Molecular Imprint Membranes Prepared by the Phase Inversion

Oct 1, 1997 - Molecular imprinting membranes of theophylline (THO) were prepared by phase inversion with poly(acrylonitrile-co-acrylic acid) (P(AN-co-...
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Langmuir 1997, 13, 5396-5400

Molecular Imprint Membranes Prepared by the Phase Inversion Precipitation Technique. 2. Influence of Coagulation Temperature in the Phase Inversion Process on the Encoding in Polymeric Membranes Hong Ying Wang, Takaomi Kobayashi,* Takahiro Fukaya, and Nobuyuki Fujii Department of Chemistry, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata 940-21, Japan Received February 4, 1997X Molecular imprinting membranes of theophylline (THO) were prepared by phase inversion with poly(acrylonitrile-co-acrylic acid) (P(AN-co-AA)). The copolymer-dimethyl sulfoxide solution with THO template was coagulated in water at various temperatures. It was found that the decrease of coagulation temperature caused an increase in THO binding to the THO-imprinted sites of the resultant membrane. The results for the 1H-NMR of the membrane before template extraction showed that the template amount in the membrane coagulated at 10 °C is higher than that at 40 °C. The IR analysis of P(AN-co-AA) membranes indicated that the effective binding of THO is due to hydrogen bonds between the THO and OH group of nondimerized carboxylic acid segments. For caffeine, which is structurally close to the THO template, less effective binding to the membranes was obtained. It was discussed that the high binding selectivity of THO solute to the copolymer membrane arises from the formation of the imprinted sites, which consist of free OH group of carboxylic acid segments for hydrogen bonds with the substrate.

Introduction The molecular imprinting technique has been found to be an effective means of encoding information in bulk material on a molecular scale.1,2 The procedure involves incorporation of small amounts of template molecule in polymerization medium. After polymerization, the template molecule is removed from the polymer. Thus, the extraction procedure leaves the functionalized size of the template molecule in the polymer network.3 According to the method, most of molecular recognition polymers were prepared to be practically applied in separations of chiral compounds4 and amino acid derivatives5 and in drug assay.6 Most of the molecular imprinted polymers were mainly concerned with chromatographic uses, because the rigid gel matrices are suitable for only the applications. Little was known about a class of the membrane made of molecular imprinted polymers. More recently, we developed a new molecular imprinting technique for porous membrane materials using poly(acrylonitrile-co-acrylic acid) (P(AN-co-AA), Chart 1).7,8 The phase inversion process of the copolymer was applied to encode the information of template molecules, theophylline (THO) (Chart 2). Polymer solidification in water took place in the presence of template, and hence, the resultant polymeric membrane contains volumetric size of the template. Here, the acrylonitrile residues work as membrane formation residues and acrylic acid (AA) residues interact with template molecules to fix the THO template in the membrane. Previous imprinting studies have focused on studies of the effect of the template concentraX Abstract published in Advance ACS Abstracts, September 1, 1997.

(1) Mosbach, K. Trends Biochem. Sci. 1994, 19, 9. (2) Wulff, G. Molecular Interaction in Bioseparations; Ngo, T., Eds.; Plenum Press: New York, 1993; p 363. (3) Andersson, I. L.; Ekberg, B.; Mosbach, K. Molecular Interaction in Bioseparations; Ngo, T., Eds.; Plenum Press: New York, 1993; p 383. (4) Sellergren, B.; Shea, K. J. J. Chromatogr., A 1993, 654, 17. (5) Sellergren, B.; Ekberg, B.; Mosbach, K. J. Chromatogr. 1985, 347, 1. (6) Vlatakis, G.; Andersson, L. I.; Muller. R.; Mosbach, K. Nature 1993, 361, 645. (7) Kobayashi, T.; Wang, H. Y.; Fujii, N. Chem. Lett. 1995, 927. (8) Wang, H. Y.; Kobayashi, T.; Fujii, N. Langmuir 1996, 12, 4850.

