Photophysical and spectroscopic studies of indigo derivatives in their keto and leuco forms

Article abstract of DOI:10.1021/jp049076y

A comprehensive spectroscopic and photophysical study of the keto and leuco forms of indigo and three other ring-substituted derivatives in solution was performed. The characterization involves absorption, fluorescence, and triplet-triplet absorption spectra, making it possible to obtain the quantum yields for fluorescence (φ_F), singlet-triplet intersystem crossing (φ_ISC)> internal conversion (τ_IC), and lifetimes for fluorescence (τ_F) and triplet decay (τ _T). For the case of the keto forms, pulse radiolysis experiments have revealed the existence of a triplet acceptor (from energy transfer from different donors) for the indigo, purple, and indirubin compounds. It is shown that with the keto form the major deactivation pathway involves internal conversion from the lowest singlet excited state to the ground state whereas with the leuco form there is competition between internal conversion, triplet formation, and fluorescence deactivation processes. Furthermore, leuco forms present much higher Stokes shifts compared with keto ones, suggesting an excited-state geometry different from the ground-state geometry, possibly involving rotational photoisomerization.

Full text of DOI:10.1021/jp049076y

J. Phys. Chem. A 2004, 108, 6975-6981
6975
Photophysical and Spectroscopic Studies of Indigo Derivatives in Their Keto and Leuco
Forms
J. Seixas de Melo,*,† A. P. Moura,† and M. J. Melo‡
Chemistry Department, UniVersity of Coimbra, 3004-535 Coimbra, Portugal, and Departamento de
ConserVac¸a˜o e Restauro and ReQuimte, Faculdade de Cieˆncias e Tecnologia, UniVersidade NoVa de Lisboa,
Quinta da Torre, 2829-516 Monte de Caparica, Portugal
ReceiVed: March 1, 2004; In Final Form: June 1, 2004
A comprehensive spectroscopic and photophysical study of the keto and leuco forms of indigo and three
other ring-substituted derivatives in solution was performed. The characterization involves absorption,
fluorescence, and triplet-triplet absorption spectra, making it possible to obtain the quantum yields for
fluorescence (φF), singlet-triplet intersystem crossing (φISC), internal conversion (φIC), and lifetimes for
fluorescence (τF) and triplet decay (τT). For the case of the keto forms, pulse radiolysis experiments have
revealed the existence of a triplet acceptor (from energy transfer from different donors) for the indigo, purple,
and indirubin compounds. It is shown that with the keto form the major deactivation pathway involves internal
conversion from the lowest singlet excited state to the ground state whereas with the leuco form there is
competition between internal conversion, triplet formation, and fluorescence deactivation processes.
Furthermore, leuco forms present much higher Stokes shifts compared with keto ones, suggesting an excited-
state geometry different from the ground-state geometry, possibly involving rotational photoisomerization.
Introduction
The explanation of the blue of indigo and its derivatives is
an intriguing and fascinating subject and was explored with great
Indigo blue is one of the earliest and most popular dyestuffs
known to man. Its use dates back to the Egyptian and Roman
civilizations. Indigo (see Chart 1) is also one of the most stable
organic dyes, which explains not only its wide use in antiquity
and premodern times but also its longevity as a colorant. The
stability of indigo is also the reason it was used in oil paintings
by some of the great masters of the 17th and 18th centuries,
such as Rubens.1,2 In De bello Gallico, Julius Caesar describes
the warrior skin paintings of his Gaulish adversaries as being
obtained from a blue juice; their faces were frightening and they
believed that by this dyeing, the skin paintings would protect
them and turn them into invulnerable warriors! Tyrian purple
(see Chart 1) is another example of a ring-substituted derivative
of indigo and is one of the most important and powerful ancient
dyes. This was derived from the Mediterranean shell fish of
the genus Purpura, whose exhaustive extraction led to the first
historically reported ecological disaster.3,4 Surprisingly, in the
very beginning of the 21st century, indigo is still a important
modern dye. Making use of the concepts of green chemistry,
the production of indigo by microorganisms is being actively
investigated.5,6
The indigo derivatives are generally known as vat dyes and
are insoluble in water in their oxidized colored form, the keto
species.3,4,7 A common characteristic of these vat dyes is the
presence of one or more carbonyl groups that, when treated with
a reducing agent in the presence of an alkali, form a water-
soluble species known as the leuco form. This form is the water-
soluble intermediate used in the dyeing process.8,9 The most
well-known dye of this family, indigo blue, is still used to color
the world-famous blue jeans.9,10
detail and ingenuity during the 1970s and 1980s.11-14 It has
been found that the fundamental chromophore of the indigo dyes
includes the central double bond (connecting the two rings),
together with the nitrogen and carbonyl groups.13 Substitution
at different ring positions of indigo is likely to promote
significant shifts in both the long-wavelength visible and UV
bands.15,16
Despite the importance of indigo, few conclusive studies have
been carried out to clarify and rationalize the photochemistry
and photophysics of this molecule, particularly of its leuco form.
