214 Langmuir 2003, 19, 214-217

A Study of Molecular Adsorption of Bromothymol Blue

by Optical Waveguide Spectroscopy
Zhi-mei Qi,†,‡ Naoki Matsuda,*,† Jose Santos,† Kiminori Itoh,§
Akiko Takatsu,| and Kenji Kato|
Nanoarchitectonics Research Center, National Institute of Advanced Industrial Science and
Technology, Tsukuba 305-8565, Japan, Graduate School of Environment and Information
Sciences, Yokohama National University, Yokohama 240-8501, Japan, and Metrological
Laboratory of Japan, National Institute of Advanced Industrial Science and Technology,
Tsukuba 305-8563, Japan
Received August 16, 2002. In Final Form: November 8, 2002

Optical waveguide spectroscopy of aqueous bromothymol blue (BTB) solutions confirmed that neutral
BTB molecules could easily adsorb from both acidic and basic solutions onto hydrophobic surfaces and that
the molecular BTB adsorption from basic solution, causing transient nonequilibrium between the molecular
and anionic BTB concentrations, was accompanied with the conversion of BTB- anions into BTB molecules
in the solution.

Optical waveguide (OWG) spectroscopy as a powerful

technique for in situ detection of molecular interactions
at the liquid/solid interface is of considerable interest.1-7
An OWG spectrometer constructed with a white light
source, a waveguide transducer, and a spectrally resolved
photodetector can provide much information such as
amount, orientation, and conformation of the adsorbate
at the interface as well as the adsorption kinetics of the
analyte. A broadband OWG spectrometer also allows for
simultaneous investigation of different chromophores
having different behaviors at the interface. In this Letter,
typical OWG spectroscopy of multiple chromophores was
performed using aqueous bromothymol blue (BTB) solu-
tions as samples that contain both chromophores of yellow
BTB molecules and blue BTB- anions. Anions of BTB and Figure 1. Schematic diagram of the OWG spectrometer used.
other pH indicator dyes have been recognized as surface-
inactive chromophores for glass plates and metal oxide information on interfacial behaviors of the neutral dye
thin films whose surfaces are generally negatively charged. molecules coexisting in aqueous solutions with their anions
As a result, basic solutions of these dyes are often used could be available in the reported works perhaps due to
as bulk absorbents for characterizing the evanescent incapabilities of the sensors generally working at a single
absorbance sensitivity of OWG sensors.7-10 However, no wavelength of 633 nm. The present study not only
characterized the OWG used but also revealed the
* To whom corresonpdence should be addressed. E-mail:
naoki-matsuda@aist.go.jp
dependence of interfacial behavior of molecular BTB on
† Nanoarchitectonics Research Center, National Institute of the surface properties and the solution pH. A somewhat
Advanced Industrial Science and Technology. similar study was reported by Eisenthal and co-workers
‡ On leave from the State Key Laboratory of Transducer that focused on investigation of the behaviors of phenols
Technology, Institute of Electronics, Academia Sinica, Beijing and anilines at the air/water interface by a technique of
100080, People’s Republic of China. the second harmonic generation.11,12 In earlier work,13 pure
§ Graduate School of Environment and Information Sciences,
BTB thin films with a refractive index of 1.69 at 633 nm
Yokohama National University.
| Metrological Laboratory of Japan, National Institute of Ad- have been fabricated by vacuum evaporation and spin-
vanced Industrial Science and Technology. coating techniques and used for optical sensing of ammonia
(1) Piraud, C.; Mwarania, E.; Wylangowski, G.; Wilkinson, J.; gas. This study demonstrated that the immersion of
O’Dwyer, K.; Schiffrin, D. J. Anal. Chem. 1992, 64, 651. hydrophobic glass slides into aqueous BTB solutions could
(2) Kato, K.; Takatsu, A.; Matsuda, N. Chem. Lett. 1995, 437. result in formation of molecular BTB monolayers onto
(3) Ross, Susan E.; Seliskar, Carl J.; Heineman, William R. Anal.
Chem. 2000, 72, 5549. the substrates.
