Methodology of rapid-scan ATR-IR spectroscopy in conjunction with Stopped-Flow technique (SF-ATR-IR)
To enable real-time monitoring of the heterogeneous Fenton reaction, an approach coupling in situ IR technique with an adapted stopped-flow method is developed. The conventional stopped-flow technique is a rapid mixing method developed for homogeneous reactions. In this method, two or more reactants are quickly mixed in an observation chamber, and the flow is then abruptly stopped to allow the detector to monitor the reaction kinetics starting from the moment of mixing. However, this typical setup is not suitable for heterogeneous reactions. Here, we adopted the concept of "stopped-flow" but modified it to accommodate the nature of the heterogeneous Fenton-like reaction, where the reaction is initiated by the contact between the Fenton catalyst and PMS (or PDS).
To enable surface-specific monitoring, we first deposited the Fenton catalyst onto an ATR diamond crystal by evacuating the catalyst suspension (Fig. 1 and Supplementary Fig. 1), which serves as a role analogous to the observation chamber in the conventional setup. Given that the detection depth of ATR-IR is limited to just a few micrometers above the crystal surface, only reactions occurring on the deposited catalyst layer are detected. Therefore, the catalyst film thickness is controlled within 1-2 μm to ensure effective signal capture (Supplementary Fig. 2a, b). On the other hand, a digitally controlled stepping syringe pump was utilized to precisely inject PMS or PDS solutions. We then used a syringe pump to inject the PMS solution, abruptly stopping it at the catalyst-coated surface, ensuring that only the surface reaction is "observed". This modified stopped-flow configuration allows us to selectively track the interfacial reaction between PMS and the catalyst in real-time.
Prior to PMS/PDS injection, 10 μL of distilled water was added to the catalyst membrane. This serves a dual purpose: it acts as a buffer layer for the PMS/PDS drops and mitigates baseline variations in the IR spectrum, which could arise from the transition between a gas-solid interface and a water-solid interface when PMS/PDS is applied directly to the dry catalyst membrane. Upon injection of a drop of PMS/PDS (approximately 10 μL), the PMS/PDS molecules diffuse through the buffer layer, abruptly "stopping" at the catalyst membrane's surface to initiate the Fenton-like reaction and ensure that only the surface reaction is "observed".
The operation of the syringe pump is synchronized with the IR spectrometer, triggering the injection of PMS/PDS in response to the forward movement of the spectrometer's mirror. Consequently, IR data acquisition begins simultaneously with the PMS/PDS injection from the syringe. The data collection occurs in rapid-scan mode, capturing IR spectra with a maximum time resolution of 60 ms per spectrum. Importantly, the ATR-IR technique only detects signals within 1-2 μm above the ATR crystal surface; therefore, only when PMS/PDS molecules diffuse close to the catalyst membrane can they be observed in the IR spectra. This SF-ATR-FTIR technique thus provides valuable insights specifically focused on the surface reactions between PMS/PDS and Fenton catalysts.
Noteworthy, to observe the weak IR signals of Fe-oxo and peroxide intermediates, the operating conditions of ATR-IR are carefully optimized. The use of an oxidation-resistant diamond crystal prevents Fe-oxo species from reacting with the ATR crystal. Additionally, the use of a mid-band MCT detector, with a cutoff at 625 cm, enhances sensitivity for detecting the low-frequency Fe-oxo band. The entire IR spectroscopy sample chamber is continuously purged with dry argon to minimize atmospheric water vapor interference and improve the signal-to-noise ratio (Supplementary Fig. 2c). All of these optimizations, combined with the flexibility to introduce various catalysts and reagents, make this technique applicable to a wide range of reaction types -- whether heterogeneous or homogeneous -- including mixing-triggered reactions such as the Fenton reaction, enzyme catalysis, and catalytic organic synthesis.
