Pitfalls in Probing Singlet Oxygen by Electron Paramagnetic Resonance Spectroscopy in Engineered Environmental Systems

Sterically hindered amine (SHA)-based electron paramagnetic resonance spectroscopy (EPR) is still widely used to detect singlet oxygen (¹O₂) in engineered environmental systems. Nevertheless, the reliability and interpretability of EPR are limited by the insufficient knowledge of the mechanism of SHA transformation to the nitroxide radical (the ¹O₂ indicator). Here, we systematically investigate the pitfalls and limitations of EPR in detecting ¹O₂. We find that multiple non-¹O₂ species, including sulfate radical (SO₄^•-), hydroxyl radical (HO^•), iodate radical (IO₃^•), high-valent iron, direct electron transfer (DET), etc., could drive the SHA-to-nitroxide radical transformation, hence severely interfering with ¹O₂ detection. The nitroxide radicals are generated via two distinct patterns dependent on the steric hindrance of non-¹O₂ species. One is SO₄^•-/IO₃^•/DET-driven single electron transfer with H₂O and ground-state O₂ participation. The other is HO^•/high-valent iron-mediated hydrogen atom abstraction and radical coupling/oxygen-rebounding. Unexpectedly, pH, rather than ¹O₂, governs the EPR results by modulating the SHA probe distributions and controlling the deprotonation processes, further contributing to the invalidity of EPR in probing ¹O₂ under environmental-relevant conditions. This study uncovers the fundamental flaws of EPR for ¹O₂ detection, enabling a more accurate interpretation of EPR data for ¹O₂ identification.