Full Paper

Binding of Methylene Blue to Polyelectrolytes

Containing Sulfonate Groups

Ignacio Moreno-Villoslada,* Cristian Torres, Felipe González,

Toshimichi Shibue, Hiroyuki Nishide

The interaction between methylene blue (MB) and poly(sodium 4-styrenesulfonate) (PSS),
poly(sodium vinylsulfonate) (PVS), and the more hydrophobic poly[sodium 2-(N-acrylamido)-
2-methyl-propanesulfonate] (PAMPS), is investigated. The main driving forces for the inter-
action with PSS are supposed to be short-range aromatic/aromatic interactions, which explain
the smaller dissociation constant, the resistance to the cleaving effect of NaCl, and the
prevention of MB self-aggregation around the macromolecules under a moderate excess of
the polymer. On the con-
trary, as a consequence
of long-range inter-
actions, a higher local
concentration of MB
around PAMPS and, more
significantly, around PVS
results in MB self-aggre-
gation that can be
quenched in the presence
of NaCl.

Introduction interest in recent years. The characteristic differences

Methylene blue (MB) is an interesting molecule with
important staining, aggregative, and redox properties. As a
staining material it has been used in medicine for
differential staining of tissues[1] and in sentinel node
mapping.[2] The aggregative properties of MB and its
analogous structures have been the subject of renewed
I. Moreno-Villoslada, C. Torres, F. González
Instituto de Quı́mica, Facultad de Ciencias, Universidad Austral de
Chile, Casilla 567, Valdivia, Chile
Fax: þ56 63 293520; E-mail: imorenovilloslada@uach.cl
T. Shibue
Materials Characterization Central Laboratory, Waseda
University, Tokyo 169-8555, Japan
H. Nishide
Department of Applied Chemistry, School of Science and
Engineering, Waseda University, Tokyo 169-8555, Japan
between the electronic absorption spectra of monomers,
dimers, and higher aggregates have been used to distin-
guish them.[3–8] MB interacts with different materials
such as colloids,[8–12] metallic surfaces,[13] silica,[14–16]
nano-objects,[17,18] polymers,[19] cyclodextrins,[20–22] and
form complexes with other low-molecular-weight mole-
cules.[22–24] Its state of aggregation may change as a
consequence of these interactions. Useful applications
can be projected on the basis of these aggregation
properties such as the colorimetric determination of
anionic hydrocolloids, nano-objects, or specific DNA
sequences.[17–19,24–27] Besides, MB interacts with nucleic
acids, proteins, and lipids and induces photosensitized
reactions after photoactivation. It generates singlet oxygen
very efficiently, causing photooxidative damages in
biological systems, including strand breakage in DNA.
When cationic organic dyes are adsorbed on negatively
charged polyelectrolytes (PELs), aggregation of the dyes

Macromol. Chem. Phys. 2009, 210, 1167–1175

ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/macp.200900042 1167
 I. Moreno-Villoslada, C. Torres, F. González, T. Shibue, H. Nishide

