Aggregation of surfactant squaraine dyes in aqueous solution and microheterogeneous media: Correlation of aggregation behavior with molecular structure

Article abstract of DOI:10.1021/ja9523911

The synthesis of several amphiphilic squaraine dyes and a study of their aggregation behavior and photophysics are reported. The several different squaraines are found to give spectrally blue-shifted aggregates in aqueous and mixed aqueous-organic solutio

Full text of DOI:10.1021/ja9523911

2584
J. Am. Chem. Soc. 1996, 118, 2584-2594
Aggregation of Surfactant Squaraine Dyes in Aqueous Solution
and Microheterogeneous Media: Correlation of Aggregation
Behavior with Molecular Structure
Huijuan Chen,† Mohammad S. Farahat,† Kock-Yee Law,*,§ and
David G. Whitten*,†
Contribution from the NSF Center for Photoinduced Charge Transfer, Department of Chemistry,
UniVersity of Rochester, Rochester, New York 14627, and Xerox Corporation, Wilson Center for
Research and Technology, 800 Phillips Road, 114-39D, Webster, New York 14580
ReceiVed July 19, 1995X
Abstract: The synthesis of several amphiphilic squaraine dyes and a study of their aggregation behavior and
photophysics are reported. The several different squaraines are found to give spectrally blue-shifted aggregates in
aqueous and mixed aqueous-organic solution and in microheterogeneous media (bilayer vesicles). While in some
cases an intermediate dimer can be detected in the monomer to aggregate conversion process, in others direct conversion
of monomer to aggregate is observed. The aggregation number can be determined together with the equilibrium
constant and thermodynamic parameters for some of the squaraines in different environments. In several cases the
aggregation number is found to be ca. 4. The finding of a strong induced circular dichroism signal when the aggregate
(but not dimer or monomer) is generated in the presence of a chiral host (or counterion) suggests that the aggregate
is chiral. From these results and molecular simulations indicating that an extended monolayer of some of the squaraines
adopts a glide or herringbone lattice we propose a chiral “pinwheel” structure for the unit aggregate and suggest that
extended aggregate structures or crystals may be a mosaic of these unit aggregates. In contrast to the monomers,
which are strongly fluorescent, the squaraine dimers and aggregates are nonfluorescent and have extremely short
exciton lifetimes, as indicated by transient spectroscopy.
Introduction
of Kasha and Hochstrasser11,12 and Kuhn and co-workers13,14
predict shifts similar to those observed in the crystals for
molecular arrangements in aggregates, it is reasonable to assume
that many squaraine aggregates exhibiting similar absorption
spectra in microcrystals,7-10 films,3,15,16 or other media17 may
show similar intermolecular arrangements. Interestingly, we
have found that several surfactant or amphiphilic squaraines
form stable monolayer films at the air-water interface that can
be transferred to optically transparent rigid supports.15,18 Not
surprisingly we find that most of the freshly prepared Lang-
muir-Blodgett (LB) films of these squaraines show blue-shifted
or “H” aggregates which would be anticipated if the molecules
are arranged in the film in a simple “card-pack” array. However,
in a number of cases we have found that heating of these films
results in their conversion to a red-shifted aggregate, a process
which may be reversed by heating the product films in a humid
atmosphere.3,15 We have also carried out Monte Carlo simula-
tions on some of these squaraine amphiphiles which suggest
that the most stable arrangement in a monolayer film is a glide
or “herringbone” lattice in which a more complex arrangement
than a simple card pack is present.15,19 Calculations of the
exciton shifts for such a structure predict a blue-shift for the
Squaraine dyes are intriguing compounds both due to their
unusual electronic structures as well as to their properties as
semiconductors and photogeneration materials in electropho-
tography.1 Most applications using squaraine derivatives em-
ploy them as solids, usually microcrystalline, in which there is
clear indication that the squaraine chromophores are ag-
gregated.1,2 Several studies have shown that squaraines can exist
as different kinds of aggregates, with at least two prominent
limiting forms commonly occurring.3-5 In a recent study, the
type of aggregation for a number of squaraines has been shown
to depend on the structure of the chromophores in mixed-solvent
experiments.6 Studies of crystalline solids show, for example,
that bis(4-methoxyphenyl)squaraine exists in the solid as a “card
pack” array in which the planar squaraines are stacked verti-
cally.7 This structure exhibits a spectrum strongly blue-shifted
compared to that of the monomer in dilute solution.2 In contrast,
bis(2-methyl-4-(dimethylamino)phenyl)squaraine forms crystals
in which the squaraine chromophores are in a “slipped stack”
arrangement; in this case the crystals show a prominent transition
strongly red-shifted compared to the solution monomer.8 The
behavior of other solid squaraines usually shows one of these
characteristic spectroscopic featuressblue-shifted corresponding
to an “H” aggregate or red-shifted characteristic of a “J”
aggregate, respectively.9,10 Since the classical exciton treatments
(5) Das, S.; Thanulingam, T. L.; Thomas, K. G.; Kamat, P. V.; George,
M. V. J. Phys. Chem. 1993, 97, 13620.
(6) McKerrow, A. J.; Buncel, E.; Kazmaier, P. M. Can. J. Chem. 1995,
73, 1605.
* Authors to whom correspondence should be addressed.
† University of Rochester.
(7) Farnum, D. G.; Neuman, M. A.; Suggs, W. T., Jr. J. Cryst. Mol.
Struct. 1974, 4, 199.
(8) Wingard, R. E. IEEE Ind. Appl. 1982, 1251.
(9) Tristani-Kendra, M.; Eckhardt, C. J. J. Chem. Phys. 1984, 81, 1160.
(10) Bernstein, J.; Goldstein, E. Mol. Cryst. Liq. Cryst. 1988, 164,
213.
§ Xerox Corporation.
X Abstract published in AdVance ACS Abstracts, March 1, 1996.
(1) Law, K. Y. Chem. ReV. 1993, 93, 449.
(2) Law, K. Y. J. Phys. Chem. 1988, 92, 4226.
(3) Liang, K.; Law, K. Y.; Whitten, D. G. J. Phys. Chem. 1994, 98,
13379.
(11) Kasha, M.; Rawls, H. R.; El-Bayoumi, M. Pure Appl. Chem. 1965,
11, 371.
(4) Buncel, E.; McKerrow, A.; Kazmaier, P. M. J. Chem. Soc., Chem.
Commun. 1992, 1242.
(12) Hochestrasser, R. M.; Kasha, M. Photochem. Photobiol. 1964, 3,
317.
0002-7863/96/1518-2584$12.00/0 © 1996 American Chemical Society

Surfactant Squaraine Dyes in Aqueous Solution
J. Am. Chem. Soc., Vol. 118, No. 11, 1996 2585
nol, ethyl acetate, hexane, methylene chloride, chloroform, dimethyl
sulfoxide, and concentrated hydrochloric acid were certified grade from
Fisher. All solvents for spectroscopic studies were spectroscopic grade
from Fisher or Aldrich. Suitable water was obtained by passing purified
in-house distilled water through a Millipore-RO/UF water purification
system. Deuterated solvents were purchased from MSD Isotopes or
Cambridge Isotope Laboratories.
Melting points were taken on a Mel-Temp II melting point apparatus
and were uncorrected. Potassium phosphate solutions (10 mM, both
monobasic and dibasic) were used to adjust the pH to 7.5 in aqueous
solution studies in the cases when it was necessary. The pH values
were measured with an Orion Research digital ionalyzer/501 pH meter.
Infrared spectra were obtained on a Matheson Galaxy 6020 FTIR.
Proton NMR spectra were recorded on a General Electric QE300 MHz
spectrometer using deuterated solvent locks. FAB mass spectra were
measured at the Midwest Center of Mass Spectrometry. Elemental
analyses were performed by Galbraith Laboratories, Inc. (Knoxville,
TN) or Desert Analytics (Tucson, AZ). Absorption spectra for both
solution and solid state studies were obtained on a Hewlett-Packard
8452A diode array spectrophotometer. The circular dichroism study
was carried out on a JASCO J-710 spectropolarimeter. Fluorescence
spectra were recorded on a SPEX Fluorolog-2 spectrofluorometer and
are uncorrected.
Bilayer vesicles were prepared according to reported procedures.24
Solutions of squaraine or mixtures of squaraine and phospholipid in
chloroform were transferred to a scintillation vial; the solvent was
evaporated by flushing with nitrogen, the residue in the vial was dried
in a vacuum desiccator overnight. An appropriate volume of Mili-Q
water was added to the vial, and the temperature was controlled at
∼70 °C for 10 min in order to hydrate the residue. The mixture was
then sonicated with a Cell Disrupter W220F from Heat Systems-
Ultrasonics, Inc. (setting 6.5, 35 w) for three 5 min periods with an
intervening 5 min delay for cooling. The stock vesicle solution was
centrifuged for 30 min at 3500 rpm using a Fisher Scientific centrifuge;
the top portion of solution was carefully taken out with a pipette. Size
extrusion experiments for vesicle systems were performed with an
extruder through CoStar Nuclepore Polycarbonate (PC) filters. Light
scattering measurements were carried out at 20 °C, and all samples
were routinely filtered through a 1.2 µm nylon syringe filter before
data acquisition.25
allowed transition similar to that actually observed in the freshly
prepared LB films.19
In recent studies of aggregates of other chromophores, most
notably those formed from trans-stilbene20,21 and trans-azoben-
zene,22 we have found that while extended aggregates occur,
there is a relatively small “unit” aggregate structure in several
cases that is cyclic, chiral, and relatively stable. These studies
to date suggest that even extended arrays of aggregate are
probably best described as mosaics of the unit aggregate. For
both the trans-stilbene aggregate and the corresponding trans-
azobenzene, the unit structure can be thought of as a piece of
a larger glide lattice. Hence it seemed of interest to examine
the aggregates formed from amphiphilic squaraines under
conditions which might favor formation of a “unit” aggregate.