In 1994, George Wyman nicely reviewed the state of the art
for the spectroscopy of indigo and its derivatives known until
then, showing that several mysteries regarding indigo’s behavior
were still left to be explained.17 Because of the complicated
nature of the spectroscopy of indigo, Wyman reported that even
though he had not been working with these compounds for more
than 15 years, little or nothing had been done during that
period.17 Because of misleading results for indigo’s fluores-
cence,17,18 most of the work reported was for thioindigo
derivatives.17,19-21 In contrast to the behavior of the heteroatom
derivatives,19,21 the remarkable stability of indigo and its ring-
substituted derivatives has been attributed to intramolecular
hydrogen bonding between the two adjacent carbonyl and N-H
groups (see Chart 2), which keeps the molecule in a trans planar
configuration, preventing the photochemical cis-trans isomer-
ization.18,22 The internal conversion from the first singlet excited
state to the ground state has been shown to be much faster than
the other S1 deactivation processes (fluorescence, intersystem
crossing, and photochemistry). This was attributed, by some
authors, to fast intramolecular transfer of one or both protons
from nitrogen to oxygen.18 However, other groups later con-
cluded that this could not be the case.23
* To whom correspondence should be addressed. E-mail: sseixas@ci.uc.pt.
Fax: 351 239 827703.
Contrary to the behavior of the usual keto form of indigo22
† University of Coimbra.
‡ Universidade Nova de Lisboa.
and several of its derivatives,3,17,19-21,23 little attention has been
10.1021/jp049076y CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/04/2004

6976 J. Phys. Chem. A, Vol. 108, No. 34, 2004
Seixas de Melo et al.
CHART 1
CHART 2
Fluorescence decays were measured using a home-built
TCSPC apparatus with a D2/N2 filled IBH 5000 coaxial flash
lamp as the excitation source, Jobin-Yvon monochromator,
Philips XP2020Q photomultiplier, and Canberra instruments
TAC and MCA.31 The fluorescence decays were analyzed using
the method of modulating functions implemented by Striker.32
The fluorescence decays of the keto forms of the compounds
were obtained with picosecond resolution in an apparatus
described elsewhere.33
paid to the leuco forms24,25 and in particular to their photo-
physical properties. The present work aims to give more insight
on the spectroscopic and photophysical properties of some
indigo derivatives including indigo itself, purple, indigocarmine,
and indigo’s structural isomer indirubin (see Chart 1), in
particular the behavior of the corresponding leuco forms. A
better knowledge of the photophysics of the keto-leuco system
will permit a better understanding of the factors that influence
indigo photodegradation and also its lifetime as a painting
material and dye.
Triplet-singlet difference absorption spectra and yields were
obtained using an Applied Photophysics laser flash photolysis
equipment pumped by a Nd:YAG laser (Spectra Physics) with
excitation wavelengths of 355 nm (leuco forms) and 535 nm
(keto forms) as described elsewhere.34 First-order kinetics was
observed for the decay of the lowest triplet state. The transient
spectra (300-700 nm) were obtained by monitoring the optical
density change at 5-10 nm intervals, averaging at least 10
decays at each wavelength. Also, depending on the wavelength
region studied, two different photomultipliers were used:
Hamamatsu IP28 and Hamamatsu R928.
Experimental Section
Indigo was prepared according to the procedure elsewhere
described8 or purchased from Aldrich and used as received.
Purple was obtained following a published procedure.26,27
Indirubin was synthesized following the procedure described
in ref 28. The crystals were washed first with a solution of
aqueous methanol (1:1.5) and then with methanol. Indigocar-
mine Merck was used as received.
The solvents used were of spectroscopic or equivalent grade.
Ethanol and methanol were previously dried over CaO and then
distilled. Dimethylformamide (DMF) was distilled and left over
molecular sieves prior to and after use.
Indigo and its derivatives indirubin and purple are considered
vat dyes, meaning that being water-insoluble (except indigo-
carmine), prior to the dying process they must be converted
into a water-soluble form (leuco form), which is usually obtained
by reducing the dye with a strong reducing agent (sodium
dithionate in alkaline media) or alternatively through electro-
chemical reduction.29
Preparation of the Leuco Species. To a solution of the dye
in DMF (ethanol, methanol and benzene were also used as
solvents), submitted to constant and gentle bubbling with Ar,
2-3 drops of a concentrated sodium dithionate/NaOH solution
(0.15 g of sodium dithionate in 10 mL of a 1 M solution of
sodium hydroxide) were added. The resulting solution was
mixed, left bubbling for more 20 min, and then sealed.
Absorption and fluorescence spectra were recorded on Shi-
madzu UV-2100 and Jovin-Yvon Spex Fluorog 3-2.2 spectro-
photometers, respectively. Fluorescence spectra were corrected
for the wavelength response of the system. The fluorescence
quantum yield for the keto forms of the compounds were
determined using indigo (φF ) 0.001 in dichoroethane22). For
the leuco forms of the compounds the fluorescence quantum
yields were determined using quaterthiophene (φF ) 0.18 in
dioxane30) and quinquethiophene (φF ) 0.36 in dioxane30) as
standards.
In all cases the signal was assigned to a triplet state because
(i) it decayed by first-order kinetics with microsecond lifetimes
and (ii) in these experiments other possible transients, such as
radical ions, were not produced on photolysis. Additionally for
the keto forms these were found to be quenched by oxygen.
The triplet molar absorption coefficients for the leuco forms
of the indigo derivatives in DMF were determined by the singlet
depletion technique, according to the well-known relationship35
(ꢀS)(∆ODT)
ꢀT )
(1)
∆ODS
where both ∆ODS and ∆ODT are obtained from the triplet
transient absorption spectra, and triplet formation quantum yields
were derived from these and actinometry with benzophenone.
