EPR Analysis of Fe3+ and Mn2+ Complexation ... - ACS Publications

Article Cite This: J. Phys. Chem. A XXXX, XXX, XXX−XXX

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EPR Analysis of Fe3+ and Mn2+ Complexation Sites in Fulvic Acid Extracted from Lignite Zoltán Klencsár*,† and Zoltán Köntös‡ †

Pitvar u. 11., 1141 Budapest, Hungary IOI Investment Zrt., Fehérvári út 108-112, 1116 Budapest, Hungary

‡

S Supporting Information *

ABSTRACT: Lignite is a rich source of humic substances such as humic and fulvic acids that are natural chelating agents with multifold applications in fields ranging from agriculture to biomedicine. Associations of heterogeneous molecular components constitute to their complex and still unresolved structure. In this work we utilize Xband electron paramagnetic resonance (EPR) spectroscopy to characterize Fe3+ and Mn2+ complexation sites in fulvic acid (FA) extracted from lignite. EPR signals of FA−Fe3+ and FA−Mn2+ complexes are identified and investigated in detail under various conditions by the means of a newly developed program code and associated analysis method that yields an accurate description of the low-field (geff ≈ 10−3) range EPR signal by assuming discrete distributions in the axial (D) and rhombic (λ) zero field splitting (ZFS) parameters associated with Fe3+ complexation sites. The results refer to the presence of FA−Fe3+ complex structures with either low (|D| ≈ 0.26 cm−1) or high (|D| ≥ 1.0 cm−1) axial ZFS parameters along with a broad distribution in λ. Outersphere, [Fe(OH2)6]3+ based complexes are found to be characterized with λ = 1/3 along with lower axial ZFS values, in accordance with a distorted octahedral ligand configuration.

1. INTRODUCTION

Humic and fulvic acids are still heterogeneous constituents that are distinguished on the basis of their group property of solubility in water: humic substances that are soluble in water at all pH levels are termed f ulvic acids (FAs), whereas those that precipitate under strongly acidic conditions (pH ≈ 2) are termed humic acids (HAs),5,8 which definition also doubles as a recipe for the separation of these humic components. Depending on their origin and method of extraction, these weak organic acids can display a variability in their properties as well as in their compositional and structural details accessible to experimental (chemical, spectroscopic, etc.) techniques that defies simple explanation in terms of a specific molecular structure and associated physical and chemical characteristics.9 Instead, according to an emerging view of humic substances, they can be seen as self-assembling supramolecular associations of relatively low molecular mass heterogeneous components, stabilized by hydrophobic effects, weak dispersion forces and hydrogen bonds.10,11 On account of the presence of hydrophobic domains in humic substances, they are hypothesized to be susceptible to self-organization of their hydrophobic and amphiphilic constituents into micellar structures.10 In this respect fulvic acids, whose typical apparent (supra)molecular size (mean weight, MW, in the order of 103 Da) is way below

The composition and structure of coal and its constituents has long been the subject of scientific interest1−3 especially from the point of view of their formation, transformations and possible applications. For practical purposes coal and its heterogeneous derivative products are usually characterized by terms that refer to common or average (group) properties of a multitude of individual constituting molecular species. Organic matter in coal can originate from the biopolymer lignin of plant tissue residues and possibly also from other sources of polyphenols during the coalification process.4,5 As the latter proceeds, organic matter gradually loses from its water, oxygen and hydrogen content, resulting in a higher-rank coal that is enriched in fixed carbon. In case of humic substances, that can account for a considerable fraction of organic matter in low-rank coals, the loss of oxygen and hydrogen associated with coalification causes gradual depletion of mainly the carboxyl, methoxy, and carbonyl groups.6,7 Thus, lignites, while displaying a lower heating value than higher-rank coals, are at the same time a potentially richer source of carboxyl-containing humic substances such as humic and fulvic acids. The latter are highly valuable ingredients as they display remarkable physical, chemical, and biological properties that make them suitable for a diverse range of applications in industry, agriculture, environmental fields as well as biomedicine.5 © XXXX American Chemical Society

Received: January 15, 2018 Revised: March 6, 2018

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DOI: 10.1021/acs.jpca.8b00477 J. Phys. Chem. A XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry A that characteristic to humic acids (MW in the order of 104−105 Da), are believed to behave differently from HAs, by being less influenced by the hydrophobic effect due to their more prominent hydrophilic character. Nevertheless, observation of polydispersity in FAs suggests that their smaller molecular components can still form molecular associations, which are kept dispersed in water due to their multitude of acidic functional groups.10 The supramolecular structure of these associations and the exact nature of the intermolecular links and the pathways leading to the association of the elemental humic components must be intimately related with the structure of the individual molecules, and particularly also with the number, nature and conformational arrangement of their functional groups (−OH, −COOH, etc.) attached. The latter may also shape the resulting supramolecular structures by promoting the formation of inner- or outer-sphere metal coordination complexes with the involvement of several of the elemental humic molecules as coordinating ligands. The ability to interact and establish different complexes with various metal ions is considered as one of the most important characteristics of humic substances12 that are also regarded as one of the most powerful natural organic chelating agents.5 Fulvic acid samples extracted from different natural sources have indeed long been known to include complexes for example with the transition metals of Fe3+, Mn2+, Cu2+, and VO2+ (vanadyl ion).13,14 These complexed metals may serve as messengers about the presence, structure and transformations of their host entities such as individual molecules and their realized molecular associations that serve as mono- or polydentate ligands in the formed complexes. Paramagnetic transition metal ions, such as those mentioned above, can reflect attributes of their surroundings via their electronic system that is perturbed by electric and magnetic interactions taking place respectively with nearby electronic charges of ligands and magnetic moments of, e.g., other paramagnetic ions. Electron paramagnetic resonance (EPR) spectroscopy is one of the most sensitive methods able to detect such perturbations, and consequently it has been widely used to detect and characterize paramagnetic complexes also of fulvic acids.12−17 The method was successfully applied to elucidate the oxidation state, the symmetry and type of the coordination sites of various metal ions complexed by humic substances15,17,18 and to investigate the stability of the complexes in question.13 On the basis of corresponding studies,15,17 FAs tend to establish outer-sphere complexes with Mn2+ in the form of Mn(H2O)62+ units of high symmetry bonded to ligand groupsprovided by FA moleculesmainly via electrostatic interaction, though an influence of pH and temperature on the formation of inner-sphere FA−Mn2+ complexes have been noted.13 In contrast, FAs were found to bind Fe3+, Cu2+, and VO2+ ions as inner sphere complexes (with possible involvement of various numbers of water ligands, see Figure S1) with the direct bonding between the metal ion and the FA ligand groups displaying some degree of covalency.13 It is rather common that EPR spectra of HAs and FAs show well discernible signals associated with complexed high-spin (HS) Fe3+ ions,12−14,17 and consequently iron is a promising candidate for reflecting differences in the structure of humic substances via its EPR signal. Simultaneous application of EPR and 57Fe Mössbauer spectroscopies for the study of Fe3+ binding sites in humic substances (HAs and FAs) extracted from soils of various origin12 led to the conclusion that in

