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Atomic Force Microscopic Images of Solvated Polymer Brushes Hiroo Iwata, Isao Hirata, and Yoshito Ikada* Research Center for Biomedical Engineering, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan Received November 18, 1996. In Final Form: February 19, 1997
In recent years, the structure and dynamics of polymer chains on surfaces have been the subject of considerable theoretical and experimental activity, as reviewed by Milner.1 Theoretical studies2-4 and computer simulations5 have predicted that the polymer chains attached to a surface at relatively high coverage and exposed to a good solvent stretch along the direction normal to the surface, like brushes. Surface observation performed using sophisticated instruments, such as a surface plasmon oscillation apparatus6 and a surface force apparatus,7 supports this prediction. An atomic force microscope (AFM) is another powerful tool used to directly explore surface topography of brushes in a solvent. However, its application to study the structure of solvated synthetic polymer chains immobilized on a surface is quite limited.8 In this work, we employed a Nuclepore membrane as a substrate for surface grafting of water-soluble polymer chains. This membrane is made of polycarbonate and has straight cylindrical pores perpendicular to the surface. The nominal pore diameter, thickness, and membrane diameter were 0.2 µm, 10 µm, and 25 mm, respectively. To covalently immobilize high densities of polymer chains on the membrane surface, graft polymerization of acrylic acid was allowed to initiate from peroxides introduced onto the surface by low-temperature plasma exposure.9 For the graft polymerization, the membrane was treated by Ar plasma under 0.04 Torr for 10 s and then immersed in a 10 wt % aqueous solution of acrylic acid in a glass ampule. After vigorous degassing, the ampule was sealed and kept at 60 °C for a given period of time to decompose the peroxides on the surface to initiate graft polymerization. Following graft polymerization of acrylic acid, the ungrafted poly(acrylic acid) (PAA) was removed from the grafted membrane by extraction with deionized water at 60 °C. The surface density of grafted PAA was determined by the dye-staining method reported elsewhere with slight modification.10 The dye employed in this study was toluidine blue O. Stoichiometric dye sorption to carboxylic groups of PAA was confirmed using a PAA gel. The graft density was calculated to be 0.80 µg/cm2, assuming that graft polymerization occurred uniformly on both the membrane surface and the wall of the pores. The molecular weight of the grafted PAA was assumed to be the same as that of the ungrafted PAA which was * To whom correspondence should be addressed. Phone: +8175-751-4115. Fax: +81-75-751-4144. E-mail: yyikada@ medeng.kyoto-u.ac.jp. (1) Milner, S. T. Science 1991, 251, 905. (2) Alexander, S. J. Phys. (Paris) 1977, 38, 977. (3) de Gennes, P. G. Macromolecules 1980, 13, 1069. (4) Milner, S. T.; Witten, T. A.; Cates, M. E. Macromolecules 1988, 21, 2610. (5) Chakrabarti, A.; Toral, R. Macromolecules 1990, 23, 2016 (6) Taunton, H. J.; Toprakciaglu, C.; Fetters, L. J.; Klein, J. Macromolecules 1990, 23, 571. (7) Tassin, J. F.; Siemens, R. L.; Tang, W. T.; Hadziioannou, G.; Swalen, J. D.; Smith, B. A. J. Phys. Chem. 1989, 93, 2106. (8) Stipp, S. L. Langmuir 1996, 12, 1884. (9) Suzuki, M.; Kishida, A.; Iwata, H.; Ikada, Y. Macromolecules 1986, 19, 1804. (10) Uchida, E.; Uyama, Y.; Ikada, Y. Langmuir 1993, 9, 1121.