is sometimes observed. In addition to the electrostatic In this paper we will show, by the use of UV-vis and
1
forces which bind the opposite charges of the polyanion H NMR spectroscopy, as well as DF, photon correlation
and cation to one another, the self-organization of dye spectroscopy (PCS), and laser Doppler anemometry (LDA),
aggregates is promoted by forces such as dispersion forces a comparative study of the binding of MB to several anionic
due to the interaction between the p-systems of the dyes PELs that contain sulfonate groups, such as poly(sodium
and hydrophobic forces which may involve the details of 4-styrenesulfonate) (PSS), poly(sodium vinylsulfonate) (PVS),
the polyanion. For aggregates to form and be stable, the sum or the more hydrophobic poly[sodium 2-(N-acrylamido)-
of these forces must be larger than the electrostatic 2-methylpropanesulfonate] (PAMPS), and the results will
repulsion between the positive charges on the dye be interpreted under the scope of MB self-aggregation and
molecules. the different forces involved in its binding to the PELs.
In our laboratory, we are pointing out the importance of
aromatic/aromatic (ar/ar) interactions on the control of
the properties of aromatic low-molecular-weight mole- Experimental Part
cules by interaction with PELs containing aromatic rings.
Reagents
Thus, the luminescent,[28–30] redox,[31] acid/base,[29,30,32]
and binding properties [29–31,33] of various substrates are Commercially available PSS (Aldrich, synthesized from the para-
modified by this method. Ar/ar interactions are one of substituted monomer, Mw ¼ 70 000), PVS (Aldrich), PAMPS (Aldrich,
the principal noncovalent forces governing molecular Mw ¼ 2 000 000), ethylene glycol (EG, Aldrich), and MB (Synth) were
used to prepare solutions in deionized distilled water. The
recognition and biomolecular structure. Studies show that
structures of MB and the different PELs are shown in Figure 1.
the preferential binding mode of MB to DNA is the
The pH was adjusted with minimum amounts of NaOH and HCl.
symmetric intercalation of MB between successive base
pairs, involving ar/ar interactions.[25] The dual relevance of
these interactions concerning the structural and functional
aspects motivates the development of synthetic systems Equipment
whose structures and properties may be tuned by ar/ar Distilled water was deionized in a Simplicity Millipore deionizer.
interactions. The pH was controlled on Hanna pH ¼ 211 and Horiba F-15 pH
Diafiltration (DF) has emerged as a suitable technique to meters. UV-vis experiments were performed at 293 K in a Helios
evaluate the interactions between low-molecular-weight g spectrophotometer. 1H NMR measurements were made in JNM
species (LMWS) and PELs.[34–38] This is a separation Lambda500 (JEOL, 500 MHz) and AVANCE 600 (Bruker, 600 MHz)
technique that discriminates molecules as a function of spectrometers. The unit used for diafiltration studies consisted of a
their size. The rate of filtration of the LMWS can be related to filtration cell (Amicon 8010, 10 mL capacity) with a magnetic
the strength of its interaction with the PEL, which is unable stirrer, a regenerated cellulose membrane with a molecular weight
cut-off of 5 000 or 10 000 Da (Ultracel PLCC, 25 mm diameter), a
to pass through the DF membrane. The main magnitudes
reservoir, a selector, and a pressure source. The size and zeta
managed in DF analyses are the filtration factor (F), defined
as the ratio between the volume in the filtrate and the
constant volume in the DF cell, the concentration in
the filtrate of the LMWS under study (cfiltrate LMWS ), the
concentration of free LMWS in the cell solution (cfree LMWS ),
the concentration of LMWS reversibly bound to the PEL
rev
bound diss
PEL
(cLMWS ), the apparent dissociation constant (KLMWS ),
free rev
bound
defined as the ratio cLMWS =cLMWS , the diafiltration
parameters km, j, u, and v, and the polymer concentration
in mole of monomeric units per liter (cP). km and j
parameters (the absolute value of the slope of the curve
ln cfiltrate
LMWS versus F in the absence and in the presence of
the PEL, respectively) are related with the strength of the
interaction, while v and u are related with the amounts of
LMWS reversibly or irreversibly bound to the polymer,
respectively. By irreversibly bound we consider molecules
bound in processes that may be reversible with an apparent
dissociation constant that tend to zero at the conditions of
the experiment. The parameters u and v are calculated
from the ordinate at the origin when plotting ln cfiltrate LMWS
versus F. Figure 1. Molecular structures of MB and the different PELs.

Macromol. Chem. Phys. 2009, 210, 1167–1175

1168 ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/macp.200900042
Binding of Methylene Blue to Polyelectrolytes Containing . . .

