Reactivity of dpph in the oxidation of catechol and catechin

Article abstract of DOI:10.1002/kin.20542

A kinetic study of the reduction of pyrocatechol and catechin by dpph radical has been carried out in various ratios of CH ₃OH/H₂O mixed solvent at pH 5.5-7.5, μ = 0.10 M [(n-Bu)₄N]ClO₄, and T = 25°C. The rate constants of oxidation in aqueous solvent, k_H2O, were obtained from the extrapolation of the linear plots of the specific rate constants k vs. % H ₂O plots at each pH value. A linear relationship between k _H2O and 1/[H⁺] was observed for both flavonoids with k_H2O = k₁K_a1/[H⁺], where K _a1 was the first acid dissociation constant on the catechol ring and k₁ is the rate constant of the oxidation of the mononegative species HX^-. The values of k₁ obtained from the slopes of the plots are (8.2 ± 0.2) × - 10⁵ and (6.1 ± 0.1) × - 10⁵ M^-1 s^-1 for pyrocatechol and catechin, respectively. The analysis of the reaction on the basis of Marcus theory for an outer-sphere electron transfer reaction yielded a value of 3.7 × - 10³ M^-1 s^-1 for the self-exchange rate constant of dpph/dpphH couple.

Full text of DOI:10.1002/kin.20542

Reactivity of DPPH· in the
Oxidation of Catechol and
Catechin
WEI-LIN CHEN, WEI-SHUEN LI, PING-JOU FU, ANDREW YEH
Department of Chemistry, Tunghai Christian University, Taichung, Taiwan, ROC
Received 3 August 2010; revised 4 November 2010; accepted 4 November 2010
DOI 10.1002/kin.20542
Published online 20 January 2011 in Wiley Online Library (wileyonlinelibrary.com).
ABSTRACT: A kinetic study of the reduction of pyrocatechol and catechin by dpph• radical
has been carried out in various ratios of CH3OH/H2O mixed solvent at pH 5.5–7.5, μ = 0.10
M [(n-Bu)4N]ClO4, and T = 25◦C. The rate constants of oxidation in aqueous solvent, kH O
,
2
were obtained from the extrapolation of the linear plots of the specific rate constants k vs.
% H2O plots at each pH value. A linear relationship between kH O and 1/[H+] was observed
2
for both flavonoids with kH O = k1 Ka1/[H+], where Ka1 was the first acid dissociation con-
2
stant on the catechol ring and k1 is the rate constant of the oxidation of the mononegative
species HX−. The values of k1 obtained from the slopes of the plots are (8.2
and (6.1
0.2) × 105
0.1) × 105 M−1 s−1 for pyrocatechol and catechin, respectively. The analysis of
the reaction on the basis of Marcus theory for an outer-sphere electron transfer reaction
yielded a value of 3.7 × 103 M−1 s−1 for the self-exchange rate constant of dpph•/dpphH
couple. ꢀ 2011 Wiley Periodicals, Inc. Int J Chem Kinet 43: 147–153, 2011
C
activity involves a consecutive two-electron oxidation
of catechol ring (or B ring) of the flavonoids to form
the corresponding quinones [4,5]. Owing to the low
solubility of most of the flavonoids in water, stud-
ies of the redox chemistry of the flavonoids usually
are conducted in organic solvents or organic/aqueous
mixed solvents, and the dpph• (2,2ꢁ-diphenyl-1-picryl-
hydrazyl) is used as the oxidizing agent. The stable
free radical dpph• has been widely used as an oxidiz-
ing agent in organic solvent, particularly in the study of
the antioxidation properties of phenols with methanol
or ethanol as the most common solvent [6,7]. In the
present work, we reported the results of our kinetic
study of the oxidation of the catechin and the catechol
by the dpph• radical in various ratios of CH3OH/H2O
mixed solvent. The intrinsic reactivity of the dpph• in
the aqueous solution would be investigated on the ba-
sis of Marcus theory. The structures of the compounds
under study are shown in Scheme 1.
INTRODUCTION
Flavonoids are a group of naturally occurring polyphe-
nolic compounds, which are present ubiquitously in
plants and thus are part of our daily diet. The interest
in flavonoids has grown during the past decade because
many studies have demonstrated that these compounds
possess bioactive potential, which is beneficial to hu-
man health [1,2]. Owing to their polyphenolic nature,
their potential biological effects are thought to arise
from their antioxidation activity, particularly their abil-
ity to scavenge free radicals [3]. It has been proposed
previously that the mechanism of this antioxidation
Correspondence to: Andrew Yeh; e-mail: adyeh@thu.edu.tw.
Contract grant sponsor: National Science Council of the Repub-
lic of China.
Supporting Information is available in the online issue at
www.wileyonlinelibrary.com.
c
ꢀ
2011 Wiley Periodicals, Inc.