humic substances one can distinguish among three different binding sites of iron: one octahedral site associated with weakly bound and easily reduced Fe3+ situated presumably at the outer surfaces of humic molecules, and two different binding sites at which Fe3+ is strongly bound and protected against chemical reduction and complexation. On the basis of the analysis of 57 Fe Mössbauer spectra of initial and reduced HAs,12 one of the strongly bound sites was associated with tetrahedral and the other with octahedral coordination of Fe3+. Tetrahedral coordination can be inferred from the relatively low 57Fe isomer shift (being below 0.2 mm/s12) of the corresponding quadrupole doublet component of the HA Mössbauer spectra. In contrast with the case of HAs, FA−Fe3+ complexes have been found to display only higher isomer shift values,19−21 which suggests that in FA−Fe3+ complexes iron typically forms octahedral coordination complexes with FA (and possibly water) molecules. The possibility to derive information about host molecules of Fe3+ ions by measuring and fitting their EPR signal was recently exploited in a study of the structure of ionic liquids.22 In the present work we follow a similar path of analysis in relation with Fe3+ and Mn2+ containing fulvic acid samples extracted from lignite, with the aim to explore the structural information that can be extracted from the corresponding X-band EPR spectra. We also establish a novel method of analysis and interpretation of the Fe3+ signal occurring in fulvic acid X-band EPR spectra, which can assist to detect and interpret changes in the local environments of Fe3+ ions, especially in samples where these environments are characterized by a distribution in the symmetry and/or the strength of the ligand field sensed by iron ions.

2. EXPERIMENTAL SECTION The fulvic acid used for the experiments was extracted from a lignite sample collected from a surface mine located in the Transdanubian area of Hungary. The raw material was extracted by alkaline at pH ≈ 10. Following acidic precipitation of HA components, the obtained liquid was filtrated and purified, and the water was evaporated. The crude yellow material was then recrystallized from water. The obtained powder (referred to in the followings as “FA powder”) was then utilized for the experiments either directly or in the form of a solution. Reagent grade chemicals were applied throughout the sample preparation procedures. For assessing the Mn concentration level of the FA sample with EPR spectroscopy measurements, for comparison purposes standard solutions were prepared by dissolving 10.5 mg of MnCl2·4H2O in 4 mL of distilled water (resulting in a Mn concentration of ∼13.3 mmol/L), and by preparing from the solution thus obtained corresponding diluted versions with 1/5, 1/25 and 1/125 concentration levels of the original. The obtained solutions were then compared with an FA solution prepared by dissolving 19.8 mg of FA powder in 0.4 mL of distilled water (sample “FSA”). To assess the spectral component associated with complexed Mn2+, FA solution (sample “FS0”) was prepared by adding 15.9 mg FA powder to 1 mL distilled water, and mixing the solution thus obtained with 0.1 mL of a solution (“MNS”) containing 1.28 mg Mn(NO3)2·4H2O in 1.1 mL distilled water. From the solution (sample “FS1”) thus obtained, a further sample (sample “FS2”) was derived by adding to it an additional 0.1 mL of the MNS solution. B


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describe the zero field splitting (ZFS) of energy levels, and reflect the symmetry of the ligand field the ion is situated in,26,30 whereas the last term describes hyperfine magnetic interaction with the nucleus, AN being the hyperfine interaction tensor, and I the vector of Ix, Iy and Iz nuclear spin operators. The latter term plays an important role in the case of Mn2+, but we neglect it for Fe3+. (From the isotopic composition of natural iron only 57Fe has a nonzero, I = 1/2 nuclear spin, and from the point of view of EPR spectroscopy the corresponding hyperfine magnetic interaction can usually be neglected on the basis of the relatively low, ∼ 2.14% abundance of this isotope in natural samples. For brevity we also omit here the nuclear Zeeman interaction and the nuclear quadrupole interaction that could have been added for the case of 55Mn.) EPR signal shapes of Mn2+ and Fe3+ can often be well described with a simplified version of the Hamiltonian given above: the g and AN tensors can be taken to be isotropic, and the ZFS term may be restricted to terms (q ≥ 0 and even26) associated with orthorhombic symmetry29 and as a further approximation to the second order terms only. The resulting Hamiltonian has the form31−33

In order to investigate the effect of FA on the state of Mn2+ in solution, four samples were prepared in the form of 0.250 mL solutions of 0.048 mg Mn(NO3)2·4H2O, each containing a different amount of FA powder dissolved: MFS0 was prepared without FA, MFS1 and MFS3 respectively with 1 mg and 3 mg of FA powder dissolved, and MFS5 with 2 mg of additional FA powder dissolved in MFS3. X-band EPR measurements were carried out with a Bruker ElexSys E500 X-band spectrometer at various temperatures in the temperature range of 140−340 K, on powdered fulvic acid samples as well as on solutions. EPR measurements performed for the sake of Mn concentration level comparisons were performed at room temperature on solution samples loaded into capillary. Unless otherwise noted, the conditions of EPR measurements involved a modulation frequency of 100 kHz, modulation amplitude of 5 G, microwave power of 20.7 mW and microwave frequency of f ≈ 9.3 GHz. The spectra were scanned in several ranges including 100−10900 G (wide range) with sweep time of ∼84 s, 100−2900 G (including the geff ≈ 10−3 range referred to in the followings as the “low-field range”) and 2000−4600 G (including the geff ≈ 3−1.5 range referred to in the followings as the “central range”) each with a sweep time of ∼42 s. (The geff effective spectroscopic splitting factor is used here to refer to typical EPR signal ranges associated with corresponding applied external magnetic field values on the basis of the relation geff = hf/BμB where h is the Planck constant, μB is the Bohr magneton and B is the applied magnetic field.) In each case the spectra (being proportional to dA/dB, i.e., the first derivative of the power of microwave absorption with respect to the applied magnetic field), were recorded in 2048 data channels, and the number of sweeps/ spectra accumulated were either 4, 8, or 16 as needed to reduce the spectral noise to an acceptable level. The magnetic field axis of the spectra were rescaled to f 0 ≈ 9.33 GHz (i.e., abscissa values were multiplied by f 0/f) for analysis and further processing. (Though this transformation is strictly valid only for pure Zeeman splitting, in our case f was always close enough to f 0 for its application being well justified for our purposes.) Detailed analysis of the low-field range EPR signal was performed by fitting the corresponding EPR spectrum range, as measured at 140 K, on the one hand for 15.9 mg FA powder, on the other hand for 0.2 mL of the FA solution sample “FSA”. Theoretical EPR spectrum calculations and fitting were carried out as detailed in the next section, on an Intel Core i7 6700K processor based PC.

⎤ ⎡ 1 Η = μB BgS + D⎢Sz 2 − S(S + 1)⎥ + E(Sx 2 − Sy 2) ⎦ ⎣ 3 + gNμ N ANSI

where D = can model the effects of an axial distortion to a perfect octahedral ligand field, E = B22 introduces effects related to rhombic distortion, and the nuclear gyromagnetic factor gN and the nuclear magneton μN are introduced in order to have AN measured in magnetic flux density units in the case of 55Mn, for which gN(55Mn) ≈ 1.38.34 In order to make use of the standardization28 of the ZFS Hamiltonian, instead of eq 2 we use the spin Hamiltonian in the following form: ⎤ ⎡ 1 Η = μB gBS + D⎢Sz 2 − S(S + 1) + λ(Sx 2 − Sy 2)⎥ ⎦ ⎣ 3 + gNμ N ANSI

⎤ ⎡ 1 ΗFe = μB gBS + D⎢Sz 2 − S(S + 1) + λ(Sx 2 − Sy 2)⎥ ⎦ ⎣ 3 (4)

