pH Dependence of the Interaction between Rhodamine B and the

J. Phys. Chem. B 2006, 110, 11809-11812

11809

pH Dependence of the Interaction between Rhodamine B and the Water-Soluble Poly(sodium 4-styrenesulfonate) Ignacio Moreno-Villoslada,*,† Marle´ n Jofre´ ,† Vı´ctor Miranda,† Rodrigo Gonza´ lez,† Tamara Sotelo,† Susan Hess,† and Bernabe´ L. Rivas‡ Instituto de Quı´mica, Facultad de Ciencias, UniVersidad Austral de Chile, Casilla 567, ValdiVia, Chile, and Departamento de Polı´meros, Facultad de Ciencias Quı´micas, UniVersidad de Concepcio´ n, Casilla 160-C, Concepcio´ n, Chile ReceiVed: March 9, 2006; In Final Form: April 19, 2006

The binding of rhodamine B (RB) to the polyanion containing aromatic groups poly(sodium 4-styrenesulfonate) (PSS) is studied by separation and spectroscopic techniques at pH between 2 and 7. Significant binding is found at pH below 5, together with a red-shift of the RB maximum of absorbance to 564 nm, and RB fluorescence quenching. The dependence of the pH is related with protonation of RB molecules. Fluorescence quenching is a consequence of a more hydrophobic environment and may occur on territorially or sitespecifically bound molecules, and/or on self-aggregated molecules in a hydrophobic polymer domain. Remarkably, the basicity of RB is increased by the influence of the polymer.

1. Introduction Long-range electrostatic interactions between polyelectrolytes and low-molecular-weight species (LMWS) have been extensively studied.1-8 These interactions are generally described by considering the counterion condensation theory of G. S. Manning. Experimental and theoretical results of investigations involving several polyanions such as poly(sodium 4-styrenesulfonate) (PSS) and DNA have proved to be explained by the theory. However, other experiments have indicated the influence of short-range site-specific interactions. As an example, by dealing with some metal ions such as Cu2+ and polycarboxylic acids, formation of coordination bonds is found in addition to these electrostatic interactions; these short-range interactions exceed in strength the long-range electrostatic interaction. In most cases, both modes of binding occur simultaneously where one mode is the dominant. Experimentally, it is difficult to separate the effects of the two modes. On the other hand, the hydrocarbon nature of the polyanions makes hydrophobic interactions important in the overall interaction. The binding of aromatic counterions by polyions containing aromatic rings is particularly strong. Molecular association involving π-π interactions is attracting much attention nowadays. They occur in many systems containing aromatic groups such as nucleic acids, porphyrins, molecular clips, proteins, polymers, etc.9-19 and are one of the principal noncovalent forces governing molecular recognition and biomolecular structure. Several effects as a consequence of π-π interactions in biological systems have been described in recent literature such as a change on the hydrogen-bonding capacity of DNA bases18,19 or enzymatic catalysis via a raise of the pKa of the substrate.17 These interactions present a short-range electrostatic character, together with a hydrophobic contribution. Despite that fact that these interactions have been known for * Corresponding author. E-mail: [email protected]. Fax: 5663-221597. † Instituto de Quı´mica, Facultad de Ciencias, Universidad Austral de Chile. ‡ Departamento de Polı´meros, Facultad de Ciencias Quı´micas, Universidad de Concepcio´n.

more than fifty years, they are not massively incorporated in the study of synthetic model compounds. Rhodamine B (RB) is an interesting molecule with spectral luminiscence properties that make it useful as a marker, as a probe in studies of various objects including biological systems, in sensors, etc. This molecule self-aggregates in concentrations higher than 10-4 M by means of π-π interactions. The aggregation induces spectroscopic changes and affects dye lasing efficiency and photostability.20-24 The extent of aggregation depends reciprocally on the temperature of the solution and is fully reversible. As RB is a zwitterionic molecule in a wide range of pH, it is expected that long-range electrostatic interactions with water-soluble polymers (WSP) are minimized, allowing then, exploration of short-range interactions. The interactions between WSP and LMWS may be detected and quantified by means of separation and spectroscopic techniques. Diafiltration has emerged as an effective technique to study electrostatic interactions because these interactions do not produce in general spectroscopic changes. The features for diafiltration analyses and interpretation have been described elsewhere.7,25-28 The main magnitudes managed in diafiltration analyses are the filtration factor (F), defined as the ratio between the volume in the filtrate and the constant volume in the diafiltration cell, the concentration of LMWS in the filtrate (cLMWSfiltrate), the total concentration of LMWS in the diafiltration cell (cLMWScell), the concentration of free LMWS in the cell solution (cLMWSfree), the LMWS reversibly bound to the WSP (cLMWSrev-bound), the LMWS irreversibly bound to the WSP (cLMWSirrev-bound), retention (R), defined as the fraction of the initial LMWS remaining in diafiltration cell, and the apparent dissociation constant (KLMWSdiss-WSP), defined as the ratio cLMWSfree/cLMWSrev-bound. Typical diafiltration profiles are obtained by plotting the logarithm of cLMWSfiltrate versus F. The slope of the profile (parameter j) gives an idea of the rate of filtration of the LMWS, which is related with KLMWSdiss-WSP, following28

