# Chemical Kinetic Method for Active-Site Quantification in Fe-N-C Catalysts and Correlation with Molecular Probe and Spectroscopic Site-Counting Methods

Jason S Bates, Jesse J Martinez, Melissa N Hall, Abdulhadi A Al-Omari, Eamonn Murphy, Yachao Zeng, Fang Luo, Mathias Primbs, Davide Menga, Nicolas Bibent, et al.

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To cite this version:

Jason S Bates, Jesse J Martinez, Melissa N Hall, Abdulhadi A Al-Omari, Eamonn Murphy, et al. Chemical Kinetic Method for Active-Site Quantification in Fe-N-C Catalysts and Correlation with Molecular Probe and Spectroscopic Site-Counting Methods. Journal of the American Chemical Society, 2023, 145 (48), pp.26222-26237. 10.1021/jacs.3c08790. hal-04740668

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Distributed under a Creative Commons Attribution 4.0 International License
Chemical Kinetic Method for Active-Site
Quantification in Fe-N-C Catalysts and Correlation
with Molecular Probe and Spectroscopic Site-
Counting Methods

Jason S. Bates<sup>a**</sup>, Jesse J. Martinez<sup>a</sup>, Melissa N. Hall<sup>a</sup>, Abdulhadi A. Al-Omari<sup>b</sup>, Eamonn

Murphy<sup>c</sup>, Yachao Zeng<sup>d</sup>, Fang Luo<sup>e</sup>, Mathias Primbs<sup>e</sup>, Davide Menga<sup>f</sup>, Nicolas Bibent<sup>g</sup>, Moulay

Tahar Sougrati<sup>g</sup>, Friedrich E. Wagner<sup>h†</sup>, Plamen Atanassov<sup>c</sup>, Gang Wu<sup>d</sup>, Peter Strasser<sup>e</sup>, Tim-

Patrick Fellinger<sup>f,i</sup>, Frédéric Jaouen<sup>g</sup>, Thatcher W. Root<sup>b</sup>, and Shannon S. Stahl<sup>a*</sup>

<sup>a</sup>Department of Chemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA

<sup>b</sup>Department of Chemical and Biomolecular Engineering, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA

<sup>c</sup>Department of Chemical and Biomolecular Engineering, National Fuel Cell Research Center, University of California, Irvine, California 92697, USA

<sup>d</sup>Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, New York 14260, USA

<sup>e</sup>The Electrochemical Catalysis, Energy and Materials Science Laboratory, Department of Chemistry, Technical University Berlin, 10623 Berlin, Germany

<sup>f</sup>Chair of Technical Electrochemistry, Department of Chemistry and Catalysis Research Center, Technische Universität München (TUM), 85748 Garching, Germany

<sup>g</sup>ICGM, Univ. Montpellier, CNRS, ENSCM, Montpellier, France

<sup>h</sup>Department of Physics, Technische Universität München (TUM), 85748 Garching, Germany

<sup>i</sup>Bundesanstalt für Materialforschung und -prüfung (BAM), 12203 Berlin, Germany

*Corresponding authors. Emails: stahl@chem.wisc.edu, jsb9vw@virginia.edu
**Abstract**

Mononuclear Fe ions ligated by nitrogen ($FeN_x$) dispersed on nitrogen-doped carbon (Fe-N-C) serve as active centers for electrocatalytic $O_2$ reduction and thermocatalytic aerobic oxidations. Despite their promise as replacements for precious metals in a variety of practical applications, such as fuel cells, the discovery of new Fe-N-C catalysts has relied primarily on empirical approaches. In this context, the development of quantitative structure–reactivity relationships and benchmarking of catalysts prepared by different synthetic routes and by different laboratories would be facilitated by the broader adoption of methods to quantify atomically dispersed $FeN_x$ active centers. In the present study, we develop a kinetic probe reaction method that uses the aerobic oxidation of a model hydroquinone substrate to quantify the density of $FeN_x$ centers in Fe-N-C catalysts. The kinetic method is compared with low-temperature Mössbauer spectroscopy, CO pulse chemisorption, and electrochemical reductive stripping of NO derived from $NO_2^-$ on a suite of Fe-N-C catalysts prepared by diverse routes and featuring either the exclusive presence of Fe as $FeN_x$ sites or the coexistence of aggregated Fe species in addition to $FeN_x$. The $FeN_x$ site densities derived from the kinetic method correlate well with those obtained from CO pulse chemisorption and Mössbauer spectroscopy. The broad survey of Fe-N-C materials also reveals the presence of outliers and challenges associated with each site quantification approach. The kinetic method developed here does not require pretreatments that may alter active-site distributions nor specialized equipment beyond reaction vessels and analytical instrumentation (e.g., NMR).
Introduction

Catalysts consisting of iron incorporated into nitrogen-doped carbon (Fe-N-C) are leading alternatives to precious metal electrocatalysts for the O₂ reduction reaction (ORR) in fuel cells,¹⁻⁴ and they are increasingly being used for the thermochemical aerobic oxidation of organic molecules.⁵⁻⁷ Mononuclear Fe species ligated at nitrogen-containing defects (FeNₓ) are widely recognized as the active centers for ORR ⁸⁻¹⁰ electrocatalysis and the aerobic oxidation of alcohols¹¹,¹² and hydroquinones.¹³ However, the high-temperature pyrolysis methods that are typically used to synthesize Fe-N-C catalysts often form Fe aggregates and carbon structures that bury FeNₓ centers in locations that are inaccessible to reacting molecules (Figure 1A).⁷ Such active-site diversity is common in heterogeneous catalysts and highlights the need for accurate quantification of the density of accessible FeNₓ species (i.e., the number of sites per catalyst mass), particularly when they are present within a mixture of other Fe-based species. The accurate measurement of the site density of catalytically relevant FeNₓ centers enables assessing the intrinsic reactivity of the FeNₓ centers, i.e., their turnover frequencies (TOF), in critical and emerging applications. TOF measurements, in turn, facilitate the comparison, benchmarking, and reproducibility of catalysts synthesized by different routes and by different researchers.¹⁴⁻¹⁷ While the accuracy of methods used to assess FeNₓ site densities is still debated, ¹⁸⁻²⁰ these methods are being used to guide synthetic design of Fe-N-C electrocatalysts.²¹⁻²³ In contrast, similar methods have seldom been applied to the study or development of Fe-N-C catalysts used in thermal catalytic applications.⁷ The work presented herein bridges these different fields and provides a foundation for characterization of catalysts use in either electrochemical or thermal applications.

Widely adopted strategies to quantify FeNₓ site densities in Fe-N-C catalysts consist of spectroscopic methods or chemical titrations with probe molecules. ⁵⁷Fe Mössbauer spectroscopy
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Comparison of methods to quantify FeNₓ site density. (A) Challenges for quantification of catalytically relevant FeNₓ sites due to active-site diversity in Fe-N-C catalysts, (B) previously reported molecular probe methods to quantify FeNₓ site density, and (C) proposed kinetic probe reaction for FeNₓ quantification, where FeNₓ are exemplarily illustrated as planar tetpyridinic FeN₄ sites.</figcaption></figure>

is a bulk method that detects all Fe species in a material and distinguishes between them based on their hyperfine parameters. These spectroscopic fingerprints identify the phases of Fe aggregates²⁴⁻²⁶ and distinguish mononuclear species with different oxidation states from one another,²⁷ and their peak areas can be interpreted quantitatively at low temperatures (4–10 K).²⁸ Low temperatures are also necessary to identify the presence of superparamagnetic Fe species.²⁶,²⁹ Dodelet and coworkers showed that the ORR reactivity of five Fe-N-C catalysts correlates poorly with the room-temperature Mössbauer peak area of a mononuclear species,
although the linearity of the correlation is improved when weighted by the BET surface area of each catalyst.³⁰ Thus, while Mössbauer spectroscopy provides clear insights into Fe speciation in a material, it does not provide direct information about the accessibility of the different Fe centers.⁷,¹⁸ *In situ* approaches that combine a catalytic stimulus or the adsorption of a probe molecule with Mössbauer spectroscopy show promise,¹⁰,³¹,³² but they have not yet been applied in the context of active-site quantification.

Chemical titrations rely on the selective binding of a molecular probe to FeNₓ sites with a known stoichiometry so that their adsorbed quantity can be directly related to the FeNₓ site density. Reported titrants for FeNₓ species include CO,³³ NO,³⁴ and CN⁻³⁵ (Figure 1B). In each case, specific conditions are required for titrant exposure and catalyst pretreatment to ensure that the saturation coverage of the titrant on FeNₓ centers is accurately quantified.

CO pulse chemisorption (Figure 1B-i) quantifies the adsorption of gas-phase CO to FeNₓ centers at 193 K.³³ Subambient temperatures are needed because CO adsorbs weakly to FeNₓ centers and begins to desorb at ~260 K.³³ Strasser and coworkers have shown that a pretreatment at 873 K is required to ensure that oxygenated species adsorbed to FeNₓ centers under ambient conditions are desorbed, leaving FeNₓ vacant and able to bind CO.³⁶ The FeNₓ site density determined by CO pulse chemisorption has been found to correlate well with the ORR reactivity of a series of Fe-N-C catalysts synthesized using polyaniline combined with other N-containing additives as nitrogen sources.³⁷

Nitrosyl species bound to FeNₓ centers can be quantified by electrochemical reductive stripping in acidic aqueous electrolyte (Figure 1B-ii).³⁴ Quantitative formation of NO–FeNₓ species occurs at open-circuit potentials after saturation with NO₂⁻ under neutral aqueous conditions, followed by exposure to acidic aqueous conditions to convert NO₂⁻ to NO according to the equation: NO₂⁻–FeNₓ + H⁺ → NO–FeNₓ + H₂O.³⁴,³⁸ Prior to the formation and
quantification of NO–FeNₓ species, Fe-N-C catalysts are bound to an electrode within a Nafion film and undergo extensive redox cycling in acidic aqueous electrolyte.³⁴,³⁹

CN⁻ titration occurs in a divided electrochemical cell with a graphite working electrode containing a stirred slurry of Fe-N-C in an Ar-purged acidic aqueous electrolyte (Figure 1B-iii).³⁵ The measured uptake of CN⁻ is correlated with the fractional decrease of the ORR current (0.85 V<sub>RHE</sub>, 0.1 M HClO₄ electrolyte) relative to a pristine Fe-N-C catalyst and extrapolated to the CN⁻ uptake where the ORR current becomes zero, reflecting the saturation of FeNₓ with CN⁻. To ensure irreversible CN⁻ adsorption, O₂ is purged from the cell under flowing Ar and the cell is polarized at 0.5 V<sub>RHE</sub> to strip oxygen-derived species from Fe-N-C. The cell is then stabilized at 1.0 V<sub>RHE</sub> before CN⁻ is added at this condition.