S0743-7463(97)00114-5 CCC: $14.00

Chart 1

Chart 2

tion on molecular imprinting of P(AN-co-AA) membranes and recognition of THO or caffeine (CAF) (Chart 2) by the copolymer membrane. However, no studies have followed the coagulation process, which would influence the molecular imprint characteristics. Thus, there are insufficient data to identify important trends of the molecular imprint technique. Since in the phase inversion process the polymer gelation condition such as temperature of the coagulation medium influences highly the resultant membrane properties,9-13 we here report the influence of coagulation bath temperature on the imprinting process. For copolymer transforming from cast solution to the solid membrane, a dimethyl sulfoxide (DMSO) cast solution of P(AN-co-AA) was coagulated in the presence of the THO template at various coagulation temperatures. We placed the emphasis on the characterization of the THO imprinted membranes prepared by changing coagulation temperature in the phase inversion process of P(AN-co-AA). (9) Mulder, M. Basic Principles of Membrane Technology; Kluwer Academic Publishers: Dordrecht, 1991; pp 83-86. (10) Loeb, S.; Milstein, F. Paper presented before First European Symposium on Fresh Water from the Sea, Athens, May 31-June 3, 1962. (11) Loeb, S.; Milstein, F. Summary Report No. 2369, Aerojet-General Corporation, Azusa, CA, October 1962. (12) Kesting, R.; Barsh, M. K.; Vincent, A. J. Appl. Polym. Sci. 1965, 9, 1873. (13) Bottino, A.; Capannelli, G.; Munari, S. J. Appl. Polym. Sci. 1985, 30, 3009.

© 1997 American Chemical Society

Molecular Imprint Membranes

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Experimental Methods Materials. All reagents used in the experiments were of reagent grade unless otherwise described. THO and CAF were products of Tokyo Kasei and used without purification. Water used in experiments was distilled and then purified by passing through ion exchange resin before use. P(AN-co-AA) was synthesized according to previous methods.7,8 Chart 1 shows the chemical structure of the copolymer (Y denotes the fraction of AA segments containing COOH unit). Identification of the copolymer was carried out with a 1H-NMR spectrometer (JNMGX 270 FT-NMR; DMSO-d6) as reported.7 The fraction, 0.145, of AA units in the copolymer was calculated from the integrals of total aliphatic protons for polyacrylonitrile (PAN) and AA segments. The molecular weight of the resultant copolymer was estimated as 7 × 104 according to the literature using a viscometric method in dimethylformamide solution.14 Membrane Preparation and Characterization. According to the previous method, phase inversion precipitation of P(ANco-AA) was carried out to prepare the porous membranes.7,8 Here, DMSO and water were selected as cast solvent and coagulation medium of the copolymer, respectively. A DMSO cast solution containing 10 wt % P(AN-co-AA) and 4.7 wt % THO template was prepared at 50 °C. The solution was then spread on a glass plate at about 100 µm thickness and coagulated in water at various temperatures in the range of 10-40 °C. The coagulated copolymer membrane was kept overnight in water in order to remove DMSO. The resulting membrane was washed with 0.1 wt % acetic acid (AcOH) aqueous solution for extraction of the template. Then the membrane was rinsed with a large excess of water. For morphology observation of the resultant membranes, a scanning electron micrograph (SEM) (JXA-733, Jeol) was used. The sample preparation was similarly carried out as previously reported.8 Fourier transform infrared (FT-IR) spectra of the membrane were measured with FT-IR spectrometer (FT-IR 8200, Shimadzu). For the FT-IR measurement, the copolymer membrane was obtained by spreading the cast on a glass plate at about 3-5 µm thickness and then the solution was immersed in water to coagulate the copolymer at various temperatures. The sample preparation for IR measurements was carried out according to a pervious report.8 A 1H-NMR spectrometer (JNM GX270 FT-NMR; DMSO-d6) was used to analyze the membrane coagulated with the template. In order to study the substrate uptake of THO and CAF, heterogeneous batch experiments were carried out. The membrane with weight (W) (g) was equilibrated in 3.6 µM THO or CAF aqueous solution for 24 h at 30 °C. Then, the THO or CAF concentration of bulk solution was estimated by using a highperformance liquid chromatography (CCPD, Toyo Soda, Co.) equipped with a UV-vis detector (UV 8000, monitored at 270 nm) with a TSKgel-ODS column. The value of the substrate binding to the THO-imprinted membrane, [Sb] (µmol/g of membrane), was calculated by the following equation

