21576

J. Phys. Chem. B 2006, 110, 21576-21581

Complex Formation between Rhodamine B and Poly(sodium 4-styrenesulfonate) Studied by

1H-NMR
Ignacio Moreno-Villoslada,*,,| Rodrigo Gonza lez, Susan Hess, Bernabe L. Rivas,
Toshimichi Shibue, and Hiroyuki Nishide|
Instituto de Qumica, Facultad de Ciencias, UniVersidad Austral de Chile, Casilla 567, ValdiVia, Chile,
Facultad de Ciencias Qumicas, UniVersidad de Concepcio n, Casilla 160-C, Concepcio n, Chile, and Materials
Characterization Central Laboratory and Department of Applied Chemistry, School of Science and
Engineering, Waseda UniVersity, Tokyo 169-8555, Japan
ReceiVed: June 27, 2006; In Final Form: July 26, 2006

A 1H NMR study is presented for the binding of rhodamine B (RB) to the polyanion containing aromatic
groups poly(sodium 4-styrenesulfonate) (PSS), which is also evidenced by diafiltration. 1H NMR spectra
showed an accentuated upfield shift of proton H6 of the benzoic ring of RB at pH 7, indicating the stacking
of RB onto PSS. The corresponding structure is proposed which is in accordance to Hunter and Sanders
rules. At pH 2, an upfield shift of the xanthene protons of RB would indicate a highly condensed state for
this molecule.

1. Introduction
Molecular association involving aromatic-aromatic interactions is attracting much attention nowadays.1-13 As a result of
the planar geometries of aromatic molecules, the molecular
surface/volume ratio is high compared to that of spherical
particles. Then, the aggregation of aromatic groups in water
may produce the release of higher amounts of surface-solvating
water molecules and, consequently, an increase in the favorable
entropic and enthalpic contributions to the free energy by means
of classical and nonclassical hydrophobic effects.1 In addition
to these solvent contributions, site-specific interactions such as
short-range electrostatic interactions, hydrogen bond formation,
- interactions, or cation- interactions may also contribute
to the free energy and define the geometry of the complexes.
These aromatic-aromatic interactions are found in water in
many systems such as nucleic acids, proteins, porphyrins,
semiconductors, molecular clips, and so forth1-13 and are one
of the principal noncovalent forces governing molecular recognition and biomolecular structure. In particular, they are
important in the stabilization of DNA and its association with
intercalators. A change in the hydrogen bonding capacity of
DNA bases as a consequence of - interactions has been
described in recent literature.4,5 The vast majority of X-ray
crystal structures of protein complexes with small molecules
reveal bounding interactions involving aromatic amino acid side
chains of the receptor and/or aromatic and heteroaromatic rings
of the ligand. A raise of the pKa of substrates by means of -
interactions with enzymes has been proposed as a mechanism
of enzymatic catalysis.6
We have recently found a smart system composed by poly(4-sodium styrenesulfonate) (PSS) and rhodamine B (RB).14 The
behavior of this system is pH dependent. Lowering the pH from
5 to 3, we have observed an abrupt change in the binding
* FAX: 56-63-221597. E-mail: imorenovilloslada@uach.cl.
Universidad Austral de Chile.
Universidad de Concepcio
n.
Materials Characterization Central Laboratory, Waseda University.
| Department of Applied Chemistry, Waseda University.

Figure 1. Molecular structures of RB and PSS.

constant and in the light absorption and emission properties of

RB. Moreover, the changes described witness an increase of
the RB pKa. This behavior is not found for other polyanions
that do not contain aromatic rings,15 so the aromatic ring of
PSS plays an important role in the overall interaction.
NMR is a useful tool to probe the stacking of aromatic rings.
The effect of stacking the rings is to place one ring in the
shielding cone of the second, resulting in upfield shifts of 1H
resonances for the stacked rings. In this paper, we analyze the
binding of RB to PSS by 1H NMR and diafiltration and will
propose a geometry for the -stacking of RB onto PSS.
2. Experimental Section
2.1. Reagents. Commercially available PSS (Aldrich, synthesized from the para-substituted monomer) and RB (Sigma)
were used to prepare the solutions in deionized distilled water
or D2O (Acros, 99.8% d-content). The structures of RB and
PSS are shown in Figure 1. The pH was adjusted with minimum
amounts of NaOH and HNO3 or DCl (Acros, +99% d-content).
2.2. Equipment. The unit used for diafiltration studies
consisted of a filtration cell (Amicon 8010, 10 mL capacity)
with a magnetic stirrer, a regenerated cellulose membrane with
a MWCO of 10 000 Da (Ultracel PLGC, 25 mm diameter), a
reservoir, a selector, and a pressure source. Distilled water was
deionized in a Simplicity millipore deionizer. The pH was
controlled on Quimis Q 400 M2 or Horiba F-15 pH meters.
UV-vis experiments including analyses of the diafiltered

