On the electrooxidation mechanism of quercetin glucosides at glassy carbon electrode

Journal of Electroanalytical Chemistry 640 (2010) 23–34

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On the electrooxidation mechanism of quercetin glucosides at glassy carbon electrode Danuta Zielinska a, Boguslaw Pierozynski a,*, Wieslaw Wiczkowski b a b

Department of Chemistry, Faculty of Environmental Management and Agriculture, University of Warmia and Mazury in Olsztyn, Plac Lodzki 4, 10-727 Olsztyn, Poland Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10 Street, P.O. Box 55, 10-747 Olsztyn, Poland

a r t i c l e

i n f o

Article history: Received 22 July 2009 Received in revised form 11 December 2009 Accepted 21 December 2009 Available online 4 January 2010 Keywords: Quercetin glucosides Electrooxidation mechanism Glassy carbon electrode UV–VIS spectroscopy HPLC–DAD–ESI–MS analysis Impedance spectroscopy

a b s t r a c t The present paper reports electrochemical (cyclic voltammetry and a.c. impedance) and spectroscopy (UV–VIS and HPLC–MS) studies of the process of adsorption and electrooxidation of two quercetin glucosides, namely: quercetin 3-O-b-glucoside (Q 3-glc) and quercetin 40 -O-b-glucoside (Q 40 -glc) at a glassy carbon electrode, in 0.1 M sodium acetate–acetic acid buffer in 90% methanol solution. The resulted information provided new insights into the mechanism of electrooxidation for two structurally different glucoside derivatives of quercetin, onto the surface of glassy carbon electrode. In addition, associated chargetransfer resistance (derived for all individual oxidation and electrosorption steps) and capacitance parameters are discussed and compared to those recently presented for quercetin aglycone. Ó 2010 Elsevier B.V. All rights reserved.

1. Introduction Flavonoids are polyphenolic compounds that are widely distributed in fruits, vegetables and beverages. Over 90% of flavonoids occur in various forms of glycosides [1]. The presence of a glycoside group significantly increases the polarity of a flavonoid molecule, which is an essential aspect in a storage system for the plant cells’ vacuoles. In recent years, extensive research activities have been initiated on biological activity of flavonoids which constitute an important part of human diet. It has been found that flavonoids could possibly act as protective agents against chronic diseases, yielding potential therapeutic applications [2–4]. For example, an anti-tumour promoting activity of flavonoids has been reported, in relation to their chemopreventive action in human carcinogenesis [5]. It is also commonly agreed that these beneficial effects of flavonoids originate from their antioxidant properties. These compounds are either hydrogen or electron-donating antioxidants, which are able to scavenge a reactive oxygen species [6,7]. Reducing properties of flavonoids are strongly enhanced by the presence of multiple hydroxyl groups (attached to aromatic rings of molecules), as well as by their ability to effectively delocalize the resulting phenoxyl radical species [8].

* Corresponding author. Tel.: +48 89 523 4177; fax: +48 89 523 4801. E-mail address: [email protected] (B. Pierozynski). 1572-6657/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2009.12.021

The structure–activity relationship (SAR) studies of flavonoids [9–13] have shown that the existence of an o-dihydroxyl group in ring denoted as B (see structure 1 in Fig. 1 below) and the presence of a 2,3-double-bond (in conjunction with a 4-oxo function in ring C) are essential for an effective, free radical scavenging activity to occur. In addition, a similar role is assigned to a 3-hydroxyl group present in the heterocyclic ring C. On the other hand, additional hydroxyl groups (present at positions 5 and 7 in ring A – see Fig. 1 again) appear to be less important when an antioxidant activity of the molecule is concerned. Nonetheless, all these structural features significantly contribute to the increasing stability of an aroxyl radical, i.e. to the antioxidant activity of the flavonol skeleton. Flavonols constitute a key group of flavonoids, which predominantly occur as various glycosides (mainly: glucosides, galactosides, rhammosides and rutinosides) [14]. Quercetin, one of the most abundant flavonols of human diet, has hydroxyl groups at: 3, 30 , 40 , 5, and 7 positions (see structure 1 in Fig. 1 below). High concentrations of quercetin can be found in onion, where it is present in four predominant forms, namely: quercetin aglycone (no sugar moieties are attached to the molecule), quercetin 3,40 -di-O-bglucoside, quercetin 3-O-b-glucoside, quercetin 40 -O-b-glucoside and isorhamnetin 40 -glucoside. However, several other forms of diglycoside and monoglycoside conjugates have been reported to exist in onion in small amounts, as well [15]. Interestingly, quercetin itself [16,17] was found to be a much more efficient chainbreaking antioxidant than its monoglucoside derivatives.

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D. Zielinska et al. / Journal of Electroanalytical Chemistry 640 (2010) 23–34

Fig. 1. Molecular structures of: 1-quercetin, 2-quercetin 3-O-b-glucopyranoside (Q 3-glc) and 3-quercetin 40 -O-b-glucopyranoside (Q 40 -glc).