Comparative studies of the approaches described above are limited,⁷,¹⁸,³⁵ but those that are available highlight drawbacks associated with each technique. A cross-laboratory study comparing CO pulse chemisorption and NO stripping observed that NO quantified 12–39% of the sites quantified by CO on a series of four Fe-N-C catalysts. This discrepancy was ascribed to different accessibility of active centers for the two techniques.¹⁸ For example, the high-temperature pretreatment used before CO pulse chemisorption may redisperse aggregated species to atomically dispersed species,⁴⁰ and a gas-phase titrant may not access the same number of active centers as liquid-phase catalytic reactions. The 1:1 binding stoichiometry of CO to FeNₓ also remains an assumption. On the other hand, the Nafion film configuration used for NO stripping may alter the accessibility of FeNₓ binding sites, and the redox cycling pretreatment that is used may influence their distribution. Additionally, converting the stripping charge to a quantity of NO–FeNₓ species requires assignment of a specific reduction product, and both 5 e⁻ (NH₄⁺)³⁴ and 3 e⁻ (NH₂OH)⁴¹ products have been proposed. Bae et al. quantified the same number of active centers on one Fe-N-C catalyst regardless of whether CO, NO (assuming 3 e⁻
reduction of NO), or CN⁻ was used,³⁵ but the generality of this observation for other catalysts was not probed.

Methods that expand beyond probe molecules and spectroscopy have also been developed. Fellinger and coworkers circumvented the challenges associated with probe molecules by synthesizing an Fe-N-C catalyst containing predominantly FeNₓ centers, then systematically leached Fe from the material to generate a series of three catalysts with varying FeNₓ contents that correlated linearly with their ORR current density (0.70–0.80 V$_{RHE}$, 0.1 M HClO₄).⁴² While this strategy was effective for determining the ORR turnover frequency of the active centers in a model material, the extension of this approach to materials that possess a mixture of active site structures has not yet been demonstrated. In another approach, Elbaz and coworkers applied Fourier-transform alternating current voltammetry to quantify the charge putatively associated with the Fe<sup>II/III</sup>Nₓ redox transition.⁴³ This technique was applied to two commercially available Fe-N-C catalysts and estimated ~60% of the active-site density quantified by NO stripping.⁴³ Lastly, Yu and coworkers have employed scanning electrochemical microscopy in combination with redox probes to quantify FeNₓ centers,⁴⁴,⁴⁵ but this strategy has not been adopted by other groups to date. The different conditions and disagreement among active-site quantification techniques, together with the specialized equipment and/or experimental challenges associated with certain techniques, motivate the development of other methods that could aid in the comparison of Fe-N-C catalysts prepared by different synthetic routes and by different research groups.

Here, we develop a new kinetic approach to quantify FeNₓ centers (exemplarily illustrated as planar tetapyridinic FeN₄ sites in **Figure 1C**) and compare it directly with low-temperature ⁵⁷Fe Mössbauer spectroscopy, CO pulse chemisorption, and reductive stripping of NO derived from NO₂⁻. This kinetic approach relies on quantifying the initial rate of hydroquinone (HQ) oxidation
to a quinone by aerobic oxidation in an aqueous slurry-phase semibatch reactor. The method uses an easily synthesized water-soluble, quinone derivative$^{46,47}$ and requires no pretreatments to the Fe-N-C catalyst. The initial rate reflects surface reactions at FeNₓ centers but not at Fe aggregates, as shown by our previous work.$^{13,48}$ The synthesis of Fe-N-C catalysts containing FeNₓ sites on solvent-accessible surfaces using recently developed approaches$^{13,49}$ therefore enables estimation of the intrinsic turnover frequency (TOF) of HQ oxidation. The HQ oxidation TOF, in turn, permits calculation of the FeNₓ site density on any material solely from its initial HQ oxidation rate. This kinetically quantified FeNₓ site density correlates well with values obtained from CO pulse chemisorption and Mössbauer spectroscopy for a suite of Fe-N-C catalysts with diverse synthetic origins, bulk Fe contents, and Fe speciation. The straightforward nature of this approach should make it a compelling complement to existing site-quantification methods.

## Results and Discussion

**Assessing the Intrinsic Reactivity of FeNₓ Centers in Fe-N-C Catalysts.** Fe-N-C catalysts containing predominantly FeNₓ centers at solvent-accessible surfaces were synthesized to quantify the intrinsic reactivity of FeNₓ centers. Fe-N-C catalysts with these attributes can be synthesized via recently reported strategies to metalate vacant macrocyclic Nₓ binding sites on nitrogen-doped carbon scaffolds (Figure 2A).$^{13,49,50}$ A metal-organic framework composed of Zn²⁺ nodes with imidazolate linkers, ZIF-8, was synthesized by reported methods$^{51}$ and used as the precursor for the N-doped carbon. ZIF-8 was treated under flowing N₂ at 1323 K to yield a nitrogen-doped carbon material containing both ZnNₓ centers and vacant Nₓ binding sites.$^{13,52}$ This material was contacted with a solution of FeCl₂ in DMF at 423 K under N₂ to effect the metalation of vacant Nₓ centers with Fe (Figure 2A). The use of solution-phase conditions ensures that Fe species are deposited on solvent-accessible surfaces of the material. Two
different concentrations of Fe were used to synthesize two materials with different bulk Fe contents (0.1 wt% and 0.4 wt%). After metalation treatments, washing, and recovery of the solids, the materials were treated under flowing N₂ at 873 K for 2 h in order to ensure the complete dispersion of Fe species,¹³ and to facilitate comparisons of reactivity with CO pulse chemisorption measurements that require a pretreatment under the same conditions.³⁶ The two catalysts synthesized by the metalation of ZIF-8-derived nitrogen-doped carbon are referred to as xFe-N-Cm-ht, where x refers to the Fe wt% quantified by ICP-OES, and “m-ht” indicates that metalation followed by a heat treatment was the synthetic route.

<figure><img src="image_4.png" /><figcaption>**Figure 2.** (A) Preparation of atomically dispersed Fe-N-C catalysts via solution-phase metalation and heat treatment. Planar tetpyridinic sites are shown as an example. (B) <sup>57</sup>Fe Mössbauer spectra (10 K) of 0.1Fe-N-Cm-ht (top) and 0.4Fe-N-Cm-ht (bottom). Data (open points) are fit with a sum (black line) of components assigned to Fe<sup>III</sup>Nₓ (D1, green), Fe<sup>II</sup>Nₓ (D2, blue), FeCl₂·4H₂O (D3, red), and FeOₓ clusters (S1, gray). (C) Concentration of quinone formed as a function of contact time on 0.4Fe-N-Cm-ht (purple) and 0.1Fe-N-Cm-ht (blue). The Fe-free analog (ZIF-8-C) is shown in red. Conditions: 30 °C, 1.1 bar O₂, 0.5 M H₂SO₄, [HQ]₀ = 50 mM, [Q]₀ = 1 mM. Lines reflect regressed fits constrained through the origin. (D) Correlation of the Fe-catalyzed HQ</figcaption></figure>
oxidation rate (per g$_{catalyst}$) with the density of FeN$_{x}$ centers quantified from the Mössbauer spectra in (B) combined with the bulk Fe content measured by ICP-OES, according to eq 1. The corrected rate reflects the difference between the rate measured in (C) and the rate measured on ZIF-8-C, as expressed in eq 2. The solid line reflects the best-fit linear regression to the three data points, where the slope is defined as TOF$_{FeNx}$. The shaded region bounded by the dashed lines represents the statistical error associated with TOF$_{FeNx}$.

The Fe speciation of the two catalysts was characterized by low-temperature Mössbauer spectroscopy (10 K). The Mössbauer spectra (Figure 2B) are predominantly composed of a single doublet component referred to as “D1,” which is assigned to a mononuclear Fe$^{III}$N$_{x}$ species.²⁷ Other minor components include “D2,” which is assigned to an Fe$^{II}$N$_{x}$ species,²⁷ “D3,” a high-spin Fe$^{II}$ species that corresponds to FeCl$_{2}$.4H$_{2}$O remaining from the synthesis,⁵² and a sextet assigned to FeO$_{x}$ aggregates (see section 3.1 and Figures S1 and S2 in the Supporting Information for additional discussion of fitting and parameters). The relative peak areas of each component in Mössbauer spectra can be interpreted to quantify the relative molar fraction of bulk Fe in the material when the Lamb–Mössbauer factors of all species are equivalent. Sougrati et al. showed that this is a reasonable assumption for D1, D2, α-Fe, and γ-Fe species only at low temperatures (5–10 K).²⁸ With this assumption, the FeN$_{x}$ active-site density can be calculated according to eq 1:

$$N_{FeN_x,Moss} = N_{Fe,bulk} \times \frac{A_{D1}}{100} \quad (1)$$

where $N_{Fe,bulk}$ (mol g$^{-1}$) is the total Fe content quantified by ICP-OES, and $A_{D1}$ reflects the percentage of total area assigned to D1 in the Mössbauer spectrum. Applying this analysis to the two Fe-N-C$^{m-ht}$ materials gives active-site densities of 19 ± 9 µmol g$^{-1}$ and 48 ± 11 µmol g$^{-1}$, where the standard errors result from least-squares fitting of the Mössbauer spectra. Similar analysis, accounting for the area of D1+D2 Fe sites, is presented in section 6 of the Supporting Information. We assume that all FeN$_{x}$ species assigned as active sites by Mössbauer spectroscopy in the Fe-N-C$^{m-ht}$ materials reside in pore environments that are accessible to the
DMF-solvated $FeCl_2$ precursor used for metalation. These quantitative active-site densities facilitate direct correlations with catalytic reactivity.

