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Coacervation of Hydrophobically Modified Polyanions by Association with Nonionic Surfactants in Water Akihito Hashidzume,* Takeshi Ohara, and Yotaro Morishima Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan Received May 28, 2002. In Final Form: September 12, 2002 Complexes of random copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate and N-dodecylmethacrylamide (DodMAm) (poly(A/Dx), where x denotes the mol % content of DodMAm) and n-dodecyl hexa(ethylene glycol) monoether (C12E6) exhibited a liquid-liquid phase separation (coacervation) when poly(A/Dx) with x ≈ 50 mol % and C12E6 were mixed in water in the presence of a sufficiently high concentration of added NaCl ([NaCl]). Phase diagrams obtained by turbidimetric titrations indicated that coacervation occurred only within a limited concentration regime both for the polymer and surfactant. Coacervation occurred more easily at higher [NaCl]; [NaCl] should be higher than ca. 0.2 M at 25 °C for coacervation to occur. The cloud points of a mixture of poly(A/D48) and C12E6 at varying [NaCl] suggested that the dominant effects of added NaCl were the shielding effect of electrostatic repulsion between sulfonate moieties in poly(A/D48) at lower [NaCl] and an enhancement of hydrophobic interaction caused by added salt at higher [NaCl]. From the intra- and interpolymer nonradiative energy transfer and quasielastic light scattering data, it was concluded that as the concentration of either of the three components (i.e., polymer, surfactant, and salt) is increased toward a phase boundary for coacervation, the size of poly(A/D48)-C12E6 soluble complexes, in which C12E6 micelles act as cross-linking junctions of different polymer chains, increases gradually at first and then abruptly near the phase boundary, ending up with coacervation at a higher concentration of the component. On the basis of these experimental data, we discussed a possible mechanism for the coacervation of poly(A/D48) with C12E6.
Introduction Interactions between water-soluble polymers and surfactants have been a subject of increasing interest in the last two decades because of their biological and technological importance.1 With a trend of increasing importance of hydrophobically modified water-soluble polymers in recent years,2 their interactions with surfactants have been attracting growing interest.3 There is a general tendency that hydrophobic modifications strengthen the polymer-surfactant interaction by providing hydrophobic sites to which surfactants bind preferentially. A characteristic feature in these polymer-surfactant systems is the competition between self-association of polymer hydrophobes and hydrophobic interactions between polymer hydrophobes and surfactants. The formation of micellelike microdomains by the self-association of polymer hydrophobes may lead to a situation where polymer hydrophobes are less available for interaction with surfactants. (1) (a) Kwak, J. C. T. Polymer-Surfactant Systems; Surfactant Science Series Vol. 77: Marcel Dekker: New York, 1998. (b) Jo¨nsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactants and Polymers in Aqueous Solution; Wiley & Sons: Chichester, 1998. (c) ColloidPolymer Interactions. From Fundamentals to Practice; Farinato, R. S., Dubin, P. L., Eds.; Wiley & Sons: New York, 1999. (2) (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.; Springer-Verlag: 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 Vol. 248; American Chemical Society: Washington, DC, 1996. (d) Laschewsky, A. Adv. Polym. Sci. 1995, 124, 1-86. (3) (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, 14991500. (d) Winnik, F. M.; Regismond, S. T. A.; Goddard, E. D. Colloids Surf., A 1996, 106, 243-247.
Chart 1. Chemical Structures of Poly(A/Dx) and C12E6
In an earlier paper, we reported on the interaction of random copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate and N-dodecylmethacrylamide (DodMAm) (poly(A/Dx), where x denotes the mol % content of DodMAm, Chart 1) with n-dodecyl hexa(ethylene glycol) monoether (C12E6, Chart 1) in 0.2 M NaCl aqueous solutions studied by fluorescence and quasielastic light scattering techniques.4 These polymers form micellelike aggregates predominantly through intrapolymer selfassociation of polymer-bound dodecyl groups.5-8 The (4) Hashidzume, A.; Mizusaki, M.; Yoda, K.; Morishima, Y. Langmuir 1999, 15, 4276-4282. (5) Yamamoto, H.; Mizusaki, M.; Yoda, K.; Morishima, Y. Macromolecules 1998, 31, 3588-3594. (6) Yamamoto, H.; Morishima, Y. Macromolecules 1999, 32, 74697475. (7) Yamamoto, H.; Hashidzume, A.; Morishima, Y. Polym. J. 2000, 32, 745-752.
