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Ultraltration Membranes Composed of Highly CrossLinked Cationic Polymer Gel: the Network Structure and Superior Separation Performance
Qifeng Wang, Sadaki Samitsu, and Izumi Ichinose*
Hydrophilic polymer gels are attractive materials for a wide range of applications in the life sciences and bioengineering. The gels are environmentally responsive and have high potential in numerous applications such as drug delivery, sensors, actuators, absorbents, and scaffolds. The transport of ions and molecules through hydrated polymer gels has been investigated by many researchers, as has the bioafnity and mechanical properties of these gels.[1] Poly(4-vinylpyridine) (P4VP) forms typical hydrated polymer gels. This polymer is readily cross-linked with alkyl dihalide at room temperature via quaternization of the pyridine groups. Cross-linkers of various size and shape can be used, resulting in P4VP gels with diverse structures and properties. Thin lms made of cross-linked poly(vinylpyridine) gels have been widely investigated as a means of applying stimuli-evoked optical, impedance, and volume changes to sensors and biodevices.[2] P4VP has intrinsic selective permeability for gases,[3] and the network structures of these gels can be controlled by controlling the extent of cross-linking. Therefore, this polymer has also been studied for its utility in gas separation, desalination, and pervaporation membranes.[4] However, like most polymer gels, P4VP gels are mechanically weak, and the gel membranes, which are generally supported on a microltration membrane, have thicknesses of a few to several tens of micrometers. In order to be practical, the separation layer needs to be as thin as possible to realize high liquid/gas uxes, which requires that the gels are mechanically stable. We here report ultraltration membranes with thicknesses of several tens to hundreds of nanometers, prepared from P4VP gels by means of a two-step cross-linking of the polymer chains. The detailed structure of the polymer network was characterized using spectroscopic techniques and permeation and rejection experiments. Furthermore, the gel membranes were demonstrated to distinguish between water-soluble proteins of different molecular masses. The methodology for preparing ultrathin ultraltration membranes of the cross-linked P4VP gel is shown in Figure 1. First, P4VP was cross-linked with 1,3-dibromopropane (DBP) in dimethyl sulfoxide (DMSO), then the resulting weakly gelled solution was diluted with water (Figure 1a). To form P4VP gel membranes, we used an extremely at sacricial layer of metal hydroxide nanostrands. Without this layer, the gel readily penetrates into the submicrometer pores of the microltration membrane, resulting in decreased permeability to water. The nanostrands spontaneously form in dilute aqueous solution of copper, zinc, or cadmium nitrate (or chloride) upon neutralization of the solution with base (see Supporting Information).[5] In the present study, cadmium hydroxide nanostrands with a diameter of 1.9 nm and lengths of a few micrometers were ltered on a polycarbonate (PC) membrane lter (Figure 1b).[6] The resultant ultrathin nanobrous layer guaranteed the uniform ow of water required to form a P4VP gel layer with a constant thickness. Subsequently, a certain volume of the diluted P4VP gel solution was ltered. At this stage, the P4VP gel layer was not mechanically stable and signicantly swelled in water. Therefore, the membrane was immersed in ethanol to extract the water, then was further subjected to cross-linking with 1,3-dibromopropane (DBP) to x the densied network of the polymer chains. Ethanol is a good solvent for P4VP, while water is a poor solvent. On the other hand, once P4VP is quaternized, ethanol becomes a poor solvent and water becomes a good one. The nal gel layer was more stable than the gel layer prior to cross-linking and swelling was signicantly reduced. The effects of this two-step cross-linking process were conrmed using bulk P4VP gels (Figure 1c). When a disc-shaped piece of cross-linked P4VP gel was placed in water, its size approximately doubled, whereas the disc shrunk by half if it was immersed in ethanol. Following the second cross-linking with DBP, shrinkage upon immersion in ethanol was marginal, and the extent of swelling in water was halved compared to the original disc. The process by which P4VP gel membranes are formed was investigated by scanning electron microscopy (SEM). Figure 2a shows an image of a nanostrand layer prepared on a PC membrane with 0.2-m pores. When a cross-linked P4VP gel solution was ltered through this membrane, a thin layer of P4VP gel with a thickness of 300 15 nm formed on the nanostrand layer (Figure 2b). After dissolving the nanostrands, the gel layer contracted to 135 30 nm upon treatment with ethanol and subsequent cross-linking (Figure 2c). The thickness of the P4VP gel membranes was easily tuned by choosing the appropriate ltration volume of P4VP gel solution (see Supporting Information). The SEM images shown in Figure 2b,c do not represent the morphology of wet P4VP gel membranes since