The φT value was obtained by comparing the ∆OD at 525 nm
of a benzene solution of benzophenone (the standard) and of
an aqueous solution of the compound (optically matched at the
laser excitation wavelength) using the following equation:36
Benzophenone
TT
Indigo derivative
∆OD
max
ꢀ
φTIndigo derivative
)
φBTenzophenone
Benzophenone
Indigo derivative
TT
ꢀ
∆OD
max
(2)
Benzophenone
TT
Benzophenone
- 1
- 1
with ꢀ
) 7200 M
cm
and φT
) 1.35
Pulse radiolysis experiments were carried out at the Free
Radical Research Facility, Daresbury, U.K., using 200 ns to 2
µs high-energy electron pulses from a 12 MeV linear accelerator
(LINAC), which were passed through solutions in a 2.5 cm
optical path length quartz cuvette attached to a flow system.
All solutions were bubbled with argon for 30-40 min before
experiments were taken. More details on the experimental setup
and other experiments details can be found elsewhere.37,38

Indigo Derivatives
J. Phys. Chem. A, Vol. 108, No. 34, 2004 6977
Figure 2. Fluorescence emission spectra in DMF for the keto (A) and
leuco (B) forms of the compounds studied. T ) 293 K.
Figure 1. Absorption spectra of the keto (full line) and leuco (dashed
line) forms of indigo obtained in DMF at T ) 293 K.
TABLE 2: Spectroscopic and Photophysical Data for the
Keto and Leuco (in Parentheses) forms of the studied
compounds in DMF solution at T ) 293 K
TABLE 1: Absorption Maxima and Extinction Coefficients
for the Keto and Leuco (in Parentheses) Forms of the
Compounds Studied in DMF Solution at T ) 293 K
a
b
λ
fluo(max)
(nm)
φF
τF
(ns)
kF
(ns-1
kNR
(ns-1
compd
indigo
λ
abs(max) (nm)
ꢀmax (M-1 cm-1
)
compd
indigo
)
)
610
(442)
601
(442)
618
(446)
546
22140
(17400)
12680
(13050)
8080
(17250)
9340
653
(523)
640
0.0023
(0.348)
0.0071
(0.225)
0.0015
(0.292)
0.14
0.0164 7.12
(3.15) (0.111) (0.207)
0.323 0.022 3.07
(3.77) (0.060) (0.206)
0.110 0.0136 9.08
(3.50) (0.083) (0.202)
purple
purple
(526)
indigocarmine
indirubin
indigocarmine 661
(529)
645
(486)
indirubin
0.00027 0.030 0.009
(0.0145) (1.78) (0.008) (0.554)
33.3
(390)
(24770)
b
a kF ) φF/τF. kNR ) (1 - φF)/τF.
Results
or no fluorescence17,18 was reported. The φF values for indigo,
Tyrian purple, and indigocarmine are on the order of 10-3, while
that for indirubin is an order of magnitude lower. Also, the
fluorescence lifetimes were found to be short, ranging from
0.110 to 0.323 ns for the indigotin type compounds, and even
lower for indirubin (0.030 ns) (Table 2). This indicates that
fluorescence is a nonefficient deactivation pathway for excited
indigo derivatives in their keto forms. In fact, the calculated
radiative rate constants are about 107 s-1 for indigotin-type
compounds and 106 s-1 for indirubin. On the other hand, the
radiationless rate constants display very high values, on the order
of 109 s-1 for the indigotin type molecules and 1010 s-1 for
indirubin. Therefore, the radiative constants for indigo, Tyrian
purple, and indigocarmine are 2 orders of magnitude lower than
the sum of the rate constants for the radiationless processes
(kNR). In the case of indirubin, the difference is now 4 orders
of magnitude (Table 2)! The values of the radiationless rate
constants are unusually high, which is consistent with molecules
possessing excited-state proton transfer in contrast to what was
suggested with some indigotin derivatives, where apparently this
process is not viable.23
For the leuco forms, the emission maxima are in the range
520-530 nm, i.e., a much narrower interval, with the exception
of the leuco form of indirubin, which is found to be ca. 30 nm
blue-shifted [λmax(em) ) 486 nm] relative to the indigotin
compounds (see Table 2). The fluorescence quantum yields for
the leuco indigo derivatives are now ca. 2 orders of magnitude
higher than those of the corresponding keto forms, with
fluorescence lifetimes now found on a nanosecond time scale
(Table 2 and Figure 3). Note that the radiative (kF) and
radiationless rate (kNR) constants have approximately the same
order of magnitude because of a decrease in the kNR and increase
in the kF values, turning fluorescence emission into an efficient
and competitive process for the deactivation of the excited state.
In this case, indirubin is no exception (although there is a
significant difference between the kF and kNR values), follow-
1. Absorption Spectra. Keto Forms. The UV-vis absorption
spectra of the four compounds (Chart 1) in dimethylformamide
(DMF) display structurally common bands for indigo, purple,
and indigocarmine consisting of a sharp band in the visible
region with a slight inflection or shoulder at shorter wavelengths.