Since the two types of aggregates generated in the films show
quite different photophysical properties which should reflect
very different photogeneration efficiencies,2,23 it would be of
interest to correlate how stability and structure of the aggregates
correlates with changes in structure of the component squaraine
molecule. Accordingly we have synthesized a series of
squaraine amphiphiles which should vary chiefly in how they
can assemble in films and related microheterogeneous media.
In the present paper we report a study of these squaraines in
aqueous and mixed aqueous-organic solutions and in micro-
heterogeneous media such as aqueous phospholipid vesicles.
In all of these solution media we can observe the transition
between monomer and aggregate; together these studies provide
a picture of the “unit aggregate structure” for the squaraines,
which is likely the precursor for the extended aggregate in
crystals or LB films. Since these solution aggregates can be
easily studied by conventional solution techniques, these studies
also provide a useful picture of the structure and photophysical
properties of the squaraine aggregates. Our results indicate that
the squaraine unit aggregate may be a similar cyclic chiral
structure to those observed with other rod-like chromophores
incorporated into amphiphile structures.21,22
Fluorescence Lifetime Measurements. Time-correlated single
photon counting (TCSPC) experiments were carried out on an instru-
ment consisting of a mode-locked Nd:YLF laser (Quantronix 4000
Series) operating at 76 MHz as the primary laser source. The second
harmonic (KTP crystal) of the Nd:YLF laser was used to synchronously
pump a dye laser (Coherent 700) circulating Rhodamine 6G in ethylene
glycol as the gain medium. The pulsewidth of the dye laser was
typically 8 ps, as determined by auto-correlation, and was cavity
dumped at a rate of 1.9 MHz. The dye laser was tuned to the desired
wavelength for sample excitation (575-605 nm). Photon flux of laser
pulses were typically less than 1011 photons/cm2. Emission from the
sample was collected by two convex lenses and focused at the entrance
slit of a Spex 1681 monochromator (0.22 m) and was detected by a
red-sensitive multichannel plate (MCP) detector (Hamamatsu R3809U-
01). The single photon pulses from the MCP detector were amplified
and used as the start signal for a time-to-amplitude converter (TAC,
EG&G Ortec), while the signal from a photodiode, detecting a small
fraction of the dye laser output, was used as the stop signal for the
TAC. The start and stop signals for the TAC were conditioned before
entering the TAC by passing through two separate channels of a
constant fraction discriminator (CFD, Tennelec TC 454). The output
of the TAC was connected to a multichannel analyzer (MCA) interface
board (Norland 5000) installed inside a 486DX2 personal computer.
The MCA was controlled by software from Edinburgh Instruments
(Edinburgh, UK). The same software was used to carry out the
deconvolution of the data and exponential fitting using the nonlinear
least squares method. Measurements were made on air saturated
samples at the magic angle (55°).
Experimental Section
Materials and General Techniques. Synthetic reagents were
generally purchased from Aldrich Chemical Company and used as
received unless specifically stated. R-, â-, and γ-cyclodextrins (99+%),
L,D-R-alanine (99%), potassium phosphate (monobasic and dibasic,
99%), dioctadecyldimethylammoniumbromide were purchased from
Aldrich. L-R-Dimyristoyl-phosphatidylcholine (DMPC, 99+%) and D,
L, and DL-R-dipalmitoylphosphatidylcholine (DPPC, 99%) were pur-
chased from Sigma and tributyl orthoformate from Pfaltz & Bauer.
Diethyl ether (anhydrous), N,N-dimethylformamide, 2-propanol, metha-
(13) V. Czikkely, H.; Fo¨rsterling, H. D.; Kuhn, H. Chem. Phys. Lett.
1970, 6, 11.
(14) V. Czikkely, H.; Fo¨rsterling, H. D.; Kuhn, H. Chem. Phys. Lett.
1970, 6, 207.
(15) Chen, H., Herkstroeter, W. G.; Perlstein, J.; Law, K. Y.; Whitten,
D. G. J. Phys. Chem. 1994, 98, 5138.
(16) Law, K. Y.; Chen, C. C. J. Phys. Chem. 1989, 93, 2533.
(17) Das, S.; Thomas, K. G.; George, M. V.; Kamat, P. V. J. Chem.
Soc., Faraday Trans. 1992, 88, 3419.
(18) Chen, H.; Law, K. Y.; Perlstein, J.; Whitten, D. G. Manuscript in
preparation.
(19) Chen, H.; Law, K. Y.; Perlstein, J.; Whitten, D. G. J. Am. Chem.
Soc. 1995, 117, 7257.
(20) Song, X, Geiger, C.; Furman, I.; Whitten, D. G. J. Am. Chem. Soc.
1994, 116, 4103.
(21) Song, X.; Geiger, C.; Leinhos, U.; Perlstein, J.; Whitten, D. G. J.
Am. Chem. Soc. 1994, 116, 10340.
(22) Song, X.; Perlstein, J.; Whitten, D. G. J. Am. Chem. Soc. In press.
(23) Kim, Y. S.; Liang, K.; Law, K. Y.; Whitten, D. G. J. Phys. Chem.
1994, 98, 984.
(24) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biophys. Acta
1985, 55, 812.
(25) The authors thank Dr. Thomas Whitesides for the light scattering
measurements.
Transient Absorption Measurements. A mode-locked Nd:YAG
laser (Continuum PY61C) with 25-35 ps 1064 nm fundamental pulses
(10 Hz) was used as the pump and the probe sources for transient
absorption measurements. The fundamental was passed through a

2586 J. Am. Chem. Soc., Vol. 118, No. 11, 1996
Chen et al.
computer controlled variable optical delay line (Velmex) to achieve
the desired delay between pump and probe beams. The second
harmonic wavelength of the laser (532 nm) was used as the excitation
source for most experiments. For experiments requiring longer
excitation wavelengths, Raman shifting of the 532 nm beam in acetone-
d6 was used to obtain 600 nm excitation pulses. The excitation beam
diameter was ∼2 mm. After the delay line the fundamental pulse was
focused into a 10 cm cell with quartz windows containing H2O/D2O
(1:1) for white light continuum generation. The white light was
collimated after the cell and focused at one end of a 200 µm fused
silica optical fiber. The white light was recollimated after exiting the
second end of the optical fiber and was split 50/50 by a cube
beamsplitter after passing through a polarization descrambler. One
beam was used as the reference, and the other was used as the
interrogation beam passing through the sample cell after reducing the
beam diameter to ∼1.5 mm by a lens. The two beams were
recollimated and focused at the ends of a bifurcated fiber optic pair
that was attached to a SPEX 270 monochromator. The spectra of the
white light continuum for the sample and reference beams with and
without excitation were detected after passing through the monochro-
mator by a cooled dual diode array detector (Princeton Instruments
ST121/DDA512), and corresponding differential absorption spectra at
the set delay time were calculated, displayed, and stored on disk by a
BASIC program.
For kinetics measurements, the second output of the monochromator
was used and the monitoring beams were detected by two miniphoto-
multiplier tubes (Hamamatsu HC120) and digitized by a LeCroy digital
oscilloscope before being available to a BASIC program for data
manipulation and storage. Sample solutions in 1 cm pathlength cells
were used with typical pulse energies of 100-500 µJ, except when
nonlinear laser intensity dependence was observed. In latter cases pulse
energies of <50 µJ (<2 × 1015 photo/cm2 at 532 nm) were used. The
instrument response was 35-40 ps (FWHM) depending on the mode-
locking dye used. The spectra showed evolution in time of ∼4 nm/ps
(positive chirp) due to the use of the fiber optic piece for delivery of
the white light continuum to the sample. The positive chirp caused
the shorter wavelengths of the spectra to appear before the longer
wavelengths; however, it did not affect the single wavelength kinetics
measurements. Prolonged and repetitive experiments for vesicle
solutions at the highest laser intensities used led to a decrease in the
ground state absorption (permanent bleaching) for 8814DHA and
88DHT. Reported data are for fresh vesicle solutions of these dyes
with minimum permanent bleaching (<2%).
Synthesis of Squaraines. 4-(N-methyl-N-(carboxypropyl)amino)-
phenyl-4′-(N,N-dimethylamino)phenylsquaraine (1114A). N-methyl-
N-(carboxypropyl)aniline (prepared according to a previous procedure,15
0.53 g, 2.76 mmol), 2-propanol (25 mL), and tributyl orthoformate (2
mL) were placed in a three-necked, 100 mL flask with a magnetic
stirbar. The mixture was stirred and brought to reflux under nitrogen
atmosphere. A solution containing 1-(p-dimethylaminophenyl)-2-
hydroxycyclobutene-3,4-dione (previously prepared,26 0.30 g, 1.38
mmol) in ∼2 mL of DMSO was added evenly through a pressure
equalizing funnel over a 3-h period. The mixture was kept at reflux
for an additional 2 h after the addition was completed. A blue
precipitate was isolated by filtration. After washing the precipitate with
cold 2-propanol and ether, the blue solid was vacuum dried. Pure
squaraine 1114A was separated from the crude product by dissolving
in hot chloroform with a Soxhlet extractor: 0.15 g (yield 28%) blue
solid was obtained after drying in vacuum oven (70 °C), mp 245-246
°C; 1H NMR (DMSO-d6) δ 8.14-8.12 (dd, 4H), 6.82-6.70 (dd, 4H),
3.57 (t, 2H), 3.20 (s, 6H), 3.12 (s, 3H), 2.32 (t, 2H), 1.81 (m, 2H); IR
(KBr) 1589 cm-1, 1734 cm-1; UV-visible λmax (CHCl3) 628 nm, ꢀmax
) 2.95 × 105 M-1 cm-1; FAB mass obsd m/z 393.1983 (M + H),
mass calcd for C23H24O4N2 392.1836.