The aerobic oxidation of a sulfonated hydroquinone (Figure 2C) was chosen as a catalytic probe reaction. This reaction was chosen because it is mechanistically well-understood and rates can be measured under kinetically limiting conditions⁴⁸ and because it is catalyzed by $FeN_x$ centers.¹³ Additional discussion of transport limitations can be found in section 4.2 of the Supporting Information. The conditions of the measurement shown in Figure 2C define initial reaction rates that reflect the quasi-equilibrated adsorption of $O_2$ at $FeN_x$ sites followed by kinetically relevant hydrogen-atom transfer from a physisorbed HQ molecule present on the doped carbon surface at saturation coverages.⁴⁸ The initial rate of HQ oxidation (per $g_{catalyst}$) was measured in semibatch reactors on $Fe-N-C^{m-ht}$ catalysts and their N-C precursor. The rate on the nominally Fe-free N-C precursor was 11–24% of the rate catalyzed by the $Fe-N-C^{m-ht}$ catalysts and was subtracted from these rates to give the rate catalyzed by $FeN_x$ sites. These corrected HQ oxidation rates correlate linearly with the $FeN_x$ active-site density of the $Fe-N-C^{m-ht}$ catalysts (Figure 2D, solid line), according to eq 2:

$$r_{HQ,corr} = r_{HQ,Fe-N-C} - r_{HQ,N-C} = TOF_{FeN_x} \times N_{FeN_x,Moss} \quad (2)$$

where $r_{HQ,Fe-N-C}$ (mol g⁻¹ s⁻¹) is the HQ oxidation rate measured on the Fe-N-C catalyst, $r_{HQ,N-C}$ (mol g⁻¹ s⁻¹) is the HQ oxidation rate measured on the N-C precursor, $r_{HQ,corr}$ (mol g⁻¹ s⁻¹) represents the corrected HQ oxidation rate attributed to Fe species, and $TOF_{FeN_x}$ (s⁻¹) is the turnover frequency of HQ oxidation catalyzed by $FeN_x$ sites. The value of $TOF_{FeN_x}$, 1.1 ± 0.3 s⁻¹, reflects the intrinsic reactivity of the surface $FeN_x$ sites in these two $Fe-N-C^{m-ht}$ catalysts.

The definition of $TOF_{FeN_x}$ provides an opportunity to interrogate the behavior of Fe-N-C catalysts that contain a mixture of Fe species. In these cases, the HQ oxidation rate could reflect contributions from these other species, e.g., $FeO_x$ and γ-Fe aggregates, reflected by eq 3:
$$r_{HQ,corr} = TOF_{FeN_x} \times N_{FeN_x} + TOF_{FeO_x} \times N_{FeO_x} + TOF_{\gamma-Fe} \times N_{\gamma-Fe} + \dots \quad (3)$$

where $TOF_i$ (s⁻¹) and $N_i$ (mol g⁻¹) reflect the turnover frequency and surface site density of any species $i$. In the limiting case where $TOF_{FeN_x}$ is much larger than the TOF of any other species, the HQ oxidation rate can be used to calculate the $FeN_x$ site density on any Fe-N-C material according to eq 4:

$$N_{FeN_x,HQ} = r_{HQ,corr}/TOF_{FeN_x} \quad (4)$$

$FeO_x$ species¹⁰,⁵³ and $Fe/Fe_3C$ species⁵⁴ show low ORR reactivity on their own, especially in acidic medium, and our prior study showed that the coexistence of aggregated Fe species with $FeN_x$ species on Fe-N-C catalysts leads to a lower HQ oxidation rate (per total Fe) than on Fe-N-C catalysts containing only $FeN_x$ species.¹³ These observations are consistent with the assumptions that lead to eq 4. We will further probe these assumptions by comparing $FeN_x$ site densities calculated using eq 4 with alternative active-site quantification methods based on molecular probes. This approach begins with 0.4Fe-N-C$^{m-ht}$ as an example.

**Quantification of FeNₓ Active Sites in Fe-N-C Catalysts using Molecular Probes.** The methods of CO pulse chemisorption and electrochemical stripping of NO derived from $NO_2^-$ were used to obtain independent estimates of the $FeN_x$ active-site density of 0.4Fe-N-C$^{m-ht}$. CO pulse chemisorption was performed in a gas-phase flow-through setup at 195 K after a pretreatment at 873 K in flowing He (Figure 3A), as previously reported.³⁶ Reduced CO peak areas (m/z = 28 detected by mass spectrometry) in the first two pulses reflect CO adsorption to $FeN_x$ centers with a cumulative uptake of 46 µmol g⁻¹, while equal peak areas observed for the third through tenth pulses indicate saturation of the $FeN_x$ sites (Figure 3A). A control experiment with the ZIF-8-C material prior to metalation shows only 3 µmol g⁻¹ uptake (Figure S12, Supporting Information), confirming that $FeN_x$ centers adsorb CO in 0.4Fe-N-C$^{m-ht}$. We
consider this uptake (3 µmol g⁻¹) to be the minimum error associated with the CO pulse chemisorption measurement.

The electrochemical stripping of NO derived from $NO_2^-$ was measured on a Nafion-bound film of 0.4Fe-N-C$m^{m-ht}$ deposited onto a rotating-disc electrode following protocols adapted from Kucernak and coworkers.<sup>34,39,55</sup> The material was exposed to a 0.125 M $NO_2^-$ solution and then the adsorbed $NO_2^-$ was converted to NO by exposure to $H^+$ in a buffered electrolyte solution (0.5
<figure><img src="image_5.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_6.png" /></figure>

Potential / V<sub>RHE</sub>

Current Density / mA cm<sup>-2</sup>

<figure><img src="image_7.png" /><figcaption>C.</figcaption></figure>

<figure><img src="image_8.png" /></figure>
**Figure 3.** Probe molecule titrations of FeNₓ sites in 0.4Fe-N-Cm²·ht. (A) CO pulse chemisorption profile (195 K). (B) The upper plot shows cyclic voltammograms measured under Ar-saturated electrolyte (10 mV s⁻¹, 0.5 M acetate buffer pH 5.2) before (blue) and after (red) NO poisoning, and after NO stripping (green), and the lower plot shows the calculated stripping current density (*i*<sub>stripping</sub> − *i*<sub>unpoisoned</sub>) and potential as a function of time derived from the CV measurements. The orange line indicates the integration baseline. See Supporting Information, Section 3.3 for alternative analysis approaches and discussion. (C) Comparison of FeNₓ active-site densities obtained by hydroquinone oxidation kinetics (HQ, red), CO pulse chemisorption (CO, blue), electrochemical stripping of NO derived from NO₂⁻ (NO, green), and ⁵⁷Fe Mössbauer spectroscopy (Möss, yellow).

M acetate, pH = 5.2, **Figure 3B**). The electrochemical ORR kinetic current density (0.8 V<sub>RHE</sub>) decreased by a factor of 12 after NO poisoning (Figure S28b, Supporting Information), which is consistent with adsorption of NO to FeNₓ active centers, rendering them inactive. The adsorbed NO was electrochemically stripped from FeNₓ centers by executing a cyclic voltammetry scan between 0.4 V<sub>RHE</sub> and −0.3 V<sub>RHE</sub> under Ar. The baseline current density measured under the same CV conditions prior to the measurement of ORR reactivity (blue curve, **Figure 3B**) was subtracted from a stripping CV measured following NO poisoning (red curve, **Figure 3B**) to yield the stripping current density associated with NO–FeNₓ species (black curve, **Figure 3B**). The accuracy of this measurement is highly sensitive to the reproducibility of the baseline CV scan. A reliable baseline is essential to ensure that the stripping charge reflects only NO stripping and not currents associated with capacitance changes or other redox events in the film. It can be observed that the green CV in **Figure 3B** labeled “Recovered”, collected following NO stripping, does not match the blue “Unpoisoned” CV. Through control experiments measuring the CVs of films lacking FeNₓ centers such as those containing carbon black and ZIF-8-C, we concluded that such confounding factors were causing the “Recovered” CV to be poorly reproducible, so we chose to use the “Unpoisoned” CV as the reference to calculate the NO stripping current density. Further discussion of these challenges and comparisons of alternative baseline and stripping CV calculations can be found in section 3.3 of the Supporting Information.
The integration baseline shown in orange in **Figure 3B** was shifted vertically in the analysis with the intent to correct for baseline differences that persisted between the stripping and unpoisoned CVs. This baseline shift was not required for all materials investigated in this work but was required in this case, further illustrating the challenges associated with the NO stripping method. The integrated stripping charge of 7.3 C g⁻¹ is converted to a molar quantity of 25 µmol g⁻¹ NO by assuming that the NO reacts with 3 H⁺ and 3 e⁻ to form NH₂OH as reported previously³⁵,⁴¹ (site density values assuming 5 e⁻ to form NH₄⁺ are reported in Table S3 in the Supporting Information). The NO reduction stoichiometry/product identity adds one additional complicating feature of this measurement.