In the case of indirubin the shoulder is absent. A representative
spectrum is shown in Figure 1 for the leuco and keto forms of
indigo. The wavelength maxima shifts are in the order indirubin
(546 nm) < purple (601 nm) < indigo (610 nm) < indigocar-
mine (618 nm) (Table 1). The molar absorption coefficients,
Table 1, with the exception of indigo (22 140 M-1 cm-1) are
low (∼10 000 M-1 cm-1).
Leuco Forms. Adding some drops of an aqueous solution of
sodium dithionite/sodium hydroxide to the DMF solution of the
dyes leads to the formation of their leuco forms (Figure 1). The
structures of these leuco forms have been clearly established
by NMR spectrometry and deuterium substitution.24,25 The
absorption spectra of the leuco forms of the compounds are blue-
shifted ca. 150 nm relative to their keto counterparts (Table 1
and Figure 1). In contrast to what was observed for the keto
forms, with the exception of indirubin, the absorption maxima
for the three indigotin-based compounds are very similar, ∼443
nm (Table 1). For the molar absorption coefficients (when
compared to values for the keto species) for indigo, a lower
value (17 400 M-1 cm-1) is observed. For Tyrian purple there
are no relevant changes (13 050 M-1 cm-1), whereas for
indigocarmine and indirubin the values doubled (17 250 and
24 770 M-1 cm-1, respectively).
2. Photophysical Properties. Fluorescence Emission Spectra
and Lifetimes. The fluorescence spectra of the compounds both
for their keto and leuco forms are shown in Figure 2. For the
four compounds studied, the emission wavelength maximum
is in the region 640-661 nm. The very low values of the
fluorescence quantum yields, φF, obtained for the keto forms
of the indigo derivatives (Table 2), are in agreement with
previous findings, where either very low values22 were observed

6978 J. Phys. Chem. A, Vol. 108, No. 34, 2004
Seixas de Melo et al.
Figure 4. Phosphorescence spectra of the leuco form of indigocarmine
in methanol at T ) 77 K (a few drops of the sodium dithionate/NaOH
aqueous solution were added to induce the formation of the leuco form).
Phosphorescence Emission Spectra. In the case of the of the
water-soluble indigocarmine, it was possible to make a low-
temperature luminescence study of the leuco form in methanol
and thus to obtain information on the nature of the lowest triplet
state. Figure 4 presents the phosphorescence spectrum of a
methanol solution of indigocarmin. A very weak band is
observed with a maximum at around 520 nm. A lifetime of 143
ms was obtained, and the excitation spectra collected with λem
) 520 nm mirror the absorption spectra of the leuco form of
indigocarmine. The proximity of the fluorescence (S1 state) and
phosphorescence (T1 state) emission bands at ∼529 and 520
nm, respectively, seems to explain the high efficiency of the
S1 ' T1 process in the leuco forms of indigocarmine when
compared to the low efficiency of this process for the keto
species. Note that the small blue shift of the phosphorescence
relative to the fluorescence band results from the fact that the
former is taken in a rigid matrix whereas the fluorescence
spectrum was obtained at room temperature.
Triplet State Characterization by Laser Flash Photolysis and
Pulse Radiolysis. Keto Forms. In general the triplet-triplet
transient absorption spectra observed for the keto forms of the
compounds studied are very weak or negligible, in agreement
with negative results from previous studies on these systems.
This is not, however, the case for diacyl and rigid trans and cis
indigo analogues, where triplet transients could be obtained and
characterized at low temperature under direct excitation,40 or
for thioindigo ring-substituted compounds, where it has been
found that the triplet state plays a role in the cis f trans
isomerization of these compounds.41
In the present study, the observation of a transient absorption
generated from direct excitation of the compound was only
possible for purple in DMF solution (Figure 5). The spectrum
is vibrationally resolved, and although weak, it was reproducible
(Figure 5).
Attempts to obtain transient triplet-triplet absorption spectra
under various conditions were made. Pulse radiolysis energy
transfer experiments using biphenyl and naphthalene as triplet
donors to indigo, purple, and indirubin were performed (Figure
6). In all these cases weak signals were obtained, and although
quenching of the donors were observed, the absorptions were
not intense enough to allow quantitative interpretation of the
data. Moreover, in the pulse radiolysis experiments, benzene
had to be used as a solvent, and under these conditions the
solubility of the dyes is very low, reducing the sensitivity of
the signals.
Figure 3. Fluorescence decays and pulse instrumental response
obtained for the keto and leuco forms of indigocarmine in DMF at T
) 293 K. The experimental conditions and obtained decay times are
shown as insets in the figure. Also shown are the weighted residuals,
autocorrelation functions (A.C.) and the ø2 values for a better judgment
of the quality of the fits.
ing the same trends as the indigotin derivatives, even though
the observed fluorescence quantum yields are lower and the
lifetimes shorter.
For the keto forms of the indigo derivatives, the observed
Stokes shifts (taken as the difference between the maxima of
the absorption and emission spectra) are small, around 40 nm,
with the exception of indirubin whose value is ∼100 nm (see
Tables 1 and 2). It is interesting to note that the leuco forms of
the indigo derivatives present more pronounced Stokes shifts
(∆SS) than the keto forms. With the exception of indirubin,
which has an identical shift of 100 nm in the leuco and keto
forms, the Stokes shifts for the leuco species are about 81-84
nm (∼3500 cm-1).