km j j e KLMWSdiss-WSP e m 1-j k -j

10.1021/jp061457j CCC: $33.50 © 2006 American Chemical Society Published on Web 05/26/2006

(1)

11810 J. Phys. Chem. B, Vol. 110, No. 24, 2006

Moreno-Villoslada et al.

Figure 1. Molecular structures of RB and PSS.

where km is the slope obtained in blank experiments (performed in the absence of the WSP), and provided that j e km e 1. This constant allows calculating and comparing the relative strength of the macromolecule toward binding the LMWS. From the ordinate at the origin in the profiles, the concentration of LMWS irreversibly bound to the WSP may also be calculated. 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. Thus, the concentration of LMWS in the diafiltration cell in every instant may be adjusted to a function

cLMWScell ) cLMWScell-init[u + V exp(-j‚F)]

Figure 2. UV-Vis absorbance of 2‚10-6 M aqueous solution of RB: (a) in the absence of PSS at pH 6, (b) in the presence of 2‚10-5 M PSS at pH 6, (c) in the absence of PSS at pH 2, (d) in the presence of 2‚10-5 M PSS at pH 2, and their respective fluorescence spectra.

(2)

where u and V are parameters, provided that u + V ) 1, and init referring to initial values (F ) 0). In blank experiments, j is substituted by km. To better visualize the LMWS irreversibly bound to the WSP, R may be plotted versus F. In this paper, the interactions between PSS and RB are studied by diafiltration and UV-vis spectroscopy of absorbance and fluorescence as a function of the pH. 2. Experimental 2.1. Reagents. Commercially available poly(sodium 4-styrenesulfonate) (PSS) (Aldrich, synthesized from the parasubstituted monomer) was fractionated by diafiltration over a membrane of a molecular weight cutoff (MWCO) of 10 000 Dalton (Biomax, 63.5 mm diameter), first in the presence of 0.10 M NaCl and then in the absence of the electrolyte. NaCl (Scharlau) and RB (Sigma) were used to prepare the solutions. The structures of RB and PSS are shown in Figure 1. The pH was adjusted with minimum amounts of NaOH and HNO3. 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 Dalton (Ultracel PLGC, 25 mm diameter), a reservoir, a selector, and a pressure source. The pH was controlled on a Quimis Q 400 M2 pH meter. UV-Vis experiments including analyses of the diafiltered solutions were performed in a Helios γ spectrophotometer at 20 °C and 1 cm of path length. Fluorescence was measured in a Kontron SFM25 spectrophotometer. 2.3. Procedure for Diafiltration. Solutions in twice-distilled water (10 mL) were prepared containing one or more of the following components: PSS (polymeric molecular weight fraction over 10 000 Da) (2.0‚10-4 M in monomeric units) and RB (1.0‚10-4 M) at pH 2, 4.5, and 7. The solutions were placed into the diafiltration cell. The pH of the aqueous solution contained in the reservoir was 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 cutoff of 10 000 Da under a total pressure of 3 bar, keeping constant the solution volume in the cell by creating a continuous flux of

Figure 3. Position of the maximum absorbance of a RB 2‚10-6 M aqueous solution as a function of the pH: (]) in the absence of PSS, ([) in the presence of 2‚10-5 M PSS.

liquid through the cell solution from the reservoir (around 0.008 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 RB concentrations analyzed by UV-vis spectroscopy. No macromolecule was found in the filtrate. Calibration curves (absorbance ) 107 842 [RB] at pH 2, absorbance ) 106 385 [RB] at pH 4.5, absorbance ) 108 174 [RB] at pH 7) were obtained at 554 nm for pH 4.5 and 7, and 558 nm for pH 2, in a range of RB concentrations between 1.0‚10-6 and 1.0‚10-5 M, with square linear regression factors of 1.00. Blank experiments were performed with the same procedure in the absence of the WSP. At least one replicate is done for every experiment. 2.4. Fluorescence Measurements. Fluorescence was measured for 2‚10-6 M RB solutions at several pH values ranging between 2 and 6 in the presence and in the absence of the WSP. The excitation wavelength was 540 nm, with a voltage of 380 V. Fluorescence intensity is given in arbitrary units (a.u.). 3. Results and Discussion. 3.1. UV-Vis Analyses. The UV-vis spectrum of a 2‚10-6 M RB solution changes with the pH in a range between 2 and 7, as can be seen in Figure 2. Because of protonation,23 at pH 2, the maximum of absorbance is red-shifted 4 nm (λmax ) 558 nm). Figure 3 shows the shifting of the maximum as a function of the pH. This shifting may serve as a method to analyze the pKa of RB at this concentration, which is approximately 3, in coincidence with values reported in the literature (3.22 at RB