148
CHEN ET AL.
5'
B
2'
OH
OH
NO
6'
2
6
NO
2
8
OH
H
HO
O
C
5
4
O N
2
N N
O N
2
N N
A
6
OH
NO
NO
2
2
OH
OH
3
dpph
(oxidized form)
dpphH (reduced form)
( ) -catechin
+
Pyrocatechol
Scheme 1 Structures of pyrocatechol, catechin, dpph•, and dpphH.
EXPERIMENTAL
A total disappearance of the absorption was found up
to the 2:1 ([dpph•]/[flavonoids]) ratio titration, and the
λ = 515 nm absorption began to show up upon further
addition of dpph•, as shown in Fig. 2. The results indi-
cated an overall two-electron stoichiometric oxidation,
similar to that observed by oxidation with transition
metal complexes [9,10]. The similarity of behavior be-
tween pyrocatechol and catechin implied that the ox-
idation went to the catechol ring with the formation
of quinone products. The reaction therefore can be ex-
pressed as Eq. (1), where H2X and X are the flavonoids
and the corresponding quinone products.
Materials and Instrumentation
(+)-Catechin (Sigma, St. Louis, MO, USA), catechol
(Fluka Chemie GmbH, Buchs, Switzerland), and dpph•
(Aldrich, St. Louis, MO, USA) were purchased and
used without further purification. The doubly distilled
deionized water was obtained by passing houseline
distilled water through a Barnsted NANO water pu-
rification system. UV–vis spectra were measured on
a Hewlett–Packard HP 8453 diode-array spectropho-
tometer. Electrochemistry was performed on a PAR
273 A potentiostat/galvanostat system as described be-
fore [8].
2dpph• + H2X −→ 2dpphH + X
(1)
Kinetic Measurements
Kinetics of Oxidation
The kinetic studies for the oxidation of catechin and
catechol in CH3OH/H2O mixed solvent at various pH
were carried out on the Hi-Tech CH-61 stopped flow
spectrophotometer. The experiments were measured
under pseudo-first-order conditions at an ionic strength
of 0.10 M with the reductants in excess. The reac-
tions were monitored by following the disappearance
of dpph• at its band maximum (λ = 515 nm). The
pseudo-first-order rate constants were obtained from
Plots of |At – A | vs. time were linear for at least three
∞
half-lives under conditions where [H2X] ꢀ 10[dpph•].
The observed rate constants, kobs, were found to in-
crease linearly with the flavonoid concentration at con-
stant pH, as shown in Figs. 3 and 4. Therefore, at
constant pH, the rate law will be
d[dpph•]
−
= 2k[dpph•][H2X]
(2)
dt
the slopes of the linear least-square fits of ln|At − A |
∞
vs. time plots.
The second-order rate constants, k, were obtained
fromtheslopes oftheone-parameterlinearleast-square
fits of the kobs vs. [H2X] plot, and the results are pre-
sented in Tables I and II. The relationship of k vs.
% H2O is linear at all pH for both pyrocatechol and
catechin, as shown in Fig. 5.
RESULTS AND DISCUSSION
Stoichiometry of Oxidation
Owing to the high π-conjugation of the molecule, the
The intercepts represent the rate constants in the
dpph• exhibits a characteristic absorption at λmax
=
pure methanol (kMeOH), and the rate constants kH O in
2
515 nm (εmax = 1.16 × 104 M−1 cm−1), which is ab-
sent in the absorption spectra of the flavonoids and
the product solution of oxidation, as shown in Fig. 1.
Taking advantage of this strong absorption and the
decoloration of the solution upon oxidation, the stoi-
chiometry of the reactions was determined by the spec-
trophotometric titrations of the flavonoids with dpph•
by monitoring the spectral changes at this wavelength.
an aqueous solution can be calculated from the slopes
and the intercepts of the plots. The values are presented
in Table III.
Since it has been proved that the rate-determining
step for the oxidation of flavonoids involves the first
electron oxidation to form the semiquinone radical in-
termediate [9,10], the mechanism of the reaction based
on the stoichiometry and the kinetic results therefore
International Journal of Chemical Kinetics DOI 10.1002/kin