The small Stokes shifts and the mirror-image relationship (see
Figures 1 and 2A) between the absorption and emission bands
of the keto forms favor the hypothesis that identical conforma-
tional rigidity and structure are found in both the ground and
singlet-excited states. This does not, however, occur with the
leuco forms, where the much more pronounced Stokes shift
suggests an excited-state species that is structurally different
from the ground-state species. In addition, these show Franck-
Condon (F.-C.) forbidden band shapes in the absorption spectra,
which suggests, in terms of the F.-C. principle,39 differences in
the relative location of potential energy minima of ground and
excited states. This once more indicates that there is a marked
change in structure between the ground and first excited singlet
state, where some considerable degree of conformational
mobility (conformational isomerism) is possible (see Discus-
sion).
Triplet states can be selectively produced42 by energy transfer
following pulse radiolysis of benzene (Bz) solutions of indigotin
or indirubin in the presence of appropriate sensitizers (biphenyl,

Indigo Derivatives
J. Phys. Chem. A, Vol. 108, No. 34, 2004 6979
Figure 5. Transient triplet-triplet spectra obtained by direct excitation
of purple in DMF at T ) 293 K. Each data point is the result of an
average of 15 shots.
Figure 7. Transient triplet-triplet absorption spectra obtained for (A)
the leuco forms of the four studied compounds and (B) for leuco-
indirubin obtained with different delay times after laser flash at T )
293 K.
naphthalene itself was found to decay with a lifetime of 62 µs
(decay collected at 420 nm). When biphenyl was used as a
donor, the same weak signal at around 670 nm was found
(Figure 6A), again indicating the formation of the transient triplet
that is observed under the experimental conditions reported in
Figure 5 (direct excitation of purple).
For indigo, similar experiments were performed using either
biphenyl or naphthalene as donors (Figure 6B). In the case of
biphenyl acting as the donor, a very weak signal between 450
and 700 nm together with a strong maximum at ∼360 nm
(biphenyl absorption) was observed in this situation. Although
the biphenyl triplet lifetime decreases in the presence of indigo
(τT ) 16 µs in the presence vs τT ) 56 µs in the absence),
which is a clear sign that energy transfer from the biphenyl
donor to indigo is occurring, the weakness of the signal
precludes determination of the molar extinction coefficient for
the generated transient. With naphthalene as the donor, similar
signals were obtained in the 440-700 nm region (Figure 6B),
although now the strong maximum is found at 420 nm (Np
triplet maxima). Again, a decrease in the naphthalene triplet
lifetime is obtained in the presence of indigo (τT ) 24.5 µs in
the presence vs τT ) 62 µs in the absence), indicating once
more that energy transfer from the naphthalene donor to indigo
acceptor is occurring.
Figure 6. Spectral data observed following pulse radiolysis of argon
saturated solutions of (A) purple (2.38 × 10-6 M) and biphenyl (10-2
M) in benzene and of (B) indigo (8 × 10-6 M) and naphthalene (10-2
M) in benzene at T ) 293 K.
SCHEME 1
Leuco Forms. In contrast to what was observed for the keto
compounds, for the leuco forms of the four compounds studied,
intense and clear triplet signals were obtained. The triplet
absorption spectra for the leuco forms of the compounds are
presented in Figure 7. A strong depletion in the singlet
absorption region followed by intense signals in the 450-600
nm visible region can be observed. The triplet maxima (∼540
nm) and band shapes seem to be approximately identical for
the compounds with the exception of indirubin, which possesses
a much broader band with a blue-shifted maximum (∼446 nm).
Triplet intersystem crossing yields are usually parameters whose
absolute value contains a degree of uncertainty that depends,
among other things, on the method and techniques used.35,44 In
the present case, these were determined by the singlet depletion
technique,35,44 through eqs 1 and 2 (see Experimental Section).
The criterion for the use of this technique implies absence of
significant triplet absorption in the depletion region. In our case,
since the depletion bands are strong and their maxima identical
to those obtained in ground-state absorption spectra (see Table
1), this seems to indicate that little, or no triplet, is absorbing
in the singlet depletion region. It is therefore possible to obtain
the extinction coefficients for the triplet absorptions (eq 1), such
that with the subsequent use of actinometry with benzophenone
Bp, or naphthalene, Np, for example). A better understanding
of the reactions occurring can be seen in Scheme 1. This is
subject to the kinetically demanded concentration ratio [Bz] .
[Bp(Np)] . [indigotin].43
An argon saturated solution of purple was studied by pulse
radiolysis in benzene solution in the presence of naphthalene
(0.01 M) as the triplet sensitizer. Following pulse radiolysis,
an initial absorption at 420 nm due to the naphthalene triplet
was replaced by a new absorption at around 670 nm. The decay
time for the naphthalene triplet (62 µs) was essentially identical
to the grow-in of the new absorption at 640 nm (with a rise
time of 61 µs), indicating that energy transfer from naphthalene
to purple occurred. Under the same experimental conditions

6980 J. Phys. Chem. A, Vol. 108, No. 34, 2004
Seixas de Melo et al.