Recently, a number of quercetin oxidation products have been reported, based on the results of chemical [18,19], enzymatic [20], radical [7,21] and electrochemical [22–25] oxidation studies of quercetin. Although these studies provide a direct evidence as to what active sites on the molecule possess an antioxidant activity, the exact mechanism of quercetin oxidation (resulting in a wide range of products) still appears ambiguous [25]. So far, limited information on the electrochemical behaviour of quercetin glucosides at glassy carbon (GC) electrode can be found in available literature [26,27], where likely the closest attention was given by researchers to rutin (3’,4’,5,7-tetrahydroxyflavone3-b-D-rutinoside) in Refs. [28] through [30]. Additionally, it has also been shown [24,29] that both quercetin and rutin, as well as their electrooxidation products could become strongly adsorbed on the surface of the glassy carbon electrode. Consequently, it appears that an adsorption intermediate step is likely to play a significant role in the kinetics and the mechanism of the process of electrooxidation of quercetin and its glycosides on the GC surface. The aim of this work was to elucidate the mechanism of electrochemical oxidation for two structurally-different quercetin glucosides, namely quercetin 3-O-b-glucoside (Q 3-glc) and quercetin 40 -O-b-glucoside (Q 40 -glc) at the glassy carbon electrode surface. This work utilizes a combination of electrochemical techniques (cyclic voltammetry and a.c. impedance spectroscopy) with carried-out spectral characterization of possible oxidation products, in order to gain new insights into electrochemical properties of these important, biologically active molecules. In addition, application of the a.c. impedance spectroscopy method has allowed us to derive the kinetic parameters of successive Faradaic oxidation steps for these two glucosides and to compare these parameters with those recently reported for quercetin aglycone in Ref. [24].

2. Experimental 2.1. Solutions and chemical reagents Quercetin 3-O-b-glucoside and quercetin 40 -O-b-glucoside chemicals were from Extra-synthese (Genay, France). An HPLC grade methanol (Merck KGaA, Darmstadt, Germany) and analytical grade sodium acetate, and acetic acid (POCH – Polish Chemical

Compounds, Poland) chemicals were used. For the chromatographic analysis, an HPLC grade acetonitrile (also from Merck KGaA) and trifluoroacetic acid (Sigma–Aldrich, Germany) were also used. All solutions were prepared using high quality water from the Millipore Direct-Q3 UV ultra-pure water purification system which supplies water of superior quality (18.2 MX cm resistivity). Stock, 1  103 M solutions of flavonoid compounds were prepared in the HPLC grade methanol. For all experiments, a supporting electrolyte was 0.1 M sodium acetate – acetic acid buffer in 90% CH3OH, and the concentration of both quercetin derivatives in the supporting electrolyte was 0.25  103 M for all CV and impedance measurements. The experiments were carried-out at two pH values: 5.0 and 7.5. All electrolysis experiments were performed at pH 7.5, where initial concentrations of glucosides were 5.0  105 M.

2.2. Equipment and experimental methodology 2.2.1. Cyclic voltammetry and a.c. impedance measurements A micro-electrochemical cell (with a total electrolyte volume of 0.2 mL), made all of Teflon, was used during the course of this work. The cell comprised three electrodes: a glassy carbon working electrode (BAS MF-2012, 3 mm diameter), an Ag/AgCl (3.5 M KCl) reference (RE) and a Pt (0.5 mm diameter coiled Pt wire) counter electrode (CE). Prior to each experiment, the cell was taken apart, rinsed with Millipore ultra-pure water and methanol. The GC working electrode was carefully hand-polished before each experiment with 0.05 lm alumina-water paste (BAS CF-1050), using BAS (MF-1040) polishing cloth. Then, the electrode was carefully rinsed with ultra-pure water and finally with methanol. The cyclic voltammetry experiments were performed at a potential sweep-rate of 50 mV s1. All a.c. impedance and cyclic voltammetry measurements were conducted at room temperature by means of the Solartron 12608 W full electrochemical system, consisting of 1260 frequency response analyzer (FRA) and 1287 electrochemical interface (EI). For impedance measurements, the generator provided an output signal of known amplitude (5 mV) and the frequency range was typically swept between 1.0  105 and 5.0  102 Hz. All potentiostatic impedance measurements were preceded by

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one-minute potential holds at the respective potential values. Each impedance frequency scan was followed by recording a full CV profile and a complete electrode cleaning procedure (as described above). The instruments were controlled by ZPlot 2.9 or Corrware 2.9 software for Windows (Scribner Associates, Inc.). Presented here results were obtained through analysis of representative series of experimental data (3–5 impedance measurements were usually carried-out at each potential value). Data analysis was

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performed with ZView 2.9 or Corrview 2.9 software package, where the impedance spectra were fitted by means of a complex, non-linear, least-squares immitance fitting program, LEVM 6 (written by J.R. Macdonald in Ref. [31]. 2.2.2. UV–visible spectral analysis UV–VIS absorption spectra were recorded for initial and preelectrolyzed (at the constant potential of 0.55 V and 1.00 V),

Fig. 2. (a) Cyclic voltammograms for glassy carbon electrode in 0.1 M acetate–acetic acid buffer solution (in 90% methanol), in the presence of 0.25  103 M Q 3-glc, at pH 5.0. The numbers 1 through 5 shown within the graphs correspond to the order of the recorded scans. The arrows on the first run indicate the potentials for which the impedance spectroscopy measurements were performed and (b) As in Fig. 2a, but for pH 7.5.

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5  105 M solutions of Q 3-glc and Q 4’-glc species, at pH 7.5. The process of electrolysis was carried-out in a simple, beaker-type (20 mL) cell, where a large surface-area (ca. 300 cm2) carbon fibre (CF) electrode (Hexcel 12 K AS4C fibre tow [32]) was used as a working electrode. The progress of electrolysis was controlled by recording the spectra after: 30, 60, 120, 180 and 240 min following the electrolysis start up. The UV-160 IPC spectrophotometer, with CPS Controller (Shimadzu, Japan) was employed to perform the UV–visible spectrophotometric measurements.

