.
  3. Create a 250 µg/mL MHET working standard using the 1000 µg/mL MHET stock standard and the 1:1 solution of UPW and THF. Example: Pipetted 2.5 mL of 1000 µg/mL MHET stock standard and 7.5 mL of 1:1 UPW and THF solution.
  4. Create the following calibration standards using the 250 µg/mL MHET standard working solution and UPW as the diluent.

Example calibration curve preparation for MHET (Click to enlarge)

Bis(2-hydroxyethyl) terephthalic acid (BHET) preparation

  1. Weigh out neat BHET standard and create a 1000 µg/mL BHET stock standard using tetrahydrofuran (THF). Example: Weighed 9,500 µg (9.5 mg) of powdered BHET and added 9.5 mL of THF.
  2. Create a 250 µg/mL BHET working standard using the 1000 µg/mL stock standard and THF. Example: Pipetted 2.5 mL of 1000 µg/mL stock standard and 7.5 mL of THF.
  3. Create the following calibration standards using the 250 µg/mL BHET standard working solution and THF as the diluent.


Example calibration curve preparation for BHET (Click to enlarge)


Sample Preparation
  • Samples must be filtered through a 0.2 µm or smaller filter prior to injection on the UHPLC
  • Samples expected to be over the linear 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




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 weight the calibration with a 1/x weighting.

Calibration verification standards
  • A calibration verification standard (CVS) is a standard from the calibration 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

Protocol references
Erickson, E., Gado, J.E., Avilán, L. et al. Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity. Nat Commun 13, 7850 (2022). https://doi.org/10.1038/s41467-022-35237-x