The combined extract was washed with water, dried over MgSO4,
evaporated to remove solvent, and isolated by vacuum distillation. Pure
N,N-dioctylaniline was isolated as a clear liquid: yield 5.48 g (65%);
1
bp 170-172 °C at 2 mmHg; H NMR (CDCl3) δ 7.25 (t, 2H), 6.75
(m, 3H), 3.32 (t, 4H), 1.62 (m, 4H), 1.35(m, 20H) and 0.92 (t, 6H).
1-Chloro-2-(p-dioctylaminophenyl)cyclobutene-3,4-dione. 1-Chloro-
2-(p-dioctylaminophenyl)cyclobutene-3,4-dione was synthesized ac-
cording to the procedure used by Wendling et al.28 A solution of 1,2-
dichlorocyclobutene-3,4-dione (1.19 g, 7.9 mmol, freshly prepared
according to a reported procedure29) and N,N-dioctylaniline (2.5 g, 7.9
mmol) in 8 mL of methylene chloride was added dropwise to a
methylene chloride solution of the catalyst (1.05 g, 7.9 mmol of AlCl3
in 30 mL CH2Cl2) at reflux. The mixture was refluxed for an hour
after addition. The product solution was poured into 50 mL of ice
water containing 2 drops of concentrated HCl. The organic layer was
separated, washed with water, dried over MgSO4, filtered, and
evaporated. The pure product (1.05 g) was obtained as light yellow
crystals by flash column chromatography on silica gel (70-230 mesh),
with ethyl acetate/hexane ) 1:20 as eluent: yield: 30.5%; mp 79-80
1
°C; IR (KBr) 1795, 1608, 1572 cm-1; H NMR (CDCl3) δ 8.15 (d,
2H), 6.73 (d, 2H), 3.41 (t, 4H), 1.64 (m, 4H), 1.30 (m, 20H) and 0.90
(t, 6H); UV-visible λmax (CHCl3) 416 nm.
1-(p-Dioctylaminophenyl)-2-hydroxycyclobutene-3,4-dione. 1-Chlo-
ro-2-(p-dioctylaminophenyl)cyclobutene-3,4-dione (1.0 g, 2.31 mmol)
was suspended in 50 mL of 18% HCl solution. The mixture was
brought to reflux for 2 h and was cooled to room temperature. The
top organic layer, a yellow liquid, was decanted into a round-bottomed
flask. After evaporation of the solvent, a brown residue was obtained.
The brown solid was washed with water (3 × 20 mL) and was
recrystallized from a mixture of DMSO (∼3 mL) and water (20 mL).
A light yellow solid was isolated by filtration, washed with ether (3 ×
5 mL), and vacuum dried at 60 °C overnight: yield 0.93 g (98%); mp
1
199-200 °C; IR (KBr) 1768 cm-1, 1577 cm-1; H NMR (DMSO-d6)
δ 7.84 (d, 2H), 6.61 (d, 2H), 3.26 (t, 4H), 1.48 (m, 4H), 1.24 (m, 20H)
and 0.83 (t, 6H); FAB mass obsd m/z 414.3012 (M + H), mass calcd
for C26H40O3N 414.3008.
4-(N-Methyl-N-(carboxypropyl)amino)phenyl-4′-(N,N-dioctylami-
no)phenylsquaraine (8814A). N-Methyl-N-(carboxypropyl)aniline
(0.47 g, 2.42 mmol) was reacted with 1-(p-dioctylaminophenyl)-2-
hydroxycyclobutene-3,4-dione (0.50 g, 1.21 mmol) in the presence of
tributyl orthoformate (1.8 mL) in 2-propanol (25 mL) for ∼3 h. A
blue precipitate was isolated by filtration. After having been washed
with 2-propanol and ether, the blue solid was vacuum dried and purified
by recrystallization from MeOH/CH2Cl2: yield 0.25 (35%); mp 202-
203 °C dec; 1H NMR (CDCl3) δ 8.38-8.35 (dd, 4H), 6.82-6.70 (dd,
4H), 3.59 (t, 2H), 3.43 (t, 4H), 3.16 (s, 3H), 2.50 (t, 2H), 2.04 (m,
2H), 1.66 (m, 4H), 1.35-1.40 (m, 20H) and 0.90 (t, 6H); IR (KBr)
1588 cm-1, 1735 cm-1; UV-visible λmax (CHCl3) 634 nm, ꢀmax ) 3.12
× 105 M-1cm-1. Anal. Calcd (C37H52O4N2): C, 75.47; H, 8.90; N,
4.76; O, 10.87. Found: C, 75.51; H 8.81; N, 4.57.
N,N-Bis(carboxypropyl)aniline. Aniline (5.0 g, 54 mmol) and ethyl
4-bromobutyrate (19.0 g, 120 mmol) in 50 mL of butanol were heated
at ∼100 °C for 16 h in the presence of 8.0 g of sodium carbonate and
0.10 g of iodine. The reaction mixture was then cooled to room
temperature, dissolved in water, and extracted with ether. The
combined extract was washed with water, dried over MgSO4, evaporated
to remove solvent, and isolated by vacuum distillation. Pure N,N-di-
(ethyl 4-butanoate)aniline was isolated as an oily liquid: Yield 8.7 g
(50%); bp 167-170 °C at 2 mmHg; 1H NMR (CDCl3) δ 7.25 (t, 2H),
6.75 (m, 3H), 4.168(q, 4H), 3.37 (t, 4H), 2.38 (t, 4H), 1.95 (m, 4H)
and 1.26 (t, 6H).
The ester product (8 g, 24.9 mmol) was then hydrolyzed in 150 mL
of 5% KOH and refluxed for 2 h. After cooling to room temperature,
the reaction mixture was washed with ether (2 × 60 mL), the aqueous
layer was then adjusted to pH 5.5 with concentrated hydrochloric acid,
4-(N-methyl-N-(carboxypropyl)amino)phenyl-4′-(N,N-dibutyl-
amino)phenylsquaraine (4414A) was reported in a previous study.15
N,N-Dioctylaniline. N,N-Dioctylaniline was prepared according to
the procedure used by Desai.27 Aniline (2.5 g, 27 mmol), bromooctane
(18.2 g, 94 mmol), and 80 mL of butanol were heated at ∼100 °C in
the presence of 4.4 g of sodium carbonate and 0.12 g of iodine for 24
h. The reaction mixture was then cooled to room temperature. The
crude product was dissolved in water and then was extracted with ether.
(26) Law, K. Y.; Bailey, F. C. Can. J. Chem. 1993, 71, 494.
(27) Desai, R. D. J. Indian Inst. Sci. 1924, 7, 235.
(28) Wendling, L. A.; Koster, S. K.; Murry, J. E.; West, R. J. Org. Chem.
1977, 42, 1129.
(29) De Selms, C. D.; Fox, C. J.; Riordan, R. C. Tetrahedron Lett. 1970,
781.
(30) Kunitake, T. Angew. Chem., Int. Ed. Engl. 1992, 31, 709.

Surfactant Squaraine Dyes in Aqueous Solution
J. Am. Chem. Soc., Vol. 118, No. 11, 1996 2587
extracted with ether (3 × 60 mL), dried over MgSO4, and evaporated
to remove solvent. N,N-Bis(carboxypropyl)aniline was obtained as a
solid with a yield of 6.1 g (96%): mp 80-82 °C; 1H NMR (CDCl3) δ
7.26 (t, 2H), 6.76 (m, 3H), 3.40 (t, 4H), 2.45 (t, 4H) and 1.96 (m, 4H).
4-(N,N-Bis(carboxypropyl)amino)phenyl-4′-(N,N-dioctylamino)-
phenylsquaraine (8844A2). N,N-Bis(carboxypropyl)aniline (0.64 g,
2.42 mmol) was reacted with 1-(p-dioctylaminophenyl)-2-hydroxycy-
clobutene-3,4-dione (0.50 g, 1.21 mmol) in 2-propanol (20 mL) in the
presence of tributyl orthoformate (1.8 g) for ∼4 h. A blue precipitate
was isolated by filtration. After washing with 2-propanol and ether,
the blue solid was vacuum dried and was purified by recrystallization
aniline (0.18 g, 0.80 mmol) was reacted with 1-(p-dibutylaminophenyl)-
2-hydroxycyclobutene-3,4-dione (0.20 g, 0.48 mmol) in glacial acetic
1
acid (25 mL) for ∼3 h: yield 0.24 (80%); mp 238-240 °C dec; H
NMR (DMSO-d6) δ 12.48 (s, 1H), 7.85-7.82 (d, 2H), 6.88-6.85 (d,
2H), 5.91 (s, 2H), 3.49-3.47 (m, 6H), 3.12 (s, 3H), 2.30-2.26 (t, 2H),
1.77-1.73 (m, 2H), 1.54 (m, 4H), 1.27-1.24 (m, 20H) and 0.85 (t,
6H); IR (KBr) 3435 cm-1, 1600 cm-1, 1197 cm-1; UV-visible λmax
(CHCl3) 634 nm, ꢀmax ) 2.25 × 105 M-1 cm-1; FAB mass obsd m/z
620.3825; mass calcd for C37H52O6N2 620.3824. Anal. Calcd C, 71.57;
H, 8.45; N, 4.51. Found: C, 71.12; H 8.46, N, 4.28.
