Precipitation of a Water-Soluble ABC Triblock Methacrylic

with time, pH, polymer concentration, salt type and concentration, and presence of a protein .... were prepared by diluting 100 times a 1% stock solut...
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Langmuir 1999, 15, 1613-1620

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Precipitation of a Water-Soluble ABC Triblock Methacrylic Polyampholyte: Effects of Time, pH, Polymer Concentration, Salt Type and Concentration, and Presence of a Protein Costas S. Patrickios,*,† Leo R. Sharma, Steven P. Armes, and Norman C. Billingham School of Chemistry, Physics, and Environmental Sciences, University of Sussex, Falmer, Brighton, East Sussex BN1 9QJ, U.K. Received June 20, 1997. In Final Form: October 6, 1998 The solution behavior of a low molecular weight ABC triblock methacrylic polyampholyte with the structure (dimethylaminoethyl methacrylate)8-(methyl methacrylate)12-(methacrylic acid)16 was investigated by turbidimetry. The variation of the optical density at 420 nm of dilute polyampholyte solutions with time, pH, polymer concentration, salt type and concentration, and presence of a protein was explored. Polyampholyte precipitation was fast and occurred around the isoelectric pH. The size of the aggregates increased with increasing salt concentration and was independent of polyampholyte concentration. High salt concentrations suppressed polyampholyte precipitation. The precipitation in the presence of various electrolytes at a concentration of 0.2 M was insensitive to the anion type for a series of potassium halides and also to the divalent sulfate anion, but was greatly increased by the divalent calcium cation and was completely suppressed by the anionic surfactant sodium dodecyl sulfate. The polyampholyte formed insoluble complexes with a basic protein, chicken egg lysozyme, in the pH region between the isoelectric points of the two reactants. The results of this study will be useful for designing the extraction of solutes, such as heavy metals and proteins, via polyampholyte electrostatic complexation; moreover, they will facilitate the development of efficient strategies for polyampholyte recovery and recycling.

Introduction Polyampholytic colloidal particles carry chemical groups which can be positively and negatively charged. In nature, polyampholytes are encountered in both the inorganic and biological fields. Silver iodide sols and metal oxides, such as aluminum oxide, are two families of polyampholytic inorganic materials.1 Proteins and nucleic acids are the two types of ampholytic biomacromolecules.2 Polyampholytes have also been made synthetically, one example being polymeric latex3 and the other example being random linear polymers.4 The latter type of synthetic polyampholytes was studied extensively during the 1950s and 1960s.5 Recent work on random polyampholytes has been conducted by the groups of Candau6-11 and McCormick.12-15 Hahn et al.16-20 prepared and character* Author to whom correspondence should be addressed. † Present address: Department of Natural Sciences, School of Pure and Applied Sciences, University of Cyprus, P.O. Box 537, 1678 Nicosia, Cyprus. (1) Hunter, R. J. Foundations of Colloid Science; Oxford University Press: New York 1986; Vol. 1 pp 170, 225, 438, 598. (2) Lehninger, A. L. Principles of Biochemistry; Worth Publishers: New York, 1988; pp 104-107. (3) Healy, T. W.; Homola, A.; James, R. O.; Hunter, R. J. Faraday Discuss. 1978, 65, 156-163. (4) Alfrey, T.; Morawetz, H.; Fitzgerald, E. B.; Fuoss, R. M. J. Am. Chem. Soc. 1950, 72, 1864-1865. (5) Katchalsky, A.; Miller, I. R. J. Polym. Sci. 1954, 14, 57-68. (6) Corpart, J.-M.; Selb, J.; Candau, F. Polymer 1993, 34, 38733886. (7) Corpart, J.-M.; Candau, F. Macromolecules 1993, 26, 1333-1343. (8) Skouri, M.; Munch, J. P.; Candau, S. J.; Neyret, S.; Candau, F. Macromolecules 1994, 27, 69-76. (9) Neyret, S.; Ouali, L.; Candau, F.; Pefferkorn, E. J. Colloid Interface Sci. 1995, 176, 86-94. (10) Neyret, S.; Candau, F.; Selb, J. Acta Polym. 1996, 47, 323-332. (11) Ohlemacher, A.; Candau, F.; Munch, J. P.; Candau, S. J. J. Polym. Sci., Part B: Polym. Phys. 1996, 34, 2747-2757. (12) McCormick, C. L.; Johnson, C. B. Macromolecules 1988, 21, 686.