To validate the capability of our spectroscopy technique for dynamically identifying heterogeneous metal-oxo species, we conducted a study using Fe-N-C, a single-atom catalyst with Fe-N coordination. Single-atom Fe-N-C catalysts have attracted significant attention in energy- and environment-related applications and have recently emerged as promising Fenton catalysts. These catalysts are typically fabricated by stabilizing monodispersed metal atoms on nitrogen-doped carbon substrates (M-N-C) through nitrogen-coordination (MN). This approach allows for precise tuning of the MN structure to control the types of reactive species generated during Fenton or Fenton-like processes. Of particular interest is the possible formation of metal-oxo species on M-N-C catalysts, as suggested by prior studies employing PMSO-probe experiments. The formation of surface metal-oxo on single-atom catalysts would offer additional advantages: on conventional catalysts with adjacent metal sites, the synergistic interaction between two adjacent metal-oxo species makes them prone to rapid consumption through reaction with water, limiting their effective utilization in pollutant removal. In contrast, the isolated nature of metal sites in single-atom catalysts prevents such undesirable interactions, effectively shielding the metal-oxo species from premature decay. This extends the lifetime of metal-oxo species, allowing them to serve as more efficient oxidants for pollutant removal rather than being wasted in reactions with the aqueous matrix. Therefore, exploring the formation and dynamic kinetics of metal-oxo species on single-atom catalysts holds profound significance.
The typical single-atom Fe catalyst was synthesized via an adapted method from literature: the Fe(Phen) precursors were first templated by nano-MgO, followed by calcination under argon gas and removal of templates by nitric acid leaching to obtain Fe-N-C (Fig. 2a). Scanning electron microscopy (SEM) images revealed that the resulting Fe-N-C featured a pleated and porous structure (Supplementary Fig. 3), while energy-dispersive X-ray spectroscopy (EDS) mapping confirmed the uniform distribution of Fe, N, and C elements (Fig. 2b). X-ray diffraction (XRD) analysis showed no diffraction peaks corresponding to crystalline or aggregated Fe (Supplementary Fig. 4). High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images further verified that Fe atoms were atomically dispersed across the carbon matrix without signs of clustering (Fig. 2c, d). This evidence underscored the effective synthesis of single-atom Fe catalysts with uniform dispersion.
The chemical state of Fe was analyzed using X-ray photoelectron spectroscopy (XPS). The high-resolution Fe 2p spectrum revealed that Fe primarily exists in the Fe state, with a peak-area ratio of 61.6% (Supplementary Fig. 5). Additionally, the N 1 s spectrum displayed an Fe-N peak at 399.13 eV, confirming the presence of Fe in the N-coordinated Fe-N form. The K-edge X-ray absorption near-edge structure (XANES) spectrum of Fe showed that the absorption edge of Fe-N-C closely aligns with that of standard iron phthalocyanine (FePc), indicating a predominant Fe oxidation state, consistent with the XPS findings (Fig. 2e). Extended X-ray absorption fine structure (EXAFS) spectroscopy was utilized to probe the coordination environment of Fe in Fe-N-C. The spectrum exhibited a prominent peak at ~1.5 Å, corresponding to the Fe-N first coordination shell. Notably, the absence of Fe-Fe peaks at ~2.2 Å (metallic Fe) or ~2.7 Å (Fe-Fe in FeO) further validated the atomic dispersion of Fe within the Fe-N-C matrix (Fig. 2f). The EXAFS wavelet transformation of Fe-N-C displayed a maximum intensity at ~4.0 Å, associated with the Fe-N interaction (Supplementary Fig. 6), confirming the atomically dispersed nature of Fe without detectable crystalline metallic phases. Finally, curve-fitting analysis of the EXAFS data indicated an Fe-N coordination number of approximately 3.8 (Supplementary Fig. 7, Supplementary Table 1), affirming the presence of the Fe-N structure.
The active species generated during the Fenton-like reaction between Fe-N-C and PMS was monitored in situ using SF-ATR-FTIR. As discussed above and shown in Fig. 3a, due to the detecting scope of ATR-IR that is limited to the near-surface region of the ATR crystal (or deposited catalyst membrane), there is an inherent delay between the injection of PMS and the observation of PMS or its reaction intermediates in the IR spectra, arising from the time required for PMS to diffuse from the syringe pump to the ATR crystal. To estimate this diffusion duration, we first conducted a controlled experiment on a bare diamond ATR crystal.