[Sb] ) (Cb - Ca)V/W

(1)

where Cb and Ca are the mole concentration (µM) of THO or CAF substrate before and after the equilibrium, respectively. V (L) is the volume of bulk equilibrium solution.

Results and Discussion Influence of Coagulation Temperature on THO Uptake to the THO-Imprinted Membranes. In the phase inversion method, the resultant membrane properties depend strongly on the preparation conditions, such as coagulation temperature.12,13 It is known that the phase inversion process involves two processes: one is solvent exchange in polymer environment and another is polymer coagulation in nonsolvent. The condition of the coagulation medium influences mainly the rate of solvent exchange between cast solvent and nonsolvent. The nonsolvent penetration into the polymer-cast solution medium becomes lower at low temperature, because of (14) Onyon, P. F. J. Phys. Soc. 1959, 37, 315.

Figure 1. THO and CAF amounts taken up into the THOimprinted membranes coagulated at various temperatures of water. Used 3.6 µM concentration of (O) THO and (0) CAF substrates was equilibrium experiments.

high viscosity and low solubility. Consequently, polymer coagulates slowly at low temperature. For PAN, it is known that the polymer segments coagulate strongly in water nonsolvent due to the dipole interaction between CN groups.15 In the present study, the temperature of the water medium was changed in order to examine the dependence of coagulation temperature on substrate binding into the THO-imprinted membranes. After extraction of the template from the membranes, the uptake experiments were carried out. Figure 1 shows plots of THO amounts, [Sb]T, bound to the THO-imprinted copolymer membranes versus coagulation temperature. The data of CAF amounts, [Sb]C, are also shown in the figure. For CAF substrate, the data show that the THO-imprinted membranes have very few bindings. On the other hand, the THO substrate is bound highly to the THO-imprinted membranes. Note that the uptake of the THO substrate strongly depends on the coagulation temperature. The decrease of the coagulation temperature causes an increase in the THO taken into the copolymer membrane. For the membranes prepared at 10 and 40 °C, [Sb]T is 1.25 and 0.26 (µmol/g of membrane), respectively. The value of the former is about 5 times that of the latter. The increase of the THO binding may be due to the resultant membrane coagulated in low temperature having a number of THOimprinted sites for only THO substrate. Membranes Coagulated at Various Water Temperatures. In order to interpret the effective uptake of THO into the membrane prepared in low coagulation temperature, SEM photographs for the various membranes were taken. Figure 2 shows the photographs of the cross section of the membranes coagulated in water at 10, 23, and 30 °C. As reported previously,16,17 PAN and its copolymer membranes have an asymmetric structure, which consists of a dense top layer supported by a porous sublayer with a finger-like structure. The SEM morphology suggests that PAN transforming from cast solution to the solid state occurs quickly in water-poor solvent. In this work, the SEM pictures show that P(AN-co-AA) membranes have similar asymmetric structure to that of PAN. In total cross section with about 80 µm thickness, the finger-like macrovoids and thin top layer are present. As the coagulation temperature decreases, the top layer thickness, which is shown with arrows, is clearly increased. (15) Matsuura, T. Synthetic Membranes and Membrane Separation Processes; CRC Press, Inc.: Boca Ratan, FL, 1994; pp 11-31. (16) Kobayashi, T.; Miyamoto, T.; Nagai, T.; Fujii, N. J. Appl. Polym. Sci. 1994, 52, 1519. (17) Kobayashi, T.; Nagai, T.; Ono, M.; Wang, H. Y.; Fujii, N. Eur. Polym. J. 1997, 33, 1191.

