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Molecularly imprinted organic–inorganic hybrid membranes for selective separation of phenylalanine isomers and its analogue
Separation and Purification Technology 68 (2009) 97–104
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Molecularly imprinted organic–inorganic hybrid membranes for selective separation of phenylalanine isomers and its analogue Hong Wu a,b,∗ , Yanyan Zhao a , Mingcheng Nie a , Zhongyi Jiang a a b
Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072, China
a r t i c l e
i n f o
Article history: Received 6 February 2009 Received in revised form 3 April 2009 Accepted 10 April 2009 Keywords: Optical resolution Molecularly imprinted hybrid membrane Sodium alginate Silane d,l-Phenylalanine
a b s t r a c t A novel kind of molecularly imprinted organic–inorganic hybrid membrane, using sodium alginate (SA) and 3-aminopropyltriethoxysilane (APTES) as the organic and inorganic phase, respectively, was prepared and investigated for the optical resolution of chiral isomers. The hybrid membrane was prepared in an aqueous environment with the presence of d-phenylalanine (d-Phe) as imprinting templates, and the silica phase was introduced in the alginate matrix by sol–gel process of APTES. It was found by FTIR and SEM–EDX characterizations that strong covalent bonds were formed between the organic and inorganic phases, and silica was homogenously distributed in the matrix. These molecularly imprinted SA–APTES hybrid membranes showed chiral separation ability towards the d,l-Phe isomers with a maximum selectivity (˛D/L ) of 1.8 at 40% APTES content. The recognition mechanism was tentatively analyzed and discussed based on permeation and binding experiments. The separation ability of the above hybrid membranes toward d-Phe and its analogue, d-tyrosine (d-Tyr), was also investigated, and a separation factor of up to 2.9 was achieved. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Most important artificial pharmaceutical compounds produced by chemical reactions are chiral. The two enantiomers exist as either a right-handed or a left-handed molecule with almost identical physical and chemical properties but probably quite different physiological activities. One enantiomer may be effective as a drug, while the other is ineffective or even toxic [1]. Chiral separation has become an indispensible process for all the chiral drug productions and needs to be more precise and efficient to meet health and drug safety requirements. Due to their analogical structure and property, the optical resolution for enantiomers remains one of the most difficult separation tasks [2]. Current isomer separation methods mainly include recrystallization, chromatography, enzymatic resolution and membrane separation. Membrane separation process shows advantages over the other techniques in productivity, operational conditions and cost [3]. The invention of molecular imprinting technology further promotes the design and development of novel membranes specifically for efficient chiral separation. There has been an increasing interest in the synthesis and study of molecularly imprinted polymers (MIPs) used as syn-
∗ Corresponding author at: Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China. Tel.: +86 22 27890882; fax: +86 22 23500086. E-mail address: [email protected] (H. Wu). 1383-5866/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2009.04.014
thetic affinity materials [4]. Molecular imprinting is a technique to introduce molecular recognition sites for a specific analyte in a synthetic polymer for selective separation or concentration of target molecules. This technique involves the formation of complexes between imprinting molecules (templates) and functional monomers/polymers based on the following three interactions: covalent bonds, non-covalent interactions or metal ion coordinations [5]. The monomer/polymer–template complexes are then fixed by polymerization and/or crosslinking. Removal of the templates finally results in cavities with a shape, structure and functional groups complementary to the templates. MIPs have been widely studied in liquid chromatography, solid phase extraction and facilitated catalysis [6]. Employment of molecular imprinting technique in