10.1021/la020493g CCC: $22.00 © 2002 American Chemical Society Published on Web 11/02/2002
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polymer micelles formed from poly(A/Dx) of x g 30 mol % have a particularly compact nanostructure where polymer chains are highly folded.5-7 The compact nanostructure is unfolded by interaction with C12E6, forming polymermicelle complexes that are soluble in water.4 We found that a liquid-liquid phase separation (i.e., coacervation) occurred in the case of poly(A/Dx) with a high content of dodecyl groups (e.g., x ≈ 50 mol %) when mixed with C12E6 in the presence of a sufficiently high concentration of added NaCl. Coacervation is a phenomenon in which a colloid solution separates into two immiscible liquid phases upon addition of a precipitant or another colloid solution.9,10 In general, one of the two liquid phases contains a larger fraction of colloid species, and the other is relatively dilute. The former phase is called “coacervate” and the latter “supernatant”.10 When a colloid species undergoes liquid-liquid phase separation because of a decrease in solubility, it is termed “simple coacervation”11 or “segregative phase separation”.12 When two or more colloid species interact to undergo coacervation, it is called “complex coacervation”13 or “associative phase separation”.12 Although coacervation is interesting because of its widespread technical applications14 and some fascinating biological implications,15 it is an underexamined subject of investigation. In this paper, we report on the phase behavior for aqueous solutions of poly(A/Dx) and C12E6 in addition to further characterization of poly(A/Dx)-C12E6 soluble complexes. On the basis of experimental data, we discuss a possible mechanism for the coacervation of poly(A/Dx) with C12E6. Experimental Section Materials. Random copolymers of sodium 2-(acrylamido)-2methylpropanesulfonate and DodMAm used in this work were prepared by free-radical polymerization of mixtures of 2-(acrylamido)-2-methylpropanesulfonic acid (AMPS) and DodMAm in N,N-dimethylformamide in the presence of 2,2′-azobis(isobutyronitrile) at 60 °C for 18 h, followed by neutralization with aqueous NaOH.16 For fluorescence study, naphthalene and pyrene doubly labeled and naphthalene or pyrene singly labeled polymers (poly(A/Dx/Np/Py), poly(A/Dx/Np), and poly(A/Dx/Py), respectively) were employed (Chart 2).5 The doubly labeled polymer was prepared by quaterpolymerization of AMPS, DodMAm, N-(1naphthylmethyl)methacrylamide (NpMAm),17 and N-(1-pyre(8) Hashidzume, A.; Yamamoto, H.; Mizusaki, M.; Morishima, Y. Polym. J. 1999, 31, 1009-1014. (9) (a) Beijerinck, M. W. Zentralbl. Bakteriol. Parasitenkd. 1896, 2, 627. (b) Beijerinck, M. W. Kolloid-Z. 1910, 7, 16-20. (10) Bungenberg de Jong, H. G.; Kruyt, H. R. Kolloid-Z. 1930, 50, 39-48. (11) Bungenberg de Jong, H. G. In Colloid Science; Kruyt, H. R., Ed.; Elsevier: Amsterdam, 1949; Vol. II, pp 232-258. (12) (a) Piculell, L.; Lindman, B. Adv. Colloid Interface Sci. 1992, 41, 149-178. (b) Piculell, L.; Lindman, B.; Karlstro¨m, G. In PolymerSurfactant Systems; Kwak, J. C. T., Ed.; Surfactant Science Series Vol. 77; Marcel Dekker: New York, 1998; pp 65-141. (13) Bungenberg de Jong, H. G. In Colloid Science; Kruyt, H. R., Ed.; Elsevier: Amsterdam, 1949; Vol. II, pp 335-432. (14) (a) Kayes, J. B. J. Pharm. Pharmacol. 1977, 29, 163-168. (b) Goddard, E. D. J. Soc. Cosmet. Chem. 1990, 41, 23-49. (c) Burgess, D. J. In Macromolecular Complexes in Chemistry and Biology; Dubin, P., Bock, J., Davis, R. M., Schulz, D., Thies, C., Eds.; Springer-Verlag: Berlin, 1994; pp 285-300. (d) Gander, B.; Blanco-Prı´eto, M. J.; Thomasin, C.; Wandrey, C.; Hunkeler, D. In Encyclopedia of Pharmaceutical Technology, 2nd ed.; Swarbrick, J., Boylan, J. C., Eds.; Marcel Dekker: New York, 2002; Vol. 1, pp 481-495. (15) Oparin, A. I. The Origin of Life on the Earth, 3rd ed.; Oliver & Boyd: London, 1957. (16) Morishima, Y.; Kobayashi, T.; Nozakura, S. Polym. J. 1989, 21, 267-274. (17) Morishima, Y.; Tominaga, Y.; Nomura, S.; Kamachi, M. Macromolecules 1992, 25, 861-866.
Hashidzume et al. Chart 2. Chemical Structures of the Labeled Polymers
Table 1. Characteristics of Polymers Used in This Study polymer code
x (mol %)a
Mw × 10-4 b
Mw/Mnb
poly(A/D37) poly(A/D48) poly(A/D40/Np/Py)c poly(A/D41/Np)d poly(A/D43/Py)e
37 48 40 41 43
6.5 1.5 1.1 2.6 8.3
2.7 1.7 2.7 2.1 3.7
a Mol % content of DodMAm in polymers determined by elemental analysis (N/C ratio). b Determined by gel permeation chromatography in methanol containing 0.20 M LiClO4. Molecular weights were calibrated by poly(ethylene glycol) standards. c The contents of NpMAm and PyMAm were 4 and 1 mol %, respectively. d The content of NpMAm was 1 mol %. e The content of PyMAm was 1 mol %.