Rhodamine B and Poly(sodium 4-styrenesulfonate)

J. Phys. Chem. B, Vol. 110, No. 24, 2006 11811

Figure 4. Ratio fluorescence at 572 nm (a.u.)/absorbance at 540 nm of a RB 2‚10-6 M aqueous solution as a function of the pH: (0) in the absence of PSS, (9) in the presence of 2‚10-5 M PSS.

concentration e 10-5 M and ionic strength I f 0).23 Interestingly, the presence of 2‚10-5 M of PSS produces a more pronounced red-shift in acidic conditions, reaching a λmax of 564 nm (see Figure 2). As can be seen in Figure 3, this shift is pH dependent, indicating the existence of a protonation equilibrium. This behavior has been reported in the literature for the absorption of RB in micelles,22 vesicles,29 and latex particles.30 The red-shift of the maximum of absorbance is due to association of the protonated RB to the WSP. The associated RB presents an increase on the apparent pKa, reaching a value of approximately 4. Fluorescence measurements have been made in order to explore fluorescence quenching by the interaction of RB with PSS. The fluorescence spectra at pH 2 and 6 in the presence and in the absence of PSS are shown in Figure 2. There is no significant overlap of emission and excitation light scattering, and it can be seen that smaller fluorescence intensity is found at pH 2 in the presence of PSS. As the absorbance at the excitation wavelength decreases both by decreasing the pH and by interaction with PSS, the ratio fluorescence/absorbance at the respective emission (572 nm) and excitation (540 nm) wavelengths is calculated. This magnitude is proportional to the fluorescence quantum yield. The results have been plotted as a function of the pH in Figure 4. No significant quenching is observed for RB alone. On the contrary, in the presence of PSS, its fluorescence is clearly quenched depending on the pH. The pattern of fluorescence quenching as a function of the pH correlates with the λmax shift in UV-vis absorbance, indicating protonation equilibria. The fluorescence quenching may be due to an internal movement of the ethyl groups of the LMWS enhancement in a more hydrophobic environment22 or to RB self-association induced by the WSP. 3.2. Diafiltration. Diafiltration results (see Table 1) confirm the change on the behavior of RB induced by the polymer as a function of the pH. The interaction of RB with the diafiltration

Figure 5. Diafiltration profiles of a RB 1‚10-4 M aqueous solution in the absence of PSS (open symbols) and in the presence of 2‚10-4 M PSS (filled symbols) (see Table 1 for linear adjustments): (], [) pH 2, (0, 9) pH 4.5, (4, 2) pH 7; diafiltration profile of a RB 1‚10-5 M aqueous solution in the absence of PSS (/); simulation of the diafiltration profile of a RB 1‚10-4 M aqueous solution interacting with a WSP with an apparent dissociation constant of 0.06 (O).

system is nearly negligible due to the high values of V and km parameters, (absolute absence of interaction is obtained for V ) km ) 1). As can be seen in Figure 5 and Table 1, the interaction of RB with the WSP at pH 7 is less intense compared with the interaction at pH 2 and 4.5. The diafiltration profiles plotted in Figure 5 show that, at pH 7, the elution profile of RB is very similar to that of the corresponding blank experiment, while at pH 2 and 4.5, the rate of filtration of RB decreases drastically. While at pH 7, quantitative elution is observed, at pH 4.5, a fraction of the initial RB appears irreversibly bound to PSS (57%). The other 43% is reversibly bound to PSS with an apparent dissociation constant 1 order of magnitude smaller than at pH 7, as can be seen in Table 1. At pH 2, more than an 80% of the initial RB is retained by the WSP during the diafiltration run. A flat elution profile is obtained (Figure 5). When apparent dissociation constants are smaller than 0.1, flat elution profiles are found and the diafiltration technique loses sensibility in order to obtain accurate constants. For instance, a simulation is made in Figure 5 for a situation where all RBs are reversibly bound to the polymer with a KRBdiss ) 0.06. However, to discard that the observed elution profile is affected by the effect of the diafiltration membrane at this low free LMWS concentration regime, a blank experiment is done at a RB concentration of 1‚10-5 M, showing the expected decay. Electrostatic interactions are sensible to the ionic strength, and in this sense we have already observed a decrease in the binding capacity of polymers as PSS at low pH, which involves relatively high ionic strength.31-33 Then, the binding of RB to the polymer at pH 2 is strongly stabilized. 3.3. Binding of RB. RB undergoes complex self-aggregation equilibria that may include the formation of dimers, trimers, or higher-order aggregates, together with protonation equilibrium.