membrane preparation produces molecularly imprinted membranes (MIMs), highlighting the possibility of more precise separation by membranes. A number of researchers have carried out systematic investigations on the molecularly imprinted membranes prepared in non-aqueous solutions (organic solvents) through covalent-bond or noncovalent-bond approaches followed by phase inversion process [7]. However, in case of biomolecular imprinting, an aqueous imprinting process is obviously much preferred owing to the much less solubility of the templates and to minimize the risk of losing bioactivities in organic solvents. In aqueous environment, the nonspecific interactions (mainly hydrogen bonding) between the templates and the functional monomers/polymers may become much weaker due to the presence of water. How to realizing and
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maintaining the imprinting of biomolecules in the membrane is a challenge for MIM development [8]. Molecularly imprinted inorganic materials, usually prepared by sol–gel process, have advantages in keeping the structure of the imprinted cavities unchanged after removal of the templates owing to their rigid nature compared to flexible organic polymers [9–11]. However, the fact that it is rather difficult or even impossible to remove templates inside the inorganic matrix greatly confines its use only to surface imprinting. Moreover, the frangible nature of inorganic materials which makes it hard to prepare large-area membrane pieces also limits their practical application. Developing organic–inorganic hybrid membranes may provide a solution to satisfy the requirements from the two aspects: unchanged imprinted cavity and good film-forming property. Hybrid membranes have shown extraordinary properties in pervaporative separation area with improved physical and mechanical properties arising form the synergism of the two kinds of building blocks [12,13]. Alginate, a polysaccharide extracted from seaweeds, is a biodegradable, biocompatible and non-toxic polymer with good film-forming property. Besides, its natural chiral environment and the large number of carboxyl groups which can assemble with such template molecules as amino acids through forming static interactions makes it an attractive material for the preparation of optical resolution membranes. But alginate alone cannot fulfill this task successfully since imprinted cavities would not be well retained due to its severe swelling and eventual dissolution in aqueous solutions. One way to overcome this drawback is by chemical crosslinking. The commonly used crosslinkers for alginate include glutaraldehyde [14], phosphoric acid [15] and divalent cations (e.g. Ba2+ , Ca2+ ) [16]. Herein, 3-aminopropyltriethoxysilane, a silane-coupling agent with a 3-aminopropyl and three ethoxysilane groups, was used as an efficient crosslinking agent for alginate modification as well as to introduce inorganic silica phases. A novel molecularly imprinted hybrid membrane, denoted as SA–APTES for simplicity, was prepared by using sodium alginate (SA) as polymer material and APTES as not only a precursor for introducing inorganic component into organic polymer matrix through sol–gel process but also a crosslinking agent to strengthen the mechanical strength of SA membrane and thus reduce its swelling degree in aqueous solutions. One of the target enantiomers for separation, d-phenylalanine (d-Phe), was used as the template molecule. The binding and permeation property and mechanism of the membranes for chiral resolution of the d,l-Phe aqueous mixtures as well as d-Phe and d-Tyr mixture was investigated and analyzed.
