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Effects of Anions on the Aggregation of Charged Microgels Yi Hou,† Changqian Yu,† Guangming Liu,† To Ngai,‡ and Guangzhao Zhang*,† Hefei National Laboratory for Physical Sciences at Microscale, Department of Chemical Physics, UniVersity of Science and Technology of China, Hefei, China, and Department of Chemistry, The Chinese UniVersity of Hong Kong, Shatin, N.T., Hong Kong, China ReceiVed: December 25, 2009; ReVised Manuscript ReceiVed: February 6, 2010
In the present study, we have prepared poly(N-isopropylacrylamide-co-vinylbenzyl trimethylammonium chloride) (NIPAM-co-VT) and poly(N-isopropylacrylamide-co-sodium acrylate) (NIPAM-co-SA) thermally sensitive microgels, which are positively and negatively charged, respectively. By use of laser light scattering (LLS), we have investigated the temperature-induced aggregation of microgels in the presence of salts with the same cation but different anions. The studies demonstrate that neither kosmotropic nor chaotropic anions can lead to the aggregation of NIPAM-co-VT microgels. No obvious specific anion effects can be observed in the phase transition of NIPAM-co-VT microgels. However, kosmotropic and chaotropic anions lead to the aggregation of NIPAM-co-SA microgels. The ordering of anions based on the aggregation temperature at the same salt concentration is consistent with the typical Hofmeister series. The aggregation is thought to be determined by the indirect interfacial effects of anions near the microgel surface. Additionally, the aggregate size of microgels increases with salt concentration for the kosmotropic anions. Introduction Specific ion effects exist in various biological and chemical systems.1,2 The first observation of specific ion effects was reported in 1888, when Hofmeister defined a series of cations and anions according to the relative ability of ions for the aggregation and precipitation of egg-white protein in aqueous solutions.3 Afterward, the studies on specific ion effects extended to other areas such as colloid and interface science,4 macromolecular systems,5 medicine,6 and oil recovery.7 However, the nature of the effects still remains elusive even for the most studied system, the protein aggregation induced by salts in aqueous solution.8 Generally, anions have received more attention than cations since they have more pronounced ion-specific effects in various systems.9–11 The so-called Hofmeister series for anions is ClO4> SCN- > I- > ClO3- > NO3- > Br- > Cl- > HCO3- > CH3COO- > F- > H2PO4- > SO42- > CO32-.12–16 Note that the order of this sequence depends on testing methods.17 On the basis of the anion-specific effects on the viscosity of aqueous solutions, the anions were categorized as kosmotropes or chaotropes.18 The anions on the left of Cl- are usually defined as chaotropes because of their weak interactions with water molecules, whereas the anions on the right of Cl- called kosmotropes are strongly hydrated by water molecules.12 The extent of hydration increases with the anions from left to right in this sequence. It is suggested that protein aggregation or the decrease in solubility of protein is associated with the electroneutrality of protein and the indirect interfacial effects of strongly hydrated anions (kosmotropes) near the surface of protein. In contrast, the increase of protein solubility appears to be the result of direct binding of weakly hydrated anions (chaotropes) to the protein surface.19 On the other hand, the charge groups on protein * To whom correspondence should be addressed. † University of Science and Technology of China. ‡ The Chinese University of Hong Kong.