potential of the colloidal systems were determined by PCS and LDA,
respectively, in a Zetasizer Nano-ZS (Malvern Instruments).
Procedures
Conventional procedures have been followed. Details for experi-
mental conditions can be found in the Figure captions. UV-Vis: In
order to have absorbances in a range of 0.1–1.0, the optical path
length was set between 1 and 10
2 cm for solutions containing MB
at concentrations ranging between 10
5 and 10
3 M. MB concen-
tration in most experiments was chosen to be 10
5 M in order to
provide good sensitivity and negligible self-association. Diafiltra-
tion: Solutions in twice distilled water (10 mL) were prepared
containing one or more of the following components: PEL
(polymeric molecular weight fraction over 10 000 Dalton), NaCl
0.1 M, and MB, at pH ¼ 7. The solutions were placed into the
diafiltration cell. The pH and the NaCl concentration of the aqueous
solution contained in the reservoir were adjusted to the same value
as in the cell solution. The filtration runs were carried out over a
regenerated cellulose membrane with a molecular weight cut-off of
5 000 Da under a total pressure of 3 bar, keeping constant the
solution volume in the cell by creating a continuous flux of liquid
through the cell solution from the reservoir (around 0.005 mL  s
1).
Vigorous stirring is held in order to minimize concentration
polarization and fouling. Filtration fractions (ranging between
6.0 and 8.0 mL) were collected and MB concentrations analyzed by
UV-Vis spectroscopy. No macromolecule was found in the filtrate.
A calibration curve (Absorbance ¼ 7.4  104 [MB]) was obtained at
665 nm at pH ¼ 7 in a range of MB concentrations between
5.0  10
7 and 1.0  10
5 M, with a square linear regression factor of
1.00. Blank experiments were performed with the same procedure,
in the absence of the PEL. At least one replicate is done for every
experiment. More specific experimental conditions are given in
Table 1. The MB concentration was chosen to be 2  10
5 M in order
to provide good sensitivity for filtration fraction analyses. The
diss
PEL
diafiltration profiles, as well as the KLMWS as it is defined in
this work, are dependent on the polymer concentration, so for the
different systems a concentration of 4  10
5 M was chosen in order
to obtain good sensitivity and diafiltration profiles that make
possible the observation of the different binding between the
different systems.
1
H NMR (D2O, 25 8C, 10
3 M MB, see Figure 1 for assignments):
d ¼ 3.12 (s, methyl), 6.87 (s, H4), 7.08 (d, H2), 7.44 (d, H1).
Other 1D 1H NMR spectra shown here were obtained for dilute
MB solutions in the range of the sensitivity of the technique
(2  10
4 M) in order to avoid precipitation with the PELs. 2D
diffusion-ordered spectroscopy (DOSY) experiments were made on
the 600 MHz spectrometer under a stimulated echo sequence
employing bipolar gradients and a longitudinal eddy current delay.
Diffusion delays in the range 20–100 ms and gradient durations in
the range 2–6 ms were searched for 10
3 M MB and 10
2 M PSS
solutions, to obtain appropriate curves (25 points) for inverse
Laplace transformation. The same procedure was done including
EG as a probe reference in order to discard the change in the
viscosity produced by the polymer. Nuclear Overhauser effect
spectroscopy (NOESY) experiments were performed on the
600 MHz spectrometer and recorded at room temperature with
mixing times of 200 ms. PCS: experiments were done at 25 8C, in
disposable sizing cuvettes. At least three replicates were done of
each measurement. Results meeting the criteria provided by the
manufacturer were taken as significant of nanoparticle formation.
The solutions analyzed contained 2  10
4 M MB and were the
same used for 1H NMR studies. LDA: The zeta potential of the
nanoparticles found was measured in electrophoretic cells where a
potential of  150 mV was established.
Results and Discussion
MB in the Absence of any PEL
The UV-Vis spectrum of MB in water shows two bands
around 610 and 666 nm (see Figure 2).[3] The ratio between
the intensities of both bands increases by increasing the MB
concentration, related with an increasing probability for
the dye to undergo self-association by means of H-type

Table 1. Results for diafiltration of 2  10
5 M MB solutions at pH ¼ 7 in the presence of different additives. For linear adjustments: y ¼ ln
hcfiltrate
MB i; x ¼ F; R2 ¼ linear regression factor.

Experiment cP [NaCl] v u j km diss
PEL a)

KMB Linear adjustment R2

mol  L
1 mol  L
1

MB-01 – – 0.7 0.3 – 0.81 – y ¼
0.81x
11.2 0.99

MB-NaCl-01 – 0.1 0.8 0.2 – 0.79 – y ¼
0.79x
11.0 1.00
PVS -MB-01 4  10
5 – 0.6 0.4 0.57 – 1.6  0.3 y ¼
0.57x
11.6 0.98
PVS -MB-NaCl-01 4  10
5 0.1 0.8 0.2 0.81 – !1 y ¼
0.81x
10.8 1.00
PAMPS -MB-01 4  10
5 – 0.6 0.4 0.42 – 0.80  0.8 y ¼
0.42x
12.0 0.95
PAMPS -MB-NaCl-01 4  10
5 0.1 0.7 0.3 0.81 – !1 y ¼
0.81x
11.0 1.00
PSS -MB-01 4  10
5 – 0.7 0.3 0.09 – 0.10  0.01 y ¼
0.09x
13.5 0.60
PSS -MB-NaCl-01 4  10
5 0.1 0.8 0.2 0.51 – 1.2  0.3 y ¼
0.51x
11.5 0.97

j m
a) diss
PEL
KMB is calculated according to 1
j
diss
PEL
 KMB  kkm
jj .