The hot solution was filtered and a known amount of APTES was added into the filtrate followed by addition of a small amount of 1 mol/L ammonia solution. The mixture was vigorously stirred for 12 h before 80 mg of d-Phe template was added. The solution was stirred for another 12 h. The resulting homogeneous solution was cast onto a clean glass plate with the aid of a casting knife. The membrane was allowed to dry at room temperature for 48 h first and then annealed at 60 ◦ C for 2 h. The dried membrane was peeled off from the glass plate and immersed in a crosslinking bath containing 10 vol.% of GA, 0.05 vol.% of concentrated HCl in water–acetone (30:70) mixture at 40 ◦ C for 24 h. After crosslinking, the membrane was washed repeatedly with deionized water to remove any possible residual of HCl and GA. For the extraction of templates, the membrane was washed by or immersed in deionized water until no d-Phe molecules could be detected by HPLC (Agilent 1100). Finally, the membrane was either dried at room temperature or in a hot air oven at 65 ◦ C. The relative mass content of APTES to SA was varied by 30, 40, 50, and 60%, and the resulting membranes were designated correspondingly as SA–APTES-30, SA–APTES-40, SA–APTES-50, SA–APTES-60. The resulting membrane was transparent in appearance and self-standing with a thickness of 30–40 m. Non-imprinted control membrane was prepared in the same way without adding any template molecules during preparation. 2.3. Membrane characterizations FT-IR spectra of the plain SA and SA/APTES were scanned in the range of 4000–600 cm−1 on a Nicolet 5DX instrument equipped with horizontal attenuated total reflectance accessories. The surface and cross-section morphologies of the hybrid membranes were observed by scanning electron microscopy (SEM) and the distribution of silica in the SA polymer domains was observed by EDX Si-mapping analysis attached to the SEM (Philips XL30E SEM). Membranes were frozen in liquid nitrogen, broken, and sputtered with gold before observation. 2.4. Binding experiments Static binding experiments were conducted to evaluate the recognition property of the membranes toward the target molecule. The imprinted and control membrane samples were, respectively, immersed in the isomer mixture of d,l-Phe with a concentration of 0.5 mmol/L for each isomer at room temperature for 12 h. The concentration of d-Phe and l-Phe in the solution was monitored
2. Experimental 2.1. Materials SA was purchased from Shanghai Tianlian Fine Chemical Corporation (Shanghai, China). APTES was purchased from Fluka (USA). d,l-Phe (>98%) and d-tyrosine (d-Tyr, ≥99%) from Sigma–Aldrich Co.(USA) were used as purchased. Glutaraldehyde (GA) (50% content in water), ammonia (25% content), hydrochloric acid (HCl) (35% content) and acetone were supplied by Tianjin Jiangtian Chemical Corporation (Tianjin, China). Acetic acid and methanol of HPLC grade were purchased from Tianjin Guangfu Chemical Corporation. Deionized water was used throughout this study. 2.2. Membrane preparation The SA solution with a concentration of 1.6 wt% was prepared by dissolving 0.8 g of SA in water at 60 ◦ C under stirring for 6 h.
Fig. 1. FT-IR spectra of SA and SA–APTES membranes.
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by analyzing with an HPLC (Agilent 1100) equipped with a UV detector and a Regis ChirosilSCA(−) column (150 mm × 4.6 mm), using methanol–water–acetic acid solution as mobile phase. The uptake (Q, mol/g dry membrane) of d,l-Phe and the adsorption selectivity (˛S ) were calculated according to the following two equations: Qi =
(Cfi − Cei )V , m
˛S =
(CfD − CeD )/(CfL − CeL ) CeD /CeL
i = D or L
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was calculated as follows: ˛D =
˛D/L ˛S
where ˛D/L represents the separation factor obtained by separation experiments described in the following section. 2.5. Separation experiments
where Cfi and Cei are the initial and equilibrium concentrations of d,l-Phe, respectively, m is the weight of dry hybrid membrane sample and V is the volume of solution. According to the solution–diffusion mechanism, commonly used to describe the transportation behavior of molecules across dense membrane, the diffusion selectivity (˛D ) of the membrane
To evaluate the separation performance of the imprinted membrane towards two isomers, permeation tests were conducted. The membrane sample was mounted tightly between two chambers (20 mL for each chamber) in a diffusion cell. A mixture of d,l-Phe with a total concentration of 1 mmol/L (0.5 mmol/L for each isomer) was added into the feeding chamber while water was added into the receiving chamber. The concentration change of d,l-Phe in the receiving chamber was monitored and determined by HPLC. The permeability coefficient P (cm2 /s) and the separation factor (˛D/L )
Fig. 2. Schematic depiction of the formation of d-Phe imprinted SA–APTES hybrid membrane.
