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Mechanistic insight into chromium(VI) reduction by oxalic acid in the presence of manganese(II)
Accepted Manuscript Title: Mechanistic insight into chromium(VI) reduction by oxalic acid in the presence of manganese(II) Author: Katarzyna Wrobel Alma Rosa Corrales Escobosa Alan Alexander Gonzalez Ibarra Manuel Mendez Garcia Eunice Yanez Barrientos Kazimierz Wrobel PII: DOI: Reference:
S0304-3894(15)00523-3 http://dx.doi.org/doi:10.1016/j.jhazmat.2015.06.066 HAZMAT 16921
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
5-2-2015 6-5-2015 30-6-2015
Please cite this article as: Katarzyna Wrobel, Alma Rosa Corrales Escobosa, Alan Alexander Gonzalez Ibarra, Manuel Mendez Garcia, Eunice Yanez Barrientos, Kazimierz Wrobel, Mechanistic insight into chromium(VI) reduction by oxalic acid in the presence of manganese(II), Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2015.06.066 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mechanistic insight into chromium(VI) reduction by oxalic acid in the presence of manganese(II)
Katarzyna Wrobel, Alma Rosa Corrales Escobosa, Alan Alexander Gonzalez Ibarra, Manuel Mendez Garcia, Eunice Yanez Barrientos, Kazimierz Wrobel*
Chemistry Department, Division of Natural and Exact Sciences, University of Guanajuato, L. de Retana 5, 36000 Guanajuato, Mexico
*Corresponding author: Phone: +52 473 7327555, e-mail: [email protected]
Abstract
Over the past few decades, reduction of hexavalent chromium (Cr(VI)) has been studied in many physicochemical contexts. In this research, we reveal the mechanism underlying the favorable effect of Mn(II) observed during Cr(VI) reduction by oxalic acid using liquid chromatography with spectrophotometric diode array detector (HPLC-DAD), nitrogen microwave plasma atomic emission spectrometry (HPLC-MP-AES), and high resolution mass spectrometry (ESIQTOFMS). Both reaction mixtures contained potassium dichromate (0.67 mM Cr(VI)) and oxalic acid (13.3 mM), pH 3, one reaction mixture contained manganese sulfate (0.33 mM Mn(II)). In the absence of Mn(II) only trace amounts of reaction intermediates were generated, most likely in the following pathways: (1) Cr(VI)→Cr(IV) and (2) Cr(VI)+Cr(IV)→2Cr(V). In the presence of Mn(II), the active reducing species appeared to be Mn(II) bis-oxalato complex (J); the proposed reaction mechanism involves a one-electron transfer from J to any chromium compound containing Cr=O bond, which is reduced to Cr-OH, and the generation of Mn(III) bisoxalato complex (K). Conversion of K to J was observed, confirming the catalytic role of Mn(II). Since no additional acidification was required, the results obtained in this study may be helpful in designing a new, environmentally friendly strategy for the remediation of environments contaminated with Cr(VI).
Keywords: Chromium(VI); Oxalic acid; Manganese(II); Liquid chromatography - nitrogen microwave plasma atomic emission spectrometry (HPLC-MP-AES); High resolution molecular mass spectrometry (ESI-QTOFMS)
1. Introduction
The reduction pathway and reaction efficiency of hexavalent Cr(VI) to trivalent Cr(III) both depend on the identity and availability of reducing agents, pH conditions, and on the presence of co-reducing species [1; 2]. Mechanistic studies carried out under specific reaction conditions are important in organic chemistry because intermediate Cr(V) or Cr(IV) species act as oxidants or catalysts of various reactions [3; 4; 5; 6]. In particular, oxalic acid has served as a prototype molecule to study the oxidation of organic compounds by chromic acid. The catalytic role of Mn(II) in these reactions has also been reported [7; 8]. Reduction of Cr(VI) at physiological
conditions is of high biological relevance because intermediate species formed during the process promote oxidative stress and DNA damage [9; 10; 11; 12]. Remediation of contaminated environments is another area in which reduction mechanisms need to be understood in detail. For example, by ensuring the efficient conversion of Cr(VI) into less harmful Cr(III) and the subsequent stabilization or elimination of the latter by sorption on a solid phase or as Cr(OH)3 precipitate [13; 14].
The feasibility of organic acids as electron donors during Cr(VI) reduction has been studied in abiotic model systems and soils [15; 16; 17; 18; 19], and to a lesser extent, in microbiological cultures [20]. It has been demonstrated that citric, tartaric, salicylic, ascorbic, and oxalic acids are capable of slow reduction at slightly acidic conditions (pH 3-5). However, this process becomes accelerated in the presence of suitable co-reducing agents and/or redox-cycling transition metal ions [14; 18; 21; 22; 23]. Specifically, catalytic action of divalent manganese (Mn(II)) during Cr(VI) reduction by citric, ascorbic, and tartaric acids has been reported in abiotic models and in soils at pH < 5 [14; 23; 24; 25; 26; 27].
In early mechanistic studies of Cr(VI) reduction, UV/Vis spectrophotometry and electron paramagnetic resonance spectroscopy were used as the primary analytical tools [4; 5; 7; 8; 27; 28; 29; 30]. In further developments, nuclear magnetic resonance, X-ray absorption spectroscopy and mass spectrometry (MS) were integrated for structural characterization of reaction intermediates [1; 11; 31; 32; 33; 34]. However, as far as we are aware, MS has not been used so far for the elucidation of the role of Mn(II) during Cr(VI) reduction by organic acids. In general terms, electrospray ionization - mass spectrometry (ESI-MS) enables the unequivocal identification of charged and polar species, based on exact mass measurements and molecular fragmentation patterns. Unlike other techniques capable of structural characterization, mass spectrometry offers fast and simultaneous analysis of various short-lived, reactive intermediates present in the reaction mixture with no requirement for their prior separation and/or isolation. Thanks to the exceptional selectivity of high resolution MS, the solution can be introduced by direct infusion and intensity changes of individual ions are monitored simultaneously and in real time for various species. In the soft ESI source, intact ions from the solution are transferred to the gaseous phase, whereas polar species are converted into molecular ions without alteration of existing covalent
bonds and strong, non-covalent interactions [35; 36]. Furthermore, hyphenation of liquid chromatography with nitrogen microwave plasma atomic emission spectrometry (HPLC-MPAES) seems to be a helpful complement of ESI-MS in studies of reaction mechanisms that involve different metal/metalloid species. In particular, HPLC-MP-AES might allow for quantitative evaluation of chromium and manganese in species generated during the course of the reaction, practically in real time.
We undertook this research in order to obtain a mechanistic insight into Cr(VI) reduction by oxalic acid in the presence of divalent manganese. Even though this reaction had been studied in many physicochemical contexts, the precise mechanism responsible for the observed favorable effect of manganese remains unclear [7; 8; 19; 37]. In this regard, a combination of UV/Vis spectrophotometry, liquid chromatography with spectrophotometric and MP-AES detection, and direct infusion high resolution ESI-MS were used here for the first time. Consistent yet complementary data obtained by ESI-MS and HPLC-MP-AES allowed us to unravel the reaction mechanism, in which Mn(II) catalyzes Cr(VI) reduction in the presence of oxalic acid without additional acidification. The results obtained may be helpful in designing new, environmentally friendly strategies for the remediation of Cr(VI)-polluted sites.
2. Experimental
2.1. Reagents and samples
All chemicals used throughout the experiment were of analytical reagent grade: deionized water (18.2 MΩ cm, Labconco, USA) and LC-MS-grade methanol (Sigma, Milwaukee, USA). Potassium dichromate, manganese sulfate, oxalic acid, and formic acid were from Sigma.
Stock solutions of hexavalent chromium (0.20M Cr(VI)), divalent manganese (0.25M Mn(II)) and oxalic acid (1.0M) were prepared from the above reagents and deionized water. The reaction mixtures were obtained by pipetting 200 µL of oxalic acid solution to a Falcon tube containing 10 mL of deionized water which was then vortexed, 50 µL Cr(VI) was added and the solution
vortexed again, followed by the addition of an aliquot of Mn(II) (0 – 80 µL) as appropriate. Finally the volume was brought to 15 mL with deionized water. The solutions were analyzed immediately and in successive runs over the time period up to 2 h.
The reaction mixture M1 contained 0.67 mM Cr(VI) and 13.3 mM oxalic acid and M2 contained an additional 0.33 mM Mn(II); the pH values in these solutions were in the range 2.93.1.
2.2. UV/Vis spectrophotometry, HPLC-DAD and HPLC – MP-AES
Absorption spectra (250 – 700 nm) of the reaction mixtures were acquired using a Spectronic 3000 Diode Array Milton Roy spectrophotometer (resolution 0.35 nm, Milton Roy Inst. Co.). Freshly prepared mixtures were also analyzed by reversed phase high performance liquid chromatography with diode array spectrophotometric detection (HPLC series 1200, Agilent Technologies); starting from preparation of the solution, successive injections (20 µL) were repeated at 10 min intervals for 1 hour. The chromatographic column was Luna C18 (250 x 4.6 mm, 5µm, Phenomenex); isocratic elution was carried out with 0.1% v/v formic acid in 0.5% v/v methanol mobile phase, at a flow rate 0.8 mL/min and detection in the wavelength range 250-700 nm. The above chromatographic system was coupled on-line to nitrogen microwave plasma atomic emission spectrometer equipped with MiraMist® Teflon nebulizer and single-pass glass cyclonic spray (MP-AES 4100, Agilent Technologies), as described previously [38]. The following instrument operating conditions were used: nebulizer pressure 220 kPa, nitrogen pressure 140 psi, viewing position 0, integration time 1 s, wavelengths 425.433 nm and 403.076 nm, for chromium and manganese specific detection, respectively.