(4) Bradshaw, J. T.; Mendes, S. B.; Saavedra, S. S. Anal. Chem. Figure 1 shows the configuration of the OWG spec-
2002, 74, 1751. trometer used. White light from a 150 W xenon lamp
(5) Qi, Z.-M.; Matsuda, N.; Yoshida, T.; Asano, H.; Takatsu, A.; Kenji,
K.; Opt. Lett. 2002, 27, 2001. passing through a glass fiber (200 µm in diameter with
(6) Santos, J. H.; Matsuda, N.; Qi, Z.-M.; Takatsu, A.; Kato, K. IEICE
Trans. Electron. 2002, E85-C, 1275. (10) Kuhn, K. J.; Burgess, L. W. Anal. Chem. 1993, 65, 1390.
(7) Doherty, W. J.; Donley, C. L.; Armstrong, N. R.; Saavedre, S. S. (11) Bhattacharyya, K.; Castro, A; Sitzmann, E. V.; Eisenthal, K.
Appl. Spectrosc. 2002, 56, 920. B. J. Chem. Phys. 1988, 89, 3376.
(8) Degrandpre, M. D.; Burgess, L. W.; White, P. L.; Goldman, D. (12) Castro, A.; Bhattacharyya, K.; Eisenthal, K. B. J. Chem. Phys.
S. Anal. Chem. 1990, 62, 2012. 1991, 95, 1310.
(9) Tsunoda, K.; Itabashi, H.; Akaiwa, H. Bull. Chem. Soc. Jpn. (13) Qi, Z.-M.; Yimit, A.; Itoh, K.; Murabayashi, M.; Matsuda, N.;
1992, 65, 581. Takatso, A.; Kato, K. Opt. Lett. 2001, 26, 629.

10.1021/la0264217 CCC: $25.00 © 2003 American Chemical Society

Published on Web 12/04/2002
Letters
Figure 2. Absorbance spectra of the acidic and basic BTB
solutions measured with the hydrophilic and hydrophobic glass
waveguides (A, acidic; B, basic; Hl, hydrophilic; Hb, hydrophobic;
gray line, TE mode; black line, TM mode).
a 100-µm core) is coupled into a slab waveguide by focusing
the light on a 5-mm 45° LaSF8 prism via a lens. With
another prism and fiber the light is coupled out of the
waveguide and then conducted to a CCD detector (S-2300,
Soma Optics, Ltd., Tokyo, Japan). A computer connected
to the CCD detector is used for data collection and
processing. A Teflon plate with a rectangular hole (2 cm
× 0.5 cm × 1 cm) as a sample cell is mounted onto the
waveguide. A polarizer is fixed in front of the input prism
coupler to select transverse electric (TE) and transverse
magnetic (TM) polarization states of the guided light. Slab
waveguides were fabricated by immersing soda-lime glass
slides (76 mm × 26 mm × 1 mm) into molten KNO3 at 400
°C for 30 min. After K+-Na+ ion exchange the glass
waveguides are highly hydrophilic. Some of them were
modified to be hydrophobic by dipping the waveguides
into the absolute toluene containing 1% (wt/wt) octa-
decyltrichlorosilane for 1 h. The silanized waveguides have
a water contact angle of >70°. The glass waveguides as
transducers of the spectrometer give a transmission
spectrum ranging from 450 to 800 nm. The acidic (0.08
mM, pH 4.80) and basic (0.96 mM, pH 9.18) BTB solutions
were prepared using deionized water and tetraborate
buffer solution as solvents, respectively. An absorption
spectrum of a solution sample can be determined from
the equation A ) log(IR/Is), where IR and IS are output
intensities at a fixed wavelength measured with the
solvent and its solution in the cell, respectively.