surfaces are all from the derivatives of the weakly hydrated ammonium group and the strongly hydrated carboxylate group.20 According to the law of matching water affinities proposed by Collins,21 only oppositely charged ions of equal water affinity spontaneously form inner sphere ion pairs, which controls the ions binding to the charge groups on protein surface. The ion-protein interactions and their subsequent influences on the aggregation of proteins are very complex and difficult to elucidate clearly since the protein surface is composed of both weakly hydrated (positively charged) and strongly hydrated (negatively charged) groups. In other words, if we can study their effects separately, we should look more into the ionspecificities in the protein aggregation. In the present work, we have prepared poly(N-isopropylacrylamide-co-sodium acrylate) (NIPAM-co-SA) and poly(Nisopropylacrylamide-co-vinylbenzyl trimethylammonium chloride) (NIPAM-co-VT) microgels by dispersion polymerization, which have strongly hydrated (negatively charged) acrylate groups and weakly hydrated (positively charged) trimethylammonium groups on their surfaces, respectively. The microgels with poly(N-isopropylacrylamide) (PNIPAM) moiety are thermally sensitive. By use of laser light scattering (LLS), we have investigated the temperature-induced aggregation of microgels in the presence of salts with the same cation (Na+) but different anions. We hope the present study can help us to understand the anion-specificities in the aggregation of proteins and other macromolecules. Experimental Section Materials. N-isopropylacrylamide (NIPAM) from Kohjin was recrystallized three times from a benzene/n-hexane mixture prior to use. Sodium acrylate (SA) and vinylbenzyl trimethylammonium chloride (VT) from Aldrich was used as received. N,Nmethylenebisacrylamide (MBA) from Sinopharm was purified by recrystallization from methanol. Potassium persulfate (KPS) from Sinopharm was recrystallized from deionized water. Na2CO3, Na2SO4, NaH2PO4, NaCl, NaBr, NaNO3, NaI, and
10.1021/jp9121694 2010 American Chemical Society Published on Web 02/25/2010
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NaSCN were all AR grade (Sinopharm) and used without further purification. Other regents were all used as received. Preparation of Microgels. The details about the preparation of microgels can be found elsewhere.22 Typically, NIPAM (1.011 g, 0.009 mol), SA (0.094 g, 0.001 mol), or VT (0.212 g, 0.001 mol) and MBA (0.077 g, 0.0005 mol) were dissolved in deionized water (190 mL) in a 500 mL three-necked flask. After the solution was purged with bubbling nitrogen for 30 min, 0.027 g of KPS in 10 mL of deionized water was introduced. The mixture was stirred under nitrogen bubbling at 70 °C for 8 h. The resultant solution was dialyzed for 1 week against deionized water using a semipermeable membrane with a molar mass cutoff of 3500 g/mol to remove the unreacted monomers. The pH for NIPAM-co-VT microgel solution after dialysis was about 6.1. Microgel dispersions for all measurements were prepared with Millipore water instead of a buffer so that no additional ions were introduced. Zeta potentials of NIPAM-coSA and NIPAM-co-VT microgels measured on a Malvern Zetasizer Nano ZS90 are -26.3 and 6.4 mV, respectively. The zeta-potentials were obtained from mobility distribution of the microgels by using the Smoluchowski approximation.23 Laser Light Scattering. A commercial spectrometer (ALV/ DLS/SLS-5022F) equipped with multi-τ digital time correlation (ALV5000) and a cylindrical 22 mW UNIPHASE He-Ne laser (λ0 ) 632 nm) as the light source was used. In static LLS,24 we can obtain the weight-average molar mass (Mw) and z-average root-mean-square radius of gyration (〈Rg〉) of particles in a dilute solution from the angular dependence of the excess absolute scattering intensity or Rayleigh ratio Rvv(q) by Zimm plot. For large aggregates, Mw and 〈Rg〉 were obtained by Guinier plot.25 In dynamic LLS,26 the measured intensity-intensity time correlation function G(2)(q,t) in the self-beating mode can transfer into line-width distribution G(Γ) by Laplace inversion. For a pure diffusive relaxation, G(Γ) is related to the translational diffusion coefficient D by (Γ/q2)Cf0, qf0 f D, and further to the hydrodynamic radius Rh via the Stokes-Einstein equation, Rh ) kBT/6πη0D, where kB, T, and η0 are the Boltzmann constant, absolute temperature and solvent viscosity, respectively. The sample was allowed to stand for 2-3 h at each temperature so that the system was in equilibrium. The precision of temperature is (0.05 °C. The concentration of microgels in each solution is 5.0 × 10-5 g/mL in all LLS experiments. Results and Discussion First, we have examined the effects of anions on NIPAMco-VT microgels with weakly hydrated (positively charged) trimethylammonium groups on their surfaces. Figure 1 shows the temperature dependences of 〈Rg〉 and 〈Rh〉 of the microgels in the presence of chaotropic and kosmotropic anions, where the concentration of each salt solution is 0.1 M. Initially, either 〈Rh〉 or 〈Rg〉 of the microgels gradually decreases. As temperature increases to and over the lower critical solution temperature (LCST ∼32 °C), they sharply decrease and level off. However, the molar mass (Mw) does not vary with temperature (shown in inset). The facts indicate that NIPAM-co-VT microgels do not aggregate but shrink; that is, neither chaotropic nor kosmotropic anions can lead to the aggregation of NIPAM-co-VT microgels. Actually, the aggregation of NIPAM-co-VT microgels is controlled by two factors. As temperature increases, the hydrophilicity of PNIPAM moiety decreases, driving the microgels to aggregate.27,28 On the other hand, the electrostatic repulsions between the positively charged microgel surface prevent the microgels aggregation, which can be screened by the counterions. The microgels do not aggregate at a temperature above
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Figure 1. Temperature dependences of the average radius of gyration 〈Rg〉 and average hydrodynamic radius 〈Rh〉 of NIPAM-co-VT microgels in the presence of chaotropic and kosmotropic anions, where the concentration of each salt is 0.1 M. The inset shows the temperature dependence of the molar mass (Mw).