ized alternating polyampholytes. The synthesis and characterization of diblock polyampholytes was not accomplished until the 1970s.21-23 As an extension of the work on diblock polyampholytes, we recently synthesized24,25 amphiphilic ABC triblock polyampholytes using group transfer polymerization (GTP).26-28 The new feature in these ABC triblock copolymers is the presence of a neutral hydrophobic block in addition to the presence of the two oppositely charged blocks. Experimental techniques such as light scattering,24,29 aqueous gel permeation chromatography25,30 and (13) McCormick, C. L.; Johnson, C. B. Macromolecules 1988, 21, 694. (14) McCormick, C. L.; Salazar, L. C. Macromolecules 1992, 25, 1896. (15) Mumick, P. S.; Welch, P. M.; Salazar, L. C.; McCormick, C. L. Macromolecules 1994, 27, 323-331. (16) Hahn, M.; Ko¨tz, J.; Linow, K.-J.; Philipp, B. Acta Polym. 1989, 40, 36-43. (17) Hahn, M.; Ko¨tz, J.; Ebert, A.; Schmolke, R.; Philipp, B.; Kudaibergenov, S.; Sigitov, V.; Bekturov, E. A. Acta Polym. 1989, 40, 331-335. (18) Ko¨tz, J.; Hahn, M.; Philipp, B. Acta Polym. 1989, 40, 401-404. (19) Ko¨tz, J.; Hahn, M.; Philipp, B.; Kudaibergenov, S.; Sigitov, V.; Bekturov, E. A. Acta Polym. 1989, 40, 405-408. (20) Hahn, M.; Jaeger, W.; Schmolke, R.; Behnisch, J. Acta Polym. 1990, 41, 107-112. (21) Kamachi, M.; Kurihara, M.; Stille, J. K. Macromolecules 1972, 5, 161-167. (22) Kurihara, M.; Kamachi, M.; Stille, J. K. J. Polym. Sci., Polym. Chem. Ed. 1973, 11, 587-610. (23) Varoqui, R.; Tran, Q.; Pefferkorn, E. Macromolecules 1979, 12, 831-835. (24) Patrickios, C. S.; Hertler, W. R.; Abbott, N. L.; Hatton, T. A. Macromolecules 1994, 27, 930-937; 2364. (25) Patrickios, C. S.; Lowe, A. B.; Armes, S. P.; Billingham, N. C. J Polym. Sci., Part A: Polym. Chem. 1998, 36, 617-631. (26) Webster, O. W.; Hertler, W. R.; Sogah, D. Y.; Farnham, W. B.; RajanBabu, T. V. J. Am. Chem. Soc. 1983, 105, 5706-5708. (27) Sogah, D. Y.; Hertler, W. R.; Webster, O. W.; Cohen, G. M. Macromolecules 1987, 20, 1473-1488. (28) Dicker, I. B.; Cohen, G. M.; Farnham, W. B.; Hertler, W. R.; Laganis, E. D.; Sogah, D. Y. Macromolecules 1990, 23, 4034-4041.

10.1021/la970662a CCC: $18.00 © 1999 American Chemical Society Published on Web 02/11/1999

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Figure 1. Chemical formulas of the units of the triblock polyampholyte. The positive charge comes from the protonated 2-(dimethylamino)ethyl methacrylate (DMAEMA) units, and the negative charge comes from the deprotonated methacrylic acid (MAA) units. The neutral methyl methacrylate (MMA) units make the polymer more hydrophobic.

