Cooperative Binding of Nonionic Surfactant to Hydrophobically

Nov 13, 2006 - because of their biological and technological importance.1 With ... (TX, Scheme 1), to p(A/C12(x)) with a wide range of x (5-60 mol %)...
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Langmuir 2007, 23, 2191-2197

2191

Cooperative Binding of Nonionic Surfactant to Hydrophobically Modified Polyanions as Studied by Frontal Analysis Continuous Capillary Electrophoresis Akihito Hashidzume,*,† Shin-ichi Watanabe,† and Yotaro Morishima‡ Department of Macromolecular Science, Graduate School of Science, Osaka UniVersity, Toyonaka, Osaka 560-0043, Japan, and Faculty of Engineering, Fukui UniVersity of Technology, 6-3-1 Gakuen, Fukui, Fukui 910-8505, Japan ReceiVed August 10, 2006. In Final Form: NoVember 13, 2006 The binding of a nonionic surfactant, Triton X-100 (TX), to amphiphilic copolymers of sodium 2-(acrylamido)2-methylpropanesulfonate and N-dodecylmethacrylamide (C12) (p(A/C12(x)), where x denotes the mol % content of C12) was investigated by frontal analysis continuous capillary electrophoresis (FACCE) combined with dynamic light scattering focusing on the effect of the hydrophobe content on the binding in a wide range of x (5-60 mol %). From binding isotherms obtained from FACCE data, the binding was found to be cooperative in the whole range of x. Furthermore, a significant change in the binding behavior, i.e., cooperativity, was found to occur in a relatively narrow range of x (38-50 mol %), which is attributable to a change in the self-association behavior of p(A/C12(x)) in this x range.

Introduction Interactions between water-soluble polymers and surfactants have been a subject of increasing interest in the past two decades because of their biological and technological importance.1 With a trend of increasing importance of hydrophobically modified water-soluble polymers (amphiphilic polymers) in recent years,2 their interactions with surfactants have attracted growing interest.3 There is a general tendency that the hydrophobic modification of a water-soluble polymer strengthens the polymer-surfactant interaction by providing the polymer with hydrophobic sites to which surfactants bind preferentially. Since the polymersurfactant interaction takes place competing with self-association of polymer hydrophobes, the formation of micelle-like microdomains from the self-association of polymer hydrophobes may lead to a situation where polymer hydrophobes are less available for interaction with surfactants. Therefore, the level of the hydrophobic modification is an important parameter for the polymer-surfactant interaction. * To whom correspondence should be addressed. Tel/Fax: +81-6-68505462. E-mail [email protected]. † Osaka University. ‡ Fukui University of Technology. (1) For example: (a) Polymer-Surfactant Systems; Kwak, J. C. T., Ed.; Surfactant Science Series; Marcel Dekker: New York, 1998; Vol. 77. (b) Jo¨nsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactants and Polymers in Aqueous Solution; Wiley and Sons: Chichester, 1998. (c) Colloid-Polymer Interactions. From Fundamentals to Practice; Farinato, R. S.; Dubin, P. L., Eds.; Wiley and Sons: New York, 1999. (2) For example: (a) McCormick, C. L.; Armentrout, R. S.; Cannon, G. C.; Martin, G. G. In Molecular Interactions and Time-Space Organization in Macromolecular Systems; Morishima, Y., Norisuye, T., Tashiro, K., Eds.; SpringerVerlag: Berlin, 1999; pp 125-139. (b) Bock, J.; Varadaraj, R.; Schulz, D. N.; Maurer, J. J. In Macromolecular Complexes in Chemistry and Biology; Dubin, P. L., Bock, J., Davies, R. M., Schulz, D. N., Thies, C., Eds.; Springer-Verlag: Berlin, 1994; pp 33-50. (c) Hydrophilic Polymers. Performance with EnVironmental Acceptability; Glass, J. E., Ed.; Advances in Chemistry Series 248; American Chemical Society: Washington, DC, 1996. (d) Laschewsky, A. AdV. Polym. Sci. 1995, 124, 1-86. (3) For example: (a) Winnik, F. M.; Ringsdorf, H.; Venzmer, J. Langmuir 1991, 7, 905-911. (b) Winnik, F. M.; Ringsdorf, H.; Venzmer, J. Langmuir 1991, 7, 912-917. (c) Goddard, E. D.; Leung, P. S. Langmuir 1992, 8, 1499-1500. (d) Winnik, F. M.; Regismond, S. T. A.; Goddard, E. D. Colloids, Surf. A 1996, 106, 243-247. (4) Hashidzume, A.; Mizusaki, M.; Yoda, K.; Morishima, Y. Langmuir 1999, 15, 4276-4282.