the LCST of microgels, indicating that the latter interactions dominate the former interactions even in the presence of the added salts. In other words, the added anions cannot effectively screen the surface charges on the microgel surface. This is understandable because only oppositely charged ions of equal water affinity can spontaneously form inner sphere ion pairs according to the law of matching water affinities.21 The Jones-Dole viscosity B coefficient of ammonium is -0.007, which is exactly the same as the value of Cl- (B ) -0.007), that is, the trimethylammonium cation and the chloride anion can form the most stable ion pairs in comparison with other anions.20,29,30 Probably for this reason, the zeta-potential value for NIPAM-co-VT microgels is much smaller than that for NIPAM-co-SA microgels before addition of the salt, as shown in the Experimental Section. The strongly hydrated kosmotropic anions with more large values of B coefficient resist forming ion pairs with the charge groups on the microgel surface since the positively charged trimethylammonium groups are weakly hydrated.21 Consequently, the added kosmotropic anions cannot effectively screen the surface charges and result in the electroneutrality of microgels as indicated by the small changes in zeta potential after the addition of salts (data not shown). On the other hand, even if the weakly hydrated chaotropic anions form ion pairs with the trimethylammonium groups to some extent on the microgel surface, they can also directly bind to the weakly hydrated area or hydrophobic area on the microgel surface, which will lead to “salting-in” effects for the microgels. In any event, because neither of them can effectively result in the electroneutrality of microgels, chaotropic and kosmotropic anions cannot lead to the aggregation of NIPAM-co-VT microgels at the present salt concentration. Note that it was reported that the presence of a salt can influence the LCST of linear PNIPAM chains.5,31 In the present case, it is clear that the ions do not have any significant effects on the PNIPAM moiety in the microgels. Otherwise, the microgels should aggregate when the salts are introduced. This is because the salt concentration used here is much lower than the reported
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Figure 3. Temperature dependences of the molar mass (Mw) of NIPAM-co-SA microgels in the presence of different chaotropic anions, where the concentration of each salt is 0.1 M.
Figure 2. Temperature dependences of the average radius of gyration 〈Rg〉 and average hydrodynamic radius 〈Rh〉 of NIPAM-co-SA microgels in the presence of different chaotropic anions, where the concentration of each salt is 0.1 M.