fluorescence spectroscopy24,29 have indicated that the extra, hydrophobic block leads to aqueous solution micellization. It has also been determined that micellization is disrupted24 at both pH extremes because of excessive charge accumulation, in agreement with the results of a simple thermodynamic model on block copolymer association.31 These polyampholytes precipitated in dilute32-34 and concentrated24,35 solutions in the absence24,32-35 and presence32-34 of proteins in a pHdependent fashion. Moreover, they showed a high affinity for adsorption onto anion-exchange columns,36 making them appropriate displacers for protein purification by anion-exchange displacement chromatography.37 While our previous work covered the characterization of various polyampholytes, in this investigation, we focus on one polymer and study in detail the effect of different parameters on precipitation in dilute solution. These parameters are time, pH, polymer concentration, salt type and concentration, and presence of a protein. Experimental Section Materials. The ABC triblock polyampholyte was synthesized by GTP26-28 via sequential monomer addition as reported earlier.24,25 The three types of units in the polymer, shown in Figure 1, are 2-(dimethylamino)ethyl methacrylate (DMAEMA), methyl methacrylate (MMA), and methacrylic acid (MAA). DMAEMA is positively charged at pH lower than 8, MMA is neutral and hydrophobic, and MAA is negatively charged at pH higher than 5. Polyampholyte latex based on styrene, MAA, and DMAEMA was prepared following the procedures of Homola and (29) Chen, W.-Y.; Alexandridis, P.; Su, C.-K.; Patrickios, C. S.; Hertler, W. R.; Hatton, T. A. Macromolecules 1995, 28, 8604-8611. (30) Patrickios, C. S.; Strittmatter, J. A.; Hertler, W. R.; Hatton, T. A. J. Colloid Interface Sci. 1996, 182, 326-329. (31) Patrickios, C. S. J. Phys. Chem. 1995, 99 (48), 17437-17441. (32) Patrickios, C. S.; Jang, C. J.; Hertler, W. R.; Hatton, T. A. In Macro-ion Characterization. From Dilute Solutions to Complex Fluids, Schmitz, K. S., Ed.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994; Vol. 548, pp 257-267. (33) Patrickios, C. S.; Hertler, W. R.; Hatton, T. A. Biotechnol. Bioeng. 1994, 44 (9), 1031-1039. (34) Nath, S.; Patrickios, C. S.; Hatton, T. A. Biotechnol. Prog. 1995, 11 (1), 99-103. (35) Patrickios, C. S.; Hertler, W. R.; Hatton, T. A. Fluid Phase Equilib. 1995, 108, 243-254. (36) Patrickios, C. S.; Gadam, S. D.; Cramer, S. M.; Hertler, W. R.; Hatton, T. A. In Macro-ion Characterization. From Dilute Solutions to Complex Fluids, Schmitz, K. S., Ed.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994; Vol. 548, pp 144-153. (37) Patrickios, C. S.; Gadam, S. D.; Cramer, S. M.; Hertler, W. R.; Hatton, T. A. Biotechnol. Prog. 1995, 11 (1), 33-38.

Patrickios et al. James38 and Harding and Healy.39 The weight and number average diameters of the latex were determined to be 433 ( 78 nm and 391 ( 72 nm, respectively. The sodium hydroxide and hydrochloric acid standard solutions, inorganic salts, citric acid, boric acid, and chicken egg lysozyme were purchased from Sigma-Aldrich, U.K. Methods. Preparation of Solutions. Most of the experiments were carried out in 0.01 M citric acid/phosphate buffers, except for the experiments with lysozyme in which a 0.01 M citric acid/ phosphoric acid/boric acid buffer was used to cover a higher pH range. The former was prepared by mixing 0.1 M solutions of citric acid and dipotassium phosphate in a 2:1 volume ratio and diluting 10 times with distilled water in a volumetric flask to give a solution with pH 3.3. In the experiments where the salt type and concentration were varied, the appropriate amount of solid salt was added to the buffer before the final dilution. The latter was prepared in a similar way by mixing 0.1 M solutions of citric acid, phosphoric acid, and boric acid in a volume ratio 1:1:2 and diluting 10 times, resulting in a solution of pH 2.7. Fresh polyampholyte solutions of concentration typically 0.01% were prepared by diluting 100 times a 1% stock solution in water with the 0.01 M buffer solution. Polyampholyte solution (2 mL) was transferred to a disposable cuvette which also served as the precipitation reactor. Typically, 15 cuvettes were used in a single set of experiments which covered a pH range from 3 to 9. pH Adjustments. The pH of the solutions in the cuvettes was adjusted by the addition of 33 µL of KOH or HCl of the appropriate concentration. Each cuvette was then inverted twice to ensure complete mixing. Optical Density. The optical density was measured at 420 nm on a double-beam UV-vis Lambda 2S Perkin-Elmer spectrophotometer. The intermediate wavelength of 420 nm was chosen because it was sufficiently low to provide enough energy for reasonable scattering and because it was relatively high to avoid saturation of the spectrophotometer. Two disposable cuvettes filled with buffer were used as the reference and blank cuvettes for the measurements. Dynamic Light Scattering. The hydrodynamic size of the particles was measured by dynamic light scattering using a photon correlation spectrometer (PCS, Malvern 7032 series) equipped with a helium-neon laser operating at a wavelength of 633 nm. Kinetic Measurements. After the pH adjustment and mixing, the optical density at 420 nm was measured at approximately 10 s intervals using the Lambda 2S spectrometer. In a separate experiment, the hydrodynamic size was determined at 130 s intervals using the PCS instrument. Steady-State Measurements. The optical density of the solutions at 420 nm was measured 30 min after the pH adjustment and mixing. We had previously established in the kinetic experiments that the value of the optical density levels off before 30 min. In the steady-state experiments, the pH typically covered the range between 3 and 9, the polymer concentration was varied between 0.002% and 0.02%, and the salt concentration ranged from 0.0 to 1.0 M. Moreover, the effect of the following additives at a concentration of 0.2 M was investigated: potassium chloride, potassium bromide, potassium iodide, potassium sulfate, calcium chloride, and sodium dodecyl sulfate. pH Measurements. The pH of the solutions in the cuvettes was measured after the spectroscopic measurement using a Corning PS30 portable pH meter. The same instrument was used for the hydrogen ion titration of 5 mL of a 1% polyampholyte solution, where the pH was varied from 2 to 12 by the dropwise addition of 0.5 M KOH standard solution. Protein Experiments. Chicken egg lysozyme (0.025 g) was dissolved in 25 g of the citric acid/phosphoric acid/boric acid (PCB) buffer, and the resulting 0.1% solution was mixed with an equal volume of a 0.02% polyampholyte solution, resulting in a mixture with protein and polyampholyte concentrations of 0.05% and 0.01%, respectively. After measuring the optical density at 420 nm 30 min after the pH adjustment, the solutions were (38) Homola, A.; James, R. O. J. Colloid Interface Sci. 1977, 59, 123134. (39) Harding, I. H.; Healy, T. W. J. Colloid Interface Sci. 1982, 89, 185-201.

