Towards reliable quantification of hydroxyl radicals in the Fenton reaction using chemical probes

Quantification of hydroxyl radical concentration using two chemical probes was assessed through the Fenton reaction. The probes were 1,2-benzopyrone (coumarin) for fluorescence and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) for electron spin resonance (ESR). The corresponding hydroxylated species, namely 7-hydroxycoumarin (7HC) and 2-hydroxy-5,5-dimethyl-1-pyrroline-N-oxide (DMPO-OH adduct), were monitored by fluorescence and ESR-spin trapping techniques, respectively. The experiments were designed according to the theoretical conditions determined for stable fluorescence and EPR signals. The results demonstrate that: the optimal [chemical probe] : [H₂O₂] ratio predicted by a simplified quasi-steady-state model was in good agreement with the optimal [chemical probe] : [H₂O₂] ratio observed experimentally for [H₂O₂] : [Fe²⁺] = 10, and the proper adjustment of the [chemical probe] : [H₂O₂] ratio at a given concentration of the Fenton's reagent improves the detected amount of hydroxyl radicals. Finally, using DMPO required a higher concentration compared to coumarin to yield the same amount of ˙OH detected but resulted in a more reliable probe for detecting ˙OH under the consideration of this study.