For samples with powder geometry the calculation of the EPR signal on the basis of eq 3 requires consideration of the orientation of the applied magnetic field with respect to that of the individual paramagnetic centers. As both the energy levels and the transition probabilities will depend on this orientation, the calculation of the EPR spectrum shape requires an integration over all possible orientations, which can be realized in practice as a numerical integral over a dense enough set of orientations associated with points on the surface of a sphere.27,35,36 With the aim to perform an accurate analysis and decomposition of the low-field range EPR signal obtained for our FA powder sample (Figure 1), a corresponding custom code was developed and fitted to the MossWinn software37,38 that provided the nonlinear least-squares fitting functions necessary to find the optimal parameter values and decom-

k

∑ ∑ k = 2,4 q =−k

(3)

where λ = E/D is the rhombicity parameter whose value can be restricted to the standard interval of [0−1/3] with λ = 0 corresponding to axial symmetry and λ = 1/3 to maximal rhombic distortion.28 The Hamiltonian used for Fe3+ is the same as above, but without the last term:

3. COMPUTATIONAL DETAILS Apart from the hyperfine magnetic interaction responsible for the magnetic splitting of a typical Mn2+ EPR signal,23 on account of their common high-spin 3d5 (L = 0) electronic state, the EPR signal of Mn2+ and Fe3+ can both be well modeled with the same spin Hamiltonian, namely24−29 Η = μB BgS +

(2)

3B02

Bkq Okq + SAN I (1)

where the first term describes electronic Zeeman interaction with B being the flux density vector of the externally applied magnetic field, g being the gyromagnetic tensor and S being the vector of the Sx, Sy and Sz effective spin operators; Oqk and Bqk in the second term are the extended Stevens operators and the associated ZFS parameters,24,25,27−29 respectively, which C


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isotropic g tensor enables further simplification of eq 5, leading to Pij = g 2 (|⟨i| cos(θ ) cos(ϕ)Sx + cos(θ ) sin(ϕ)Sy − sin(θ )Sz|j⟩|2 + |⟨i| sin(ϕ)Sx − cos(ϕ)Sy|j⟩|2 )

with g = gx = gy = gz. In order to sum the contribution of individual paramagnetic centers to the theoretical powder EPR spectrum, we consider (θ,ϕ) angle pairs associated with points picked on the surface of a sphere according to the scheme put forward in the work of Alderman et al.39 While this choice is the same as that made in the work of Morin et al.,27 our further solution deviates from the one described in the latter work by not searching for the resonance magnetic field values, but rather calculating and summing up absorption contributions for each of the considered magnetic field orientations. While this method (which we prefer to call in the followings as the “direct method”) may be considered as unfeasible on account of it being considerably slower (computationally more intensive) than that tracing the resonance magnetic field values as a function of field orientation,27 it is also free of the complications and necessary approximations associated with the latter method.27,36 Consequently, it is more straightforward to judge the accuracy of the calculated theoretical spectrum in the case of the direct method where the accuracy is mainly limited by noise introduced through the discretization of the continuous manifold of possible magnetic field orientations. In our approach, for a given B applied magnetic field magnitude, for each magnetic field orientation, represented by a (θ,ϕ) angle pair, we numerically solve the eigenproblem of the Hamiltonian given either by eq 3 (for Mn2+) or by eq 4 (for Fe3+) for S = 5/2, and for each of the corresponding possible electronic transitions we determine transition probabilities according to eq 6. Then, by assuming a Lorentzian-shaped energy dependence of the absorption spectrum of a single electronic transition,36 for the given B we calculate an absorption contribution of the selected transition as

Figure 1. Wide range X-band EPR spectra of 15.2 mg fulvic acid powder measured at different temperatures by applying modulation amplitude of 2 G and microwave power of 10.4 mW.

position details of the EPR signal in question. The code was developed according to the following theoretical guidelines. For the case when the microwave magnetic field vector is perpendicular to the static magnetic field applied (corresponding to our experimental setup), the dependence of the transition probabilities on the orientation of the external magnetic field with respect to the g tensor eigensystem is treated in the work of van Veen,36 and corresponding formulas can also be found in related works.27,35 Following van Veen,36 we calculate the relative transition probabilities between the electronic states ⟨i| and ⟨j| as

Aij (B , θ , ϕ) =

Pij(B , θ , ϕ) Γ 2π (εij(B , θ , ϕ) − hf0 )2 + Γ 2/4

(7)

where εij is the energy difference between the ith and jth energy levels, and Γ is the full width at half-maximum of the Lorentzian, which parameter is treated as a fit parameter in order to account for homogeneous as well as moderate inhomogeneous broadening of the absorption spectrum. The relative absorption value at applied field B is then calculated as A (B ) =

Pij = |⟨i|gx cos(θ) cos(ϕ)Sx + gy cos(θ) sin(ϕ)Sy

∑ ∑ Aij(B , θ , ϕ) θ ,ϕ i,j

(8)

where the summation over θ and ϕ considers discrete magnetic field orientations selected according to the scheme put forward by Alderman et al.39 The number of orientations that is necessary to consider in order to reduce spectral noise (occurring due to discretization) to an acceptable level depends on the value of Γ: for Γf = Γ/h = 0.6 GHz 16384, whereas for Γf = 0.3 GHz 65536 discrete orientations over one spherical octant was found to be necessary to take into account in order to reach a low-enough level of discretization error introduced into the final theoretical powder EPR spectrum. The latter is

− gz sin(θ )Sz|j⟩|2 + |⟨i|gx sin(ϕ)Sx − gy cos(ϕ)Sy|j⟩|2

(6)

(5)

where θ and ϕ are the polar and azimuthal angles of B in the g tensor eigensystem (here assumed to coincide with that of the ZFS Hamiltonian), and gx, gy, and gz are the diagonal elements of the g tensor in the same system. (In the above expression we have omitted a constant factor and made an obvious correction compared to eq (A8) in van Veen’s work.36) Assuming an D


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In addition, fitting was performed by assuming a distribution in the D and λ values in a way that relied exclusively on the precalculated spectra, without the need to calculate spectra for in-between values of D and λ. On the one hand, for each of the different paramagnetic Fe3+ centers hypothesized we have assumed a moderate Gaussian distribution in D by calculating