2.2.3. HPLC–DAD–ESI–MS analysis Chromatographic separation was performed before and after the electrolysis by means of the Shimadzu HPLC–DAD–ESI–MS (Kyoto, Japan) system. The system consisted of the following elements: two LC-10 ADVP pumps, a DAD detector (SPD-M10 AVP) set at: 294, 360 and 367 nm, an MS detector (QP8000a), an autosampler set to 5 lL injection (SIL-10 ADVP), a column oven (CTO10 ASVP) and the system controller (SCL-10 AVP). The Cadenza CD-C18 column (3 lm, 2.0  250 mm) from Imtakt (Japan) was used to separate the flavonoids. All analyses were performed at 45 °C, with the flow rate of 0.2 mL/min, in a gradient system composed of two mobile phases: (A) water:trifluoroacetic acid (99.9:0.1; v/v) and (B) acetonitrile:trifluoroacetic acid (99.9:0.1; v/v). Gradients were as follow: 21–35–80–21–21% B, at the gradient time, tG = 0–25–45–49–60 min. All scan measurements were performed using a negative electrospray ionization (ESI–MS) mode in the quadrupole mass spectrometer, with the following settings: heater temperature of N2 gas set at 230 °C, a CDL voltage set at 50 V, a probe voltage set at 4.0 kV, a nebulizer gas (N2) flow of 4.0 L/min and the defragmentation voltage set at 45 V. The pseudomolecular ion ([M– H] = 463 m/z), related to Q 3-glc and Q 4’-glc molecules, was monitored during the process of electrolysis, based on the spectral maxima, taken at k1 = 360 and k2 = 367 nm, respectively. The same

system parameters were applied for the identification of in situ formed new compound(s).

3. Results and discussion 3.1. Electrooxidation of Q 3-glc at glassy carbon electrode by cyclic voltammetry The cyclic voltammetric behaviour of 0.25  103 M solution of Q 3-glc (see structure 2 in Fig. 1) at the GC electrode in 0.1 M acetate–acetic buffer (at pH 5.0 and pH 7.5) is presented in Fig. 2a and b, correspondingly. The reversible oxidation peak observed in Fig. 2a (denoted there as peak 1 and centred at Ep = 0.38 V) and the less reversible oxidation peak observed for pH 7.5 in Fig. 2b (centred at Ep = 0.24 V) correspond to electrooxidation of the 30 ,40 -dihydroxyl (catechol) group in ring B of the Q 3-glc molecule. This peak results from a two-electron, two-proton transfer reaction of the catechol moiety to form an o-quinone species (see product 1a in Scheme 1). An observed peak-to-peak potential separation (DEp) for the Q 3-glc molecule at pH 5.0 was estimated between 34 and 44 mV (see an inset to Fig. 2a), which was within a range for a quasi-reversible process. The above is in fairly good agreement with previous studies on the electrochemical, cyclic voltammetry behaviour of quercetin and rutin [22–24,30]. Please, note that the peak-current potential related to the reversible oxidation of the catechol group for Q 3-glc is shifted by ca. 70 mV towards more positive potentials (for pH 5.0) and by some 90 mV for pH 7.5, as compared to that recorded for quercetin in Ref. [24], under the same experimental conditions. This finding is also in good agreement with work of Volikakis and Efstathiou [33] who showed that electrochemical oxidation of flavonoids containing large number of free hydroxyl groups proceeded at significantly lower potential ranges than that for molecules having large substituents (e.g. carbohydrate moieties).

Scheme 1. The proposed mechanism for electrochemical oxidation of Q 3-glc molecule on the surface of GC electrode.

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When the potential range for cycling is limited to the first oxidation peak (see insets to Fig. 2a and b), the reduction peak for 30 ,40 -o-quinone (denoted in the insets and in Fig. 2a as peak 10 ) appears at ca. Ep0 = 0.35 V (at pH 5.0) and at ca. Ep0 = 0.20 V, at pH 7.5. Thus, the 30 ,40 -o-quinone species becomes reversibly reduced on the GC surface to form surface-adsorbed quercetin (see Scheme 1, reduction steps: 1a–1 and 1b–1c). On the other hand, when the potential range for cycling is extended to ca. 1.2 V, the reversibility of the low potential oxidation process becomes significantly hindered at pH 7.5 (see Fig. 2b).

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Another oxidation peak (denoted as peak 2 in Fig. 2a and b) and centred at Ep = 1.06 V (at pH 5.0), and at ca. Ep = 0.96 V for pH 7.5 corresponds to an irreversible redox reaction, involving the 5,7dihydroxyl group at ring A of the Q 3-glc molecule (see product 1b in Scheme 1). A similar, irreversible redox reaction was described by Ghica and Brett in Ref. [30], for the process of oxidation of rutin. The above oxidation phase (see reaction step 1a to 1b in Scheme 1) is a one-electron, one-proton charge-transfer reaction. It is strongly believed [34] that the electron-donating ability of the resorcinol moiety OH groups (5-OH and 7-OH groups in ring

Fig. 3. (a) Cyclic voltammograms for glassy carbon electrode in 0.1 M acetate–acetic acid buffer solution (in 90% methanol), in the presence of 0.25  103 M Q 40 -glc, at pH 5.0 (other details as in Fig. 2a) and (b) As in Fig. 3a, but for pH 7.5.

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Scheme 2. The proposed mechanism for electrochemical oxidation of Q 40 -glc molecule on the surface of GC electrode.

A) strongly depends on their mutual acid/base properties. Based on the derived pKa values for various poly hydroxyflavonols in Ref. [34], it is the 7-OH group that has significantly stronger electron donating properties (pKa(5-OH) » pKa(7-OH)).