ABC Triblock Methacrylic Polyampholyte

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Figure 2. Experimental and theoretical hydrogen ion titration curves of the polyampholyte carrying 16 acidic groups with pKa ) 5.4 and 8 basic groups with pKb ) 8.0. centrifuged for 15 min at 13 000 rpm. A two-phase system composed of an optically clear supernatant and a solid precipitate was formed. The supernatant was then transferred to a quartz cuvette and analyzed for protein by measuring the absorbance at 280 nm to calculate the amount of dissolved (not precipitated) protein present. A linear calibration curve had been constructed to convert the absorbance at 280 nm to protein concentration and hence calculate the amount of dissolved protein.

Results and Discussion There is a fair amount of work in the literature on amphoteric latex precipitation.3 We have also contributed some studies on block polyampholyte precipitation.24,32-35 Polyampholyte precipitation is dictated by the balance between the attractive and repulsive forces between the particles or chains. The van der Waals and hydrophobic forces are the attractive forces and favor precipitation. These forces are not pH-dependent. In contrast, electrostatic repulsive forces (due to the net charge of the polyampholytesimportant far from the isoelectric pH), which oppose precipitation, are pH-dependent because the charge of the weakly basic and weakly acidic groups of the polyampholyte varies with the pH. To the attractive forces can be added the electrostatic attraction between the oppositely charged blocks (most important near the isoelectric pH where the positive and negative charges balance), which is also pH-dependent. Precipitation initiates at a critical pH where the attractive forces have just overcome the electrostatic repulsion. It must be noted that precipitation is not simply a thermodynamic balance of forces but also has a kinetic component: differentiation between precipitation and nonprecipitation also depends on the time frame of the observation. In principle, every solution should precipitate if sufficient time is allowed. For the purposes of this study, the time frame of observation is set within 30 min. Hydrogen Ion Titration. As will be presented later, pH has a dramatic effect on the precipitation of the polyampholyte. This is due to the composition of the polyampholyte of weakly basic and weakly acidic groups whose charge varies with pH. Thus, the pH dependence of the polyampholyte charge will be invaluable for explaining the precipitation results. Figure 2 presents the experimental and theoretical hydrogen ion titration curves of the polyampholyte. The experimental curve was obtained by titrating 5 mL of a 1% polyampholyte solution in distilled water (no added salt) from pH 2 to 12 using a standard solution of KOH, 0.5 M. The net charge was calculated from the experimental points by assuming a polyampholyte isoelectric point of 5.4. The isoelectric point of 5.4 was consistent with a theoretical calculation from

Figure 3. Time dependencies of optical density at 420 nm determined by turbidimetry, and aggregate size determined by dynamic light scattering of a 0.01% solution of the triblock polyampholyte at pH 6.0: (a) linear plot and (b) doublelogarithmic plot.