TABLE 3: Transient Triplet-Triplet Parameters [Extinction Coefficients (ETT), Wavelength Maxima (λT fT ), Triplet Lifetimes
(τT), and Intersystem Crossing Triplet Yields (OT)] and Internal Conversion Yield (OIC) Together with thmeaxRate Constants
Associated with These Processes and the Fluorescence Process (OF in Table 2) for the Leuco Forms of the Indigo Derivatives in
DMF at T ) 293 K
1
n
T1fTn
max
compd
ꢀTT (M-1cm-1
)
λ
(nm)
τT (µs)
φT
φIC
kF (ns-1
)
kIC (ns-1
)
kISC (ns-1
)
indigo
purple
indigocarmine
indirubin
12 210
18 900
16 240
23 130
540
15
20
18
80
0.125
0.190
0.250
0.171
0.527
0.585
0.458
0.815
0.111
0.060
0.083
0.008
0.167
0.155
0.131
0.458
0.040
0.050
0.070
0.096
538
540
446
(eq 2) it is possible to obtain the intersystem crossing triplet
yields (see Table 3).
the excited-state cis-trans isomerization and photochromic
properties of these derivatives.45 This loss of planarity involves
the two carbons of the central bond that adopt a slightly
pyramidal bonding geometry and undergo a hybridization
change from sp2 toward sp3.45
Discussion
The main experimental results obtained for the systems under
study, both in their leuco and keto forms, are given in Tables
1-3. For the leuco forms, the complete photophysical charac-
terization has been possible, and the complete set of results is
summarized in Tables 2 and 3.
Concerning the explanation for the absolute prevalence of
the radiationless deactivation pathway in the colored keto forms
of the indigo type compounds, we comment on some of the
arguments discussed in the literature, in particular on the data
reported by Elsaesser et al.23 In this work, on the basis of a
pioneer picosecond infrared study, it was concluded that proton
transfer did not take place in the S1 state of the indigo derivative
4,4′,7,7′-tetramethylindigo (TMI). TMI was chosen, in place of
indigo, because of its higher solubility in organic nonpolar
solvents and also because, as said by the authors, the four methyl
groups prevent the formation of intermolecular H bridges. On
the basis of the results obtained for TMI, it was stated that “the
results give convincing proof that proton transfer does not occur
in the first electronic state of indigo.”23 This is the reference
paper that has been used to exclude ESPT as being responsible
for the high photostability of indigo.22 Moreover, in this paper
for TMI in water, the reported value for the lifetime of S1 is 30
ps, and for the fluorescence quantum yield it is 0.0014.23 While
in the case of the fluorescence quantum yield the values are in
reasonable agreement with our data for indigo (Table 2), the
same cannot be said for the fluorescence lifetime. If, as claimed,
the introduction of the four methyl groups avoids intermolecular
hydrogen bridge formation, the decrease from 140 ps (Table 2
for indigo) to 30 ps23 could be considered to be due to a more
efficient internal conversion in the case of the substituted indigo.
Yet in our case, a value of 30 ps was obtained only for indirubin
(see Table 2). This low lifetime,23 which is consistent with that
of indirubin, could point to a nonplanar structure for TMI and
therefore suggests that some caution should be taken concerning
the generalization of the absence of ESPT from TMI to the
indigo molecule.
Another important seminal work on the photophysics of
indigo, based on picosecond transient absorption studies, was
published by Kobayashi et al.18 Their main conclusions are in
agreement with our results, namely, that “the absence of
fluorescence of indigo can be explained in terms of a very fast
internal conversion from the lowest excited singlet state (S1) to
the ground state (S0)”.18 Here, the word “absence” should instead
be read as “very little” as attested by our data. In that work it
was proposed that ESPT could enhance the internal conversion
process.18 They observed in water (a bad solvent for the keto
form of indigo) a value of 270 ps for the relaxation of indigo
to the ground state, although the repopulation of its ground state
was found to be characterized by a lifetime of 1.5 ns.18 To
explain such observations, the authors have suggested the decay
of the singlet state to an intermediate state having a lifetime
270 ps, which was followed by decay to the ground state within
1.5 ns.18 However, the nature of this intermediate was not clearly
established.18
After determination of both the fluorescence quantum yields
and the intersystem crossing triplet yields, it is now possible to
establish the balance between all the yields and rate constants
for the deactivation processes occurring with the leuco forms
of the indigo derivatives (see Table 3). For the leuco species,
the data in Table 3 show that with the exception of indirubin
the fluorescence, intersystem crossing (kISC), and internal
conversion (kIC) rate constants are of the same order of
magnitude, meaning that these processes compete for the
excited-state deactivation. It is also observed that the level and
kind of substitution in the fundamental indigotin moiety
determine the balance between the different deactivation
processes.11 The introduction of bromine (purple) and sulfur
(sulfoxide group in indigocarmin) increases the intersystem
crossing triplet yield as a consequence of the classical heavy
atom effect.39 There is also a decrease in the fluorescence
quantum yield and no significant change in the internal
conversion quantum yield with this pattern of substitution,
indicating that the increase in φT is made at the expense of the
fluorescence channel (see Tables 2 and 3). A similar conclusion
can be drawn from the rate constant data. The results obtained
for these leuco species are in contrast with the general behavior
found with the keto forms (Table 2), where the radiationless
channels rule the deactivation processes.17,22
The different behavior found for indirubin in its keto and
leuco forms clearly shows that the structure influences the
spectroscopic and photophysical properties of these compounds
(Tables 2 and 3). Rotational photoisomerization could occur
with indirubin, and this would explain the high Stokes shifts
values observed. In fact, note that in the case of indigotin
compounds, intramolecular hydrogen bonding between the
amine hydrogen and the carbonyl oxygen (Chart 2A) possibly
avoids rotation around the carbon-carbon central bond, whereas
in the case of indirubin, intramolecular hydrogen bonding can
be established in one individual indene ring (Chart 2B) liberating
the molecule to freely rotate around the carbon-carbon central
bond. The same line of argument is valid for the possibility of
rotational isomerization to occur with the leuco form of the
indigo derivatives (absence of intramolecular hydrogen bond-
ing). In fact, it seems reasonable to associate the larger ∆SS
values in the leuco forms with some degree of rotation around
the central CdC bond. This phenomenon has been observed
for indigo derivatives with bulky groups on the nitrogen atom,
such as N,N′-dimethylindigo45 or N,N′-diacetylindigo46 where
this kind of substitution destroys the planarity of the indigo
structure, in particular around the double bound, thus favoring
Without excluding ESPT, a possible explanation for the fast

Indigo Derivatives
J. Phys. Chem. A, Vol. 108, No. 34, 2004 6981
(2) Novotna´, P.; Boon, J. J.; vand der Horst, J.; Paca´kova´, V. Color.
