Name: Quantification of Polybutylene Adipate Terephthalate-based Micro- and Nano-plastics from Soil Using Proton Nuclear Magnetic Resonance Spectroscopy (Video) | JoVE Journal
Uploaded: 2025-06-06T12:45:00.000+00:00
Duration: 5 min 5 s
Description: Learn to quantify PBAT micro- and nano-plastics in soil using proton NMR spectroscopy. This method enables reliable environmental monitoring and advances biodegradable plastic research.

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Thank you to the USDA-NIFA for funding this project through award number 2020-67019-31167 to SMS and to the University of Tennessee Strategic Planning Research Initiatives (SPRINT) program for an internal grant DGH and SMS. The funders had no role in study design, data collection, analysis, interpretation, report writing, or the decision to submit the article for publication.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

We propose a method for solvent extraction of PBAT from soil coupled with 1H-NMR to quantify PBAT in the extract. Key elements of the extraction process include the extraction technique and equipment, the solvent used to extract, and the time requirements. We chose to use an extraction technique that requires relatively simple and inexpensive equipment (glass jars, glass beads, and a shaker table) compared to the Soxhlet extraction and accelerated solvent extraction (ASE) demonstrated by Nelson et al.<sup clas...

Methods to measure MP contamination in soil are necessary to understand the scope of plastic contamination in global soils1, sources of plastic contamination2, and potential solutions3. Agricultural soils are uniquely exposed to plastic contamination: more than 15 million metric tons of plastic are used in agriculture each year as of 20214, including 2.5 million tons of plastic mulch5. Plastic mulch is used in close contact with soil, reapplied one or more times per year6, and can be difficult to fully remove from soil after their useful life7. One key area where MP measurement methods are needed is in the assessment of biodegradable plastic products like biodegradable plastic mulches for use in vegetable systems8.
Biodegradable-in-soil plastic products are promising alternatives to conventional agricultural plastics because they could eliminate plastic contamination of soil by plastic mulching if they work as intended. In 2022, less than 1 million metric tons of biodegradable plastic were produced globally, with rapid growth expected for the industry9. The four most common biodegradable polymers, polylactic acid (PLA), polymerized starch, polyhydroxyalkanoates (PHA), and polybutylene adipate terephthalate (PBAT), are all used commercially or experimentally in agricultural biodegradable plastic mulches10. Despite their promise, the degradation of these biodegradable plastic products in field conditions is variable11. While some studies of biodegradable plastic mulch degradation in the field have focused on macroplastic fragments11, assessing the complete degradation of plastic materials requires the ability to recover MPs and NPs from soil. Microplastic quantification from soil is also important to assess the potential for negative impacts on soil ecosystems from MP pollution12.
Some commonly used techniques for MP identification and quantification include visual analysis, micro-Fourier-transformed infrared spectroscopy (&#181;FTIR), micro-Raman spectroscopy (&#181;Raman), gas chromatography-mass spectroscopy (GCMS including pyrolysis GCMS and thermal extraction desorption GCMS), and thermogravimetric analysis13. Other developing techniques include near-infrared spectroscopy for in situ MP measurement in soils14, fatty acid methyl esterification extraction for GCMS analysis15, and nuclear magnetic resonance spectroscopy16. As many as 90% of studies quantifying MPs in soil used visual analysis (alone or paired with other techniques) to identify plastic particles, while 77% used FTIR, Raman, or GCMS spectroscopy techniques17. Developing and harmonizing a diverse variety of MP quantification techniques can help expand the scientific community&#39;s ability to answer diverse microplastic research questions18. Three generic approaches exist to prepare soil samples to quantify microplastics: 1) separating unchanged individual microplastic particles from the soil (ex. by density separation), 2) extracting transformed plastic or polymer material (ex. by dissolution), or 3) analyzing bulk soil. Both &#181;FTIR and &#181;Raman spectroscopy require individual MPs to be separated from the soil before they can be chemically identified19 while pyro-GCMS may be performed on isolated plastic particles separated from soil or bulk soil20. Separating biodegradable MPs from soil can be difficult because some digestions used to remove soil organic matter can degrade or otherwise chemically alter biodegradable polymers, including PBAT21. Micro-Raman and &#181;FTIR spectroscopy both also have a spatial resolution limit: particles must be larger than 10-20 &#181;m for &#181;FTIR and 1 &#181;m for &#181;Raman (if individual particles this small can be prepared for analysis)19,22. These techniques can provide chemical identification of MP polymers, and spectroscopic imaging can be employed to measure MP size22. All types of pyro-GCMS are limited in that samples are destroyed during analysis.
NMR has been used successfully to characterize soil constituents and contaminants23, to quantify MPs16,24,25,26, and to assess the degradation of PBAT and another polymer, polystyrene27,28. When run under quantitative parameters, 1H-NMR spectroscopy produces spectra where the area of each spectral peak is directly proportional to the number of contributing hydrogens in the sample; this allows for quantification of the constituent components in a sample29. NMR is a valuable analytical technique for quantifying some MPs, including PBAT, within soil because it allows for simultaneous quantification and identification, it is suitable for complex and impure mixtures, and it does not require chemically identical reference standards30,31. NMR spectroscopy of a solvent extract could quantify MPs or NPs smaller than those that can be processed for &#181;FTIR, &#181;Raman, or GCMS quantification. Despite these advantages, quantitative NMR approaches still provide lower sensitivities than destructive mass spectroscopy-based approaches32.
The proposed method, diagramed in Figure 1, describes a workflow to process and analyze soil samples containing PBAT - including nano-sized PBAT plastics. In the method, PBAT polymer is extracted from soil samples by shaking the soil with a mixture of chloroform and methanol. The solvent extracts containing PBAT are dried and then redissolved in deuterated chloroform with an added internal calibrant. NMR spectroscopy is conducted on the extracts using quantitative parameters. The resulting spectra are analyzed to quantify PBAT by comparing the area of fitted peaks corresponding to hydrogen atoms of the PBAT and calibrant molecules. This method applies the solvent extraction and proton nuclear magnetic resonance spectroscopy (1H-NMR) approach demonstrated by Nelson et al.33. The goal was to employ a method suitable to process environmental scale samples (~100 g of soil) and to process a number of samples in parallel without specialized extraction equipment.