nylmethyl)methacrylamide (PyMAm).18 The singly labeled polymers were prepared by terpolymerization of AMPS, DodMAm, and either of NpMAm or PyMAm. The details of the procedures for the polymer synthesis were reported elsewhere.19 Compositions of the nonlabeled copolymers were determined by elemental analysis (N/C ratio), and those of the labeled polymers were determined by elemental analysis and UV absorption spectroscopy. Mw and Mw/Mn values were determined with a JASCO GPC-900 system equipped with Shodex Asahipak GF-7M HQ columns in combination with a JASCO UV-975 detector using a 0.20 M LiClO4 solution in methanol as the eluent with 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 polymers used in this study. The contents of DodMAm (i.e., x) are in the range of 37-48 mol %. The range of Mw is (1.5-8.3) × 104, and that of Mw/Mn is 1.7-3.7. n-Dodecyl hexa(ethylene glycol) monoether (C12E6, Chart 1) was used as supplied by Nikko (18) Morishima, Y.; Tominaga, Y.; Kamachi, M.; Okada, T.; Hirata, Y.; Mataga, N. J. Phys. Chem. 1991, 95, 6027-6034. (19) Morishima, Y.; Nomura, S.; Ikeda, T.; Seki, M.; Kamachi, M. Macromolecules 1995, 28, 2874-2881.
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Chemical Co. without further purification. NaCl (Wako Pure Chemicals) was used without further purification. Milli-Q water was used for all measurements. Measurement. (a) Preparation of Polymer Solutions. For all measurements, polymer solutions were prepared as follows: Solid polymer samples (recovered by a freeze-drying technique) were dissolved into pure water at 90 °C with vigorous stirring for 15 min. After cooling the solutions to room temperature, a predetermined amount of NaCl was added to the polymer solutions to adjust ionic strength.7 (b) Turbidimetric Titration. A typical procedure of turbidimetric titrations is as follows: A solution of 10.0 g/L poly(A/D48) (20.0 mM DodMAm unit) containing 0.60 M NaCl was added to a solution of 10.0 mM C12E6 containing 0.60 M NaCl (initial volume, 2.0 mL) in a 1 cm path length quartz cuvette using a microburet with stirring, and turbidity (reported as 100 - %T) was measured with a JASCO V-520 spectrophotometer at 420 nm at 25 °C. Intervals for mixing were fixed to 5 min for stabilization (we confirmed that turbidity reaches a constant value within this time interval). (c) Cloud Point Measurements. Cloud points were determined with a Brinkmann PC920 probe colorimeter equipped with a 1 cm path length fiber optics probe monitored at 450 nm. The heating rate was regulated to 0.5 °C/min around the cloud point. The maximum error in cloud point values was calculated to be 0.5% from at least three determinations. (d) Nonradiative Energy Transfer (NRET). Fluorescence spectra were recorded in the wavelength range of 300-550 nm on a Hitachi F-4500 spectrophotometer for 0.050 g/L poly(A/ D40/Np/Py) in 0.20 M NaCl and for the mixture of 0.10 g/L poly(A/D41/Np) and 0.025 g/L poly(A/D43/Py) in 0.40 M NaCl at varying concentrations of added C12E6. The intensities of naphthalene and pyrene fluorescence were estimated at 326 and 395 nm, respectively. The contribution from direct pyrene excitation was corrected by subtracting from each spectrum the emission spectrum of the corresponding pyrene singly labeled polymer of the same pyrene concentration. (e) Quasielastic Light Scattering (QELS). The distribution of the relaxation times and the apparent hydrodynamic radius (Rh) were measured with an Otsuka Electronics Photal DLS-7000 light scattering spectrometer equipped with an Ar ion laser (output power ) 50 mW at λ ) 488 nm) and an ALV-5000 multiτ-digital time correlator. All measurements were performed at 25 °C. The distributions of relaxation times and Rh for soluble complexes of poly(A/D48) with C12E6 were measured varying the scattering angle. To obtain the relaxation time distribution, the inverse Laplace transform analysis was performed by conforming the REPES algorithm.20
g(1)(t) )
∫τA(τ) exp(-t/τ) d ln τ
(1)
where τ is the relaxation time and g(1)(t) is the normalized autocorrelation function. Apparent values of Rh were calculated using the Einstein-Stokes relation, Rh ) kBT/(6πηD), where kB is Boltzmann’s constant, T is the absolute temperature, η is the solvent viscosity, and D is the diffusion coefficient determined by QELS. Sample solutions were prepared by adding a titrant (an aqueous solution of poly(A/D48), C12E6, or NaCl) to an initial solution (an aqueous solution of C12E6, poly(A/D48), or a mixture of the polymer and surfactant) using a microburet. The initial solution and titrant were filtered prior to preparation of sample solutions using a 0.2 µm pore size disposable membrane filter. (f) Absorption Spectra. Absorption spectra were recorded with a JASCO V-520 spectrophotometer by use of a 1 cm path length quartz cuvette. (g) Steady-Shear Viscosity. The steady-shear viscosities of a coacervate were measured at 25 °C on a RheoLogica DynAlyser 100 stress-control rheometer equipped with a cone and plate. The radius of the cone is 40 mm, and the angle between the cone and plate is 4.0°. A coacervate sample was prepared from 7.0 g/L poly(A/D48) and 14 mM C12E6 in 0.46 M NaCl and recovered by removing the supernatant with a pipet. (20) (a) Jakesˇ, J. Czech. J. Phys. 1988, B38, 1305-1316. (b) Jakesˇ, J.; Sˇ teˇpa´nek, P. Czech. J. Phys. 1990, 40, 972-983.