the copolymer composition40 and also coincided with the midpoint of the pH region of precipitation in the experiments without added salt (see below). The theoretical titration curve was based on the polyampholyte composition of 16 acidic and 8 basic groups and used simple dissociation equilibria without consideration of intramolecular electrostatic interactions. To take into account these intramolecular interactions in a simplified manner, effective values of the dissociation constants (pK) were used as calculated in a previous study.41 Thus, the pK of the MAA unit was taken as 5.4 (compared to the monomer value of 4.6), and that of DMAEMA unit as 8.0 (compared to the monomer value of 8.6). There is good agreement between the experimental and theoretical titration curves. The greatest discrepancy is observed in the low-pH region (pH < 5) and can be attributed to the strong electrostatic interactions within the negatively charged acidic block (which is the major component). The electrostatic attractive interactions between blocks can also affect the titration, but probably to a lesser degree than the intrablock interaction: the latter is of shorter range and stronger, and should be more important in determining the ease by which a proton is captured or released by the polymer. The sigmoidal portion (more visible in the theoretical curve) at low pH (pH < 6.5) is due to the titration of the acidic units, and the sigmoidal portion at high pH (pH > 6.5) is due to the titration of the basic units. The polyampholyte has a net negative charge above the isoelectric pH of 5.4 and a net positive charge below the isoelectric pH. Effect of Time. Figure 3a shows the time dependencies of the optical density at 420 nm and aggregate size of a 0.01% polyampholyte solution at pH 6.0 without added salt as determined by spectrophotometry and dynamic (40) Patrickios, C. S. J. Colloid Interface Sci. 1995, 175, 256-260. (41) Merle, Y. J. Phys. Chem. 1987, 91, 3092-3098.

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Figure 4. Effect of KCl concentration on the kinetics of precipitation of 0.01% solutions of the triblock polyampholyte at pH 5.0 as followed by turbidity at 420 nm: (a) linear plot and (b) double logarithmic plot.

light scattering, respectively. Both the solution turbidity and aggregate size increased rapidly at short times and more slowly at later times, indicating that interparticle association is nearing completion. The similar trends in these two quantities indicate that the turbidity is a good measure of aggregate size. This is consistent with the known linear dependence of turbidity on the crosssectional area of the scattering particles.42 It should be noted, however, that the particle size seems to increase more slowly than the turbidity. For optically nonabsorbing particles, the optical density is proportional to the particle size raised to some power n which is usually 6.33 By examining the power-law correlation of the particle size and the optical density, we can examine whether the aggregation mechanism is diffusion-limited or reaction-limited. While for diffusionlimited aggregation, the logarithm of the particle size increases linearly with the logarithm of time, for reactionlimited aggregation, the logarithm of the particle size increases linearly with time.43 The linearity in the doublelogarithmic plot in Figure 3b implies that the mechanism of aggregation is diffusion-limited rather than reactionlimited. This means that most of the collisions were effective under the experimental conditions and that the combination of polyampholyte particles was mainly due to these sticking encounters as described by the Smolukowski theory.44 Figure 4a shows the effect of potassium chloride concentration on the kinetics of precipitation of 0.01% solutions of the triblock polyampholyte at pH 5.0 as followed by optical density. At low salt concentrations, 0.0 and 0.2 M KCl, there was a rapid increase in the optical (42) Hiemenz, P. C. Principles of Colloid and Surface Chemistry, 2nd ed.; Marcel Dekker: New York; 1986; pp 271, 273. (43) Lin, M. Y.; Lindsay, H. M.; Weitz, D. A.; Ball, R. C.; Klein, R.; Meakin, P. Nature 1989, 339, 360-362. (44) Probstein, R. F. Physicochemical Hydrodynamics; Butterworths: Boston, 1989; p 236.