Noteworthy, the commercially available PMS is not solely a purified sample of potassium hydrogen persulfate (KHSO); it also contains redox-inert KHSO and KSO. Therefore, we need to identify characteristic IR bands for each component to separately estimate their near-surface concentrations. These characteristic bands should be distinct and not overlap with those of other components. As shown in Supplementary Fig. 8a, the standard IR spectrum of the PMS mixture reveals several distinct bands. Notably, the characteristic IR bands for persulfates appear at 1250 and 1060 cm, corresponding to the S-O (or S = O) vibrations of the SO moiety, as well as the peroxyl (O-O) bond at 880 cm. For the other two components, KHSO exhibits IR bands at 1193 and 1050 cm, while KSO shows a band at 1100 cm. To avoid interference among the components, we used the intensity of the 1250, 1193 and 1100 cm bands to represent HSO, HSO and SO, respectively.
On the bare diamond ATR crystal, as illustrated in Fig. 3b, the IR bands corresponding to HSO, HSO and SO, became detectable 0.6 s after syringe injection. The kinetics of all PMS components showed a similar pattern (Fig. 3d and Supplementary Fig. 8b, solid symbols): their intensities sharply increased until stabilizing around 18.0 s, indicating that PMS diffusion had reached equilibrium. However, when Fe-N-C was deposited on the diamond crystal, as shown in Fig. 3c and d (empty symbols), the observation delay for the HSO band at 1250 cm extended by an additional 0.5 s. Following this delay, the increase in intensity was slower. Notably, after peaking at 12.0 s, the intensity began to decline and was completely depleted at approximately 100.0 s. This behavior suggested that HSO was actively reacting with the Fe-N-C catalyst, with the apparent intensity of the 1250 cm band reflecting the difference between HSO that diffused to the surface and that consumed in the reaction. In contrast, the intensities of HSO and SO exhibited a monotonic increase, reaching higher equilibrium values than on the bare diamond. This accelerated increase was attributed to the generation of HSO and SO from the reaction between HSO⁻ and the Fe-N-C catalyst.
Significantly, almost concurrently with the appearance of the HSO band, a new band at 833 cm emerged (Fig. 3c), closely matching the frequency of surface Fe = O reported in the literature. As shown in the kinetics (Fig. 3e, f), the IR bands of HSO (1250 cm) began to decay at approximately 12 s, marking the point where the consumption of near-surface HSO outpaced its supplement from the bulk solution. Complete depletion of the 1250 cm band was observed at 230.16 s, suggesting the exhaustion of HSO in both the near-surface region and the bulk system. In contrast, the 833 cm band exhibited a delayed decay, initiating at approximately 66 s, with complete disappearance occurring at 234.12 s (Fig. 3e, f). The extended existence of Fe = O, lasting an additional 3.96 s beyond the depletion of HSO, highlights its long lifetime on the Fe-N-C on the scale of seconds. This result provided the experimental identification of the lifetime of surface metal-oxo generated during the Fenton reaction.
The effect of pH on the lifetime of Fe = O was also examined, given that Fe = O can degrade via reaction with H to form Fe. As shown in Supplementary Fig. 8c-e, adjusting the pH of the Fenton-like system from approximately 2 (the original value used in the above experiments) to 7 using NaOH led to a slight increase in the Fe = O lifetime from 3.96 s to 4.08 s, an increase of only 0.12 s, about 3% of the original lifetime. This indicates that although Fe = O is slightly more stable under neutral conditions, the overall extension is minimal, suggesting that pH has a limited impact on its lifetime (Supplementary Fig. 8c-e).
To further consolidate the assignment of the IR band corresponding to surface Fe = O on Fe-N-C, we conducted an O isotope labeling experiment. Due to the lack of commercially available O-labeled persulfate, we opted to use O-labeled water (HO) instead. Since surface Fe = O is produced from the oxidation of persulfate, it implies that the oxo O originates from persulfate rather than water. Consequently, the O labeling of water should have a minimal impact on the source of the oxo groups. However, previous studies have indicated that an oxygen exchange can occur between Fe = O and water. When using PMSO as a molecular probe to detect Fe = O, Jiang et al. found that, when O-PMS and HO are employed, the oxidation of PMSO by Fe = O results in the formation of mixed-labeled PMSOO. This observation raises the possibility that following the formation of Fe = O from O-PMS, an oxygen exchange with HO may lead to the formation of Fe = O and the subsequent oxidation products of PMSOO (Fig. 4a).