Figure 1. Turbidimetric titration for 10.0 mM C12E6, adding 10.0 g/L poly(A/D48) in 0.60 M NaCl at 25 °C. The initial volume of 10.0 mM C12E6 was 2.0 mL.
Figure 2. Turbidimetric titration for 7.5 g/L poly(A/D48), adding 50.0 mM C12E6 in 0.30 M NaCl at 25 °C. The initial volume of 7.5 g/L poly(A/D48) was 2.0 mL.
Results and Discussion Phase Behavior for a System of Poly(A/D48), C12E6, NaCl, and Water. (a) Turbidimetric Titration. Phase behavior for aqueous mixtures of poly(A/D48) and C12E6 was investigated by turbidimetric titration at 25 °C. Figure 1 shows an example of turbidimetric titration data with addition of a solution of 10.0 g/L poly(A/D48) to 2.0 mL of a 10.0 mM C12E6 solution at a constant NaCl concentration ([NaCl]) of 0.60 M. At small volumes of the added titrant (vt), turbidity increases only slightly with increasing vt. As vt is further increased, turbidity commences to increase abruptly at vt ≈ 0.3 mL and levels off when vt > 0.4 mL. The turbid solution separates into a highly viscous layer in the bottom and a much less viscous layer on the top on standing for several hours, which indicates that the increased turbidity observed is due to coacervation. The titration data were fitted with two straight lines as indicated in Figure 1, and from their cross point, a value of vt for the onset of an abrupt increase in turbidity was determined to be 0.32 mL. From this onset vt, the onset concentration of poly(A/D48) (Cp) for coacervation was calculated to be 1.37 g/L at a C12E6 concentration ([C12E6]) of 8.63 mM.21 Figure 2 shows a typical example of turbidimetric titration data with addition of 50.0 mM C12E6 to 2.0 mL of 7.5 g/L poly(A/D48) at [NaCl] ) 0.30 M. Turbidity commences to increase abruptly at vt ≈ 0.2 mL, this turbidity increase corresponding to the onset of coacervation. The onset vt was determined to be 0.25 mL from the cross point of the two straight lines drawn in the figure, and then, from this vt, a value of [C12E6] for the onset of coacervation was calculated to be 5.52 mM at Cp (21) In this turbidimetric titration, as vt is increased, [C12E6] decreases. Thus, [C12E6] at the onset Cp was also calculated using the volume and [C12E6] of the initial solution, Cp of the titrant, and the onset vt.
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Figure 4. [NaCl]c for the coacervation as a function of [dodecyl group] at [C12E6]/[DodMAm unit] ) 0.80 (O), 1.00 (0), and 1.20 (4). Figure 3. Phase diagram for the mixture of poly(A/D48) and C12E6 in the presence of varying concentrations of NaCl at 25 °C. [NaCl] ) 0.30 (O), 0.40 (0), and 0.60 M (4).
) 6.67 g/L. In the vt range of 0.27-1.0 mL, turbidity is higher than 90%. It was confirmed that the high turbidity arose from coacervation. However, as vt is increased beyond 1.05 mL, turbidity decreases abruptly in a narrow vt region and then decreases gradually. This observation indicates that the coacervates formed are redissolved at vt > 1.05 mL, resulting in a one-phase state. About two dozens sets of onset Cp and [C12E6] for coacervation were determined by turbidimetric titrations at varying [NaCl]. The Cp values were converted into the molar concentrations of DodMAm units in poly(A/D48) ([DodMAm unit]), and the [C12E6] values were plotted against [DodMAm unit] at varying [NaCl] in Figure 3. This figure exhibits that coacervation occurs only within a limited concentration regime for both the polymer and surfactant, indicating that the coacervation is due to polymer-surfactant complex formation. If we represent the coacervation region in terms of the ratio of concentrations of [C12E6] and [DodMAm unit] ([C12E6]/[DodMAm unit]), coacervation occurs only when the [C12E6]/[DodMAm unit] ratios are 0.40-2.1, 0.25-2.4, and 0.16-3.0 at [NaCl] ) 0.30, 0.40, and 0.60 M, respectively. The coacervation region is wider at higher [NaCl], indicating that a high ionic strength is a favorable condition for coacervation to occur. Turbidimetric titrations were also carried out adding a 5.0 M NaCl solution to aqueous solutions containing poly(A/D48) and C12E6 at varying [C12E6]/[DodMAm unit] ratios of 0.80, 1.00, and 1.20. Values of [NaCl] ([NaCl]c) for the onset of coacervation were plotted as a function of dodecyl group concentration ([dodecyl group] ) [DodMAm unit] + [C12E6]) in Figure 4. At [dodecyl group] < 1 mM, [NaCl]c decreases sharply with increasing [dodecyl group]. On the other hand, at [dodecyl group] > 1 mM, [NaCl]c decreases slightly and then becomes virtually constant. The data in this figure suggest that [NaCl] should be higher than ca. 0.2 M at 25 °C for coacervation to occur at any Cp and [C12E6]. The effect of the content of dodecyl groups (i.e., x) in poly(A/Dx) on the phase behavior was examined using poly(A/D37) in comparison with poly(A/D48). Turbidimetric titration data (not shown) using 10.0 g/L poly(A/ D37) and 50.0 mM C12E6 as an initial solution and a titrant, respectively, indicated that a much higher [NaCl] (≈0.8 M) was required for coacervation to occur, the coacervation region shifting toward a higher [NaCl] region. Thus, it is concluded that a higher content of the hydrophobe in poly(A/Dx) is favorable for coacervation to occur.
