# Kinetics and Mechanism of the Reaction between Chromium(III) and 3,4-Dihydroxy-Phenyl-Propenoic Acid (Caffeic Acid) in Weak Acidic Aqueous Solutions

Authors: Vladimiros Thoma; Konstantinos Tampouris; Athinoula L. Petrou
Journal: Bioinorganic Chemistry and Applications
 (2008)

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## Abstract

Our study of the complexation of 3,4-dihydroxy-phenyl-propenoic acid by chromium(III) could give information on the way that this metal ion is available to plants. The reaction between chromium(III) and 3,4-dihydroxy-phenyl-propenoic acid in weak acidic aqueous solutions has been shown to take place by at least three stages. The first stage corresponds to substitution (Id mechanism) of water molecule from the Cr(H2O)5OH2+ coordination sphere by a ligand molecule. A very rapid protonation equilibrium, which follows, favors the aqua species. The second and the third stages are chromium(III) and ligand concentration independent and are attributed to isomerisation and chelation processes. The corresponding activation parameters are ΔH2(obs)≠ = 28.6 ± 2.9 kJ mol−1, ΔS2(obs)≠ = −220± 10 J K−1mol−1, ΔH3(obs)≠ = 62.9 ± 6.7 kJ mol−1 and ΔS3(obs)≠ = −121± 22 J K−1mol−1. The kinetic results suggest associative mechanisms for the two steps. The associatively activated substitution processes are accompanied by proton release causing pH decrease.


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## Body

## 1. Introduction

Polyphenols are widely found   in plants and are
present in all plant-derived systems [1, 2]. Hydroxy-cinnamates, especially
caffeic acid (see Figure  1) and its derivatives, are widely distributed and their
presence in fruit juices is due to their easy extractability. Foods containing
polyphenols undergo enzymatic and nonenzymatic browning due to autoxidation
reactions [4, 5].
Figure 1
Caffeic acid.
Initial oxidation of compounds like caffeic
acid results in the formation of their quinone form [6, 7], which, being strongly
electrophilic, undergoes nucleophilic attack [9, 10]. The rest of the reaction in
which the quinone participates is the same, no matter if the quinone has been
produced enzymatically or chemically.The above reaction takes place under acidic
conditions and is catalysed in the presence of metals [12, 13]. Food processing that
includes  treatment in alkaline
conditions results in decreased nutritional value due to phenol oxidation
reactions with amino acids and proteins through  their nitrogen [15].Under alkaline conditions, caffeic acid reacts
rapidly with oxygen [16]. Under acidic conditions, the nonenzymatic reaction is
slow [17].Researchers [18] have found that the oxidation
of caffeic acid is extremely rapid at pH ≥ 8 and the pH dependency along with the
relatively low and constant Arrhenius activation energies at all pH's indicate
that the phenolate ions are also involved in the reaction.The rate of the reaction is increased by
increasing pH (4.56× 10−7 s−1 at pH 4.0
and 1.67 ×10−5 s−1 at pH
8.0, both at 35°C) and temperature (2.12 × 10−6 s−1 at 5°C
and 1.67 × 10−5 s−1 at 35°C,
both at pH 8.0; 1.39 × 10−7 s−1 at 5°C
and 2.29 × 10−6 s−1 at 35°C,
both at pH 5.0) [18]. The Arrhenius activation energy was found to be 49.0±6.4 kJ mol−1 at pH 8.0
and the phenolate anion concentration was proposed to be the controlling factor
of the rate of autoxidation [18].Our goal was to study the kinetics and
mechanism of the complexation of caffeic acid with chromium(III) under weak
acidic aqueous conditions that exclude the autoxidation of the ligand.Caffeic acid, being a degradation product of
humic acids, can be found in soils and nutrient solutions where plants grow.
The study of the complexation of caffeic acid by metal ions, in our case by
chromium(III), could give information on the way that metal ions are available
to plants.