calculated via numerical differentiation of the A(B) curve once it is obtained for a dense-enough set of B values. For the calculations a parallel algorithm was developed that is able to distribute the job of spectrum calculation for data points associated with different B external magnetic field magnitudes over the available processor cores/threads. By proceeding as given above, in the case of Fe3+ the calculation of an EPR spectrum in the range of 0−12000 G in 2401 points (i.e., with a magnetic field step of 5 G) took respectively ∼2.5 and ∼10 min for Γf = 0.6 and 0.3 GHz on the applied PC. In order to demonstrate the capabilities of our code, for comparison purposes we used it to recreate theoretical spectra corresponding to examples taken from the literature36 (see Figures S2 and S3 in the Supporting Information). A calculation time of several minutes for a single theoretical spectrum makes efficient fitting of EPR parameters unfeasible. However, for the case of 56Fe the simplified Hamiltonian of eq 4 enables one to overcome this difficulty by precalculating EPR spectra on a dense enough grid of the possible and reasonable (D, λ) value pairs. Namely, the standard value range of λ is constrained to [0,1/3], and the value of |D| can also be constrained to the range of [0 cm−1, 1 cm−1] with good approximation. (For |D| = 1 cm−1 the corresponding term is already the leading term of the Hamiltonian for the relevant magnetic field range, and further increase in |D| has only a minor effect on the corresponding X-band EPR spectrum in the temperature- and magnetic field range of interest. See Figures S4−S6 for examples in this regard.) In addition, as at the applied temperatures HFe in eq 4 does not allow one to determine the sign of D for powder samples (D and −D resulting in the same spectrum in the high temperature limit), it is enough to take into account D values from the range of, e.g., [−1 cm−1,0 cm−1] only. Consequently, theoretical EPR spectra were calculated for 101 equidistant D values selected in the latter range (i.e., with a step size of 0.01 cm−1), and for each selected D value for 34 equidistant λ values in the range of [0,0.33] (i.e., with a step size of 0.01; note that the difference between spectra with λ = 0.33 and λ = 1/3 is negligible for our purposes), resulting in 3434 theoretical functions. The spectra were calculated for g = gs ≈ 2.002319, in the magnetic field range of 0−12000 G with a step size of 5 G, by assuming a microwave frequency of 9.33 GHz. Such sets of spectrum collections were calculated for three different absorption widths: Γf = 0.3, 0.6 and 1.2 GHz, providing us with altogether 10302 precalculated EPR spectrum functions for fitting. As our experiments were all performed at relatively high (≥140 K) sample temperatures, the theoretical spectra were all calculated in the high-temperature limit. The functions thus derived enable us to perform a relatively fast fitting of experimental spectra to the weighted sum of a multitude of theoretical EPR spectra associated with a corresponding multitude of different Fe3+ paramagnetic centers each characterized with its own EPR parameters of D, λ, g, Γf, and relative weight. Though the spectra were derived only for g = gs, they can still be used to fit the isotropic g factor as well, as according to eq 4 a change in g can be modeled (apart from an amplitude factor) simply by a corresponding rescaling of the B axis of the precalculated spectra. Theoretical spectra associated with Γf absorption widths in the range of 0.3−1.2 GHz can be modeled with acceptable accuracy by taking the suitably weighted average of two corresponding precalculated spectra (one with the closest lower and one with the closest higher absorption width).

Q (Dc , σD , λ , g , Γf ) ⎡ (D − D )2 (−D − D )2 ⎤ n ∑i = 0 q(Di , λ , g , Γf )ai⎢exp − i 2 c + exp − i 2 c ⎥ 2σD 2σD ⎣ ⎦ = ⎡ (Di − Dc)2 (−Di − Dc)2 ⎤ n ∑i = 0 ai⎢exp − + exp − ⎥⎦ 2σD2 2σD2 ⎣ (9)

(

(

) ( ) ) ( )

where a0 = 1/2 and ai = 1 for i > 0, q(Di,λ,g,Γf) refers to an EPR function calculatedas described abovefrom the corresponding precalculated theoretical EPR functions of q(Di,λ,gs,Γf/GHz ∈ [0.3,0.6,1.2]), Di = −i/100, and n > 0, and for i = n + 1 the sum of exponential functions inside the square brackets is smaller than 1/1000. The Dc center of the distribution can take on any value, but for i > 100, q(Di,λ,g,Γf) = q(−Di,λ,g,Γf) is approximated with q(D100,λ,g,Γf). On the other hand, we have assumed a pi = p(λi = i/100) (i = 0−33) free-form discrete distribution in the rhombicity 33 parameter λ. The pi ≥ 0 (∑i = 0 pi = 1) values (leading to an optimum least-squares fit of the experimental EPR spectrum) were determined via the method of Hesse and Rübartsch40 (originally developed for the determination of hyperfine magnetic field distributions on the basis of Mössbauer spectra) as implemented in the MossWinn program.37,41 Combination of the Gaussian distribution in D with the free-form discrete distribution in λ leads then to an EPR spectrum shape of 33

Y (Dc , σD , g , Γf ) =

(Dc , σD , λi , g , Γf ) ∑ pQ i i=0

(10)

3+

for paramagnetic Fe centers characterized by a ZFS parameter of D ≈ Dc with a strain of σD. For the description of the lowfield range of the measured EPR spectra multiple such Y functions (each complemented with a variable multiplicative factor for the adjustment of its relative weight) could then be assumed in order to account for Fe3+ centers with different Dc values.

4. RESULTS AND DISCUSSION Figure 1 displays EPR spectra of 15.2 mg of the powdered fulvic acid sample at selected temperatures. In the spectra one can distinguish two main signals: the one in between 667 G (geff ≈ 10) and ∼2000 G with a sharp peak at B ≈ 1550 G (geff ≈ 4.3) and another ranging roughly from 2500 to 4500 G with a sextet feature centered at around B ≈ 3300 G (geff ≈ 2) (the shallow signal at around ∼9 kG appears due to ambient air surrounding the FA powder in the sample holder quartz tube). Microwave absorption peaks in the former signal range are typical for dilute paramagnetic HS Fe3+ ions situated in a ligand or crystal field whose symmetry is notably different from cubic (such as axial or rhombic), with the sharp peak at geff ≈ 4.3 being a well-known fingerprint for HS Fe3+ experiencing a rhombic ligand/crystal field.12,14,42 The sextet signal centered at geff ≈ 2 is a fingerprint of paramagnetic Mn2+ ions subject to hyperfine magnetic interaction with their I = 5/2 spin 55Mn nucleus.13,15,43 On the basis of Figure 1 we can also notice the E


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The Journal of Physical Chemistry A complete lack of the free radical signal often observed in humic materials due to organic free radicals of the semiquinone type,12−14 which may in part be a consequence of the radical spin quenching effect of metal complexation as found for the case of humic acids.44 In the latter case, complexation of Mn2+ by HA was found to be accompanied by a strong quenching of the free radical spins, detected as a decrease in the magnitude of the corresponding EPR signal. A Mn concentration of as low as 0.2 mmol/g (HA) was found to be enough to diminish the free radical EPR signal to nonmeasurable levels.44 In order to find an approximation of the Mn concentration level of our FA sample, we compared the Mn2+ EPR absorption signal intensity (Ia, equivalent to the second integral of the corresponding EPR spectrum component, but obtained via fitting in our case) of an FA solution with that of standard solutions of MnCl2·4H2O. For the latter solutions, Ia was found to display a near linear dependence on the Mn concentration (Figure S7). The spectrum of FA solution (Figure S7) could be well fitted with two subcomponents: a sextet signal associated with Mn2+, and a single Lorentzian derivative (geff ≈ 2.11, Γpp ≈ 91 G) attributed to Fe3+. By taking into account the second integral of only the Mn2+ component, on the basis of the straight line slope fitted to the Ia data obtained for the standard solutions it follows, that our FA solution had a Mn concentration of ∼0.15 mmol/L, and consequently 1 g of our FA sample contains ∼0.003 mmol of Mn (∼0.17 mg). This is considerably lower than the value found to be necessary for a full quenching of organic free radicals in humic acids.44 However, by considering that in fulvic acids a typical concentration of the number of free radicals can be estimated as being in the order of 1017 radical spins in 1 g FA (i.e., roughly 1 free radical in 6000 FA molecules),13 we can still expect to have above 1 order of magnitude more Mn2+ ions than free radicals in our FA sample, which could play a role in the quenching of the free radical signal. Assuming an FA molar mass of 1000 g/mol13, in our FA sample there is on average 1 Mn2+ ion for every 330 FA molecules. By examining the temperature dependence of the amplitudes of the different signals in the EPR spectrum of FA powder (Figure 1), one can observe a decrease in signal amplitude with increasing temperature. As in a paramagnet the EPR absorption signal intensity (Ia) should follow the temperature dependence of Curie’s law, a decrease in Ia with increasing temperature is the expected behavior in our sample. Provided that the shape of a paramagnetic EPR signal does not change appreciably with temperature, the same temperature dependence should be reflected in the as-measured EPR signal amplitudes as well. To check whether Fe3+ and Mn2+ indeed behave as paramagnets in our FA sample, we have drawn selected peak-to-peak (pp) amplitudes (Δpp) as a function of temperature as displayed in Figure 2. In the case of Mn2+, a fit to the Curie−Weiss law reveals θ ≈ 0 (i.e., compliance with Curie’s law), so that the absorption behavior of Mn2+ corresponds well to that of a paramagnet. In contrast, the temperature dependence of the Δpp amplitude of the Fe3+ signal at geff ≈ 4.3 requires θ ≈ − 200(±30) K along with an additive constant (−b) to fit the data well. A possible reason for this could be a change of the signal shape with temperature. A temperature dependent signal broadening is indeed evidenced in the Γpp peak-to-peak width of the geff ≈ 4.3 signal, which shows an increasing trend with an average rate of ∼0.22 G/K in the studied temperature interval. By drawing and fitting Γpp2Δpp (which is proportional to the second integral area in the case of a Lorentzian derivative) as a