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Figure 2. SEM photographs of the cross section of the copolymer membranes coagulated at (a) 10, (b) 23, and (c) 30 °C.

The thickness for 10 °C is about 10 µm, while that for 30 °C is about 1 µm. The difference strongly suggests that coagulation of P(AN-co-AA) is influenced by the temperature of the water medium. It is well-known that the membrane structure is mainly determined by liquid-liquid demixing in the phase inversion process.18 In P(AN-coAA) morphology at 10 °C coagulation temperature, the liquid-liquid demixing is delayed relative to that at high temperature. This is supported by the result for a thick top layer with spongelike structure as shown in Figure 2a. However, at high coagulation temperature, for example, 30 °C, the SEM photograph shows that the solvent exchange between DMSO and water occurs quickly, because the membrane has significant asymmetric structure with a very thin top layer. This results from very fast solvent exchange and strong coagulation of PAN segments. When a P(AN-co-AA)-cast solution contains THO templates, the PAN segments coagulate together with the template. As a result, the segments fix the template volumetric size in the membrane coagulated. Thus, the THO-imprinted sites are formed after the extraction of the template by washing with AcOH solution.7,8 It is considered that, during coagulation, some template molecules are soluble into water medium. Therefore, this process may lower the formation of the imprinted sites in the membrane coagulated. In contrast, there are THO template molecules, which interact with AA segments by hydrogen bonds and remain in the membrane. The THO template is responsible for the imprinted site formation. Comparison of 1H-NMR spectra was made for the copolymer membrane coagulated at 10 and 40 °C, as shown in Figure 3. Evidently, for 10 °C coagulation, the resonance peaks at 3.55 and 8.08 ppm are for THO template in the membrane. The additional peaks near 1.7-2.3 ppm come from methylene protons of copolymer backbone. The signal for CH protons of the copolymer overlaps with the 3-3.4 ppm peak for water. For 40 °C coagulation, the 3.55 ppm resonance peak of the methyl group for the THO template is very weak relative to that for 10 °C coagulation. The difference in the spectra indicates that the amounts of the THO template in the coagulated membrane depend upon the temperature of the water bath. The amounts of the THO template solubilized into water medium are lowered with the decrease of the coagulation temperature, because of an reduction of solubility of THO into water. (18) Reference 9, pp 58-68.

Figure 3. 1H-NMR spectra of P(AN-co-AA) membranes in DMSO-d6 measured at 50 °C: (a) 10 °C and (b) 40 °C coagulation temperature for the phase inversion of P(AN-co-AA). Samples without the template extraction were used.

As reported previously,8 THO substrate binds to P(ANco-AA) membrane due to hydrogen bonding between the THO and COOH group of the copolymer. To confirm the interaction of P(AN-co-AA) with THO, we measured IR spectra of the THO-imprinted membranes coagulated in water at various temperatures. Figure 4 shows FT-IR spectra of the copolymer membranes prepared at 10 and 40 °C coagulation temperatures. The IR spectra are for P(AN-co-AA) membranes before the THO extraction without washing by AcOH solution. Therefore, the template molecules still remain in the membranes coagulated. The assignments of IR bands are given in Table 1. We can find the characteristic IR peaks of CN stretching at 2242 cm-1 and CH bending at 1450 cm-1 for PAN segments.19 For AA segments of the copolymer, CdO stretching near 1730 cm-1 is useful for the characterization.20 At 2937 cm-1, a sharp peak for CH bend of PAN segments appears. Furthermore, it is noted that the spectra have the OH stretching vibration in the 25003600 cm-1 region. It is known that there are two kinds of OH stretching in the wavenumber region.21 One is from carboxylic acid dimers of AA segments (Scheme 1, a), and the IR peaks appear near around 2500-3300 cm-1. (19) Yamadera, R. Koubunshi Kagaku 1964, 21, 362. (20) Lee, Y. M.; Oh, B. J. Membr. Sci. 1995, 98, 183.

