Oxidation pathways of natural dye hematoxylin in aqueous solution

Article abstract of DOI:10.1135/cccc2010096

The oxidation mechanism of hematoxylin was studied in phosphate buffers and 0.1 M KCl by cyclic voltammetry and UV-Vis spectroscopy under deaerated conditions. The redox potential of hematoxylin in buffered solution strongly depends on pH. A two electron oxidation is preceded by deprotonation. The homogeneous rate of deprotonation process of hematoxylin in 0.1 M phosphate buffer is k_d = (2.5 ± 0.1) × 10⁴ s ^-1. The cyclic voltammetry under unbuffered conditions shows the distribution of various dissociation forms of hematoxylin. The dissociation constants pK₁ = 4.7 ± 0.2 and pK₂ = 9.6 ± 0.1 were determined using UV-Vis spectroscopy. The final oxidation product was identified by gas chromatography with mass spectrometry detection as hemathein. The distribution of oxidation products differs under buffered and unbuffered conditions. The dye degradation in natural unbuffered environment yields hemathein and hydroxyhematoxylin, which is absent in buffered solution.

Full text of DOI:10.1135/cccc2010096

Oxidation Pathways of Natural Dye Hematoxylin
1097
OXIDATION PATHWAYS OF NATURAL DYE HEMATOXYLIN IN
AQUEOUS SOLUTION
b
a2
Romana SOKOLOVÁa1,*, Ilaria DEGANO , Magdaléna HROMADOVÁ
,
Jana BULÍČKOVÁa3, Miroslav GÁL and Michal VALÁŠEK
a4
c
a
J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v.v.i.,
Dolejškova 3, 182 23 Prague, Czech Republic; e-mail: sokolova@jh-inst.cas.cz,
hromadom@jh-inst.cas.cz, bulickov@jh-inst.cas.cz, gal@jh-inst.cas.cz
Department of Chemistry and Industrial Chemistry, University of Pisa,
Via Risorgimento 35, 56100 Pisa, Italy; e-mail: ilariad@dcci.unipi.it
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i.,
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: valasek@uochb.cas.cz
1
2
3
4
b
c
Received August 2, 2010
Accepted September 14, 2010
Published online November 9, 2010
The oxidation mechanism of hematoxylin was studied in phosphate buffers and 0.1 M KCl
by cyclic voltammetry and UV-Vis spectroscopy under deaerated conditions. The redox po-
tential of hematoxylin in buffered solution strongly depends on pH. A two electron oxida-
tion is preceded by deprotonation. The homogeneous rate of deprotonation process of
hematoxylin in 0.1 M phosphate buffer is kd = (2.5 0.1) × 104 s–1. The cyclic voltammetry
under unbuffered conditions shows the distribution of various dissociation forms of
hematoxylin. The dissociation constants pK1 = 4.7
0.2 and pK2 = 9.6
0.1 were deter-
mined using UV-Vis spectroscopy. The final oxidation product was identified by gas chro-
matography with mass spectrometry detection as hemathein. The distribution of oxidation
products differs under buffered and unbuffered conditions. The dye degradation in natural
unbuffered environment yields hemathein and hydroxyhematoxylin, which is absent in buf-
fered solution.
Keywords: Hematoxylin; Oxidation; Dye degradation; Cyclic voltammetry.
Degradation process of flavonoid compounds used as colorants in historical
tapestries is a serious problem for the conservation and restoration of the
art objects. Hematoxylin 1 and hemathein 2 (Scheme 1) are the principal
chromophores of Hematoxylon campechianum and Hematoxylon brasiletto,
two species also known as “logwood”, originating from South America. The
original color of medieval textiles is transformed by ambient unbuffered
aerobic environment and its color fades away or changes1,2. It is highly de-
sirable to find possible degradation pathways and decomposition products
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114
© 2010 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2010096