## 2. Experimental Results

### 2.1. Reagents and Materials

The reagents used were all of analytical grade.
Caffeic acid (Alfa
Aesar), used as received, was
dissolved in dilute (0.1 M) solution of KOH in
concentrations ranging from 0.0025 to 0.01 M. Stock solutions (0.2 M) of Cr(III) were
prepared from Cr(NO3)3⋅9H2O. The
ionic strength was adjusted using KNO3. The solutions were used immediately after their
preparation to avoid transformation and decomposition reactions. The addition
of the chromium(III) solution kept the pH below 4 due to its acidic hydrolysis. All
kinetic experiments were performed at pH values below 4 in the presence of air.
At 21°C, the starting pH of the solutions was 2.3.


### 2.2. Kinetic Experiments

The kinetics of the substitution of water
molecules in the coordination sphere ofCr(H2O)63+  by caffeic acid
were followed at 537 nm, where the biggest absorbance difference between the
final product and the initial (t=0) mixture of Cr(H2O)63+/caffeic acid
exists. The absorbances were recorded on a Hitachi Model 100-60
spectrophotometer and the electronic spectra were recorded on a Varian Cary 3E
spectrophotometer.First-order rate constants were estimated with
a nonlinear least-squares fit.Pseudofirst-order conditions (excess of
Cr(III), i.e., acidic solution) were employed for all of the kinetic
experiments, whereas experiments in excess of ligand (caffeic acid) were not
possible to be performed due to its oxidation in the alkaline solution of the
reaction mixture. Experiments at temperatures higher than 45°C
were also avoided due to the acceleration of the ligand's
decomposition/autoxidation.The plots ofln(A∞−At), where  A∞ and At are absorbances after the completion of the reaction
and at time t, against time were found to be nonlinear (see Figure  2); they are
curved at short reaction times and have constant slope at large reaction times.
Figure 2
A typical nonlinear plot ofln(A∞−At) versus time. Conditions: [caffeic acid]0 =
0.01 M, [Cr(III)]0 = 0.08 M, 1 cm cell, T = 303 K, I = 0.5 M.
The rate constants were calculated assuming two
consecutive first-order steps according to methods found in the literature [19, 20].A reaction sequence consisting of two
first-order (or pseudofirst-order) steps(1)A→k2B→k3C
admits of two mathematical solutions; the sets
of rate constants are such that the fast and slow kinetic steps are
interchanged [19, 21, 22].The linear second part (long-time) gives a
slope of−k3 and
thus the k3(obs)  values were obtained. The rate constants k2(obs) for A → B step were evaluated by the method of Weyh