<table><tbody><tr><td>1,4-dimethoxybenzene</td><td>Arcos Organics 99%+</td><td>AC115411000&amp;nbsp;</td><td>1 mg per sample</td></tr><tr><td>Amber glass bottle with PTFE lined lid (1 L)</td><td>Kimble</td><td>5223253C-26</td><td>reusable</td></tr><tr><td>Chloroform; trichloromethane</td><td>Fisher Chemical</td><td>AA43685M6&amp;nbsp;</td><td>90 mL per sample; Fisher Optima</td></tr><tr><td>Deuterated chlorform; trichloro(deuterio)methane</td><td>Sigma Aldrich</td><td>1034200025</td><td>1 mL per sample; minimum 99.8% deuterated; stabilized with silver</td></tr><tr><td>Glass beads (3 mm diameter)</td><td>Propper Manufacturing</td><td>3000600</td><td>20 per sample</td></tr><tr><td>Glass extraction jars with PTFE lined lid (~250 mL volume)</td><td>Kimble</td><td>5510858B</td><td>2 per sample, reusable</td></tr><tr><td>Graduated cylinder, 1 L, polypropylene</td><td>Nalgene</td><td>3662-1000</td><td>reusable</td></tr><tr><td>Graduated cylinder, 500 mL, polypropylene</td><td>Nalgene</td><td>3662-0500</td><td>reusable</td></tr><tr><td>Methanol</td><td>Fisher Chemical</td><td>A412-4&amp;nbsp;</td><td>20 mL per sample; certified ACS</td></tr><tr><td>Micropipette wth range of 0.5 - 1 mL</td><td>Fisher Scientific</td><td>3123000063</td><td>reusable</td></tr><tr><td>NMR spectrometer</td><td>Bruker</td><td>n/a</td><td>500 MHz instrument</td></tr><tr><td>NMR tube (7 inch height, high-throughput)</td><td>Wilmad</td><td>WG-1000-7</td><td>1 per sample</td></tr><tr><td>Platform shaker</td><td>Eppendorf, Excella E5&amp;nbsp;</td><td>M1355-0000</td><td>reusable</td></tr><tr><td>Polyethylene pipette tip (10 mL volume)</td><td>Eppendorf</td><td>22492098</td><td>1 per sample, single use</td></tr><tr><td>Polypropylene micropipette tips (1 mL volume)</td><td>Fisher Scientific</td><td>02-707-510</td><td>3 per sample, single use</td></tr><tr><td>Semi-microbalance</td><td>Mettler Toledo</td><td>30532226</td><td>reusable</td></tr></tbody></table>