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were calculated by the following equations: Pi =
Ki Vd A(CFi − CRi )
˛D/L =
i = L, D
PD PL
where Ki is the concentration change rate of the receiving solution obtained by calculating the slope value of the diffused analyte concentration–time curve, V is the volume of solution, A is the effective membrane area, d is the membrane thickness and (CFi − CRi ) is the concentration difference between feeding and receiving chambers. The separation ability of the hybrid membranes for separating d-Phe and its analogue, d-Tyr, was also investigated by permeation tests. The experimental procedure was the same as that described above except that l-Phe was replaced by d-Tyr. 3. Results and discussion 3.1. Formation of SA/APTES hybrid molecularly imprinted membranes The chemical structure of the SA–APTES hybrid membranes was analyzed by FT-IR spectra in the range of 4000–650 cm−1 as shown in Fig. 1. The absorption peaks appeared at 1602 cm−1 and 1412 cm−1 were assigned to the asymmetric and symmetric stretching of carboxylate salt groups on sodium alginate [17,18]. The absorption bands of the amino groups (–NH2 ) attributed to the addition of APTES, which should have appeared at 1596 cm−1 , was overlapped with the peak of carboxylate groups. The overlapped peak of –COOH and –NH2 was weakened with the increase of relative mass content of APTES added to the membrane. This was due to the chemical reaction happened between the –COOH group of SA and the –NH2 group of APTES, resulting in a new peak appeared at 1486 cm−1 which was attributed to the amide covalent bonds (–CO–NH–). The absorption peaks at 1000–1100 cm−1
were attributed to both the C–O groups on SA and the formation of Si–O–Si bonds (1095 cm−1 ) as a result of condensation of the silanol groups (Si–OH) after hydrolysis of APTES. The formation of –CO–NH– bonds was expected to remarkably improve the compatibility between the organic and the inorganic components and thus benefit the maintenance of the structure of the imprinted cavities. A possible process for the formation of the imprinted hybrid membrane was presented and illustrated schematically in Fig. 2. The difficulty in imprinting under aqueous condition lies in the weakening effect of water on the formation of noncovalent interactions between the template and the polymer since water would compete with the templates for functional groups of the polymer to form hydrogen bonds. The abundance of carboxyl groups and hydroxyl groups on SA was supposed to enhance the interactions between template and functional polymer to form pre-assemble complex. Besides, the amino groups that remained after hydrolysis of APTES might also interact with the carboxyl groups on the amino acid templates, thus making additional favorable contribution to keeping the structure of the imprinting sites after silica condensation. A portion of the residual carboxyl groups as well as hydroxyl groups of SA would be crosslinked by forming covalent bonds with the amino groups and the silanol groups of the hydrolyzed APTES during sol–gel process. A dense organic–inorganic network was thus fabricated. In addition, the propyl chain connecting the amino group and the silicon was thought to produce a transition area between the inorganic silica phase and the organic SA chains. After removal of templates, the imprinted cavities with distinct shape, size and chemical functionality remained in the hybrid matrix and the specific recognition sites were formed. 3.2. Membrane morphology and silica distribution The SEM images of the membrane surface of the SA–APTES hybrid membrane were shown in Fig. 3. The SEM images (Fig. 3(a)–(c)) confirmed the dense structure of the hybrid membranes prepared. The transparent appearance of the hybrid membranes was a macroscopic indication of the good compatibil-
Fig. 3. SEM images of imprinted SA–APTES hybrid membrane surface: (a) SA–APTES-40; (b) SA–APTES-50; (c) SA–APTES-60; (d) SA–APTES-40 with a SA/d-Phe mass ratio of 6. The SA/d-Phe mass ratio of (a)–(c) membrane was 10.