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density in the first 2 min of the experiment which leveled off after 10 min, similar to the experiment of Figure 3 at pH 6.0. At 1.0 M KCl, the optical density did not increase in the first minute, after which it increased steadily and leveled off after 30 min. The presence of this lag time, which suggests polymer reorganization (possibly compression of initial aggregates: notice that the initial optical density is not zero) before aggregation, does not allow comparison of the initial slopes of aggregation for the different electrolyte concentrations. DLVO theory dictates that the initial rate of aggregation is higher at higher electrolyte concentrations, and this has been verified experimentally with silver halide sols in the presence of barium nitrate.45 The final value of the optical density at high salt concentration is higher than that at low salt concentration, indicating larger aggregate size in the former case. This is consistent with a greater driving force for aggregation, in agreement with the DLVO theory. Figure 4b replots the data in Figure 4a in double logarithmic axes, illustrating more clearly the lag time in the high salt concentration solution. The curves at 0.0 and 0.2 M KCl are linear in the double-logarithmic plot, as in Figure 3b. Moreover, the portion of the curve at 1.0 M KCl after the lag time is also linear. These observations again suggest a diffusion-limited aggregation mechanism. We also examined the effect of potassium chloride concentration on the kinetics of precipitation of polyampholyte styrene latex carrying MAA and DMAEMA ionic groups. Latex (0.005%) solutions at 0.0 and 1.0 M KCl were studied at pH 5.5 and revealed a trend similar to that observed with the triblock polyampholyte. There was a 1 min lag time in the onset of precipitation of the high salt concentration latex solution, and there was good linearity between the optical density and time in the double-logarithmic plot. One difference with the latex precipitation was that the optical density in both the low and high salt concentration solutions started to decrease after 10 min, probably due to sedimentation. This can be attributed to the larger size of latex particles compared with the precipitating polyampholyte chains. The existence of a 1 min lag time in the precipitation of the 1.0 M KCl solutions of both the triblock polyampholyte and the polyampholyte styrene latex suggests that this lag time may not be related to the micellar nature of the triblock polyampholyte, but it is in general a characteristic of organic polymeric ampholytes as opposed to inorganic crystals. Effect of Polyampholyte Concentration. The effect of varying the copolymer concentration on the optical density in the pH range 3-7 was investigated and compared to that of amphoteric latex. Using these data, a phase diagram was constructed to examine the stability of the polyampholyte solution as a function of pH. Another diagram was also produced in which the optical density was plotted against polymer concentration at selected values of pH. Figure 5a shows the effect of copolymer concentration on turbidity without added salt 30 min after the pH adjustment. Each curve has a trapezoidal shape, consisting of a plateau and two rather steep edges. The height of the trapezium depends on the copolymer concentration. The pH values corresponding to the edges of the trapezium are the critical pHs at which the transition from complete dissolution to precipitation occurs. The lower critical pH of 3.8 will be referred to as the acidic critical pH. The higher critical pH is 6.8 and will be referred to as the (45) Reerink, H.; Overbeek, J. Th. G. Discuss. Faraday Soc. 1954, 18, 74-84.

ABC Triblock Methacrylic Polyampholyte

Figure 5. Effect of triblock polyampholyte concentration on the precipitation of its solutions without added salt as a function of pH as followed by the optical density at 420 nm, 30 min after the pH adjustment: (a) original data, (b) phase diagram, and (c) turbidity as a function of polymer concentration at three different pH values.

basic critical pH. The acidic and basic critical pHs are equally spaced from the polyampholyte isoelectric point of 5.4. The shape of each curve is not perfectly trapezoidal, and it exhibits two maxima and one minimum. The two maxima occur 1 pH unit on either side of the isoelectric point. In the acidic region, there is an excess of positive charges, and similarly, in the basic region, there is an excess of negative charges. The presence of charge in colloidal particles creates an energy barrier to coagulation. Thus, the optical density would be expected to increase as we approach the isoelectric point where the energy barrier to coagulation is reduced because the net charge approaches zero. The trend in Figure 5a, however, is exactly the opposite, with a local minimum in the optical density at the isoelectric point within the range 5.3-5.5. A possible explanation for this observation is that the aggregates at the two maxima are larger than the neutral aggregates at the isoelectric point because of Coulombic repulsion. As the excess charge is very small (about 2.5 according to the experimental hydrogen ion titration curve of Figure 2), the electrostatic repulsion is too weak to inhibit aggregation but sufficient to increase the spacing of the components of an aggregate compared with the situation at the isoelectric point. The observation of the