Based on multiple injections, the mean retention time with respective standard deviation is given for each chromatographic signal throughout the text.
2.3. ESI-QTOFMS
A high resolution, electrospray ionization - quadrupole-time of flight mass spectrometer maXis impact ESI-QTOFMS equipped with Data Analysis 4.1 (Bruker Daltonics) was used with sample introduction by direct infusion (3 µL/min). ESI was operated in negative mode with ion spray voltage 3000 V, dry gas 4 L/min, drying temperature 180 °C and nebulizing gas pressure 0.3 bar. Spectra of the reaction mixture were acquired in the m/z range 80-800, acquisition was repeated seven times over a 90 min period. For internal mass-lock calibration, the molecular ion of H2Cr2O7 was used (m/z 216.85379).
3. Results and discussion
3.1. Direct UV/Vis spectrophotometric measurements and HPLC-DAD results
In the first approach, a series of solutions containing 0.67 mM Cr(VI) and oxalic acid in molar excess with respect to chromium up to 50:1 were prepared. The pH values in these solutions were in the range 2.9 - 3.1; UV/Vis absorption spectra of these solutions showed two bands (350 nm, 445 nm) with no change of their shape nor intensity during at least four hours, in agreement with the earlier finding that the reduction of Cr(VI) by oxalic acid is kinetically favored only in acidic media (pH < 1.6) [5]. Different concentrations of Mn(II) were added to dichromate solution to cover the range of Cr:Mn molar ratio from 16:1 to 1:2; these solutions presented identical spectral profile as Cr(VI) with no change in successively recorded spectra. Finally, different aliquots of Mn(II) were added to the mixture Cr(VI) + oxalic acid to obtain Cr:Mn molar ratios 16:1; 8:1; 4:1; 2:1; 1:1; 1:2 and UV/Vis spectra were recorded. In accordance with earlier studies [7; 37], Mn(II) caused a time-dependent decrease of Cr(VI) absorption signal and the reduction kinetics was clearly favored by increasing the concentration of Mn(II) (changes of absorbance at 350 nm during the course of the reaction are presented in Fig. 1S in the Electronic Supplementary Material, ESM). In further experiments, the mixture M2 containing 0.67 mM Cr(VI), 13.3 mM oxalic acid and 0.33 mM Mn(II) was used. The composition of M2 (molar ratio of oxalic acid-to-Cr-to-Mn 20:2:1, pH 3.0) was selected in order to promote reaction kinetics and also to facilitate the detection of intermediate species by different instrumental techniques. It was verified by the UV/Vis absorption profile of M2 that it presented gradual changes (Fig. 2S, ESM); in particular, the Cr(VI) signal at 350 nm decreased promptly and was
not detected after 1 h, whereas changes of absorbance at 295 nm and 445 nm suggested the formation of at least two chromium intermediates [12; 28]. Spectral overlapping of the different, poorly absorbing species Cr(III), Cr(IV), Cr(V) hindered positive detection of Cr(III) [39].
Despite the evident utility of the above results for confirmation of Cr(VI) reduction in the presence of oxalic acid and Mn(II) these experiments did not allow for any mechanistic interpretation. To enhance analytical capability, chromatographic separation with diode array spectrophotometric detection was used. It should be emphasized that mild conditions were applied, thus giving priority to preserving species’ identity and not their baseline separation (see Experimental).
In the first approach, the mixture containing Cr(VI) + oxalic acid (M1) and the Cr(VI) solution were analyzed, the respective chromatograms registered at 350 nm (Fig. 1a). The main chromatographic peak eluted at 5.05 ± 0.01 min and the absorption spectrum at apex was characteristic for Cr(VI) (Fig. 1a,e). As compared to the pure dichromate solution, two additional minor signals were observed in the analysis of M1, with retention times 4.09 ± 0.03 min and 4.46 ± 0.04 min (Fig. 1a). Based on the absorption spectra at apex (Fig. 1c,d), these species correspond to the intermediate oxidation states generated during Cr(VI) reduction [12; 28], in line with direct spectrophotometric measurements (Fig. 2S, ESM). Since both minor peaks remained practically unchanged for at least 2h, it is clear that Cr(VI) reduction by oxalic acid was initiated immediately, but intermediates generated at this stage could not be further reduced. Indeed, the formation of a long-lived Cr(V) intermediate species during Cr(VI) reduction by oxalic acid has been reported and Cr(VI)-to-Cr(V) reduction was considered a rate-limiting step [4; 28; 40]. On the other hand, reduction to Cr(IV) [41], formation of Cr(V) from Cr(VI) and Cr(IV) [42] as well as generation of mixed-valence chromium intermediates were discussed in other studies [8; 43].
Typical chromatograms obtained for the reaction mix M2 at 2-3 min, 15 min and 60 min from its preparation are shown in Fig. 1b (detection at 350 nm). As can be observed in this Figure, the elution profile was similar as described above for M1 (the number, position and shape of chromatographic signals), yet intensities of individual peaks changed over the time. The chromatographic signal corresponding to Cr(VI) (retention time 5.05 ± 0.02 min) was decreasing
in successive injections and disappeared after 60 min. In the first injection, two earlier peaks in Fig. 1b presented identical absorption spectra as those registered without Mn(II) (Fig. 1c,d), yet their intensities were lower (Fig. 1a,b). Despite incomplete separation, it can be observed that the two weak signals presented different intensity changes in repeated analysis; the first (4.09 ± 0.03 min) slowly increased over the time but the second (4.46 ± 0.04 min) initially increased and then diminished (Fig. 1b). At the same time, spectra acquired at the apex of the two peaks presented gradual changes. These results suggest that the final product of reduction (Cr(III)) probably coeluted with reaction intermediate in the first of two poorly resolved peaks, whereas the second chromatographic signal contained only intermediate chromium species. It is also clear that Mn(II) activated the reduction of intermediate chromium forms. Our results are consistent with many earlier studies reporting capability of Mn(II) for the reduction of both, Cr(IV) and Cr(V) oxidation states [8; 30; 43].
To examine Cr(III) formation, the chromatograms shown in Fig. 1b were registered at 540 nm; two signals were obtained with retention times 2.57 ± 0.01 min and 4.06 ± 0.03 min, respectively; their intensities gradually increased in successive injections (Fig. 2a). The absorption spectra acquired from the chromatogram run 60 min after preparation of M2 (Fig. 2a), were identical and showed two maxima (415 nm, 570 nm, Fig. 2b,c) consistent with previously reported spectra of Cr(III) in oxalate solution [44]. It should be emphasized that the second chromatographic signal of lower intensity (4.06 ± 0.03 min, Fig. 2a) was also present when the detection wavelength was set at 350 nm (4.09 ± 0.03 min, Fig. 1b), confirming a co-elution of the minor Cr(III) product with element intermediates.
Absorption spectra acquired in the chromatographic system, as well as the changes of signal intensities over time, were consistent with the results obtained in direct spectrophotometric measurements. This indicates that the chromatographic process had negligible effect on the reduction process and species identity. Despite better selectivity of HPLC-DAD as compared to direct UV/Vis measurements, species eluting between 4 min and 4.5 min could not be unequivocally assigned. Furthermore, spectrophotometric measurements did not enable the detection of manganese compounds.
3.2. HPLC-MP-AES results
Chromatograms obtained for M2 in six successive injections are presented in Fig. 3. For chromium emission line (425.433 nm), four chromatographic signals were observed with the retention times 2.50 ± 0.01 min, 4.18 ± 0.02 min, 4.42 ± 0.02 min, 5.68 ± 0.02 min (Fig. 3a), which resembled the elution profile obtained with UV/Vis detection (Fig. 1a,b; Fig. 2a). However, chromatographic signals were slightly delayed with respect to HPLC-DAD due to differences in the instrumental set-up.
For manganese specific detection at 403.076 nm, two chromatographic peaks were eluted with retention time 2.30 ± 0.02 min and 4.58 ± 0.03 min, respectively (Fig. 3b). As shown in Fig. 3c, chromatographic signals acquired for Cr and Mn in this same chromatographic run did not overlap, which indicates that the two elements were not present together in any of the species formed during Cr(VI) reduction. This finding is relevant, because the mechanism is responsible for the favorable effect of Mn(II) that has yet to be clarified, although some authors suggest the formation of intermediate species composed of oxalate, Cr and Mn [7; 37; 45].
For peak assignment, chromatograms of individual standard solutions of Cr(VI), Cr(III) and Mn(II) in the presence of oxalic acid were obtained. Each of them showed only one chromatographic peak, which remained unchanged during several injections. Based on the retention times obtained for the above standards, Cr-containing signals eluting at 2.50 ± 0.01 min and 5.68 ± 0.02 min were assigned to Cr(III) and Cr(VI), respectively; whereas two other poorly separated signals in Fig. 3a (4.18 ± 0.02 min, 4.42 ± 0.02 min) would correspond to Cr intermediates. One chromatographic peak was obtained by analyzing Cr(III) + oxalic acid standard mix (2.50 min) but two Cr(III) peaks were detected in M2 (Fig. 2, Fig. 3), which suggests that the second, minor compound (4.42 ± 0.02 min) was formed only as a reduction product. For manganese, the first peak eluting at 2.30 ± 0.02 min (Fig. 3b) corresponded to Mn(II).