In an earlier paper, we reported on the interaction of random copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate and N-dodecylmethacrylamide (C12) (p(A/C12(x)), where x denotes the mol % content of C12, Scheme 1) with n-dodecyl hexa(ethylene glycol) monoether (C12E6) in 0.2 M NaCl aqueous solutions studied by fluorescence and dynamic light scattering (DLS) techniques.4 These polymers form micelle-like aggregates predominantly through intrapolymer associations of dodecyl hydrophobes.5-8 The polymer micelles formed from p(A/C12(x)) of x g ca. 40 mol % have a particularly compact nanostructure where polymer chains are highly folded.5,6,8 The compact nanostructure is unfolded by interaction with C12E6, forming polymer-surfactant complexes that are soluble in water.4 More recently, we found that a liquid-liquid phase separation occurred in the case of p(A/C12(x)) with a high content of C12 (e.g., x ≈ 50 mol %) when mixed with C12E6 in the presence of a sufficiently high concentration of added NaCl.9 In this work, we have focused on the effect of the hydrophobe content on the binding of a nonionic surfactant, Triton X-100 (TX, Scheme 1), to p(A/C12(x)) with a wide range of x (5-60 mol %). We employed a frontal analysis continuous capillary electrophoresis (FACCE) technique,10 in conjunction with a DLS technique, to investigate the polymer-surfactant interaction. TX is a UV-active surfactant advantageous for FACCE. FACCE is a powerful tool to investigate the association equilibrium of colloidal species because it allows one to obtain the concentrations of bound and free species in a short time period using a small amount of samples. FACCE has been used mainly for binding equilibrium of protein-polymer systems.11-20 To the best of our (5) Yamamoto, H.; Mizusaki, M.; Yoda, K.; Morishima, Y. Macromolecules 1998, 31, 3588-3594. (6) Yamamoto, H.; Morishima, Y. Macromolecules 1999, 32, 7469-7475. (7) Hashidzume, A.; Yamamoto, H.; Mizusaki, M.; Morishima, Y. Polym. J. (Tokyo, Jpn.) 1999, 31, 1009-1014. (8) Yamamoto, H.; Hashidzume, A.; Morishima, Y. Polym. J. (Tokyo, Jpn.) 2000, 32, 745-752. (9) Hashidzume, A.; Ohara, T.; Morishima, Y. Langmuir 2002, 18, 92119218. (10) Gao, J. Y.; Dubin, P. L.; Muhoberac, B. B. Anal. Chem. 1997, 69, 29452951. (11) Hallberg, R. K.; Dubin, P. L. J. Phys. Chem. B 1998, 102, 8629-8633.

10.1021/la062379u CCC: $37.00 © 2007 American Chemical Society Published on Web 12/22/2006

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Hashidzume et al. Table 1. Characteristics of Polymers Used in This Study

Scheme 1. Chemical Structures of p(A/C12(x)) and TX

polymer code

x (mol %)a

Mw × 10-4 b

Mw/Mnb

p(A/C12(5)) p(A/C12(18)) p(A/C12(28)) p(A/C12(38)) p(A/C12(50)) p(A/C12(60))

5 18 28 38 50 60

2.2 5.0 2.2 2.1 5.0 5.3

2.5 3.7 2.9 2.9 1.9 1.9

a

Mol % content of C12 in copolymers determined by elemental analysis (N/C ratio). b Determined by GPC in methanol containing 0.20 M LiClO4. Molecular weights were calibrated with poly(ethylene glycol) standards.