Technol. 2003, 119, 121.
(3) Clark, R. J. H.; Cooksey, C. J.; Daniels, M. A. M.; Withnall, R.
EndeaVour 1993, 17, 191.
(4) Cooksey, C. J. Molecules 2001, 6, 736.
(5) Berry, A.; Dodge, T. C.; Pepsin, M.; Weyler, W. J. Ind. Microbiol.
Biotechnol. 2002, 28, 127.
(6) Padden, A. N.; Dillon, V. M.; John, P.; Edmonds, J.; Collins, M.
D.; Alvarez, N. Nature 1998, 396, 225.
internal conversion rate constants values lies in the small energy
difference between the ground and lowest singlet excited state.
In fact, as is well-known from the golden rule for radiationless
transitions, a small value in the S1-S0 energy gap favors
nonradiative vs radiative decay pathway by increasing the
vibrational overlap (Franck-Condon integrals)39 of the wave
functions for the nonradiative modes of these two states, which
in consequence gives rise to indigo’s blue color and also to its
magnificent photostability.
(7) Se´quin-Frey, M. J. Chem. Educ. 1981, 58, 301.
(8) Seixas de Melo, J.; Barroso, M. Bol. Soc. Port. Quim. 2000, 81,
66.
Conclusions
(9) Brown, T. M.; Cooksey, C. J.; Dronsfield, A. T. Educ. Chem. 2001,
38, 69.
(10) Balfour-Paul, J. Indigo; British Museum Press: London, 2000.
(11) Klessinger, M. Angew. Chem., Int. Ed. Engl. 1980, 19, 908.
(12) Klessinger, M.; Lu¨ttke, W. Tetrahedron 1963, 19 (Suppl. 2), 315.
(13) Wille, E.; Lu¨ttke, W. Angew. Chem., Int. Ed. Engl. 1971, 10, 803.
(14) Pfeifer, G.; Otting, W.; Bauer, H. Angew. Chem., Int. Ed. Engl.
1976, 15, 52.
(15) Sadler, P. W. Spectrochim. Acta 1960, 16, 1094.
(16) Sadler, P. W. J. Org. Chem. 1956, 21, 316.
(17) Wyman, G. M. EPA News Lett. 1994, 50, 9.
(18) Kobayashi, T.; Rentzepis, P. M. J. Chem. Phys. 1979, 70, 886.
(19) Wyman, G. M.; Zarnegar, B. M.; Whitten, D. G. J. Phys. Chem.
1973, 77, 2584.
(20) Wyman, G. M.; Zarnegar, B. M. J. Phys. Chem. 1973, 77, 831.
(21) Wyman, G. M.; Zarnegar, B. M. J. Phys. Chem. 1973, 77, 1204.
(22) Miliani, C.; Romani, A.; Favaro, G. Spectrochim. Acta, Part A 1998,
54, 581.
(23) Elsaesser, T.; Kaiser, W.; Lu¨ttke, W. J. Phys. Chem. 1986, 90, 2901.
(24) Voss, G. J. Soc. Dyers Colour. 2000, 116, 87.
(25) Voss, G.; Schramm, W. HelV. Chim. Acta 2000, 83, 2884.
(26) Cooksey, C. J. Dyes History Archaeol. 1994, 13, 7.
(27) Clark, R. J. H.; Cooksey, C. J. New J. Chem. 1999, 323.
(28) Clark, R. J. H.; Cooksey, C. J.; Daniels, M. A. M.; Withnall, R.
Educ. Chem. 1996, 16.
(29) Roessler, A.; Crettenand, D.; Dossenbach, O.; Marte, W.; Rys, P.
Electrochim. Acta 2002, 47, 1989.
The photophysical properties of indigo and three of its
selected derivatives have been studied in their oxidized (keto)
and reduced (leuco) forms. For the first time, detailed singlet
and triplet excited-state studies are presented for the leuco forms,
including fluorescence (spectra, quantum yields, and lifetimes),
triplet absorption spectra, intersystem crossing triplet yields, and
triplet lifetimes. As a consequence of this, it has been possible
to obtain for the leuco species all the excited-state rate constants,
allowing a full photophysical characterization of these systems.