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two maxima is not unique to the particular polyampholyte under study; another polyampholyte with composition (DMAEMA)12-(MMA)12-(MAA)12 presented the same distinctive curve centered at its isoelectric point of 6.6.33 As it will be shown later, the same two maxima and one minimum also appear in the turbidity-pH profile of the amphoteric latex. The data shown in Figure 5a can be used to construct Figure 5b,c. Figure 5b is a phase diagram of the polyampholyte in the pH-polymer concentration space, without added salt. The broken line is drawn at the polyampholyte isoelectric point of 5.4. The filled circles in Figure 5b are drawn at the pHs of nonzero optical density in Figure 5a, whereas the open circles indicate optically clear solutions. The transition from complete solubility to polyampholyte precipitation occurs at the same critical pHs, irrespective of polymer concentration. This arises from the constancy of the acidic and basic critical pHs observed in Figure 5a. Thus, polymer concentration does not affect polyampholyte stability, which is understandable considering that polymer concentration cannot affect the charge of the polyampholyte. It is noteworthy that the precipitation pH range of the same copolymer at higher concentrations, 1%37 and 5%,35 without added salt, was between 4 and 6.5, very similar to the range observed in the dilute solutions studied here. Figure 5c shows the effect of polyampholyte concentration on optical density 30 min after the adjustment at pH 4.6, 5.4, and 6.2. The turbidity of the polyampholyte increases linearly with polymer concentration. Considering the linear dependence of turbidity on aggregate concentration and on aggregate cross-sectional area,42 this trend suggests that at higher polymer concentrations more aggregates of the same (average) size are formed. We also performed steady-state (turbidity was recorded 30 minutes after pH adjustment) precipitation experiments with amphoteric latex at two different latex concentrations, 0.0005% and 0.005%, without added salt. The pH dependence of the optical density of the latex solutions at both concentrations was again trapezoidal, similar to the triblock polyampholyte precipitation profile. The optical density of the higher concentration latex was 10 times greater than that of the lower concentration latex, consistent with a linear optical density-concentration law. The higher concentration latex solutions (0.005%) precipitated around the isoelectric point of 5.3 (between 4 and 7), but the lower concentration solutions (0.0005%) precipitated at a somewhat lower pH range centered at pH 5.0. The details of the precipitation profile of the higher concentration solution were very similar to those of the triblock polyampholyte, having two maxima and one minimum. No maxima and minima were detected in the precipitation profile of the lower concentration solutions, probably because of the very low values of the optical density, which was around 0.015. This is similar to the precipitation profile of the lowest concentration (0.002%) triblock polyampholyte solution in Figure 5a whose optical density was around 0.020 and no maxima or minima were visible. These similarities in the pH dependence of the precipitation of solutions of the triblock polyampholyte and the amphoteric polystyrene latex again suggest that no extra features are introduced by the micellar nature of the triblock polyampholyte material. Effect of Salt Concentration. The effect of varying the salt concentration between 0.0 and 1.0 M KCl on the optical density of 0.01% triblock polyampholyte solutions in the pH range 3-7 was investigated and compared to that of the amphoteric latex. These data were used again

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Figure 6. Effect of KCl concentration on the precipitation of 0.01% solutions of the triblock polyampholyte as a function of pH as followed by the optical density at 420 nm, 30 min after the pH adjustment: (a) Original data, (b) phase diagram, and (c) turbidity as a function of the salt concentration at pH 5.2.

to construct the phase diagram as well as a plot of optical density versus salt concentration at a selected value of pH. Figure 6a shows the effect of KCl concentration on the turbidity of 0.01% triblock polyampholyte solutions 30 min after the pH adjustment. Again, the general shape of the curves is trapezoidal, with the exception of the 1.0 M KCl curve, which is very narrow and triangular. An increase in the salt concentration reduces the pH of the precipitation region. The acidic critical pHs lie in the range 3.5-4.5, and the basic critical pHs lie in the range 5.5-6.5. Figure 6b is a phase diagram constructed from the turbidity data in Figure 6a. Similar to Figure 5b, filled circles indicate conditions of precipitation, and open circles show polyampholyte solubility. As the salt concentration increases, the values of the acidic critical pHs increase, and the values of the basic critical pHs decrease, leading to a narrowing of the precipitation region around the isoelectric point. Corpart and Candau7 also found that a high salt concentration promotes the dissolution of their high molecular weight random polyampholytes based on strongly acidic and strongly basic groups. In contrast, Healy et al.3 observed the opposite trend in amphoteric latex precipitation. These workers found that as the cesium

Patrickios et al.