Figure 2. Peak-to-peak signal amplitude (Δpp) of selected signals of the Fe3+ (geff ≈ 10−3) and Mn2+ (geff ≈ 2) signal range in the X-band EPR spectra of fulvic acid powder as a function of temperature. The actual derivation of the quantities depicted are shown in the inset. Solid lines through the points represent least-squares fitting curves of the displayed equations.

function of temperature for the geff ≈ 4.3 signal, the result becomes reconcilable with Curie’s law in the sense that fixing b = 0 (Figure 2) results in θ ≈ 0 inside the error of the fit (which is not the case for Δpp alone). It then appears that the behavior of Fe3+ in FA can also be reconciled with a paramagnetic state. It has been established43 that the Aav average hyperfine splitting (i.e., the average distance measured in Gauss) between neighboring peaks of the Mn2+ sextet reflects, approximately, the degree of ionicity of the bonds involving the manganese ion. Negative ligands of Mn2+ can establish covalent bonding by donating electrons to states involving the 4s state, by which they perturb the excited ionic state 3s13d54s1 that is responsible for the hyperfine splitting. For H2O and F− ligands Aav = 98 G was obtained as representing the ionic state. As the hyperfine splitting in a covalent state is 1 order of magnitude smaller (and can therefore be neglected as a first approximation), with increasing bond covalency the hyperfine splitting decreases, and the numerical value of Aav given in G can be approximately taken as the degree of ionicity expressed in percent;43 for O2− ligands, for example, an Aav range of 80−90 G is obtained, referring to 80−90% ionicity (and 20−10% covalency) of the corresponding bonds. For the MnCl2 solutions, we obtained (Figure S7) a splitting of ∼95.7 G that is close to the ionic limit andin accordance with previous findings45may also be attributed to an octahedral coordination of Mn2+ with water molecules, i.e. to the hexaaqua ion of [Mn(OH2)6]2+. The average hyperfine splitting of Mn2+ is only marginally smaller in the FA solution (Figure S7), where we find a value of Aav = 94.6(6) G. This suggests that Mn2+ ions exist in the form of F


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The Journal of Physical Chemistry A [Mn(OH2)6]2+ in the FA solution, which supports the view that in aqueous systems Mn2+ forms outer-sphere complexes with fulvic acids.15,17 The average Mn2+ hyperfine splitting evaluated as a function of temperature (Figure 3) for the sextet EPR signal observed

Turning now our attention to the EPR signal in the low-field range, we note that in general Fe3+ and Mn2+ may both contribute to the low- and central field spectral ranges.23−25,31−33,47 However, on account of its lower charge, in similar complexes Mn2+ is expected to experience milder ligand field effects, and consequently lower D values in eq 3, than Fe3+. In X-band it therefore usually shows an EPR signal that is most intense in the geff ≈ 2 region of the spectrum. In order to assess the possible contribution of Mn2+ to the low-field range signal in the case of our FA sample, we measured the FA solutions “FS0”, “FS1”, and “FS2” having zero, one and two units of added Mn(II) nitrate, respectively, at 140 K. As the amount of added Mn(II) nitrate increases, the corresponding spectra (Figure 4) show an increase in the

Figure 3. Average hyperfine splitting between neighboring absorption peaks of the sextet of Mn2+ in FA powder as a function of temperature. The average was calculated by fitting the Mn2+ sextet signal with 6 Voigt derivative functions superimposed on a broad Voigt derivative function as background, and taking one-fifth of the distance of the fitted center positions of the first and sixth peaks. The solid line through the points represents the least-squares fitting curve of the displayed expression.

for the FA powder sample (Figure 1) is markedly different from that obtained for the FA solution. On the one hand, for the powder sample Aav remains below 89.3 G at all the applied temperatures, referring to a higher degree of average covalency of Mn2+ bonds. It is then clear that in the dried powder sample Mn2+ is not typically present as hexaaqua ion, but rather with one or more of its ligands replaced by functional groups of FA complexing sites, such as oxygen of phenolic and/or carboxylic hydroxyl groups. The oxygen ligand(s) thus replacing water and bonding to Mn2+ could increase the overall covalency of Mn2+ bonds leading thereby to a reduction of Aav toward the 80−90 G range as observed. The shape of temperature dependence of Aav in FA powder (Figure 3) suggests that it is reduced with increasing temperature due to a thermally activated process. Assuming a single excited state characterized by an energy of ε that is considerably larger than kBT at all the applied temperatures, the Aav data of Mn2+ in FA powder was successfully fitted by taking into account the corresponding Boltzmann factor as depicted on Figure 3. From the fit, we observed ε ≈ 0.12 eV with a relative error of ∼10%. This energy value is equivalent with ∼970 1/cm lying in the infrared range, suggesting that the activation energy is associated with a molecular vibration mode of the Mn(II)−FA complex, whose gradual set in with increasing temperature leads to an increased average covalency of the Mn2+ bonds. As metal−oxygen systems typically display stretching frequencies in the order of 1000 1/cm,46 it is plausible to attribute the thermally activated decrease in Aav (Figure 3) to the excitation of stretching vibrational modes of Mn2+ bonds with oxygen groups of FA molecules.