Figure 5. Cloud point for the mixture of 2.0 g/L poly(A/D48) and 50.0 mM C12E6 as a function of [NaCl].
(b) Cloud Point Measurements. The phase behavior of the mixture of poly(A/D48), C12E6, and NaCl depends on temperature. For example, an optically clear single-phase solution of a mixture of 2.0 g/L poly(A/D48), 50.0 mM C12E6, and 0.10 M NaCl at 25 °C undergoes coacervation when temperature is raised to 42 °C. An increase in turbidity at this temperature is sufficiently abrupt to define a critical temperature for the onset of coacervation as a cloud point. The cloud point was found to depend on the concentration of added salt. Figure 5 exhibits an example of the dependence of the cloud point on the salt concentration for a mixture of 2.0 g/L poly(A/D48) and 50.0 mM C12E6. In the range [NaCl] < 0.03 M, the cloud point decreases abruptly with increasing [NaCl] presumably because of the shielding effect of electrostatic repulsion between sulfonate moieties in poly(A/D48). The shielding of interpolymer electrostatic repulsion favors associations of two or more polymers with the same micelle, the micelle playing a role of a junction of polymer chains. At [NaCl] g 0.05 M, the cloud point decreases gradually with increasing [NaCl] presumably because of an enhancement of hydrophobic interaction due to changes in the water structure caused by added salt22 in addition to the electrostatic shielding effect. At higher [NaCl] (greater than ca. 0.5 M), the enhancement of hydrophobic interaction may be a dominant effect. Characterization of Poly(A/Dx)-C12E6 Soluble Complexes. (a) NRET. Coacervation may be preceded by the formation of polymer-micelle soluble complexes. If that is the case, the formation of soluble complexes prior to coacervation and a transition from the soluble complexes to coacervates should be important processes to be investigated for clarification of a mechanistic aspect of (22) Collins, K. D.; Washabaugh, M. W. Q. Rev. Biophys. 1985, 18, 323-422.
Coacervation of Hydrophobically Modified Polymers
coacervation. Therefore, as a basis for an understanding of coacervation mechanisms, we performed characterization of poly(A/Dx)-C12E6 complexes soluble in water. For characterization of associative polymers, NRET between an energy donor and an energy acceptor covalently attached to the same polymer chain or different polymer chains provides a sensitive tool to probe into the association behavior of the polymer.23 Since NRET between two chromophores occurs through dipole-dipole interactions between an excited energy donor and a ground-state acceptor, the efficiency of NRET from a donor to an acceptor depends most significantly on their separation distance. When donors and acceptors are covalently attached to the same polymer chain, the efficiency of NRET may be related to a conformational change of the polymer chain. On the other hand, in mixed solutions of polymers labeled with either a donor or an acceptor, the efficiency of NRET between the two labels may correspond to the extent of interpolymer association. In general, naphthalene and pyrene are useful for an energy donor and acceptor, respectively, because this donor/acceptor pair has a large spectral overlap, and naphthalene can be almost selectively excited at a wavelength near 290 nm. In addition, this pair provides an appropriate “spectroscopic ruler” for a study of changes in association behavior on the scale of few nanometers (i.e., the Fo¨rster radius is R0 ) 2.86 nm for transfer from 1-methylnaphthalene to pyrene24). The occurrence of NRET is experimentally indicated by an increase in pyrene fluorescence with excitation of naphthalene. Polymers with lower DodMAm contents (≈40 mol %) were employed to prepare poly(A/Dx)-C12E6 complexes as homogeneous systems for NRET measurements. Naphthalene and pyrene labels are hydrophobic, but it was confirmed that these labels had little or no effect on the association behavior of poly(A/Dx) when x was sufficiently high (g30 mol %) and the loading level of the labels was sufficiently low (e5 mol %).5 Figure 6a shows the intensity ratio of pyrene fluorescence to naphthalene fluorescence (IPy/INp) in the fluorescence spectra for 0.050 g/L poly(A/D40/Np/Py) (Chart 2) ([DodMAm unit] ) 0.084 mM) plotted as a function of [C12E6] at [NaCl] ) 0.20 M. IPy/INp decreases sharply with