knowledge, however, FACCE has not been employed for studies of binding equilibria in polymer-surfactant systems, although there have been a few examples of polymer-surfactant interaction as studied by CE.21,22 Experimental Section Materials. Copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate and N-dodecylmethacrylamide (C12) used in this work (p(A/C12(x)), where x denotes the mol % content of C12, Scheme 1) were prepared by radical copolymerization of 2-(acrylamido)-2methylpropanesulfonic acid and C12 in N,N-dimethylformamide in the presence of 2,2’-azobis(isobutyronitrile) at 60 °C for 18 h, followed by neutralization with aqueous NaOH.23 Compositions of the copolymers were determined by elemental analysis (N/C ratio). Weight average molecular weights (Mw) and ratios of weight to number average molecular weight (Mw/Mn) were determined with a JASCO GPC-900 system equipped with two Shodex Asahipak GF-7M HQ columns in combination with a JASCO UV-975 detector using a 0.20 M LiClO4 solution in methanol as eluent at a flow rate of 1.0 mL/min, and molecular weights for the polymers were calibrated with poly(ethylene glycol) standards (Scientific Polymer Products, Inc.). Table 1 lists the characteristics of copolymers used in this study. The contents of C12 (i.e., x) are in the range of 5-60 mol %. Mw ranges (2.1-5.3) × 104, and Mw/Mn ranges 1.9-3.7. Triton X-100 (TX, Scheme 1) was purchased from Nakalai Tesque and used as received. Milli-Q water was used for all measurements. Other reagents were used without further purification. Measurements. Preparation of Polymer Solutions. A stock solution of 2 g/L p(A/C12(x)) was prepared by dissolving each solid polymer sample (recovered by freeze-drying) in a borate buffer (the ionic strength (I) of 0.05 and pH 8.2), prepared by adding NaOH and H3BO3 to Milli-Q water, at 70 °C with vigorous stirring for 15 min. A stock solution of 20 mM TX was prepared by dissolving TX in the borate buffer. The stock solutions were stored overnight at (12) Gao, J. Y.; Dubin, P. L.; Muhoberac, B. B. J. Phys. Chem. B 1998, 102, 5529-5535. (13) Gao, J. Y.; Dubin, P. L. Biopolymers 1999, 49, 185-193. (14) Hattori, T.; Hallberg, R.; Dubin, P. L. Langmuir 2000, 16, 9738-9743. (15) Hattori, T.; Kimura, K.; Seyrek, E.; Dubin, P. L. Anal. Biochem. 2001, 295, 158-167. (16) Seyrek, E.; Dubin, P. L.; Tribet, C.; Gamble, E. A. Biomacromolecules 2003, 4, 273-282. (17) Girard, M.; Turgeon, S. L.; Gauthier, S. F. J. Agric. Food Chem. 2003, 51, 6043-6049. (18) Seyrek, E.; Hattori, T.; Dubin, P. L. Methods Mol. Biol. 2004, 276, 217228. (19) Hattori, T.; Bat-Aldar, S.; Kato, R.; Bohidar, H. B.; Dubin, P. L. Anal. Biochem. 2005, 342, 229-236. (20) Pouliquen, G.; Tribet, C. Macromolecules 2006, 39, 373-383. (21) Collet, J.; Tribet, C.; Gareil, P. Electrophoresis 1996, 17, 1202-1209. (22) Polozova, A.; Winnik, F. M. Langmuir 1999, 15, 4222-4229. (23) Morishima, Y.; Kobayashi, T.; Nozakura, S. Polym. J. (Tokyo, Jpn.) 1989, 21, 267-274.

room temperature for equilibration. Sample solutions for DLS and FACCE measurements were prepared by mixing the stock solutions of p(A/C12(x)) and TX, and the borate buffer, fixing the polymer concentration (Cp) at 1 g/L. All the sample solutions were equilibrated overnight and then filtered using a 0.2 µm pore size disposable membrane filter prior to measurement. Dynamic Light Scattering (DLS). Distributions of the relaxation times and apparent hydrodynamic radii (Rh) were measured with an Otsuka Electronics Photal DLS-7000 light scattering spectrometer equipped with an Ar ion laser (output power ) 50 mW at 488 nm) and an ALV-5000 multi τ digital time correlator at 25 °C. The distributions of relaxation times and Rh were measured at varying scattering angles. To obtain the relaxation time distribution, the inverse Laplace transform analysis was performed by conforming the REPES algorithm.24 g(1)(t) )

∫ τA(τ) exp(- τt ) d ln τ

(1)

where τ is the relaxation time and g(1)(t) is the normalized autocorrelation function. The relaxation time distributions are given as a τA(τ) versus log τ profile with an equal area. Apparent values of Rh were calculated using the Einstein-Stokes relation, Rh ) kBT/ (6πηD), where kB is Boltzman’s constant, T is the absolute temperature, η is the solvent viscosity, and D is the diffusion coefficient determined by DLS. The details of DLS instrumentation and theory are described in the literature.25 Frontal Analysis Continuous Capillary Electrophoresis (FACCE). FACCE measurements were performed with a Beckman P/ACE 5510 instrument using a cartridge equipped with a bare fused silica capillary (Restek, i.d. ) 50 µm). The total length of the capillary was 27 cm, and the effective separation length (from inlet to detection window) was 20 cm. A borate buffer solution (I ) 0.05, pH 8.2) was used as the run buffer. The capillary was conditioned by flushing 0.1 M NaOH and water successively at 20 psi for 1 min followed by washing with the borate buffer prior to use. After injection of a solution of neutral marker, mesityl oxide, for 2 s, the capillary inlet end was transferred to a sample vial to initiate sample introduction and separation by applying a constant voltage of 10 kV at 25 °C. The sample signal was detected by UV absorption at 214 nm. The details of FACCE instrumentation are described in the literature.10