10.1021/jp0640169 CCC: $33.50 2006 American Chemical Society

Published on Web 10/05/2006

Complex Formation Between RB and PSS

J. Phys. Chem. B, Vol. 110, No. 43, 2006 21577

Figure 2. 1H NMR aromatic region spectra of solutions in D2O at pH 7.5 (1) and 2.2 (2) of different RB concentrations: (a) 110-2 M; (b) 510-3
M; (c) 110-3 M; (d) 510-4 M.

solutions were performed in a Heios spectrophotometer at

293 K and 1 cm of path length. 1H NMR measurements were
made in JNM-Lambda500 (JEOL, 500 MHz) and AVANCE600
(Bruker, 600 MHz) spectrometers.
2.3. Procedures. Details for diafiltration procedures may be
found elsewhere,16-18 and experimental conditions are provided
in the figure captions. 2D diffusion-ordered spectroscopy
(DOSY) experiments were made 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 in
10-3 M RB and 10-2 M PSS solutions, to obtain appropriate
curves (25 points) for inverse Laplace transformation. The same
procedure was done by taking well-resolved signals of RB as
reference in mixtures of 10-3 M RB and variable amounts of
PSS.
3. Results and Discussion
3.1. RB Alone. RB is a zwitterionic molecule which
undergoes self-aggregation at concentrations higher than 10-4
M. Its pKa at concentrations lower than 10-5 M is 3.2. Figure
2 shows the aromatic region of solutions at four different RB
concentrations and pHs higher and lower than the pKa. According to Burghrdt et al.,19 assignments of the signals are related
to the RB structure shown in Figure 1. Protonation of RB
produces (a) a higher withdrawing effect on the benzoic ring
and (b) a decrease in the electron density of the carboxylic group
situated directly over the xanthene ring. This may explain the
downfield shift of protons H3, H4, and H5 in the benzoic
group in the protonated molecule. On the other hand, the
xanthene protons are upfield-shifted at increasing RB concentrations, due to - stacking. A structure for the dimer at [RB]
) 210-3 M was deduced by other authors19 showing a faceto-face stacking of the xanthene groups. At higher concentrations, higher-order associations may be found.
3.2. RB-PSS at pH 7. The interaction of RB with PSS at
this pH is evidenced by diafiltration. Diafiltration features are
described elsewhere.16-18 The main magnitudes managed in
diafiltration analyses are the filtration factor (F), defined as the

Figure 3. Diafiltration profiles of an RB 110-4 M aqueous solution

at pH 7 in the absence of PSS (+) and in the presence of different PSS
concentrations: ([) 210-4 M; (]) 410-4 M; (9) 610-4 M; (0) 810-4
M; (2) 110-3 M; (4) 210-3 M; (see Table 1 for linear adjustments).

ratio between the volume in the filtrate and the constant volume
in the diafiltration cell, the concentration in the filtrate of the
low-molecular-weight species under study (LMWS) (cLMWSfiltrate), the concentration of free LMWS in the cell solution
(cLMWSfree), the concentration of LMWS reversibly bound to the
water-soluble polymer (WSP) (cLMWSrev-bound), the apparent
dissociation constant (KLMWSdiss-WSP), defined as the ratio
cLMWSfree/cLMWSrev-bound, the diafiltration parameters km, j, u, and
V, and the polymer concentration in moles per liter of monomeric
units (cP). km and j parameters (the absolute value of the slope
of the curve ln cLMWSfiltrate versus F in the absence and in the
presence of the WSP, respectively) are related to the strength
of the interaction, while V and u are related to the amounts of
LMWS reversibly or irreversibly bound to the polymer,
respectively. Results of diafiltration of a 10-4 M solution of
RB at pH 7 shown in Figure 3 and Table 1 indicate no
significant interaction of RB with the diafiltration cell components (km close to 1). On the other hand, the amounts of RB
irreversibly bound to PSS are negligible (u values close to 0),
and the fraction of RB reversibly bound to PSS increases with

21578 J. Phys. Chem. B, Vol. 110, No. 43, 2006

Moreno-Villoslada et al.