1098
Sokolová, Degano, Hromadová, Bulíčková, Gál, Valášek:
of the original dyes. Degradation products formed during the aging process
can be used as fingerprints for the determination and restoration of the
original color.
SCHEME 1
Chemical structures of hematoxylin 1 and hemathein 2
A detailed study of the electrochemical behavior of 1 is missing in the lit-
erature, although the electrochemistry and electrocatalytic activity of the
hematoxylin-modified carbon paste electrode towards the electrocatalytic
oxidation of NADH were reported3. Recently, compound 1 was used as
chemical sensor in modified carbon paste electrode, glassy carbon electrode
and multi-wall carbon nanotube modified glassy carbon electrode for the
determination of some analytes4. Hemathein 2 was used as a pH-sensitive
redox active compound in an amperometric pH-sensing biosensor5,6. The
positive shift of anodic peak of cyclic voltammograms of 2 on n-eicosane-
graphite composite electrode with decreasing pH was reported6. Since com-
pounds 1 and 2 contain the phenolic group it is expected that the oxi-
dation of phenols is relevant for the present study. The anodic oxidation
pathways of phenols, polyphenols and hydroquinones have been exten-
sively studied7–15. Their oxidation is generally followed by complicated se-
quence of coupled chemical reactions. The quinones formed after the
oxidation of hydroquinones can undergo the nucleophilic addition of the
solvent and a trihydroxyderivative is developed. Its further oxidation
causes the formation of hydroxyquinone16. The influence of pH on electro-
chemical response of polyphenols and hydroquinones was previously re-
ported7,8,11,13. The aim of this study is the elucidation of oxidative processes
in buffered solution of different pH. We will identify the final oxidation
products and possibly search for factors, which influence their distribution.
Natural degradation of 1 and 2 proceed in an unbuffered environment.
Hence, we will look also for effects caused by the absence of buffering me-
dium.
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

Oxidation Pathways of Natural Dye Hematoxylin
1099
EXPERIMENTAL
Reagents
Hematoxylin 1 was purchased from Fluka. The reagents used as supporting electrolytes such
as potassium chloride and chemicals for preparation of phosphate or phosphate/citrate buff-
ers (KH2PO4, NaH2PO4, NaOH, citric acid) were of reagent grade. Phosphate buffers (pH
2.5–12) were prepared at constant ionic strength. The solutions were prepared with
ultrapure water (Millipore). All the solvents were Carlo Erba (Milan, Italy) HPLC grade ex-
cept from ethyl acetate (AcOEt), Anal R, BDH. Hexadecane and 2,4-dihydroxybenzo-
phenone, used as internal standards, and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)
containing 1% trimethylchlorosilane were purchased from Sigma (Milan, Italy). Stock solu-
tions of analytes in methanol were prepared from 1 from Fluka and 2 (approx. 85%) from
Sigma (Milan, Italy). All reagents and chemicals were used without further purification.
Techniques
Electrochemical measurements were done using an electrochemical system for cyclic
voltammetry. It consisted of a fast rise-time potentiostat interfaced to a personal computer
via an IEEE-interface card (AdvanTech, model PCL-848) and a data acquisition card
(PCL-818) using 12-bit precision. Cyclic voltammetry was also conducted using a PGSTAT 12
AUTOLAB potentiostat (Ecochemie, The Netherlands). A three-electrode electrochemical cell
was used with an Ag|AgCl|1 M LiCl reference electrode (with potential 0.210 V vs SHE) sepa-
rated from the test solution by a salt bridge. The working electrodes were platinum electrode
(diameter 0.8 mm) and glassy carbon electrode (diameter 0.7 mm). The auxiliary electrode
was cylindrical platinum net. Oxygen was removed from the solution by passing a stream of
argon. The oxidation products of 1 were prepared by exhaustive electrolysis of 1.4 × 10–3
–
2.54 × 10–3 M solutions on a Pt wire or on a carbon paste electrode.
Products were identified using a Trace GC gas chromatograph (Thermo Electron Corpora-
tion, USA) equipped with a programmable temperature vaporizing (PTV) injection port and
a mass spectrometric detector based on an ion trap analyzer (Polaris Q, Thermo Electron
Corporation, USA). The PTV injector was in the CT ‘splitless with surge’ mode at 280 °C
with a surge pressure of 100 kPa, and the mass spectrometer parameters were: electron im-
pact ionization (70 eV), ion source temperature 230 °C, scan range m/z 50–700 and interface
temperature 280 °C. Chromatographic separation was performed on a DB-5MS chemically
bonded fused silica capillary column (J & W Scientific, Agilent Technologies) with stationary
phase 5%phenyl–95%methylpolysiloxane, and of dimensions 0.25 mm i.d., 0.1 µm film
thickness, 25 and 30 m length. The gas chromatographic conditions were as follows: initial
temperature 57 °C, 2 min isothermal, then ramped at 10 °C/min up to 200 °C, 3 min iso-
thermal, then ramped at 20 °C/min up to 300 °C and then isothermal for 20 min. The car-
rier gas was He (purity 99.9995%), at a constant flow rate of 1.2 ml/min. The peak
assignment was based on comparison with analytical reference compounds and materials,
with library mass spectra (NIST 1.7) and with mass spectra reported in the literature.
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