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ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mcp-journal.de 1169

Figure 2. Normalized UV-Vis spectra at pH ¼ 7 of MB solutions in
the absence of NaCl (continuous lines) and in the presence of 0.1 M
NaCl (dotted lines) at concentrations of (a, a0 ) 10
5 , (b, b0 ) 10
4 ,
and (c, c0 ) 10
3 M.
contacts. Thus, the band at 666 nm is called the monomer
band, while the band at 610 nm is called the dimer band.
The 1H NMR spectrum of this molecule is described in the
experimental section.
The effect of the addition of 0.1 M NaCl to the solutions
can be also seen in Figure 2. The ratio between the
intensities of the dimer and monomer bands increases, a
fact that is interpreted as a higher tendency of MB to form
aggregates at these conditions.
MB in the Presence of PVS
Among the series presented in this work, PVS is expected to
exhibit a pure PEL character, i.e., the main forces involved in
the interaction with MB are long-range electrostatic forces.
A large literature [18,19,25] together with our own observa-
tions are concordant with the existence of large aggregates
of MB on the environment of polyanions, revealed by a
sharp decrease on the intensity of the monomer band, and
the appearance of a band at around 570 nm (see Figure 3)
that we name polyMB band. The decrease on the monomer
band is noticeable even in the presence of PVS concentra-
tions lower than equimolar (in sulfonate groups) with
respect to MB. For equimolar polymer concentrations or
higher with respect to the dye, the polyMB band appears
clearly, and its position do not change appreciably by
increasing the polymer concentration. According to the
exciton theory, the position of this band will depend on
the average length of the aggregates, so that we can
conclude that the binding of the dye is cooperative and
Macromol. Chem. Phys. 2009, 210, 1167–1175
1170 ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
I. Moreno-Villoslada, C. Torres, F. González, T. Shibue, H. Nishide
Figure 3. UV-Vis spectra of a 10
5 M MB aqueous solution in the
absence of any PEL (a), and in the presence of PVS at concen-
trations of 2.5  10
7 (b), 5  10
6 (c), 1  10
5 (d), 5  10
5 (e),
4  10
4 (f), and 1  10
3 M (g), and in the presence of 0.1 M NaCl
and PVS at concentrations 1  10
5 (h) and 4  10
4 M (i).
the low-molecular-weight molecules (LMWM) do not
distribute randomly on the polymer surface, but forming
large aggregates. The formation of these aggregates is
justified by a local higher concentration of the dyes around
the polyanions due to long-range electrostatic interactions,
and the tendency of MB to self-aggregate. Thus, in the
environment of the polymer, MB aggregates are strongly
charged and may produce precipitates with the comple-
mentarily charged PVS at high concentrations and degree
of neutralization: these MB aggregates may behave as
supramolecular polymers,[39,40] and interpolymer com-
plexes may be formed. At dilute conditions and low degree
of neutralization (PVS 10
3 M, MB 10
4 M), PVS/MB aggre-
gates are found in the nanometric scale, as detected by PCS
and LDA. The nanoaggregates formed have an apparent
mean diameter of 127 nm (with a polydispersity index of
0.293) and showed a zeta potential of
34.5 mV. Molecules
confined in nanoparticles are difficult to see by 1H NMR
spectroscopy, since they undergo fast relaxation. It is
shown in Figure 4 the aromatic region for MB alone at a
concentration 2  10
4 M (Figure 4a), and in the presence of
10 times PVS (Figure 4b). It can be seen that the signals of
MB disappear in the presence of the polymer, corresponding
to a highly aggregated state.
Long-range electrostatic interactions are easy to quench
by the addition of other electrolytes in excess, so NaCl could
be used to cleave the interaction between MB and PVS. This
can be observed in Figure 3 where UV-Vis experiments
show the recuperation of the spectra of pristine MB by the
addition of the single electrolyte in the presence of
the polymer. NMR experiments show the recuperation
of the MB signals (Figure 4c) by the addition of NaCl, in
DOI: 10.1002/macp.200900042
Binding of Methylene Blue to Polyelectrolytes Containing . . .

Figure 5. DF profiles of a 2  10
5 M MB solution in the absence

of added NaCl (left) and in the presence of 0.1 M NaCl (right):
in the absence of any PEL (, *), in the presence of 4  10
5 M
PVS (^, ^), in the presence of 4  10
5 M PAMPS (&, &), and in
the presence of 4  10
5 M PSS (~, ~); see Table 1 for linear
adjustments.