N-Methyl-3,5-dimethoxyaniline. 3,5-Dimethoxyaniline (6.9 g, 45
mmol), methyl iodide (6.4 g, 45 mmol), and 50 mL of THF were stirred
at room temperature in the presence of 3.4 g of sodium acetate for 10
h. The crude product was dissolved in water and then was extracted
with ether. The combined extract was washed with water, dried over
MgSO
1
from DMSO/CH2Cl2: yield 0.21 (26.3%); mp 183-184 °C dec; H
NMR (CDCl3) δ 8.40-8.36 (dd, 4H), 6.90-6.75 (dd, 4H), 3.55 (t,
4H), 3.43 (t, 4H), 2.50 (t, 4H), 1.96 (m, 4H), 1.68 (m, 4H), 1.35-1.40
(m, 20H) and 0.90 (t, 6H); IR (KBr) 1587 cm-1, 1735 cm-1; UV-
visible λmax (CHCl3) 638 nm, ꢀmax ) 2.21 × 105 M-1 cm-1. Anal.
Calcd (C40H56O6N2): C, 72.70; H, 8.54; N, 4.24; O, 14.53. Found:
C, 72.34; H, 8.58; N, 3.99.
4, evaporated to remove solvent, and isolated by column
chromatography on silica gel (230-270 mesh), with ethyl acetate/
hexane ) 1:4 (v/v) as eluent. Pure N-methyl-3,5-dimethoxyaniline was
1
isolated as a white solid: yield 2.63 g (35%); H NMR (CDCl3) δ
N-(Ethyl 4-butanoate)-3,5-dimethoxyaniline. 3,5-Dimethoxy-
aniline (10.1 g, 65.9 mmol), ethyl 4-brombutyrate (4.95 g, 25.4 mmol),
and 80 mL of butanol were heated at ∼70 °C in the presence of 3.0 g
of sodium acetate for 10 h. The reaction mixture was then cooled to
room temperature. The crude product was dissolved in water and then
was extracted with ether. The combined extract was washed with water,
dried over MgSO4, evaporated to remove solvent, and isolated by
column chromatography on silica gel (70-230 mesh), with ethyl
acetate/hexane ) 1/2 as eluent. Pure N-(ethyl 4-butanoate)aniline was
5.90 (s, 1H), 5.82 (s, 2H), 3.76 (s, 6H), 2.83 (s, 3H), 1.96 (m, 2H).
N-Methyl-N-(8-bromooctyl)-3,5-dimethoxyaniline. N-methyl-3,5-
dimethoxyaniline (0.87 g, 5.2 mmol), 1,8-dibromooctane (4.2 g, 15.6
mmol), and 50 mL of butanol were heated at ∼90 °C in the presence
of 0.52 g of sodium bicarbonate for 16 h. The crude product was
dissolved in water and then was extracted with ether. The combined
extract was washed with water, dried over MgSO4, and evaporated to
remove solvent, and the product was isolated by column chromatog-
raphy on silica gel (230-270 mesh), with ethyl acetate/hexane ) 1/20
as eluent. N-methyl-N-(8-bromooctyl)-3,5-dimethoxyaniline (1.86 g)
1
isolated as a white solid: yield 4.82 g (73.6.%); mp 42-43 °C; H
NMR (CDCl3) δ 5.96 (s, 1H), 5.92 (s, 2H), 4.14 (m, 2H), 3.78 (s, 6H),
3.18 (t, 2H), 2.43 (t, 2H), 1.96 (m, 2H), 1.27 (t, 3H).
1
was isolated as a solid: yield (54.8%); H NMR (CDCl3) δ 5.89 (s,
3H), 3.80 (s, 6H), 3.42 (t, 2H), 3.28 (t, 2H), 2.92 (s, 3H), 1.86 (m,
2H), 1.34-1.60 (m, 10H).
N-Methyl-N-(ethyl 4-butanoate)-3,5-dimethoxyaniline. N-(ethyl
4-butanoate)-3,5-dimethoxyaniline (4.8 g, 18.0 mmol), methyl iodide
(2.55 g, 18.0 mmol), and 30 mL of tetrahydrofuran were heated at
∼50 °C in the presence of 1.48 g of sodium acetate for 10 h. The
reaction mixture was then cooled, poured into 50 mL of water, and
then extracted with ether. The combined extract was washed with
water, dried over MgSO4, evaporated to remove solvent, and isolated
by column chromatography on silica gel (70-230 mesh), with ethyl
acetate/hexane ) 1/8 as eluent. Pure N-methyl-N-(ethyl 4-butanoate)-
aniline was isolated as an oily liquid: yield 3.26 g (62.0%); bp 152-
8-{N-(3,5-Dihydroxyphenyl)-N-methylamino}octyltrimethyl am-
monium Bromide. N-methyl-N-(8-bromooctyl)-3,5-dimethoxyaniline
(1.5 g), was hydrolyzed by refluxing in 50 mL of a 1:1 mixture of
acetic acid and hydrobromic acid (47-49%) for 2 h. After having
been cooled, the reaction mixture was washed with ethyl acetate
(2 × 50 mL), and the aqueous layer was adjusted to pH 6 with
concentrated sodium hydroxide, extracted with ethyl acetate (3 × 50
mL), dried over MgSO4, and evaporated to remove the solvent. An
oily liquid (N-methyl-N-(8-bromooctyl)-3,5-dihydroxyaniline) was
obtained (yield: 0.83 g, 60%). The oily liquid was dissolved in 10
mL of THF and kept at 0 °C. Trimethylamine was then added to the
above solution. The reaction mixture was sealed and stirred at room
temperature for 5 h. A white precipitate was formed and isolated by
filtration, washed with THF, and dried in vacuum (40 °C): yield: 0.78
1
153 °C/0.05 mmHg; H NMR (CDCl3) δ 5.92 (s, 3H), 4.15 (q, 2H),
3.80 (s, 6H), 3.35 (t, 2H), 2.93 (s, 3H), 2.35 (t, 2H), 1.92 (m, 2H),
1.27 (t, 3H).
N-Methyl-N-(carboxypropyl)-3,5-dihydroxyaniline. N-Methyl-N-
(ethyl 4-butanoate)aniline (3 g, 10.7 mmol) was hydrolyzed by refluxing
in 50 mL of a 1:1 mixture of acetic acid and hydrobromic acid (47-
49%) for 2 h. After cooling to room temperature, the reaction mixture
was washed with ethyl acetate (2 × 70 mL), the aqueous layer was
then adjusted to pH 5.5 with concentrated sodium hydroxide, extracted
with ethyl acetate (3 × 70 mL), dried over MgSO4, and evaporated to
remove solvent. N-Methyl-N-(carboxypropyl)-3,5-dihydroxyaniline was
obtained as an oily liquid with a yield of 2.06 g (86%): 1H NMR
(DMSO) δ 8.78 (s, 2H), 5.55 (d, 3H), 3.17 (t, 2H), 2.75 (s, 3H), 2.20
(t, 2H) and 1.65 (m, 2H).
1
g (80%); mp 196 °C; H NMR (MeOD) δ 5.72 (s, 2H), 5.66 (s, 1H),
3.34 (t, 2H), 3.20 (t, 2H), 3.11 (s, 9H), 2.84 (s, 3H), 1.76 (m, 4H),
1.50-1.60 (m, 4H), 1.40 (m, 4H).
Squaraine Quaternary Ammonium Bromide 88DHT. Similar to
the synthesis of 4414DHA, 1-(p-N,N-dioctylaminophenyl)-2-hydroxy-
3,4-dione (0.15 g, 0.50 mmol) was reacted with 8-{N-(3,5-dihy-
droxyphenyl)-N-methylamino}octyltrimethylammonium bromide (0.23
g, 0.60 mmol) in boiling glacial acetic acid for 2 h. A blue precipitate
formed after the reaction mixture was cooled down to room temperature.