The FeNₓ site densities quantified by CO and NO titrations are compared in **Figure 3C** with those derived from HQ oxidation and Mössbauer spectroscopy. The HQ-based site density calculated by eq 4 uses the value of TOF<sub>FeNx</sub> estimated in **Figure 2C**, and thus agrees with the Mössbauer site density by definition; however, this relationship will not be guaranteed for catalysts made by alternative synthetic routes (*vide infra*). The FeNₓ site density quantified by CO closely agrees with that estimated by the HQ and Mössbauer methods, whereas NO quantifies a lower FeNₓ site density (52–55% of the other methods). Lower active-site densities quantified by NO are consistent with a previous cross-laboratory study,¹⁸ and these differences may have chemical or procedural origins. Chemically, (i) there may be reduced active-site accessibility in the ionomer-containing film used to contact the catalyst with an electrode, or (ii) FeNₓ active centers may degrade during the extensive redox cycling pretreatments used before NO stripping. Procedurally, key assumptions may not be met, including (i) that the baseline used to correct the stripping data accurately leads to the quantification of NO stripping charge and no other spurious contributions, and (ii) that the stripping event reflects a well-defined stoichiometric reduction event to NH₂OH (or NH₄⁺) rather than a mixture of products. Next, we
extend these comparisons to include a suite of Fe-N-C catalysts synthesized by diverse routes and containing different distributions of Fe<sup>II</sup>N<sub>x</sub> and Fe<sup>III</sup>N<sub>x</sub> species in addition to different proportions of FeN<sub>x</sub> sites coexisting with a variety of aggregated Fe species.

**Quantification of FeN<sub>x</sub> Active Sites in Fe-N-C Catalysts Synthesized by Diverse Methods.** Twelve additional Fe-N-C catalysts were synthesized by different routes by six different laboratories and acquired from one fuel cell catalyst company to supplement the Fe-N-C<sup>m-ht</sup> catalysts described above (Table 1). Fe-N-C materials are generally named “xFe-N-C[descriptor]”, where the leading x denotes the bulk Fe content quantified by ICP-OES.<sup>56</sup> Adventitious air was included in solution-phase metalation treatments to intentionally deposit FeO<sub>x</sub> species alongside FeN<sub>x</sub> species in xFe-N-C<sup>ma-ht</sup> materials (“ma” = metalation with air). A 0.3Fe-N-C<sup>HCl-m-ht</sup> material was synthesized by the solution-phase metalation (cf. Figure 2A) of HCl-treated ZIF-8-derived carbon. Another Fe-N-C material was synthesized by a FeCl<sub>3</sub>/LiCl eutectic salt melt metalation of a Zn-N-C material prepared via salt templating pyrolysis of 1,2-dicyanobenzene within a ZnCl<sub>2</sub>/LiCl mixture, followed by HCl washing and flash pyrolysis, and is denoted 4.7Fe-N-C-ST, where “ST” reflects the salt templating procedure used by the team at BAM (Bundesanstalt für Materialforschung und -prüfung).<sup>57</sup> A 1.5Fe-Phen-C catalyst was synthesized by the pyrolysis of a carbon-supported [Fe(Phen)<sub>3</sub>]<sup>2+</sup> complex. A 1.2Fe-PANI-C catalyst was synthesized by the pyrolysis of a carbon-supported Fe/polyaniline mixture. A second catalyst was synthesized by the pyrolysis of a mixture of activated carbon support, Fe precursor, and polyaniline, and is denoted 5.7Fe-N-C-TUB (TUB = Technical University Berlin).<sup>58</sup> A 0.5Fe-N-C-UCI catalyst (UCI = University of California, Irvine) was synthesized by the impregnation of an Fe/nicarbazin mixture into a sacrificial nanoporous silica template via pyrolysis and acid etching.<sup>59,60</sup> A commercial fuel cell catalyst was obtained from Pajarito Powder, denoted 0.5Fe-N-C-PAJ.<sup>61</sup> Two xFe-N-C-UB catalysts were prepared by the pyrolysis
of an Fe₂O₃@ZIF-8 composite under different gas compositions, namely 10% H₂/Ar (0.9Fe-N-C-UB; UB = University at Buffalo) and Ar (0.8Fe-N-C-UB).⁶² A 0.8Fe-N-C-CNRS catalyst was synthesized by the pyrolysis of a ball-milled mixture of ZIF-8, Fe(OAc)₂, and 1,10-phenanthroline under Ar at 1323 K (CNRS = Centre National de la Recherche Scientifique).⁸

<table><caption>**Table 1. Summary of Fe-N-C catalysts investigated in this work.**</caption><thead><tr><th rowspan="2">Fe-N-C catalyst</th><th colspan="2">Bulk Fe Content<sup>a</sup></th><th rowspan="2">Brief Description</th></tr><tr><th>/ wt%</th><th>/ 10<sup>-5</sup> mol g<sup>-1</sup></th></tr></thead><tbody><tr><td>0.1Fe-N-C<sup>m-ht</sup></td><td>0.1</td><td>2.1</td><td>Solution-phase metalation and heat treatment<sup>13</sup></td></tr><tr><td>0.4Fe-N-C<sup>m-ht</sup></td><td>0.4</td><td>6.6</td><td>Solution-phase metalation and heat treatment<sup>13</sup></td></tr><tr><td>1.3Fe-N-C<sup>ma-ht</sup></td><td>1.3</td><td>24</td><td>Solution-phase metalation (adventitious air) and heat treatment<sup>13</sup></td></tr><tr><td>8.4Fe-N-C<sup>ma-ht</sup></td><td>8.4</td><td>150</td><td>Solution-phase metalation (Fe(OAc)<sub>2</sub> precursor, adventitious air) and heat treatment<sup>13</sup></td></tr><tr><td>0.3Fe-N-C<sup>HCl-m-ht</sup></td><td>0.3</td><td>5.8</td><td>Solution-phase metalation of HCl-treated ZIF-8-C and heat treatment<sup>13</sup></td></tr><tr><td>4.7Fe-N-C-ST</td><td>4.7</td><td>84</td><td>Eutectic salt melt metalation and heat treatment<sup>57</sup></td></tr><tr><td>1.5Fe-Phen-C</td><td>1.5</td><td>26</td><td>Pyrolysis of C-supported [Fe(Phen)<sub>3</sub>]<sup>2+</sup><sup>13</sup></td></tr><tr><td>1.2Fe-PANI-C</td><td>1.2</td><td>22</td><td>Pyrolysis of C-supported Fe/polyaniline<sup>13</sup></td></tr><tr><td>5.7Fe-N-C-TUB</td><td>5.7</td><td>102</td><td>Pyrolysis of C-supported Fe/polyaniline<sup>58</sup></td></tr><tr><td>0.5Fe-N-C-UCI</td><td>0.5</td><td>8.9</td><td>Sacrificial support method (SSM)<sup>59,60</sup></td></tr><tr><td>0.5Fe-N-C-PAJ</td><td>0.5</td><td>8.6</td><td>SSM – manufactured by Pajarito Powder<sup>61</sup></td></tr><tr><td>0.9Fe-N-C-UB</td><td>0.9</td><td>16</td><td>Pyrolysis of Fe₂O₃@ZIF-8 composite in 10% H₂/Ar<sup>62</sup></td></tr><tr><td>0.8Fe-N-C-UB</td><td>0.8</td><td>15</td><td>Pyrolysis of Fe₂O₃@ZIF-8 composite in Ar<sup>62</sup></td></tr><tr><td>0.8Fe-N-C-CNRS</td><td>0.8</td><td>14</td><td>Pyrolysis of ZIF-8 mixed with Fe(OAc)<sub>2</sub> and 1,10-phenanthroline in Ar at 1323 K<sup>8</sup></td></tr></tbody></table>

The FeNₓ active-site density of all fourteen Fe-N-C catalysts (N<sub>FeNx,HQ</sub>) was estimated using the kinetic HQ oxidation approach and compared with alternative quantification approaches. CO pulse chemisorption data (N<sub>FeNx,CO</sub>) were measured on thirteen catalysts, NO stripping profiles (N<sub>FeNx,NO</sub>) were measured on thirteen catalysts, and Mössbauer spectra (N<sub>FeNx,Möss</sub>) were measured on eleven catalysts. These site densities are compared for each catalyst in **Figure 4A**. In a few cases, there was not sufficient material to employ all four characterization methods. We note that although the value of N<sub>FeNx,Möss</sub> is determined using the area of the D1 component according to

<footnote><sup>a</sup>Quantified by ICP-OES.⁵⁶</footnote>
eq 1, we have also included a full analysis with D1+D2 in Section 6 of the Supporting Information. We will focus on each materials class in turn, then assess broader trends in the comparison between active-site quantification approaches.

<figure><img src="image_9.png" /><figcaption>**Figure 4.** Comparison of kinetic, spectroscopic, and probe-molecule methods to quantify the density of FeNₓ centers in the Fe-N-C catalysts investigated in this work. FeNₓ quantities are compared based on (A) a bulk site density basis (mol g_catalyst⁻¹) and (B) a site utilization basis normalized to the bulk Fe content of each material. Hydroquinone oxidation (red bars), CO pulse chemisorption (blue bars), electrochemical NO stripping (green bars), and ⁵⁷Fe Mössbauer spectroscopy (yellow bars) are compared. The ⁵⁷Fe Mössbauer spectra were measured at 10 K on xFe-N-C^m-ht and 1.3Fe-N-C^ma-ht and at 4.2 K for all other reported values. HQ oxidation error bars reflect the propagated standard error of the rate measurement (±20%) and TOF value (±0.3</figcaption></figure>
$s^{-1}$) used to calculate $N_{FeNx}$. CO pulse chemisorption error bars reflect the larger of either the error associated with sample mass (±10%) or the error expected from background adsorption determined on an Fe-free ZIF-8-C material (3 µmol g$^{-1}$, see Figure S12). Electrochemical NO stripping error bars reflect the error derived from control experiments (5 µmol g$^{-1}$, see section 3.3.2 of the Supporting Information). Mössbauer spectroscopy error bars reflect the statistical error of the least-squares fitting procedure. Alternative analyses based on D1+D2 as the Mössbauer active-site density can be found in Section 6 of the Supporting Information.

