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Testing, quantification, in situ characterization and calculation simulation for electrocatalytic nitrate reduction

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Abstract

The electrocatalytic nitrate reduction reaction (NO3RR) has emerged as a promising approach for sustainable nitrogen management, enabling the selective conversion of nitrate into targeted nitrogen-containing compounds, such as ammonia and hydroxylamine. However, the efficiency and selectivity of the NO3RR are highly dependent on the physicochemical properties of the electrocatalysts, necessitating a standardized and comprehensive characterization protocol. Here we provide a detailed methodology for the structural, chemical, electronic and electrochemical characterization of the materials used in the NO3RR. We outline procedures for evaluating catalyst morphology, composition and redox states, as well as methodologies for quantifying reaction products to determine nitrate conversion efficiency and selectivity. To track catalyst evolution and reaction pathways under reaction conditions, we present real-time monitoring strategies that capture structural changes, key reaction intermediates and electronic transformations associated with chemical bond formation and cleavage. In addition, we incorporate theoretical calculations to comprehensively evaluate the reaction pathways and their interplay with the electronic structures of electrocatalysts, providing deeper mechanistic insights into the reaction kinetics, active site evolution and selectivity-determining factors. This Protocol is designed for researchers in electrocatalysis, environmental chemistry and energy conversion, offering a reproducible workflow for catalyst assessment. The step-by-step methodology ensures reliable data collection and interpretation, enabling direct comparisons across different catalysts and facilitating the development of more efficient NO3RR catalysts. The entire workflow requires ~8–10 days, depending on sample preparation and measurement duration.

Key points

  • Ex situ characterization of the materials and products of electrochemical reactions provides valuable information but does not track intermediates and their evolution. This Protocol integrates bulk and surface characterization techniques with in situ/operando spectroscopies, electrochemical measurements and density functional theory calculations to better understand the reaction mechanism.

  • We describe the characterization workflow for the analysis of electrocatalytic nitrate reduction; this workflow can be adapted to study other electrocatalytic processes.

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Fig. 1: Schematic of the physicochemical and functional characterization techniques for the electrocatalytic NO3RR.
Fig. 2: 1H NMR spectra for ammonia-14N quantification.
Fig. 3: UV–Vis absorption spectroscopy for indophenol blue spectrophotometry.
Fig. 4: UV–Vis absorption spectroscopy for ammonia quantification.
Fig. 5: UV–vis calibration curve for NH3 determination via Nessler’s reagent spectrophotometry.
Fig. 6: UV–Vis absorption spectroscopy for nitrite-N quantification using diazotization spectrophotometry.
Fig. 7: In situ XAS analysis of the RuCo catalyst under electrochemical conditions.
Fig. 8: Assembly of the Raman cell and in situ electrochemical Raman measurement.
Fig. 9: In situ XRD analysis of the RuCo catalyst under electrochemical conditions.
Fig. 10: Gold plating on silicon ATR crystal and in situ electrochemical ATR-FTIR measurements.
Fig. 11: Electrochemical DEMS setup and product analysis.
Fig. 12: In situ electrochemical EPR setup and hydrogen radical detection.
Fig. 13: Quantification of 15NH4+ using 1H NMR spectroscopy.
Fig. 14: Free energy diagram and reaction intermediates for NO3RR on Pd and Bi1Pd.
Fig. 15: Minimum energy pathway and activation energies for N–O bond-breaking steps in NO3RR.
Fig. 16: Electrochemical performance and mechanistic insights into NO3RR on Ru-Co catalysts.

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Data availability

The data in this Protocol are available in the supporting primary research papers or the Mendeley database (https://doi.org/10.17632/dz3zy7vb3c.2)56. The Origin files can be opened using https://www.originlab.com/viewer/ if Origin is not installed on your computer.

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Acknowledgements

We acknowledge financial support from the High-Level Talent Start-Up Fund of Huaibei Normal University (grant no. 03106369 to D.M.), the Excellent Scientific Research and Innovation Team of the Education Department of Anhui Province (grant no. 2024AH010027 to D.M.) and the National Natural Science Foundation of China (grant nos. 22271213 to B.Z. and 52225308 to L.-M.L.). K.D. acknowledges financial support from the Chinese CSC Scholarship Program. Moreover, we extend our gratitude to X. Wang from Gaossunion Corporation for illustrating the schematic diagram of the in situ electrochemical electrolytic cell.

Author information

Author notes

  1. These authors contributed equally: Kai Dong, Shuhe Han, Yanan Li.

Authors and Affiliations

  1. School of Physics and Electronic Information, Huaibei Normal University, Huaibei, China

    Kai Dong, Zhongliao Wang, Yongchao Yao, Xin Wang & Dongwei Ma

  2. School of Chemical Engineering, The University of Adelaide, Adelaide, South Australia, Australia

    Kai Dong & Xiaogang Sun

  3. Department of Chemistry, School of Science, Tianjin University, Tianjin, China

    Shuhe Han & Bin Zhang  (张兵)

  4. Department of Applied Physics, The Hong Kong Polytechnic University, Hung Hom, Hong Kong

    Shuhe Han

  5. College of Chemistry, Sichuan University, Chengdu, China

    Yanan Li

  6. Anhui Province Industrial Generic Technology Research Center for Alumics Materials, Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei, China

    Zhongliao Wang & Xin Wang

  7. School of Physics, Beihang University, Beijing, China

    Chuang Xue & Li-Min Liu

  8. School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore

    Haobo Li

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Contributions

K.D. was primarily responsible for writing the protocol. S.H. contributed to the development of methodologies for product analysis, in situ electrochemical XRD, in situ electrochemical XAS and isotopic labeling techniques. Y.L. developed the electrochemical mass spectrometry methodology. Z.W. established the theoretical calculation framework. X.S., Y.Y. and X.W. contributed to protocol refinement and data validation. C.X. and H.L. participated in discussions on theoretical calculations. D.M., L.-M.L. and B.Z. provided overall oversight and contributed to discussions on protocol development.

Corresponding authors

Correspondence to Dongwei Ma, Li-Min Liu or Bin Zhang  (张兵).

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Nature Protocols thanks Uttam Kumar Ghorai and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Han, S. et al. Nat. Catal. 6, 402–414 (2023): https://doi.org/10.1038/s41929-023-00951-2

Wang, Y. et al. Angew. Chem. Int. Ed. 59, 5350–5354 (2020): https://doi.org/10.1002/anie.201915992

Zhang, G. et al. Angew. Chem. Int. Ed. 62, e202300054 (2023): https://doi.org/10.1002/anie.202300054

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Dong, K., Han, S., Li, Y. et al. Testing, quantification, in situ characterization and calculation simulation for electrocatalytic nitrate reduction. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01289-8

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