and Hamm
[20]
using the consecutive rate equation
(2)A∞−At=a2e−k2(obs)t+a3e−k3(obs)t,
where a2 and a3 are
dependent upon the rate constants and the extinction coefficients. The values
of a2e−k2(obs)t  were obtained from Δ=A∞−At−a3e−k3(obs)t at various times t (see Figure  2). So, lnΔ=constant−k2(obs)t  and the k2(obs) values were derived from the slope of the plots of lnΔ
versus t for small values of t. A typical plot is shown in Figure 3.Figure 3
A typical plot of lnΔ
versus time. Conditions: [caffeic acid]0 = 0.01 M, [Cr(III)]0 = 0.08 M, 1 cm cell, T = 303 K, I = 0.5 M.The experimental data show a curvature of theln(A∞−At) versus t plot at all
temperatures for various (excess) chromium(III) concentrations. The assumption
of the existence of two consecutive steps for the under-study reaction and the
computation of  k2(obs) and  k3(obs) values fits well with
the experimental data. The analysis of the data gives a small value for ΔH2(obs)≠  and a very big absolute value for ΔS2(obs)≠  suggesting that a composite reaction takes
place; this could be a fast equilibrium followed by the k2 slow step
[19]. Contribution of uncomplexed Cr(III) species in the absorbance values,
mainly due to the excess of Cr(III), does not cause problems in the graphs
(see Figures 2 and 3) because it is included in both A∞ and At and is eliminated due to the subtraction of At from A∞.Table1 gives the k2(obs) and k3(obs) values for the reaction at various temperatures (298 K, 303 K, 308 K, and 310 K).Table 1
Values ofk2(obs) and k3(obs) at various temperatures.
k2(obs)× 104 (s−1)k3(obs) × 105 (s−1)T (K)2.02.82982.64.83033.06.33083.48.4310The activation parametersΔH2(obs)≠,    ΔS2(obs)≠ and ΔH3(obs)≠, ΔS3(obs)≠, corresponding
to k2(obs) and k3(obs), respectively, are calculated from
the linear Eyring plots (see Figure  4) and are given in Table 2.Table 2
Activation parameters for stepsA→k2B and B→k3C.
ΔH2(obs)≠ (kJ mol−1)ΔS2(obs)≠ (J K−1mol−1)ΔH3(obs)≠ (kJ mol−1)ΔS3(obs)≠ (J K−1mol−1)28.6±2.9−220 ± 1062.9±6.7−121 ± 22Figure 4
The Eyring plots fork2(obs) and k3(obs) at the temperatures studied.At times longer than four to five half-lives of
the B→ C step, a reaction involving oxidation of the
ligand sets in, causing anomalous further absorbance changes. The recorded
values of A∞ used for Figures 2, 3, and all other graphs were very close to the true
values since they were also obtained by plotting A=f(t); it was thus possible
to confirm the completion of the reaction.