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ity between the polymer and the silica phases. When the APTES content reached 50%, some silica nanoparticles could be observed on the membrane surface by SEM, and when this content further increased to 60%, the particles became larger, revealing an increasing aggregation of the silica under higher concentrations. In this study, the APTES content was kept lower than 60%. The amount of templates used for imprinting is a key factor that influences the amount of imprinting cavities and final selectivity. The effect of template content on the separation factor was investigated by decreasing the polymer/template ratio from 20, 10 to 6 (data not shown). It had been found that the separation selectivity increased first notably with the increase of the template amount (ratio decreasing from 20 to 10), and then level off, little change in selectivity was found when the ratio further decreased from 10 to 6. However, the quality of the membrane at higher template content was rather poor. Fig. 3(d) showed the SEM image of the imprinted SA–APTES-40 membrane surface with a higher SA/d-Phe mass ratio of 6. Some diamond- or rectangle-shaped delves could be clearly observed on the surface. These defects resulted from the crystallization of the amino acids during evaporation of solvent due to the high concentration of templates. The higher the template content was, the bigger the crystal particles became, leading to big defects after template removal. Taking both selectivity and membrane quality into consideration, a ratio of 10 was applied for membrane preparation. Fig. 4 showed the EDX Si-mapping image of the hybrid membrane. A uniform dispersion of silica domains (the bright spots) inside the SA matrix was observed, revealing a successful introduction of inorganic phase in organic polymer at molecular level by APTES sol–gel process. 3.3. Effect of APTES content on the enantioselectivity The separation factors (enantioselectivity) of SA–APTES hybrid membranes prepared with different contents of APTES and dried at room temperature were shown in Fig. 5. Both the imprinted and control membranes under study showed a preferential permeation for d-Phe over l-Phe. Pre-experiments showed that when APTES content was low (<30%), almost no selective ability was found due to the loose and too flexible structure. With the increase of APTES content from 30 to 60%, the selectivity of the imprinted hybrid membrane increased first and then decreased. For the imprinted membrane, besides the hybrid structure, the imprinting effect and the maintenance of the imprinted cavity were another
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Fig. 5. Effect of APTES content on the enantioselectivity of imprinted and control SA–APTES hybrid membranes dried at room temperature (d,l-Phe mixture with a concentration of 0.5 mmol/L for each isomer).
two determining factors for efficient chiral recognition. This chiral recognition ability was enhanced by increasing the APTES content since the inorganic phase helped in maintaining the structure of the cavities. However, when the APTES content was higher than 40%, too much more carboxyl groups on SA would be consumed by crosslinking with APTES, resulting in a weaker ability to form template–polymer complexes and thus leading to a poorer imprinting effect. Therefore, there was an optimal content of inorganic phase in the hybrid membrane to achieve a balance between structure maintenance and pre-assembly formation. Moreover, the compatibility between the organic and the inorganic phases became poorer as the APTES content increased up to 60% (Fig. 3(c)). Regarding the imprinted SA–APTES hybrid membranes prepared and dried at room temperature, the highest selectivity of 1.8 was achieved at an APTES content of 40%. 3.4. Binding isotherms Equilibrium adsorption experiments were conducted to investigate the binding behavior of the imprinted SA–APTES(D)-40 membrane for d-Phe. The equilibrium adsorption data were fitted, respectively, according to the Langmuir model and Freundlich model as shown by the following two equations: Langmuir model :
Ce 1 Ce + = Q Qmax bQmax
where Ce is the equilibrium of final concentration of d-Phe in solution, Q is the adsorption capacity of d-Phe adsorbed on the membrane at equilibrium concentration, Qmax is the maximum adsorption capacity, and b is the Langmuir constant. Freundlich model :
1/n
Q = Qf Ce
where Qf and n are the two Freumdlich constants. The linearized plots of Ce /Q versus Ce and ln Q versus ln Ce , plotted based on the above two equations respectively, were shown in Fig. 6. The calculated results were listed in Table 1. It can be Table 1 Langmuir and Freundlich isotherm constants of imprinted SA–APTES-40 membrane. Langmuir
Fig. 4. EDX Si-mapping image of SA–APTES-40 membrane.
Freundlich
Qmax (mmol/g)
b (L/mmol)
RL
Qf (mmol/g)
n
RF
0.0073
4.71
0.992
0.0064
2.90
0.972
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Fig. 6. Adsorption isotherms of imprinted SA–APTES-40 membrane for d-Phe fitted by (a) Langmuir model and (b) Freundlich model.