1100
Sokolová, Degano, Hromadová, Bulíčková, Gál, Valášek:
In order to perform GC-MS analysis, electrolysis products were derivatized with a silyl-
ating agent BSTFA. Derivatization conditions were: 10 µl of 2,4-dihydroxybenzophenone (so-
lution in isopropanol, internal standard IS1) were added to the sample, the solution was
dried and 30 µl of the derivatization agent BSTFA in 50 µl of AcOEt were added, the reaction
took place at 60 °C for 30 min in closed glass vials. Just before injection, 10 µl of
hexadecane (solution in isooctane, internal standard IS2) and 150 µl of AcOEt were added,
2 µl of the final solution were injected in the GC system.
A diode-array UV-Vis spectrometer Agilent 8453 with 1.0 cm quartz cuvettes was used for
recording of the absorption spectra. The absorption spectra of 1 × 10–5 M hematoxylin in
phosphate/citrate buffer in the pH range 2.5 to 11.0 were recorded for 50 s after mixing of
solution in the deaerated cell.
RESULTS AND DISCUSSION
Acid-Base Equilibria
The protonation reactions play an important role in the electrochemistry of
phenols and quinones. For the elucidation of an overall mechanism it is
necessary to determine, which dissociation form is present in the solution
at a given pH. The dissociation constants of hematoxylin 1 pK1 = 3.82 and
pK2 = 6.88 were reported by Masoud et al.17, who used spectrophotometry.
Zare et al.3 published the apparent dissociation constant pK′ = 8.0 of 1 in-
corporated into the carbon paste. The compound 2 has values of pK1
=
6.70 18 and pK2 = 6.86 19 determined by spectrophotometric methods. Since
0.15
A
O
2
0.2
0.1
0.0
0.10
A
0.05
Ar
0
5000
10000
t, s
300
400
500
600
λ, nm
FIG. 1
The change of UV-Vis absorption spectra of 2 × 10–5 M hematoxylin in phosphate buffer at
pH 7 under the air oxygen. The inset shows the dependence of absorbance at 560 nm on time
under argon atmosphere (᭹) and under air oxygen (᭺)
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

Oxidation Pathways of Natural Dye Hematoxylin
1101
we found strong influence of the air oxygen on the absorption spectra of
the compound 1 (Fig. 1), we evaluated the dissociation constants of 1 by re-
cording the absorption spectra in phosphate and phosphate/citrate buffers
under strictly deaerated conditions. The absorption spectrum of 1 at pH 2.5
is characterized by the bands with absorption maxima at 237 and 289 nm.
The band at 237 nm increases during the change of pH to higher values
and slightly undergoes a blue shift. The height of the band at 289 nm does
not change. The band with the absorption maximum at 204 nm and a
small shoulder at 237 nm occurs in the absorption spectrum of 1 in phos-
phate buffers at pH higher than 6.5. The band with absorption maximum
at 560 nm appears at this pH value and increases with increasing pH until
the value of pH 10.4; the band at 204 nm decreases significantly during
the same change of pH and the isosbestic point is found at 220 nm. The pH
dependence of the maximum absorbance of bands changing due to the
acid-base equilibrium is related to pK by the Eq. (1)18
Amax − A
A − Amin
pH = pK + log
(1)
where Amax and Amin are the maximum and minimum absorbances mea-
sured at the maximum and minimum pH values, respectively. The plots of
log [(Amax–A)/(A–Amin)] vs pH are linear with the intercept equal to pK and
the corresponding slopes 1. The pK1 = 4.7 0.2 was estimated from the
change of absorption at 232 nm, which was the only change in absorption
spectra in acid region recorded in the range of 190 to 700 nm up to pH 7
(Fig. 2a). The limited accuracy of pK1 estimation is caused by uncertainty in
the deconvolution of overlapping absorption bands. The determination of
pK2 = 9.6 0.1 is considerably more precise. It is obtained from the change
of the absorbance at 204 and 560 nm (Fig. 2b), where straight lines have
slopes +1 and –1, respectively, and the point of their intersection is well
defined. The increase of the absorption band at 560 nm indicates that a di-
anion of 1 is formed. This band corresponds to a chromophore similar to
the anion of the compound 2 reported by Bettinger et al.18.
The absorption band at 289 nm in solution of the compound 1 at pH 9 is
accompanied by a shoulder at 305 nm. This shoulder slightly increases with
increasing pH. The band at 560 nm shifts to lower wavelength at pH above
10.4. The values of pK1 and pK2 were used for comparing the concentration
of different dissociation forms of the compound 1 under our experimental
conditions. The distribution diagram of various forms is given in Fig. 3. The
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