Results DLS. In this study, Cp was fixed at 1 g/L, practically a lower limit for DLS measurements, because we intended to minimize the effect of the formation of complexes containing plural polymer chains. On the basis of our previous studies,6,7,9 it can be anticipated that, when Cp ) 1 g/L, the polymer-surfactant complex formed at higher TX concentrations contains only one polymer chain. Figure 1 shows examples of histograms of relaxation time distributions obtained by DLS measurements for (24) (a) Jakes, J. Czech. J. Phys. 1988, B38, 1305-1316. (b) Jakes, J.; Stepa´nek, P. Czech. J. Phys. 1990, 40, 972-983. (25) Dynamic Light Scattering: the Method and Some Applications; Brown, W., Ed.; Monographs on the Physics and Chemistry of Materials; Oxford University Press: New York, 1993.

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Figure 2. Apparent Rh as a function of Cs for mixtures of 1 g/L p(A/C12(x)) and TX and for polymer-free TX in a borate buffer (I ) 0.05, pH ) 8.2): x ) 5 (circle), 18 (square), 28 (triangle), and 38 (diamond) mol % (a); x ) 50 (open circle) and 60 (open square) mol %, and polymer-free TX (close circle) (b).

p(A/C12(x)) of x ) 28 and 60 mol % in the absence and presence of TX at varying concentrations raging from 1 to 10 mM. Plots of average relaxation times against squares of scattering vectors exhibited a good linear relationship passing through the origin (data not shown), indicating that these relaxation modes are due to translational diffusion. Using the diffusion coefficients obtained from the slopes of the straight lines, apparent values of Rh were calculated using the Einstein-Stokes equation, and thus, estimated Rh values are indicated in Figure 1. In the absence of TX (i.e, Cs ) 0 mM), the histogram exhibits an unimodal distribution and the apparent Rh for p(A/C12(28)) is 5.0 nm (Figure 1a). In the presence of TX, Rh increases gradually from 5.6 to 7.8 nm as Cs is increased form 1 to 5 mM. Since the critical micelle concentration (cmc) of TX have been reported to be ca. 0.2-0.4 mM,26 this observation indicates that p(A/C12(28)) interacts with TX to form polymer-micelle complexes. As will be discussed in the following subsection on FACCE, a considerable fraction of free TX micelles coexists at Cs g 5 mM, and thus, the apparent Rh of 7.3 nm is the mean value of those for complexes and free TX micelles. Values of Rh for the free micelles are estimated to be 2-4 nm. In the case of p(A/C12(60)), the histogram also exhibits a unimodal distribution in the absence of TX but the apparent Rh is much larger than that for p(A/C12(28)), as can be compared in Figure 1a and b. Our previous studies demonstrated that p(A/C12(x)) of x > ca. 50 mol % had a considerable tendency for interpolymer association to form micelle-like aggregates

comprised of plural polymer chains.6,8 Therefore, the relaxation mode in Figure 1b in the absence of TX is ascribable to interpolymer aggregates. Upon addition of 1 mM TX, apparent Rh decreases to 7.6 nm, suggestive of dissociation of the interpolymer aggregates.6,27 As Cs is further increased up to 5 mM, TX micelles are bound to the polymer chain, resulting in a slight increase in apparent Rh. Similar to the case of p(A/ C12(28)), at Cs g 5 mM, a significant amount of free TX micelles coexist and thus the Rh obtained is the mean value for those of complexes and free TX micelles. DLS measurements indicated that relaxation modes were also due to translational diffusion for the other polymers. The apparent Rh values obtained are plotted against Cs in Figure 2. Figure 2a indicates that apparent Rh values for p(A/C12(x)) of x ) 5-38 mol % are weakly dependent on Cs. In the cases of p(A/C12(x)) of x ) 5 and 18 mol %, Rh decreases slightly upon addition of a small amount (0.1-1 mM) of TX, implying that binding of TX induces slight folding of the polymer chain. In the Cs region of 1-5 mM, apparent Rh increases because of complexation of p(A/C12(x)) of x ) 5 and 18 mol % with TX micelles. In the case of p(A/C12(x)) of x ) 28 mol %, apparent Rh is practically constant at Cs e 1 mM, but apparent Rh increases with increasing Cs from 1 to 5 mM because of the complex formation. A similar tendency was observed for the polymer with x ) 38 mol %. In the case of p(A/C12(x)) of x ) 60 mol %, in contrast, apparent Rh is strongly dependent on Cs, as can be seen in Figure 2b. When 1 mM TX is added, Rh decreases markedly from 18 to 7.6 nm, indicating that binding of TX micelles causes dissociation of the interpolymer aggregates.6 As Cs is increased from 1 to 5 mM, Rh increases slightly because of the further binding of TX micelles. The practically same trend was found for the polymer with x ) 50 mol %. FACCE. Figure 3a and b shows examples of FACCE data for mixtures of 1 g/L p(A/C12(x)) of x ) 28 and 60 mol % and varying concentrations of TX. The FACCE data in this figure

(26) van Os, N. M.; Haak, J. R.; Rupert, L. A. M. Physico-Chemical Properties of Selected Anionic, Cationic and Nonionic Surfactants; Elsevier: Amsterdam, 1993.