Molecular Imprint Membranes

Figure 4. FT-IR spectra of the THO-imprinted membrane coagulated at 10 and 40 °C without the THO template extraction. Table 1. FT-IR Results of P(AN-co-AA) Membrane cm-1

segments

3520

AA

3216 2600 2937 2242 1730 1450 1280 847

AA AA AN, AA AN AA AN AA AN

band assignment19-24 OH stretching: free (unhydrogen bond) COOH group OH stretching: dimerized COOH groups OH stretching: dimerized COOH groups CH stretching CN stretching CdO stretching: CH2 bending: PAN backbone C-O stretching: C-C(dO)-O CH2 rocking

Scheme 1

Another is from free carboxylic acid of the copolymer, and the peak appears near the 3520 cm-1 region.22,23 Because of the dimerization of the COOH group, this kind of COOH group may not contribute to form hydrogen bonding with THO templates. In contrast, the presence of the free OH group may be suitable for hydrogen bonding formation with THO in the uptake experiments. Thus, the free OH groups of COOH segments (Scheme 1, c) create the THO recognition sites in the copolymer matrix. Previous data of viscosity for the cast solution containing various template concentrations indicated that the addition of THO to the copolymer-DMSO cast solution causes a low viscosity of the copolymer-DMSO cast solution.8 This is due to the reduction of the COOH dimers, since the THO added binds to the COOH segments (Scheme 1, b). Figure 5 shows FT-IR spectra of the THO-imprinted membranes obtained after the THO extraction. In the (21) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds, 4th ed.; John Wiley & Sons Inc.: New York, 1991; p 183. (22) Herzberg, G. Molecular Spectra and molecular structure, II. Infrared and Raman Spectra of Polyatomic Molecules; Van Nostrand Reinhold Co.: New York, p 334. (23) Reference 20, pp 174-175. (24) Yamadera, R. Koubunshi Kagaku, 1964, 21, 362.

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Figure 5. FT-IR spectra of the THO-imprinted membranes with the THO extraction. The coagulation was done in 10 and 40 °C. Dashed line is for the spectra of P(AN-co-AA) membranes obtained without the template extraction.

figure, the spectra were shown in the wavenumber region of 2000-4000 cm-1. The dashed lines are from the spectra of the corresponding membrane without extracting. The extraction was confirmed by measuring with 1H-NMR for the membrane. After the extraction of the template, the membranes show no resonance peak for the template in the NMR spectra. For IR measurements, the spectra difference between the membranes without and with the template removal is remarkable in the low coagulation temperature. Note in the figure that the IR peak intensity near 3520 cm-1 for OH stretching of free COOH groups increases by the removal of the THO, when the membrane is coagulated in the low temperature. At 40 °C coagulation, these spectra show no significant difference in the membranes prepared with and without the template extraction. The spectral data show the sharp IR peak near 2242 cm-1 for the CN group of PAN segments. The IR intensity for CN stretching is independent of the template extraction from membrane. So, we calculated peak intensity ratio (OH/CN) of each OH stretching to the 2242 cm-1 CN stretching for the membranes. Here, the results for OH stretching of COOH dimers near 2600 cm-1 was used for the calculation, because the 3216 cm-1 OH stretching for the dimerized COOH groups is close to the 3520 cm-1 OH stretching of free COOH group. Figure 6 shows values of OH/CN for P(AN-co-AA) membranes coagulated at each temperature. It is clear that the values obtained at 10 °C coagulation show a large difference in both membranes with and without the template extraction. The results for the IR spectra show that the concentration of the free OH group in the membrane increases in the low coagulation temperature. In another OH stretching peak for COOH dimers, the values of OH/CN are almost independent of coagulation temperature. As a result, it is reasonable to be considered that the free OH group for COOH segments is responsible for the THO-imprinted sites, because the sites can interact with THO solute via hydrogen bonds. Selectivity of the THO-Imprinted Membrane. A significant difference between the THO and CAF uptake is presented in Figure 1. Therefore, in order to estimate the selective binding to the various THO-imprinted membranes prepared by each coagulation temperature, we calculated the selectivity factor, RTHO/CAF, for THO and CAF, which is defined in eq 2