one,5,31 and the interactions between the added ions and the charge groups on the microgel surface dominate the interactions between the ions and PNIPAM moiety. Then, we turned to the effects of anions on the aggregation of NIPAM-co-SA microgels with strongly hydrated (negatively charged) carboxylate groups on their surfaces. Since NIPAMco-SA and NIPAM-co-VT microgels have the same mole percentage of PNIPAM in them, the influences of the added salts on PNIPAM moiety can also be neglected. Figure 2 shows the temperature dependences of 〈Rg〉 and 〈Rh〉 of NIPAM-coSA microgels in the presence of different chaotropic anions, where the concentration of each salt solution is also 0.1 M. It shows that either 〈Rg〉 or 〈Rh〉 decreases as temperature increases at a temperature below the LCST, indicating the shrinking of NIPAM-co-SA microgels. As temperature increases to and over the LCST, 〈Rh〉 and 〈Rg〉 increase with temperature except for NaSCN solution, indicating the aggregation of microgels. As we know, NIPAM-co-SA microgels do not aggregate but shrink upon heating in a salt-free solution due to the electrostatic repulsions between the charged surfaces.32,33 Thus, the aggregation observed here is actually induced by the added ions. In Figure 3, it can be seen that the molar mass (Mw) does not vary with temperature below the LCST but increases at temperatures above the LCST for NaCl, NaBr, and NaNO3, further indicating the aggregation of microgels. Figure 3 also shows that NaI only leads NIPAM-co-SA microgels to form smaller aggregates in comparison with other anions, and the addition of NaSCN almost does not induce the aggregation of microgels. For all the five salts, when the salt concentration is lower than 0.1 M, NIPAM-co-SA microgels show a temperature dependence similar to that in the salt-free solutions (data not shown), further indicating that the aggregation of microgels is induced by the added salts. Figure 4 shows the temperature dependences of 〈Rg〉 and 〈Rh〉 of NIPAM-co-SA microgels as a function of the concentration of a typical kosmotropic anion (SO42-). The microgels are
Figure 4. Temperature dependences of the average radius of gyration 〈Rg〉 and average hydrodynamic radius 〈Rh〉 of NIPAM-co-SA microgels as a function of [SO42-].
individuals at the low temperatures as indicated by the small 〈Rh〉 and 〈Rg〉. As temperature increases to the LCST, 〈Rh〉 and 〈Rg〉 sharply increase, indicating the aggregation of NIPAMco-SA microgels. Furthermore, the aggregate size increases with SO42- concentration ([SO42-]) for the same temperature, implying that the increasing salt concentration can significantly enhance the extent of aggregation. At the same time, Figure 5 shows that the molar mass (Mw) of aggregates in the presence of SO42- is much larger than those for chaotropic anions (Figure 3). Clearly, the kosmotropic anion SO42- is more able to induce the aggregation of NIPAM-co-SA microgels than the chaotropic anions. Similar phenomena were also observed for other kosmotropic anions such as CO32- and H2PO4- (shown in the Supporting Information). Figure 6 shows the aggregation temperature (Ta) of NIPAMco-SA microgels at the same salt concentration of 0.1 M for different anions including kosmotropes and chaotropes. It is evident that the aggregation temperature increases as the anions transit from kosmotropes to chaotropes, indicating that the kosmotropic anions can more efficiently promote the aggregation of NIPAM-co-SA microgels than the chaotropic anions. Also,
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Figure 5. Temperature dependences of the molar mass (Mw) of NIPAM-co-SA microgels as a function of [SO42-].
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Figure 7. Salt concentration (Cs) dependence of the aggregation temperature (Ta) of NIPAM-co-SA microgels in the presence of SO42-, CO32-, and H2PO4-.
Figure 8. Schematic illustration of the aggregation of NIPAM-co-SA microgels induced by anions.
Figure 6. The aggregation temperature (Ta) of NIPAM-co-SA microgels for different anions, where the concentration of each salt is 0.1 M.