3.2. Electrooxidation of Q 40 -glc at glassy carbon electrode by cyclic voltammetry The Q 40 -glc molecule (see structure 3 in Fig. 1) is structurally different from the Q 3-glc molecule, as its 40 -OH group in ring B is substituted by the glucose moiety (otherwise conjugated at the 3-OH position for Q 3-glc). The cyclic voltammograms for Q 40 glc on the GC electrode, present at 0.25  103 M in 0.1 M acetate–acetic buffer, at pH 5.0 and 7.5 are shown in Fig. 3a and b, respectively. The main difference in the cyclic voltammetry behaviour of Q 40 -glc and Q 3-glc species is the presence of an additional oxidation peak, which appears in the CVs of the former glucoside at an intermediate potential range (see peak 2 in Fig. 3a and b). Thus, the first irreversible oxidation peak, centred at Ep = 0.47 V (for pH 5.0) and at Ep = 0.34 V for pH 7.5 (see peak 1 in Fig. 3a and b, and in insets to these figures) corresponds to electrooxidation of the 30 -OH group (ring B) of the Q 40 -glc molecule (see product 2a in Scheme 2). It can be observed in Fig. 3a and b that the peak-current potential values for peak 1 have significantly shifted to more positive potentials, as compared to the case of the Q 3-glc molecule, recorded in Fig. 2a and b. The above can be explained in terms of increasing difficulty upon oxidation of the 30 -OH group brought about by the presence of the glucose moiety, located at the 40 position [33]. The second oxidation peak (denoted as peak 2 in Fig. 3a and b) is centred at ca. Ep = 0.80 V and at 0.70 V, for pH 5.0 and 7.5, correspondingly. This peak is related to oxidation of the 3-OH group attached to the ring C (see products 2b and 2c in Scheme 2). Again, the recorded peak-current potential values for peak 2 (for both pHs) were considerably higher (by some 200–250 mV) than those recently obtained for quercetin aglycone (see Fig. 2a and b in Ref. [24]).

The third oxidation peak (denoted as peak 3 in Fig. 3a and b) is centred at ca. Ep = 1.08 V and at 0.98 V, for pH 5.0 and 7.5, respectively. This peak corresponds to the process of electrooxidation of the resorcinol moiety of ring A (see product 2e in Scheme 2). Interestingly, the peak-current potentials for peak 3, recorded for Q 40 glc at pH 5.0 and 7.5, compare very-well with the voltammetric locations of peak 2 for Q 3-glc in Fig. 2a and b. Similar potential range (ca. 0.90–1.20 V vs. Ag/AgCl) was reported as characteristic for the process of oxidation of 5,7-dihydroxyflavones by many other authors, including Ghica and Brett [30], and Hendrickson et al. [35]. It has to be stated however that for potentials more po-

Fig. 4. Complex-plane impedance plots for the process of electrooxidation of Q 3glc on GC electrode in contact with 0.1 M sodium acetate - acetic acid buffer in 90% CH3OH solution, recorded for pH 5.0 at 293 K, for the stated potential values. The solid lines correspond to representation of the data according to the equivalent circuits shown in Figs. 4a and c of Ref. [24].

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Table 2 Resistance and capacitance parameters for the process of electrochemical reactivity of Q 3-glc and Q 40 -glc quercetin derivatives at glassy carbon electrode, obtained by finding the equivalent circuits which best fitted the impedance data, as recently shown in Ref. [24] (Fig. 4cc in Ref. [24]). The electrolyte was 0.1 M CH3COONa/ CH3COOH10.1016/j.jelechem.2009.12.021 acetate buffer solution in 90% CH3OH (at pH 7.5) and the concentration of both quercetin derivatives was 0.25  103 M. Q 3-glc E (mV)

RF1 (X cm2)

200a 260a 300c RF3 800c 1000c 1100c

0

Fig. 5. As in Fig. 4 but for Q 4 -glc (the result obtained at 475 mV involves the charge-transfer step coupled with diffusion of Q 40 -glc species to the GC surface).

sitive than ca. 1.20 V additional Faradaic processes could also proceed on the GC electrode (also see rising of the background current, observed in Figs. 2a and b and 3a and 3b for potentials Pca. 1.00 V). These processes are primarily concerned with electrooxidation of the GC surface, which finally (at even more positive potentials) leads to anodic bulk evolution of oxygen.

3.3. Impedance characterization of the process of electrooxidation of Q 3-glc and Q 40 -glc molecules at glassy carbon electrode In this work, a.c. impedance experiments were carried-out in order to evaluate the mechanism of electrooxidation processes for two quercetin derivatives (Q 3-glc and Q 40 -glc molecules) that take place on the surface of glassy carbon electrode. In addition, an important aspect of this work was to compare obtained

106 Cdl (F cm2 su1)

494 ± 42 81.9 ± 5.3 6732 ± 525 (X cm2) 5595 ± 178 3090 ± 51 2668 ± 104

106 Cdl

u

79.3 ± 4.1 91.9 ± 1.0 114.5 ± 3.1 (F cm2 su1 1 ) 45.8 ± 1.0 53.3 ± 0.8 51.9 ± 1.6

0.750 ± 0.007 0.836 ± 0.007 0.777 ± 0.005 u1 0.841 ± 0.004 0.821 ± 0.002 0.886 ± 0.005

Q 40 -glc E (mV) 250c 300c 350c 400c

RF1 (X cm2) 14,755 ± 384 4698 ± 66 2592 ± 59 7982 ± 386

106 Cdl (F cm2 su1) 49.9 ± 0.6 40.7 ± 0.7 36.3 ± 1.0 44.2 ± 1.7

u 0.846 ± 0.002 0.859 ± 0.003 0.871 ± 0.004 0.829 ± 0.006

900c 980c 1050c 1150c

RF3 (X cm2) 2983 ± 66 2180 ± 55 2833 ± 91 2084 ± 42

106 Cdl (F cm2 su1 1 ) 34.8 ± 1.0 46.4 ± 1.3 58.1 ± 1.8 64.2 ± 1.6

u1 0.858 ± 0.005 0.840 ± 0.004 0.830 ± 0.005 0.852 ± 0.004

a As circuit ‘c’, but coupled with diffusion of the Q 3-glc species to the GC electrode surface.

impedance results with those recently published on the impedance behaviour of quercetin aglycone at GC electrode in Ref. [24]. It should be stressed here that a comprehensive literature search conducted by the authors of this work has revealed only two relevant papers (see work by He et al. in Ref. [36] and that by Mulazimoglu et al. in Ref. [37]) that involved application of the electrochemical impedance spectroscopy technique in studies of electrochemical behaviour of quercetin on GC or GC-wax electrode. Nevertheless, discussion of the impedance results presented in the above-mentioned papers was not particularly comprehensive.