Dr. Q. Wang, Dr. S. Samitsu, Prof. I. Ichinose Organic Nanomaterials Center National Institute for Materials Science 11 Namiki, Tsukuba, Ibaraki 3050044, Japan E-mail: ICHINOSE.Izumi@nims.go.jp Prof. Dr. I. Ichinose Japan Science and Technology Agency CREST, 5 Sanbancho, Chiyodaku, Tokyo 102-0075, Japan

DOI: 10.1002/adma.201100475

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Figure 1. a) Structure and photograph of cross-linked P4VP gel and photograph of the diluted solution. b) Scheme for the preparation of a two-step cross-linked P4VP gel membrane. c) Photographs of disc-shaped P4VP gels and the expanded/shrunken gels in water and in ethanol, the P4VP gel further cross-linked with DBP, and the cross-linked gel expanded in water.

the specimens had been dried in vacuum. Indeed, specimens prepared by a freeze-dry technique had double the thickness (310 10 nm, Figure 2d) of specimens dried at room temperature (135 nm, Figure 2b). Freeze-dried gel membranes had very at and smooth cross sections. Filtration experiments provide a tool for gaining insight into the internal structures of cross-linked gels. When the pore sizes of a hydrophilic gel are in the range of a few nanometers, the

Figure 2. Cross-sectional SEM images of a cadmium hydroxide nanostrand layer (a), a P4VP gel layer formed on the nanostrand layer (b), two-step cross-linked P4VP gel membrane (c, d). Inset in (a) shows the surface morphology of the nanostrand layer. The gel membranes were prepared from 500 L of 0.23 mg mL1 P4VP gel solution. Image (d) was obtained from a freeze-dried specimen. The scale bars are 200 nm.

membrane has the potential to be used as an ultraltration membrane, which usually has pores in the range 1 to 100 nm. The permeation rate of water increases inversely to the thickness of the membrane and, in the case of ultrathin gel membranes, the permeation rate is very sensitive to changes in pore size distribution. This makes it easy to characterize the network structure of cross-linked hydrogels under certain conditions. The pores in P4VP gel membranes were rst investigated by studying what size proteins could be retained by the membranes. The results are summarized in Table 1 for three membranes with different thicknesses: P4VP-100 (39 nm), P4VP-250 (78 nm), and P4VP-500 (135 nm). When an aqueous solution of cytochrome c (Cyt.c) was ltered in dead-end mode using a P4VP-100 membrane at pH 4.23, the protein concentration in the permeate (2.16 mL) decreased to 4.7% (95.3% rejection) and the concentration in the remaining feed (7.84 mL) increased about 27%. From this increase in the upper stream, we calculated that 78.4% of the Cyt.c was rejected at the surface of the membrane. Furthermore, the ltration rate (ux) at a pressure difference of 80 kPa was calculated to be 466 L m2 h1 based on ltration time, the volume of the ltrate, and the effective membrane area after considering the porosity of the substrate (10%). This value is one to two orders of magnitude greater than those of commercial ultraltration membranes with similar separation properties. The separation (i.e., the rejection of molecules above a certain molecular size) properties of the membranes increased with increasing pH of the test solution. In contrast, the rejection calculated from the upper stream decreased signicantly at pH 7.52 and higher. This is because Cyt.c, which has an isoelectric point of 10.2, begins to electrostatically adsorb onto the outermost surface of the positively charged gel membrane under neutral and alkaline conditions. The rejection of Cyt.c by the P4VP-250 membrane was higher than 96%