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Wang et al. Table 2. Selective Factor, rTHO/CAF, for the THO-Imprinted Membranes Obtained by Various Coagulation Temperatures coagulation temp (°C)

[Sb]T (µmol/g of membrane)

[Sb]C µmol/g of membrane)

RTHO/CAF

10 15 30 40

1.25 0.85 0.48 0.26

0.024 0.030 0.026 0.023

52 28 18 11

Chart 3

Figure 6. IR peak ratio of OH stretching at (b, O) 3520 and (2, 4) 2600 cm-1 to the 2937 cm-1 CN stretching for the copolymer membranes coagulated at various temperatures. Closed and open symbols are for the membranes without and with the template extraction, respectively.

RTHO/CAF ) [Sb]T/[Sb]C

(2)

The resultant value of RTHO/CAF for each membrane is listed in Table 2. A high selectivity factor is obtained for the THO-imprinted membrane coagulated at 10 °C. The value means that the binding amount of THO is 52 times that of CAF. The comparison also means that the sites in the membrane interact with hydrogen on the 7-nitrogen atom of THO, since CAF, which is structurally close to the THO template, has a methyl group on the 7-nitrogen atom instead of the H group. Therefore, the CAF molecule has less effective binding than the THO molecule to the imprint sites. Consequently, the free COOH group of P(AN-coAA) shows the important role on the selectivity of the THO-imprinted membrane. Furthermore, two substrates, 2-hydroxyethyltheophylline (HETHO) and uracil (Chart 3), were used to evaluate binding amounts for the THO-imprinted membrane coagulated at 10 °C. The selectivity factor, R, for THO and their substrates was calculated as well as CAF using eq 2. Here, HETHO has a hydroxyethyl group instead of the H group of THO. Uracil has H groups on N-atom and two carbonyl groups in the chemical structure as well as THO. However, the framework structure of uracil is different than that of THO. Table 3 shows binding amounts and R for these substrates. For HETHO, the binding to the THO-imprinted membrane is much lower than that for THO, while that is a little higher than that for CAF. Hence, to interact with the THO-imprinted sites of the membrane for HETHO having a hydroxyl group is easier than for CAF having a methyl group on the N-atom. For uracil, which has both carbonyl groups and -NH groups in the molecular framework, the binding amount is almost same as that for HETHO and smaller than that for THO. The result for uracil indicates that the THOimprinted membrane encodes volumetric size of THO

Table 3. Binding Amounts and the Selective Factor of the THO-Imprinted Membrane for Different Substratesa substrate

binding amounts (µmol/g of membrane)

R

THO CAF HETHO Uracil

1.25 0.024 0.32 0.40

1 52 3.9 3.2

a The THO-imprinted membrane used was prepared at 10 °C coagulation temperature.

template in the imprint sites of the membrane. As a result, the THO-imprinted membrane shows sharp recognition for the THO molecule. Conclusions It was found that the coagulation temperature in the phase inversion of P(AN-co-AA) influences strongly the THO-imprinted site formation. The decrease of the coagulation temperature caused the increase of THO taken into the membrane. The results for THO or CAF uptake experiments showed that THO-imprinted sites in the membrane take effectively only THO. The substrate uptake behavior of the copolymer membrane was supported by characterizations of P(AN-co-AA) membranes by means of 1H-NMR, SEM, and FT-IR measurements. The IR data suggested strongly that nondimerized COOH segments are responsible for the effective THO uptake into the membrane through hydrogen bonds between free COOH and THO. The selectivity of the THO-imprinted membrane was confirmed by using CAF, HETHO, and uracil. Acknowledgment. This research was partly supported by Grant-in-Aid for Scientific Research (C) (09650757) of the Ministry of Education, Science, Sports and Culture, Japan. LA970114X