CV was used to monitor the electrochemical properties of the three groups of phenolic compounds: hydroxycinnamic acids, benzoic acids, and flavonoids. Specifically, caffeic and ferulic acids (hydroxycinnamic acids), gallic, syringic and vanillic acids (benzoic acids) and catechin, epicatechin, quercetin 3-O-rutinoside and quercetin (flavonoids), were monitored.
First, the electrochemical conditions have been optimized in gallic and caffeic acids, catechin and quercetin 3-O-rutinoside. Subsequently, with the optimized conditions, the electrochemical behavior of nine individual model solutions and the electrochemical behavior of 36 binary model solutions (with two phenolic compounds) were measured.
3.1.1. Optimization of Electrochemical Conditions
First, conditions such as pH, concentration and scanning rate for cyclic voltammetry measurement were studied in four phenolic compounds (gallic and caffeic acids, catechin, and quercetin 3-O-rutinoside) for optimization.
Electrochemical measurements were taken at different pH (3.0 and 7.0), phenolic concentrations (1.0 and 0.5 g/L) and scanning rates (100 and 50 mV/s).
Table 1 shows the electrochemical parameters (E
pa, E
pc, I
pa, I
pc, E°, and ΔE) extracted from the cyclic voltammetry curves of the phenolic compounds. Different conditions show significant effects (
p < 0.05) among electrochemical behaviors. Specifically, pH differences in the solutions showed a significant effect on I
pa, E
pa, and E°, as well as differences in the scanning rate on I
pa and I
pc.
Gallic acid, adjusted to pH 3.0, showed two well-defined anodic peaks (E
pa1 = 0.414 V and E
pa2 = 0.786 V) and one cathodic peak (E
pc1 = 0.350 V). The last cathodic peak does not appear at pH 7.0 (
Figure 1a), indicating that at this pH, the oxidation product is not reduced on the glassy carbon electrode [
14].
Furthermore, the E
pa values at pH 7.0 are displaced with respect to pH 3.0. Yakovleva et al. (2007) [
15] observed that an increase in the pH of the electrolytic solution leads to a decrease in the E
pa value, caused by a decrease in the degree of antioxidant protonation and a change in the charge of the molecule to negative values. These pH differences in the electrolyte solutions also showed significant differences (
p < 0.05) on the other electrolyte parameters: E
pc, E°, and ΔE values of the first peak and E
pa values of the second peak.
Additionally, considering the gallic acid concentration (1.0 and 0.5 g/L) and the scanning rate (50 and 100 mV/s) for the measurement of cyclic voltammetry, no significant changes in E
pa and E
pc values were observed. The I
pa value increases with increasing gallic acid concentration and scanning rate, but this relationship is not linear when higher concentrations of phenolic are used [
1,
16].
The electrochemical behavior of caffeic acid at different pH, concentrations, and scanning rates is shown in
Table 1. As can be seen in
Figure 1b, the voltammogram corresponding to caffeic acid at different pH levels shows a single anodic peak with a corresponding cathodic peak, which is related to the mechanism of oxidation of catechol groups. Significant differences (
p < 0.05) were found in the potential values of the anodic and cathodic peaks (E
pa and E
pc) and the potential midway between the half peak potential (E°) according to the pH solution. The E
pa values depend on the pH of the electrolyte solution, which changes from 0.439 to 0.301 V at pH 3.0 and pH 7.0, respectively. These potential values are consistent with previous studies, considering the expected 59 mV per pH unit shift in the potential [
17].
The concentration of caffeic acid (1.0 and 0.5 g/L) did not show significant differences in the values of the electrochemical parameters studied. Regarding the scanning rate for the measurement of cyclic voltammetry (100 and 50 mV/s), significant differences (p < 0.05) are observed in Ipc, being higher at 100 mV/s.