For quantitative evaluation of element species at different stages of Cr(VI) reduction, the following experiment was performed: 15 mL of M1 was prepared and introduced repeatedly to
the chromatographic system with MP-AES detection for one hour (4 injections). Then a small aliquot (20 µL) of Mn(II) stock standard was added, yielding a final manganese concentration of 0.33 mM. The M2 obtained was analyzed for a total of 80 min at 10 min intervals. In Figure 3d, the results are presented as concentrations of Mn and Cr found in each chromatographic signal in successive chromatographic runs (time scale on X-axis). The detailed description of quantification procedure is given in the ESM, Fig. 3S, Fig. 4S.
It can be observed in Fig. 3d that the initial concentration of Cr(VI) remained practically unchanged in the absence of Mn(II), and only trace amounts of Cr intermediates were detected (two poorly separated minor peaks of Cr in Fig. 3 were integrated for quantification). After addition of Mn(II), the reduction process was activated immediately and completed after 1 h. At the same time, the concentration of Cr(III) was increasing gradually (signal at 2.50 ± 0.01 min); for the two poorly separated signals (4.18 ± 0.02 min, 4.42 ± 0.02 min, Fig. 3a), similar tendency was observed yet the increase was much less pronounced which confirms that intermediate species were quantified together with the second, minor Cr(III) product (Fig. 1b, Fig. 2a). The percentage recovery of Cr in each chromatographic run, calculated as a sum of concentrations found in each peak with respect to the concentration introduced on-column (0.67 mM), was in the range 80 - 113%, indicating acceptable reliability of analytical data.
The initial decrease of Mn(II) concentration (2.30 ± 0.02 min, Fig. 3b,d) was accompanied by formation of the second element species (4.58 ± 0.03 min, Fig. 3b,d) but after one hour, there was a tendency toward Mn(II) retrieval and decrease of the second species – a clear indication of the intermediate character of the latter. When two manganese forms were present, the emission intensity between their respective chromatographic signals was higher than the baseline (Fig. 3b), which suggests that intermediate species might be unstable during the chromatographic process. Based on the integration of two Mn signals, the percentage recovery was 65 - 102%; these relatively lower values as compared to Cr should be ascribed to continuous element elution mentioned above.
Taken together, the results obtained in this part of study demonstrate that Mn(II) is required for fast and efficient reduction of Cr(VI) by oxalic acid at pH 3. The reduction process involves
generation of Cr intermediate species and, at least two Cr(III) compounds are formed as the final products. During the course of the reaction, Mn(II) activates the reduction of Cr intermediate species; by doing so, it is converted to a different form (probably Mn(III)) which seems to return to the initial Mn(II) at the end of reduction process. No species containing two elements was detected.
3.3. ESI-QTOFMS results
For the elucidation of molecular structures involved in Cr(VI) reduction, the reaction mixture was prepared identically as for HPLC-MP-AES and it was analyzed by high resolution mass spectrometry with direct infusion. Changes of abundance for each m/z signal were followed by repeated measurements; the first two of them were performed for Cr(VI) + oxalic acid mix M1 (2-3 min after preparation and at 10 min), then Mn(II) was added yielding M2 and mass spectra were acquired in five successive measurements between 10 and 90 min. Two first spectra obtained in the absence of Mn(II) did not present important differences. However, after the addition of Mn(II), a gradual change of intensity over time was observed for several m/z values and new signals corresponding to manganese species appeared (ESI-QTOFMS spectra in the m/z ranges 90-350, 226-234 and 154-157 are presented in Fig. 5Sa-c, ESM). Eleven signals were selected for further analysis; in Table 1 their respective molecular formulas are presented together with theoretical and experimental m/z values of molecular ions (Data Analysis 4.1); mass error was in the range 0.18 – 2.39 ppm, which confirms accurate species assignment. For each molecular ion, its abundance was acquired during the course of the reaction and the results are presented in Fig. 4 and Fig. 5. It can be observed in Fig. 4a that in M1 there were three hexavalent compounds in equilibrium: chromate (major species, A), Cr(VI) mono-oxalato complex (B) and dichromate (C). After Mn(II) addition, abundances of A, B, C promptly decreased in agreement with HPLC-DAD (Fig. 1b) and HPLC-MP-AES (Fig. 3d) results. As indicated from the very beginning, chromatographic resolution of individual species was not intended and A, B, C in the aforementioned Figures eluted always in form of one broad chromatographic peak. It has also been mentioned before that only one chromatographic peak was observed for the solution of Cr(VI) and oxalic acid.
Two pentavalent chromium intermediates, Cr(V) bis-oxalato complex (D) and Cr(V) monooxalato complex (E), were detected in M1 but their abundances in mass spectra did not change until manganese was added to the solution. Then, the formation of the two intermediates increased and after about 20 min, both species started to disappear gradually (Fig. 4b), also in agreement with data presented in the previous sections. In particular, ESI-MS results confirmed the presence of two Cr(V) intermediates in poorly resolved chromatographic peaks eluting between 4.0 and 4.5 min.
Detection and identification of tetravalent chromium (compound F) is noteworthy, because it proved that the reduction process under study involves three one-electron transfers. This task was challenging for many reasons; Mn(II) had been reported as a catalyst of Cr(IV) reduction [8; 30] and Cr(IV) is generally considered a short-life intermediate[1], hence only minute amounts could be expected in the reaction mixture. Furthermore, compounds E and F differ only by one hydrogen atom and their isotopic patterns partly overlap; difficulties in the detection of Cr(IV) by ESI-MS had been highlighted before [1]. In Fig. 4c, change of molecular ion intensity over the time is presented for compound F (Cr(IV) mono-oxalato complex); whereas Figure 6S in the ESM shows how the intensity of molecular ion at m/z 172.91837 was measured.
The three Cr(III) compounds detected ( G, H, I) were identified as Cr(III) bis-oxalato, Cr(III) tri-oxalato and Cr(III) mono-oxalato complexes, respectively; abundancies of molecular ions presented a gradual increase in the M2 mix (Fig. 4d) consistent with HPLC-DAD (Fig. 2a) and HPLC-MP-AES (Fig. 3a,d) data. It should be noted, however, that only two Cr(III) forms were observed before, most likely due to insufficient selectivity of analytical tools and low concentrations of H and I.
The compounds J and K were identified as Mn(II) bis-oxalato complex and Mn(III) bisoxalato complex, respectively (Table 1, Fig. 5). According to HPLC-MP-AES results (Fig. 3d), the J signal appearing after Mn(II) addition first decreased and then recovered at the end of the reduction process (Fig. 5). The abundance change for compound K resembled that observed for the intermediate form of Mn in HPLC-MP-AES analysis (Fig. 3d). However, relative intensity of K in mass spectra was much higher with respect to Mn intermediate in MP-AES chromatograms
(compare Fig. 5 and Fig. 3d). This difference should be ascribed to partial degradation of Mn intermediates during the chromatographic process (continuous Mn elution between two well defined peaks), whereas direct infusion and soft ionization source in mass spectrometry analysis apparently had negligible effect on the integrity of K.
3.4. Proposed mechanism of Cr(VI) reduction
In the M1 mix, only small amounts of Cr(V) and Cr(IV) intermediates were generated and it was deduced that the oxalic acid itself is capable of reducing Cr(VI) to Cr(IV) by two-electron transfer with the release of CO2 and that Cr(V) could be then generated from the mixed Cr(VI)Cr(IV) species [8; 43]. However, the progression of the reduction process was not observed (the reaction scheme is presented in Fig. 7S, ESM). In the M2 solution, the active reducing species appeared to be Mn(II) bis-oxalato complex (J), as shown in the reaction mechanism in Fig. 6. It should be noted that the complexation of Mn(II) by small organic ligands such as citrate or oxalate had been proposed as a prerequisite for electron transfer during the reduction of Cr(VI) [21; 23; 27; 37]. Formation of Mn(III) bis-oxalato complex K accompanied by increasing abundances of Cr(V) and Cr(IV) intermediates (Fig. 6, 7) suggest that one electron was transferred from J to chromium species B, D or E, converting Cr=O contained in their structures to Cr-OH and causing oxidation of divalent Mn species J to trivalent K. The ability of Mn(II) for reduction of Cr(VI), Cr(V) and Cr(IV) had been observed in other studies [2; 43; 46], it is also relevant that favorable Mn(II) effect during chromium(VI) reduction by organic acids was reported to be more pronounced at lower acidities [47], which confirms that disruption of the – OH group in Mn(II) bis-oxalato complex was required for electron transfer (pH 3). The unique species of Cr(IV) detected by ESI-MS corresponded to compound F which, unlike structures suggested for Cr(IV) intermediates before [1; 40], did not present a Cr=O bond. It was expected that Mn(II) would act equally in all one-electron transfers, so the conversion of F into X by the loss of H2O, was proposed. As uncharged and non-polar compound, X could not be confirmed by ESI-MS but its reduction product (I) was detected (Table 1, Fig. 4d). An esterification reaction between oxalic acid and an –OH group in I would yield compound G - the main Cr(III) species detected by ESI-MS. This is consistent with the earlier study, where quantitative evaluation of total consumption of oxalic acid indicated its requirement not only for Cr(VI) reduction but also
for Cr(III) complexation [5]. Generation of different Cr(III) oxalato complexes during Cr(VI) reduction has also been reported elsewhere [7].