increasing [C12E6] in the region [C12E6] < 0.3 mM and then remains almost constant at [C12E6] > 0.3 mM. These intrapolymer NRET data indicate that highly collapsed polymer chains in the aggregates of poly(A/D40/Np/Py) are unfolded by complexation with C12E6, and the extent of the unfolding increases as [C12E6] is increased. Figure 6b shows IPy/INp for a mixture of 0.10 g/L poly(A/D41/Np) and 0.025 g/L poly(A/D43/Py), where the total concentration of DodMAm unit is 0.22 mM, plotted as a function of [C12E6] at [NaCl] ) 0.40 M. As [C12E6] is increased, IPy/INp increases at [C12E6] < 0.3 mM, reaches a maximum, and then decreases at [C12E6] > 0.3 mM. These interpolymer NRET data indicated that with increasing [C12E6], a tendency for interpolymer association increases, reaches a maximum, and then decreases. From these observations, we can conclude as follows: When a small amount of C12E6 is added, the polymers are bound to C12E6 micelles by association of polymer-bound dodecyl (23) (a) Webber, S. E. Chem. Rev. 1990, 90, 1469-1482. (b) Winnik, F. M. Polymer 1990, 31, 2125-2134. (c) Ringsdorf, H.; Simon, J.; Winnik, F. M. Macromolecules 1992, 25, 5353-5361. (d) Ringsdorf, H.; Simon, J.; Winnik, F. M. Macromolecules 1992, 25, 7306-7312. (e) Hu, Y.; Kramer, M. C.; Boudreaux, C. J.; McCormick, C. L. Macromolecules 1995, 28, 7100-7106. (f) Kramer, M. C.; Steger, J. A.; Hu, Y.; McCormick, C. L. Macromolecules 1996, 29, 1992-1997. (24) Berlman, I. B. Energy Transfer Parameters of Aromatic Compounds; Academic Press: New York, 1973.
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Figure 6. IPy/INp as a function of [C12E6] for 0.05 g/L poly(A/ D40/Py/Np) in 0.20 M NaCl (a) and for the mixture of 0.10 g/L poly(A/D41/Np) and 0.025 g/L poly(A/D43/Py) in 0.40 M NaCl (b) at 25 °C.
groups with the micelle core (i.e., comicellization of polymer dodecyl groups with the surfactants), the micelle acting as a cross-linking junction of different polymer chains in the interpolymer complex. As [C12E6] is increased, the number of the interpolymer complexes increases. However, as [C12E6] is further increased beyond a certain level, the number of polymer chains bound per micelle decreases, and the number of interpolymer complexes decreases. A similar tendency has been observed for a number of systems in which hydrophobically modified water-soluble polymers interact with surfactants.25 (b) QELS. Changes in the hydrodynamic size for the soluble complexes were monitored by QELS. Figure 7 shows relaxation time distributions in QELS measured at a scattering angle of 90° for a mixture of 5.0 g/L poly(A/D48) and C12E6 of varying concentrations at [NaCl] ) 0.20 M. Apparent hydrodynamic radii (Rh) were calculated from the Stokes-Einstein relation using diffusion coefficients estimated from the linear plot of Γ versus q2. Values of Rh thus calculated are indicated in the figure. In the absence of C12E6, a unimodal distribution was observed, and Rh for poly(A/D48) was calculated to be ca. 11 nm. As [C12E6] is increased, Rh for soluble complexes increases. When [C12E6] is increased up to ca. 3.7 mM, coacervation occurs, and a peak appears in a region of much longer relaxation times. The apparent Rh for this peak was calculated to be ca. 4.3 µm. The peak observed in the longer relaxation time region may be due to droplets of coacervate. On the other hand, the peak with a smaller Rh in Figure 7e may be due to soluble complexes in the supernatant. Figure 8 shows Rh for poly(A/D48)-C12E6 soluble com(25) For example: (a) Tanaka, R.; Meadows, J.; Williams, P. A.; Phillips, G. O. Macromolecules 1992, 25, 1304-1310. (b) Nystro¨m, B.; Thuresson, K.; Lindman, B. Langmuir 1995, 11, 1994-2002. (c) Loyen, K.; Iliopoulos, I.; Olsson, U.; Audebert, R. Prog. Colloid Polym. Sci. 1995, 98, 42-46. (d) Seng, W. P.; Tam, K. C.; Jenkins, R. D.; Bassett, D. R. Langmuir 2000, 16, 2151-2156.
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Figure 9. Steady-state viscosity as a function of shear rate for a coacervate prepared from 7.0 g/L poly(A/D48) and 14 mM C12E6 in 0.46 M NaCl.
Figure 7. Relaxation time distributions for the mixture of poly(A/D48) and C12E6 with varying [C12E6] in 0.20 M NaCl at 25 °C. Sample solutions were prepared by adding 10.0 mM C12E6 to 5.0 g/L poly(A/D48); [C12E6] ) 0 (a), 0.98 (b), 1.9 (c), 2.8 (d), and 3.7 mM (e).