REACTIVITY OF DPPH• IN THE OXIDATION OF CATECHOL AND CATECHIN
149
2.5
2
1.5
1
a
b
0.5
0
d
c
200
300
400
500
600
700
800
Wavelength (nm)
Figure 1 Absorption spectra of flavonoids and dpph•: a, dpph•; b, pyrocatechol; c, catechin; and d, product solution after
oxidation.
1.0
(a)
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5
[dpph]/[pyrocatechol]
1.0
0.8
0.6
0.4
0.2
0.0
(b)
0
1
2
3
4
5
[dpph]/[(+)-catechin]
Figure 2 Stoichiometric plots for the titration of flavonoids by dpph•: (a) pyrocatechol and (b) catechin.
fast
can be proposed as shown in Eqs. (3)–(8), with the rate
law as shown in Eq. (9)
dpph• + H2X•+ −→ dpph•− + X + 2H+ (6)
fast
dpph• + HX• −→ dpph•− + X + H+
(7)
(8)
H2X ꢁ H+ + HX−
(3)
(4)
(5)
fast
4dpph•− + 4H+ −→ 4dpphH
k0
dpph• + H2X −→ dpph•− + H2X•+
k0[H+] + k1Ka1
kH O
=
(9)
k1
2
dpph• + HX− −→ dpph•− + HX•
[H+]
International Journal of Chemical Kinetics DOI 10.1002/kin

150
CHEN ET AL.
4
3
2
1
0
1.4
(a)
1.2
(b)
9:1
8:2
7:3
6:4
9:1
8:2
7:3
6:4
1
0.8
0.6
0.4
0.2
0
0
0.001
0.002
0.003
0
0.0005 0.001 0.0015 0.002 0.0025 0.003
[Pyrocatechol], M
[Pyrocatechol], M
12
9
40
30
20
10
0
(c)
(d)
9:1
8:2
7:3
6:4
9:1
8:2
7:3
6:4
6
3
0
0
0.001
0.002
0.003
0
0.001
0.002
0.003
[Pyrocatechol], M
[Pyrocatechol], M
Figure 3 kobs vs. [pyrocatechol] plots: pH (a) 6.00, (b) 6.50, (c) 7.00, (d) 7.51; μ = 0.10 M [(C4H9)4N]ClO4, and T = 25◦C.
According to this rate law, a linear relationship with
slope k1Ka and intercept k0 between k vs. 1/[H+] plots
is expected. However, nice linear plots of k vs. 1/[H+]
passing through origin were observed, as shown in
Fig. 6. The results imply that k1Ka1 ꢂ k0[H+], and
Eq. (9) can be reduced to Eq. (10)
k1Ka1
[H+]
kH O
=
(10)
2
0.8
6
5
4
3
2
1
0
(b)
(a)
0.6
0.4
0.2
9:1
9:1
8:2
7:3
6:4
8:2
7:3
6:4
0
0
0.0005
0.001
0.0015
0.002
0.0025
0
0.0005
0.001
0.0015
0.002
0.0025
[(+)-Catechin], M
[(+)-Catechin], M
20
15
10
5
80
(c)
(d)
60
40
20
0
9:1
8:2
7:3
6:4
9:1
8:2
7:3
6:4
0
0
0.0005
0.001
0.0015
0.002
0.0025
0
0.0005
0.001
0.0015
0.002
0.0025
[(+)-Catechin], M
[(+)-Catechin], M
Figure 4 kobs vs. [catechin] plots: pH (a) 5.48, (b) 6.48, (c) 7.01, (d) 7.48; μ = 0.10 M [(C4H9)4N]ClO4 and T = 25◦C.
International Journal of Chemical Kinetics DOI 10.1002/kin