Figure 2 shows absorption spectra of the acidic (A) and
basic (B) BTB solutions obtained with the hydrophilic (Hl)
and hydrophobic (Hb) glass waveguides. In the A-Hl
spectra the TE- and TM-polarized absorbances are nearly
zero in the wavelength range 470-720 nm. However, the
A-Hb spectra exhibit relatively large absorbances at
wavelengths below 520 nm, which, compared with the
A-Hl spectra, reveals that the detected absorbances are
attributed to absorption of the molecular BTB adlayer
rather than that of the bulk solution. In the present case
the bulk solution absorption could not be detected due to
a low bulk concentration as well as a weak evanescent
field. From the findings it is concluded that neutral BTB
Langmuir, Vol. 19, No. 2, 2003 215
molecules can be immobilized from aqueous BTB solution
onto hydrophobic rather than hydrophilic glass. This is
because the attractive force between the hydrophobic
surface and the hydrophobic groups (i.e., benzene rings
and -CH3) of the BTB molecule is larger than the solvation
force between water and its hydrophilic groups such as
-SO3-, -Br, and -OH. In addition, it was observed from
the A-Hb spectra that at a fixed wavelength (<520 nm)
the TM-polarized absorbance is larger than that for TE.
This discrepancy does not imply orientation of the
molecular BTB adlayer and just results from the fact that
the TM mode has an evanescent field stronger than that
of the TE mode.
A larger evanescent power of the TM mode guided in
the ion-exchanged glass waveguide was confirmed by OWG
spectroscopy of the basic BTB solution. The B-HI and B-Hb
spectra in Figure 2 exhibit an absorbance band in the
wavelength range 520-670 nm, which is attributed to
absorption of the anionic BTB in the bulk solution. It is
evident that with the TM mode, the peak absorbance at
617 nm is larger than that with the TE mode, independent
of the OWG surface properties. The comparison indicates
that the TM mode contributes a larger evanescent power
to the bulk absorbent than the TE mode. This difference
has been demonstrated to result from the positive bi-
refringence, nTM - nTE > 0, of potassium ion exchanged
waveguides induced by compressive stress in the ion-
exchanged layers.14
In the wavelength range 470-520 nm, the TE- and TM-
polarized absorbances are present in the B-Hb spectra
but disappear in the B-Hl spectra. On the basis of the
findings above, this significant difference results from the
molecular BTB adsorption from the basic solution onto
the hydrophobic waveguide. By use of the Henderson-
Hasselbalch equation: pH ) pKa + log(CB/CA), where pKa
for BTB in aqueous solution is 7.1 and CA and CB are bulk
concentrations of the BTB molecule and its anion. CA in
the basic BTB solution is calculated to be 8 µM, which is
10 times as low as that in the acidic BTB solution. However,
the detected absorbances at wavelengths e520 nm in the
A-Hb and B-Hb spectra are relatively close to each other.
To confirm this extraordinary result, the absorbance
spectra of several samples having the same pH values
(4.80 or 9.18) but different dye concentrations were
investigated using the silanized glass waveguides. The
filled circles in Figure 3 show the TM-polarized absorb-
ances at 470 nm as a function of the calculated CA in pH
9.18 BTB solutions. The absorbance rapidly increases with
increasing CA up to 2 µM and then moves toward
saturation with further increase in CA. The filled squares
in Figure 3 show the case of pH 4.80 BTB solutions, and
two lines represent the best fits to the Langmuir isotherm.
At the same CA the absorbance of the basic BTB solution
is much larger than that of the acidic one, suggesting that
the number of neutral BTB molecules adsorbed from the
basic solution is much greater than that from the acidic
one in the case of the same CA. The difference between
molecular BTB adsorption from the acidic and basic
solutions is considered to result from the conversion of
BTB- anions into BTB molecules in the basic solution. It
is known from the Henderson-Hasselbalch equation that
CA and CB in a given aqueous BTB solution are determined
by the solution pH. Neutral BTB molecules are the
predominant chromophore in acidic BTB solutions, and
thus the molecular BTB adsorption from acidic solutions
should have a negligible effect on the equilibrium between
CA and CB. It is not such a case for basic BTB solutions
(14) Qi, Z.-M.; Itoh, K.; Murabayashi, M. Appl. Opt. 2000, 39, 5750.
