Arlette Danelle Djitieu Deutchouaab,
Yannick Ngueumaleua,
Rossel Wendji Liendjia,
Sarrah Sonita Poungoue Hangaa,
Bruno Boniface Ngueloa,
Gustave Kenne Dedzo*a and
Emmanuel Ngamenia
aLaboratory of Analytical Chemistry, Faculty of Sciences, University of Yaoundé I, B. P. 812, Yaoundé, Cameroon. E-mail: kennegusto@yahoo.fr
bDepartment of Processing and Quality Control of Aquatic Products, Institute of Fisheries and Aquatic Sciences at Yabassi, University of Douala, B. P. 7236, Douala, Cameroon
First published on 3rd January 2024
2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a stable organic free radical widely used in various fields as a model free radical. There is scarce information about the stability of this compound in the chemical environments in which it is used. Side reactions between DPPH and other species can alter the precision of experiments that use DPPH, such as the evaluation of antioxidant properties amongst others. Following recent investigations highlighting reactions between DPPH and metal cations or Lewis acids, a quantitative reaction between DPPH and Fe3+ in acetonitrile was studied in the present work. UV-Vis spectroscopy and electrochemistry were used to monitor the reaction. The results obtained indicate a fast and multistep reaction between Fe3+ and DPPH that can be simplified as a simple redox reaction with the formation of Fe2+ and DPPH+. The reaction mechanism proposed follows complex steps involving two competing phenomena: a disproportionation which accelerates the reaction and an oxidation process that slows it down through DPPH regeneration.
For a typical electrochemical experiment, the initial concentration of DPPH in the electrolytic solution was set at 0.1 mM and controlled amounts of Fe3+ were added to the solution using stock solutions prepared in acetonitrile. The signals were recorded at the stationary electrode by cyclic voltammetry at 50 mV s−1 or by linear sweep voltammetry at 50 mV s−1 on a rotating electrode (900 rpm).
(1) |
Fig. 1 UV-Vis spectra of 0.1 mM DPPH in 0.03 M TBAHFP acetonitrile solution, in the presence of controlled amounts of Fe(ClO4)3 (0 mM, 0.02 mM, 0.05 mM, and 0.10 mM). |
This result indicates a quantitative chemical reaction between Fe3+ and DPPH in acetonitrile. A similar result was recently obtained while reacting DPPH with Cu2+.12 Fig. S1† presents a series of UV-Vis spectra of DPPH recorded in the presence of increasing amounts of Cu2+ and demonstrates that Cu2+ can oxidize DPPH to yield DPPH+ with subsequent formation of Cu+. It thus seems that Fe3+ reacts similarly with DPPH in acetonitrile. However, the plot of the residual percentage of DPPH (determined from the intensity of the band at 519 nm) as a function of the amount of Fe3+ added followed a different trend from the linear one obtained with Cu2+ (see the inset of Fig. 1).
For Fe3+, a fast and linear decrease in the concentration range 0 mM to 0.02 mM was followed by a gradual less important decrease of the DPPH percentage at higher concentrations of Fe3+. The slope of the linear section (−3051 ± 77% mM−1) was approximately 3 times greater than that obtained in the presence of Cu2+ (−1097 ± 9% mM−1). This indicates that the reactivity of DPPH in acetonitrile in the presence of Cu2+ or Fe3+ does not follow the same mechanism.
The electrochemical behavior of DPPH in the presence of Fe3+ was studied in order to better understand the mechanism involved during the reaction between these two chemical species. The electrochemical signal of DPPH in acetonitrile, obtained using cyclic voltammetry at a stationary electrode, is characterized by two fast and reversible monoelectronic systems (Fig. 2). These systems were assigned to the reversible reduction of DPPH to DPPH− (EPC = 0.017 V and EPA = 0.095 V) and the reversible oxidation of DPPH to DPPH+ (EPA = 0.640 V and EPC = 0.560 V).16–18
Fig. 2 also depicts the DPPH signal at a rotating electrode, showing two well-defined waves (oxidation at E1/2 = 0.60 V and reduction at E1/2 = 0.05 V) confirming the oxidizing and reducing properties of the DPPH radical.
The signals recorded on a rotating glassy carbon electrode in the presence of controlled amounts of Fe3+ in 0.1 mM DPPH solution (Fig. 3) reveal several findings.