REACTIVITY OF DPPH• IN THE OXIDATION OF CATECHOL AND CATECHIN
151
Table I Specific Rate Constants for the Oxidation of Pyrocatechol by dpph•at pH 6.0–7.5 in CH3OH/H2O Solventa
pH
CH3OH/H2O Ratio
k(M−1 s−1
)
pH
CH3OH/H2O Ratio
k (M−1 s−1
)
6.00
9:1
8:2
7:3
6:4
9:1
8:2
7:3
6:4
(7.60 0.09) × 101
(1.51 0.02) × 102
(2.21 0.02) × 102
(2.86 0.02) × 102
(1.76 0.02) × 102
(3.87 0.01) × 102
(5.25 0.02) × 102
(6.90 0.01) × 102
7.00
9:1
8:2
7:3
6:4
9:1
8:2
7:3
6:4
(6.45 0.09) × 102
(1.24 0.02) × 103
(1.73 0.02) × 103
(2.21 0.09) × 103
(3.42 0.05) × 103
(5.0 0.1) × 103
(6.0 0.2) × 103
(7.5 0.1) × 103
6.50
7.51
aμ = 0.10 M [(C4H9)4N]ClO4, T = 25◦C.
Table II Specific Rate Constants for the Oxidation of Catechin by dpph• at pH 5.5–7.5 in CH3OH/H2O Solventa
pH
CH3OH/H2O Ratio
k(M−1 s−1
)
pH
CH3OH/H2O Ratio
k (M−1 s−1
)
5.48
9:1
8:2
7:3
6:4
9:1
8:2
7:3
6:4
(5.25 0.02) × 101
(7.65 0.02) × 101
(1.14 0.02) × 102
(1.63 0.05) × 102
(3.66 0.07) × 102
(6.15 0.02) × 102
(8.65 0.02) × 102
(1.26 0.09) × 103
6.97
9:1
8:2
7:3
6:4
9:1
8:2
7:3
6:4
(1.11 0.02) × 103
(2.12 0.03) × 103
(3.01 0.05) × 103
(3.74 0.05) × 103
(4.66 0.07) × 103
(8.4 0.1) × 103
6.48
7.49
(1.05 0.03) × 104
(1.37 0.03) × 104
aμ = 0.10 M [(C4H9)4N]ClO4, T = 25◦C.
With pKa1 of the catechol (9.23 [11]) and catechin
(8.79 [12]), the values of k1, obtained from slopes
the reaction follows Eqs (11)–(14):
ꢀ
k12
=
=
k11k22K12f12W12
[ln K12 + (w12 − w21)/RT ]2
(11)
of the one parameter linear least-square fits of kH O
2
vs. 1/[H+] plots are (8.2
0.2) × 105 and (6.1
ln f12
4[ln(k11k22/1022) + (w11 + w22)/RT ]
0.1) × 105 M−1 s−1 for pyrocatechol and catechin, re-
spectively, at μ = 0.10 M [(n-Bu)4N]ClO4 and T =
25◦C.
(12)
W12 = exp[−(w12 + w21 − w11 − w22)/2RT ]
(13)
According to Marcus theory for the outer-sphere
electron transfer reaction, the specific rate constant of
8.00E + 03
(a)
1.60E + 04
1.20E + 04
8.00E + 03
4.00E + 03
0.00E + 00
(b)
6.00E +
03
4.00E + 03
2.00E + 03
0.00E + 00
0
10
20
30
40
50
0
10
20
30
40
50
%H2O
%H O
2
pH6.00
pH6.50
pH7.00
pH7.51
pH5.48
pH6.48
pH7.01
pH7.48
Figure 5 k vs. % H2O plots: (a) pyrocatechol and (b) catechin.
International Journal of Chemical Kinetics DOI 10.1002/kin

152
CHEN ET AL.
4.00E + 04
3.00E + 04
2.00E + 04
1.00E + 04
0.00E + 00
2.00E + 07
1/[H+]
1.00E + 07
3.00E + 07
Catechin
4.00E + 07
0.00E + 00
Pyrocatechol
Figure 6 kH O vs. 1/[H+] plots.
2
zizj e2
hydrogen electrode (NHE)), in fair agreement with the
reported value of 0.47 V measured in methanol [13].
With reduction potentials of flavonoids and dpph• and
self-exchange rate constants of flavonoids, the self-
exchange rate constant of dpph•/dpphH couple can be
calculated. The values are 4.1 × 103 M−1 s−1 based
on the results of pyrocatechol and 3.3 × 103 M−1 s−1
based on the results of catechin. Taking the average
of the two, the self-exchange rate constant of dpph•
wij
=
√
(14)
DSaij (1 + βaij μ)
where k11 and k22 are self-exchange rate constants of
dpph• and flavonoids, k12 and K12 are rate constants
and equilibrium constants of Eq. (14). zi and zj are
charges of reacting species. Since neutral species are
involved in both reactants and products in Eq. (14), this
leads to zizj = 0, and the work term in Marcus theory
(W12) can therefore be excluded for our system.
therefore is k11 = 3.7 × 103 M−1 s−1
.
Although the dpph• compound has been widely
used as an oxidizing agent in the oxidation of phe-
nols, most of the work has been concentrated in the
investigation of the antioxidant properties of the phe-
nols [6,7,14–18], and little effort has been devoted to
the reactivity study of dpph•. Our results may provide
important information for the redox reactions involv-
ing this compound, particularly in the investigation of
the antioxidation property of the flavonoid compounds.
Equations (11) and (12) can thus be simplified as
ꢀ
k12
=
=
k11k22K12f12
(15)
(16)
(ln K12)2
4 ln(k11k22/1022)
ln f12
The reduction potentials and self-exchange rate
constants for HX./HX− couples are 0.52 V, 1.2 ×
109 M−1 s−1 and 0.59 V, 1.6 × 1010 M−1 s−1 for
pyrocatechol and catechin, respectively [9]. The re-
duction potential for dpph•/dpphH couple, measured
in 6:4 (CH3OH/H2O) solvent, is 0.45 V (vs. normal
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