Are We Truly Accurately Quantifying HO • Using Benzoic Acid Hydroxylation in Engineered HO • -Producing Systems?

The hydroxyl radical (HO•), which is one of the most reactive intermediates, plays a significant role in numerous environmental chemical processes in both natural and engineered systems. Given its extremely short lifetime and low steady-state concentration, aromatic hydroxylation probes such as benzoic acid (BA) are commonly used for indirect detection, relying on the formation of stable and long-lived hydroxylation products. However, BA-derived signals may not originate from HO•, leading to a potential misinterpretation. Here, we systematically tested three representative engineered HO•-producing systems (e.g., UV/H₂O₂, homogeneous Fenton, and heterogeneous Fenton) to evaluate the reliability of BA hydroxylation signals for HO• quantification. It was revealed that BA can form a radical cation (BA•+) via electron transfer, followed by rapid hydrolysis that generates hydroxybenzoic acid. Moreover, BA and its oxidation products interfere with the reaction process by competing for photons in photochemical systems, altering the reactivity of Fe(III)/Fe(II) species through complexation in homogeneous systems, and blocking catalytic active sites in heterogeneous systems. These confounding factors compromise the validity of aromatic hydroxylation in identifying HO•. Overall, this study emphasizes the importance of understanding probe-induced interferences to ensure a more reliable characterization of HO•-involved processes.