Fe-N-C catalysts derived from solution-phase metalation approaches (black squares and circles, **Figure 4A**) and a eutectic salt melt metalation strategy (4.7Fe-N-C-ST, yellow pentagon, **Figure 4A**) generally show agreement between $N_{FeNx,HQ}$ and $N_{FeNx,CO}$. NO stripping quantified fewer FeNₓ sites than HQ and CO in several cases. Mössbauer spectroscopy shows wider but nonsystematic variations: in the case of 8.4Fe-N-C<sup>ma-ht</sup>, $N_{FeNx,Möss}$ is smaller than the values given by the other methods (44–83%); and in the case of 1.3Fe-N-C<sup>ma-ht</sup>, $N_{FeNx,Möss}$ is higher by a factor of 1.7–2.4. The Mössbauer spectrum of 8.4Fe-N-C<sup>ma-ht</sup> (Figure S4) shows that the majority of Fe is present as FeOₓ (97 ± 6%), which leads to a large error in the deconvoluted area that reflects the FeNₓ site fraction (3 ± 3%). The Mössbauer spectrum of 1.3Fe-N-C<sup>ma-ht</sup> shows that over 50% of the Fe in the material is present as FeOₓ (Figure S3), but the D1 doublet feature assigned to Fe<sup>III</sup>Nₓ is not subject to large fitting errors (32 ± 3%). However, the presence of magnetically ordered FeOₓ aggregates in this material suggests that there might also be nanosized FeOₓ aggregates, which can behave as superparamagnetic species even at 10 K and lower.<sup>26,63–65</sup> Such species show a D1-like signal rather than a sextet feature, causing the FeNₓ site density by eq 1 to be overestimated. These cases illustrate two drawbacks of Mössbauer spectroscopy: (i) its accuracy becomes poor when FeNₓ is a minority species in the spectrum, and (ii) small FeOₓ aggregates may contribute to D1 peak area identified as FeNₓ species. In contrast, CO chemisorption has been shown to be insensitive to FeOₓ sites,<sup>19,33,36,37</sup> and the HQ and CO methods give site densities within reasonable agreement for these materials. This similarity between FeNₓ site densities quantified by HQ oxidation and CO pulse chemisorption suggests that FeOₓ sites that coexist with FeNₓ centers do not contribute significantly to HQ oxidation.
Close agreement between $N_{FeNx,HQ}$ and $N_{FeNx,CO}$ is also observed for materials prepared by pyrolysis of material containing a molecular Fe complex (1.5Fe-Phen-C, open circle in **Figure 4A**) and from an Fe/polyaniline/carbon mixture (1.2Fe-PANI-C and 5.7Fe-N-C-TUB, open circle and blue triangle in **Figure 4A**, respectively). The Mössbauer spectra of these materials show peaks for metallic and carbidic Fe, and in the case of the PANI-derived materials, $Fe_3S_4$ (**Figure S6–S8**). The values of $N_{FeNx,NO}$ for these materials deviate from $N_{FeNx,HQ}$ and $N_{FeNx,CO}$ by factors of 0.5–1.8, without a systematic deviation. Mössbauer spectroscopy agrees well with the other methods in the case of 1.5Fe-Phen-C, but it estimates a higher $FeN_x$ site density than the other methods when applied to the polyaniline-derived catalysts. The value of $N_{FeNx,Möss}$ is higher than $N_{FeNx,HQ}$ by a factor of 2 for 1.2Fe-PANI-C and by a factor of 3.7 for 5.7Fe-N-C-TUB. The Mössbauer spectrum (Figure S8) of 5.7Fe-N-C-TUB is broader than those measured on other materials and shows a minority of D1 species, resulting in a larger error (12 ± 12% D1). So, subsequent analyses will be considered both with and without this outlier. The close agreement between HQ and CO in these cases is consistent with their selectivity for $FeN_x$ quantification, without interference from Fe metal, carbide, and sulfide particles.

The 0.5Fe-N-C-UCI and 0.5Fe-N-C-PAJ materials are synthesized via a similar silica- templating approach.⁶⁶,⁶⁷ The $FeN_x$ active-site densities of 0.5Fe-N-C-PAJ (red star, **Figure 4A**) quantified by all four approaches are similar, within error, whereas the kinetic method quantified higher active-site densities than CO and NO on 0.5Fe-N-C-UCI (green diamond, **Figure 4A**). Potential reasons for this disagreement include (1) that the intrinsic reactivity of some $FeN_x$ centers is higher in 0.5Fe-N-C-UCI than in the metalated materials used to determine $TOF_{FeNx}$, (2) that $FeN_x$ centers become occluded or degraded by pretreatments prior to probe-molecule measurements (873 K for CO, redox cycling for NO), or (3) that probe molecules do not saturate all $FeN_x$ centers under the conditions of the measurement.
The xFe-N-C-UB and 0.8Fe-N-C-CNRS catalysts were synthesized by variations on an approach based on the pyrolysis of the ZIF-8 metal-organic framework. The 0.8Fe-N-C-CNRS catalyst (orange circle, **Figure 4A**) shows good agreement between active-site densities quantified by HQ, CO, and Mössbauer, but not NO. In contrast, the values of $N_{FeNx,HQ}$ are higher than $N_{FeNx,CO}$ by factors of 1.6–2.0 on xFe-N-C-UB catalysts (blue squares, **Figure 4A**). Further, the value of $N_{FeNx,NO}$ matches $N_{FeNx,CO}$ in one case (0.8Fe-N-C-UB) but is significantly lower in the other case (0.9Fe-N-C-UB). The Mössbauer spectrum (Figure S10, Supporting Information) of 0.8Fe-N-C-UB estimates an $FeN_x$ active-site density that lies between those of the kinetic and probe-molecule methods. Among these Fe-N-C materials, NO consistently estimates a lower $FeN_x$ active-site density than the other methods as observed in other studies.¹⁹ Potential reasons for these deviations have been discussed above, and one additional reason could be that accurate estimates for the Fe-free N-C HQ oxidation rate were not available on these materials, which would lead to an overestimate of the active-site density by HQ oxidation (cf. eq 2 and eq 4, where $r_{HQ,N-C} = 0$).

The $FeN_x$ active-site density can also be expressed as an Fe utilization after normalization to the total bulk Fe content in each material.³³ The Fe utilization value is crucial to assess the reasonableness of an active-site quantity, as values >1 likely suggest problems with the measurement or underlying assumptions of the method. All Fe utilization values shown in **Figure 4B** fall between 0–1, indicating that Fe is a reasonable source of all of the sites counted by the protocols used in this study. Kucernak and coworkers have shown that gas-phase NO also adsorbs at sites not associated with Fe on Fe-N-C surfaces,⁶⁸ consistent with another report where gas-phase NO exposure estimated a ~2x higher site density than NO derived from $NO_2^-$ exposure.⁵² In fact, Fe utilization values >1 and as high as 6 have gone unnoticed in NO titration
data.⁶⁹⁻⁷¹ These considerations highlight how Fe utilization should serve as a first diagnostic for unselective titration or other methodological artifacts.

**Comparison of FeNₓ Active-Site Quantification Methods.** Correlations between N<sub>FeNₓ,HQ</sub> and each of the other three active-site quantification methods are shown as parity plots in **Figure 5**. The remaining correlations between the other methods can be found in Figure S48 of the Supporting Information. The deviation from parity is assessed as the mean absolute error (MAE) and the mean percentage error (MPE), where smaller values indicate better agreement between the
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 5.** Parity plots comparing spectroscopic and probe-molecule FeNₓ site quantification methods with the kinetic method. The dashed lines indicate equality between the two values. All x-axes reflect kinetically quantified FeNₓ site densities derived from hydroquinone oxidation rates and the intrinsic TOF of FeNₓ sites (1.1 ± 0.3 s⁻¹). The y-axes reflect (A) CO pulse chemisorption, and (B) stripping of NO derived from NO₂⁻, and (C) Mössbauer spectroscopy. The mean absolute error (MAE) and mean percentage error (MPE) associated with each correlation are calculated according to eq S9 and S10 in the Supporting Information and shown in the plot area. The two xFe-N-C^m-ht materials (black squares) are omitted from the MAE and MPE values in (C) because they were used to estimate TOF<sub>FeNx</sub> (Figure 2D) and so N<sub>FeNx,HQ</sub> and N<sub>FeNx,Möss</sub> are not independently determined. The MAE and MPE values in (C) without the outlier 5.7Fe-N-C-TUB are reported in pale red text.</figcaption></figure>
two active-site quantification methods (for more details, see section 5 of the Supporting Information). Data points that lie in the lower-right of the parity plots indicate overcounting by HQ and/or undercounting by the other method, whereas data points in the upper-left reflect the inverse.

The strongest correlation between two FeNₓ active-site quantification methods is observed with the HQ oxidation and CO pulse chemisorption techniques (Figure 5A, MAE = 1.4, MPE = 26%). The correlation between HQ and NO is rather poor (Figure 5B, MAE = 2.3, MPE = 53%). Mössbauer spectroscopy also shows worse agreement with HQ oxidation than does the CO pulse chemisorption technique (Figure 5C, MAE = 2.1, MPE = 38%), even after removal of the 5.7Fe-N-C-TUB outlier (MAE = 1.6, MPE = 32% after removal of 5.7Fe-N-C-TUB). Three outliers in Figure 5A fall in the lower-right of the parity plot because CO may undercount or because HQ oxidation may overcount active sites, whereas the inverse is the case for 4.7Fe-N-C-ST (yellow pentagon). NO generally estimates fewer FeNₓ active-sites than HQ or CO (Figure 5B and S48A), which is consistent with prior reports¹⁸ and inconsistent with the hypothesis that NO adsorbs to Fe aggregates.⁵³ The poor NO correlation may be due, in part, to challenges in obtaining a reproducible baseline as discussed in section 3.3.2 of the Supporting Information. The correlation between Mössbauer spectroscopy and HQ oxidation in Figure 5C does not show systematic outliers.⁷² The major outlier, 5.7Fe-N-C-TUB, and the two data points that are the next-farthest from parity (xFe-N-Cma-ht, black circles) illustrate two major challenges with Mössbauer spectroscopy, which were noted above: (i) that large errors result when FeNₓ centers are a minority species (<10% in 8.4Fe-N-Cma-ht, Figure S4) which causes the data point to appear in the lower right of Figure 5C, and (ii) that sufficiently small FeOₓ clusters may not become magnetically split into a sextet feature and would be misidentified as FeNₓ species (as in 1.3Fe-N-Cma-ht, Figure S3 and 5.7Fe-N-C-TUB, Figure S8), causing the data points to appear in the
upper left of **Figure 5C**. These quantitative comparisons based on a suite of Fe-N-C catalysts indicate that the kinetic active-site quantification strategy developed here correlates well with CO pulse chemisorption and, in most cases, with Mössbauer spectroscopy.