1102
Sokolová, Degano, Hromadová, Bulíčková, Gál, Valášek:
undissociated form of hematoxylin 1 is marked as AH2, while the first
dissociation leads to the anion of 1 AH– and further dissociation yields
dianion of 1 A2–.
1.0
a
pK = 4.7 0.2
0.5
1
0.0
–0.5
–1.0
3
b
4
5
6
3.0
pK = 9.6 0.1
2
2.0
1.0
0.0
–1.0
–2.0
7
8
9
10
pH
FIG. 2
Plot of log [(Amax – A)/(A – Amin)] versus pH. 1 × 10–5 M hematoxylin in phosphate/citrate
buffer (a): 232 (᭺) nm and in phosphate buffer (b): 204 (᭹), 560 (᭺) nm
–
AH
AH
2
2–
A
0.001
+
H
0.000
4
5
6
7
8
9
10
pH
FIG. 3
The distribution of dissociation forms of hematoxylin calculated for total analytical concentra-
tion of hematoxylin 1.2 × 10–3 mol l–1
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

Oxidation Pathways of Natural Dye Hematoxylin
1103
Voltammetry in Buffered Solutions
Cyclic voltammetry of the compound 1 in acidic solution shows one quasi-
reversible two-electron oxidation wave and a corresponding cathodic wave
(Fig. 4). As the pH of the solution increases, the peak potential of anodic
wave is shifted to less positive values and at highest pH the oxidation wave
splits into two overlapping peaks. A complete loss of reversibility occurs at
pH > 6.9, indicating that under these conditions the primarily generated
product is not stable. The concentration of 1 does not have an influence on
the potential of the anodic peak. This proves an absence of a bimolecular
chemical process. The increase of the anodic peak current with the increas-
ing pH is given in the inset of Fig. 4. The peak currents of 1 in the phos-
phate buffer at pH 4.9 (curve 1 in Fig. 4) are linearly dependent on the
square root of the scan rate. A similar linear dependence on the square root
of the scan rate in the range of 0.005 to 1 V s–1 holds for all pH values. Thus
the oxidation process is diffusion controlled over the whole pH range. The
anodic peak potential shifts towards more positive values with an increase
of the scan rate by 20 mV per decade. From these observations one can con-
clude that an acidobasic equilibrium participates significantly in the mech-
anism of oxidation20
ArHp
p H+ + Arp–
.
(2)
2.0
1
2
3
0.0
–2.0
6
5
4
–4.0
–6.0
4
3
5
5
6
7
8
9 10 11 12 13
pH
0.5
0.0
–0.5
–1.0
E, V
FIG. 4
Cyclic voltammogram of 1.2 × 10–3 M hematoxylin in phosphate buffer on Pt microelectrode
at pH 4.9 (1), 5.6 (2), 5.9 (3), 6.9 (4) and 12.5 (5) and at the scan rate of 0.05 V s–1. Cyclic
voltammograms of pH 6.3, 6.5 and 7.2 are not shown. The dependence of the anodic peak cur-
rent on pH is shown in the inset
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