the ordering of anions based on the aggregation temperature agrees well with the typical Hofmeister series,12–16 indicating that the specific anion effects dominate the microgel aggregation. It is known that Na+ (B ) 0.086) has the greatest tendency to bind to carboxylates (B ) 0.052) forming ion pairs in comparison with other monovalent cations including K+ (B ) -0.007), Li+ (B ) 0.150), and Cs+ (B ) -0.045).20 Thus, the added salts would lead the surface of microgels to be closer to the electroneutrality, which can be confirmed by a ∼ 75% drop in their zeta potentials (absolute value) after the addition of salts compared with the original value (shown in the Supporting Information). Accordingly, the aggregation of NIPAM-co-SA microgels ought to be determined by the indirect interfacial effects by anions near the microgel surface since all the salts have the same cation of Na+. In general, the interfacial water near the surface of a solute has three different layers: the first layer immediately adjacent to the solute surface is the solvation layer, the second layer is the transition layer, and the third layer is the bulk surface.12 In the present system, the strongly hydrated kosmotropic anions would insert into the third water layer and compete for water molecules in the second layer, making them unavailable to help the first water layer in solvating the microgel surface.19 In other words, the more hydrated anions would make the bulk solution a more poor solvent for the microgels, thereby resulting in a lower aggregation temperature. Likewise, for the weakly hydrated chaotropic anions, the less hydrated anions would make the bulk solution a more good solvent for the microgels and can more easily bind to the nonpolar or hydrophobic surface (“salting in” effects), leading to a higher aggregation temperature.19 Almost no aggregation of NIPAMco-SA microgels is observed in NaSCN solution, further indicating that the aggregation is governed by the indirect
interfacial effects described above. This is why the ordering of anions based on the aggregation temperature of NIPAM-coSA microgels in different salt solutions is consistent with the typical Hofmeister series. To further understand the interactions between the kosmotropic anions and the NIPAM-co-SA microgels, we have also examined the aggregation temperature (Ta) of NIPAM-co-SA microgels at different concentrations of SO42-, CO32-, and H2PO4-. In Figure 7, it can be seen that Ta linearly decreases with the anion concentration. It is known that the surface tension of water at the hydrophobic/aqueous solution interface varies linearly with salt concentration for simple inorganic salts up to moderate concentrations.34 This is similar to the phenomena occurring at the nonpolar/aqueous solution interface, where the added kosmotropes raise the surface tension of the bulk solvent, leading the microgel surface to minimize its solvent accessible surface area.19 This makes the solubility of microgels and the aggregation temperature decrease. Additionally, the polarization of water molecules in the first hydration shell of a macromolecular solute is also linearly dependent on the salt concentration.5 Therefore, the linear relationship can be taken as an indicator that the kosmotropic anions can raise the surface tension and polarize the water molecules in the first hydration layer on the microgel surface. Note that the order of Ta for these anions has dependence of salt concentration. The specific anion effects that cause the Ta difference are more pronounced at higher salt concentrations as the interactions between microgels are dominated by the long-range nonspecific electrostatic forces and the short-range ion-specific forces at the low and higher salt concentrations, respectively. The phase transition of NIPAM-co-VT microgels in the presence of different salts does not exhibit obvious specific anion effects, whereas the ordering of anions based on the aggregation temperature of NIPAM-co-SA microgels in different salt solutions follows the typical Hofmeister series. Considering that a protein contains both carboxylate and ammonium groups, the
Effect of Anions on Aggregation of Charged Microgels aggregation of proteins in the presence of salts with different anions but the same cation should be determined by the molecular interactions related to carboxylate group. Namely, ion-carboxylate interactions play a more important role in the specific anion effects regarding the protein aggregation than the ion-ammonium interactions. Figure 8 illustrates the aggregation of NIPAM-co-SA microgels induced by the anions. Conclusion The study on the aggregation of NIPAM-co-VT and NIPAMco-SA microgels in the presence of different anions leads to the following conclusions: Kosmotropic and chaotropic anions cannot lead NIPAM-co-VT microgels to aggregate because neither of them can effectively result in the electroneutrality of microgels. In contrast, kosmotropic and chaotropic anions lead NIPAM-co-SA microgels to aggregate. The aggregation is determined by the indirect interfacial effects of anions near the microgel surface, and the ordering of anions based on the aggregation temperature is consistent with the classical Hofmeister series. The present study suggests that the specific anion effects in the protein aggregation should be dominated by the carboxylate group related interactions. Acknowledgment. The financial support of the National Distinguished Young Investigator Fund (20725414) and the Ministry of Science and Technology of China (2007CB936401) is acknowledged. Supporting Information Available: Temperature dependence of 〈Rg〉 and 〈Rh〉 of NIPAM-co-SA microgels under different concentrations of CO32- and H2PO4-. Zeta potentials (ζ) of NIPAM-co-SA microgels before (H2O) and after the addition of different anions. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Kunz, W.; Nostro, P. Lo.; Ninham, B. W. Curr. Opin. Colloid Interface Sci. 2004, 9, 1. (2) Parsegian, V. A. Nature 1995, 378, 335. (3) Hofmeister, F. Arch. Exp. Pathol. Pharmakol. 1888, 24, 247.