At the end of reduction process, the recovery of Mn(II) from Mn(III) was observed by HPLCMP-AES (Fig. 3d) and by ESI-MS (Fig. 5), confirming that trivalent Mn species is unstable, reactive and appears only in the presence of an electron acceptor (B, D, E, X). The proposed reaction pathway (Fig. 6) points to the catalytic role of divalent manganese, often observed in earlier studies, yet without clarifying the precise reaction mechanism [2; 8; 27; 42; 43]. Importantly, formation of any mixed Cr-Mn species during the course of the reaction was excluded.
4. Conclusions
A combination of analytical techniques based on different physicochemical principles, has provided a mechanistic insight into the role played by divalent manganese during reduction of chromium(VI) by oxalic acid in aqueous solution (pH 3). Direct UV/Vis spectrophotometric measurements and HPLC-DAD results indicated that Mn(II) is needed for fast and efficient reduction; in the reaction mix M2, the reduction was completed within 1 h and generation of at least two Cr intermediates and two final Cr(III) products was observed. By integrating MP-AES detection, quantification of chromium and manganese in chromatographically separated compounds was achieved and generation of any intermediate containing two elements was excluded. By direct infusion of the reaction mixture to ESI-QTOFMS, mass spectra were acquired in real time during the course of reaction and eleven compounds participating in the reduction process were identified (A-K).
Even though oxalic acid itself was capable of initiating the reduction process with the formation of small amounts of Cr(V) and Cr(IV) intermediates, further reduction in M1 mix did not occur. After the addition of Mn(II), generation of Mn(II) bis-oxalato complex (J) facilitated reduction by one-electron transfer from J to any chromium compound containing Cr=O bond, which results in its reduction to Cr-OH and generation of Mn(III) bis-oxalato complex (K). At
the end of the reduction process, conversion of K to J was observed, confirming catalytic role of Mn(II).
Finally, the results obtained in this research suggest that oxalic acid, in combination with Mn(II), may be an interesting option for remediating contaminated environments because the reduction of Cr(VI) in this system does not require strong acidic conditions, and the final oxidation product (CO2) is relatively harmless.
Acknowledgements
The financial support from National Council of Science and Technology, Mexico (CONACYT), projects 178553, 123732 is gratefully acknowledged. The authors thankfully acknowledge the support from the University of Guanajuato, project 408/2014. The authors also wish to thank the Directorate for Research Support and Postgraduate Programs at the University of Guanajuato for their assistance in the proof-reading of the English-language version of this article.
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2-ethyl-2-
hydroxybutanoato(2-/1-) complexes, Inorg. Chem. 43 (2004) 1046-1055. [32] E. Gaggelli, F. Berti, N. D'Amelio, N. Gaggelli, G. Valensin, L. Bovalini, A. Paffetti, L. Trabalzini, Metabolic pathways of carcinogenic chromium, Environ. Health Perspect. 110 Suppl 5 (2002) 733-738. [33] E. Gaggelli, N. D`Àmelio, N. Gaggelli, G. Valensin, L. Bovalini, A. Paffetti, L. Trabalzini, Inferences on the nature of Cr(V) or Cr(IV) species formed by reduction of dichromate by a bovine liver homogenate: NMR and mass-spectrometric studies, Bioinorg. Chem. Appl. 1 (2003) 285-298. [34] V.A. Okello, S. Mwilu, N. Noah, A. Zhou, J. Chong, M.T. Knipfing, D. Doetschman, O.A. Sadik, Reduction of hexavalent chromium using naturally-derived flavonoids, Environ. Sci. Technol. 46 (2012) 10743−10751. [35] M.N. Eberlin, Electrospray ionization mass spectrometry: a major tool to investigate reaction mechanisms in both solutions and the gas phase, Eur. J. Mass Spectrom. (Chichester, Eng) 13 (2007) 19-28. [36] L.S. Santos, Online mechanistic investigations of catalyzed reactions by electrospray ionization mass spectrometry: A tool to intercept transient species in solution, Eur. J. Org. Chem. 2008 (2008) 235-253. [37] Kabir-ud-Din, K. Hartani, Z. Khan, Effect of micelles on the oxidation of oxalic acid by chromium(VI) in the presence and absence of manganese(II), Colloids Surf A 193 (2001) 1-13. [38] A.R. Corrales Escobosa, K. Wrobel, E. Yanez Barrientos, S. Jaramillo Ortiz, A.S. Ramirez Segovia, K. Wrobel, Effect of different glycation agents on Cu(II) binding to human serum albumin studied by liquid chromatography, nitrogen microwave plasma atomic emission spectrometry, inductively coupled plasma mass spectrometry and high resolution molecular mass spectrometry, Anal. Bioanal. Chem. (2014) DOI 10.1007/s00216-014-8335-1..
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Table 1 Assignment of chromium- and manganese- containing compounds, based on the exact mass measurements carried out in the reaction mixture by direct infusion ESI-QTOFMS.
m/z Compound
neutral
Oxidation state
experimental
theoretical
∆, ppm
formula
Cr
A
116.92826
116.92854
2.39
H2CrO4
VI
B
188.91324
188.91328
0.23
H2C2CrO7
VI
C
216.85379
216.85379
0
H2Cr2O7
VI
D
243.89499
243.89529
1.22
C4CrO9
V
E
171.91046
171.91054
0.49
HC2CrO6
V
F
172.91870
172.91837
1.91
H2C2CrO6
IV
G
227.90020
227.90037
0.76
HC4CrO8
III
H
317.89574
317.89568
0.18
H3C6CrO12
III
I
155.91560
155.91563
0.19
HC2CrO6
III
J
231.90574
231.90542
1.36
H2C4MnO8
II
K
230.89791
230.89769
0.96
HC4MnO8
III
Mn
Figure captions:
Fig. 1.
HPLC-DAD results obtained for the reaction mixtures at different time periods from their preparation. (a) 0.67 mM Cr(VI) (____) and M1 mix (----): chromatograms acquired after 2-3 min with the detection at 350 nm. (b) M2 mix: chromatograms acquired immediately (……), after 15 min (---) and 60 min (____) from its preparation at 350 nm. (c); (d); (e) - Absorption spectra recorded at maxima of the chromatographic peaks in (a) eluting at 4.09 min; 4.46 min; 5.05 min, respectively.
Fig. 2.
HPLC-DAD results obtained for the reaction mixture M2 at different time periods from its preparation. (a) chromatograms acquired immediately (……), after 15 min (---) and 60 min (____); detection wavelength 540 nm.
(b); (c) - Absorption spectra recorded at maxima of the chromatographic peaks eluting at 2.57 min; 4.06 min, respectively; reaction time 60 min.
Fig. 3. HPLC-MP-AES results obtained for the reaction mixture M1, converted after 1 h to M2 by Mn(II) addition. (a) chromatograms acquired for Cr (425.433 nm) immediately after Mn(II) addition (____) and after 15 min (----), 30 min (- . - ), 60 min (……). (b) chromatograms acquired for Mn (403.076 nm), immediately after Mn(II) addition (____) and after 15 min (- .. -), 30 min (----) 60 min (…...). (c) chromatogram acquired 1 h after Mn(II) addition with specific detection of Cr (----) and Mn (____). (d) Concentrations corresponding to each chromatographic peak in successive injections: (- -) – Cr(VI) signal tret = 5.68 min; (-
-) – Cr(III) signal tret = 2.50 min; (- -) –
Cr(intermediate) signal tret = 4.18 min + tret = 4.42 min; (- -) – Mn(II) signal tret = 2.30 min; (- -) – Mn(intermediate) signal, tret = 4.58 min.
Fig. 4. Molecular mass spectrometry results: molecular structures of eight compounds chromium (A-K) and changes in their abundance over time. (a) A, B, C. (b) D, E. (c) F. (d) G, H, I
Fig. 5. Molecular mass spectrometry results: molecular structures of two compounds of manganese (J, K) and changes in their abundance over the time of reaction.
Fig. 6.
Proposed mechanism of Cr(VI) reduction by oxalic acid in the presence of Mn(II).