Each FeNₓ active-site quantification technique can be further compared on several qualitative bases: the conditions of the measurement, the fundamental and practical challenges that these conditions engender, and the generality of the method for other MNₓ sites (M ≠ Fe), as summarized in **Table 2**. The fundamental limitations have been described in detail in the foregoing discussion and are summarized as follows: (i) some methods may not accurately differentiate FeNₓ from other Fe species in some cases (Mössbauer), (ii) pretreatment and measurement conditions can cause active-site distributions to change (CO, NO), (iii) errors can arise associated with materials limitations such as low FeNₓ fraction (Mössbauer) or lack of data on an Fe-free analog (HQ), and (iv) inaccuracies due to unexpected deviations from fundamental assumptions such as active-site saturation (CO, NO) or intrinsic reactivity of sites (HQ). The methods included in **Table 2** alongside HQ oxidation were selected based on their reported ability to quantitatively distinguish accessible FeNₓ sites and their breadth of adoption in the field. Other characterization techniques, such as X-ray absorption spectroscopy, are commonly used, but they lack the ability to accurately quantify FeNₓ in the presence of Fe species and to distinguish accessible from inaccessible sites, and are therefore not included.⁷,⁷³,⁷⁴
<table><caption>Table 2. Qualitative Comparison of FeNₓ Active-Site Quantification Methods in this Study.</caption><thead><tr><th></th><th>Mössbauer spectroscopy</th><th>CO pulse chemisorption</th><th>Stripping of NO derived from NO<sub>2</sub><sup>-</sup></th><th>HQ oxidation kinetics</th></tr></thead><tbody><tr><th>Pretreatment conditions</th><td>Vacuum-ambient pressure, ≤10 K</td><td>873 K, flowing He</td><td>Nafion film, aqueous acetate buffer (pH 5.2), potential cycling (1.0 to 0.3 V<sub>RHE</sub>) under Ar and O<sub>2</sub></td><td>None</td></tr><tr><th>Measurement conditions</th><td>Vacuum-ambient pressure, ≤10 K</td><td>195 K, flowing He, CO pulsing</td><td>Aqueous acetate buffer (pH 5.2), NO<sub>2</sub><sup>-</sup> adsorption followed by conversion to NO, stripping from 0.4 to -0.3 V<sub>RHE</sub></td><td>Stirred slurry, 303 K, 0.5 M H<sub>2</sub>SO<sub>4</sub>, 50 mM HQ, 1 atm O<sub>2</sub></td></tr><tr><th>Fundamental Challenges</th><td><ul><li>Struggles to differentiate FeN<sub>x</sub> from very small FeO<sub>x</sub> clusters</li><li>Large errors when FeN<sub>x</sub> is a minor species</li></ul></td><td><ul><li>High-T pretreatment can influence active-site distribution</li><li>Measurement conditions are very different from catalytic conditions</li></ul></td><td><ul><li>Reproducibility of baseline</li><li>Potential cycling pretreatment can influence active-site distribution</li><li>Ionomer film may influence site accessibility</li></ul></td><td><ul><li>Overcounts if rate of Fe-free analog is not quantified</li><li>Inaccurate if FeN<sub>x</sub> structural variations impact HQ oxidation TOF</li></ul></td></tr><tr><th>Practical Challenges</th><td><ul><li>Costly instrumentation</li><li>Not broadly available</li><li>Long acquisition times (days) and high sample quantities or costly <sup>57</sup>Fe precursors</li></ul></td><td><ul><li>Costly instrumentation</li><li>Requires gas-phase characterization expertise</li><li>Medium throughput (~2-3 analyses per day)</li></ul></td><td><ul><li>Inexpensive</li><li>Requires electrochemical expertise</li><li>Medium throughput (~1-2 analyses per day)</li></ul></td><td><ul><li>Inexpensive</li><li>Requires no specialized equipment</li><li>Parallelizable for high throughput</li></ul></td></tr><tr><th>Generality for other MN<sub>x</sub></th><td>Mössbauer-active elements are very limited (e.g., <sup>57</sup>Fe, <sup>119</sup>Sn)</td><td>Fe, Sn, Co, Mn reported,<sup>33,75</sup> but CO does not adsorb to NiN<sub>x</sub>,<sup>58</sup> others are untested</td><td>Reported for Fe, Co, Ni, Sn, Sb, Bi, and Mn,<sup>34,55,76</sup> but only extensively validated for Fe</td><td>Untested; requires accurate TOF<sub>MN<sub>x</sub></sub> determination for each new M and intrinsic reactivity that is above N-C</td></tr></tbody></table>

Practical limitations are also important to consider for adoption of these methods. Mössbauer spectroscopy and CO pulse chemisorption require costly spectroscopic or gas-phase analytical instrumentation, whereas NO electrochemical stripping and HQ oxidation use comparatively simple equipment. This study shows that no single active-site quantification technique should be
employed on its own, but rather at least two should be compared and validated for accurate
comparison of catalysts with one another. The inclusion of HQ oxidation kinetics as a new
method should facilitate active-site quantification by laboratories lacking specialized materials
characterization equipment. HQ oxidation also provides a rapid screening approach to down-
select materials for more costly or time-intensive characterization, because rates can be measured
in parallel in a short amount of time and do not require large sample quantities (5–10 mg). This
advantage could accelerate the iterative process of materials synthesis and pair well with recently
developed high-throughput synthesis approaches.⁷⁷

In addition, the prospects for generalizing these methods to metals other than Fe are
important as new catalytic applications arise. Mössbauer spectroscopy shows limited generality
due to the nature of the Mössbauer effect, whereas the probe molecule and kinetic methods show
promise (Table 2). It is worth noting, however, that the specificity of Mössbauer spectroscopy
can be an advantage when Fe is present in bimetallic or multimetallic catalysts.⁷⁸ Some
limitations have been encountered with CO pulse chemisorption. For example, NiNₓ centers are
not titrated,⁵⁸ presumably because they do not adsorb CO strongly enough. This requirement may
be somewhat limiting and could also apply to the NO-stripping technique if some MNₓ sites do
not adsorb NO with sufficient strength. This hypothesis is consistent with recent data from
Kucernak and coworkers that show a lack of a clear NO stripping peak on materials containing
Zn, Ni, Sn, Sb, Bi, and Mn.⁵⁵ Active-site saturation requirements do not limit HQ oxidation, but
generality may be limited by different considerations. Each new metal would require the value of
TOF<sub>MNx</sub> to be determined again, which in turn requires sufficiently well-defined materials to
establish reliable benchmarks. The extension of metalation strategies to other metals may
provide this opportunity. It may also be anticipated that some metals possess a lower intrinsic
reactivity that cannot be accurately distinguished from the background reactivity of the metal-
free analog; this case is already apparent for the ZnNₓ centers likely present in the Fe-free
analogs of ZIF-8-derived catalysts studied in this work.

Conclusions

The aerobic oxidation of a sulfonated hydroquinone molecule serves as a catalytic probe
reaction for the kinetic quantification of mononuclear FeNₓ active centers in Fe-N-C catalysts.
The intrinsic turnover frequency of hydroquinone oxidation is assessed using Fe-N-C catalysts
that contain solvent-accessible FeNₓ centers synthesized by metalation strategies and
characterized by low-temperature Mössbauer spectroscopy. The hydroquinone oxidation
turnover frequency enables the calculation of the FeNₓ active-site density of a suite of Fe-N-C
catalysts with varying synthetic provenance and Fe speciation. The collection of fourteen Fe-N-C
materials compared in this study encompasses a broader – and thus more representative –
sampling of the diversity of Fe-N-C catalysts than previous active-site benchmarking efforts¹⁹
and, thereby, reveals deeper insights into the materials characteristics and fundamental
challenges that cause active-site quantification approaches to deviate from one another. These
broad comparisons of kinetic, probe-molecular, and spectroscopic approaches to quantify FeNₓ
centers show that the kinetic method correlates well with CO pulse chemisorption and
Mössbauer spectroscopy but does not correlate well with electrochemical NO stripping. Yet, no
single quantification strategy is yet suitable to be considered accurate for every Fe-N-C catalyst,
highlighting the importance of cross-validation with more than one technique. The 1:1
correlations between three different active-site quantification approaches (HQ, CO, Mössbauer)
on more than ten different Fe-N-C materials are unprecedented in the Fe-N-C literature. These
correlations provide the basis for the expanded use of active-site quantification in any study that
measures the rate of a catalytic reaction on Fe-N-C catalysts, so that reaction rates can be
properly compared on a per-site basis.

The kinetic approach to quantify FeNₓ centers developed here not only complements existing methods but also offers attractive unique features. The absence of a pretreatment that may alter the state of the catalyst expands the range of catalysts whose site densities can be quantified accurately, such as catalysts synthesized using mild temperatures or those that have undergone extensive aging in a device or other treatments that cause deactivation.<sup>10,53,79</sup> The relative ease of applying the kinetic quantification method should also facilitate its use for rapid screening in conjunction with materials synthesis efforts and drive the broader adoption of active-site benchmarking in areas where it is seldom employed.<sup>7</sup>

**Methods**

**Materials Synthesis**

The 2,3,5,6-tetrakis(propylsulfanyl-3'-sulfonate)-1,4-hydroquinone tetrasodium salt (HQ) used in this study was prepared according to a previously reported protocol.<sup>46,47</sup> The synthetic protocol is straightforward, but samples of this material can also be made available to others. For groups interested in obtaining a sample, contact the corresponding author (S. S. Stahl).