(27) Upon addition of a small amount of TX (e1 mM), a significant decrease in the scattering intensity was observed in the DLS measurements. This observation supports dissociation of interpolymer aggregates formed from p(A/C12(60)).

Figure 1. Histograms of relaxation time distributions obtained by DLS measurements for 1 g/L p(A/C12(28)) (a) and p(A/C12(60)) (b) in a borate buffer (I ) 0.05, pH ) 8.2) in the presence of varying concentrations of TX.

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Hashidzume et al.

Figure 3. FACCE data (a and b) and migration time distributions (c and d) obtained by differentiation of the FACCE data for 1 g/L p(A/C12(28)) (a and c) and p(A/C12(60)) (b and d) in a borate buffer (I ) 0.05, pH ) 8.2) in the presence of varying concentrations of TX.

were differentiated to obtain distributions of migration times, which correspond to conventional capillary electropherograms (Figure 3c and d).28-30 In Figure 3c and d, signals at a migration time of ca. 1.8 min are due to electrically neutral species, mesityl oxide (neutral marker) or free micelles and unimers of TX. In the absence of TX (i.e., Cs ) 0 mM), the signals around 5.0 and 5.5 min are due to p(A/C12(x)). In the presence of TX, the signals following the peaks for free TX are ascribable to complexes between the polymer and TX micelles. As Cs is increased, the migration time for the complexes decreases, indicating that the electrophoretic flow decreases. Previously, Collet et al.21 have reported that the electrophoretic flow of hydrophobically modified poly(sodium acrylate) decreases upon complexation with nonionic and zwitterionic surfactants because of an increase in the average friction of the monomers and bound surfactant micelles. Thus, the decrease in the electrophoretic flow may be due to the increase in the average friction of the monomers and bound TX micelles. It should be noted here that, in the case of p(A/C12(28)) at Cs g 3 mM, the signals due to the complexes are bimodal and considerably broad. Differentiated FACCE data indicated that the migration time distribution was broader for p(A/C12(x)) of smaller x (data not shown), suggesting that the binding of TX micelles to the polymer is affected by the hydrophobe content in p(A/C12(x)). However, no clear explanation for the bimodal and broad migration time distribution can be offered at the present time. Electrophoretic mobility (µ) can be calculated from

µ)

( )

lL 1 1 V ts t0

(2)

where l is the length of capillary between the anode and the detector, L is the total capillary length, V is the applied voltage, and ts and t0 are the migration times for sample and the neutral marker, respectively.31 For all the p(A/C12(x)), the average values of µ (µ j ) were calculated using average values of ts (ths), which (28) Staggemeier, B.; Huang, Q. R.; Dubin, P. L.; Morishima, Y.; Sato, T. Anal. Chem. 2000, 72, 255-258. (29) Zhang, B.; Hattori, T.; Dubin, P. L. Macromolecules 2001, 34, 67906794. (30) Zhang, B.; Kirton, G. F.; Dubin, P. L. Langmuir 2002, 18, 4605-4609.

can be calculated as

ht s )

∫t t ∫t t

2

tA′(t) dt

2

A′(t) dt

1

(3)