quantification of polybutylene adipate terephthalate-based micro- and nano-plastics from soil using proton nuclear magnetic resonance spectroscopy

Quantification of Polybutylene Adipate Terephthalate-based Micro- and Nano-plastics from Soil Using Proton Nuclear Magnetic Resonance Spectroscopy

A method to recover and quantify micro- and nano-plastics (MPs and NPs) formed in the soil during biodegradation is needed to accurately assess the degradation and environmental impact of biodegradable plastic products. The presence of MPs and NPs in soil may alter soil properties like aggregation behavior or have toxic effects on soil biota. Existing MP recovery methods are not always suitable for measuring biodegradable polymers like polybutylene adipate terephthalate (PBAT); some common digestion procedures with acids or oxidizers can destroy PBAT-based biodegradable MPs. Identification methods like micro-FTIR and micro-Raman spectroscopy are also limited by the minimum size of particles that can be recovered and analyzed. Therefore, this method was developed to extract and quantify PBAT from soil to assess the mass fraction of MPs and NPs in the soil without chemically transforming PBAT. In the protocol, a chloroform-methanol solution is used to selectively extract PBAT from the soil. The solvent is evaporated from the extract, and then the extract is redissolved in deuterated chloroform. The extract is analyzed by proton nuclear magnetic resonance spectroscopy (1H-QNMR) under quantitative parameters to quantify the amount of PBAT in each sample. Solvent extraction efficiencies for PBAT range from 76% in a shady loam soil to 45% in an Elkhorn sandy loam soil. PBAT recovery may be reduced for photo-oxidized materials compared to pristine ones and may be reduced in soils with high clay content. Extraction efficiencies do not depend on PBAT concentration within the test range, but lower extraction efficiencies were observed for NPs than for MPs. PBAT quantification results were comparable to the quantification of plastic degradation by measuring cumulative soil respiration in a laboratory incubation study.

To assess the efficacy of this method in quantifying PBAT polymer from the soil, calibration curves were constructed by extracting PBAT from spiked samples created from three different soils. For each of the three soils (details in Table 3), the soil was passed through a 2 mm sieve and then air dried. Spiked samples were created by adding 0, 9, 18, 27, or 36 mg of PBAT-based MP to 100 g of dry soil (5 replicates of each). This is equivalent to 0, 63, 126, 189, or 252 mg/kg of PBAT polymer. MPs were made ...

A method to quantify micro- and nano-plastics originating from polybutylene adipate terephthalate in soil using nuclear magnetic resonance spectroscopy is described here. This technique improves on existing methodology because it extends to quantification of nanoplastics and it can easily be adapted for processing environmental samples in bulk.

A method to recover and quantify micro- and nano-plastics (MPs and NPs) formed in the soil during biodegradation is needed to accurately assess the ...

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Learn to quantify PBAT micro- and nano-plastics in soil using proton NMR spectroscopy. This method enables reliable environmental monitoring and advances biodegradable plastic research.

Quantification of Polybutylene Adipate Terephthalate-based Micro- and Nano-plastics from Soil Using Proton Nuclear Magnetic Resonance Spectroscopy (Video) | JoVE Journal

Quantification of Polybutylene Adipate Terephthalate-based Micro- and Nano-plastics from Soil Using Proton Nuclear Magnetic Resonance Spectroscopy

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