Figure 4. EPR spectra of FA solution FS0, and of the same solution with one (FS1) and two (FS2) units of Mn(II) nitrate solution added. The spectra were recorded at 140 K.

geff ≈ 2 range Mn2+ sextet signal as expected, and a moderate decrease of the low-field range signal amplitude due to dilution of the original FS0 solution. However, part of the low-field range signal at around 1350 G displays increasing relative amplitude with respect to the rest of the range. Mn(II) nitrate added to the FA-solution apparently leads to an enhancement of the EPR signal in the low-field range. In order to separate the signal of the additional Mn2+ ions, the low-field range spectrum of FS1 was scaled such that its main peak (at around B ≈ 1550 G) closely matched the corresponding peak of FS2, and the scaled spectrum thus obtained was subtracted from the spectrum of FS2 (Figure 5). The difference curve can be fitted with a sextet of equidistant Lorentzian derivatives, yielding Aav ≈ 90(1) G, Γpp ≈ 267.3(2) G and Bc ≈ 1559(1.3) G as the center of the six lines. While Aav equals (inside error) with the average 55Mn hyperfine splitting (∼89.24 G) derived for the FA powder sample at T = 140 K on the basis of the geff ≈ 2 sextet signal (Figure 3), the value of Bc is equivalent to geff ≈ 4.28, i.e. the position where a fully rhombic (λ = 1/3) signal should appear on the basis of eq 4. Consequently, the low-field range signal gradually enhanced as a result of the addition of Mn(II) G


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Figure 5. (a) EPR spectra of solutions FS2 (overlapping circles) and FS1 (solid line) with the latter spectrum being scaled such that its main peak (at around B ≈ 1550 G) closely matches the corresponding peak of FS2. (b) Difference between FS2 and the scaled version of FS1 (circles) and its best fit with a sextet of equidistant Lorentzian derivatives (solid line) yielding the parameters Aav ≈ 90 G, Γpp ≈ 267.3 G, and Bc ≈ 1559 G as the center of the six lines (equivalent to geff ≈ 4.28). The curved baseline was accounted for by a slope and a superimposed broad Lorentzian derivative. Figure 6. EPR spectra of Mn(II) nitrate solutions of (a) MFS0, (b) MFS1, (c) MFS3, and (d) MFS5, where the numbers indicate the mass (in mg) of FA powder dissolved in solutions corresponding to that of MFS0. The spectra were recorded at 140 K.

nitrate to the FS0 solution can be attributed to Mn2+ complexed by FA in a fully rhombic ligand field configuration. The interaction of FA molecules with Mn2+ ions in the Mn(II) nitrate solution is reflected in the EPR spectra of samples MFS 0−5 as displayed in Figure 6. Without FA, the frozen Mn(II) nitrate solution results in a geff ≈ 2 range Mn2+ signal where the sextet structure is masked by heavy broadening of the EPR peaks due to magnetic interactions between close lying Mn2+ ions (sample MFS0, Figure 6a). Increasing amounts of FA dissolved in Mn(II) nitrate solution results in gradual narrowing of the EPR peaks (Figure 6b−d), reflecting the reduction of magnetic interaction effects due to the spatial separation of Mn2+ ions caused by the formation of FA−Mn2+ complexes. Clearly, there was an abundant presence of active Mn2+ complexation sites in the applied amount of FA molecules. By considering that the MFS solutions each contained ∼0.19 μmol of Mn2+ along with approximately ∼1, 3, and 5 μmol of FA molecules (for MFS1, MFS3, and MFS5, respectively, by assuming an FA molar mass of 1000 g), in these solutions there were at least 5 FA molecules present for each Mn2+ ion, which can account for the throughout complexation of all Mn2+ ions, as well as for the associated increase of Mn− Mn distances due to the spatial separation caused by the sufficiently large FA molecules. (Note that the native Mn2+ concentration of FA is at least 1 order of magnitude smaller in comparison.) The low-field range EPR spectra of the same solutions are displayed in Figure 7. With the increase of FA concentration one can observe the increase of the signal amplitude associated with Fe3+ as expected, but on the basis of Figure 7b (in comparison with Figure 1) it is clear that the peak emerging at ∼1300 G (geff ≈ 5) must belong to FA-complexed Mn2+ ions that originate from the Mn(II) nitrate solution. This provides

clear evidence for the need to consider rhombic FA−Mn(II) complexation sites beside those associated with near octahedral ligand environment (contributing to the sextet signal at geff ≈ 2). Note that in comparison with MFS1, in the spectra of MFS3 and MFS5 (Figure 7, parts c and d) one can observe only a moderate increase/narrowing in the corresponding signal, indicating that the complexation of Mn2+ has been largely completed, and there are thus no more Mn2+ ions available in the solution for being complexed by the surplus FA molecules. In order to see how corresponding native Mn2+ ions may contribute to the full range of the EPR spectrum of the original FA sample, we have calculated a Mn2+ EPR spectrum according to the Hamiltonian of eq 3, with the parameters g = 2.00232, D = −0.045 cm−1, λ = 0.33333, AN = −23.75 T, and Γf = 0.6 GHz, and compare the result with the full range spectrum of FA powder on Figure 8. The calculated curve accounts well for the signal feature apparent in between 1200 and 1400 G, as well as for further spectrum features such as the “plateau” at around 2400 G, and the broadening of the central (geff ≈ 2) absorption. We note that the chosen hyperfine interaction constant AN = −23.75 T corresponds to the Aav ≈ 89.24 G average hyperfine splitting on the basis of the relation g μB AN ≈ s A av ≈ 2661A av gN μ N (11) where gN ≈ 1.38 was substituted for the case of 55Mn. The broadening of the EPR signal in the central range, and the simultaneous appearance of relatively sharp sextet peaks suggest the presence of a strain in the D ZFS parameter of Mn2+, H


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Figure 7. Narrow range EPR spectra of Mn(II) nitrate solutions of (a) MFS0, (b) MFS1, (c) MFS3, and (d) MFS5, where the numbers indicate the mass (in mg) of FA powder dissolved in solutions corresponding to that of MFS0. The signal associated with FA-complexed Mn2+ ions (originating mainly from Mn(II) nitrate) situated in a rhombic ligand environment is marked with an arrow on the spectrum of MFS1 (b). The spectra were recorded at 140 K.

up the Mn2+ sextet to have equal value in the spectra, but allowing the amplitudes of the sextets to change independently during the fit. The contributions of high-spin Fe3+ ions to the spectra were taken into account according to eq 10, i.e. by assuming a Gaussian strain in the value of D and a free-form discrete distribution in the value of λ. Application of a single Y(Dc, σD, g, Γf) function (eq 10) to account for the Fe3+ part of the spectrum provided a fair fit (Figures S9, S10) of the spectra (with close lying Fe3+ parameter values obtained for FA powder and FSA frozen solution, see Table S1), though still with clear residual deviations between theory and experiment. A considerably improved fit (Figure 9) could be obtained by assuming the presence of two different types of Fe3+ ligand field environments: one characterized with a lower (|D| ≈ 0.26 cm−1) and one with a higher (|D| ≥ 1.0 cm−1) axial ZFS parameter. For the latter the fit preferred negligible values of strain in D, and consequently this component was fitted by assuming |D| = 1.0 cm−1 without any strain (σD = 0). Dc and g values were assumed to be the same in FA powder and FSA frozen solution, whereas σD, Γf and the relative weight of the low-D (|D| ≈ 0.26 cm−1) and high-D (|D| ≥ 1.0 cm−1) components were allowed to vary

referring to a multitude of ligand environments experienced by complexed Mn2+ ions. The accurate description of the full signal range of the FA powder EPR spectrum would therefore require the assumption of a distribution in the D ZFS parameter. Using the developed code this computationally very intensive fitting procedure was not feasible with the computing power we had available. Therefore, in the followings the contribution of Mn2+ to the low-field range signal will be taken into account with a sextet having parameters equal to those (Aav ≈ 90 G and Bc ≈ 1559 G) derived on the basis of the difference between the FS2 and the scaled FS1 spectrum (Figure 5). We then aimed at a consistent decomposition of the low-field range EPR signal of FA powder and FSA frozen solution at 140 K (Figure 9) without considering the g ≈ 2 signal range. The effect of the latter on the low-field range signal was nevertheless taken into account by a smoothly curved background (Figure S8) modeled by the superposition of a cubic polynomial and a broad Voigt derivative function whose parameters were fitted together with those of the actual low-field range signal. A simultaneous fit of the two spectra (Figure 9) was carried out by constraining the Γpp width of the Lorentzians building I