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simultaneously produced during graft polymerization. The average molecular weight of the PAA was found to be 2.2 × 106 Da, when determined from the intrinsic viscosity method.11 This indicated that the average degree of polymerization of grafted PAA was 30 000 units. The AFM apparatus employed in this study was the OLYMPUS NV2000 (OLYMPUS, Tokyo, Japan) with a scanner S30W specialized for AFM observation under water. The PAA-grafted membrane was immersed in buffered solutions of various pHs in a liquid cell and analyzed with a AFM in the constant repulsive force mode using a silicon nitride probe. A V-shaped cantilever having a nominal spring constant of 0.02 N m-1 was used. Although the tapping mode is superior to the contact mode for observing the image of an easily deformable surface, our system was not provided with the tapping mode. As will be discussed in connection with AFM images shown in Figure 2, the force applied on the cantilever was kept as low as possible when the distinct outermost surface structure of the grafted layer could be observed. The surface was scanned under the tip at a scanning speed of 5.0 µm/s in the scan direction and 9.8 nm/s in the perpendicular direction. The scanning area of 5 µm × 5 µm was observed in all cases with 512 × 512 data points collected per image, corresponding to a data pixel size of 9.8 nm × 9.8 nm. Buffered solutions were prepared using 0.7 M sodium barbital, 0.7 M sodium acetate, 8.5 wt % sodium chloride, and 0.1 M hydrochloric acid following Michelis’ method.12 Figure 1 shows scanning electron microscopic (SEM) photographs of the surface of the nontreated membrane and the membrane grafted with PAA in the dry state. Some defects such as overlapping pores and several dents were observed even on the nontreated surface, but most of the pores had diameters of approximately 0.2 µm, as described in the Nuclepore catalog. There was no distinct difference in pore diameter between the nontreated and the grafted membranes. Even after PAA grafting, the grafted membrane maintained its original pore structure in the dry state. PAA is soluble in neutral and alkaline aqueous media, and hence the grafted PAA will form a highly hydrated soft layer on the membrane surface when the polymer chains are brought into contact with these media. AFM images of the PAA-grafted membrane in water are expected to greatly depend on the force applied to the cantilever because the grafted PAA chains form a highly soft layer. Figure 2 shows a typical example of the effect of the force applied to the cantilever on the AFM image of the membrane surface at pH 8.0. The AFM image of the PAA-grafted membrane observed under a 10 nN force resembled the SEM image. However, pore images became less distinctive with decreasing applied forces on the cantilever. In fact, clear pores were undetectable on the membrane when a 1.0 nN force was applied to the cantilever, and only hill and valley structures were observed. This suggests that the cantilever tip has penetrated into the hydrated PAA grafted layer to scan the perforated substrate surface at the higher applied force. Since the AFM image of membranes drastically changed depending on the applied force as demonstrated above, we kept the force applied on the cantilever as low as possible in order to produce a distinct outermost surface structure of the grafted layer. PAA carries a number of carboxyl groups on the side (11) Takahashi, A.; Hayashi, N.; Kagawa, I. Kogyo Kagaku Zasshi (J. Chem. Soc., Ind. Chem. Sec.) 1956, 60, 1059. (12) Chemical Handbook; Kagaku Binnran Kisohenn, II, Ed.; Japanese Society of Chemistry: Maruzen, Japan, 1975; p 1495.
© 1997 American Chemical Society
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Figure 1. Scanning electron micrographs of the untreated Nuclepore membrane and the PAA-grafted membrane. No distinct differences was observed in the surface morphology between these two membranes in the dry state: (A) untreated Nuclepore membrane; (B) PAA-grafted Nuclepore membrane with a graft density of 0.80 µg/cm2
Figure 2. Effect of the force applied to the cantilever on the atomic force microscopic image of the grafted Nuclepore membrane with the graft density 0.80 µg/cm2 in pH 8.0 buffered solution. The 3-D images of the same area on the membrane were represented to display the effect of the force applied on the cantilever. Parts b and d show 2-D section analyses between the arrows indicated in the 3-D images in parts a and b, respectively: (a and b) 1.0 nN; (c and d) 10 nN.