TABLE 1: Results for Diafiltration of 10-4 M RB Solutions at pH 7 and Different PSS Concentrationsa
expt

cP (M)

RB-03
PSS -RB-01
PSS -RB-02
PSS -RB-03
PSS -RB-04
PSS -RB-05
PSS -RB-06

210-4
410-4
610-4
810-4
110-3
210-3

0.91
0.85
0.85
0.92
0.94
0.95
0.94

0.09
0.15
0.15
0.08
0.06
0.05
0.06

0.63
0.49
0.41
0.37
0.29
0.18

km
0.84

KRBdiss-PSS b

linear adjustments
for the experimental data

R2

2.4 ( 0.5
1.2 ( 0.2
0.8 ( 0.1
0.7 ( 0.8
0.5 ( 0.05
0.2 ( 0.03

y ) -0.84x - 9.5
y ) -0.63x - 9.6
y ) -0.49x - 9.9
y ) -0.41x - 10.0
y ) -0.37x - 10.1
y ) -0.29x - 10.4
y ) -0.18x - 10.9

1.00
0.99
0.99
0.99
0.99
1.00
1.00

a
For linear adjustments: y ) ln cRBfiltrate; x ) F; R2 ) linear regression factor. b KRBdiss-PSS is calculated following (j/1 - j) e KLMWSdiss-WSP
e (kmj/km - j).

Figure 4. 1H NMR aromatic region spectra of solutions in D2O at pH 7 of (a) RB 110-3 M; (b-d) RB 110-3 M in the presence of different PSS
concentrations: (b) 0.510-3 M, (c) 210-3 M, (d) 610-3 M; (e) PSS 110-2 M.

polymer concentration (decreasing values of j), which is

reflected in the KRBdiss-PSS values.
The association of RB with PSS is seen by 1H NMR by a
general broadening and upfield shift of the RB bands (see
Figures 4 and 5). This behavior is not found in analogous
systems including polyelectrolytes with no aromatic groups, such
as poly(sodium vinyl sulfonate) (PVS).15 In Figure 4, some
representative spectra are shown for constant amounts of RB
in the presence of variable amounts of PSS, while in Figure 5,
the increase of the chemical shifts of the different RB aromatic
protons for a set of experiments is shown as a function of PSS
concentration. The upfield shift, and consequently the binding
of RB to PSS, increases with increasing the WSP concentration
but at a decreasing rate, due to a decreasing free RB in solution.
This indicates an equilibrium binding in accordance to diafiltration observations. The only exception to the general upfield
shift is the H3 signal, and we interpret this fact as a higher

Figure 5. Upfield shift of RB aromatic signals ([RB] ) 110-3 M in

D2O at pH 7) as a function of PSS concentration: (]) H3; (O) H4;
(4) H5; (0) H6; ([) H4H5; (9) H1H8; (2) H2H7.

Complex Formation Between RB and PSS

J. Phys. Chem. B, Vol. 110, No. 43, 2006 21579

Figure 6. COSY spectrum of the aromatic region of a RB 110-3 M/PSS 210-3 M solution in D2O at pH 7.

tendency of RB to protonate in the presence of PSS as evidenced

by the change on its pKa.14 H4 and H5 have the same chemical
shift for RB alone, but they are differently shifted in the presence
of the polymer. To assign each signal, a COSY experiment has
been done for the sample corresponding to spectrum c in Figure
4. The COSY spectra shown in Figure 6, allowed us to assign
the signal at 7.53 ppm to H4, since a clear correlation is seen
with H3. The correlation between H5 and H6 also appears
clearly, which confirms the overlapping of H6 with H1H8 at
this PSS/RB ratio, following the upfield shift tendency evidenced
in Figure 5. On the other hand, the signals for H4 and H5
overlap with those of the polymer protons appearing around
7.4 ppm, and for some PSS/RB ratios, they were not clearly
distinguishable. To obtain the respective chemical shifts, COSY
and DOSY experiments are useful. DOSY experiments at pH
7 showed diffusion coefficients for a 10-2 M PSS solution of
0.910-10 m2 s-1 and of 910-10 m2 s-1 for a 10-3 M RB
solution. For mixtures of 10-3 M RB and variable amounts of
PSS at pH 7, the diffusion delays and gradient durations were
selected by observing the well-resolved peaks H3 and H6 of
RB. This allowed us to obtain the 1D spectra at small gradient
amplitudes, where all the signals are recuperated, and medium
gradient amplitudes, where the signals for RB are lost, but not
those of PSS, since the diffusion coefficient of RB is higher
than that of PSS. Then, subtracting the latter 1D spectra from
the former, the chemical shifts for H4 and H5 become
observable. It can be seen in Figures 4 and 5 that the upfield
shift in the RB signals is more accentuated for H6 and H5
protons (0.6 ppm for H6), which witnesses the stacking of RB
onto PSS by means of - interactions. Due to these -
interactions, the pKa of RB changes,14 as pointed out by other
authors considering other substrates.6
To propose a structure for the stacking of RB onto PSS at
pH 7, we search to satisfy the following criteria: (a) explain
the high shift value of H6, (b) conserve the symmetry, (c) avoid
unacceptable steric hindrances, (d) provide an adequate charge
disposition, and (e) be in accordance with Hunter-Sanders