It was shown that completely different spectroscopic and
photophysical parameters are presented by the two forms (leuco
and keto). The comparison between the photophysical properties
of the leuco and keto forms revealed that for all the keto forms
studied the radiationless rate constants are lower by ca. 1 order
of magnitude than those found for the leuco forms. As a
consequence, fluorescence and triplet state formation are, in the
case of the leuco forms, alternatives to the internal conversion
and efficient deactivation channels for the singlet excited state.
In the case of the keto forms, the very low energy difference
between the ground and first excited singlet states (which will
enable significant overlap of the vibrational levels of the two
states) seems to be the main contributory factor to the high
values observed for the radiationless rate constants. It cannot,
however, be excluded that excited-state proton transfer could
play a part in the observed photostability.
(30) Becker, R. S.; Seixas de Melo, J.; Macanita, A. L.; Elisei, F. J.
Phys. Chem. 1996, 100, 18683.
Furthermore, the high Stokes shift observed in the leuco
systems, as well as for indigo’s structural isomer indirubin, in
comparison with the small values for the keto forms suggests a
lack of rigidity, in the excited state, for the former structures
when compared with the rigid structure of the keto form.
The differences observed between the two forms suggest that
the well-known photostability of the keto forms of indigo may
not be present with the reduced species.
(31) Seixas de Melo, J.; Fernandes, P. F. J. Mol. Struct. 2001, 565-
566, 69.
(32) Stricker, G.; Subramaniam, V.; Seidel, C. A. M.; Volkmer, A. J.
Phys. Chem. B 1999, 103, 8612.
(33) Lima, J. C.; Abreu, I.; Brouillard, R.; Mac¸anita, A. L. Chem. Phys.
Lett. 1998, 298, 189.
(34) Seixas de Melo, J.; Silva, L. M.; Kuroda, M. J. Chem. Phys. 2001,
115, 5625.
(35) Carmichael, I.; Hug, G. L. J. Chem. Phys. Ref. Data 1986, 15, 1.
Even if the enigma of cis-indigo, as described by George
Wyman,17 is yet to be solved, we hope that our results will
contribute to a better understanding of these dazzling molecules.
(36) Kumar, C. V.; Qin, L.; Das, P. K. J. Chem. Soc., Faraday Trans.
2 1984, 80, 783.
(37) Holder, D. J.; Allan, D.; Land, E. J.; Navaratnam, S. Presented at
the European Particle Accelerator Conference, Paris, 2002 (available at
http://www.frrf.dl.ac.uk/MOPLE116.pdf).
Acknowledgment. Financial support from FCT (Portuguese
Science Foundation) is acknowledged. J.S.M. acknowledges
Prof. A. Mac¸anita (IST) and Dr. J. C. Lima (FCT-UNL) for
letting him use the picosecond TCSPC equipment to obtain the
decay times of the keto forms of the compounds studied, Dr.
S. Navaratnam (Free Radical Research Facility, Daresbury
Laboratory) for his assistance with the pulse radiolysis experi-
ments, and finally Prof. Hugh Burrows for a critical reading of
the manuscript. S. Moura is gratefully acknowledged for the
synthesis of 6,6′-dibromoindigo. The second-year graduate
Conservation students I. Silva, M. Sousa, and S. Sequeira, as
well as Dr. A. J. Parola, are gratefully acknowledged for the
indirubin sample.
(38) Burrows, H. D.; Seixas de Melo, J.; Forster, M.; Guntner, R.; Scherf,
U.; Monkman, A. P.; Navaratnam, S. Chem. Phys. Lett. 2004, 385, 105.
(39) Becker, R. S. Theory and Interpretation of Fluorescence and
Phosphorescence; Wiley-Interscience: New York, 1969.
(40) Gorner, H.; Pouliquen, J.; Kossanyi, J. Can. J. Chem. 1987, 65,
708.
(41) Kirsch, A. D.; Wyman, G. M. J. Phys. Chem. 1977, 81, 413.
(42) Monkman, A. P.; Burrows, H. D.; Miguel, M. D.; Hamblett, I.;
Navaratnam, S. Chem. Phys. Lett. 1999, 307, 303.
(43) Burrows, H. D.; Seixas de Melo, J.; Serpa, C.; Arnaut, L. G.;
Miguel, M. d. G.; Monkman, A. P.; Hamblett, I.; Navaratnam, S. Chem.
Phys. 2002, 285, 3.
(44) Seixas de Melo, J.; Burrows, H. D.; Svensson, M.; Andersson, M.
R.; Monkman, A. P. J. Chem. Phys. 2003, 118, 1550.
(45) Miehe, G.; Su¨sse, P.; Kupcik, V.; Egert, E.; Nieger, M.; Kunz, G.;
Gerke, R.; Knieriem, B.; Niemeyer, M.; Lu¨ttke, W. Angew. Chem., Int.
Ed. Engl. 1991, 30, 964.
References and Notes
(1) van Eikema Hommes, M. Paper presented at the 20th Annual
Meeting of Dyes in History and Archaeology, Amsterdam, 2001.
(46) Grimme, G.; Grimme, S.; Jones, P. G.; Boldt, P. Chem. Ber./Recl.
1993, 126, 1015.