FIGURE 1
FIGURE 2
Absorbance, mAU, 540 nm
6
a)
5
b
4
c 3 2 1 0 0
4 Time, min
6
50
Absorbance, mAU
b)
40
Absorbance, mAU
2
30 20 10
c)
40 30 20 10 0
0 300
400 500 Wavelength, nm
600
300
400 500 600 Wavelength, nm
FIGURE 3
a)
Emission intensity 10 3
Emission intensity 10 3
55 45 35 25 15
12.5 7.5 2.5
5 0
2
4 Time, min
6
0
8
0.80
b)
8
Concentratiom, mM
Emission intensity 10 3
c)
17.5
6 4 2
2
4 Time, min
6
8
d)
0.60 0.40 0.20 0.00
0
2
4 Time, min
6
8
0
50
100 Time, min
150
FIGURE 4 20
a)
40
Intensity 10 3
Intensity 10 3
50
30 20 10
16 12 8 4
0
0 0
30
60
90
0
Time, min - -
A
- -
B
-
-
O O
O
Cr(VI) OH OH
-
-
H2Cr2O7
O
O
Intensity 10 3
2 1
-
E
O O
O
O
(V) Cr OH
O
O
40
3
-
-(V) Cr O
90
D
O O
O
c)
4
30 60 Time, min
C
O
O H2CrO4
Intensity 103
b)
O
O
d)
30 20 10 0
0 0
30 -
60 Time, min -
0
90
30
F
-
-
60 Time, min
G
90
-
-
H
O O HO O
O
OH
O
O
O
OH
O
OH
(III) Cr
Cr (IV)
O
O
O
O O
O
O
O
OH
O HO (III) Cr O O O
- ■O
O
O
O
I
(III) Cr OH
FIGURE 5
Intensity 10 3
30 20 10 0 0
30
60 Time, min
90
J
-
O
O
O
O
O
-
-
(II) Mn
O
O
H
K
O
Mn (III)
O
H
O
O O
-
O
H O
O O
FIGURE 6
O
O
O
O
H
(II)
Mn
O
O
O
O (III)
Cr OH
HO
O
O
O
Cr
OH O
HO OH O
O O
(V)
OH
+
Mn
(VI)
O OH
O
O
O
O - H2O
J
K
B (A,C)
E O
O
O
O
H
Mn
(V)
O
(IV)
Cr
+
O
O
O
O
J
K
Cr O
OH
OH
HO
OH (II)
O
O
O
E
F - H2O
X O
O
O
O
H
(II)
Mn
O
O
O
K +
(IV)
Cr
O
O
O
O
O
(III)
Cr
HO
O
OH
J
O
X
O
O Cr
OH
I
O
Cr
+ oxH2 O
O
HO
O (III)
(III)
O
I
O
O
G
O
+
H2O
S0304-3894(15)00523-3 http://dx.doi.org/doi:10.1016/j.jhazmat.2015.06.066 HAZMAT 16921
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
5-2-2015 6-5-2015 30-6-2015
Please cite this article as: Katarzyna Wrobel, Alma Rosa Corrales Escobosa, Alan Alexander Gonzalez Ibarra, Manuel Mendez Garcia, Eunice Yanez Barrientos, Kazimierz Wrobel, Mechanistic insight into chromium(VI) reduction by oxalic acid in the presence of manganese(II), Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2015.06.066 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mechanistic insight into chromium(VI) reduction by oxalic acid in the presence of manganese(II)
Katarzyna Wrobel, Alma Rosa Corrales Escobosa, Alan Alexander Gonzalez Ibarra, Manuel Mendez Garcia, Eunice Yanez Barrientos, Kazimierz Wrobel*
Chemistry Department, Division of Natural and Exact Sciences, University of Guanajuato, L. de Retana 5, 36000 Guanajuato, Mexico
*Corresponding author: Phone: +52 473 7327555, e-mail: [email protected]
Abstract
Over the past few decades, reduction of hexavalent chromium (Cr(VI)) has been studied in many physicochemical contexts. In this research, we reveal the mechanism underlying the favorable effect of Mn(II) observed during Cr(VI) reduction by oxalic acid using liquid chromatography with spectrophotometric diode array detector (HPLC-DAD), nitrogen microwave plasma atomic emission spectrometry (HPLC-MP-AES), and high resolution mass spectrometry (ESIQTOFMS). Both reaction mixtures contained potassium dichromate (0.67 mM Cr(VI)) and oxalic acid (13.3 mM), pH 3, one reaction mixture contained manganese sulfate (0.33 mM Mn(II)). In the absence of Mn(II) only trace amounts of reaction intermediates were generated, most likely in the following pathways: (1) Cr(VI)→Cr(IV) and (2) Cr(VI)+Cr(IV)→2Cr(V). In the presence of Mn(II), the active reducing species appeared to be Mn(II) bis-oxalato complex (J); the proposed reaction mechanism involves a one-electron transfer from J to any chromium compound containing Cr=O bond, which is reduced to Cr-OH, and the generation of Mn(III) bisoxalato complex (K). Conversion of K to J was observed, confirming the catalytic role of Mn(II). Since no additional acidification was required, the results obtained in this study may be helpful in designing a new, environmentally friendly strategy for the remediation of environments contaminated with Cr(VI).
Keywords: Chromium(VI); Oxalic acid; Manganese(II); Liquid chromatography - nitrogen microwave plasma atomic emission spectrometry (HPLC-MP-AES); High resolution molecular mass spectrometry (ESI-QTOFMS)
1. Introduction
The reduction pathway and reaction efficiency of hexavalent Cr(VI) to trivalent Cr(III) both depend on the identity and availability of reducing agents, pH conditions, and on the presence of co-reducing species [1; 2]. Mechanistic studies carried out under specific reaction conditions are important in organic chemistry because intermediate Cr(V) or Cr(IV) species act as oxidants or catalysts of various reactions [3; 4; 5; 6]. In particular, oxalic acid has served as a prototype molecule to study the oxidation of organic compounds by chromic acid. The catalytic role of Mn(II) in these reactions has also been reported [7; 8]. Reduction of Cr(VI) at physiological
conditions is of high biological relevance because intermediate species formed during the process promote oxidative stress and DNA damage [9; 10; 11; 12]. Remediation of contaminated environments is another area in which reduction mechanisms need to be understood in detail. For example, by ensuring the efficient conversion of Cr(VI) into less harmful Cr(III) and the subsequent stabilization or elimination of the latter by sorption on a solid phase or as Cr(OH)3 precipitate [13; 14].
The feasibility of organic acids as electron donors during Cr(VI) reduction has been studied in abiotic model systems and soils [15; 16; 17; 18; 19], and to a lesser extent, in microbiological cultures [20]. It has been demonstrated that citric, tartaric, salicylic, ascorbic, and oxalic acids are capable of slow reduction at slightly acidic conditions (pH 3-5). However, this process becomes accelerated in the presence of suitable co-reducing agents and/or redox-cycling transition metal ions [14; 18; 21; 22; 23]. Specifically, catalytic action of divalent manganese (Mn(II)) during Cr(VI) reduction by citric, ascorbic, and tartaric acids has been reported in abiotic models and in soils at pH < 5 [14; 23; 24; 25; 26; 27].
In early mechanistic studies of Cr(VI) reduction, UV/Vis spectrophotometry and electron paramagnetic resonance spectroscopy were used as the primary analytical tools [4; 5; 7; 8; 27; 28; 29; 30]. In further developments, nuclear magnetic resonance, X-ray absorption spectroscopy and mass spectrometry (MS) were integrated for structural characterization of reaction intermediates [1; 11; 31; 32; 33; 34]. However, as far as we are aware, MS has not been used so far for the elucidation of the role of Mn(II) during Cr(VI) reduction by organic acids. In general terms, electrospray ionization - mass spectrometry (ESI-MS) enables the unequivocal identification of charged and polar species, based on exact mass measurements and molecular fragmentation patterns. Unlike other techniques capable of structural characterization, mass spectrometry offers fast and simultaneous analysis of various short-lived, reactive intermediates present in the reaction mixture with no requirement for their prior separation and/or isolation. Thanks to the exceptional selectivity of high resolution MS, the solution can be introduced by direct infusion and intensity changes of individual ions are monitored simultaneously and in real time for various species. In the soft ESI source, intact ions from the solution are transferred to the gaseous phase, whereas polar species are converted into molecular ions without alteration of existing covalent
bonds and strong, non-covalent interactions [35; 36]. Furthermore, hyphenation of liquid chromatography with nitrogen microwave plasma atomic emission spectrometry (HPLC-MPAES) seems to be a helpful complement of ESI-MS in studies of reaction mechanisms that involve different metal/metalloid species. In particular, HPLC-MP-AES might allow for quantitative evaluation of chromium and manganese in species generated during the course of the reaction, practically in real time.
We undertook this research in order to obtain a mechanistic insight into Cr(VI) reduction by oxalic acid in the presence of divalent manganese. Even though this reaction had been studied in many physicochemical contexts, the precise mechanism responsible for the observed favorable effect of manganese remains unclear [7; 8; 19; 37]. In this regard, a combination of UV/Vis spectrophotometry, liquid chromatography with spectrophotometric and MP-AES detection, and direct infusion high resolution ESI-MS were used here for the first time. Consistent yet complementary data obtained by ESI-MS and HPLC-MP-AES allowed us to unravel the reaction mechanism, in which Mn(II) catalyzes Cr(VI) reduction in the presence of oxalic acid without additional acidification. The results obtained may be helpful in designing new, environmentally friendly strategies for the remediation of Cr(VI)-polluted sites.
2. Experimental
2.1. Reagents and samples
All chemicals used throughout the experiment were of analytical reagent grade: deionized water (18.2 MΩ cm, Labconco, USA) and LC-MS-grade methanol (Sigma, Milwaukee, USA). Potassium dichromate, manganese sulfate, oxalic acid, and formic acid were from Sigma.
Stock solutions of hexavalent chromium (0.20M Cr(VI)), divalent manganese (0.25M Mn(II)) and oxalic acid (1.0M) were prepared from the above reagents and deionized water. The reaction mixtures were obtained by pipetting 200 µL of oxalic acid solution to a Falcon tube containing 10 mL of deionized water which was then vortexed, 50 µL Cr(VI) was added and the solution
vortexed again, followed by the addition of an aliquot of Mn(II) (0 – 80 µL) as appropriate. Finally the volume was brought to 15 mL with deionized water. The solutions were analyzed immediately and in successive runs over the time period up to 2 h.
The reaction mixture M1 contained 0.67 mM Cr(VI) and 13.3 mM oxalic acid and M2 contained an additional 0.33 mM Mn(II); the pH values in these solutions were in the range 2.93.1.