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Table 1. Separation performance of P4VP gel membranes.

pHb) Flux [L m2 h1] P4VP-100a) Rejection [%] Feed Cyt.c 2.28 4.23 6.04 7.52 9.48 Mb Hb Ts--CD TMPyP DPX
a)

P4VP-250a) Flux [L m2 h1] Rejection [%] Feed 263 392 344 536 290 377 380 - 364 - 89.2 95.7 75.5 32.2 - 77.4 90.8 - 51.4 - Permeate 98.2 99.4 96.8 98.8 100.0 95.5 98.4 - 96.9 - 196 305 270 413 277 349 339 215 202 216
1

P4VP-500a) Flux [L m2 h1] Rejection [%] Feed 100.0 99.1 88.5 42.8 - 82.8 96.7 5.6 95.2 99.4 Permeate 99.1 99.3 99.2 99.2 100.0 98.6 99.7 35.4 99.0 97.6

Permeate 88.8 95.3 93.8 98.2 100.0 95.9 97.7 - 79.9 -

309 466 399 614 305 393 395 - 638 -

76.0 78.4 64.9 17.2 - 64.9 87.0 - 13.3 -

3.94 3.83 - - -

P4VP-100, P4VP-250, and P4VP-500 denote membranes prepared from 100 L, 250 L, and 500 L of P4VP gel solution (0.23 mg mL ). Their average thicknesses were 39 nm, 78 nm, and 135 nm, respectively. The effective membrane area was 2.84 cm2. The ux obtained at a pressure difference of 80 kPa was normalized with the 10% porosity of PC membrane; b)The pH of protein solutions was adjusted with HCl and NaOH. The pH of organic dyes was not adjusted. Cyt.c (20 g mL1); Mb (20 g mL1); Hb (20 g mL1); Ts--CD, (0.1 mM); TMPyP (5 M); DPX (10 M).

between pH 2.28 and 9.48, as calculated from the concentration of Cyt.c in the permeate. The much thicker P4VP-500 membrane rejected Cyt.c by more than 99% at pH values of 4.23 and lower, as calculated from the feed. This indicates that Cyt.c does not adsorb onto the membrane surface. The ux at pH 4.23 was 305 L m2 h1, which is very high for an ultraltration membrane. Furthermore, P4VP gel membranes rejected more than 95% of myoglobin (Mb) and hemoglobin (Hb), demonstrating that these cross-linked gel membranes may be useful for concentrating proteins, at least within a certain pH range, without signicant ltration loss. Since quaternized P4VP coatings have been used for forming permanent antibacterial surfaces,[7] the membranes described above demonstrate signicant potential for medical applications. The molecular dimensions of Cyt.c are 2.5 2.5 3.7 nm3. Therefore, the P4VP-100 membrane should have some pores larger than this at pH 2.28, since the membrane rejected less than 90% of Cyt.c. On the other hand, the pores in the thickest membrane (P4VP-500) are no more than 3.7 nm in width. To evaluate the pore size of P4VP-500, we examined the rejection of a neutral -cyclodextrin derivative, mono-6-O-(ptoluenesulfonyl)--cyclodextrin (Ts--CD). Analysis of the permeate showed that 35.4% was rejected by the membrane (Table 1); since the molecular width of this cyclodextrin derivative is about 1.7 nm (including the hydration water),[6b,8] this membrane should have many pores of 1.52.0 nm diameter. Overall, the polymer network of the cross-linked P4VP gel seems to have a pore size distribution in the range 2.0 1.0 nm. If the membrane charge density is higher than that of the solution, the same charged ions are electrostatically repelled and permeation is suppressed by the membrane.[9] This was also observed for positively charged P4VP gel membranes. For example, the rejection of a cationic dye, meso-tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP, 1.7 1.7 nm2), was 99.0%, and that of a relatively small organic compound, p-xylene-bis(N-pyridinium