Figure 8. Rh as a function of Cp for soluble complexes of poly(A/D48) with C12E6 in 0.40 M NaCl at 25 °C. Sample solutions were prepared by adding 10.0 g/L poly(A/D48) to 10.0 mM C12E6.
plexes as a function of Cp. Rh for the complexes increases gradually from 6.8 to 17 nm with increasing Cp from 0 to 1.30 g/L and then increases markedly to 32 nm with increasing Cp to 1.73 g/L. When Cp was increased up to a certain level (Cp ≈ 1.9 g/L), coacervation occurred and a new peak was observed in a region of much longer relaxation times. The apparent value of Rh for the peak which might be due to droplets of coacervate was calculated to be ca. 1.7 µm. Furthermore, Rh for a mixture of 3.56 g/L poly(A/D48) ([DodMAm unit] ) 7.14 mM) and 7.14 mM C12E6 increased remarkably from 10 to 38 nm with increasing [NaCl] from 0.13 to 0.20 M. At a certain [NaCl] (≈0.26 M), coacervation occurred, and a peak that might be due to droplets of coacervate appeared in a region of much longer relaxation times (Rh ≈ 4.1 µm) (data not shown). These QELS data indicate that as the concentration of either of the three components (i.e., polymer, surfactant, and salt) is increased toward a phase boundary for coacervation, the size of poly(A/D48)-C12E6 soluble complexes increases gradually at first and then abruptly
near the phase boundary, ending up with coacervation at a higher concentration of the component. Steady-Shear Viscosity for the Coacervate. The coacervate observed in the present study was a slightly turbid and highly viscous liquid, the volume of which was less than 10% of the total solution. From absorption spectra of the coacervate observed after diluting with water, it was estimated that the coacervate phase contained more than 90% of the total polymers by weight. The polymer concentration in the coacervate phase corresponds to ca. 5 wt % or higher, although we were unable to estimate the concentration of the surfactant in the coacervate. Thus, we are interested to study rheological properties of this dense fluid of the coacervate. Steady-shear viscosities of the coacervate prepared from 7.0 g/L poly(A/D48) and 14.0 mM C12E6 in 0.46 M NaCl are plotted in Figure 9 as a function of the shear rate. The viscosity of the coacervate at a shear rate lower than 2 s-1 was ca. 3 orders of magnitude higher than that of the supernatant obtained (ca. 7 × 10-3 Pa s). Coacervates may be best understood as having a large network structure of polymer-micelle complexes that are formed via interpolymer associations of polymers with micelles which act as junctions; there must be a sufficiently large number of polymers interacting per micelle. As the shear rate is increased up to ca. 2 s-1, the viscosity decreases gradually, but a remarkable shear-thinning was observed at a shear rate greater than ca. 3 s-1. In the higher shear rate region, an increase in the shear rate may cause a significant fragmentation of the interpolymer junctions by micelles, sheared into polymer-micelle complexes with a smaller size. Mechanism of the Coacervation of Poly(A/D48) and C12E6. A conceptual illustration of a proposed model for coacervation is presented in Figure 10. In the absence of C12E6, poly(A/D48) forms collapsed micellelike aggregates predominantly through intrapolymer hydrophobic associations in aqueous media. When a small amount of C12E6 is added, the polymer interacts with the surfactant to form poly(A/D48)-C12E6 complexes which can be viewed as mixed micelles of the amphiphilic polymer and the surfactant. As a result, the polymer chains adopt a less compact conformation. Since the micelles act as crosslinks between different polymer chains, an increase in [C12E6] leads to an increase in the size of the poly(A/D48)C12E6 complexes. As the number of cross-links is increased beyond a certain level, the size of the complexes increases abruptly to form a large network structure, resulting in coacervation. However, if the surfactant concentration is further increased, the number of polymers bound to one micelle will decrease, and eventually there will be only
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Figure 10. A conceptual illustration for poly(A/D48)-C12E6 coacervation.
one polymer in a micelle and all cross-linking effects disappear (Figure 2). The concentration of added NaCl is important for poly(A/D48)-C12E6 coacervation. As shown in Figure 4, coacervation does not occur at [NaCl] lower than ca. 0.2 M at 25 °C. At lower [NaCl], interpolymer electrostatic repulsion may disturb polymer chains to interact with the same micelle. At [NaCl] > ca. 0.2 M at 25 °C, however, the number of polymers per micelle (i.e., the number of junctions) may become sufficiently large for coacervation to occur. As [NaCl] is further increased, the added NaCl promotes the formation of water structure to enhance hydrophobic interaction, and thus coacervation occurs more easily. In addition, added NaCl may eliminate an entropy loss upon confining polymer chains to the coacervate phase. In a one-phase solution of a polyelectrolyte, counterions are distributed in the whole solution. In a phase-separated state with a polyelectrolyte in one phase, however, the counterions are confined to the polymer phase, leading to a significant entropy loss. At high [NaCl], on the other hand, this entropy contribution is eliminated and hence phase separation will occur without entropy penalty. Consequently, coacervation occurs favorably at sufficiently high concentrations of added NaCl. Temperature is also an important factor to affect coacervation of the poly(A/D48)-C12E6 system. Figure 5 shows that coacervation occurs even at [NaCl] < 0.2 M at higher temperatures, which means that the poly(A/D48)C12E6 coacervation occurs more easily at higher temperatures. These observations may be explained as follows: With increasing temperature, the hydration of ethylene oxide units in C12E6 decreases,26 making C12E6 molecules more hydrophobic. Thus, as temperature is increased up to a certain level, a C12E6 micelle may interact hydrophobically with a number of polymer chains surpassing the electrostatic repulsion between different polymer chains. In addition, with increasing temperature, the size of C12E6 micelles increases.27 When two or more poly(A/ D48) molecules interact with a micelle of a larger size, interpolymer electrostatic repulsion may be smaller. The mixture of poly(A/D48) with C12E6 undergoes coacervation instead of a solid-liquid phase separation (i.e., precipitation). A critical difference between the coacervation and precipitation is the extent of dehydration of the polymer-rich phase; coacervates maintain a sig(26) (a) Tiddy, G. J. Phys. Rep. 1980, 57, 1-46. (b) Mitchell, D. J.; Tiddy, G. J.; Waring, L.; Bostock, T.; McDonald, M. P. J. Chem. Soc., Faraday Trans. 1 1983, 79, 975-1000. (c) Meguro, K.; Ueno, M.; Esumi, K. In Nonionic Surfactants. Physical Chemistry; Schick, M. J., Ed.; Surfactant Science Series Vol. 23; Marcel Dekker: New York, 1987; pp 109-183.