## 2.1. Reagents and Materials

The reagents used were all of analytical grade.
Caffeic acid (Alfa
Aesar), used as received, was
dissolved in dilute (0.1 M) solution of KOH in
concentrations ranging from 0.0025 to 0.01 M. Stock solutions (0.2 M) of Cr(III) were
prepared from Cr(NO3)3⋅9H2O. The
ionic strength was adjusted using KNO3. The solutions were used immediately after their
preparation to avoid transformation and decomposition reactions. The addition
of the chromium(III) solution kept the pH below 4 due to its acidic hydrolysis. All
kinetic experiments were performed at pH values below 4 in the presence of air.
At 21°C, the starting pH of the solutions was 2.3.


## 2.2. Kinetic Experiments

The kinetics of the substitution of water
molecules in the coordination sphere ofCr(H2O)63+  by caffeic acid
were followed at 537 nm, where the biggest absorbance difference between the
final product and the initial (t=0) mixture of Cr(H2O)63+/caffeic acid
exists. The absorbances were recorded on a Hitachi Model 100-60
spectrophotometer and the electronic spectra were recorded on a Varian Cary 3E
spectrophotometer.First-order rate constants were estimated with
a nonlinear least-squares fit.Pseudofirst-order conditions (excess of
Cr(III), i.e., acidic solution) were employed for all of the kinetic
experiments, whereas experiments in excess of ligand (caffeic acid) were not
possible to be performed due to its oxidation in the alkaline solution of the
reaction mixture. Experiments at temperatures higher than 45°C
were also avoided due to the acceleration of the ligand's
decomposition/autoxidation.The plots ofln(A∞−At), where  A∞ and At are absorbances after the completion of the reaction
and at time t, against time were found to be nonlinear (see Figure  2); they are
curved at short reaction times and have constant slope at large reaction times.
Figure 2
A typical nonlinear plot ofln(A∞−At) versus time. Conditions: [caffeic acid]0 =
0.01 M, [Cr(III)]0 = 0.08 M, 1 cm cell, T = 303 K, I = 0.5 M.
The rate constants were calculated assuming two
consecutive first-order steps according to methods found in the literature [19, 20].A reaction sequence consisting of two
first-order (or pseudofirst-order) steps(1)A→k2B→k3C
admits of two mathematical solutions; the sets
of rate constants are such that the fast and slow kinetic steps are
interchanged [19, 21, 22].The linear second part (long-time) gives a
slope of−k3 and
thus the k3(obs)  values were obtained. The rate constants k2(obs) for A → B step were evaluated by the method of Weyh
and Hamm
[20]
using the consecutive rate equation
(2)A∞−At=a2e−k2(obs)t+a3e−k3(obs)t,
where a2 and a3 are
dependent upon the rate constants and the extinction coefficients. The values
of a2e−k2(obs)t  were obtained from Δ=A∞−At−a3e−k3(obs)t at various times t (see Figure  2). So, lnΔ=constant−k2(obs)t  and the k2(obs) values were derived from the slope of the plots of lnΔ
versus t for small values of t. A typical plot is shown in Figure 3.Figure 3
A typical plot of lnΔ
versus time. Conditions: [caffeic acid]0 = 0.01 M, [Cr(III)]0 = 0.08 M, 1 cm cell, T = 303 K, I = 0.5 M.The experimental data show a curvature of theln(A∞−At) versus t plot at all
temperatures for various (excess) chromium(III) concentrations. The assumption
of the existence of two consecutive steps for the under-study reaction and the
computation of  k2(obs) and  k3(obs) values fits well with
the experimental data. The analysis of the data gives a small value for ΔH2(obs)≠  and a very big absolute value for ΔS2(obs)≠  suggesting that a composite reaction takes
place; this could be a fast equilibrium followed by the k2 slow step
[19]. Contribution of uncomplexed Cr(III) species in the absorbance values,
mainly due to the excess of Cr(III), does not cause problems in the graphs
(see Figures 2 and 3) because it is included in both A∞ and At and is eliminated due to the subtraction of At from A∞.Table1 gives the k2(obs) and k3(obs) values for the reaction at various temperatures (298 K, 303 K, 308 K, and 310 K).Table 1
Values ofk2(obs) and k3(obs) at various temperatures.
k2(obs)× 104 (s−1)k3(obs) × 105 (s−1)T (K)2.02.82982.64.83033.06.33083.48.4310The activation parametersΔH2(obs)≠,    ΔS2(obs)≠ and ΔH3(obs)≠, ΔS3(obs)≠, corresponding
to k2(obs) and k3(obs), respectively, are calculated from
the linear Eyring plots (see Figure  4) and are given in Table 2.Table 2
Activation parameters for stepsA→k2B and B→k3C.
ΔH2(obs)≠ (kJ mol−1)ΔS2(obs)≠ (J K−1mol−1)ΔH3(obs)≠ (kJ mol−1)ΔS3(obs)≠ (J K−1mol−1)28.6±2.9−220 ± 1062.9±6.7−121 ± 22Figure 4
The Eyring plots fork2(obs) and k3(obs) at the temperatures studied.At times longer than four to five half-lives of
the B→ C step, a reaction involving oxidation of the
ligand sets in, causing anomalous further absorbance changes. The recorded
values of A∞ used for Figures 2, 3, and all other graphs were very close to the true
values since they were also obtained by plotting A=f(t); it was thus possible
to confirm the completion of the reaction.