Figure 7. Proposed structure for the stacking of RB onto PSS at

pH 7.

rules.13 The proposed geometry for the interaction is shown

in Figure 7, where an edge-to-face stacking occurs between
the RB electron-deficient benzoic ring (edge) and the PSS
electron-rich benzenosulfonate ring (face), according to
Hunter and Sanders rules. The contacting edge should contain
H6 and H5, so that the negatively charged carboxylic group
is placed at the opposite side of the negatively charged sulfonate
group. Moreover, if the xanthene group of RB adopts an
opposite disposition with respect to the polymer backbone
avoiding steric hindrances, its positive charge would be located
between the two negative charges, stabilizing the symmetrical
structure.
3.3. RB-PSS at pH 2. The study of this system at acidic
pH is more complex. Diafiltration experiments showed a
quantitative binding yet at a PSS/RB ratio of 2/1 and low
concentrations.14 Increasing the absolute concentration of both
components results in precipitate formation, but by increasing
the PSS/RB ratio from PSS/RB ) 4, precipitation is avoided.
Precipitation is due to a charge balance between the negatively
charged PSS and the positively charged protonated RB.
On the other hand, fluorescence is quenched for PSS/RB
ratios under 20, but it is visibly conserved at PSS/RB ) 100,
so fluorescence quenching is not due to the binding of RB to
PSS itself. This may mean that, at moderate and low PSS/RB
ratios, RB is found in a highly condensed state, which may be
avoided under high PSS/RB ratios. Although experiments are

21580 J. Phys. Chem. B, Vol. 110, No. 43, 2006

Moreno-Villoslada et al.

Figure 8. 1H NMR aromatic region spectra of solutions in D2O of RB 3.410-3 M and PSS 210-2 M at different pHs: (a) 6.9; (b) 5.2; (c) 4.6;
(d) 4.1; (e) 2.2.

being done by other techniques in order to clarify this behavior,

we show here the results obtained by 1H NMR.
1H NMR also supports the above hypothesis, since by
increasing the pH, the signal/noise ratio decreases dramatically,
so that the aromatic signals of RB are lost at 297 K at a PSS/
RB ratio of 6, even with an increase in the RB concentration to
3.410-3 M. This can be seen in Figure 8. Increasing the pH
produces an upfield shift of the xanthene protons. On the
contrary, the signals of the RB benzoic ring are downfield
shifted. The formation of higher-order structures in which RB
is compacted within the polymer domain is congruent with these
facts. The downfield shifting of H6 and H5 may indicate a
reaccommodation of the polymer. Note that all the signals are
upfield-shifted with respect to those found for RB selfassociation at acidic pH and high concentrations (see Figure
2). Lost signals at pH 2.2 and 297 K are recovered at 343 K
with small changes in the chemical shifts.
Conclusions
RB binds to PSS at pH 7 by means of - interactions
according to Hunter and Sanders rules. 1H NMR shows the
interaction between the benzoic ring of RB and the aromatic
ring of PSS in an edge (RB) to face (PSS) geometry. Diafiltration experiments also evidence the interaction between the
polyanion and the zwitterionic molecule. At pH 2, an upfield
shift of the xanthene protons of RB would indicate a highly
condensed state for this molecule, which is in accordance with
previous observations of fluorescence quenching.

Acknowledgment. The authors thank Fondecyt (grants no.

1030669 and no. 1060191) and the 21COE program Practical
Nano-Chemistry at Waseda University from MEXT, Japan,
for financial support.
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