1

where t is the migration time, A′(t) is the differentiated FACCE electropherogram, and t1 and t2 are the migration times at which the signal due to the polymer-micelle complexes starts and ends in the differentiated FACCE electropherogram, respectively. It should be noted here that the hts is an apparent average value because both p(A/C12(x)) and TX are UV active and because of how the composition in the complex depends on migration time is unknown. Therefore, µ j is also apparent one. All the µ j values obtained are negative because the polymer-micelle complexes are negatively charged. Values of - µ j are plotted as a function of Cs in Figure 4. For all the polymers examined, as Cs is increased from 0.2 to 7 mM, - µ j decreases gradually from ca. 3 × 10-4 cm2 V-1 s-1, presumably because of the increase in the average friction of the monomers and bound TX micelles.21 At Cs g 7 mM, - µ j levels off at ca. 1.6 × 10-4 cm2 V-1 s-1 for x ) 5 and 18 mol % and at ca. 1.0 × 10-4 cm2 V-1 s-1 for x ) 28-60 mol %. These observations indicate that the binding of TX micelles to p(A/C12(x)) is practically saturated at Cs g 7 mM. Binding Isotherms for p(A/C12(x))-TX. Using the signal intensities (absorbances) in the FACCE data (Figure 3a and b), the total concentrations of TX molecules existing as free (i.e., unbound) micelles and free molecules (unimers) in the bulk phase (Cs,f) and of TX molecules in polymer-bound micelles (Cs,b) were calculated.32 The value of Cs,f was calculated from the abrupt increase in absorbance at ca. 1.8 min, which corresponded to the signal due to free TX unimers and micelles in the differentiated FACCE data. The value of Cs,b was calculated (31) Li, S. F. Y. Capillary Electrophoresis. Principles, Practice, and Applications; Journal of Chromatography Library; Elsevier: Amsterdam, 1992; Vol. 52. (32) Since this study is dealing with polymer-micelle interaction, the concentrations of free and bound TX micelles should be determined. Unfortunately, however, it is practically impossible to determine the aggregation numbers of the bound TX micelles, and thus, it was not possible to obtain the concentrations of the bound TX micelles.

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Discussion

Figure 4. Electrophoretic mobility (-µ j ) as a function of Cs for mixtures of 1 g/L p(A/C12(x)) and TX in a borate buffer (I ) 0.05, pH ) 8.2): x ) 5 (circle), 18 (square), 28 (triangle), and 38 (diamond) mol % (a) and x ) 50 (circle) and 60 (square) mol % (b).

Figure 5. Binding isotherms for mixtures of 1 g/L p(A/C12(x)) and TX in a borate buffer (I ) 0.05, pH ) 8.2): x ) 5 (circle), 18 (square), 28 (triangle), and 38 (diamond) mol % (a) and x ) 50 (circle) and 60 (square) mol % (b). The best-fitted curves using the Hill model (eq 4) are also drawn.

from the difference between the absorbance due to free TX unimers and micelles and that in the flat region at longer migration times by subtracting the absorbance due to p(A/C12(x)) itself. For each data point, FACCE was measured three times and the errors of Cs,f and Cs,b were confirmed to be less than 5%. The values of Cs,b are plotted as a function of Cs,f in Figure 5 to obtain binding isotherms. For all the polymers examined, Cs,b increases significantly in a relatively narrow Cs,f range of 10-1-100 mM, indicative of cooperative binding of TX to p(A/C12(x)). The onset of the cooperative binding is in fair agreement with the cmc of TX (≈0.2-0.4 mM),26 a manifestation of the binding occurring between the polymer and TX micelles.

Analysis of the Binding Isotherms. It should be important to discuss the binding of TX micelles to p(A/C12(x)) taking into account the self-association behavior of p(A/C12(x)) as a function of x. In the absence of TX, polymer chains of p(A/C12(x)) with higher x adopt a folded chain conformation due to hydrophobic self-associations of dodecyl groups within the polymer chain, where hydrophobic microdomains are surrounded, and hence “protected”, by loops of charged segments.5,6,8 Our previous studies on the binding of C12E6 micelles to p(A/C12(x))4,7,9 made it clear that the polymer chains became unfolded upon micelle binding to the polymer and that the chain unfolding allowed further binding of micelles in a cooperative manner. This cooperativeness was inferred to be due to an increase in the availability of dodecyl hydrophobes for the micelle binding. Thus, taken together with the sigmoidal nature of the binding isotherms in Figure 5, we anticipated the same type of cooperativeness for the binding of TX micelles to p(A/C12(x)). To the best of our knowledge, however, there have been no specific models that can be applied to such cooperative polymer-micelle binding. In the case of cooperative binding of a small molecule to a polymer having a number of binding sites, such as the binding of an ionic surfactant molecule to an oppositely charged polyelectrolyte,33-37 the binding of the small molecule at one site increases the affinity for the molecule at adjacent sites. To account for such binding cooperativity, two common models are often applied;38,39 the Hill model40 and the Zimm-Bragg model.41 However, these models are not apparently suitable to apply to the binding of TX micelles to p(A/C12(x)) because the cooperativeness for this system is due to a change in the polymer conformation upon micelle binding. Despite the fact that the origin for the cooperativity is different, we attempted to fit the two models to our binding data for the p(A/C12(x))-TX system. Then, we found surprisingly good fits as shown in Figure 5 (with the Hill model) and in Figure S1 in the Supporting Information (with the Zimm-Bragg model). The binding of nonionic surfactants to amphiphilic random copolymers of R-L-amino acids has recently been reported.42 This peptide-micelle system is similar to our p(A/C12(x))-TX system in that the driving force for the micelle binding is hydrophobic interaction between hydrophobic parts of the polymer and micelles and that the micelle binding induces a conformational change of the polymer. It is also reported that in the peptide-micelle system, the micelle binding is cooperative and the Hill model is preferred for analysis of the binding isotherms.42 According to the Hill model, Cs,b is given as40