The blue solid was obtained by filtration and was washed with ether,
dried in vacuum (50 °C), and further purified by recrystalization from
4-(N-methyl-N-(carboxypropyl)amino)phenyl-4′-(N,N-dibutyl-
amino)-2′,6′-dihydroxyphenylsquaraine (4414DHA). N-Methyl-N-
(carboxypropyl)-3,5-dihydroxyaniline (0.22 g, 0.98 mmol) was reacted
with 1-(p-dibutylaminophenyl)-2-hydroxycyclobutene-3,4-dione (0.25
g, 0.83 mmol) in glacial acetic acid (25 mL) for ∼3 h. A blue
precipitate was isolated by filtration. After having been washed with
ether, the blue solid was vacuum dried and purified by recrystallization
1
methanol and hexane: yield 0.18 g (64%); mp 166-168 °C dec; H
NMR (CDCl3) δ 8.07-8.04 (d, 2H), 6.68-6.71(d, 2H), 5.79 (s, 2H),
3.68 (m, 4H), 3.45 (s, 9H), 3.40-3.45 (m, 4H), 3.17 (s, 3H), 1.80-
1.30 (m, 36H), 0.92 (t, 3H). IR (KBr) 3459, 1642, 1599, 1183 cm-1
;
;
1
from acetic acid: yield 0.23 (54.8%); mp 278-280 °C dec; H NMR
UV-visible λmax (CHCl3) 634 nm, ꢀmax ) 1.78 × 105 M-1 cm-1
+
(DMSO-d6) δ 12.48 (s, 1H), 7.88-7.86 (d, 2H), 6.89-6.86 (d, 2H),
5.91 (s, 2H), 3.59-3.40 (m, 6H), 3.13 (s, 3H), 2.31-2.26 (t, 2H), 1.77-
1.72 (m, 2H), 1.58-1.49 (m, 4H), 1.36-1.29 (m, 4H) and 0.90 (t, 6H);
IR (KBr) 3458, 1597, 1192 cm-1; UV-visible λmax (CHCl3) 634 nm,
ꢀmax ) 2.42 × 105 M-1 cm-1; FAB mass obsd m/z 508.2570; mass
calcd for C29H36O6N2 508.2573. Anal. Calcd C, 68.47; H, 7.14; N,
5.51. Found: C, 68.14; H, 7.09; N, 5.71.
HRFAB mass obsd 704.5347 (M+); mass calcd for C44H70N3O4
704.5366.
Squaraine Quaternary Ammonium Bromide 48DHT. Similar to
the synthesis of 88DHT. 1-(p-N,N-dibutylaminophenyl)-2-hydroxy-
3,4-dione (0.15 g,0.50 mmol) was reacted with 8-{N-(3,5-dihydroxy-
phenyl)-N-methylamino}octyltrimethylammonium bromide (0.23 g,
0.60 mmol) in boiling glacial acetic acid for 2 h: yield of the squaraine
formation step 63%; mp 178-179 °C dec; 1H NMR (CDCl3) d 8.07-
8.04 (d, 2H), 6.68-6.71(d, 2H), 5.79 (s, 2H), 3.68 (m, 4H), 3.45 (s,
9H), 3.40-3.45 (m, 4H), 3.17 (s, 3H), 1.80-1.30 (m, 36H), 0.92 (t,
4-(N-Methyl-N-(carboxypropyl)amino)phenyl-4′-(N,N-dioctyl-
amino)-2′,6′-dihydroxyphenylsquaraine (8814DHA). Similar to the
synthesis of 4414DHA. N-Methyl-N-(carboxypropyl)-3,5-dihydroxy-

2588 J. Am. Chem. Soc., Vol. 118, No. 11, 1996
Chen et al.
Figure 1. Absorption spectra for type I squaraines in H2O (pH ) 7.5,
[dye] ) 5 × 10-6 M): (a) 1114A; (b) 4414A; (c) 8844A2; and (d)
8814A.
3H). IR (KBr) 3457 cm-1, 1640 cm-1, 1598 cm-1, 1182 cm-1; UV-
visible λmax (CHCl3) 634 nm, ꢀmax ) 1.81 × 105 M-1 cm-1; FAB mass
+
obsd (M+) 592.4120; mass calcd for C36H54N3O4 592.4114.
Results
The series of squaraine amphiphiles is shown in Chart 1. All
of these compounds contain hydrophilic head groups, either a
carboxylic acid (type I and II) or a quaternary ammonium salt
(type III) and various alkyl substituents to render them more
or less hydrophobic. Two types of squaraine chromophore are
present: in the “type I” squaraines the chromophore is a simple
diphenyl squaraine, while for the “type II” and “type III” one
of the phenyl rings is dihydroxylated to facilitate intramolecular
H-bonding to the squaraine oxygens. In addition, the “type III”
squaraines which contain a quaternary ammonium salt as the
hydrophilic head group are expected to be capable of forming
“pure” squaraine vesicles.30 All of the compounds with the
exception of 1114A form good LB films which can be
transferred to optically transparent supports; in each case the
films at the air-water interface and the freshly transferred films
show a strong absorption at 520-530 nm, blue-shifted compared
to monomer (650 nm in water). The type I squaraines, 4414A
and 8814A, give conversion to a red-shifted aggregate when
their LB films are heated. 8844A2 gives a mixture of red and
blue-shifted forms on heating, while the type II squaraines
4414DHA and 8814DHA and type III squaraines 48DHT and
88DHT also show partial conversion to the red-shifted ag-
gregate. As described in the introduction, the main focus of
the present work is the behavior of this series of squaraines in
solution and microheterogeneous media.
In Homogeneous Solution. Type I Squaraines. The type
I squaraines show only limited solubility in water; however,
concentrations on the order of 10-5-10-6 M can be obtained
in water (pH ) 7.5) for short duration (ca 1 h) by injection of
a concentrated organic solution of the squaraine into the aqueous
or partially aqueous medium in an “arrested crystallization”
process. We previously reported that 4414A forms a mixture
of monomer and dimer in water.15 The structural assignment
of the blue-shifted species absorbing at 594 nm was supported
by the finding that a similar absorbing species was obtained
when aqueous solutions of 4414A were treated with γ-cyclo-
dextrin. In contrast R- and â-cyclodextrins incorporate only
the monomer of 4414A. For 4414A a study of the monomer-
dimer equilibrium in pure water led to a determination of the
thermodynamic parameters for dimer formation of ∆H0° ) -6.84
kcal/mol and ∆S0° ) -1.83 cal/mol deg.15 As shown in Figure
1, the series of four type I squaraines shows a remarkable
contrast in the range of species present in pure water at pH )
Figure 2. Absorption spectra for squaraines in DMSO-H2O mixed
solvent ([dye] ) 5 × 10-6 M): (1) 8814A and (2) 8844A2.
7.4 ([squaraine] ) 5 × 10-6 M). For 1114A virtually only the
monomer is observed; a small amount of absorption at ca. 580
nm is ascribed to the dimer. This is reinforced by the finding
that addition of γ-cyclodextrin results in conversion of most of
the monomer to this species. In contrast, the squaraines with
larger hydrophobic alkyl group substituents exhibit prominent
transitions which can be assigned to the dimer (ca. 594 nm)
and the aggregate (530 nm). 8814A exists predominantly as
the aggregate with the same maximum (530 nm) as in the LB
film while 8844A2 shows both the dimer and the aggregate
maxima.
Addition of dimethyl sulfoxide (DMSO) to aqueous solu-
tions of the type I squaraines increases their solubility in general
and results in a preference for monomer, compared to pure
aqueous media. For 1114A only monomer is observed even at
very low (<2%) DMSO content. For the other type I squaraines
the interconversion between aggregate, dimer and mono-
mer is observed with increase in DMSO%(v/v); the behavior
of these is summarized in Table 1. Figure 2 compares the
behavior of 8844A2 and 8814A over a range of DMSO
concentrations. For the former a mixture of aggregate, dimer
and monomer is evident over a wide range, while for the latter
a clear isosbestic point is observed between aggregate and
monomer and no clear maximum corresponding to the dimer is
detectable.
Type II and III Squaraines. The addition of two hydroxyl
groups to one of the squaraine phenyl rings, such that intramo-
lecular hydrogen bonding with the squaraine oxygens can occur,
is anticipated to increase the ease of forming a card-pack
aggregate and alter the solubility of the various species
encountered above for the type I squaraines. In addition we
have found that the type II squaraines have much better long-
term stability in the presence of water compared to the type I
squaraines and other squaraine dyes, which generally decompose
upon prolonged exposure to aqueous media. This can probably
be attributed to greater resistance to nucleophilic attack by water
on the type II squaraines.31 The solubility of type II squaraines
4414DHA and 8814DHA in pure water is very low, even lower
than that of the corresponding type I squaraines, 4414A and
8814A. Similar to the type I squaraines, 4414DHA forms

Surfactant Squaraine Dyes in Aqueous Solution
J. Am. Chem. Soc., Vol. 118, No. 11, 1996 2589
Table 1. Aggregation of Squaraines in the DMSO-H2O Mixed
Solvent
aggregation
range
maximum isosbestic
b
squaraine dominant species (DMSO%)a Ia/(I +I
pointc
)
a
m
1114A
4414A
dimer (590 nm)
monomer (648 nm)
dimer (594 nm)
V
0%
<10%
21%
0%
V
yes
monomer (650 nm)
aggregate (530 nm)
5%
10%
V
8844A2
8814A
53%
no
dimer (590 nm)
monomer (650 nm)
aggregate (530 nm)
V
60%
40%
V
65%
20%
V
60%
70%
V
80%
0%
V
81%
83%
85%
25%
Figure 3. Absorption spectra for 4414DHA (6.56 × 10-6 M) in
DMSO-H2O mixed solvent at different temperatures: (a)-(f) 5, 15,
25, 35, 45, 55 °C.
yes
yes
yes
no
monomer (650 nm)
4414DHA aggregate (520 nm)
V
monomer (650 nm)
8814DHA aggregate (520 nm)
V
repulsion. In contrast, the ionic strength effect on the aggrega-
tion of type I and II squaraines is much lower than for type III.
It is important to note that for squaraines 8814A, 4414DHA,
8814DHA, and 88DHT, an equilibrium between monomer and
aggregate was observed with no detectable dimer present. The
monomer to aggregate conversion can be accomplished either
by changing the percentage of DMSO or the solution temper-
ature. One example is given in Figure 3 indicating the
monomer-aggregate conversion of 4414DHA at various tem-
peratures (5-55 °C). Due to the high stability of 4414DHA,
8814DHA, and 88DHT in DMSO-H2O mixed solvent, it is
possible to analyze the monomer-aggregate equilibrium care-
fully over a moderate temperature range for 88DHT as has been
done previously for 4414DHA and 8814DHA (see Discussion).