## 3. Discussion

The value of pKa for Cr3+/Cr(OH)2+ is about 4 [23, 24]. At pH < 4, chromium(III) complex exists mainly in its
hexa-aqua monomeric form with absorbance maxima at 575 and 410 nm. However, over the pH range 3-4 there is some
Cr(H2O)5OH2+ and the reaction with Cr(H2O)5OH2+ rather than with Cr(H2O)63+ should be
considered as taking place because of its
largely higher reactivity. All the experiments in this work were
conducted at pH values lower than 4.
Under these conditions the ligand exists mainly as a neutral molecule and a
monoanion [25, 26].Upon mixing of the reactants, an initially
violet (Craq3+)
and a brown (caffeic acid) solution, a brown-green complex, is formed. The
brown-green complex was formulated as an oxygen-bound chromium(III) compound A (Scheme 1) based on the UV/Vis spectra and the formation and subsequent
transformation (substitution) kinetics. In all the kinetic experiments, we
assume to begin with the already associated brown-green first complex A.
This suggests that the following two slow steps (k2, k3)
did not have a contribution on the first step at the temperatures studied.Scheme 1Theln(A∞−At) versus time plots, being
nonlinear, are indicative of a complex reaction, not a single-step process. The
two first-order consecutive steps (analysis described above) were found to be
independent on chromium(III) concentration (see Table   3), suggesting that a (first)
step resulting in the formation of a complex upon reaction between the reactive
forms of the reactants (Cr(H2O)5OH2+, neutral
form of caffeic acid) took place. Thus three steps at least (k1, k2,
k3) take place (Scheme 1): the first, a fast one, which was not
studied, and the last two, which result from the analysis of our data.Table 3
Dependence ofk2(obs) and k3(obs) on Cr(III) concentrations at various temperatures for the reactions A→k2B→k3C. Conditions: [caffeic acid]0 =
0.005 M,  I = 1.0 M.
k2(obs)×  104 (s−1)k3(obs)×   105(s−1)[Cr(III)]0(M)T (K)2.02.80.0182982.24.80.083032.45.10.093032.84.70.103032.94.50.113032.86.00.073083.26.60.093083.39.10.083103.78.50.083103.37.60.14310The fact that a first fast step is taking place
with a Cr(III) reacting species is not consistent with substitution rate
constants of the hexa-aqua form of chromium(III); thus the mechanistic pathway
(Scheme1) is suggested to most probably be through the hydroxy species.Because of the protonation of the ligand, the
hydroxyl and the carboxylic groups are blocked, and the attacks by
chromium(III) can take place on them by releasing protons, effect that is
measured as pH decrease of the solution (see Figure5). Experiments in excess
ligand were not performed as explained above (Experimental Section 2) due to
solubility problems and oxidation of the ligand in alkaline solutions.Figure 5
The pH versus time plots of a typical
mixture of caffeic acid/Cr(III). Conditions: [caffeic acid]0=
0.01 M, [Cr(III)]0 = 0.09 M, T = 308 K.The proposed mechanism for the formation of the
various species is shown in Scheme1. The attack of Cr(H2O)5OH2+ at the carboxylic group of caffeic acid, 1, leads to complex 2;
this results in shifting equilibrium (3)Cr(H2O)63+⇌KaCr(H2O)5OH2++H+pH<4 to the right.A very rapid protonation equilibrium which
follows step 1 favors the aqua species (complexA). In conjugate-base
mechanisms the conjugate base, though present as only a small fraction of the
total, reacts and then reprotonates as it naturally would. The reactive species
must be Cr(H2O)5OH2+ and not Craq3+.ComplexA reacts in two consecutive
steps (k2, k3) to give B and C.In Figure6, spectra of the reaction mixture at
298 K are recorded at various times after mixing starting from the brown-green
complex A. The spectra correspond to mixtures of A, B, and C.Figure 6
Spectra of a typical caffeic acid/Cr(III) mixture at various times after mixing; [caffeic acid]0 =
0.015 M, [Cr(III)]0= 0.025 M, T = 298 K, 1 cm cell. (a) 0 seconds,
(b) 1800 seconds, (c) 3600 seconds, (d) 7200 seconds, (e) 14400 seconds.The biggest absorbance difference between the
spectra ofA and C was found to be at 537 nm
(Experimental Section 2). As a result of the complexation, the absorbance maximum
was displaced to lower wavelengths (stronger ligand field, higher Dq).Thek2(obs) and k3(obs) dependence on chromium(III) concentration (see Table   3) was studied at various
temperatures in order (a) to find if a second or third chromium(III) ion is
reacting with the already formed complex A and (b) to be able to
calculate the activation parameters (ΔH≠, ΔS≠). By isolating the Cr3+/caffeic acid final complex
(complex C) in solid form from Cr(III)/caffeic acid mixtures of
various stoichiometries, we found that only one ligand molecule enters the