seen from the data in Table 1 that both the Langmuir equation and the Freundlich equation fitted well for d-Phe adsorption on the imprinted hybrid membrane within the experimental concentration range with a Langmuir correlation coefficient rL = 0.992 and a Freundlich correlation coefficient rF = 0.972. The Langmuir dimensionless constant separation factor, RL , was also calculated according to the dimensionless expression, RL = 1/(1 + bC0 ) where C0 is the initial concentration of d-Phe. RL is commonly used as an indicator in analyzing the adsorption isotherms which can be classified into four types, unfavorable, linear, favorable and irreversible, respectively corresponding to the following four different cases, RL > 1, RL = 1, 0 < RL < 1 and RL = 0 [19]. All the RL values, decreasing from 0.462 to 0.240 while the initial d-Phe concentration increased from 0.25 to 0.67 mmol/L, were lower than 1, revealing a favorable adsorption of d-Phe on the imprinted hybrid membrane. Regarding the Freundlich model, the parameter Qf and 1/n indicates the adsorption capacity and adsorption intensity, respectively. The value n = 2.9 (>1) again confirmed the favorable adsorption behavior of the imprinted hybrid membrane. In addition, Scatchard analysis was also carried out to estimate the binding performance of the imprinted membrane using the saturation binding data: Scatchard equation :
Qmax − Q Q = C0 Kd
where Kd is the equilibrium dissociation constant. The adsorption data were plotted according to the Scatchard equation as shown in Fig. 7. A straight line with a slope and intercept of −1/Kd and Qmax /Kd , respectively, was obtained, indicating that only one kind of binding sites existed in the imprinted hybrid membrane and they were homogeneous in respect to the affinity for d-Phe. A similar observation of this one kind of binding sites could be found in some other literatures [20,21], while most imprinted materials were found to possess multiple kinds of binding sites. This strong affinity was supposed to originate from the template effect in the imprinting process and the better structural maintenance attributed to the hybridizing effect via the introduction of inorganic phase. The above Scatchard analysis agreed with the results of Langmuir and Freundlich fitting where the Langmuir model fitted relatively better than the Freundlich model (rL = 0.992 > rF = 0.972). Langmuir model assumes a homogeneous binding site distribution with equal energy while the Freundlich model assumes a heterogeneous surface binding energy [22]. The non-specific adsorption not in the imprinted cavities could be ignored in the concentration range studied. The Kd and Qmax values can be calculated to be 0.226 mmol/L and 7.465 mol/g from the slope and the intercept of the Scatchard regression, respectively.
3.5. Selective binding ability towards d-Phe Binding experiments were conducted by immersing the d-Phe imprinted hybrid membranes in a d,l-Phe mixture to investigate the chiral recognition ability. The adsorption amounts for d- and l-Phe were shown in Fig. 8. Both the imprinted and control membranes showed a preferential adsorption for d-Phe over l-Phe, corresponding to the previous permeation experimental results. Compared with control membranes, d-Phe imprinted hybrid membranes showed both remarkably stronger binding ability and higher adsorption selectivity (˛S ) for d-Phe relative to l-Phe. Imprinting led to an increase in the adsorption ability for d-Phe over l-Phe by more than five times (from 1.11 to 6.14 mol/g) for SA–APTES-40 membrane and more than four times (from 1.08 to 4.63 mol/g) for SA–APTES-50 membrane compared to their corresponding control ones. Meanwhile, the adsorption selectivity of d-Phe to l-Phe for the imprinted membranes was also significantly increased by nearly two times. These adsorption data proved the efficient imprinting effect in the hybrid membranes. The improved enantioselectivity of imprinted membranes was attributed to the selective recognition and binding ability of the imprinting sites with “memory” created after the removal of templates. 3.6. Separation mechanism analysis The following two mechanisms are commonly applied in analyzing the selective transport of molecules in imprinted membranes:
Fig. 7. Scatchard plot estimate for binding nature of imprinted SA–APTES-40 membrane.
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Fig. 8. Comparison of binding properties of imprinted and control SA–APTES membranes for each isomer (d,l-Phe mixture with a concentration of 0.5 mmol/L for each isomer).