Fe-N-C materials reported in this work are generally named “xFe-N-C[descriptor]”, where the leading x denotes the bulk Fe content quantified by ICP-OES<sup>56</sup> as described in section 1 of the Supporting Information, and [descriptor] provides information about the synthetic route or institution of origin. Fe-N-C materials with low Fe loadings and the majority of Fe in atomically dispersed FeNₓ configurations were synthesized by a solution-phase metalation of ZIF-8-derived N-C following our previous report.<sup>13</sup> These materials are labeled as xFe-N-C<sup>m-ht</sup>, where “m-ht” refers to a metalation followed by a heat treatment. Fe-N-C materials with higher Fe loadings
including agglomerated Fe species were synthesized by the same approach, but under an atmosphere containing air, and are labeled xFe-N-C<sup>ma-ht</sup> ("ma" = metalation with air). An Fe-N-C material was synthesized by the solution-phase metalation of ZIF-8-derived N-C after a gas-phase HCl treatment, denoted as 0.3Fe-N-C<sup>HCl-m-ht</sup> ("HCl-m" = HCl treatment followed by metalation), following our previous report.<sup>13</sup> All materials described above were loaded into a quartz boat and treated within a horizontal tube furnace in flowing N<sub>2</sub> at 873 K (ramp rate = 10 K min<sup>-1</sup>) for 2 h, then cooled by convection to ambient temperature; this heat treatment is denoted as "ht" in sample names. Further details can be found in the Supporting Information, section 2.2.

The 1.5Fe-Phen-C material was synthesized through the pyrolysis of a carbon-supported Fe complex ligated by 1,10-phenanothroline based on methods reported in the literature<sup>80</sup> and described as "1.5Fe-Phen-C" in our previous publication.<sup>13</sup> The 1.2Fe-PANI-C material was synthesized through pyrolysis of a mixture of carbon, polyaniline, and Fe based on methods reported in the literature<sup>81,82</sup> and described as "1.2Fe-PANI-C" in our previous publications.<sup>48,13</sup>

The 4.7Fe-N-C-ST catalyst was synthesized by a FeCl<sub>3</sub>/LiCl eutectic salt melt metalation strategy reported previously.<sup>57</sup> Briefly, a Zn-N-C material was prepared by the pyrolysis of 1,2-dicyanobenzene within a ZnCl<sub>2</sub>/LiCl mixture (60 mol% LiCl) in an Ar atmosphere at 1073 K for 1 h, followed by washing with 0.1 M HCl. Then the Zn-N-C was (trans)metalated at 443 K in a eutectic FeCl<sub>3</sub>/LiCl salt melt followed by the same acid washing, and a flash pyrolysis at 1273 K for 0.33 h under Ar. The 0.5Fe-N-C UCI catalyst was synthesized by the sacrificial support method, where an iron-nicarbazin mixture is melted into the nanoporous sacrificial silica template via pyrolysis, with subsequent acid etching to remove the silica template and metallic nanoparticles. The process is reported in detail in the literature.<sup>59,60</sup> The synthesis of the 5.7Fe-N-C-TUB catalyst involved aniline polymerization, followed by pyrolysis of a mixture of activated carbon support, Fe precursor, and polyaniline based on methods reported in the literature.<sup>58</sup> The
0.9Fe-N-C-UB material was prepared through the pyrolysis of an Fe₂O₃@ZIF-8 composite under 10% H₂/Ar as reported in the literature. The Fe₂O₃@ZIF-8 composite was prepared according to previous work.⁸³ For comparison, a 0.8Fe-N-C-UB material was synthesized with an identical Fe₂O₃@ZIF-8 precursor and heat treatment, except that the pyrolysis gas was Ar. The 0.8Fe-N-C-CNRS catalyst was prepared by the pyrolysis of ZIF-8 mixed with Fe(OAc)₂ and 1,10-phenanthroline in Ar at 1323 K, as reported previously.⁸ A commercially available Fe-N-C catalyst was sourced from Pajarito Powder, LLC⁶¹ (product number PMF-011904), and is referred to as “0.5Fe-N-C-PAJ.”

## Materials Characterization

**⁵⁷Fe Mössbauer Spectroscopy.** Spectra were collected on a 1024 channel See Co model W304 resonant gamma-ray spectrometer using ⁵⁷Co on Rh foil as a gamma-ray source. Isomer shifts were referenced to α-Fe foil at room temperature. Fe-N-C samples were loaded into the sample chamber and spectra were collected under vacuum with a source velocity range of ±10 mm s⁻¹. Fe-N-C samples were cooled to 4.2–10 K using a Janis model SHI-850 cryostat controlled by a Lakeshore model 336 temperature controller. Spectra were fit with the VindaD Excel add-in.⁸⁴ Fitting details and spectra can be found in section 3.1 of the Supporting Information. All reported Mössbauer spectra were collected at the University of Wisconsin with the exception of 4.7Fe-N-C-ST (at TUM).

**CO Pulse Chemisorption.** The density of FeNₓ centers in Fe-N-C catalysts was estimated by the adsorption of CO at 195 K following previously reported methods.³³,³⁶ Fe-N-C materials were supported between two quartz wool plugs within a U-shaped quartz tube loaded onto a Micromeritics Autochem II 2920 Chemisorption instrument equipped with a residual gas analyzer (MKS Cirrus) for effluent analysis and a Micromeritics CryoCooler accessory for sub-ambient temperature control. The temperature of the bed was controlled with a quartz-sheathed
thermocouple in contact with the upper quartz wool plug. Fe-N-C materials were treated in flowing He (UHP, Airgas, 50 cm³ min⁻¹) to 873 K (10 K min⁻¹) for 0.25 h, allowed to cool to ambient temperature, then further cooled to 195 K (5 K min⁻¹) using the CryoCooler accessory. During this cooling step, a separate gas stream of 5% CO/He was introduced to a sample loop of 500 µL volume held at 383 K and continuously flowed at a rate of 50 cm³ min⁻¹. The system was allowed to dwell after cooling to 195 K for 0.25 h to allow for equilibration of the sample temperature and RGA signal, then pulsing of the CO from the sample loop was started. A pulse was executed as follows: the CO/He flow in the loop was stopped for 30 s to ensure pressure equilibration in the loop (P = 1 bar), then the 6-port valve was switched to direct the He carrier gas stream through the CO-containing sample loop; He carrier gas was allowed to flow through the sample loop for 300 s, then the 6-port valve was switched back to allow the sample loop to fill with CO/He for 270 s. This pulsing procedure was repeated ten times to ensure that a minimum of three CO peaks (m/z = 28) at the end of the experiment were of equivalent area, indicating saturation of FeNₓ sites with CO. After CO saturation was complete, the sample was allowed to dwell in flowing He at 195 K for 0.25 h, then heated to 873 K (10 K min⁻¹) and held for 0.25 h to desorb CO and with the intent to return the surface to the same state as before CO adsorption. The molar quantity of CO per pulse (Nₚulse) was calculated assuming the ideal gas law in the loop (P = 1 bar, T = 383 K, V = 500 µL, CO mole fraction = 0.05), and the RGA peak area (m/z = 28) corresponding to this quantity (Aᵣₜt) was calculated as the average peak area of the final ≥3 pulses of approximately equivalent area. The amount of CO adsorbed in each pulse was then calculated as Nᵣₐs = Nₚulse(1 - Aₚulse/Aᵣₜt), and the cumulative amount of CO adsorbed over all ten pulses to reach saturation was assumed to reflect the density of FeNₓ sites in the material. A summary of CO-titrated FeNₓ densities and all pulsing profiles can be found in section 3.2 of the Supporting Information. All reported CO pulse chemisorption data were
collected at the University of Wisconsin with the exception of 4.7Fe-N-C-ST (at Technical
University Berlin as previously reported¹⁹,³⁶).

**Reductive Stripping of NO derived from NO₂⁻.** The density of FeNₓ centers was estimated from the reductive stripping of NO derived from NO₂⁻ on a subset of Fe-N-C catalysts using previously reported methods³⁴ including a modified cleaning protocol reported later.³⁹ Fe-N-C films were deposited on a glassy carbon rotating disk electrode (RDE) with a diameter of 5 mm housed within a cylindrical PTFE shroud (Pine Research Instrumentation, E6R1 ChangeDisk), which served as the working electrode (WE). Catalyst inks composed of 5 mg of Fe-N-C with 54 µL of Nafion solution (5 wt% in mixture of lower aliphatic alcohols and water, Sigma-Aldrich), 530 µL of isopropanol (≥99.5%, Sigma-Aldrich), and 530 µL of H₂O (18.2 MΩ cm) were sonicated in an ice-water bath for 1 h. The electrode was polished with a 0.05 µm alumina suspension (BASi) on a polishing pad, then sonicated in methanol (Avantor, anhydrous) for 0.25 h. The electrode was mounted on the rotator in an inverted configuration and rotated at ~100 rpm. While rotating the electrode, an aliquot of the catalyst ink was deposited on the electrode to achieve a loading of 0.2 mg cm⁻² of catalyst on the electrode, as quantified by the volume of ink dispensed from a volumetric displacement pipet (Eppendorf, 20 µL). The film was allowed to dry at ambient temperature for >0.33 h. The WE was immersed in 15–20 mL of a 0.5 M acetate buffer (HOAc/NaOAc) at a pH of 5.2 within a 25 mL four-neck round-bottomed flask equipped with a Ag/AgCl reference electrode, graphite rod counter electrode, and PTFE gas-purge tube. The electrodes were connected to a potentiostat (BioLogic BP-300), and potentials were converted to the reversible hydrogen electrode (RHE) scale using the relationship E<sub>RHE</sub> = E<sub>Ag/AgCl</sub> + 0.059pH + 0.1976 V.