Interestingly, as shown in Fig. 4b, in the IR spectra, replacing regular HO with HO revealed a new band at 795 cm. The original 833 cm band persisted in the early stages of the reaction, but with decreased intensity compared to the scenario with only O. As the reaction progressed, the 833 cm band gradually diminished, leaving the 795 cm band as the sole observed feature. The theoretical value for the O/O isotope shift can be predicted using vibration frequency calculation equations ("Isotope shift calculation" section in Supplementary Information). If the 833 cm band is assigned to Fe=O, its O counterpart should shift down to 796 cm, closely aligning with the observed band at 795 cm. Literature also supports a theoretical shift of approximately 36 cm for the Fe-O harmonic oscillator. We also performed a control experiment mixing regular O-PMS with HO to check for the presence of O-persulfate through oxygen exchange (Supplementary Fig. 9a). However, no isotope-shifted band for persulfate was observed in the IR spectra, indicating that direct oxygen exchange does not occur between PMS and HO. In addition, we dissolved O-labeled PMS in HO and allowed the solution to stand for 24 h. High-resolution mass spectrometry (HRMS, Supplementary Fig. 9b) was then performed, and no peak corresponding to O-labeled PMS (m/z = 114.96) was detected, thereby ruling out the possibility of oxygen exchange under these conditions. Based on both infrared spectroscopy and high-resolution mass spectrometry analyses, we can confidently exclude the possibility of oxygen exchange between HO and PMS.
Therefore, the observed 795 cm band can be assigned to O-labelled Fe = O, resulting from an immediate O-exchange between Fe = O (produced from the oxidation by O-persulfate) and HO. Notably, the growth of the Fe = O signal exhibited a delayed kinetic profile compared to Fe = O. Since the direct exchange of the oxo-O in a metal-oxo double bond is generally challenging, this kinetic delay would suggest a gradual oxygen-exchange pathway: it resembles the O-exchange between water and the carbonyl group of aldehyde or ketone, which occurs via the formation of a geminal diol intermediate. The addition of HO on Fe = O generates a geminal-hydroxyl structure of Fe(OH)(OH), subsequent dehydration of this intermediate yields either Fe = O or reverts to Fe = O, due to the relatively small energy difference between the Fe-O-H and Fe-O-H bonds. As a result, multiple exchange cycles are needed to accumulate a significant amount of the Fe = O species. This requirement for repeated exchange events explains why the Fe = O signal does not emerge with the same kinetics as Fe = O, and instead appears with a noticeable delay. Nevertheless, this isotope experiment further supports our assignment of the 833 cm band to surface Fe = O. More importantly, the observed oxygen exchange indicates that this surface Fe = O species possesses a relatively long lifetime and stability in the aqueous matrix, making it a potentially high-activity species for degrading aqueous pollutants.
For comparison, we performed similar experiments with the replacement of PMS with peroxydisulfate (PDS), using the same SF-ATR-FTIR characterization method (Fig. 5a). Unlike PMS, commercial-available PDS is a pure sample that exhibits characteristic IR peaks at 1274 cm and 1050 cm, corresponding to the SO moiety in the SO group (Supplementary Fig. 10a). During the in-situ monitoring the Fenton reaction between PDS and Fe-N-C, as shown in Fig. 5a, the appearance of PDS peaks exhibited a similar delay owing to PDS diffusion. The kinetic behavior mirrored that of PMS, initially increasing, reaching a maximum, and then declining to extinction. We also observed the growth of SO and HSO peaks, demonstrating a reaction between PDS and Fe-N-C. However, no new characteristic peaks were observed in the 800 ~ 900 cm range. Replacing HO with HO did not change the characteristic peaks of PDS, and no new characteristic peaks emerged (Supplementary Fig. 10b). Therefore, no Fe = O species is produced in the PDS and Fe-N-C system.