1104
Sokolová, Degano, Hromadová, Bulíčková, Gál, Valášek:
Here ArHp denotes the natural form of the studied organic molecule and p
is the number of protons participating in the overall mechanism. Voltam-
metric results indicate that the electroactive form is Arp–, which is formed
by the dissociation of proton from the phenolic group. The presence of pro-
ton participation in the overall chemical reaction is supported by the shift
of anodic peak potential towards lower values. The number of hydrogen
ions p, participating in the chemical reaction preceding to the electron
transfer, can be estimated from the dependence of the half-wave potential
E1/2 on the pH of the solution21
∂E1/2
∂ln[H+ ]
RT
αnF
= p
(3)
where n is number of electrons involved in the electron transfer. The log-
plot analysis of semi-integrated currents yields a slope 95 mV per decade,
which is independent on pH. This indicates that the rate of the heteroge-
neous electron transfer is pH independent. The pH dependence of the half-
wave potential of semiintegrated currents gives two linear segments with
slopes 92 mV pH–1 and 28 mV pH–1. The two lines intersect at the point,
which corresponds to the apparent dissociation constant pK2′ = 6.7 (not
shown). The observed slope 2.3RT/αnF = 95 mV and the estimated slope of
the dependence of half-wave potential on the concentration of H+ accord-
ing to Eq. (3) is equal to 92 mV pH–1. This yields the number of dissociated
protons p = 0.97. Therefore for pH lower than pK1, only one hydrogen ion
participates in the rate-determining preceding deprotonation (Scheme 2).
This scheme resembles the ‘schema carre’22 (Scheme 3) and the mechanism
in buffered solution proceeds through the formation of a monoanion and
the acceptance of two electrons. The mechanism involves the formation of
phenoxy cation8,16 and the compound 2 is the final product. Therefore, the
electroactive form responsible for the main oxidation wave is an anionic
form of the compound 1.
The inset in Fig. 4 shows that the oxidation currents at pH < 7 decrease
with the decreasing pH. The oxidation current becomes controlled by
the rate of the preceding dissociation, which yields the electroactive mono-
anion or dianion of 1. The oxidation currents at pH < 7 and the determined
values of K1 and K2 enable the estimation of the rate of the homogeneous
deprotonation. For this purpose, the voltammograms were numerically
transformed by semiintegration to S-shaped waves. The limiting semi-
integrated currents yield the kd rate constant of the preceding dissociation
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

Oxidation Pathways of Natural Dye Hematoxylin
1105
(Eq. (2)) on the basis of the Koutecký theory23. The ratio of the semi-
integrated limiting kinetic current Ik to the diffusion limited semiintegrated
current Id is related to rate parameters by a tabulated function F(χ) = Ik/Id.
SCHEME 2
The oxidation of hematoxylin 1
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

1106
Sokolová, Degano, Hromadová, Bulíčková, Gál, Valášek:
SCHEME 3
The ‘schema carre’
The parameter χ is a function of a monomolecular rate constant ρ and
a corresponding equilibrium constant of the chemical reaction σ. For the
equilibrium Eq. (2) and p = 1, the substitution ρ = kd × [AH] and σ = [H+]/K
in the Koutecký equation yields the expression
χ2 [H+ ]
K[AH]t
kd = 0.58
.
(4)
The estimated value of the rate constant of dissociation in 0.1 M phosphate
buffer is kd = (2.5 0.1) × 104 s–1 (Fig. 5). The concentration of the support-
ing electrolyte does not have any influence on the shape of the oxidation
waves, which indicates an absence of the double-layer effects.
The exhaustive electrolysis at a constant potential was performed in order
to obtain the oxidation products of the compound 1. We report here the
products obtained at two different representative pH values of 5.9 and 9.0.
The electric charge required for an exhaustive electrolysis at pH 5.9 corre-
sponds to the consumption of two electrons. The products obtained by an
exhaustive electrolysis were separated and GC-MS analysis confirmed the
formation of 2 as the main product under buffered conditions. The main
peaks in the MS of derivatized hemathein-4TMS correspond to m/z values:
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

Oxidation Pathways of Natural Dye Hematoxylin
1107
588 ([M]•+), 573 ([M – CH3]+), 560 ([M – CO]•+), 545 ([M – CO – CH3]+), 471
([M – CO – OTMS]+), 73 ([TMS]•+).
The UV-Vis spectrum obtained during the electrolysis (Fig. 6) shows the
increase of bands at 292 and 442 nm with slight shoulder at 560 nm, which
indicates the formation of the compound 2. The absorption spectra of 2 at
the same pH were reported by Bettinger et al.18, giving λmax = 445 nm for 2
11
4 × 10
11
2 × 10
0
–7
0
1 × 10
–1
+
1/c , l mol
H
FIG. 5
The plot of 0.58 χ2/tK [AH] versus 1/[H+] for analytical concentration of hematoxylin 1.2 ×
10–3 mol l–1
0.1
A
0.0
300
400
500
600
λ, nm
FIG. 6
The change of UV-Vis absorption spectra of 1.1 × 10–5 M hematoxylin during the exhaustive
electrolysis at 0.55 V in phosphate buffer at pH 5.9
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