In the presence of 0.1 M NaCl, the interaction of MB with

PAMPS is also quenched, as can be seen in Figure 5 and 6, and
Figure 4. 500 MHz 1H NMR spectra in D2O of MB 2  10
4 (a), MB Table 1. Figure 6 shows that the spectrum of pristine MB is
2  10
4 and PVS 2  10
3 (b), and MB 2  10
4, PVS 2  10
3 and recuperated in the presence of the single electrolyte for
NaCl 0,1 M (c). solutions containing PAMPS. DF results shown in Figure 5
and Table 1 confirm the quenching of the long-range
interaction in the presence of 0.1 M NaCl, since negligible
accordance to the quenching of the MB/polymer interaction
differences are found in the presence or in the absence of
and disaggregation of the dye.
the polymer. However, at the conditions used for 1H NMR
More direct evidence of the quenching of the interaction
experiments, the MB signals are lost both in the presence
produced by the addition of NaCl should be found by DF.
and in the absence of NaCl.
As DF is a separation technique that discriminates small
The higher dispersant ability shown by UV-Vis spectro-
molecules from large molecules and aggregates, the
scopy, the higher binding ability seen by DF, and resistance
binding of MB to different PELs can be followed and
to the cleaving effect of NaCl detected by 1H NMR, with
quantified. This can be seen in Figure 5 and Table 1, where
respect to the situation in the presence of PVS are, according
DF profiles performed at different conditions have been
diss
PVS to our interpretation, due to the higher hydrophobicity of
plotted. In the absence of NaCl, the KMB takes a value
around 1.6; in the presence of 0.1 M of NaCl, the DF profile is
very similar to that of the blank experiment (in the absence
of the PEL), revealing the absence of interaction.

MB in the Presence of PAMPS

In contrast to PVS, PAMPS exhibits a higher hydrophobic
character due to the aliphatic branches on its pendant
groups. Thus, a contribution from hydrophobic forces on
the binding of MB to this polymer is expected. As can be seen
in Figure 6 by UV-Vis spectroscopy, the behavior of this
polymer is very similar to that of PVS, but a certain tendency
to avoid large self-aggregates of MB is found, revealed by a
shift of the polyMB band to lower energies in the presence
of a large excess of PAMPS. No nanoparticle formation is
detected by PCS at similar conditions at which they
Figure 6. UV-Vis spectra of a 10
5 M MB aqueous solution in the
were detected for PVS. Moreover, DF profiles shown in
absence of any PEL (a), and in the presence of PAMPS at concen-
Figure 5 and analyzed in Table 1 show a higher binding trations of 5  10
7 (b), 5  10
6 (c), 1  10
5 (d), 4  10
5 (e),
ability for this polymer than for PVS, as can be deduced 4  10
4 (f), and 1  10
3 M (g), and in the presence of 0.1 M NaCl
diss
PVS and PAMPS at concentrations 1  10
5 (h) and 4  10
4 M (i).
by the smaller KMB , which takes a value around 0.8.

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 I. Moreno-Villoslada, C. Torres, F. González, T. Shibue, H. Nishide

this polymer, together with the higher flexibility of the

chains supporting the sulfonate groups.

MB in the Presence of PSS

A different scenario is found for PSS. This PEL presents an
aromatic ring to which the sulfonate group is directly
attached. We have already reported the high ability of this
polymer to bind aromatic cations or zwitterions, due to a
suitable complementation of short-range electrostatic
forces, nonspecific hydrophobic interactions, and ar/ar
interactions.[28–33] UV-Vis analyses show that in the
presence of small amounts of PSS, the aggregation of MB
is also induced in the environment of the polymer, as can be
seen in Figure 7 by the disappearance of the monomer band.
But as the concentration of PSS increases from an equimolar
ratio (in sulfonate groups), the band corresponding to the
higher aggregates shifts continuously to 616 nm, revealing
the continuous formation of smaller aggregates. Moreover,
the monomer band is clearly increased by increasing the
polymer concentration, indicating high dispersion of the
dyes on the polymer domain; the increasing monomer band
is shifted 4 nm to lower energies. This may be due to the
interaction of the MB transition moment with that of the
polymer as a consequence of short-range interactions. No
nanoparticle formation is detected by PCS at the same
conditions as it was detected for PVS, which also supports
the absence of large aggregates of MB. The 1H NMR MB
signals do not disappear in the presence of PSS, but shows
Figure 8. 500 MHz 1H NMR spectra in D2O of MB 2  10
4 (a), MB
upfield shifts of about 0.4 ppm and broadening, as can be
2  10
4 and PSS 2  10
3 (b), MB 2  10
4, PSS 2  10
3, and NaCl
seen in Figure 8a and b, facts that we take as proof of the 0.1 (c), and PSS 10
2 M (d).
existence of ar/ar interactions: the effect of stacking two