Microheterogeneous Media: Bilayer Vesicle Systems.
Aggregation in Mixed Vesicles. The solubility of type I and
II squaraines (except 1114A) in water is significantly enhanced
upon addition of the phospholipid, and a clear solution of the
mixed dye and the phospholipid is obtained upon probe
sonication for a proper period of time (ca. 15 min). Generally,
the molar ratio of dye to phospholipid can reach as high as 1/1
for most type I and II squaraines with the formation of clear
and stable solutions. For example, 8814A can be incorporated
as a guest into phospholipid vesicles in water. As shown in
Figure 4, the absorption spectrum for relatively high ratios of
squaraine to phospholipid (dimyristoylphosphatidylcholine
(DMPC) or dipalmitoylphosphatidylcholine (DPPC)) shows a
predominance of the blue-shifted (520 nm) aggregate similar
to the spectra obtained in LB films or in DMSO-H2O mixtures.
At lower dye/DMPC ratios more contribution from the monomer
spectrum is observed. Similar results for 8814DHA/DMPC
mixtures are also observed.19 Light scattering and membrane
filtration experiments have indicated that these vesicles are
considerably larger than pure DMPC vesicles.19
Other type I and II squaraines except 8844A2 have similar
aggregation behavior to that of 8814DHA and 8814A in
phospholipid vesicles. Significant amounts of 8844A2 exist as
monomer even at very high dye/DMPC ratio (such as 1/5). This
is not surprising since the weak chromophore-chromophore
interaction of 8844A2 due to steric effects allows a complete
dispersion of the dye as individual monomers into the phos-
pholipid bilayers.
The coexistence of monomer, dimer, and aggregate in the
mixed vesicles makes it difficult to accurately calculate the
aggregation number for these squaraine aggregates formed in
the bilayer systems. No pure aggregate spectra can be obtained
even at very low PC/dye ratio for most of the squaraines.
However, squaraine 8814DHA can be dispersed in DMPC with
relatively high dye concentration (5 × 10-5 M) at high dye/
monomer (650 nm)
aggregate (520 nm)
48DHT
dimer (588 nm)
monomer (650 nm)
aggregate (520 nm)
V
9%
88DHT
50%
V
70%
80%
yes
monomer (650 nm)
a The minimum range of (DMSO% (v/v) required to observe the
changes from aggregate to monomer. b Ia/(Ia + Im): ratio of the
absorbance of aggregate to the sum of absorbance of aggregate and
monomer at certain DMSO%. c During the conversion from aggregate
to monomer at constant dye concentration.
monomeric (650 nm) complexes with R-cyclodextrin in water
and dimer (585 nm) with γ-cyclodextrin. In DMSO-H2O mixed
solvent 4414DHA and 8814DHA form a mixture of aggregate
(520 nm) and monomer (650 nm) the ratio of which is dependent
on the fraction of DMSO (DMSO%). Similar to 8814A, the
aggregate-monomer conversion in both cases occurs with a
clear isosbestic point indicating an equilibrium between the two
species without much, if any, population of the dimer. Table 1
summarizes the range of solution composition over which the
interconversion occurs; compared to the type I squaraines there
is a greater percentage of aggregate in the mixed solvents as
well as a persistence of aggregate to higher percentages of
DMSO.
Even though type III squaraines 48DHT and 88DHT are
designed for the study of aggregation in pure vesicles, their
aggregation behavior in DMSO-water mixed solvent is also
very interesting. Also shown in Table 1, the different behavior
of 44DHT and 88DHT due to different hydrophobic chain
length is clear: for the former a mixture of monomer, dimer,
and aggregate is obtained even at very low percent DMSO
(DMSO%) with no isosbestic point for the conversion; while
for the later only monomer and aggregate are observed with a
clear isosbestic point for the conversion and a much greater
percentage of the aggregate as well as a higher range of
DMSO%. Comparing the aggregation behavior of 48DHT with
that of 4414DHA, it appears that introducing the quaternary
ammonium salt head group in the squaraine surfactant dimin-
ishes somewhat the tendency of the squaraine to aggregate. This
is probably due to the electrostatic repulsion between the
positively charged head groups. In fact, addition of electrolytes
such as NaCl to the solution of type III squaraines in DMSO-
H2O mixed solvent dramatically enhances the aggregate forma-
tion which can be attributed to the increase in the effective
dielectric constant of the solution which reduces the electrostatic

2590 J. Am. Chem. Soc., Vol. 118, No. 11, 1996
Chen et al.
monomer and dimer are likely too far removed from the chiral
center in the phospholipid host for there to be a strong chiral
perturbation of the chromophore. The observation of a strong
signal from the aggregate suggests that the aggregate itself may
be chiral, as has been suggested recently for other systems.21,22
The polarity of the biphasic ICD spectrum for 8814DHA/DPPC
system was found to be dependent on the chirality of the host
phospholipid (L-DPPC vs D-DPPC), with no observed ICD
signal when a racemic mixture of D-DPPC and L-DPPC was
used as host.19 The ICD spectra for 8814DHA show no change
as the solutions are heated through the phase transition tem-
perature for DPPC (43 °C).32 Other type I and II squaraines
which can also be incorporated into DMPC or DPPC vesicles
show induced circular-dichroism spectra with similar biphasic
features.
Aggregation in Pure Vesicles. Both squaraines 48DHT and
88DHT form stable large vesicles upon probe sonication
(diameter 177 and 194 nm, respectively) which cannot be
extruded through 100 nm filters. In contrast to the mixed
vesicles in which a significant amount of monomer (indicated
by the monomer fluorescence at ca. 655 nm in contrast to the
nonfluorescent aggregate) still exists even at a high dye/
phospholipid ratio, the spectrum of the pure vesicles shows a
very sharp blue-shifted absorption at 520 nm which is almost
the same in shape and absorption maximum as those observed
in DMSO-H2O mixed solvents and in LB films, with virtually
no monomer present (supported by the lack of emission).
Similar to the mixed vesicles, the aggregate can be converted
to a mixture of the dimer (580 nm) and monomer (ca. 630 nm)
by addition of another quaternary ammonium salt surfactant,
dioctadecyldimethylammoniumbromide (DODAB), to the solu-
tion. The aggregate obtained from 48DHT vesicles has a
slightly broadened absorption compared with 88DHT, which
might be attributed to the fact that the hydrophobic chains (butyl
groups) in 48DHT are shorter and form less ordered bilayer
membranes than 88DHT.
Figure 4. Absorption (1) and CD (2) spectra for 8814A (1.0 × 10-5
M) in L-DMPC vesicles. Dye/DMPC: (a)-(d) 1/5, 1/10, 1/25, 1/50.
Chart 1 Squaraine Structures
No ICD signal was observed for pure vesicles of 48DHT
and 88DHT. However, the quaternary ammonium salt head
groups in these squaraines can electrostatically interact with
negatively charged species; the formation of the complex of
48DHT or 88DHT with a chiral amino acid such as R-alanine
is possible under slightly basic conditions (pH ) 8-9). Not
surprisingly, similar biphasic ICD signals which correspond to
the aggregate transition were observed for the vesicles binding
D- or L-R-alanine. It is obvious that the biphasic ICD behavior
is a general phenomenon which relates only to the squaraine
aggregate rather than the dimer or monomer species, which
reinforces the concept that the squaraine aggregates are chiral.
DMPC ratio (1/2.5) to form the pure aggregate (520 nm) whose
spectrum is the same in shape and absorption maximum to that
obtained in DMSO-H2O mixed solvent and in LB films. The
apparent extinction coefficient for the aggregate at λmax (520
nm) is ca. 1.1 × 105 M-1 cm-1. The pure monomer spectrum
is easily obtained from the very low concentration of dye in
DMPC vesicles.
Additional information concerning the aggregate structure and
aggregate incorporation into the mixed vesicles is provided by
examining the circular dichroism spectra of the 8814A/DMPC
mixtures in water. When 8814A is dispersed in L-DMPC (R
isomer) the blue-shifted aggregate shows a strong biphasic
induced circular dichroism (ICD) spectrum in the range 450-
550 nm whose crossing point coincides with the λmax of the
major exciton band (Figure 4). As shown in Figure 4, when
the ratio of DMPC to dye is increased so that appreciable
amounts of dimer and monomer are present, the ICD signal
still corresponds to the aggregate exciton with little or no signal
corresponding to the latter two species. This is reasonable since
the squaraine chromophore is achiral, and in dilute vesicles the
Fluorescence Lifetime Measurements. Fluorescence decay
measurements were carried out for 4414A and 4414DHA in a
number of solvents. The results of these measurements are
summarized in Table 4. All decays were biexponential, and
no wavelength dependence was observed on the calculated decay
parameters across the emission spectrum for all measurements
except when ethanol solvent was used. Typical fluorescence
decay curves (4414A in acetonitrile) are shown in Figure 5.