Table 1 Resistance and capacitance parameters for the process of electrochemical reactivity of Q 3-glc and Q 40 -glc quercetin derivatives at glassy carbon electrode, obtained by finding the equivalent circuits which best fitted the impedance data, as recently shown in Ref. [24] (Figs. 4aa and 4cc in Ref. [24]). The electrolyte was 0.1 M CH3COONa/CH3COOH acetate buffer solution in 90% CH3OH (at pH 5.0) and the concentration of both quercetin glucosides was 0.25  103 M. Q 3-glc E (mV) 420

a

1100c 1200c 1300c Q 40 -glc E/mV 400c 450c 475a 500c 810c 900c 1000c 1050c 1150c a

RF1 (X cm2)

106 Cdl (F cm2)

RAds (X cm2)

106 CAds (F cm2 su1)

u

23.4 ± 0.6 RF3 (X cm2) 2448 ± 56 1705 ± 33 1054 ± 20

15.9 ± 0.2 106 Cdl (F cm2 su1 1 ) 84.6 ± 1.4 70.6 ± 1.3 57.3 ± 1.2

1969 ± 47

468.1 ± 6.1

0.643 ± 0.004 u1 0.817 ± 0.003 0.863 ± 0.003 0.896 ± 0.003

RF1 (X cm2) 8024 ± 140 1279 ± 20 664 ± 31 383 ± 37 RF2 (X cm2) 4824 ± 143 6704 ± 214 RF3 (X cm2) 3496 ± 80 1982 ± 34 1294 ± 27

106 Cdl (F cm2 su1) 64.6 ± 0.9 56.4 ± 1.1 43.8 ± 1.1 44.0 ± 2.7 106 Cdl (F cm2 su1 1 ) 73.5 ± 1.7 77.1 ± 1.6 106 Cdl (F cm2 su2 1 ) 77.4 ± 1.6 78.9 ± 1.6 71.5 ± 2.0

As circuit ‘c’, but coupled with diffusion of the Q 40 -glc species to the GC electrode surface.

u 0.840 ± 0.003 0.866 ± 0.003 0.905 ± 0.004 0.884 ± 0.009 u1 0.847 ± 0.004 0.843 ± 0.004 u2 0.853 ± 0.004 0.868 ± 0.003 0.888 ± 0.004

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3.3.1. Behaviour at pH 5.0 The impedance behaviour of the process of electrooxidation of Q 3-glc and Q 40 -glc molecules at the GC electrode, in contact with 0.1 M sodium acetate–acetic acid buffer in 90% methanol solution (at pH 5.0), is shown in Figs. 4 and 5, respectively, and in Table 1 below. For the Q 3-glc molecule, the impedance spectrum exhibits two semicircles at the potential value of 420 mV, which is very close to that of the capacitive peak 1 in Fig. 2a. Thus, the smaller, high-frequency partial semicircle (see an inset to Fig. 4) corresponds to the Faradaic oxidation (parameter RF1 in Table 1) of the catechol OH groups in ring B. On the other hand, the part of a large-diameter semicircle, observed throughout the intermediate and low frequencies is related to the charge-transfer process (parameter RAds in Table 1), accompanying electrosorption of Q 3-glc on the GC electrode surface. The RF1 resistance recorded at 420 mV comes to 23.4 X cm2, which is about 8 times as large as that recently recorded value for quercetin in Ref. [24] (see Table 1 in this reference). The above is a clear illustration of a dramatic, detrimental effect that a single glucose moiety of a neighbouring ring imposes on the Faradaic oxidation process of the catechol dihydroxyl moiety. On the other hand, the adsorption resistance (RAds) and capacitance (CAds) parameters at 420 mV come to 1969 X cm2 and 468 F cm2 su1, respectively. It should be stressed here that the above was the only case when the molecule adsorption parameters were clearly discernible in the Nyquist impedance plots. Furthermore, the RF3 resistance parameter recorded in Table 1 corresponds to the process of electrooxidation of the resorcinol (dihydroxyl) group of ring A (see peak 2 in Fig. 2a and the impedance spectra recorded for potentials: 1100, 1200 and 1300 mV in Fig. 4). A capacitance dispersion effect (represented by distorted semicircles) can be observed in all Nyquist plots, reported in Figs. 4 and 5. Therefore, the constant phase element (CPE) equivalent circuit models [24] were used to characterize the electrochemical behaviour of Q 3-glc and Q 40 -glc species in this work. The CPE behaviour observed in the Nyquist plots is believed to be the effect of inhomogeneous distribution of current, caused by progressing surface non-uniformity [38,39] for the glassy carbon electrode (see also the values of dimensionless parameters u, u1 and u2 in Tables 1 and 2). The impedance behaviour of Q 40 -glc species (see Fig. 5 and Table 1) at pH 5.0 is characterized by a dramatic change in the