aromatic rings is to place one ring in the shielding cone of

Figure 7. UV-Vis spectra of a 10
5 M MB aqueous solution in the
absence of any PEL (a), and in the presence of PSS at concen-
trations of 2  10
7 (b), 2.5  10
6 (c), 1  10
5 (d), 2  10
5 (e),
8  10
5 (f), 2  10
4 (g), and 1  10
3 (h), and in the presence of
0.1 M NaCl and PSS at concentrations 1  10
5 (i) and 4  10
4 M (j).
the second, produced by the circulation of the aromatic
p-electrons, resulting in upfield shifts of 1H resonances.
Note that the peaks informed for a 10
3 M MB solution in
the experimental section are shifted upfield more than
0.1 ppm with respect to the signals shown in Figure 4a
and 8a for a 2  10
4 M MB solution. This is due to the
self-aggregation of MB by means of ar/ar interactions at
higher concentrations. However, for a 10
3 M MB solution in
the presence of 10 times PSS, the corresponding chemical
shifts for the aromatic protons of MB are equivalent to those
depicted in Figure 8b for a 2  10
4 M solution in the
presence of 10 times PSS as can be inferred from Figure 9,
indicating that the interaction of MB with PSS dominates
over the self-aggregation of the dye.
In order to reinforce the idea of MB attached to the PSS
functional groups by means of short-range ar/ar interac-
tions, DOSY and NOESY 1H NMR experiments have been
performed. DOSY experiments showed diffusion coeffi-
cients for a 10
2 M PSS solution of 9.7  10
11, and
2.1  10
9 m2  s
1 for a 10
3 M MB solution. The correspond-

Macromol. Chem. Phys. 2009, 210, 1167–1175

1172 ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/macp.200900042
Binding of Methylene Blue to Polyelectrolytes Containing . . .
Figure 9. 600 MHz 1H NMR NOESY spectra in D2O of MB 2  10
3
and PSS 10
2 M.
ing PSS/MB mixture showed a change of one order of
magnitude for the MB diffusion coefficient, achieving a
value of 9.9  10
11 m2  s
1, similar to that of PSS, and
indicating the binding to the polymer. EG was added as
probe in order to discard viscosity effects produced by the
polymer: in the presence of 10
2 M PSS, the diffusion
coefficient for EG showed a value of 2.7  10
9 m2  s
1.
NOESY experiments showed cross peaks between MB and
PSS signals, as can be seen in Figure 9, indicating
intermolecular distances smaller than 5 Å. These peaks
showed negative intensity, caused by the high molecular
weight of the complexes. In contrast, weak positive
intensities are found for NOESY experiments, performed
for MB alone. The overlapping of the aromatic signals of MB
with those of PSS makes difficult to observe cross peaks in
the aromatic region, provided that the intensities of cross
signals corresponding to MB-MB contacts and MB-PSS
contacts have different sign.
In the presence of 0.1 M NaCl, the interaction of MB with
PSS is quenched at a relative low PSS concentration, but this
interaction is resistant to the cleaving effect of the single
electrolyte, and, in the presence of a large excess of the PEL,
the interaction is conserved, as can be seen in Figure 7 by
UV-Vis spectroscopy. DF results presented in Figure 5 and
diss
PEL
Table 1 show the lowest KMB in the absence of NaCl
among the three PELs (around 0.1), and jet under a very low
excess of PSS, the interaction is still noticeable in the
presence of NaCl, showing an apparent dissociation
constant of 1.2. 1H NMR spectroscopy also shows that in
the presence of NaCl, the dyes are still bound to the polymer,
since the shifted signals are conserved, as can be seen in
Figure 8c.
Macromol. Chem. Phys. 2009, 210, 1167–1175
ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 10. Position of the maximum corresponding to MB aggre-
gates (polyMB) as a function of the PEL concentration relative to
MB concentration (10
5 M): (^) PVS; (&) PAMPS; (~) PSS.
Different Binding
The behavior found for PSS is different from the observed for
PVS and PAMPS. According to our interpretation, in the
presence of PVS, MB should form supramolecular polymers
in the environment of the PEL due to a higher local
concentration, undergoing interpolymer complexation
with the polyanion. This binding may have a cooperative
character, as a consequence of which, the polyMB band is
slightly shifted under increasing PVS concentrations.
This can be seen in Figure 10 where the position of this
band is plotted versus the PEL concentration.
PAMPS behaves very similarly to PVS. However, due to
the hydrophobicity and chain flexibility of this polymer, it
exhibits a certain dispersant ability reflected on the shift of
the polyMB band to lower energies, (see Figure 10), a
dissociation constant smaller than for PVS, as can be seen in
Table 1, and a certain resistance to the cleaving effect of
NaCl as seen by 1H NMR.
In contrast, the main driving forces for the MB/PSS
interaction have a short-range character, so the dye
molecules distribute randomly on the polymer binding
sites. MB aggregates are quenched by a small excess of PSS
by comparison with the other PELs, as can be seen in
Figure 10 by the shift of the polyMB band to lower energies.
This dispersant ability can also be followed by the increase
of the monomer band, as can be seen in Figure 11. While the
monomer band in the presence of PVS or PAMPS increases
only slightly in the presence of a large excess of the PELs, the
corresponding increase in the presence of PSS is higher and
takes place from a moderate excess of the polymer.
www.mcp-journal.de 1173