bromide) (DPX, 0.6 1.5 nm2), was 97.6%, as estimated from the permeate of P4VP-500 membranes. Water permeation through a P4VP-500 membrane was examined in the range pH 2.010.0 (see Supporting Information) at a pressure difference of 80 kPa. The ux increased with pH from 220 L m2 h1 (pH 2.0) to 332 L m2 h1 (pH 7.0) to 580 L m2 h1 (pH 10.0), with a specic increase at pH 4.0 (337 L m2 h1). Given the large ux increase at high pH, the pores in the polymer network should expand with increasing pH, probably due to hydroxide ions. The specic increase at pH 4.0 is likely explained by the protonation of free pyridine units, as reported by Mika et al.[10] Water ux increased inversely to the membrane thickness. However, the ux observed for the thinnest membrane (P4VP-100) was about three times smaller than the value expected from the thickness of the membrane. This is due to compression of the membrane by the applied pressure. In contrast, water ux through P4VP-500 membrane linearly increased with applied pressure up to at least 80 kPa and compression gradually increased between 0.1 and 0.5 MPa (see Supporting Information). Therefore, we analyzed the ux at 80 kPa using the HagenPoiseuille equation:
J =
2 gB r p

8:*J

(1)

where J is the volume ux (m3 m2 s1), is the surface porosity, rp is the average pore radius (m), is the viscosity (Pa s), is the thickness (m), is the tortuosity, and p is the pressure drop across the membrane (Pa).[11] P4VP-500 membrane had a water ux of 332 L m2 h1 at a pressure difference (p) of 80 kPa at pH 7.0. Using the viscosity of water at 20 C (1.002 103 Pa s), the thickness of freeze-dried membrane (310 nm), the presumed pore size (2.0 nm), and the tortuosity (1.25), we estimated the surface porosity of the membrane to be 28%.

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In our experimental conditions, the pyridine groups of P4VP were highly quaternized with DBP. Figure 3a shows a N 1s XPS spectrum of a P4VP gel membrane transferred onto a silicon wafer before the second cross-linking. The peaks located at 398.5 eV and 401.4 eV were assigned to the nitrogen of the neutral pyridine and that of the quaternized pyridine, respectively.[12] Deconvolution conrmed that some pyridine groups coordinate to a cadmium ion (or form hydrogen bonds with partially hydrolyzed DBP), giving a binding energy of approximately 399.8 eV.[13] The amount of quaternized pyridine was 23%, as calculated from the peak area. This ratio decreased to 17% after the second cross-linking (Figure 3b). Furthermore, the atomic ratio of carbon to nitrogen (C/N) increased from 8.43 to 9.53. The outermost membrane surface might be covered by relatively hydrophobic polymer chains after the second cross-linking since