kinetics of electrooxidation of the catechol-assigned 30 -OH group. Thus, the charge-transfer resistance parameter (RF1 in Table 1) reaches its minimum value of 383 X cm2 at 500 mV, which potential is close to that of the capacitive peak 1 in Fig. 3a. This resistance is about 16 and 132 times as large as that recorded above for Q 3glc and for quercetin aglycone [24], correspondingly. Interestingly, this reaction step becomes diffusion-controlled, at the peak-current potential of 475 mV (see the impedance spectrum for 475 mV in Fig. 5 and the corresponding Bode impedance plot in Fig. 6). The resulting diffusional Warburg parameters, RD (diffusional resistance) and T (time constant) were derived and came to 8152 ± 423 X cm2 and 4.7 ± 0.6 s, respectively. The remaining two Faradaic oxidation steps (see peak 2 and peak 3 in Fig. 3a) are concerned with electrooxidation processes of the 3-hydroxyl group (see parameter RF2 in Table 1) in ring C and the resorcinol OH group (see parameter RF3 in Table 1) at ring A, correspondingly. In both cases, a single and distorted semicircle is observed in the Nyquist impedance plots (see an inset to Fig. 5). The kinetics of these processes are much slower than those for the initial electrooxidation step (compare values of RF2 and RF3 resistance parameters with those of RF1 in Table 1). On the other hand, both RF2 and RF3 charge-transfer resistance parameters compare fairly well with the corresponding resistance parameters reported for quercetin in Ref. [24]. The above indicates that the effect of the glucose moiety (located at the 40 position at ring B) on the process of electrooxidation of unconjugated OH groups at rings C and A is rather insignificant (contrast to the effect of the glucose moiety in Q 3-glc on the behaviour of its catechol dihydroxyl group). The double-layer capacitance (Cdl) parameter, measured over the studied potential ranges, fluctuated between ca. 45 and 85 F cm2 (su1). Furthermore, it has to be stressed that for potentials negative to the capacitive peak 1 in Figs. 2a and 3a, as well as for the potential range between this peak and peak 2 in Figs. 2a and 3a, the impedance behaviour for both Q 3-glc and Q 40 -glc species was purely capacitive (with some CPE action observed). 3.3.2. Behaviour at pH 7.5 The impedance behaviour of Q 3-glc at pH 7.5 is somewhat different from that observed at pH 5.0. Thus, the charge-transfer resistance (RF1) parameter reaches its minimum of ca. 82 X cm2 at 260 mV, again a potential very close to that of the capacitive

Fig. 6. Bode phase-angle plots for the process of electrooxidation of Q 3-glc and Q 40 -glc molecules on the GC electrode, for the cases where the charge-transfer step was coupled with diffusion of Q 3-glc and Q 40 -glc species to the GC surface (other details as in Fig. 4.

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peak 1 in Fig. 2b (see Table 2). This result is in line with that obtained for quercetin in Ref. [24], where the RF1 parameter for electrooxidation of the catechol OH goups (recorded at pH 7.5) is significantly larger than that derived at pH 5.0. The above is accompanied by a considerable shift of potential range for electrooxidation of the catechol dihydroxyl group towards the less positive potentials at pH 7.5. Please also note that the presence of the

31

glucose moiety for Q 3-glc has also a significant impact on the RF1 parameter, which is about 6.7 times as large as that recently recorded for quercetin aglycone [24], under the same experimental conditions. In addition, the process of Q 3-glc electrooxidation at the GC electrode becomes diffusion-controlled at potentials (200 and 260 mV) close to that of the peak-current potential – peak 1 in Fig. 2b (see also Fig. 6). The calculated diffusional Warburg

Fig. 7. (a) UV–VIS absorption spectra for 0.05  103 M Q 3-glc (in 0.1 M acetate–acetic acid buffer solution), subjected to continuous electrolysis at a large surface-area (ca. 300 cm2) carbon fibre electrode, under the constant potential of 0.55 V vs. Ag/AgCl, at pH 7.5. Spectral tracing was repeated at: 30, 60, 120, 180 and 240 min, following the electrolysis start up. The dashed line was recorded before electrolysis commenced (arrows were placed to indicate changes in the spectra). (b) HPLC–DAD chromatogram for 0.05  103 M Q 3-glc (in 0.1 M acetate–acetic acid buffer solution), recorded at 360 nm (upper line) and 294 nm (lower line) before electrolysis. (c) As in Fig. 7b, but after 240 min of electrolysis. (d) ESI–MS (electrospray ionization-selected ion monitoring) spectrum for the negative ion of Q 3-glc ([M–H] = 463 m/z), recorded before electrolysis. (e) As in Fig. 7d, but after 240 min of electrolysis.