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Table 1. Parameters of Fe3+ and Mn2+ Spectral Components Resulting from the Best Fit of the Low-Field Range of the EPR Spectra of FA Powder and FSA Frozen Solution at 140 K.a FSA frozen parameter \ sample

FA powder

solution

T, K

140 |D| ≥ 1.0 cm‑1 Fe3+ component |D|, cm−1 1.0F |D| ≈ 0.26 cm‑1 Fe3+ component |Dc|, cm−1 0.264(2) σD, cm−1 0.088(2) 0.105(1) A0.26/A1.0 2.0(2) 2.0(1) parameters common to both Fe3+ components g 2.0052(2) Γf, GHz 0.41(2) 0.30(1) Mn2+ component Bc, G 1559F Aav, G 90F Γpp, G 146(8)

Figure 8. Theoretical X-band EPR spectrum of Mn2+ experiencing a rhombic ligand field (thick solid line) modeled (with the developed code) according to eq 3 with parameters g = 2.00232, D = −0.045 cm−1, λ = 0.33333, A = −23.75 T, and Γf = 0.6 GHz, in comparison with the spectrum (connected circles) of fulvic acid powder measured at 140 K (Figure 1). A sextet of equidistant (Aav = 89.24 G) Lorentzian derivatives superimposed on a broad Voigt derivative provides an acceptable approximation of the theoretical spectrum in the low-field range, as shown below the full range spectra in the left bottom corner of the figure. The amplitude of the full range theoretical signal is set arbitrarily in order to match well that of the measured spectrum.

a

The parameter values shown as common to both samples were assumed to be the same in the two corresponding spectra. Parameters common to both Fe3+ components were assumed to have the same value for |D| ≥ 1.0 cm−1 and |D| ≈ 0.26 cm−1. The value of the rhombicity parameter λ was assumed to be subject to distribution that is shown in Figure 10 for both samples. Numbers in parentheses denote standard error in the last digit as resulted from the fit. The letter “F” refers to a fixed parameter. A0.26/A1.0 denotes the relative area fraction of the two components. Bc denotes the center and Aav the average hyperfine splitting of the sextet signal of Mn2+, both fixed on the basis of results shown in Figure 5. Γpp is the peak-to-peak width of the individual Lorentzians contributing to the sextet. See Figures 9 and 11, as well as Figure S11 for the corresponding Fe3+ and Mn2+ spectral components.

The theoretical fit curve based on the assumption of two different types of Fe3+ ligand field environments along with the Mn2+ sextet signal very closely follows the experimental spectra throughout the applied magnetic field range (Figure 9). According to the decomposition, one clear difference between the powder and frozen solution samples is the reduced relative weight of the low-field Mn2+ sextet signal (with respect to that of the total Fe3+ signal) in the latter sample (see Figure S11). The relative weight of the low-D Fe3+ component with respect to that of the high-D one remains close to 2.0 in both samples. The normalized pi = p (λi = i/100) (i = 0..33) distributions obtained for the low-D and high-D components in the powder and frozen solution samples are shown in Figure 10, while comparison of the shapes of the corresponding signals in the two samples are depicted in Figure S11. Clearly, the spectra can only be described by the assumption of a broad range of λ rhombicity parameters for both the low-D and high-D components. This refers to a multitude of configurations possible for the formation of FA−Fe3+ complexes: differences in λ (and D) may be caused by the involvement of different FAprovided (e.g., carboxylic-, phenolic- and alcoholic hydroxyl) ligands in the complex formation, by a different number of H2O molecules contributing as ligands, as well as by differences in the geometry of the complex characterized, e.g., by various degrees and kinds of distortion in the preferred octahedral ligand environment of Fe3+. Though there may exist correlations between the p(λ) distributions associated with the low-D and the high-D

Figure 9. Low-field range EPR spectra of (a) FA powder and (b) FSA frozen solution at 140 K (discrete data points depicted with open circles), their decomposition indicated via three subcomponents (solid lines) determined via least-squares fitting (two subcomponents for Fe3+ and one for Mn2+ as indicated for FA powder), and the resulting envelope of the fit (solid line through the data points). The residual of the fit is shown below the spectra. See Figure S11 for the comparison of the shape and relative amplitude of corresponding subspectra of the powder and frozen solution samples.

independently in the two spectra. In addition, g and Γf were assumed to be the same for the low-D and high-D components. For the Fe3+ parameter values resulting from the fit, see Table 1. J


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temperature for FA powder (Figure 3). This indicates the formation of the [Mn(OH2)6]2+ hexaaqua ions, and consequently that of outer sphere FA−Mn2+ complexes on the dissolution of FA powder in water. Similarly, in the case of Fe3+ the low-D (|D| ≈ 0.26 cm−1) components may be associated with FA−Fe3+ complexes where water molecules play a significant role as ligands in the first coordination sphere of iron, whereas high-D components can be attributed to inner sphere FA−Fe3+ complexes where majority of ligands is provided by FA. In particular, according to observation (c) above, in the FA solution low-D configurations tend to form with λ ≈ 1/3, which rhombicity parameter may be attributed to outer sphere FA−Fe 3+ complexes. Namely, it has been pointed out48−50 that distortions to a perfect octahedral ligand environment (with all the six ligands being identical and consequently with both D and E in eq 2 being zero) can lead to configurations with D = 0 and E ≠ 0 being proportional to the measure of distortion. Such a situation, though seemingly not covered by the Hamiltonian of eq 3, can be shown to be equivalent49 with the standard case of λ = E/D = 1/3 and D ≠ 0, and thus to contribute to an isotropic EPR peak at g = 30/7 ≈ 4.286. The part of the low-D signal that is characterized by λ ≈ 1/3 can thus originate from outer sphere FA−Fe3+ complexes with the corresponding [Fe(OH 2)6]3+ ion displaying a distorted octahedral ligand configuration due to the influence of the FA molecule. While such configurations appear to exist also in the powdered sample, their occurrence is clearly enhanced in FA solution. Good overall consistency between the corresponding p(λ) distributions in the powder and the frozen solution samples suggests that there are FA−Fe3+ complex configurations that remain practically unchanged when FA is dissolved in water. Such configurations can therefore be regarded as having a geometry that is stabilized via the formation of the complex itself. This points to the possible role of metal ions, and particularly of Fe3+, in the formation and stabilization of supramolecular association structures of individual FA molecules. Though we have derived the p(λ) distributions solely on the basis of the low-field spectrum part of FA powder and FSA frozen solution (Figure 9), the distributions in question (Figure 10) may in principle also result in appreciable EPR signal intensity at higher magnetic fields. In order to check also possible contributions of the low-D and the high-D Fe3+ components to the compound signal at geff ≈ 2, for FA powder we have reproduced the corresponding theoretical spectra over a wider range of magnetic fields, as shown in Figure 11. It is clear that appreciable contribution to the geff ≈ 2 range signal comes only from the low-D component, but even in this case, the small signal centered at geff ≈ 2 can remain unnoticed in measured spectra, as it is easily hidden by the majority Mn2+ signal component in the same region of the spectrum (see, e.g., Figure 8). Overall the wider range theoretical Fe3+ signal is in good compliance with the measured full range spectra of FA powder, reinforcing the validity of our approach chosen for the decomposition of the low-field range spectrum parts.