216 Langmuir, Vol. 19, No. 2, 2003
Figure 3. Measured absorbance at 470 nm versus the
calculated CA in pH 9.18 and 4.80 BTB solutions. Lines show
the best fits to the Langmuir adsorption isotherm.
where neutral BTB molecules become the minority chro-
mophore. The molecular BTB adsorption from basic
solutions will decrease CA to some extent. To keep the
equilibrium between CA and CB in basic BTB solutions
with fixed pH values, the molecular BTB adsorption will
be accompanied by spontaneous conversion of BTB- anions
into BTB molecules in the solutions. As a result, a large
number of BTB- anions in basic solutions, which served
as a source to generate neutral BTB molecules for
adsorption onto the hydrophobic waveguide, resulted in
the increased absorbances in the wavelength range 470-
520 nm. The molecular BTB adsorption onto hydrophobic
glass would make the pKa value of the dye at the interface
smaller than that in aqueous solution.
The molecular BTB adsorption onto hydrophobic glass
was found to obey the Langmuir isotherm. With the surface
concentration CS in mol/cm2 and its maximum CSmax, the
Langmuir adsorption equation can be expressed as CS/
CSmax ) C/(C + R), where C is the bulk concentration and
R ) 55.5 exp(∆Gads/RT).12 From the equation it is seen
that 1/CS linearly depends on 1/C. The pseudo-Beer law
noted below indicates that the absorbance A is proportional
to CS. Therefore, a linear dependence of 1/A on 1/C exists
for the Langmuir adsorption. Using the measured ab-
sorbances in Figure 3, we did find that 1/A was a linear
function of 1/C (here C ) CA + CB). From two straight
lines in Figure 4 ∆Gads is derived to be -8.02 and -8.07
kcal/mol for molecular BTB adsorption from pH 9.18 and
4.80 solutions, respectively. As expected, both energies of
adsorption are approximately equal, independent of the
solution pH. A linear relationship also exists between 1/A
and 1/CA, from which ∆Gads is calculated to be different
for different pH (-10.81 and -8.07 kcal/mol for pH ) 9.18
and 4.80). A comparison between two calculations suggests
that not CA but C determines ∆Gads and CS of BTB
molecules adsorbed onto a given hydrophobic substrate.
After the BTB solution was removed from the sample
cell, the hydrophobic waveguide surface without retaining
any solution drop was exposed to air. Such a waveguide,
in the presence of ammonia gas, could absorb the guided
light in the wavelength range 520-680 nm (see Figure 5)
due to deprotonation of the BTB molecules immobilized
onto the waveguide. This is more evidence of the existence
of the molecular BTB adlayer on the hydrophobic glass
surface. The molecular BTB adsorption onto the silanized
TiO2 film was also observed by the OWG spectroscopy
performed with a TiO2 film coated glass waveguide. The
Letters
Figure 4. 1/A as a linear function of 1/C.
Figure 5. Absorption spectrum (a) of the molecular BTB
adlayer formed from pH 4.80 BTB solution onto the hydrophobic
glass waveguide and spectral response (b) of the adlayer to
ammonia gas.
OWG spectroscopy also confirmed the molecular adsorp-
tion of thymol blue (TB) from aqueous solutions onto
hydrophobic substrates. The molecular adsorption from
a given BTB or TB solution was found to highly depend
on the waveguide surface hydrophobicity that had a bad
reproducibility and consequently resulted in difficulty in
accurate measurement.