2.2. UV/Vis spectrophotometry, HPLC-DAD and HPLC – MP-AES
Absorption spectra (250 – 700 nm) of the reaction mixtures were acquired using a Spectronic 3000 Diode Array Milton Roy spectrophotometer (resolution 0.35 nm, Milton Roy Inst. Co.). Freshly prepared mixtures were also analyzed by reversed phase high performance liquid chromatography with diode array spectrophotometric detection (HPLC series 1200, Agilent Technologies); starting from preparation of the solution, successive injections (20 µL) were repeated at 10 min intervals for 1 hour. The chromatographic column was Luna C18 (250 x 4.6 mm, 5µm, Phenomenex); isocratic elution was carried out with 0.1% v/v formic acid in 0.5% v/v methanol mobile phase, at a flow rate 0.8 mL/min and detection in the wavelength range 250-700 nm. The above chromatographic system was coupled on-line to nitrogen microwave plasma atomic emission spectrometer equipped with MiraMist® Teflon nebulizer and single-pass glass cyclonic spray (MP-AES 4100, Agilent Technologies), as described previously [38]. The following instrument operating conditions were used: nebulizer pressure 220 kPa, nitrogen pressure 140 psi, viewing position 0, integration time 1 s, wavelengths 425.433 nm and 403.076 nm, for chromium and manganese specific detection, respectively.
Based on multiple injections, the mean retention time with respective standard deviation is given for each chromatographic signal throughout the text.
2.3. ESI-QTOFMS
A high resolution, electrospray ionization - quadrupole-time of flight mass spectrometer maXis impact ESI-QTOFMS equipped with Data Analysis 4.1 (Bruker Daltonics) was used with sample introduction by direct infusion (3 µL/min). ESI was operated in negative mode with ion spray voltage 3000 V, dry gas 4 L/min, drying temperature 180 °C and nebulizing gas pressure 0.3 bar. Spectra of the reaction mixture were acquired in the m/z range 80-800, acquisition was repeated seven times over a 90 min period. For internal mass-lock calibration, the molecular ion of H2Cr2O7 was used (m/z 216.85379).
3. Results and discussion
3.1. Direct UV/Vis spectrophotometric measurements and HPLC-DAD results
In the first approach, a series of solutions containing 0.67 mM Cr(VI) and oxalic acid in molar excess with respect to chromium up to 50:1 were prepared. The pH values in these solutions were in the range 2.9 - 3.1; UV/Vis absorption spectra of these solutions showed two bands (350 nm, 445 nm) with no change of their shape nor intensity during at least four hours, in agreement with the earlier finding that the reduction of Cr(VI) by oxalic acid is kinetically favored only in acidic media (pH < 1.6) [5]. Different concentrations of Mn(II) were added to dichromate solution to cover the range of Cr:Mn molar ratio from 16:1 to 1:2; these solutions presented identical spectral profile as Cr(VI) with no change in successively recorded spectra. Finally, different aliquots of Mn(II) were added to the mixture Cr(VI) + oxalic acid to obtain Cr:Mn molar ratios 16:1; 8:1; 4:1; 2:1; 1:1; 1:2 and UV/Vis spectra were recorded. In accordance with earlier studies [7; 37], Mn(II) caused a time-dependent decrease of Cr(VI) absorption signal and the reduction kinetics was clearly favored by increasing the concentration of Mn(II) (changes of absorbance at 350 nm during the course of the reaction are presented in Fig. 1S in the Electronic Supplementary Material, ESM). In further experiments, the mixture M2 containing 0.67 mM Cr(VI), 13.3 mM oxalic acid and 0.33 mM Mn(II) was used. The composition of M2 (molar ratio of oxalic acid-to-Cr-to-Mn 20:2:1, pH 3.0) was selected in order to promote reaction kinetics and also to facilitate the detection of intermediate species by different instrumental techniques. It was verified by the UV/Vis absorption profile of M2 that it presented gradual changes (Fig. 2S, ESM); in particular, the Cr(VI) signal at 350 nm decreased promptly and was
not detected after 1 h, whereas changes of absorbance at 295 nm and 445 nm suggested the formation of at least two chromium intermediates [12; 28]. Spectral overlapping of the different, poorly absorbing species Cr(III), Cr(IV), Cr(V) hindered positive detection of Cr(III) [39].
Despite the evident utility of the above results for confirmation of Cr(VI) reduction in the presence of oxalic acid and Mn(II) these experiments did not allow for any mechanistic interpretation. To enhance analytical capability, chromatographic separation with diode array spectrophotometric detection was used. It should be emphasized that mild conditions were applied, thus giving priority to preserving species’ identity and not their baseline separation (see Experimental).
In the first approach, the mixture containing Cr(VI) + oxalic acid (M1) and the Cr(VI) solution were analyzed, the respective chromatograms registered at 350 nm (Fig. 1a). The main chromatographic peak eluted at 5.05 ± 0.01 min and the absorption spectrum at apex was characteristic for Cr(VI) (Fig. 1a,e). As compared to the pure dichromate solution, two additional minor signals were observed in the analysis of M1, with retention times 4.09 ± 0.03 min and 4.46 ± 0.04 min (Fig. 1a). Based on the absorption spectra at apex (Fig. 1c,d), these species correspond to the intermediate oxidation states generated during Cr(VI) reduction [12; 28], in line with direct spectrophotometric measurements (Fig. 2S, ESM). Since both minor peaks remained practically unchanged for at least 2h, it is clear that Cr(VI) reduction by oxalic acid was initiated immediately, but intermediates generated at this stage could not be further reduced. Indeed, the formation of a long-lived Cr(V) intermediate species during Cr(VI) reduction by oxalic acid has been reported and Cr(VI)-to-Cr(V) reduction was considered a rate-limiting step [4; 28; 40]. On the other hand, reduction to Cr(IV) [41], formation of Cr(V) from Cr(VI) and Cr(IV) [42] as well as generation of mixed-valence chromium intermediates were discussed in other studies [8; 43].
Typical chromatograms obtained for the reaction mix M2 at 2-3 min, 15 min and 60 min from its preparation are shown in Fig. 1b (detection at 350 nm). As can be observed in this Figure, the elution profile was similar as described above for M1 (the number, position and shape of chromatographic signals), yet intensities of individual peaks changed over the time. The chromatographic signal corresponding to Cr(VI) (retention time 5.05 ± 0.02 min) was decreasing
in successive injections and disappeared after 60 min. In the first injection, two earlier peaks in Fig. 1b presented identical absorption spectra as those registered without Mn(II) (Fig. 1c,d), yet their intensities were lower (Fig. 1a,b). Despite incomplete separation, it can be observed that the two weak signals presented different intensity changes in repeated analysis; the first (4.09 ± 0.03 min) slowly increased over the time but the second (4.46 ± 0.04 min) initially increased and then diminished (Fig. 1b). At the same time, spectra acquired at the apex of the two peaks presented gradual changes. These results suggest that the final product of reduction (Cr(III)) probably coeluted with reaction intermediate in the first of two poorly resolved peaks, whereas the second chromatographic signal contained only intermediate chromium species. It is also clear that Mn(II) activated the reduction of intermediate chromium forms. Our results are consistent with many earlier studies reporting capability of Mn(II) for the reduction of both, Cr(IV) and Cr(V) oxidation states [8; 30; 43].
To examine Cr(III) formation, the chromatograms shown in Fig. 1b were registered at 540 nm; two signals were obtained with retention times 2.57 ± 0.01 min and 4.06 ± 0.03 min, respectively; their intensities gradually increased in successive injections (Fig. 2a). The absorption spectra acquired from the chromatogram run 60 min after preparation of M2 (Fig. 2a), were identical and showed two maxima (415 nm, 570 nm, Fig. 2b,c) consistent with previously reported spectra of Cr(III) in oxalate solution [44]. It should be emphasized that the second chromatographic signal of lower intensity (4.06 ± 0.03 min, Fig. 2a) was also present when the detection wavelength was set at 350 nm (4.09 ± 0.03 min, Fig. 1b), confirming a co-elution of the minor Cr(III) product with element intermediates.
Absorption spectra acquired in the chromatographic system, as well as the changes of signal intensities over time, were consistent with the results obtained in direct spectrophotometric measurements. This indicates that the chromatographic process had negligible effect on the reduction process and species identity. Despite better selectivity of HPLC-DAD as compared to direct UV/Vis measurements, species eluting between 4 min and 4.5 min could not be unequivocally assigned. Furthermore, spectrophotometric measurements did not enable the detection of manganese compounds.
3.2. HPLC-MP-AES results
Chromatograms obtained for M2 in six successive injections are presented in Fig. 3. For chromium emission line (425.433 nm), four chromatographic signals were observed with the retention times 2.50 ± 0.01 min, 4.18 ± 0.02 min, 4.42 ± 0.02 min, 5.68 ± 0.02 min (Fig. 3a), which resembled the elution profile obtained with UV/Vis detection (Fig. 1a,b; Fig. 2a). However, chromatographic signals were slightly delayed with respect to HPLC-DAD due to differences in the instrumental set-up.
For manganese specific detection at 403.076 nm, two chromatographic peaks were eluted with retention time 2.30 ± 0.02 min and 4.58 ± 0.03 min, respectively (Fig. 3b). As shown in Fig. 3c, chromatographic signals acquired for Cr and Mn in this same chromatographic run did not overlap, which indicates that the two elements were not present together in any of the species formed during Cr(VI) reduction. This finding is relevant, because the mechanism is responsible for the favorable effect of Mn(II) that has yet to be clarified, although some authors suggest the formation of intermediate species composed of oxalate, Cr and Mn [7; 37; 45].