ethanol was used as the solvent for the cross-linker. Even though only about 20% of the pyridine groups were cross-linked, the twostep cross-linked P4VP gel membranes were stable enough to be used for high-pressure ltration. Indeed, a pressure of at least 2.6 MPa could be applied to a P4VP-500 membrane formed on a PC membrane lter (see Supporting Information). Almost all the incorporated DBP seems to be used as cross-linker, since the bromo group was not detected by XPS. Since the degree of crosslinking is 17%, 1/6 of the pyridine groups are bound to other polymer chains. An average structural unit should therefore have a chain length composed of six ethylene units (1.4 nm) and have a half-length of two cross-linked pyridinium groups (0.6 nm). If 18 ethylene units and three DBPs form a ring, the diameter is calculated to be 2.5 nm. In this case, the pore size should be no more than 2.0 nm, after subtracting the width of the alkyl chain (ca. 0.5 nm). In a similar way, 24 ethylene units and four crosslinkers give a 3.3 nm ring with a maximum pore size of 2.8 nm (see Supporting Information). The polymer network in P4VP gel membranes should have such a cross-linked structure. As illustrated in Figure 3c, the pores are composed of 10 to 25 ethylene units and crosslinkers, and the pore sizes are distributed in the range 2.0 1.0 nm in the expanded conformation. The porosity should be 28%, as suggested from the analysis of water permeation. These areas are drawn with hatched lines in Figure 3c. The pores, which demonstrate effective separation performance, change size in response to pH and other external conditions. We have demonstrated that mechanically stable ultrathin P4VP gel membranes are obtained by a two-step cross-linking process. A nanobrous sacricial layer of cadmium hydroxide nanostrands is useful for the preparation of an ultrathin lter cake of the hydrophilic gel, due to the ultrane nonwoven structure of the nanostrand layer and the high and uniform water permeability of the membrane. After removal of the nanostrands and the subsequent second cross-linking, the gel layer became sufciently stable to be subjected to high-pressure ltration. An important point of this study is that evaluation of the rejection properties and water permeability of the membrane made it possible to elucidate the pore sizes and porosity of the hydrophilic gels. An understanding of the network structure is indispensable for understanding the fundamental properties of polymer gels, such as diffusion, thermal conductance, and mechanical properties. We should also stress that highly cross-linked P4VP gel membranes show excellent rejection properties for proteins and dye molecules and that the ltration rate was one to two orders of magnitude higher than that of commercial ltration membranes. Clearly, highly positively charged P4VP gel membranes demonstrate signicant advantages for the separation of small cationic molecules and will be useful for a wide range of industrial applications.

Experimental Section
Materials: Poly(4-vinyl pyridine) (P4VP, Mw = 160 000), cytochrome c (Cyt.c) from bovine heart, myoglobin (Mb) from equine heart, equine hemoglobin (Hb), meso-tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), and p-xylene-bis(N-pyridinium bromide) (DPX) were purchased from Sigma-Aldrich. Mono-6-O-(p-toluenesulfonyl)-cyclodextrin (Ts--CD), 1,3-dibromopropane (DBP), CdCl22.5H2O, 2-aminoethanol, and other chemicals were purchased from Kanto Chemical. All the chemicals were used as received without further

Figure 3. Nitrogen (N 1s) XPS spectra of P4VP gel membranes before (a) and after (b) the second cross-linking. c) Schematic illustration of network structure of cross-linked P4VP gel.

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purication. A track-etched polycarbonate (PC) membrane was purchased from Whatman. Ultrapure water of 18.2 M produced by a Millipore direct-Q system was used for the experiments. Preparation of P4vp Gel Membranes: Nanostrand solution (10 mL; see Supporting Information) was suction ltered onto a PC membrane with an effective diameter of 1.9 cm. A given volume (100 L, 250 L, and 500 L) of cross-linked P4VP solution (0.23 mg mL1) was diluted with 5 mL of water and ltered onto the PC membrane with the nanostrand layer. Then, the nanostrand layer was dissolved by the passage of 10 mL of 0.01 M aqueous HCl. The remaining P4VP gel membrane was rinsed with water, air dried, and contracted by slowly passing ethanol through the membrane for 30 min using a ltration funnel. Subsequently, a 2 mM ethanolic solution of DBP was slowly passed though the membrane for 1 h, then the solution was kept on the membrane overnight by removing the pressure difference. Finally, the DBP solution was passed through under reduced pressure, and the two-step cross-linked P4VP gel membrane was rinsed with ethanol and water. Characterization: P4VP gel membranes were characterized using a scanning electron microscope (SEM, Hitachi S-4800) after coating with a 2-nm-thick platinum layer.[14] X-ray photoelectron spectroscopy (XPS) measurements were carried out on a PHI Quantera SXM photoelectron spectrometer using a monochromatic Al KR X-ray source. The separation properties were evaluated by ltering a given volume of protein or dye solution. The changes in the concentrations of the feed and the permeate were examined by UV-vis spectroscopy, allowing calculation of the percent rejection of the solute molecules.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements
The authors thank J. P. Gong (Hokkaido University), W. Oppermann (Clausthal University of Technology), and the 21th Polymer Network Group Meeting (Goslar) for many helpful discussions. Received: February 5, 2011 Published online:

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