nificant amount of water, while precipitates are highly dehydrated. In the present case, charged polymer chains and ethylene oxide units in poly(A/D48)-C12E6 coacervates should remain sufficiently hydrated. Furthermore, significant osmotic hydration arising from Na+ counterions confined to the coacervate phase makes the coacervate “swell” with water. Therefore, in the case of the poly(A/ D48)-C12E6 system, significant dehydration does not occur upon phase separation, resulting in coacervation. Conclusion Phase behavior for mixtures of poly(A/D48) and C12E6 was studied by turbidimetric titration and cloud point measurements. The phase diagrams obtained by turbidimetric titrations (Figures 3 and 4) indicated that coacervation occurred only when the [C12E6]/[DodMAm unit] ratios were 0.40-2.1, 0.25-2.4, and 0.16 - 3.0 at [NaCl] ) 0.30, 0.40, and 0.60 M, respectively, and that [NaCl] should be higher than ca. 0.2 M at 25 °C for coacervation to occur at any Cp and [C12E6]. In addition, a higher content of the hydrophobe in poly(A/Dx) was favorable for coacervation to occur. To examine the temperature dependence of the phase behavior of mixtures of poly(A/D48), C12E6, and NaCl, the cloud points of a mixture of poly(A/D48) and C12E6 were determined at varying [NaCl] (Figure 5). At [NaCl] < 0.03 M, the cloud point decreased abruptly with increasing [NaCl] because of the shielding effect of electrostatic repulsion between sulfonate moieties in poly(A/D48). At [NaCl] g 0.05 M, the cloud point decreased gradually with increasing [NaCl] presumably because of an enhancement of hydrophobic interaction caused by added salt in addition to the electrostatic shielding effect. At higher [NaCl] (greater than ca. 0.5 M), the enhancement of hydrophobic interaction might be a dominant effect. Soluble complexes of poly(A/Dx) with C12E6 were characterized by intra- and interpolymer NRET and QELS techniques. From the intra- and interpolymer NRET data (Figure 6), we concluded as follows: When a small amount of C12E6 was added, the polymers were bound to C12E6 micelles to form interpolymer complexes, in which the micelle acted as a cross-linking junction of different (27) (a) van Os, N. M.; Haak, J. R.; Rupert, L. A. M. Physico-Chemical Properties of Selected Anionic, Cationic and Nonionic Surfactants; Elsevier: Amsterdam, 1993. (b) Balmbra, R. R.; Clunie, J. S.; Corkill, J. M.; Goodman, J. Trans. Faraday Soc. 1962, 58, 1661-1167. (c) Corti, M.; Degiorgio, V. J. Phys. Chem. 1981, 85, 1442-1445. (d) Cebula, D. J.; Ottewill, R. H. Colloid Polym. Sci. 1982, 260, 1118-1120. (e) Brown, W.; Johnsen, R.; Stilbs, P.; Lindman, B. J. Phys. Chem. 1983, 87, 45484553. (f) Glatter, O.; Fritz, G.; Lindner, H.; Brunner-Popela, J.; Mittelbach, R.; Strey, R.; Egelhaaf, S. U. Langmuir 2000, 16, 86928701.
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polymer chains. As [C12E6] was increased, the number of interpolymer complexes increased. However, as [C12E6] was further increased beyond a certain level, the number of polymer chains bound per micelle decreased, and the number of interpolymer complexes decreased. QELS data (Figures 7 and 8) indicated that as the concentration of either of the three components (i.e., polymer, surfactant, and salt) was increased toward a phase boundary for coacervation, the size of poly(A/D48)-C12E6 soluble complexes increased gradually at first and then abruptly near the phase boundary, ending up with coacervation at a
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higher concentration of the component. On the basis of these experimental data, we discussed a possible mechanism for the coacervation of poly(A/D48) with C12E6. Acknowledgment. This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists No. 12750800 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. LA020493G