To bolster the credibility of our IR findings, we conducted additional analyses using electron paramagnetic resonance (EPR) and PMSO molecular probe experiments. In EPR analysis (Fig. 5b), using 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as a trapping agent, in the PDS system, the intense signal with the typical feature of trapped SO* and *OH can be clearly observed, confirming the generation of SO* and *OH in PDS system. In contrast, these trapped radical signals were absent in the PMS system; instead, we detected the 5,5-dimethyl-2-pyrrolidinone-yloxy (DMPOX; a = 7.2 G and a = 4.1 G) signal, an oxidation product of DMPO. The absence of significant signals for trapped SO* and *OH suggests the single-electron oxidation of Fe to Fe is negligible in the PMS system, where this pathway is accompanied by the stoichiometric generation of SO* and *OH. Also, for both systems, the absence of O was also confirmed (Fig. 5c). These results confirm that the PMS and PDS systems generate distinct oxidative species, with the PMS system predominantly producing Fe = O directly.
Furthermore, the PMSO molecular probe experiment was performed. As shown in Fig. 5d, the results revealed that PMSO was selectively oxidized to PMSO in the Fe-N-C/PMS system, whereas no PMSO signal was detected in the Fe-N-C/PDS system. Given that Fe = O is known to selectively oxidize PMSO to PMSO, whereas *OH and SO* cannot, these findings are consistent with the IR and EPR results, confirming the formation of Fe = O species in the PMS system and suggesting their absence in the PDS system
Combining the results from in-situ FT-IR and EPR, we conclude that PMS effectively activates Fe-N-C, resulting in the formation of surface Fe = O species as the dominant active species. In contrast, the presence of PDS primarily generates free radicals such as SO* and *OH, with no detectable Fe = O formation. To elucidate the mechanistic differences between the PMS and PDS systems, we performed density functional theory (DFT) calculations with a focus on the detailed reaction intermediates and transition states, aiming to clarify the disparity in energy barriers between the two pathways. Our calculations revealed that the PMS system facilitates the rapid formation of the high-valent Fe = O species via a low-barrier oxygen atom transfer (OAT) process, with an overall activation free energy of 13.5 kcal/mol (Supplementary Fig. 11, Supplementary Data 1). In contrast, the PDS system follows a distinct single electron transfer (SET) mechanism. Although the initial step is highly exergonic, then owing to the more complex dimeric structure of PDS compared to PMS, the formation of Fe = O by PDS then requires involving multiple intermediates and transition states. The cumulative effect of these steps results in a significantly higher overall energy barrier along the reaction coordinate, making the formation of Fe = O in the PDS system both not only thermodynamically and kinetically unfavorable (Supplementary Fig. 12, Supplementary Data 1). As a result, the Fe = O species is effectively inaccessible under PDS conditions.
To further demonstrate the universality and reliability of our SF-ATR-FTIR technique in identifying Fe = O species, we synthesized two additional Fe-N-based single-atom catalysts (Supplementary Fig. 13; Supplementary Table 4). During in situ monitoring of their Fenton-like reactions, a characteristic IR band around 830 cm was consistently observed for both catalysts (Supplementary Fig. 14a, b). Complementary PMSO molecular probe experiments and EPR analyses also supported the presence of Fe = O. These consistent findings confirmed that the ~830 cm IR band detected by our technique is a reliable indicator of Fe = O formation (Supplementary Fig. 14c-e).
Fe = O has been recognized for its ability to facilitate an oxygen-atom transfer (OAT) process during the oxidation of organic and inorganic substrates. This characteristic makes it especially effective in oxidizing substrates that possess lone-pair electrons, for instance, the aqueous arsenic (III) (As). The aqueous As, classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC), has garnered significant attention due to its health risks, even low-level exposure to As can lead to various health issues. The preferred approach to remove As is to oxidize it to arsenate (As), which has lower toxicity and is more readily removed by physical-chemical methods. However, a significant challenge is that, in a real environmental system, aqueous As typically exists at low concentrations and coexists with large amounts of low-toxicity organic and inorganic substrates. This presence complicates the use of broad-spectrum oxidizing agents, like radicals, which cannot selectively oxidize As in the presence of interfering substrates.