The catalyst film was pretreated using sequential cyclic voltammetry (CV) scans without
electrode rotation while the electrolyte was saturated with either O₂ (>99.2%, Airgas) or Ar
(99.998%, Airgas). Pretreatment CVs were performed between 1.05 and -0.4 V<sub>RHE</sub> as follows: (a) three repetitions under O<sub>2</sub> at 5 mV s<sup>-1</sup>, (b) 20 repetitions under Ar at 100 mV s<sup>-1</sup>, (c) 10 repetitions under Ar at 10 mV s<sup>-1</sup>, then (a)-(c) again, then one final treatment described in (a). After this, (d) a linear sweep voltammogram (LSV) was recorded under O<sub>2</sub> from 1.0 to 0.3 V<sub>RHE</sub> at 5 mV s<sup>-1</sup> while rotating at 1600 rpm and is denoted the "unpoisoned" LSV. Then, under Ar, without rotation, a "pre-baseline" CV (e) was performed from 1.0 to 0.3 V<sub>RHE</sub> at 10 mV s<sup>-1</sup>, with the intent to equilibrate the system, followed by a "baseline" CV (f) from 0.4 to -0.3 V<sub>RHE</sub> at 10 mV s<sup>-1</sup>. The "baseline" CV (f) was preceded by a 30 s hold at 0.4 V<sub>RHE</sub> to further ensure equilibration and a reproducible current density. The "pre-baseline" CV (e) was used to correct LSV data, and the "baseline" CV (f) was used as the reference for the NO stripping CV (g, below).

Next, at open-circuit potential, the WE was rotated at 300 rpm and immersed in ~15 mL of an aqueous 0.125 NaNO<sub>2</sub> solution (≥97%, Sigma-Aldrich) for 300 s. The WE was then immersed in ~15 mL of H<sub>2</sub>O (18.2 MΩ cm) for 60 s, then in ~15 mL of the electrolyte solution (pH 5.2 acetate buffer) for 600 s, then in a fresh ~15 mL of H<sub>2</sub>O for 60 s. The WE was then returned to the cell under O<sub>2</sub>-saturated electrolyte and an LSV was recorded under the same conditions described in (d), referred to as the "poisoned" LSV. Then, in Ar-saturated electrolyte and without rotation, the "pre-baseline" CV (e) was recorded, followed by an equilibration for 30 s holding the potential at 0.4 V<sub>RHE</sub>, and then a CV (g) from 0.4 to -0.3 V<sub>RHE</sub> at 10 mV s<sup>-1</sup>, referred to as the "stripping" CV. Another LSV in O<sub>2</sub>-saturated electrolyte was recorded under the same conditions described in (d), referred to as the "recovered" LSV. These LSVs are used to confirm the success of NO poisoning. After measurement of the "recovered" LSV, the same poisoning steps described above were performed, and following poisoning the "pre-baseline" (e) and "stripping" (g) CVs were recorded. This stripping CV was compared with the "baseline" CV
(f) to quantify the amount of NO stripped. This second poisoning and stripping protocol was adopted to ensure that the stripping curve was not perturbed by exposure to O₂ during the “poisoned” ORR LSV, as recommended in a recent report.³⁹ The “pre-baseline” (e) and “baseline” (f) steps were repeated after this final stripping, which was recently reported by the same group to be suitable for baseline correction.⁵⁵ The FeNₓ site density (N<sub>FeNx</sub>, mol g⁻¹) was calculated according to the following equation:

$$N_{FeN_x} = \frac{Q_{strip}}{n_{strip}F} \qquad (5)$$

where Q<sub>strip</sub> (C g⁻¹) is the charge associated with the reductive stripping of NO, F is Faraday's constant (C mol⁻¹), and n<sub>strip</sub> is the number of electrons associated with the reduction of a NO–FeNₓ species. The value of n<sub>strip</sub> was assumed to be 3 according to reports that the product of NO reduction on Fe-N-C catalysts is NH₂OH,⁴¹,³⁵ that is: NO – FeNₓ + 3H⁺ + 3e⁻ → FeNₓ + NH₂OH. A step-by-step form of this procedure can be found in section 3.3.1 of the Supporting Information. A summary of NO-titrated FeNₓ densities using both 3-electron and 5-electron stripping assumptions with all possible combinations of stripping and baseline voltammograms, and all relevant LSV and CV data can be found in section 3.3.3 of the Supporting Information. All reported NO stripping data were collected at the University of Wisconsin.

## Hydroquinone Oxidation Kinetic Measurements

2,3,5,6-Tetrakis(propylsulfanyl-3'-sulfonate)-1,4-hydroquinone tetrasodium salt (HQ) was prepared according to our previous publication.⁴⁶,⁴⁷ In a typical experiment, M–N–C samples (5–20 mg) were combined with 0.5 M H₂SO₄ (10–20 mL, prepared using 18.2 MΩ cm H₂O) and mixed at 2.3×10⁴ rpm for >180 s using a stainless-steel dispersing tool (IKA S 18 N - 10 G), resulting in aggregates <50 µm. Aliquots (20–750 µL) of this well-mixed dispersion were weighed into separate disposable thick-walled borosilicate glass tubes (10 mL), and additional 0.5 M H₂SO₄ was weighed into each to give a total volume of ~750 µL. The purpose of these
preparation steps is to ensure that systematically varying and accurate quantities of catalyst <1
mg are weighed into separate tubes so that the rate can be efficiently quantified in parallel as a
function of catalyst loading at constant reaction time. The reactor tubes were loaded into a large-
capacity orbital mixer (Glas-Col) equipped with an aluminum heating block that covers the base
of the tubes (3 cm), an aluminum cooling block located 7.5 cm from the base of the tubes
controlled by a recirculating chiller, and a gas manifold sealed above the cooling block by viton
o-rings and rubber septa. The heating block and cooling block were set to 30 °C and 10 °C,
respectively, >0.5 h before loading reactor tubes. The system was pressurized with O₂ (99%,
Airgas) to 1.1 bar and evacuated then pressurized for 5 cycles to displace air, then sealed at 1.1
bar under O₂. The reaction pressure was set slightly above 1 bar to facilitate initial pressure
testing of the system prior to reaction. The assembly was mixed at a rotation rate of 1200 rpm for
>300 s before starting the reaction. A 250 µL aliquot of a 0.2 M solution of HQ in 0.5 M H₂SO₄
was injected via syringe (Hamilton) into each reactor tube through the rubber septa to initiate the
reaction, and the time of injection was noted as the initial time (t₀) for each reactor tube. After
0.33 h, the pressure was lowered to 1 bar, and the solutions were withdrawn through the septa
into disposable syringes and passed through 0.2 µm PTFE filters to complete the reaction. The
filtration time of each reaction solution was noted as the final time (t_f) to calculate the overall
reaction time (t_f – t₀). An aliquot of each product solution (~310 mg) was combined with ~330
mg of a 10–60 mM stock solution of pivalic acid (99%) in D₂O (99.9 % atom D) as an internal
standard and analyzed by ¹H NMR spectroscopy to quantify quinone products and unreacted
hydroquinone (see Figure S41 in the Supporting Information for a representative spectrum). A
catalyst-free blank was included in every run and used as the initial time reference. Mass
balances were generally calculated to be 100 ± 5%. The error associated with these rate
measurements was estimated by independent replicates in our previous work¹³ to be ± 20%. A
summary of FeNₓ densities estimated by HQ oxidation and all initial rate data can be found in
section 4.3 of the Supporting Information. All reported HQ oxidation data were collected at the
University of Wisconsin.

Note: Samples of the HQ may be obtained by contacting the corresponding author (S. S. Stahl). Further discussion of the procedure for calculating initial rates of HQ oxidation and considerations of transport limitations, which are essential for accurate site quantitation, are included in sections 4.1 and 4.2 of the Supporting Information.

## ASSOCIATED CONTENT

**Supporting Information.**

Additional experimental details, characterization data, kinetic data, and supplementary
discussions are available in the Supporting Information document.

## AUTHOR INFORMATION

**Corresponding Authors**

* Shannon S. Stahl; Email: stahl@chem.wisc.edu

* Jason S. Bates; Email: jsb9vw@virginia.edu

**Present Address**

‡Jason S. Bates: Department of Chemical Engineering, University of Virginia, Charlottesville,
Virginia 22903, USA

**Author Contributions**

The manuscript was written through contributions of all authors. All Authors have given
approval to the final version of the manuscript.

**Notes**

†Deceased during the preparation of this manuscript.
ACKNOWLEDGMENTS

The authors thank Cathy Clewett for assistance with Mössbauer spectroscopy, Fatemeh
Khamespanah for assistance with materials synthesis, and Edgard Lebrón-Rodríguez and Ivé
Hermans for access to instrumentation for CO pulse chemisorption measurements. This research
was supported by the U.S. Department of Energy, Office of Science, Basic Energy Sciences,
under award no. DE-FG02-05ER15690, and by a Ruth L. Kirschstein NRSA fellowship from the
NIH (F32GM137472, to J.S.B.). Spectroscopic instrumentation was partially supported by the
NIH (1S10 OD020022-1) and the NSF (CHE-1048642). The Mössbauer MS4 spectrometer was
supported by the Department of Chemistry, University of Wisconsin–Madison. ICP-OES
instrumentation at the Water Sciences Engineering Laboratory was supported by the University
of Wisconsin. The UCI components of this work were funded by US Department of Energy,
Office of Energy Efficiency and Renewable Energy (EERE), through the Advanced
Manufacturing Office program to Sandia National Laboratories (AOP 34920). This project
received financial support from the German Federal Ministry of Education and Research
(BMBF) through grant 03XP0251 (“KorrZellKat”). Funding by the German Research
Foundation (DFG) within the framework of the priority program SPP 2240 (research project FE
1590/1-1) is gratefully acknowledged by BAM. F. J. acknowledges financial support from the
French National Research Agency through the ANIMA project (grant number ANR-19-CE05-
0039). G. W. acknowledges partial support from the National Science Foundation (CBET -
1804326 and 2223467).

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