coordination sphere of the metal. A stoichiometry of 1:1 for the reaction of Cr(III) with caffeic acid is
proposed in accord with the elemental analyses and the consecutive first-order
isomerisation and chelation reactions in the Cr(III) center. The elemental
analyses: 28.11% C and 5.05% H correspond to the
formula [Cr(H2O)4(caffeic acid−3H) K+OH−⋅2H2O]
for which the calculated percentages for C and H are 27.49 % and 4.62%,
respectively.The assumption of two consecutive steps
suggesting transformation ofA to B and of B to C could be checked by the above studies. The independence of k2(obs) and k3(obs) on chromium(III) concentration confirms the above
assumption.The calculated activation parameters (ΔH≠, ΔS≠) obtained from the Eyring plots (temperature dependence experiments,
Figure 4) suggest structures of the activated complexes formed in the
corresponding processes (steps 2 and 3) as well as the mechanisms taking place.It was not possible to obtain the activation
parametersΔH1(obs)≠  and   ΔS1(obs)≠ since the wide range variation of temperature
needed for the above purpose could not be achieved in our experiments. A
dissociative mechanism Id is, however, expected for step 1, since
for the conjugate base Cr(H2O)5OH2+ dissociative mechanism Id is supported [27] because the strong
labilizing effect of the coordinated OH− on the trans H2O leads to a 102-103 fold rate enhancement for the
hydroxy-over the hexa-aqua ion.The values of the activation parameters for
step 2  (ΔH2(obs)≠,  ΔS2(obs)≠)
are implying that a composite reaction takes place. This consists of the fast
equilibrium (K0) followed by the slow step 2 (Scheme
1). These composite activation parameters are ΔH2(obs)≠=ΔH0+ΔH2≠ and ΔS2(obs)≠=ΔS0+ΔS2≠, where ΔH0 and ΔS0 correspond to the equilibrium (K0) and ΔH2≠ and ΔS2≠ to the
second step (k2).The values ofΔH3(obs)≠ and ΔS3(obs)≠ correspond to a single step k3.The negative values ofΔS≠ (ΔS2(obs)≠ and ΔS3(obs)≠), the
independence of k2(obs)  and k3(obs) on chromium(III) concentration, the
displacement of λmax, and the pH
decrease (see Figure  5) lead to the mechanism presented in Scheme 1, where
substitution of water molecules in the coordination sphere of Cr(III) by
caffeic acid molecule is associatively activated. The first step (k1)
is an Id replacement of water molecule by caffeic acid. The second
step is an isomerisation and the third step is a chelation. All three steps
take place with concomitant proton release. A stoichiometry of 1:1 for the reaction of
Cr(III) with caffeic acid is proposed in accord with the elemental analyses and
the consecutive first-order isomerisation and chelation reactions in the
Cr(III) center.The pH decrease (see Figure5) is due to the
release of protons upon the course of the reactions (Scheme 1).Structures of the activated complexesA≠ and B≠ are given in Scheme 2.Scheme 2
Activated complexesA≠ and B≠.The suggested chelate formation in the
transition states in Scheme2 could occur preferably by an associative
mechanism (ΔS≠<0) which has been found to be operative in reactions of Cr(III)
[28–30].
Alternatively, the original complex A could be a chelate itself, and
this could explain the formation of products containing Cr–O bonds. The
negative entropies of activation, ΔS≠, however, suggest the formation of more organised transition states
from the less well-organised reactants. Thus, complex A is not in
chelated form and the mechanism shown in Scheme 1 is preferred. In this
mechanism the phenolic groups act as internal attacking groups to the
chromium-bound H2O molecules, which are thus supplied with a proton
in the same complex (Scheme 2) and released as H3O+.An alternative structure for the final productC is chelation at the carboxylic group through the oxygens. However, if chelation
at the carboxylic group took place, a four-membered ring would result, which is
less preferable. The five-membered ring which is supported to be formed
according to the suggested mechanism (Scheme 1) is more probable. Thus we
discard this alternative.The two-step isomerisation—chelation
of the first complex that is formed,A, produces
PhO-bound chromium(III) species, characterised by UV/Vis spectra and kinetic
behaviour which are typical of other known complexes containing Cr–O bonds [28, 29].The above presented data suggest that binding
is taking place through the two phenolic groups of the ligand molecule, caffeic
acid,1. Such mode of binding (catecholic type) has been also suggested
in the reaction of dihydrocaffeic acid with chromium(III) in weak acidic
aqueous solutions [31]. The above reaction also takes place in at least three
stages (complexation, isomerisation, chelation).



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*Source: 624583-2008-07-28.xml*