The same results were obtained for catechin; the pH of the electrolyte solution changed significantly (
p < 0.05) in terms of electrochemical behavior, mainly concerning the values of the first anodic and cathodic peaks potentials (E
pa, and E
pc) and the potential midway between the half peak potential (E°) (
Table 1). The catechin voltammogram shows two main anodic peaks at 0.495 and 0.835 V and one cathodic peak at 0.261 V at pH 3.0, while at pH 7, these potentials are displaced at lower values (E
pa1 = 0.334 V, E
pa2 = 0.681 V, and E
pc1 = 0.107 V) (
Figure 1c). Regarding catechin concentration (1.0 and 0.5 g/L), only significant differences (
p < 0.05) were found in the ∆E values, providing information on the number of electrons transferred in a reversible redox reaction. The scanning rate (100 and 50 mV/s) for the measurement of caffeic acid did not show significant differences in the values of the electrochemical parameters.
The electrochemical behavior of quercetin 3-
O-rutinoside at different pH, concentrations, and scanning rates is shown in
Table 1. As can be seen in
Figure 1d, the voltammogram corresponding to quercetin 3-
O-rutinoside at different pH levels shows two anodic peaks and one cathodic peak. Significant differences (
p < 0.05) were found in the potential values of the first anodic and cathodic peaks (E
pa and E
pc) and the potential midway between the potential of the half peak (E°) according to the pH solution. In acid solutions, the potential value of the anodic and cathodic peak increases; therefore, at pH 3, the E
pa1 and E
pc1 values were 0.479 V and 0.431 V, while at pH 7, they were 0.277 and 0.212 V, respectively. The concentration of quercetin 3-
O-rutinoside and the measurement scan rate did not show significant differences in terms of the values of the electrochemical parameters studied.
From the results obtained, it is concluded that the Epa and Epc values are mainly modified by pH, whereas the concentration of the electrolyte solution and the scanning rate affect the Ipa and Ipc values. In this regard, the values of Epa and Epc depend on the nature of the electrochemical compounds (qualitative analysis), while those of Ipa and Ipc are measures of the concentration (quantitative analysis). Based on the results obtained, the measurement conditions selected were as follows: pH 3.0, 100 mV/s and 1.0 g/L.
3.1.2. Electrochemistry of Phenolic Compounds: Individual Model Solutions
The electrochemical behavior of nine phenolic compounds belonging to different phenolic groups has been evaluated under previously established conditions.
Table 2 shows the electrochemical parameters extracted from the voltammograms of the individual model solutions.
Gallic, syringic, and vanillic acids were selected as hydroxybenzoic acids. The voltammograms obtained for the three compounds present two anodic peaks and one cathodic peak. However, despite the structural similarity of these three phenolic compounds, the presence of methoxyl groups in the aromatic rings of syringic and vanillic acids makes their oxidation mechanism differ from that of gallic acid. For gallic acid, the first anodic peak and its corresponding cathodic peak are related to a characteristic reversible reaction of the –OH groups at positions 3 and 4 of the aromatic ring [
2]. For the other two acids, the first anodic peak and the cathodic peak are associated with the transfer of an electron, giving rise to the formation of the corresponding phenoxy radical. The second anodic peak is associated with a second transfer of an electron, giving rise to a carbocation that simultaneously generates the corresponding dioxobenzoic acid and a molecule of methanol via hydrolysis [
18,
19].
With respect to hydroxycinnamic acids, caffeic and ferulic acids were evaluated. The caffeic acid shows an anodic peak and a cathodic peak, which are related to the oxidation of the catechol group. However, the ferulic acid shows two anodic peaks and one cathodic peak. The first anodic peak obtained for ferulic acid is associated with the transfer of an electron to give rise to the corresponding phenoxy radical [
20]. The second anodic peak is associated with a second transfer of an electron that would give rise to a carbocation that, via hydrolysis, generates 3,4-dioxocinnamic acid and a molecule of methanol [
20].
Catechin and epicatechin show great similarity in electrochemical behavior due to their nearly identical chemical structures and the fact that they undergo the same oxidation mechanism (
Figure 2). The first oxidation peak appears at 0.495 V in both voltammograms and corresponds to the oxidation of the catechol group. The cathodic peak, corresponding to the reduction of the quinone group, appears at 0.261 V for catechin and 0.148 V for epicatechin. The second anodic peak appears at 0.835 V for catechin and 0.851 V for epicatechin and represents oxidation of the resorcinol group [
21]. However, some authors indicate that this anodic peak may correspond to the irreversible oxidation of the –OH group at position 3 of the non-aromatic ring [
14,
22].