Cs,b ) Cs,sat

(KCs,f)n 1 + (KCs,f)n

(4)

where Cs,sat is the concentration of bound TX at saturation, K is (33) Shirahama, K.; Kameyama, K.; Takagi, T. J. Phys. Chem. 1992, 96, 6817-6820. (34) Tret’yakova, A. Y.; Bilalov, A. V.; Barabanov, V. P. Vysokomol. Soedin. Ser. A 1992, 34, 86-90. (35) Satake, I.; Yang, J. T. Biopolymers 1976, 15, 2263-2275. (36) Hayakawa, K.; Kwak, J. C. T. J. Phys. Chem. 1982, 86, 3866-3870. (37) Kosmella, S.; Koetz, J.; Shirahama, K.; Liu, J. J. Phys. Chem. B 1998, 102, 6459-6464. (38) Shirahama, K. In Polymer-Surfactant Systems; Kwak, J. C. T., Ed.; Surfactant Science Series; Marcel Dekker: New York, 1998; Vol. 77, pp 143191. (39) Linse, P.; Piculell, L.; Hansson, P. In Polymer-Surfactant Systems; Kwak, J. C. T., Ed.; Surfactant Science Series; Marcel Dekker: New York, 1998; Vol. 77, pp 193-238.

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Figure 6. Parameters, Cs,sat, K, and n, obtained by fitting using the Hill model (eq 4) as a function of x for binding of TX to p(A/C12(x)).

the binding constant, and n is the Hill coefficient. The Hill coefficient, n, is a parameter for coorperativity, being unity for non-cooperative binding (i.e., Langmuir-type binding) and larger than unity for cooperative binding. Figure 5 also includes the best-fitted curves using eq 4. For all the polymers, the curves fit in well with the experimental data. The parameters, Cs,sat, K, and n evaluated from the best fit are plotted in Figure 6 as a function of x. Figure 6a suggests the presence of a maximum Cs,sat in the x region of 30-40 mol %. The binding of TX to p(A/C12(x)) is driven dominantly by hydrophobic interaction between dodecyl hydrophobes and the tail of TX. In the present FACCE measurements, the concentration of dodecyl hydrophobes (CC12) increases with x because Cp was fixed at a constant concentration of 1 g/L. The ratios of Cs,sat to CC12 were calculated (Figure S2 in Supporting Information). As x is increased from 5 to 18 mol %, the Cs,sat/CC12 ratio decreases sharply from 15.1 to 4.2. As x is further increased up to 60 mol %, Cs,sat/CC12 decreases gradually to 1.5. These observations suggest that, at x g 18 mol %, TX micelles interact with hydrophobic microdomains formed from dodecyl hydrophobes, and the folded conformation of the polymer chain remains as such even at saturation.4 This may be because hydrophobic self-association of dodecyl hydrophobes is stronger for the polymers with higher x in the complex. Figure 6b indicates that the binding constant, K, was virtually independent of x in the range of (2.9-3.5) × 103 M-1. As can be seen in Figure 6c, the Hill coefficients, n, for all the polymers are larger (40) Hill, A. V. J. Physiol. (Oxford, U.K.) 1910, 40, 190-224. (41) Zimm, B. H.; Bragg, J. K. J. Chem. Phys. 1959, 31, 526-535. (42) Sjo¨gren, H.; Ericsson, C. A.; Evenaes, J.; Ulvenlund, S. Biophys. J. 2005, 89, 4219-4233.

Hashidzume et al.