Absence of wavelength dependence is illustrated by the
superimposed decay curves obtained at different wavelengths
in Figure 5b. Squaraine compounds studied are expected to be
monomeric under the experimental conditions used for these
measurements (µM concentration) as indicated by their absorp-
tion spectra. In addition, sample dilution for 4414A in
acetonitrile and chloroform did not affect the calculated decay
constants. These results suggest that the biexponential nature
of the fluorescence decays is not caused by intermolecular

Surfactant Squaraine Dyes in Aqueous Solution
J. Am. Chem. Soc., Vol. 118, No. 11, 1996 2591
Figure 5. Fluorescence decay curves for 4414A in acetonitrile: (a)
biexponential fit and residuals for decay at 660 nm and (b) superim-
posed fluorescence decays at three different wavelengths.
effects such as aggregation (dimers and larger aggregates do
not show any measurable fluorescence).
Figure 6. Transient absorption spectra after laser excitation at 532
nm (a) and bleaching recovery at 640 nm (b) for 4414A in ethanol.
The fitted curve represents a biexponential function with time constants
of 120 and 600 ps.
Transient Absorption Measurements. Excited state life-
times for 4414A and 4414DHA were also measured in several
solvents using picosecond transient absorption (TA) spectros-
copy. The measured lifetimes were in good agreement with
those from fluorescence decay measurements. However, due
to the inherently higher signal/noise ratio associated with the
TCSPC technique, values obtained from fluorescence decays
are expected to be more reliable (Table 4). Representative
differential absorption spectra for 4414A in ethanol are shown
in Figure 6 along with a biexponential fit to the recovery of the
bleaching signal at 640 nm. The decay parameters for the
excited state absorption in the 480 nm region and ground state
bleaching band at 640 nm were identical in all cases within the
experimental accuracy of the measurements. The displacement
of the peak of the bleaching signal from 640 to ∼650 nm in
Figure 6 is due to stimulated emission. No variation in the shape
or decay constants of the TA spectra were observed as a result
of laser pump energy changes.
Transient absorption spectra were also obtained for squaraine
aggregates formed in the 88DHT and 8814DHA/DMPC aque-
ous vesicle solutions. Complex spectral evolution was observed
for these systems after the initial laser excitation. There was
also strong dependence of the shape and kinetics of the spectra
on laser pump intensity as can be observed in Figure 7 for the
8814DHA/DMPC solution. The peak of the ground state
bleaching band in these spectra is blue-shifted due to the
presence of an overlapping absorption band in the 510-580
nm range. At all laser energies examined, the absorption band
below 480 nm decayed with a deconvoluted lifetime of 30 ps.
However, bleaching recovery at 520 nm appeared nonexponen-
tial and slowed with increase in laser pump energy (Figure 8).
The excited state decay kinetics of fluorescent aggregated
stilbenes in microheterogeneous environments such as bilayer
vesicles have also been found to be nonexponential.33 Although
it was possible to obtain reasonable biexponential fitting
parameters for the decays in Figure 8, such parameters would
not be based on a valid decay mechanism. Nevertheless, an
initial recovery time of ∼30 ps was obtained for 88DHT and
8814DHA/DMPC at low laser intensity (<50 µJ/plse) and
deconvolution using an exponential model. Close examination
of the new absorption monitored at 565 nm revealed a growth
rate constant of 60 ps after deconvolution of the instrument
response (150 µJ/plse, 8814DH/DMPC). Although the mag-
nitude of this signal increased with laser pump energy, it did
not show linear or quadratic dependence on the pump energy.
Similar results were observed for an aqueous vesicle solution
of 88DHT, with the exception that no excited state absorption
was observed in the 510-580 nm region at low laser pump
energies (<50 µJ/plse).
Discussion
Aggregation of Squaraines in water and DMSO-H2O
Mixtures. For all of the amphiphilic squaraine dyes investigated
(31) Law, K.; Bailey, F. C. J. Org. Chem. 1992, 57, 3278.
(32) Gennis, R. B. Biomembranes: Molecular Structure and Function;
Cantor, C. R., Ed; Springer-Verlag: New York, 1989.
(33) Farahat, M. S.; Song, X.; Geiger, C.; Whitten, D. G. Manuscript in
preparation.
(34) Benesi, H.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703.
(35) Law, K. Y. J. Phys. Chem. 1989, 93, 5925.

2592 J. Am. Chem. Soc., Vol. 118, No. 11, 1996
Chen et al.
Figure 9. Benesi-Hildebrand plot of lnIa Versus ln(Iao - Ia) of
squaraine 4414DHA in DMSO-H2O mixed solvent with 50% (v/v)
DMSO at 308 K.
(“hydrophobic effects”). For the mixed DMSO-H2O solutions
a comparison of the entire series of compounds may be made
by examining both the range of solution composition and the
maximum extent of aggregation as indicated by the maximum
value of Ia/(Ia + Im) where Ia and Im are the absorbance values
for aggregate and monomer at the respective maximum; the
maximum value of this ratio is always obtained at the minimum
value of the percent DMSO given in Table 1. In several cases
the presence of isosbestic points during the solvent-change
mediated aggregate-monomer interconversion (Figure 2(1) and
3) suggests that there is a simple equilibrium between aggregate
of a discrete size and monomer. From the data presented in
Table 1, it is clear that for similarly substituted squaraines of
the different series, those of the type II show the highest
tendency to aggregate while those of the type I and type III
show lower but similar tendencies to aggregate. The higher
tendency to form aggregates for the type II squaraines is readily
attributed to intermolecular hydrogen bonding for the dihydroxyl
derivatives.5 In the case of the type III squaraines, some
repulsion between the quaternary head groups may account for
the slightly lower tendency to aggregate than that observed for
the similar squaraines terminating in carboxyl groups (type II).
In agreement with this postulate, we find that addition of
electrolytes such as NaCl or KNO3 in the solution enhances
the aggregation of 48DHT and 88DHT in DMSO-H2O
mixtures.
Figure 7. Time resolved spectra and laser pulse energy dependence
for 8814DHA/DMPC (1/2.5) vesicle solution.
Quantitative Analysis of Squaraine Aggregation. The
equilibrium between monomer (M) and aggregate (Agg) of order
n is represented by eq 1 and can be analyzed by the Benesi-
Hildebrand treatment34 according to eqs 2 and 3.
Figure 8. Decay of transient absorption and bleaching signals for 88DT
vesicle solution as a function of laser pulse energy.
nM ) Agg
(1)
in the present study it is clear that formation of similar spectrally
blue-shifted aggregates is a very general process in water, in
mixed aqueous organic solutions, in the presence of bilayer-
forming surfactants, and in Langmuir-Blodgett films. In
several cases a dimer can also be detected; the dimer generally
shows an absorption blue-shifted from monomer and intermedi-
ates between the monomer and aggregate. For the different
functionalized squaraines such as the “type I” squaraines, some
clear trends may be noted in the tendency to form aggregates.
As shown in Figure 1, the increase in alkyl chain length in the
series, 1114A, 4414A, and 8814A, results in an increase in the
extent of aggregation at a constant dye concentration in water
K ) [Agg]/[M]n
(2)
(3)
lnIa ) n ln (Iao - Ia) + constant
where Ia is the absorbance of Agg at any concentration and Iao
is the absorbance of pure aggregate (no monomer present). The
analyses are based on two ratios: (1) Ra, the ratio of aggregate
absorbance at λ1 (522 nm), to that at λ2 (650 nm) which can be
calculated from the pure aggregate spectrum and (2) Rm, the
ratio of monomer absorbance at the same two wavelengths,

Surfactant Squaraine Dyes in Aqueous Solution
J. Am. Chem. Soc., Vol. 118, No. 11, 1996 2593
Table 2. Equilibrium Constants and Thermodynamic Functions for
4414DHA in DMSO-H2O Mixed Solvent (50% DMSO)
temp aggregation equilibrium
∆H°
∆S°
(K)
no. (n)
constant Ka (kcal mol-1
)
(cal mol-1 k-1
)
278
288
298
308
318
4.35
4.01
4.29
4.34
4.21
5.56 × 1020
7.63 × 1018
1.05 × 1018
5.64 × 1016
2.93 × 1020
-4.20
-46.4
a Equilibrium constant in (mol-1 L)n-1
.
Table 3. Quantitative Results of the Aggregation of Squaraines in
DMSO-H2O Mixed Solvent
∆H° (kcal ∆H° (cal
squaraine DMSO%
na
Kb
mol-1
)
mol-1 k-1
)
4414DHA
8814DHA
88DHT
50
79.5
60
4.2 ( 0.2 7.63 × 1018 -42.0
5.2 ( 0.6 2.79 × 1021 -40.7
3.9 ( 0.3 1.38 × 1017 -45.5
-46.4
-37.4
-74.4
a Average aggregation number. b Equilibrium constant at 298 K in
(mol-1 L)n-1
.
Table 4. Excited State Lifetimes for 4414A and 4414DHA in
Different Media
fluorescence decaya
transient absorption
b
b
medium τ1 (ps)
f1
τ2 (ps)
4414A
45 0.06 1.06
f2
ø2
τ (ps)c
r
CH3CN
EtOH
DMSO
CHCl3
H2O
405 0.94
650 0.95 100 0.05 1.09
1080 0.94 140 0.06 1.06
2170 0.98 168 0.02 1.03
600, 120
Figure 10. Schematic representation of the aggregate structures as
viewed from above in the direction of molecular long axis: (a) chiral
pinwheel unit aggregates with arbitrary designation of “R” and “S”
enantiomers; (b), (c), and (d): glide or herringbone lattice viewed as
mosaic of “all R”, “all S” and a mixture of equal portion of “R” and
“S”, respectively.