Figure 10. P(λ) distributions derived for FA powder and FSA solution at 140 K, on the basis of the low-field range EPR signal: distribution curves a and b are associated with FA−Fe3+ complex configurations characterized by |D| ≥ 1.0 cm−1 and |D| ≈ 0.26 cm−1, respectively. The distribution curves belonging to the powder sample are marked with open circles. The distribution curves are defined only for λ = i0.01 with i = 0−33 being an integer: solid line connections between the discrete distribution data points serve as a guide to the eye only.

component fitted to the same spectrum, assessment of the reliability of the obtained distributions via Monte Carlo techniques and the good consistency found between corresponding distributions obtained for the powder and frozen solution samples allows us to make the following observations. (a) The probability of occurrence of axially symmetric configurations (λ ≈ 0) is negligibly low. (b) Ligand configurations with λ values around ∼0.12 though abundantly present in low-D configurations, appear to be missing in high-D ones, whereas the situation is reversed for λ ≈ 0.23−0.24. (c) λ ≈ 1/3 values mainly occur in low-D configurations, where their relative occurrence strongly increases with the dissolution of FA, suggesting that λ ≈ 1/3 and |D| ≈ 0.26 cm−1 are associated with the involvement of water ligands in the formation of the FA−Fe3+ complexes. (d) There is a good overall consistency between the corresponding p(λ) distributions in the powder and the frozen solution samples, which suggests that even when FA gets dissolved, there will be ligand environments of FA−Fe3+ complexes that remain intact. It is worth to mention that points a and d and the pronounced presence of Fe3+ complex configurations with λ ≈ 1 /3 along with a diminished presence of the low-field Mn2+ signal in the frozen solution sample are also consistent with the fit results based on a single p(λ) distribution (see Figures S9 and S10); i.e., these can be regarded as essential characteristics that are reflected by the spectra in a quasi model independent way. With respect to that in FA-powder, the lower amplitude of the low-field Mn2+ sextet signal in the frozen solution sample indicates that with the incorporation of further water molecules into the first coordination sphere of the FA−Mn2+ complexes the corresponding axial ZFS parameter (D) becomes reduced. Covalency is reduced as well, as revealed by the Aav average hyperfine splitting of the Mn2+ sextet signal at geff ≈ 2, which was found to be ∼94 G for the FSA frozen solution sample at 140 K, in contrast with the lower values found at the same

5. CONCLUSIONS Fulvic acid extracted from lignite includes metallic elements of iron and manganese in the form of inner-sphere and outersphere FA−metal complexes involving high-spin forms of Fe3+ and Mn2+. X-band EPR spectra of FA powder reveal K


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complexes, which may be used to follow and characterize changes in FA complex structures in response to variable conditions and chemical reactions. By using the newly developed program code it was possible to account for the low-field range of X-band EPR spectra of Fe3+ and Mn2+ containing fulvic acid powder and frozen solution samples with high accuracy, with the corresponding analysis providing detailed information about the FA−metal complexes in the frame of the applied theoretical model. The applied method may therefore open up new ways to study the structure and chemical interactions of fulvic acid complex structures on the basis of their EPR spectra.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpca.8b00477. Figures S1−S11 and Table S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(Z.Kl.) E-mail: [email protected]. Telephone: +36-705604720. ORCID

Zoltán Klencsár: 0000-0003-0175-7024

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Figure 11. Contribution of (a) the high-D (|D| ≥ 1.0 cm ) and (b) the low-D (|D| ≈ 0.26 cm−1) component to the theoretical Fe3+ EPR signal fitted to the low-field EPR spectrum range of FA powder (Figure 9a) according to the corresponding P(λ) distributions shown in Figure 10. Part c displays the sum of parts a and b, i.e. the total theoretical contribution of the Fe3+ signal to the X-band EPR spectrum of FA powder.

Author Contributions

The authors declare their contribution to this work as follows: spectroscopy and spectral analysis, Z.Kl; fulvic acid preparation and chemistry, Z.Kö. Notes

The authors declare the following competing financial interest(s): Z.Kl. declares no conflicts of interest. Z.Kö. is chairman of the Board of Directors of the company IOI Investment Zrt. that specializes in the production and sale of fulvic acid derivative products. The type of fulvic acid subject of the present research is not included in the product line of the company though, so there is no direct conflict of interest concerning the sample investigated in the work.

characteristic paramagnetic powder signals of these metallic species at around geff ≈ 2 and at lower magnetic fields extending until geff ≈ 10 with a sharp peak at geff ≈ 4.3. While the signal at geff ≈ 2 originates mainly from complexes of Mn2+ and that at geff > 4 mainly from complexes of Fe3+, complexes of both metals contribute with EPR signal intensity to both ranges. While at geff ≈ 2 the contribution of Fe3+ is rather low in comparison with the more intense Mn 2+ signal, the contribution of Mn2+ to the low-field range of the spectra must be taken into account in order to achieve proper decomposition and analysis of the Fe3+ signal. The EPR signal shape refers to the presence of a multitude of complex configurations characterized by different values of the axial (D) and rhombic (λ) ZFS parameters. In particular, the broad extension of the low-field signal to geff ≈ 10 can mainly be accounted for by a broad distribution in the value of the λ rhombicity parameter characterizing FA−Fe3+ complexes. One can distinguish between configurations with high and low |D| values for both Mn2+ and Fe3+, the high-D configurations typically associated with inner-sphere complexes and the low-D ones with complexes having several water molecules as ligands. Outer-sphere complexes with six water ligands appear to be found in a distorted octahedral geometry characterized by λ = E/D = 1/3, or alternatively by D = 0 and E ≠ 0. Stability of Fe3+ complex configurations that occur in FA powder as well as in FA frozen solution point out the possible role of complexed metals in the stabilization of supramolecular association structures of individual FA molecules. X-band EPR spectra of Fe3+ and Mn2+ complexed by fulvic acid include rich information about the structure of FA−metal

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ACKNOWLEDGMENTS The authors express their thanks to Prof. Ferenc Simon (Institute of Physics, Budapest University of Technology and Economics, Budapest, Hungary) for making available the applied spectrometer for recording the EPR spectra.

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ABBREVIATIONS AND NOTATIONS FA = fulvic acid HA = humic acid FSA = fulvic acid solution sample ZFS = zero field splitting D = axial ZFS parameter Dc = center of a Gaussian distribution of D values σD = standard deviation of a Gaussian distribution of D values E = rhombic ZFS parameter λ = rhombicity parameter, λ = E/D AN = hyperfine interaction tensor AN = isotropic hyperfine coupling constant, expressed in T units Aav = average hyperfine splitting reflected by Mn2+ sextet spectra, expressed in G units DOI: 10.1021/acs.jpca.8b00477 J. Phys. Chem. A XXXX, XXX, XXX−XXX

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Aij(B,θ,ϕ) = contribution of the electronic transition between levels i and j to the microwave absorption spectrum at applied magnetic field magnitude and orientation given by B and (θ,ϕ), respectively A(B) = microwave absorption level of a powder sample at applied magnetic field magnitude B Γ = full width at half-maximum of the Lorentzian microwave absorption profile associated with a single electronic transition Γf = same as Γ but expressed in frequency units, Γf = Γ/h where h is the Planck constant Γpp = peak-to-peak width of an apparent EPR signal Δpp = peak-to-peak amplitude of an apparent EPR signal

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