Figure 11. Absorbance of the monomer band (664 nm) of MB as a
function of the PEL concentration relative to MB concentration
(10
5 M): (^) PVS; (&) PAMPS; (~) PSS.
The binding ratio of MB to the different PELs decreases in
the order PSS > PAMPS > PVS. This can be seen comparing
diss
PEL
the DF profiles in Figure 5. The corresponding KMB
diss
PVS
shown in Table 1. KMB is nearly 20 times higher than
diss
PSS
KMB . As shown before, in the presence of 0.1 M NaCl the
interaction with PVS and PAMPS is lost, while a residual
diss
PSS
interaction is found for PSS showing a KMB of 1.2.
These differences are, then, associated with the different
contribution of the main forces involved in the interaction:
long-range electrostatic, hydrophobic, and short-range
ar/ar interactions. The different binding of MB to
these PELs, in terms of strength and mode of binding,
may be crucial for the control of MB properties and the
design of technological applications.
Conclusion
The interaction between methylene blue (MB) and the
polyanions containing sulfonate groups poly(sodium
4-styrenesulfonate) (PSS), poly(sodium vinylsulfonate)
(PVS), and the more hydrophobic poly[sodium 2-(N-
acrylamido)-2-methyl-propanesulfonate] (PAMPS), has
been investigated. Apparent dissociation constants and
sensitivity to the cleaving effect of added NaCl are found in
the order PVS > PAMPS > PSS. It is suggested that the main
driving forces for the interaction of MB with PVS are long-
range electrostatic interactions, while hydrophobic inter-
actions are also important in the case of PAMPS. Due to
these long-range interactions, a higher local concentration
of MB around these polymers results in MB self-aggrega-
Macromol. Chem. Phys. 2009, 210, 1167–1175
1174 ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
I. Moreno-Villoslada, C. Torres, F. González, T. Shibue, H. Nishide
tion; however, PAMPS exhibits a certain dispersant ability
due to its hydrophobicity and higher chain flexibility. In
contrast, the main driving forces for the interaction with
PSS are short-range aromatic/aromatic interactions, which
result in stronger binding (smaller dissociation constants),
and a resistance to the cleaving effect of added NaCl. Due to
the short-range nature of the interaction, MB molecules
may distribute randomly on the polymeric binding sites,
and the self-aggregation of the dye is prevented at a
moderate excess of the polymer.
Acknowledgements: The authors thank Fondecyt (Grant No
1090341, Chile) and the Global COE program ‘‘Practical Chemical
Wisdom’’ at Waseda University from MEXT, Japan, for financial
support.
Received: January 27, 2009; Revised: May 12, 2009; Accepted:
May 13, 2009; DOI: 10.1002/macp.200900042
Keywords: Interactions; methylene blue; polyelectrolytes; poly-
(sodium 4-styrenesulfonate)
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