We first assessed the effectiveness of the Fe-N-C/PMS system for removing 20 μM As in a deionized water matrix. By quantifying the produced As (Supplementary Fig. 15), we found a significant generation of As within just 5 min, with approximately 85.5% of As rapidly converted to As during this time (Fig. 6a). The final degradation rate of As reached 99.55% within 15 min, demonstrating the high efficiency of the Fe-N-C/PMS system in treating As. In contrast, when using the Fe-N-C/PDS system, where the ROS shifted from Fe = O to free radicals, only trace amounts of As were detected in the initial 5 min, after which the generation of As became more pronounced. A scaled-up As removal experiment (Supplementary Fig. 16) was also conducted to demonstrate the feasibility of Fe-N-C/PMS even in the enlarged system. In addition, we re-collected the Fe-N-C catalyst (Fe-N-C-r) after the As degradation experiment and performed a series of post-reaction characterizations, including EXAFS, XPS, and HAADF-STEM (Supplementary Fig. 17), to verify the stability of the catalyst. The superior performance of Fe-N-C/PMS in the oxidative removal of As can be attributed to the differing mechanisms. As shown in Fig. 6b, in the Fe-N-C/PDS system, the predominant radicals, with *OH and SO*, initiate a one-electron oxidation process that first converts As to As, this one-electron oxidation step faces a significant energy barrier of 2.40 eV, which slows the reaction in the initial stage. As a result, only a limited amount of As is generated until most As is oxidized to As. Once this occurs, the oxidation of As to As becomes energetically favorable, leading to an increase in As production as observed in Fig. 6a. In contrast, in the Fe-N-C/PMS system, the dominant ROS, Fe = O, facilitates a more efficient two-electron oxygen-atom transfer that directly transforms As to As with a much lower energy barrier of only 0.56 eV. This mechanism results in more efficient oxidative removal of As. The specific reactivity of Fe = O in As oxidation was also confirmed by our ST-ATR-FTIR measurements. First, the direct reaction between free PMS and As was excluded by the control experiment in the absence of the Fe-N-C catalyst, as the introduction of As did not cause any decay in the characteristic peak of PMS at 1250 cm within 10 min' reaction (Fig. 6c). Subsequently, when the Fe-N-C catalyst was immobilized on the ATR surface, co-injection of As and PMS led to the disappearance of the Fe = O characteristic band at 833 cm, indicating its rapid consumption by As (Fig. 6d).
We then assessed the removal of As in a real lake water matrix, sourced from the artificial lake at Beijing Olympic Park, which had a chemical oxygen demand (COD) value of 118 mg·L. Using the Fe-N-C/PMS system, the aqueous contaminants were effectively degraded, resulting in a COD reduction to 3 mg·L within 30 min (Fig. 6e). Notably, despite the presence of various interfering contaminants, the removal of As remained highly effective and a removal ratio of 96.8% was achieved. In contrast, while the Fe-N-C/PDS system also led to a significant decrease in COD, the conversion of As to As was substantially hindered compared to the distilled water experiments, with only a small amount of As detected after 30 min and an As removal ratio of only 12.5%. Similarly, the presence of humic acid and pH adjustment had minimal impact on As removal by the Fe-N-C/PMS system, highlighting its excellent resistance to environmental interferences (Supplementary Figs. 18, 19). These results underscore the superior performance of Fe-N-C/PMS in selectively removing As even in complex wastewater environments. This effectiveness stems from the specific reactivity of Fe = O towards As and the long lifetime of the Fe = O species generated in the Fe-N isolated structure. The extended stability of Fe = O ensures that the oxidative potential of PMS is utilized efficiently to remove As and other contaminants, rather than being lost to meaningless decay.
This Fe-N-C/PMS system would demonstrate strong potential for practical environmental remediation owing to its excellent recoverability, cost-efficiency, and compatibility with existing water treatment processes. The catalyst can be recovered through simple physical methods, and future development of floating-type configurations may further improve separation efficiency and operational feasibility. Its unique ability to selectively oxidize As via the formation of Fe = O, combined with the high catalytic efficiency and low metal loading requirements of a single-atom catalyst, significantly reduces material costs. Moreover, PMS is already widely used in advanced oxidation processes, and the Fe-N-C/PMS system enhances both degradation efficiency and selectivity, ensuring seamless integration with current treatment infrastructures. These features collectively position the system as a promising candidate for scalable and sustainable water treatment applications.