For peak assignment, chromatograms of individual standard solutions of Cr(VI), Cr(III) and Mn(II) in the presence of oxalic acid were obtained. Each of them showed only one chromatographic peak, which remained unchanged during several injections. Based on the retention times obtained for the above standards, Cr-containing signals eluting at 2.50 ± 0.01 min and 5.68 ± 0.02 min were assigned to Cr(III) and Cr(VI), respectively; whereas two other poorly separated signals in Fig. 3a (4.18 ± 0.02 min, 4.42 ± 0.02 min) would correspond to Cr intermediates. One chromatographic peak was obtained by analyzing Cr(III) + oxalic acid standard mix (2.50 min) but two Cr(III) peaks were detected in M2 (Fig. 2, Fig. 3), which suggests that the second, minor compound (4.42 ± 0.02 min) was formed only as a reduction product. For manganese, the first peak eluting at 2.30 ± 0.02 min (Fig. 3b) corresponded to Mn(II).
For quantitative evaluation of element species at different stages of Cr(VI) reduction, the following experiment was performed: 15 mL of M1 was prepared and introduced repeatedly to
the chromatographic system with MP-AES detection for one hour (4 injections). Then a small aliquot (20 µL) of Mn(II) stock standard was added, yielding a final manganese concentration of 0.33 mM. The M2 obtained was analyzed for a total of 80 min at 10 min intervals. In Figure 3d, the results are presented as concentrations of Mn and Cr found in each chromatographic signal in successive chromatographic runs (time scale on X-axis). The detailed description of quantification procedure is given in the ESM, Fig. 3S, Fig. 4S.
It can be observed in Fig. 3d that the initial concentration of Cr(VI) remained practically unchanged in the absence of Mn(II), and only trace amounts of Cr intermediates were detected (two poorly separated minor peaks of Cr in Fig. 3 were integrated for quantification). After addition of Mn(II), the reduction process was activated immediately and completed after 1 h. At the same time, the concentration of Cr(III) was increasing gradually (signal at 2.50 ± 0.01 min); for the two poorly separated signals (4.18 ± 0.02 min, 4.42 ± 0.02 min, Fig. 3a), similar tendency was observed yet the increase was much less pronounced which confirms that intermediate species were quantified together with the second, minor Cr(III) product (Fig. 1b, Fig. 2a). The percentage recovery of Cr in each chromatographic run, calculated as a sum of concentrations found in each peak with respect to the concentration introduced on-column (0.67 mM), was in the range 80 - 113%, indicating acceptable reliability of analytical data.
The initial decrease of Mn(II) concentration (2.30 ± 0.02 min, Fig. 3b,d) was accompanied by formation of the second element species (4.58 ± 0.03 min, Fig. 3b,d) but after one hour, there was a tendency toward Mn(II) retrieval and decrease of the second species – a clear indication of the intermediate character of the latter. When two manganese forms were present, the emission intensity between their respective chromatographic signals was higher than the baseline (Fig. 3b), which suggests that intermediate species might be unstable during the chromatographic process. Based on the integration of two Mn signals, the percentage recovery was 65 - 102%; these relatively lower values as compared to Cr should be ascribed to continuous element elution mentioned above.
Taken together, the results obtained in this part of study demonstrate that Mn(II) is required for fast and efficient reduction of Cr(VI) by oxalic acid at pH 3. The reduction process involves
generation of Cr intermediate species and, at least two Cr(III) compounds are formed as the final products. During the course of the reaction, Mn(II) activates the reduction of Cr intermediate species; by doing so, it is converted to a different form (probably Mn(III)) which seems to return to the initial Mn(II) at the end of reduction process. No species containing two elements was detected.
3.3. ESI-QTOFMS results
For the elucidation of molecular structures involved in Cr(VI) reduction, the reaction mixture was prepared identically as for HPLC-MP-AES and it was analyzed by high resolution mass spectrometry with direct infusion. Changes of abundance for each m/z signal were followed by repeated measurements; the first two of them were performed for Cr(VI) + oxalic acid mix M1 (2-3 min after preparation and at 10 min), then Mn(II) was added yielding M2 and mass spectra were acquired in five successive measurements between 10 and 90 min. Two first spectra obtained in the absence of Mn(II) did not present important differences. However, after the addition of Mn(II), a gradual change of intensity over time was observed for several m/z values and new signals corresponding to manganese species appeared (ESI-QTOFMS spectra in the m/z ranges 90-350, 226-234 and 154-157 are presented in Fig. 5Sa-c, ESM). Eleven signals were selected for further analysis; in Table 1 their respective molecular formulas are presented together with theoretical and experimental m/z values of molecular ions (Data Analysis 4.1); mass error was in the range 0.18 – 2.39 ppm, which confirms accurate species assignment. For each molecular ion, its abundance was acquired during the course of the reaction and the results are presented in Fig. 4 and Fig. 5. It can be observed in Fig. 4a that in M1 there were three hexavalent compounds in equilibrium: chromate (major species, A), Cr(VI) mono-oxalato complex (B) and dichromate (C). After Mn(II) addition, abundances of A, B, C promptly decreased in agreement with HPLC-DAD (Fig. 1b) and HPLC-MP-AES (Fig. 3d) results. As indicated from the very beginning, chromatographic resolution of individual species was not intended and A, B, C in the aforementioned Figures eluted always in form of one broad chromatographic peak. It has also been mentioned before that only one chromatographic peak was observed for the solution of Cr(VI) and oxalic acid.
Two pentavalent chromium intermediates, Cr(V) bis-oxalato complex (D) and Cr(V) monooxalato complex (E), were detected in M1 but their abundances in mass spectra did not change until manganese was added to the solution. Then, the formation of the two intermediates increased and after about 20 min, both species started to disappear gradually (Fig. 4b), also in agreement with data presented in the previous sections. In particular, ESI-MS results confirmed the presence of two Cr(V) intermediates in poorly resolved chromatographic peaks eluting between 4.0 and 4.5 min.
Detection and identification of tetravalent chromium (compound F) is noteworthy, because it proved that the reduction process under study involves three one-electron transfers. This task was challenging for many reasons; Mn(II) had been reported as a catalyst of Cr(IV) reduction [8; 30] and Cr(IV) is generally considered a short-life intermediate[1], hence only minute amounts could be expected in the reaction mixture. Furthermore, compounds E and F differ only by one hydrogen atom and their isotopic patterns partly overlap; difficulties in the detection of Cr(IV) by ESI-MS had been highlighted before [1]. In Fig. 4c, change of molecular ion intensity over the time is presented for compound F (Cr(IV) mono-oxalato complex); whereas Figure 6S in the ESM shows how the intensity of molecular ion at m/z 172.91837 was measured.
The three Cr(III) compounds detected ( G, H, I) were identified as Cr(III) bis-oxalato, Cr(III) tri-oxalato and Cr(III) mono-oxalato complexes, respectively; abundancies of molecular ions presented a gradual increase in the M2 mix (Fig. 4d) consistent with HPLC-DAD (Fig. 2a) and HPLC-MP-AES (Fig. 3a,d) data. It should be noted, however, that only two Cr(III) forms were observed before, most likely due to insufficient selectivity of analytical tools and low concentrations of H and I.
The compounds J and K were identified as Mn(II) bis-oxalato complex and Mn(III) bisoxalato complex, respectively (Table 1, Fig. 5). According to HPLC-MP-AES results (Fig. 3d), the J signal appearing after Mn(II) addition first decreased and then recovered at the end of the reduction process (Fig. 5). The abundance change for compound K resembled that observed for the intermediate form of Mn in HPLC-MP-AES analysis (Fig. 3d). However, relative intensity of K in mass spectra was much higher with respect to Mn intermediate in MP-AES chromatograms
(compare Fig. 5 and Fig. 3d). This difference should be ascribed to partial degradation of Mn intermediates during the chromatographic process (continuous Mn elution between two well defined peaks), whereas direct infusion and soft ionization source in mass spectrometry analysis apparently had negligible effect on the integrity of K.
3.4. Proposed mechanism of Cr(VI) reduction
In the M1 mix, only small amounts of Cr(V) and Cr(IV) intermediates were generated and it was deduced that the oxalic acid itself is capable of reducing Cr(VI) to Cr(IV) by two-electron transfer with the release of CO2 and that Cr(V) could be then generated from the mixed Cr(VI)Cr(IV) species [8; 43]. However, the progression of the reduction process was not observed (the reaction scheme is presented in Fig. 7S, ESM). In the M2 solution, the active reducing species appeared to be Mn(II) bis-oxalato complex (J), as shown in the reaction mechanism in Fig. 6. It should be noted that the complexation of Mn(II) by small organic ligands such as citrate or oxalate had been proposed as a prerequisite for electron transfer during the reduction of Cr(VI) [21; 23; 27; 37]. Formation of Mn(III) bis-oxalato complex K accompanied by increasing abundances of Cr(V) and Cr(IV) intermediates (Fig. 6, 7) suggest that one electron was transferred from J to chromium species B, D or E, converting Cr=O contained in their structures to Cr-OH and causing oxidation of divalent Mn species J to trivalent K. The ability of Mn(II) for reduction of Cr(VI), Cr(V) and Cr(IV) had been observed in other studies [2; 43; 46], it is also relevant that favorable Mn(II) effect during chromium(VI) reduction by organic acids was reported to be more pronounced at lower acidities [47], which confirms that disruption of the – OH group in Mn(II) bis-oxalato complex was required for electron transfer (pH 3). The unique species of Cr(IV) detected by ESI-MS corresponded to compound F which, unlike structures suggested for Cr(IV) intermediates before [1; 40], did not present a Cr=O bond. It was expected that Mn(II) would act equally in all one-electron transfers, so the conversion of F into X by the loss of H2O, was proposed. As uncharged and non-polar compound, X could not be confirmed by ESI-MS but its reduction product (I) was detected (Table 1, Fig. 4d). An esterification reaction between oxalic acid and an –OH group in I would yield compound G - the main Cr(III) species detected by ESI-MS. This is consistent with the earlier study, where quantitative evaluation of total consumption of oxalic acid indicated its requirement not only for Cr(VI) reduction but also
for Cr(III) complexation [5]. Generation of different Cr(III) oxalato complexes during Cr(VI) reduction has also been reported elsewhere [7].