1108
Sokolová, Degano, Hromadová, Bulíčková, Gál, Valášek:
and 556 nm for its anion. This behavior is analogous to the oxidation path-
way of catechol studied by Petrucci et al.24.
The absorption spectra obtained during the electrolysis of 1 in phosphate
buffer at pH 9 (not shown) show sharp increasing absorption band at
560 nm, which is consistent with the formation of 2 (confirmed as the main
oxidation product also by GC-MS analysis). The electrolysis in buffered so-
lution at pH 9 proceeded very slowly and did not reach completion. The
curve 1 in Fig. 7 shows the absorption spectrum of the solution of 1 after
the electrolysis. When the solution was subjected to contact with air fur-
ther increase of the absorption band at 560 nm was observed. In addition,
a new absorption band at 720 nm appears (curve 2 in Fig. 7). These absorp-
tion bands completely disappear in 1 h (curve 3 in Fig. 7) due to the follow-
ing chemical reaction most likely the addition of water. This effect was
found by Herath et al.25, who described the complete disappearance of
the magenta color in oxidized state within a two hour irradiation in the
presence of a photosensitizer. The process dealing with the change of ab-
sorption bands (curves 2 and 3 in Fig. 7) does not occur under deaerated
conditions.
The strong influence of air oxygen on the absorption spectra of the com-
pound 2 can explain the certain inconsistence in values of pK of 1 and 2
reported in literature. The correct values of pK of 1 and 2 reported so far
0.3
A
0.2
2
0.1
3
1
0.0
200
300
400
500
600
700
800
900
λ, nm
FIG. 7
The absorption spectra of 1.14 × 10–5 M hematoxylin after the exhaustive electrolysis at 0.25 V
in phosphate buffer at pH 9 in deaerated cell (1), in the presence of oxygen in 600 s after the
opening of the spectrophotometric cell (2) and in the presence of oxygen in 1 h after opening
of the spectrophotometric cell (3)
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

Oxidation Pathways of Natural Dye Hematoxylin
1109
in literature cannot be identified without knowledge of the exact experi-
mental conditions used. This is due to the high unstability of 2 in contact
with oxygen, as we demonstrated in Fig. 7.
Voltammetry in Unbuffered Solutions
We recall that the elucidation of a pH dependent decomposition of 1 in
buffered media may be different from processes occurring in natural en-
vironment without a presence of pH buffering medium. Therefore we will
investigate the oxidation in unbuffered media, which is not a standard pro-
cedure in organic electrochemistry. Quan et al.26 used with advantage the
difference in unbuffered and buffered solution for a complete knowledge of
the redox processes of quinones.
In unbuffered solution the cyclic voltammograms have different shapes.
The oxidation in the unbuffered solution proceeds at more positive poten-
tials than in buffered solution due to the changes in reaction layer caused
by the local change of protons concentration. The main oxidation wave of
AH– anion at 0.52 V decreases at pH higher than 7.0 (Fig. 8). This decrease
with the increasing pH is in agreement with the distribution of species in
the solution (Fig. 3). The oxidation wave at 0.043 V rises and increases with
1
0
–1
–2
–3
–4
–5
0.5
0.0
E, V
FIG. 8
Cyclic voltammogram of 1.4 × 10–3 M hematoxylin under unbuffered conditions in 0.1 M KCl
on Pt microelectrode at pH 8.0 (1), 8.7 (2), 8.7 (3; second scan), 9.1 (4) and 10.0 (5) at the scan
rate of 0.05 V s–1. The pH of solution remained unchanged after the performing of the cyclic
voltammogram
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