2100, 150
50
246 0.07
50 0.93 1.47
4414DHA
EtOH
DMSO
CHCl3
268 0.85
778 0.98
268 0.92
35 0.15 1.09
74 0.02 1.01
64 0.08 1.00
220, 30
260, 60
that it must contain attractive (bonding) interactions not possible
in the dimer. Taken together with the induced circular dichroism
observed for the aggregation of 8814DHA in DMPC vesicles
(but not for monomer or dimer) and for the “pure” vesicles of
48DHT and 88DHT in the presence of chiral R-alanine
enantiomers, this suggests that the unit aggregate is chiral. A
structure fulfilling these requirements may be the “pinwheel”
structure (Figure 10). This structure also may be extracted from
Monte Carlo simulations of monolayer clusters of the squaraine
acids such as 4414A and 4414DHA.19 In such cases the lowest
energy calculated structure which shows the best agreement
between calculated and measurable properties, such as tilt angle,
surface area per molecule, and exciton splittings calculated by
the extended dipole treatment of Kuhn and co-workers,13,14 is a
glide plane or herringbone arrangement shown schematically
in Figure 10.19 It is quite straight forward to extract the “unit
aggregate” or pinwheel structure as a primary building block
for the glide or herringbone lattice. This arrangement is very
similar to that concluded as the most likely structure for
spectrally blue-shifted aggregates observed for other aromatic
chromophores such as trans-stilbene and trans-azobenzene
derivatives,21,22 and it may well be a general supramolecular
structure from which larger crystallites or aggregates may be
constructed. We have discussed elsewhere19,21 some of the
factors which may contribute to the stability of the “pinwheel”
structure for the unit aggregate.
a Measured at or near the peak of the emission spectrum. Excitation
at 590 nm. b Fractional contribution to total intensity at measured
wavelength. c Exponential decay constants measured at the peak of the
ground state bleaching band. Excitation at 532 nm.
which can be obtained from the pure monomer spectrum. In
the case of 4414DHA, Ra and Rm are calculated to be 5.99 and
0.023, respectively. Ia and Iao at any dye concentration (Co)
can be calculated from eqs 4 and 5, while the concentrations
are determined by eqs 6 and 7. I1 and I2 are the measured
absorbance of the dye solution at λ1 (522 nm) and λ2 (650 nm),
respectively, at any concentration.
Iao ) ꢀaCo1
Ia ) Ra(I1 - RmI2)/(Ra - Rm)
[Agg] ) Ia/ꢀaCo1
(4)
(5)
(6)
(7)
[M] ) Co-n[Agg]
Figure 9 shows a Benesi-Hildebrand plot for 4414DHA in
a 50% (v/v) DMSO-H2O mixture. Table 2 summarizes values
of K, n, ∆H°, and ∆H° in the same solvent mixture over the
0
0
temperature range 278-318 K. Similar treatment of the
aggregation of three different squaraines in DMSO-H2O leads
to the aggregation numbers collected in Table 3. The aggrega-
tion number is ca. 4 in each case, suggesting that a “unit
aggregate” of the squaraines consists of four squaraine mol-
ecules. The aggregation number of 8814DHA in DMPC
vesicles is also estimated to be 3.5 ( 1. The relatively high
stability of this “unit aggregate” compared to the dimer suggests
It is interesting to note that while the pinwheel “unit structure”
is chiral, the extended herringbone lattice is achiral. This is
easily understood, as shown in Figure 10, since the same glide
lattice may be constructed from a mosaic of “all R”, “all S”, or
equal amounts of R and S unit aggregates. This leads to a simple

2594 J. Am. Chem. Soc., Vol. 118, No. 11, 1996
Chen et al.
observed in the present systems.40 In the present systems the
excited state absorption in the 400-480 nm region is attributed
to a transition from the one-exciton to the two-exciton level41
in the H-aggregates of 8814DHA and 88DHT. The more
complex spectral evolution and laser intensity dependence in
the 500-580 nm region are believed to be caused by large
exciton-exciton interactions that may lead to the formation of
biexctons.41,42
Summary
The effect of molecular structure on the aggregation of
amphiphilic squaraines in aqueous solution and in microhet-
erogeneous media has been investigated. All of the amphiphilic
squaraines form blue-shifted, “H” aggregates. The tendency
for aggregation increases as the length of the hydrocarbon chain
increases, indicative of a hydrophobic effect. While intermo-
lecular hydrogen bonding enhances the aggregation, squaraines
with quaternary ammonium head groups exhibit less tendency
for aggregation probably due to electrostatic repulsion between
the head groups. Experimentally, we show that the tendency
for aggregation increases when an electrolyte is present in the
solution. Unlike the monomeric form of squaraine, the blue-
shifted aggregates are nonfluorescent and short-lived (∼30 ps).
The photophysics of the aggregate is complex and remains to
be elucidated. Evidence is obtained that most squaraines form
aggregates without forming a dimer or a trimer first. The
aggregation number can be determined by a Benesi-Hildebrand
type analysis to be ∼4 for several squaraines. Taken together
with the induced circular dichroism observed for the aggregate
and Monte Carlo simulation results, we propose that the unit
aggregate is a tetramer with a chiral “pin-wheel” structure. The
extended aggregate observed in LB films and vesicles are simply
a mosaic of the unit aggregates. This model is supported by
observation of induced CD as a function of dye concentration
in DMPC vesicles, where ICD signal is shown to decrease at
high dye concentration.
Figure 11. 8814DHA (1.0 × 10-5 M) in DMPC vesicles with various
8814DHA/DMPC molar ratios: (1) absorption spectra and (2) plot of
the absorbance at 518 nm and the ICD intensity of the mixed vesicle
solution versus the 8814DHA/DMPC molar ratio.
prediction that as chiral unit aggregates are collected to form a
larger aggregate mosaic, there may be a decrease in the intensity
normalized ICD signal. Interestingly we find, as shown in
Figure 11, that as the conversion of unit aggregate to extended
aggregate increases (in this case for 8814DHA in DMPC as
the 8814DHA/DMPC molar ratio increases), both the overall
ICD and the normalized ICD intensity decrease after an initial
maximum is obtained.
Excited State Dynamics. The steady state spectroscopy and
photophysics of squaraines have been studied in detail by one
of the authors.35-37 The photophysics of the squaraines in the
present study are in general agreement with previous reports.
In addition, due to the higher sensitivity of the time-resolved
instrumentation used, an additional short-lived component for
the fluorescence decays is observed. This biexponential decay
suggests that there may be more than one species that contributes
to the fluorescence emission.38 Contributions from a solvent-
solute complex as well as a rotational isomer to the fluoresence
of monomeric squaraine dyes in solution have been reported.36
Further characterization of the monomeric squaraines beyond
that already presented was not carried out because the prime
objective of this work was to investigate the properties of the
aggregates.
Transient absorption spectroscopy was used to characterize
the excited state behavior of the nonfluorescent aggregated
species in bilayer vesicle solutions. The lifetime of the
aggregate, measured by monitoring the decay of absorption at
480 nm or the initial ground state bleaching recovery at 520
nm using low laser intensities, is ∼30 ps. The intensity-
dependent excited state absorption in the 510-580 nm region
suggests a process involving different excited moieties.39
Photodissiciation into monomeric squaraine, which has been
reported for other dimeric squaraines in solution,5 is not
Acknowledgment. We are grateful to the National Science
Foundation for support of this work as part of the NSF Center
for Photoinduced Charge Transfer (CHE-9120001).
JA9523911
(38) The biexponential nature of the excited state decay may also be
caused by interconversion between close-lying electronic states after the
initial excitation process, in addition to the usual deactivation channels
(Jones, G. II; Farahat, M. S. AdV. Electron Transfer 1993, 3, 1 and Rettig,
W. Topics Curr. Chem. 1994, 169, 253). In such a case, no wavelength
dependence is observed for the fluorescence decay parameters when only
one of the excited states involved is emissive (Lippert, E.; Rettig, W.;
Bonacic-Koutecky, V.; Miehe, J. AdV. Chem. Phys. 1987, 68, 1).
(39) Multiphoton ionization may be dismissed based on the lack of
quadratic dependence of the new absorption on laser intensity. Formation
of dye radicals by an excited state annihilation mechanism may also be
ruled out, since the observed spectra at long times after laser excitation
appear only as positive bands indicating an oscillator strength greater than
that of the ground statesan unlikely situation.
(40) Formation of the monomeric form would give rise to a distinctive
new absorbance peak in the 640 nm region, contrary to the observed results
(Figure 7).
(41) Observation of two-exciton bands in pump-probe experiments and
other non-linear processes have been explored both theoretically (Spano,
F. C. Chem. Phys. Lett. 1995, 234, 29 and Mukamel, S. Principles of
Nonlinear Optical Spectroscopy; Oxford University: Oxford, 1995) and
experimentally (VanBurgel, M.; Wiersma, D. A.; Duppen, K. J. Chem. Phys.
1995, 102, 20 and Horng, M. L.; Quitevis, E. L. J. Phys. Chem. 1993, 97,
12403).
(42) In order to investigate the excitonic interactions in squaraine dyes,
a model series of covalently linked dimeric squaraines has been synthesized
and their steady state and time resolved properties have been investigated.
The pump-probe spectra for these molecules show two-exciton absorption
bands, and they can be modeled using non-linear optical response theory
(see Mukamel, S. in ref 41). Farahat, M. S.; Liang, K.; Mukamel, S.;
Whitten, D. G. Manuscript in preparation.
(36) Law, K. Y. J. Phys. Chem. 1987, 91, 5184.
(37) Law, K. Y. J. Photochem. Photobiol. A 1994, 84, 123.