At the end of reduction process, the recovery of Mn(II) from Mn(III) was observed by HPLCMP-AES (Fig. 3d) and by ESI-MS (Fig. 5), confirming that trivalent Mn species is unstable, reactive and appears only in the presence of an electron acceptor (B, D, E, X). The proposed reaction pathway (Fig. 6) points to the catalytic role of divalent manganese, often observed in earlier studies, yet without clarifying the precise reaction mechanism [2; 8; 27; 42; 43]. Importantly, formation of any mixed Cr-Mn species during the course of the reaction was excluded.
4. Conclusions
A combination of analytical techniques based on different physicochemical principles, has provided a mechanistic insight into the role played by divalent manganese during reduction of chromium(VI) by oxalic acid in aqueous solution (pH 3). Direct UV/Vis spectrophotometric measurements and HPLC-DAD results indicated that Mn(II) is needed for fast and efficient reduction; in the reaction mix M2, the reduction was completed within 1 h and generation of at least two Cr intermediates and two final Cr(III) products was observed. By integrating MP-AES detection, quantification of chromium and manganese in chromatographically separated compounds was achieved and generation of any intermediate containing two elements was excluded. By direct infusion of the reaction mixture to ESI-QTOFMS, mass spectra were acquired in real time during the course of reaction and eleven compounds participating in the reduction process were identified (A-K).
Even though oxalic acid itself was capable of initiating the reduction process with the formation of small amounts of Cr(V) and Cr(IV) intermediates, further reduction in M1 mix did not occur. After the addition of Mn(II), generation of Mn(II) bis-oxalato complex (J) facilitated reduction by one-electron transfer from J to any chromium compound containing Cr=O bond, which results in its reduction to Cr-OH and generation of Mn(III) bis-oxalato complex (K). At
the end of the reduction process, conversion of K to J was observed, confirming catalytic role of Mn(II).
Finally, the results obtained in this research suggest that oxalic acid, in combination with Mn(II), may be an interesting option for remediating contaminated environments because the reduction of Cr(VI) in this system does not require strong acidic conditions, and the final oxidation product (CO2) is relatively harmless.
Acknowledgements
The financial support from National Council of Science and Technology, Mexico (CONACYT), projects 178553, 123732 is gratefully acknowledged. The authors thankfully acknowledge the support from the University of Guanajuato, project 408/2014. The authors also wish to thank the Directorate for Research Support and Postgraduate Programs at the University of Guanajuato for their assistance in the proof-reading of the English-language version of this article.
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Table 1 Assignment of chromium- and manganese- containing compounds, based on the exact mass measurements carried out in the reaction mixture by direct infusion ESI-QTOFMS.
m/z Compound
neutral
Oxidation state
experimental
theoretical
∆, ppm
formula
Cr
A
116.92826
116.92854
2.39
H2CrO4
VI
B
188.91324
188.91328
0.23
H2C2CrO7
VI
C
216.85379
216.85379
0
H2Cr2O7
VI
D
243.89499
243.89529
1.22
C4CrO9
V
E
171.91046
171.91054
0.49
HC2CrO6
V
F
172.91870
172.91837
1.91
H2C2CrO6
IV
G
227.90020
227.90037
0.76
HC4CrO8
III
H
317.89574
317.89568
0.18
H3C6CrO12
III
I
155.91560
155.91563
0.19
HC2CrO6
III
J
231.90574
231.90542
1.36
H2C4MnO8
II
K
230.89791
230.89769
0.96
HC4MnO8
III
Mn
Figure captions:
Fig. 1.
HPLC-DAD results obtained for the reaction mixtures at different time periods from their preparation. (a) 0.67 mM Cr(VI) (____) and M1 mix (----): chromatograms acquired after 2-3 min with the detection at 350 nm. (b) M2 mix: chromatograms acquired immediately (……), after 15 min (---) and 60 min (____) from its preparation at 350 nm. (c); (d); (e) - Absorption spectra recorded at maxima of the chromatographic peaks in (a) eluting at 4.09 min; 4.46 min; 5.05 min, respectively.
Fig. 2.
HPLC-DAD results obtained for the reaction mixture M2 at different time periods from its preparation. (a) chromatograms acquired immediately (……), after 15 min (---) and 60 min (____); detection wavelength 540 nm.
(b); (c) - Absorption spectra recorded at maxima of the chromatographic peaks eluting at 2.57 min; 4.06 min, respectively; reaction time 60 min.
Fig. 3. HPLC-MP-AES results obtained for the reaction mixture M1, converted after 1 h to M2 by Mn(II) addition. (a) chromatograms acquired for Cr (425.433 nm) immediately after Mn(II) addition (____) and after 15 min (----), 30 min (- . - ), 60 min (……). (b) chromatograms acquired for Mn (403.076 nm), immediately after Mn(II) addition (____) and after 15 min (- .. -), 30 min (----) 60 min (…...). (c) chromatogram acquired 1 h after Mn(II) addition with specific detection of Cr (----) and Mn (____). (d) Concentrations corresponding to each chromatographic peak in successive injections: (- -) – Cr(VI) signal tret = 5.68 min; (-
-) – Cr(III) signal tret = 2.50 min; (- -) –
Cr(intermediate) signal tret = 4.18 min + tret = 4.42 min; (- -) – Mn(II) signal tret = 2.30 min; (- -) – Mn(intermediate) signal, tret = 4.58 min.
Fig. 4. Molecular mass spectrometry results: molecular structures of eight compounds chromium (A-K) and changes in their abundance over time. (a) A, B, C. (b) D, E. (c) F. (d) G, H, I
Fig. 5. Molecular mass spectrometry results: molecular structures of two compounds of manganese (J, K) and changes in their abundance over the time of reaction.
Fig. 6.
Proposed mechanism of Cr(VI) reduction by oxalic acid in the presence of Mn(II).
FIGURE 1
FIGURE 2
Absorbance, mAU, 540 nm
6
a)
5
b
4
c 3 2 1 0 0
4 Time, min
6
50
Absorbance, mAU
b)
40
Absorbance, mAU
2
30 20 10
c)
40 30 20 10 0
0 300
400 500 Wavelength, nm
600
300
400 500 600 Wavelength, nm
FIGURE 3
a)
Emission intensity 10 3
Emission intensity 10 3
55 45 35 25 15
12.5 7.5 2.5
5 0
2
4 Time, min
6
0
8
0.80
b)
8
Concentratiom, mM
Emission intensity 10 3
c)
17.5
6 4 2
2
4 Time, min
6
8
d)
0.60 0.40 0.20 0.00
0
2
4 Time, min
6
8
0
50
100 Time, min
150
FIGURE 4 20
a)
40
Intensity 10 3
Intensity 10 3
50
30 20 10
16 12 8 4
0
0 0
30
60
90
0
Time, min - -
A
- -
B
-
-
O O
O
Cr(VI) OH OH
-
-
H2Cr2O7
O
O
Intensity 10 3
2 1
-
E
O O
O
O
(V) Cr OH
O
O
40
3
-
-(V) Cr O
90
D
O O
O
c)
4
30 60 Time, min
C
O
O H2CrO4
Intensity 103
b)
O
O
d)
30 20 10 0
0 0
30 -
60 Time, min -
0
90
30
F
-
-
60 Time, min
G
90
-
-
H
O O HO O
O
OH
O
O
O
OH
O
OH
(III) Cr
Cr (IV)
O
O
O
O O
O
O
O
OH
O HO (III) Cr O O O
- ■O
O
O
O
I
(III) Cr OH
FIGURE 5
Intensity 10 3
30 20 10 0 0
30
60 Time, min
90
J
-
O
O
O
O
O
-
-
(II) Mn
O
O
H
K
O
Mn (III)
O
H
O
O O
-
O
H O
O O
FIGURE 6
O
O
O
O
H
(II)
Mn
O
O
O
O (III)
Cr OH
HO
O
O
O
Cr
OH O
HO OH O
O O
(V)
OH
+
Mn
(VI)
O OH
O
O
O
O - H2O
J
K
B (A,C)
E O
O
O
O
H
Mn
(V)
O
(IV)
Cr
+
O
O
O
O
J
K
Cr O
OH
OH
HO
OH (II)
O
O
O
E
F - H2O
X O
O
O
O
H
(II)
Mn
O
O
O
K +
(IV)
Cr
O
O
O
O
O
(III)
Cr
HO
O
OH
J
O
X
O
O Cr
OH
I
O
Cr
+ oxH2 O
O
HO
O (III)
(III)
O
I
O
O
G
O
+
H2O