Firstly, the presence of Fe3+ was followed by a significant decrease of the reduction current (at EC1/2 = 0.05 V) and the DPPH oxidation current (at EA1/2 = 0.60 V). The formation of a DPPH+ reduction wave (at EC1/2 = 0.56 V) that increases in intensity with the concentration of Fe3+ was also observed.
Secondly, the presence of an oxidation wave at EA1/2 = 0.95 V that increases in intensity with the concentration of the metal cation was seen. This signal was similar to the oxidation wave of reduced DPPH (DPPH-H).12,16
Thirdly, a new reduction wave at 0.2 V with an intensity that increased with the concentration of Fe3+ added to the solution was observed. A similar signal was recently observed in acetonitrile during the electrochemical reduction of DPPH in the presence of Zn2+. This signal was assigned to the electrochemical formation of the compound Zn(DPPH)2.12
The cyclic voltammograms recorded under identical experimental conditions, but at a stationary electrode (see ESI, Fig. S2†), confirmed these observations.
It thus appears that the presence of Fe3+ in a DPPH solution results in the formation of DPPH+ and a new reducing compound analogous to DPPH-H. A similar result was reported by Nakanishi et al. (2014) during the reaction between DPPH and Sc3+ (disproportionation of DPPH to yield DPPH+ and Sc(DPPH)2+).13 More recently, the disproportionation of DPPH in acetonitrile in the presence of protons (to yield DPPH+ and DPPH-H) was also reported.12 Thus, the acidity of Fe3+ promotes the disproportionation of DPPH with the formation of DPPH+ and DPPH−. DPPH−, being a Lewis base, would react with Fe3+ to form a compound with structure [Fe(DPPH)n]3−n, analogous to Sc(DPPH)2+, Zn(DPPH)2 or DPPH-H according to eqn (2).
Fe3+ + nDPPH− → [Fe(DPPH)n]3−n | (2) |
Thus, DPPH disproportionation is expected to occur in the presence of Fe3+ following eqn (3).
Fe3+ + 2nDPPH → nDPPH+ + [Fe(DPPH)n]3−n | (3) |
However, the disproportionation reaction proposed in eqn (3) does not take into account the experimental data reported (variation of the absorbance at 519 nm as a function of the concentration of Fe3+ added to the solution in the inset of Fig. 1). According to eqn (3), a linear decrease was expected with a slope depending on the stoichiometry of the reaction (i.e. the experimental value of n). Moreover, considering that the value of n should be greater than or equal to 1, the DPPH percentage should be zero for n = 1, when [Fe3+] = 1/2 [DPPH], as was recently observed in acetonitrile, in the presence of HClO4.12 The experimental data show (inset of Fig. 1) that the DPPH percentage decay was not linear and the consumption of the radical compound was complete when [Fe3+] = [DPPH], suggesting an equimolar reaction. Consequently, the reaction between DPPH and Fe3+ certainly proceeds via a complex mechanism.
This could be explained by considering two main hypotheses: (i) the disproportionation of DPPH in the presence of Fe3+ is more favorable compared to a potential classical oxidation reaction as observed in the presence of Cu2+.12 This hypothesis is supported by the high acidity of Fe3+;14,15 (ii) the oxidizing properties of Fe3+ are sufficient to promote the oxidation of the reduced DPPH (DPPH− is produced during the disproportionation) to DPPH (even if bound to Fe3+), according to eqn (4).
[Fe(DPPH)n]3−n → DPPH + [Fe(DPPH)n−1]3−n | (4) |
Therefore, the reaction between Fe3+ and DPPH can be simplified by the balance of eqn (3) and (4) (eqn (5)).
Fe3+ + (2n−1) DPPH → nDPPH+ + [Fe(DPPH)n−1]3−n | (5) |
In order to identify the reaction that better reflects these experimental observations, the theoretical variations of DPPH percentages in solution were calculated based on the stoichiometry of eqn (5), as a function of the concentrations of Fe3+ added during the measurements. The values of n explored were limited to 1, 2 and 3. Fig. 4 presents the expected variations of the theoretical DPPH percentages in solution as a function of the concentration of Fe3+ added.