1110
Sokolová, Degano, Hromadová, Bulíčková, Gál, Valášek:
the increasing pH. It corresponds to the irreversible oxidation of dianion
A2–. There is no cathodic wave when the potential scan is switched at
0.15 V (curve not shown for clarity). At low pH the phenoxycation is
formed (see Scheme 2) and gradually becomes the predominant form. This
is in agreement with the previous finding that the primarily generated
product should be stabilized at pH lower than 6.9. The potential of the oxi-
dation of dianion A2– depends on the apparent pH near the electrode
surface because under unbuffered conditions the concentration of H+/OH–
is not high enough compared to the concentration of the sample11. When
the potential scan is reversed at 0.7 V (curve 1 in Fig. 8), a new reduction
wave occurs at 0.0 V. This wave is related to the product of the subsequent
chemical reactions of phenoxycation AH+ formed during the oxidation at
0.52 V. The reversible reduction of keto-form of 2 resembles the reported
reduction of benzoquinones26. We suggest that it is the reversible reduction
of related quinone moiety
A + e–
A– .
(5)
Its oxidation counterpart is visible at the second scan (curve 3 in Fig. 8). An-
other oxidation wave at 0.185 V appears at pH > 9 (curve 4 in Fig. 8). Since
the main oxidation wave is the oxidation of AH– form of 1 and the oxida-
tion of dianion A2– occurs at 0.043 V, the oxidation wave at 0.185 V corre-
sponds to the oxidation of A–, which is the oxidation product of dianion
A2– – e–
A– .
(6)
The anodic peak potential of this wave agrees with the anodic peak poten-
tial of the redox pair A/A–, which appears during the second scan.
The exhaustive electrolysis at a constant anodic peak potential was per-
formed in order to obtain the oxidation products of 1 also in the un-
buffered solution (Fig. 9). The electric charge required for an exhaustive
electrolysis at constant anodic peak potentials 0.52 V corresponds to the
consumption of two electrons as in the case of buffered solutions. The elec-
trolysis at 0.52 V yields 2 as a main product and several minor products.
One of them is hydroxyhematoxylin 3 which can be formed by nucleo-
philic addition of water to keto-form of 2 (Scheme 4). This was confirmed
by the mass spectrum of derivatized hydroxyhematoxylin-5TMS, which
yields main peaks at m/z values 678 ([M]•+), 663 ([M – CH3]+), 660 ([M –
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

Oxidation Pathways of Natural Dye Hematoxylin
1111
H2O]•+), 645 ([M – H2O – CH3]+), 588 ([M – HOTMS]•+), 573 ([M – CH3 –
HOTMS]+), 483 ([M – CH3 – 2 HOTMS]+), 73 ([TMS]•+). Its mass spectrum is
consistent with the mass spectrum of derivatized 1: 662 ([M]•+), 647 ([M –
CH3]+), 572 ([M – HOTMS]•+), 557 ([M – CH3 – HOTMS]+), 483 ([M –
HOTMS – OTMS]+), 73 ([TMS]•+). The amount of this product is relatively
small (about 7%) due to the fact that in unbuffered solution the pH of solu-
tion changes during the electrolysis towards lower values. Under these con-
ditions the enol-form of 2 prevails and the nucleophilic addition of water
to keto-form is disfavored. The absorption spectrum of the solution oxi-
SCHEME 4
The formation of hydroxyhematoxylin 3
0.0
2
1
–5.0
0.5
0.0
E, V
FIG. 9
Cyclic voltammogram of 2.54 × 10–3 M hematoxylin in 0.1 M KCl on Pt microelectrode at the
scan rate of 0.05 V s–1 before (1) and after (2) electrolysis on Pt wire at 0.52 V
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

1112
Sokolová, Degano, Hromadová, Bulíčková, Gál, Valášek:
dized at 0.6 V (Fig. 10) shows an increase in the absorption bands at 204,
289 and 440 nm. During the electrolysis a new band is formed, next to the
one at 440 nm. The new band at 389 nm indicates that a product of oxida-
tion undergoes further chemical reaction. Similar behavior was mentioned
by Papouchado et al.7 in the case of catechol in 2 M perchloric acid, where
new absorption band matches completely the profile of the oxidized
1,2,4-trihydroxybenzene. The gas chromatogram of electrolyzed 1 at 0.6 V
shows two main products at the retention time tR = 32.80 and 33.34 min
in 3:1 ratio. This result is in agreement with absorption spectra performed
during the electrolysis (Fig. 10) and the absorption band at 389 nm can be
due to the presence of compound with shorter retention time. The mass
spectrum of this derivatized product (MW 646) has main peaks at m/z
values 646, 631, 589, 571 and 483. This oxidation product was not found
under buffered conditions. The product with a longer retention time was
identified by GC-MS analysis as the compound 2.
0.1
A
0.0
300
400
500
600
λ, nm
FIG. 10
The change of UV-Vis absorption spectra of 1.1 × 10–5 M hematoxylin during the exhaustive
electrolysis at 0.6 V in 0.1 M KCl
CONCLUSIONS
The oxidation mechanism of hematoxylin depends on the pH of solution
and its pathway is given by the distribution of various dissociation forms in
solution, which can be described using the known scheme ‘carré’. We con-
firmed that a two electron oxidation is preceded by the dissociation of
hematoxylin. Hemathein is a product of oxidation in a wide range of pH
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