than unity, indicative of cooperative binding. In the case of the polymers with x ) 5-38 mol %, n values fall in the range 2.12.6, whereas, for the polymers with x ) 50 and 60 mol %, n values are larger, ranging from 3.1 to 3.4, than those for the polymers with smaller x. This anomalous dependency of the Hill coefficient on the hydrophobe content in the polymer indicates that the behavior of TX binding to p(A/C12(x)) changes considerably in a relatively narrow range of x between 38 and 50 mol %. Cooperative Binding of TX to p(A/C12(x)). Our previous studies have clarified the self-association behavior of p(A/C12(x))5-8 and the complexation behavior of p(A/C12(x)) with C12E6.4,7,9 The self-association and complexation behavior can be roughly divided into the following two regions with respect to the C12 content, x: Region 1 where x e ca. 30 mol % and Region 2 where x g ca. 40 mol %. In Region 1, the polymer chain assumes a loosely folded conformation because the dodecyl hydrophobes self-associate to form micelle-like aggregates along the polymer chain. Such micelle-like aggregates are surrounded by charged loops and thus may be referred to as “primary micellar units”. When C12E6 is added to an aqueous solution of the polymer, C12E6 micelles interact with the primary micellar units to form complexes, resulting in a slight unfolding of the polymer chain.4 In Region 2, the polymer chain collapses into a highly folded conformation because the primary micellar units associate with each other as they are not fully surrounded or “protected” by charged loops. When x is further increased beyond ca. 50 mol %, the tendency for interpolymer association becomes significant while the polymer remains as a highly folded chain. When C12E6 is added to an aqueous solution of the polymer, C12E6 micelles associate with the primary micellar units, resulting in a considerable unfolding of the polymer chain.4,9 Given that TX is a nonionic surfactant based on ethylene oxide similar to C12E6, the behavior of the binding of TX micelles to the polymer should be similar, if not the same, to that of C12E6 micelles. Therefore, on the basis of the above considerations, the cooperativity observed for the binding of TX micelles to the polymer in Region 1 may be explained in terms of a slight unfolding of the polymer chain caused by the micelle binding, the chain unfolding facilitating further binding of micelles in a cooperative manner. In Region 2, the primary micellar units are less accessible to TX micelles since the polymer chains are highly folded. However, once a TX micelle associates with the primary micellar unit, the polymer chain is unfolded so as to enhance the accessibility of TX micelles to the primary micellar units. Thus, the larger n values found in Region 2 may be explained by a considerable unfolding of the polymer chain.

Conclusion We have demonstrated that FACCE is a very useful technique to investigate association equilibria in polymer-surfactant interactions. The binding of TX micelles to p(A/C12(x)) was investigated by FACCE in conjunction with DLS. In the case of p(A/C12(x)) with x ) 5-38 mol %, apparent Rh was weakly dependent on Cs whereas it was strongly dependent on Cs for p(A/C12(x)) of x ) 50 and 60 mol %, Rh decreasing remarkably with increasing Cs at Cs e 1 mM. These findings indicate that the binding of TX to the latter polymers caused dissociation of interpolymer aggregates. When Cs was increased to 5 mM, apparent Rh increased slightly for all the polymers examined, indicative of the formation of p(A/C12(x))-TX micelle complexes. Differentiated FACCE data indicated that - µ j decreased gradually from ca. 3 × 10-4 cm2 V-1 s-1 with increasing Cs from 2 × 10-4 to

Binding of Nonionic Surfactant to Polyanions

7 mM presumably because of an increase in the average friction of the monomers and bound TX molecules upon complex j leveled off, indicating that the formation. At Cs g 7 mM, - µ binding of TX to p(A/C12(x)) was practically saturated at Cs g 7 mM. Binding isotherms obtained with Cs,f and Cs,b determined from FACCE data were found to be fitted well to the Hill model. The Hill coefficient, n, was larger than unity in the whole x range examined, indicative of cooperative binding of TX micelles to the polymer. Cs,sat exhibited a maximum in the x region of 3040 mol %. As x was increased, the Cs,sat/CC12 ratio decreased markedly from 15.1 to 4.2 in the x range of 5-18 mol %, and the ratio decreased gradually to 1.5 in the x range of 18-60 mol %. These observations indicate that when x g 18 mol %, the polymer chain continues to assume a folded conformation even at saturation of the micelle binding presumably because hydrophobic interaction between dodecyl hydrophobes in the polymer is stronger at higher x. The apparent binding constant, K, was virtually independent of x, in the range (2.9-3.5) × 103

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M-1. The value of n was in the range 2.1-2.6 in the x region of 5-38 mol %, whereas n was in the range of 3.1-3.4 at x ) 50 and 60 mol %. This change in n is attributable to the change in the self-association behavior of p(A/C12(x)) in the relatively narrow x range of 38-50 mol %. Thus, the FACCE technique readily allowed us to observe directly the cooperativity in the binding of TX to p(A/C12(x)). Acknowledgment. The authors would like to express their acknowledgment to Professor Takahiro Sato and Associate Professor Toshiyuki Shikata, Department of Macromolecular Science, Graduate School of Science, Osaka University, for fruitful discussion and suggestions. Supporting Information Available: Zimm-Bragg analysis, binding isotherms, and graph of Cs,sat/CC12 vs x. This material is available free of charge via the Internet at http://pubs.acs.org. LA062379U