The voltammograms of quercetin and quercetin 3-O-rutinoside show two anodic peaks and one cathodic peak. Both compounds undergo the same oxidation mechanism: The first anodic peak and its corresponding cathodic peak correspond to the reversible oxidation of the catechol group. The second anodic peak represents the oxidation of the resorcinol group.
E
pa values of the first peak in the anodic scan were used to classify the compounds according to their antioxidant potential. A compound with a lower E
pa value is a better antioxidant because it has a greater ability to donate electrons [
23]. Therefore, the E
pa1 order is as follows: gallic acid (0.414 V), caffeic acid (0.439 V), quercetin (0.439 V), syringic acid (0.447 V), ferulic acid (0.471 V), quercetin 3-
O-rutinoside (0.479 V), vanillic acid (0.487), catechin, and epicatechin (0.495 V). Taking this order into account, gallic acid is the most potent antioxidant. With respect to phenolic acids, gallic acid is a better antioxidant than syringic acid and vanillic acid. Among flavonols, quercetin shows a higher ability to donate electrons than quercetin 3-
O-rutinoside. Catechin and epicatechin present the same ability. Taking into account the second oxidation peak, the E
pa2 order is as follows: syringic acid (0.673 V), ferulic acid (0.754 V), and quercetin 3-
O-rutinoside (0.754), gallic acid (0.786 V), vanillic acid (0.835 V) and catechin (0.835 V), epicatechin (0.851 V) and quercetin (0.851 V). In this case, syringic acid undergoes the easiest oxidation [
23].
3.1.3. Electrochemistry of Phenolic Compounds: Binary Model Solutions
A total of 36 binary model solutions, with two phenolic compounds, were prepared to study the possible interaction between the oxidation processes of different compounds.
Table 2 shows the different combinations with their corresponding electrochemical parameters. As can be observed, all of the mixtures retained their two anodic peaks, but in general, the E
p values were different for a compound alone or mixed.
Taking into account the E
pa1 values, gallic acid was a better antioxidant alone than in combination; however, the other eight compounds are easier to oxidize when mixed with any other compound than alone (
Table 2).
Syringic, vanillic, caffeic, and ferulic acids, catechin, epicatechin, and quercetin 3-
O-rutinoside mixed with quercetin have an E
pa1 value lower than this when they are in individual solutions (
Table 2). In a previous study, ascorbic acid, caffeic acid, catechin, and hesperetin were better antioxidants in binary mixtures with quercetin [
23].
There are six mixed solutions in which the E
pa1 value is lower than the same for the two compounds in individual solutions: SYR-VAN (0.406 V), CAF-QUER (0.414 V), SYR-QUER (0.423 V), VAN-QUER (0.423 V), VAN-EPI (0.471 V), and EPI-RUT (0.471 V). Other studies indicated an oxidation potential lower for a mixture of quercetin and catechin than for both individual compounds [
23]; however, in our work, this does not occur for quercetin.
Figure 3 shows the voltammograms corresponding to the syringic and vanillic acids in individual and binary model solutions (SYR-VAN). As can be seen, the binary model solution retained its two anodic peaks corresponding to the syringic and vanillic acids measured individually, and with the E
p values lower than those of the individual model solution. Thus, in the potential zone between 0.5 and 1 V, the E
pa values were 0.673 and 0.835 V for the syringic and vanillic acids, respectively, while in the SYR-VAN solution, two peaks appear at 0.633 and 0.770 V. This fact indicates a higher antioxidant activity for the binary model solution.
A particular case is the mixed solution of caffeic acid and quercetin 3-O-rutinoside; here, the voltammogram does not show a second anodic peak related to quercetin 3-O-rutinoside. This could be due to the difference in Ipa values between these two compounds for the value of the corresponding potential (Epa2).