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parameters, RD and T for the potential of 260 mV vs. Ag/AgCl came to 6101 ± 174 X cm2 and 35.3 ± 1.5 s, correspondingly. The behaviour of the RF3 charge-transfer parameter (in reference to electrooxidation of the 5,7-dihydroxyl group at ring A) follows that already discussed above for the acidic (pH 5.0) medium. For the Q 40 -glc species, the kinetics of the Faradaic oxidation of the 30 -OH group in ring B are dramatically slower at pH 7.5 (by ca. 7 times) than those recorded at pH 5.0 (compare the RF1 parameter for Q 40 -glc at 350 mV in Table 2 with the RF1 resistance recorded for Q 40 -glc at 500 mV in Table 1). However, it has to be noted that (again) a substantial voltammetric shift (by ca. 150 mV) is observed for peak 1 in Fig. 3b towards the lower potential range, as compared to that observed in Fig. 3a. On the other hand, values of the RF3 parameter (RF2 resistance was not discernible for the experiments carried-out for Q 40 -glc at pH 7.5) in Table 2 generally follow those recorded for this molecule at pH 5.0 (Table 1). Furthermore, similar fluctuation of the Cdl parameter, as well as the CPE behaviour in the Nyquist plots, were observed for both Q 3glc and Q 40 -glc quercetin glucosides at pH 7.5. The dimensionless u parameter varied between ca. 0.75 and 0.88 (see Table 2 for details). 3.4. Electrooxidation of quercetin glucosides studied by UV–VIS spectroscopy and HPLC–DAD–ESI–MS techniques In this study, the UV–VIS spectrophotometry and HPLC–DAD– ESI–MS techniques were employed to monitor the process of electrochemical oxidation of Q 3-glc and Q 40 -glc molecules (on large surface-area CF electrode), upon bulk electrolysis trials carriedout at pH 7.5. 3.4.1. Electrooxidation of Q 3-glc at CF electrode by UV–VIS spectroscopy and HPLC–DAD–ESI–MS techniques The UV–VIS spectral changes for the Q 3-glc molecule (recorded for 0.05  103 M Q 3-glc-based buffer solution at pH 7.5) are shown in Figs. 7a and 8 below. The absorption spectra were recorded before and during the electrolysis, at two different potentials, namely: 0.55 V (potential positive to that of the peak 1 in Fig. 2b; see Fig. 7a) and at 1.00 V vs. Ag/AgCl (see the peak 2 in Figs. 2b and 8). The HPLC–DAD–ESI–MS analyses were employed to monitor the consumption of the Q 3-glc species in solution and the formation of new product(s) during the process of continuous electrolysis. For a fresh, unelectrolyzed solution (see the dashed line in Fig. 7a), the absorption spectra for Q 3-glc exhibited two strong absorption bands, centred at: 360 nm (band I) and 256 nm (band II), as well as a very weak band at 294 nm (band III). The first two bands (band I and band II) are characteristic of the B–C ring conjugation (cinnamoyl system, band I) and the A–C ring conjugation (benzoyl system, band II). On the other hand, the band III (centred at 294 nm) is considered [28] to be indicative of the quinonic structure, as well as the keto-hydroxyl tautomerism between the ring positions denoted as 4 (ring C) and 7 (ring A) for the Q 3-glc molecule, leading to a quinonic resonance structure (as suggested by Timbola et al. for quercetin in Ref. [22]). The spectral analysis of Q 3-glc-based solution, electrolyzed at 0.55 V vs. Ag/AgCl, showed a significant intensity reduction and finally a disappearance of the band I from the spectra in Fig. 7a. At the same time, the band II revealed only a slight reduction in intensity, while practically no changes were observed for intensity of the band III (see again Fig. 7a). The HPLC–DAD–ESI–MS analysis, performed before and after the electrolysis, showed significant progress in the process of electrooxidation of Q 3-glc, which appeared almost complete after 4 h of continuous electrolysis. This conclusion is based on the calculation of the peak area at 360 nm, recorded before and after the electrolysis (see Figs. 7b

Fig. 8. UV–VIS absorption spectra for 0.05  103 M Q 3-glc (in 0.1 M acetate– acetic acid buffer solution), subjected to continuous electrolysis at a large surfacearea (ca. 300 cm2) carbon fibre electrode, under the constant potential of 1.00 V vs. Ag/AgCl, at pH 7.5 (other details as in Fig. 7a).

and c). The above is supported by monitoring the pseudomolecular ion ([M–H] = 463 m/z) disappearance, related to the Q 3-glc peak eluting at 10.5 min (see Figs. 7d and e below). The UV–VIS spectral changes for the Q 3-glc molecule, recorded at the potential of 1.00 V, are shown in Fig. 8. Here, similar changes were observed to those recorded for the potential of 0.55 V. Nevertheless, the intensity reduction observed for band II in Fig. 8 is dramatic, as compared to that observed in Fig. 7a. Based on the HPLC– DAD–ESI–MS analysis, the process of CF surface electrooxidation of Q 3-glc also came to completion after 4 h of uninterrupted electrolysis (the respective HPLC–DAD chromatograms and ESI–MS-spectra are not shown). An observed intensity reduction for band I at both applied potentials (0.55 and 1.00 V) is related to the progress of electrooxidation of the catechol dihydroxyl group at ring B of the Q 3-glc molecule (see product 1a in Scheme 1 again). Generally, the intensity of band II becomes strengthened by the presence of free hydroxyl group(s) at ring A. Thus, weakening of this band (as observed at the applied potential of 1.00 V in Fig. 8) confirms that the consecutive process involves oxidation of the 5- or/and 7-OH group(s) in ring A (see again peak 2 in Fig. 2b and product 1b in Scheme 1). The above was also confirmed for rutin in Ref. [30]. On the other hand, practically no changes in the intensity of band III, at the potential of 0.55 V (see Fig. 7a), were observed. The above is a result of conjugation between the 3-OH group and the glucose moiety in ring C of the Q 3-glc molecule. Thus, it can be concluded that the tautomeric forms (as quinone-methide species) cannot be formed from o-quinone in this case. The above phenomenon is in contrary to tautomerization of o-quinone from quercetin, process that is only being possible due to the presence of an unconjugated 3-OH group on the ring denoted as C [22]. 3.4.2. Electrooxidation of Q 40 -glc at CF electrode by UV–VIS spectroscopy and HPLC–DAD–ESI–MS techniques The UV–VIS spectral changes for the Q 40 -glc molecule (recorded for 0.05  103 M Q 40 -glc-based buffer solution at pH 7.5) are presented in Figs. 9a and 10. Again, the absorption spectra were recorded before and during the electrolysis, at two different potentials, namely: 0.55 V (potential just positive to that of the peak 1 in Fig. 3b; see Fig. 9a) and at 1.00 V (potential near the peak-current value for peak 3 in Fig. 3b; see Fig. 10. Again, for a fresh solution (see the dashed line in Fig. 9a), the absorption spectra for Q 40 -glc exhibited two strong absorption