As expected, the decrease of the DPPH percentages becomes increasingly fast at higher values of n (slopes of −1000% mM−1, −3000% mM−1 and −5000% mM−1 when the values of n were 1, 2 and 3, respectively). For Fe3+ concentrations in the range 0 mM to 0.02 mM, the experimental data overlapped almost perfectly with the case of n = 2 (slope of −3051 ± 77% mM−1 instead of −3000% mM−1). This is evidence that the reaction between DPPH and Fe3+ in this domain proceeds mainly via disproportionation (4 moles of DPPH for 1 mole of Fe3+) (eqn (6)), followed by the oxidation of reduced DPPH (DPPH−) by Fe3+ (eqn (7)). The combination of these two equations yields the overall reaction eqn (8).
4DPPH + Fe3+ → 2DPPH+ + Fe(DPPH)2+ | (6) |
Fe(DPPH)2+ → Fe(DPPH)+ + DPPH | (7) |
3DPPH + Fe3+ → 2DPPH+ + Fe(DPPH)+ | (8) |
For Fe3+ concentrations higher than 0.02 mM, there was a gradual decrease of the slope of the curve, reflecting a substantial modification of the reaction mechanism. This modification tends to increase the amount of Fe3+ needed to react with DPPH. This phenomenon can be rationalized by considering the oxidation of the compound Fe(DPPH)+ by Fe3+ according to eqn (9).
Fe(DPPH)+ + Fe3+ → DPPH + 2Fe2+ | (9) |
Three hypotheses support this explanation:
(i) The reaction of eqn (9) regenerates DPPH, which would perfectly explain the decrease of the slope of the experimental curve;
(ii) This reaction would be slower than the disproportionation reaction because the DPPH− bound to Fe2+ necessarily becomes less reducing (the electron being involved in the dative bond with the metal). This also explains the over voltage associated with the oxidation of compound Fe(DPPH)+ during the electrochemical measurements (Fig. 2);
(iii) The reaction of eqn (9) occurred quantitatively when Fe(DPPH)+ was found in appreciable amounts in solution, as indicated by the shape of the curve of Fig. 3. Disproportionation thus accelerates DPPH consumption, while the redox reaction between Fe3+ and reduced DPPH slows down the entire process. By combining eqn (8) and (9) that summarize the antagonistic processes, the chemical reaction between these two compounds is finally a simple oxidation of DPPH to DPPH+ by Fe3+ (eqn (10)).
DPPH + Fe3+ → DPPH+ + Fe2+ | (10) |
This equation is perfectly in agreement with the overall stoichiometry of the reaction corresponding to 1 mole of DPPH for 1 mole of Fe3+, as depicted in the inset of Fig. 1.
To confirm this mechanism, the presence of Fe2+ in the medium after reaction between Fe3+ and DPPH was checked using the 2,2′-bipyridine (BiPy) colorimetric test. Bipy can react with Fe2+ to form a dark red complex.19,20 An excess of BiPy was thus added to a mixture containing equimolar amounts of DPPH and Fe3+ (Fig. 5). The violet solution of 0.1 mM DPPH (Fig. 5(i)) became yellow after addition of 0.1 mM Fe3+, indicating the presence of DPPH+ (Fig. 5(ii)). The solution then turns from yellow to orange-red after adding an excess of BiPy (Fig. 5(iii)). This confirmed the formation of the complex between Fe2+ and BiPy.
Therefore, it is reasonable that the transformation presented in eqn (9) is the one ideally describing the reactivity of DPPH in the presence of Fe3+ in acetonitrile. However, it is the intermediate steps (disproportionation of DPPH and oxidation of DPPH− by Fe3+) that govern the rate of the reaction. In other words, during the reaction between DPPH and Fe3+, disproportionation ensures a fast transformation of DPPH, while DPPH− oxidation slows down the entire process by regenerating DPPH, as summarized in Scheme 1.
It appears that under certain conditions, free radicals can be oxidized by metal cations such as Fe3+. In the case of the reactions involved during the antioxidant properties evaluation, Fe3+ could be mistakenly perceived as an antioxidant. Thus, antioxidant extracts containing Fe3+ would erroneously present greater antioxidant properties when determined in acetonitrile.
Footnote |
† Electronic supplementary information (ESI) available: Experimental section; Fig. S1, UV-Vis spectra of DPPH solution in presence of controlled amounts of copper perchlorate and Fig. S2, cyclic voltammograms of DPPH in presence of controlled amounts of iron(III) perchlorate. See DOI: https://doi.org/10.1039/d3ra07106e |
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