J. Phys. Chem. B, Vol. 114, No. 11, 2010 3803 (4) Lo´pez-Leo´n, T.; Santander-Ortega, M. J.; Ortega-Vinuesa, J. L.; Bastos-Gonza´lez, D. J. Phys. Chem. C 2008, 112, 16060. (5) Zhang, Y. J.; Furyk, S.; Bergbreiter, D. E.; Cremer, P. S. J. Am. Chem. Soc. 2005, 127, 14505. (6) Lo Nostro, P.; Ninham, B. W.; Milani, S.; Lo Nostro, A.; Pesavento, G.; Baglioni, P. Biophys. Chem. 2006, 124, 208. (7) Morrow, N. R.; Tang, G. Q.; Valat, M.; Xie, X. J. Pet. Sci. Eng. 1998, 20, 267. (8) Zhang, Y. J.; Cremer, P. S. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 15249. (9) Kunz, W.; Henle, J.; Ninham, B. W. Curr. Opin. Colloid Interface Sci. 2004, 9, 19. (10) Leontidis, E. Curr. Opin. Colloid Interface Sci. 2002, 7, 81. (11) v. Klitzing, R.; Wong, J. E.; Jaeger, W.; Steitz, R. Curr. Opin. Colloid Interface Sci. 2004, 9, 158. (12) Collins, K. D.; Washabaugh, M. W. Q. ReV. Biophys. 1985, 18, 323. (13) Castilho, L.; Moraes, A.; Augusto, E.; Butler, M. Aminal Cell Technology; Taylor and Francis, 2007; p 302. (14) Wuthier, U.; Simon, W. Mikrochim. Acta 1986, III, 225. (15) Zhang, Y. J.; Cremer, P. S. Curr. Opin. Chem. Biol. 2006, 10, 658. (16) Saloma¨ki, M.; Tervasma¨ki, P.; Areva, S.; Kankare, J. Langmuir 2004, 20, 3679. (17) Nostro, P. L.; Fratoni, L.; Ninham, B. W.; Baglioni, P. Biomacromolecules 2002, 3, 1217. (18) Jones, G.; Dole, M. J. Am. Chem. Soc. 1929, 51, 2950. (19) Collins, K. D. Methods 2004, 34, 300. (20) Collins, K. D. Biophys. Chem. 2006, 119, 271. (21) Collins, K. D. Biophys. J. 1997, 72, 65. (22) Zhao, Y.; Zhang, G. Z.; Wu, C. Macromolecules 2001, 34, 7804. (23) Ohshima, H.; Healy, T. W.; White, L. R. J. Colloid Interface Sci. 1982, 90, 17. (24) Chu, B. Laser Light Scattering, 2nd ed; Academic Press: New York, 1991. (25) Guinier, A.; Fournet, G. Small-Angel Scattering of X-ray; John Wiley: New York, 1955. (26) Berne, B. J.; Pecora, R. Dynamic Light Scattering; Plenum Press: New York, 1976. (27) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. (28) Peng, S. F.; Wu, C. J. Phys. Chem. B 2001, 105, 2331. (29) Krestov, G. A. Thermodynamics of SalVation: Solution and Dissolution, Ions and SolVents, Structure and Energetics; Horwood: New York, 1991. (30) Robinson, J. B., Jr.; Strottmann, J. M.; Stellwagen, E. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 2287. (31) Freitag, R.; Garret-Flaudy, F. Langmuir 2002, 18, 3434. (32) Deryaguin, B. V.; Landau, L. Acta Phys. Chem. 1941, 14, 633. (33) Verwey, E. J. W.; Overbeek, J. Th. G. Theory of the Stability of Lyophobic Colloids; Elsevier: Amsterdam, 1948. (34) Jarvis, N. L.; Scheiman, M. A. J. Phys. Chem. 1968, 72, 74.
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