TABLE 1: Results for Diafiltration of 10-4 M RB Solutions in the Absence of PSS (RB-01-RB-03) and in the Presence of 2‚10-4 M PSS (PSS-RB-01-PSS-RB-03)a experiment

pH

V

u

RB-01 RB-02 RB-03 PSS-RB-01 PSS-RB-02 PSS-RB-03

2 4.5 7 2 4.5 7

1.00 0.93 0.91

0.00 0.07 0.09

0.43 0.94

0.57 0.06

a

j

km

KRBdiss-PSSb

0.85 0.80 0.84 0.29 0.66

-

0.41 ( 0.01 2.9 ( 0.7

linear adjustments for the experimental data

R2

y ) -0.85x - 8.9 y ) -0.80x - 9.2 y ) -0.84x - 9.5

1.00 1.00 1.00

y ) -0.29x - 11.2 y ) -0.66x - 9.3

0.99 1.00

For linear adjustments: y ) ln 〈cRBfiltrate〉; x ) F; R2 ) linear regression factor. b Calculated using expression 1.

cRBirrev-bοund (M)

5.7‚10-5 6‚10-6

11812 J. Phys. Chem. B, Vol. 110, No. 24, 2006 Both the quenching of the fluorescence of RB and the red-shift of its maximum of absorbance indicates self- and/or intermolecular association. As we have seen, a correspondence between the red-shift and the fluorescence extinction and that these facts are pH dependent, we can assume that protonation is associated with self- and/or intermolecular association. Self-aggregation of protonated RB is described in the literature.23 In this sense, in the experimental conditions in this work, this protonation/ association occurs massively at pH below 5 in the presence of PSS. By combining the spectroscopic and the diafiltration results, we can give the following interpretation for RB binding to PSS: (1) The negatively charged PSS interacts electrostatically with the positively charged aminoxanthene group of RB. As the retention profiles obtained for other polyanions34 do not follow the pattern presented here, we point out the importance of the aromatic group for short-range electrostatic interaction stabilization by means of hydrophobic geometry-nonspecific interactions or geometry-specific π-π interactions. (2) As a result of the interaction of the polymer with RB, the carboxylic group of RB becomes more basic once the positive charge of the dye is canceled. At pH below 5, the polymer induces the association/protonation of RB and the spectral characteristics of the LMWS change. Several hypotheses can be handled to explain the binding of these protonated molecules based on their fluorescence quenching: (i) they are territorially or sitespecifically bound to the surface of the WSP, and fluorescence quenching is due to a more hydrophobic environment, (ii) they are territorially or site-specifically bound to the surface of the WSP, and fluorescence quenching is due to self-stacking of RB molecules on the polymer surface, and (iii) protonated RBs selfaggregate within a hydrophobic polymer domain, so that an optimal internal free space of the aromatic WSP to accommodate RB is achieved. The positive charge of the aggregates is stabilized by the negative charge of the polymer. (3) At pH higher than 5, electrostatic repulsions between the negatively charged groups (sulfonate and carboxylate) would minimize the binding of RB to the polymer. It has been reported that there is an increase of the basicity of RB up to pKa ) 4.1 because of RB self-aggregation at concentrations higher than 10-4 M,23 and even higher (pKa ) 5.7) because of RB association to sodium dodecyl sulfate micelles.22 The observed increase on the basicity of RB by means of the interaction with PSS is an important tool that may be useful for technological applications. 4. Conclusions The interaction between rhodamine B (RB) and poly(sodium 4-styrenesulfonate) (PSS) has been investigated. The binding of RB to the polyanions increases the apparent pKa of the RB. At pH 7, the binding of RB to PSS is minimized because the zwitterionic form is predominant. At pH 2 and 4.5, protonated