D. Zielinska et al. / Journal of Electroanalytical Chemistry 640 (2010) 23–34

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Fig. 9. (a) UV–VIS absorption spectra for 0.05  103 M Q 40 -glc (in 0.1 M acetate–acetic acid buffer solution), subjected to continuous electrolysis at a large surface-area (ca. 300 cm2) carbon fibre electrode, under the constant potential of 0.55 V vs. Ag/AgCl, at pH 7.5 (other details as in Fig. 7a). (b) HPLC–DAD chromatogram for 0.05  103 M Q 40 glc (in 0.1 M acetate–acetic acid buffer solution), recorded at 367 nm (upper line) and 294 nm (lower line) before electrolysis. (c) As in Fig. 9b, but after 240 min after electrolysis. (d) ESI–MS (electrospray ionization-selected ion monitoring) spectrum for the negative ion of Q 40 -glc ([M–H] = 463 m/z) for the peak eluted at 14.5 min at 367 nm, recorded after 240 min of electrolysis. (e) As in Fig. 9d, but for the peak eluted at 7.3 min at 294 nm, recorded after 240 min of electrolysis.

bands, centred at: 367 nm (band I) and 253 nm (band II), and a somewhat weaker absorption band at 294 nm (band III). A continuous, dramatic intensity reduction for bands I and II can be observed in Fig. 9a. In contrast, the intensity of band III has significantly strengthened during the course of electrolysis. The appearance of two isosbestic points (point p1 at 312 and point p2 at 277 nm in Fig. 9a) confirms that only two absorbing species, namely: the substrate (Q 40 -glc) and the main oxidation product could be present in the thin solution layer, at the CF electrode surface. The hypochromic shift of the spectral maxima from 367 to

294 nm indicates that during the process of oxidation, the B–C ring conjugation in the Q 40 -glc molecule has significantly weakened. The presence of a new oxidation product was confirmed by recording the HPLC–DAD spectra at 367 and 294 nm, prior to and after completion of electrolysis in Figs. 9b and c, respectively. This conclusion becomes strongly supported by the recorded ESI–MS spectra of the peak eluting at 7.3 min (see Figs. 9d and e). Thus, as a result of Q 40 -glc electrolysis at the CF surface, the new (cyclic ether-type) compound, with a molecular mass of 494 ([M– H] = 493 m/z) has been formed (also, see the proposed chemical

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Fig. 10. UV–VIS absorption spectra for 0.05  103 M Q 40 -glc (in 0.1 M acetate– acetic acid buffer solution), subjected to continuous electrolysis at a large surfacearea (ca. 300 cm2) carbon fibre electrode, under the constant potential of 1.00 V vs. Ag/AgCl, at pH 7.5 (other details as in Fig. 7a).

moiety (5,7-dihydroxyl group) of ring A. All oxidation steps exhibited significant pH-dependence, which was most evident in the case of the catechol (or the 30 -OH) group. Thus, facilitated deprotonation of these hydroxyl groups at pH 7.5 resulted in considerable enhancement of the charge-transfer process, as compared to that in the acidic (pH 5.0) solution. Moreover, the presence of the glucopyranoside functional group at the 40 position in ring B led to dramatic increase of the reaction resistance parameter (by ca. 132 times at pH 5.0), as compared to that recently recorded for unmodified quercetin. Hence, it is evident that there is a strict and important relationship between the molecular structure of a flavonoid molecule and its oxidation properties. Finally, application of HPLC and MS techniques for the characterization of electrooxidation processes of quercein glucosides allowed us to help determine the main paths for electrooxidation of Q 3-glc and Q 40 -glc compounds at glassy carbon electrode along with identification of the main oxidation product (a cyclic ether denoted as product 2d in Scheme 2) upon electrolysis of Q 40 -glc-based, sodium acetate–acetic acid buffer solution. References

structure of this new compound – product 2d in Scheme 2). Consequently, the process of electrooxidation of Q 40 -glc is evidenced by progressive disappearance of the main substrate peak (eluting at 14.5 min) in Figs. 9b and c. The UV–VIS spectral changes for the process of Q 40 -glc oxidation, recorded at the potential of 1.00 V, are shown in Fig. 10. Again, similar changes in the intensity of bands I and II to those recorded at the potential of 0.55 V, were observed. In addition, only very weak changes in the intensity of band III were recorded (the HPLC–DAD chromatograms and ESI–MS-spectra are not shown). Furthermore, the analysis of the ESI–MS-spectra for a new peak recorded at 294 nm (eluting at 7.3 min) confirmed the presence of the same new compound (product 2d in Scheme 2), with the molecular mass of 494 (HPLC–DAD chromatograms and ESI–MSspectra are not shown). However, the new product was present in solution at significantly smaller extent, as compared to that recorded for the lower potential (0.55 V) case. An observed intensity reduction for band I at both applied potentials (0.55 and 1.00 V) is related to progressive oxidation of the 30 -OH group in ring B of the Q 40 -glc molecule. Furthermore, significant intensity weakening for band II confirms progressive changes in ring A, including subsequent oxidation of the resorcinol moiety at the potential of 1.00 V (see also peak 3 in Fig. 3b and product 2e in Scheme 2). In addition, an observed large intensity enhancement for band III at the applied potential of 0.55 V (see Fig. 9a) indicates that (again) the B–C ring conjugation in the Q 40 -glc molecule has significantly weakened, thus favouring the formation of a new product in the absence of the 2,3-double bond in the C ring. Formation of a similar oxidation product was also proposed for quercetin, studied at GC electrode in a hydro-alcoholic solution by Timbola et al. in Ref. [22]. 4. Conclusions Electrooxidation behaviour of Q 3-glc and Q 40 -glc molecules at the surface of glassy carbon electrode was examined by means of a.c. impedance spectroscopy, cyclic voltammetry, UV–VIS spectroscopy and HPLC–DAD–ESI–MS techniques. A multi-step surface electrooxidation process commences at the 30 ,40 -OH (or 30 -OH for Q 40 -glc) group(s) at ring B, which is then followed by oxidation of the 3-OH group (ring C) and the resorcinol

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