Notes
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Figure 3. AFM image of a grafted Nuclepore membrane with the graft density 0.80 µg/cm2 in pH 3.5 buffered solution. The image area is 5 µm × 5 µm. The marked area is magnified in Figure 4.
chain which exhibit various degrees of dissociation depending on the pH of the medium. The carboxyl groups do not dissociate in an acidic solution, whereas PAA chains gain an increasing number of negative charges with increasing the pH of the buffered solution. The electrostatic interactions between intra- and intermolecules and between macroions and small ions have drastic effects on the conformation of polyelectrolyte chains. Therefore, the morphology of the PAA-grafted layer would also be expected to drastically change by varying the pH of the buffered solution. In the following measurements, AFM images were recorded under 1.0 nN to minimize deformation of the water-swollen, soft graft brushes. Figure 3 shows a large-scale AFM image of the grafted membrane in a buffered solution of pH 3.5. The AFM image of the area marked in Figure 3 was magnified in Figure 4, where other images obtained in buffered solutions of different pHs are also shown. Even though the force applied on the cantilever was as low as 1.0 nN, pores were clearly observed at pH 3.5 because the PAA chains underwent shrinkage and precipitation on the membrane surface at the low pH. In the buffered solution of pH 4.0, the pore size became slightly smaller, but the pore contour was still clearly observed. On the contrary, AFM images drastically changed in the vicinity of pH 5.2, which is the pKa of PAA,13 indicating conformational changes of grafted PAA chains. All of the pores were filled with solvated PAA-grafted chains, and furthermore the height of the pores became larger than that of the flat regions. In the buffered solution of pH 8.1, hill and valley structures were clearly apparent. (13) Nagasawa, M.; Murase, T.; Kondo, K. J. Phys. Chem. 1965, 69, 4005.
The end-to-end distance of a typical linear polymer chain is much less than 100 nm. For example, the Flory endto-end distance of a swollen polystyrene molecule with a high degree of polymerization of 6340 is 80 nm in a good solvent.6 This fact implies that a synthetic polymer molecule with a typical molecular weight will find it difficult to occupy the full space of a 100 nm radius pore to which it is grafted. As seen in Figures 2 and 4, pores were entirely occupied by grafted PAA chains, with some being squeezed out from the pores at high pHs. This can be explained in terms of polymer chain interactions, as schematically shown in Figure 5. Intra- and intermolecular ionic repulsion between the carboxyl groups of densely grafted PAA chains attached to the membrane surface through one must have induced much greater extension and asymmetric conformation. If a linear PAA chain with a degree of polymerization of 30 000 is fully extended, the thickness of the grafted PAA layer is estimated to be 3.9 µm, calculated from the degree of polymerization and the C-C bond length. This calculation suggests that the PAA chains grafted on the membrane surface can overfill the space of a 100 nm radius pore. An explanation for the elevation of the pore area at pHs 5.0 and 8.1 is schematically illustrated in Figure 5. When grafted chains that exist in and on the rim of the pores are extended, the segment density within the pore will increase as a function of (radius)-2. Ionic repulsion between anionic charges on the side chains also will be enhanced with increasing segment density. As a result, the grafted layer would swell to reduce the segment density, thereby producing an increased height of the pore area. Finally, is has been suggested that the grafted polymer
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Figure 4. Effect of the pH of buffered solutions on the atomic force microscopic images of the PAA-grafted Nuclepore membrane with a graft density of 0.80 µg/cm2. The images were obtained under 1.0 nN force applied to the cantilever. The AFM images were obtained in buffered solutions of different pHs, and the area marked in Figure 3 was magnified: (a) pH 3.5; (b) pH 4.0; (c) pH 5.0; (d) pH 8.1.
Figure 5. Schematic representation of the grafted chains on the membrane.
chains can dynamically open and close the pores in response to pH, acting as a molecular valve to regulate the filtration characteristics.14 Buffered solutions of various pHs were filtered through the grafted Nuclepore membrane with a graft density of 0.3 µg of PAA/cm2 under a pressure of 1.0 kgf/cm2 using an Amicon 8010 ultrafiltration cell (Bevery, MA). The dependence of the water filtration rate on pH is shown in Figure 6. It can be seen (14) Iwata, H.; Matsuda, T. J. Membr. Sci. 1988, 38, 185.
Figure 6. pH dependence of the water filtration rate for a PAA-grafted Nuclepore membrane with a graft density of 0.30 µg/cm2.
that the filtration rate was nearly independent of pH in the range 5-7. However, in the vicinity of the pKa of PAA (i.e. 5.2), the filtration rate sharply increased with decreasing pH. This was due to opened and closed pores at low and high pH, respectively, as explained previously. One of the interesting applications of this dynamic behavior would be an environment-sensitive membrane. LA962012X