Oxidation Pathways of Natural Dye Hematoxylin
1113
from 4.9 to 12. At pH higher than pK2, the reaction pathway leads to the
formation of a dianion, which is oxidized at lower potentials. We identified
that in an unbuffered solution also two redox steps are present, which were
clearly distinguished by cyclic voltammetry. They are in agreement with
the distribution of species in the solution. Hemathein is formed as the main
product also by oxidation of the unbuffered samples. Hydroxyhematoxylin
is a minor product, which results from nucleophilic addition of water on
keto-form of hemathein. We can conclude that the dye degradation in nat-
ural unbuffered environment yields hemathein and hydroxyhematoxylin,
which was absent in buffered solution.
This work was supported by the Czech Science Foundation (203/09/1607 and 203/08/1157) and
the Ministry of Education, Youth and Sports of the Czech Republic (COST OC140).
REFERENCES
1. Schweppe H.: Handbuch der Naturfarbstoffe. Vorkommen, Verlagsgesellschaft.
Landeberg/Lech, 1992.
2. Ferreira E. S. B., Quye A., McNab H., Hulme A. N.: Dyes in History and Archaeology 2002,
18, 63.
3. Zare H. R., Nasirizadeh N., Mazloum-Ardakani M., Namazian M.: Sens. Actuators, B 2006,
120, 288.
4. Zare H. R., Nasirizadeh N.: Sens. Actuators, B 2010, 143, 666.
5. Streꢀanský M., Pizzariello A., Streꢀanská S., Miertuš S.: Anal. Chim. Acta 2000, 415, 151.
6. Pizzariello A., Streꢀanský M., Streꢀanská S., Miertuš S.: Talanta 2001, 54, 763.
7. Papouchado L., Petrie G., Adams R. N.: J. Electroanal. Chem. Interfacial. Electrochem. 1972,
38, 389.
8. Nilsson A., Ronlan A., Parker V. D.: J. Chem. Soc., Perkin Trans. 1 1973, 2337.
9. Papouchado L., Sandford R. W., Petrie G., Adams R. N.: J. Electroanal. Chem. Interfacial.
Electrochem. 1975, 65, 275.
10. Bailey S. I., Ritchie I. M.: J. Chem. Soc., Perkin Trans. 2 1983, 645.
11. Bailey S. I., Ritchie I. M.: Electrochim. Acta 1985, 30, 3.
12. Hapiot P., Neudeck A., Pinson J., Fulcrand H., Neta P., Rolando Ch.: J. Electroanal. Chem.
1996, 405, 169.
13. Ryan M. D., Yueh A., Chen W.-Y.: J. Electrochem. Soc. 1980, 127, 1489.
14. Hotta H., Sakamoto H., Nagano S., Osakai T., Tsujino Y.: Biochim. Biophys. Acta 2001,
159.
15. Lalor G. C.: J. Soc. Dyers Colour 1962, 78, 549.
16. Morrow G. W.: Anodic Oxidation of Oxygen-Containing Compounds in Organic
Electrochemistry (H. Lund and O. Hammerich, Eds), 4th ed., p. 589. Marcel Dekker, Inc.,
New York 2001.
17. Masoud M. S., Hagaag S. S.: Indian J. Chem., Sect. A: Inorg., Phys., Theor. Anal. 1982, 21,
323.
18. Bettinger Ch., Zimmermann H. W.: Histochemistry 1991, 95, 279.
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114

1114
Sokolová, Degano, Hromadová, Bulíčková, Gál, Valášek:
19. Lalor G. C., Martin S. L.: J. Soc. Dyers Colour 1959, 75, 517.
20. Brown E. R., Sandifer J. R. in: Physical Methods of Chemistry (B. W. Rossiter and J. F.
Hamilton, Eds), Vol. II, pp. 273–314. John Wiley & Sons, Inc., New York 1986.
21. Koryta J.: Electrochim. Acta 1959, 1, 26.
22. Jacq J.: J. Electroanal. Chem. Interfacial. Electrochem. 1971, 29, 149.
23. Koutecký J.: Collect. Czech. Chem. Commun. 1953, 18, 597.
24. Petrucci R., Astolfi P., Greci L., Firuzi O., Saso L., Marrosu G.: Electrochim. Acta 2007, 52,
2461.
25. Herath A., Priyantha N., Rajapakse G., Karunaratne V., Wickramasinghe A.: J. Natl. Sci.
Foundation Sri Lanka 2007, 35, 239.
26. Quan M., Sanchez D., Wasylkiv M. F., Smith D. K.: J. Am. Chem. Soc. 2007, 129, 12847.
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 11, pp. 1097–1114