The potassium ion exchanged glass waveguides used
above have a narrow spectral bandwidth that limits the
measured absorbances of the molecular BTB adlayer to
wavelengths g470 nm. To achieve a complete electron
spectrum of the molecular BTB adlayer, the waveguide
transducer of the OWG spectrometer was changed into a
50-µm-thick glass plate that could transmit light at
wavelengths down to 360 nm (see ref 5 for details). Before
use, the plate was silanized in hexamethyldisilazane vapor
at 90 °C for 1 h. Figure 6a shows the measured TE-
polarized absorption spectrum of the basic BTB solution
(1.10 mM, pH 9.18). The spectrum includes two bands,
and the left band with a peak at 440 nm is attributed to
absorption of the molecular BTB adlayer. For comparison,
an absorption spectrum of the bulk solution measured
with a conventional UV-vis spectrometer is also shown
in Figure 6a (the BTB solution was diluted in order to
obtain a measurable spectrum). The molecular BTB
adsorption onto the hydrophobic glass plate makes the
evanescent absorption spectrum quite different in shape
from the bulk solution spectrum. Figure 6b shows the
evanescent absorbance at 440 nm versus the measuring
time (the CCD detector recorded the absorption spectrum
once every second). The steady-state absorbance was
Letters
Figure 6. Absorption spectra (a) of pH 9.18 BTB solutions
measured with the OWG and conventional UV-vis spectrom-
eters and (b) dependence of the evanescent absorbance at 440
nm on the measuring time. The waveguide used for OWG
spectroscopy was a 50-µm-thick glass plate with a silanized
surface.
detected immediately as the solution sample was intro-
duced into the cell within 1 s from t ) 0. It indicates that
the molecular BTB adsorption on the hydrophobic glass
surface is very fast.
A thin-film glass plate serving as the waveguide
transducer allows us to estimate the surface concentration
by use of the waveguide theory and the pseudo-Beer law:5
A ) ηcL, where η is the optical power fraction in the
adlayer,  is the molar absorptivity, L is the interaction
path length, and c ) CS/d (d is the adlayer thickness). In
the present case, L is 2 cm and  for molecular BTB is
1.638 × 104 M-1 cm-1 at 440 nm. To calculate η, the BTB
molecule having a stereo structure is treated as a ball
with a diameter of 1 nm and the effective refractive index
Langmuir, Vol. 19, No. 2, 2003 217
of the guided light in the glass plate was measured to be
∼1.43.5 On the basis of the TE mode in a four-layer slab
waveguide consisting of the air substrate (n ) 1), the 50-
µm glass plate (n ) 1.53), the 1-nm BTB adlayer (n )
1.69), and the water superstrate (n ) 1.333), η in the
molecular BTB adlayer is calculated to be 2.1324 × 10-5
at 440 nm. With a measured absorbance of A ) 0.44 at
440 nm, CS is determined to be 6.3 × 10-11 mol/cm2, which
occupies 38% of a closely packed BTB monolayer. This is
the largest surface concentration for the thin-film glass
plate used because the dye concentration of 1.10 mM
exceeds the value required for saturated adsorption of
BTB molecules from pH 9.18 solution (see Figure 3). Such
a low surface concentration suggests a low degree of
silanization of the glass surface.
A comparison between the measured and calculated
evanescent absorbances at 617 nm demonstrated that the
calculation of CS above was rational and accurate. At 617
nm the evanescent power fraction in water superstrate
was calculated to be 2.0272 × 10-3 and  for anionic BTB
is 3.958 × 104 M-1 cm-1. CB in the solution sample used
was derived to be 1.0922 mM. Therefore, the calculated
absorbance at 617 nm is A ) 0.1752. From Figure 6a the
measured evanescent absorbance at 617 nm is A ) 0.1728,
which is approximately equal to the calculated value. Note
that calculating CS would be difficult for potassium ion-
exchanged glass waveguides due to difficulty in accurate
determination of refractive index and depth of the ion-
exchanged layer.
In conclusion, the dependence of the interfacial behavior
of neutral BTB molecules on both the surface property
and the solution pH has been demonstrated by the OWG
spectroscopy of aqueous BTB solutions. In the meantime,
the study shows the outstanding applicability of the OWG
spectroscopy for simultaneous characterization of multiple
chromophores having different properties at the liquid/
solid interface.
Note Added after ASAP Posting. This article was
released ASAP on 12/4/2002. Changes made after posting
were as follows: last page of paper, first paragraph, line
6, equation c ) CSd was changed to c ) CS/d; last page of
paper, first paragraph, line 18, the value 6.3 × 10-9 was
changed to 6.3 × 10-11. The correct version was posted on
12/12/2002.
LA0264217