Moreno-Villoslada et al. RBs are bound to the polymer. A red-shift of the RB maximum of absorbance is observed (λmax ) 564 nm) for the bound molecules and their fluorescence is quenched, which may indicate a more hydrophobic environment for the RB molecules, and/or RB self-aggregation induction. Acknowledgment. We thank Fondecyt (grants 1030669 and 1060191) for financial support. References and Notes (1) Manning, G. S. Q. ReV. Biophys. 1978, 11, 179. (2) Manning, G. S. J. Phys. Chem. 1984, 88, 6654. (3) Dewey, T. G. Biopolymers 1990, 29, 1793. (4) Paoletti, S.; Benegas, J.; Cesa´ro, A.; Manzini, G. Biophys. Chem. 1991, 41, 73. (5) Nordmeier, E.; Dawe, W. Polymer J. 1991, 23, 1297. (6) Nordmeier, E. Macromol. Chem. Phys. 1995, 196, 1321. (7) Rivas, B. L.; Moreno-Villoslada, I. J. Phys. Chem. B 1998, 102, 6994. (8) Hao, M. H.; Harvey, S. C. Macromolecules 1992, 25, 2200. (9) Meyer, E. A.; Castellano, R. K.; Diederich, F. Angew. Chem., Int. Ed. 2003, 42, 1210. (10) Hunter, C. A.; Sanders, J. K. M. J. Am. Chem. Soc. 1990, 112, 5525. (11) Linse, P. J. Am. Chem. Soc. 1992, 114, 4366. (12) Lokey, R. S. Nature 1995, 375, 303. (13) Reek, J. N. H. J. Am. Chem. Soc. 1997, 119, 9956. (14) Brunsveld, L. Folmer, B. J. B.; Meijer, E. W.; Sijbesma, R. P. Chem. ReV. 2001, 101, 4071. (15) Rashkin, M. J.; Waters, M. L. J. Am. Chem. Soc. 2002, 124, 1860. (16) Sinnokrot, M. O.; Sherrill, C. D. J. Phys. Chem. A 2003, 107, 8377. (17) Mignon, P.; Loverix, S.; De Proft, F.; Geerlings, P. J. Phys. Chem. A 2004, 108, 6038. (18) Verse´es, W.; Loverix, S.; Vandemeulebroucke, A.; Geerlings, P.; Steyaert, J. J. Mol. Biol. 2004, 338, 1. (19) Mignon, P.; Loverix, S.; Steyaert, J.; Geerlings, P. Nucleic Acids Res. 2005, 33, 1779. (20) Lo´pez Arbeloa, I.; Ruiz Ojeda, P. Chem. Phys. Lett. 1982, 87, 556. (21) Lo´pez Arbeloa, F.; Ruiz Ojeda, P.; Lo´pez Arbeloa, I. J. Lumin. 1989, 44, 105. (22) Mchedlov-Petrossyan, N. O.; Vodolazkaya, N. A.; Doroshenko, A. O. J. Fluoresc. 2003, 13, 235. (23) Mchedlov-Petrossyan, N. O.; Kholin, Y. V. Russ. J. Appl. Chem. 2004, 77, 414. (24) Ilich, P.; Mishra, P. K.; Macura, S.; Burghrdt, T. P. Spectrochim. Acta, Part A 1996, 52, 1323. (25) Moreno-Villoslada, I.; Miranda, V.; Oyarzu´n, F.; Hess, S.; Luna, M.; Rivas, B. L. J. Chil. Chem. Soc. 2004, 49, 121. (26) Moreno-Villoslada, I.; Miranda, V.; Gutie´rrez, R.; Hess, S.; Mun˜oz, C.; Rivas, B. L. J. Membr. Sci. 2004, 244, 205. (27) Rivas, B. L.; Pereira, E. D.; Moreno-Villoslada, I. Prog. Polym. Sci. 2003, 28, 173. (28) Moreno-Villoslada, I.; Miranda, V.; Jofre´, M.; Chandı´a, P.; Villatoro, J. M.; Bulnes, J. L.; Corte´s, M.; Hess, S.; Rivas, B. L. J. Membr. Sci. 2006, 272, 137. (29) Tamai, N.; Yamazaki, T.; Yamazaki, I.; Mizuma, A.; Mataga, N. J. Phys. Chem. 1987, 91, 3503. (30) Makashima, K.; Duhamel, J.; Winnik, M. A. J. Phys. Chem. 1993, 97, 10702. (31) Moreno-Villoslada, I.; Rivas, B. L. J. Phys. Chem. B 2002, 106, 9708. (32) Rivas, B. L.; Moreno-Villoslada, I. J. Membr. Sci. 2001, 187, 271. (33) Rivas, B. L.; Moreno-Villoslada, I. J. Membr. Sci. 2000, 178, 165. (34) Data not shown.