nitrate concentration increased, the values of the acidic critical pHs decreased and the values of the basic critical pHs increased, leading to a broadening of the precipitation region with salt concentration. This difference in phase behavior with increasing salt concentration can be attributed to the low content of ionic groups and the high content of hydrophobic styrene groups on the latex surface which suppress the salting-in effect and accentuate the salting-out effect. It is noteworthy that the pH range of precipitation at higher salt concentrations shifts to below the isoelectric point of 5.4. This can be attributed to preferential anion (chloride, in this case) binding relative to cation (potassium) binding, which can lead to an effective lowering of the isoelectric point. This binding has also been observed in proteins and can shift the isoelectric point by as much as 4 pH units.46 Healy et al.3 observed a similar shift in the precipitation region of their amphoteric latex to a lower pH range at high potassium nitrate concentrations (>0.5 M) but attributed this to a specific cation hydration effect. As mentioned in the previous paragraph, the same authors3 found that the cesium nitrate concentration had a different effect on amphoteric latex precipitation: it caused a symmetrical broadening in the pH range of precipitation at high cesium nitrate concentrations (>0.1 M). Because the polyampholyte concentration does not affect the value of the polyampholyte charge, the pHsalt concentration phase diagram of the triblock polyampholyte is expected to be polyampholyte concentration independent. Indeed, the pH-KCl concentration phase diagrams at polyampholyte concentrations of 1%37 and 5%35 were very similar to that observed in the present dilute solution study. Figure 6c, also constructed from the data in Figure 6a, illustrates the effect of KCl concentration on optical density 30 min after the pH adjustment of 0.01% solutions of the triblock polyampholyte to pH 5.2. This pH was chosen because the value of the optical density remains high, even in the 1.0 M KCl polyampholyte solutions. Figure 6c shows that, at low KCl concentrations (0.0, 0.1, and 0.2 M), the optical density increases linearly with salt concentration, suggesting an increase in aggregate size. At intermediate and high salt concentrations (0.3-1.0 M), the optical density levels off, indicating that the aggregate size becomes constant. The increase in aggregate size with salt concentration can be viewed as an expansion of the aggregates when a higher salt concentration is present. This is consistent with the increase in polyampholyte solubility with salt concentration24 already observed in the phase diagram in Figure 6b, which is collectively called the “salting-in” effect. Two molecular interpretations can be given to the salting-in effect. First, the salt ions promote the hydration of the charged groups of the polymer, leading to a reduction in the activity coefficient of the polymer which, in turn, allows more copolymer to dissolve.47 Second, the salt screens the electrostatic attraction between the positively and the negatively charged units of the polyampholyte,24 thus hindering coagulation and encouraging dissolution: this mechanism is referred to as the “anti-polyelectrolyte” effect. The pH dependence of the precipitation of 0.005% solution of amphoteric latex was investigated at 0.0 and 1.0 M KCl. In the absence of salt, the latex precipitated between pH 4 and 7, as outlined earlier. In the presence (46) Velick, S. F. J. Phys. Colloid Chem. 1949, 53, 135. (47) Tanford, C. Physical Chemistry of Macromolecules; Wiley: New York, 1961; p 466.

ABC Triblock Methacrylic Polyampholyte

Figure 7. Effect of salt type at 0.2 M salt concentration on the precipitation of 0.01% solutions of the triblock polyampholyte without added salt as a function of pH as followed by the optical density at 420 nm, 30 min after the pH adjustment.

of 1.0 M KCl, the basic critical pH was lowered by 1.5 units to 5.5; the acidic critical pH was lowered too, but its exact value could not be determined as latex acidic redissolution did not occur even at pH 3, which was the lowest pH examined. This is the same behavior as that observed by Healy et al.3 for amphoteric latex in the presence of potassium nitrate. Effect of Salt Type. The effect of salt type on polyampholyte precipitation was investigated using a series of potassium halides as well as potassium sulfate, calcium chloride, and an anionic surfactant, sodium dodecyl sulfate (SDS). Figure 7 shows the effect of salt type at a 0.2 M salt concentration on the pH dependence of the optical density 30 min after the pH adjustment of 0.01% solutions of the triblock polyampholyte. The general shape of the curves is again trapezoidal with two exceptions, SDS and calcium chloride. The similarity in the shape of the turbidity profiles for all the monovalent potassium salts indicates that changing the anion type does not affect polyampholyte precipitation, and so there is no anion size or hydration effect at this salt concentration. The divalent sulfate anion was also found not to affect polyampholyte precipitation. The absence of specific monovalent anion effects observed here must be contrasted with the specific monovalent cation effects identified by Healy and co-workers3 for amphoteric latex dispersions. Corpart and Candau7 observed specific cation and anion effects in the precipitation of their high molecular weight random polyampholytes based on strongly acidic and strongly basic groups at salt concentrations above 0.3 M. The divalent calcium cation caused a large increase in the turbidity of the triblock polyampholyte solution at pH values above the isoelectric pH of 5.4. Because of its divalency, calcium can act as an ionic cross-link between polymer chains. The positively charged calcium ion attracts the negatively charged polyampholyte molecules above the isoelectric point. This hypothesis is supported by the observation that below pH 5.4 the calcium curve is the same as that of the potassium halides. This may imply that the polyampholyte can be used for the complexation and extraction of heavy multivalent metallic cations at a pH above the isoelectric point. The insoluble polyampholyte-heavy metal precipitate could be recovered by filtration and redissolved at a low pH (e.g. pH 3); the polyampholyte can then be recovered by selective precipitation near the isoelectric pH (e.g. at pH 4). In contrast to the divalent calcium behavior, the divalent sulfate does not interact with the polymer at pH