(i) facilitated permeation driven by preferential affinity and transport of the target molecules while the transport of other molecules are relatively much slower; (ii) retarded permeation due to too strong affinity binding of the target molecules while the transport of other molecules are relatively much faster until a saturation of MIP sites with target molecules is reached [23]. For SA–APTES-40 and SA–APTES-50 membranes dried at 65 ◦ C, diffusion selectivity (˛D ) was calculated according to the solution–diffusion mechanism model based on the separation factor (˛D/L ) data obtained by permeation experiments and the adsorption selectivity (˛S ) data obtained by binding experiments, and was listed in Table 2. It could be seen that both the ˛D and ˛S were higher than one, revealing that the diffusion and adsorption behavior of the hybrid membranes were both preferential toward d-isomer. The transport of the target molecules, d-Phe, across the hybrid membrane occur via a fixed carrier-mediated (“facilitated”) process by adsorbing on and desorbing from the imprinted cavities. Therefore, the transport mechanism of SA–APTES hybrid membranes for d,l-Phe isomer separation was in accordance with the first mechanism (facilitated permeation) mentioned above. This transport-selectivity mechanism was a good indication for practical application of this kind of imprinted membrane in continuous separation rather than only as a membrane adsorber for batch adsorption. Fabrication of composite membranes with a porous support and a thin imprinted skin layer might provide a possibility for continuous enantioseparation. 3.7. Separation of d-Phe and its analogue d-Tyr The separation performance of the imprinted SA–APTES hybrid membrane for d-Phe and its analogue d-Tyr was investigated. The separation factor (ˇ) was defined as the ratio of the permeability coefficients of the two molecules, ˇ = Pd-Phe /Pd-Tyr . Fig. 9 showed the separation ability of the imprinted SA–APTES membranes with various APTES contents for d-Phe and its analogue, d-Tyr. The separation factor of the control hybrid membrane for these two analogues was around 1.0 and changed little with APTES content, Table 2 The separation factor (˛D/L ), adsorption selectivity (␣S ) and diffusion selectivity (˛D ) of the imprinted and control membranes dried at 65 ◦ C. SA–APTES-40
˛D/L ˛S ˛D
SA–APTES-50
Imprinted
Control
Imprinted
Control
2.87 1.46 1.97
1.48 1.31 1.13
2.52 1.70 1.68
1.34 1.04 1.29
Fig. 9. Separation performance of imprinted SA–APTES hybrid membranes for d-Phe and its analogue d-Tyr (d-Phe and d-Tyr mixture with a concentration of 0.5 mmol/L for each amino acid).
revealing that non-imprinted SA–APTES membrane was unable to selectively separate the two D-type structural analogues only by its own chiral nature. In comparison, the separation ability was significantly enhanced by imprinting. The effect of APTES content on the separation factor, ˇ, showed a similar trend to that in separating enantiomers, i.e., ˇ increased first and then decreased with the increase of APTES content, a maximum of 2.9 appearing at 40% APTES content. There did exist an optimal organic–inorganic phase ratio where the crosslinking structure and the formation and maintenance of the imprinted cavities reached a proper balance to get the best recognition and separation performance. 4. Conclusions Molecularly imprinted SA–APTES hybrid membranes with enantioselectivity were prepared by sol–gel process in aqueous solution and successfully applied in chiral resolution of underivatized d,lPhe racemic mixture. The addition of APTES by hydrolysis and condensation significantly improved the compatibility between the organic and inorganic phase via covalent interactions, creating a dense and uniform hybrid network and thus reducing the degree of swelling of the materials in aqueous system, and finally ensuring the formation and maintenance of imprinting sites. The target molecules’ transport was facilitated by strengthening the binding
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ability of the imprinted membrane through specific interactions between the imprinting cavities and the templates, resulting in an enhanced chiral resolution ability. The adsorption behavior of the imprinted hybrid membrane fitted the Langmuir model well, suggesting only one kind of binding site existed in the membrane. In addition, these imprinted hybrid membranes also exhibited a promising separation performance for separation of D-type structural analogues. Acknowledgements We gratefully acknowledge financial supports from National Natural Science Foundation of China (No. 20306021) and the Programme of Introducing Talents of Discipline to Universities (No. B06006). References [1] T.Q. Yan, C. Orihuela, Rapid and high throughput separation technologies— steady state recycling and supercritical fluid chromatography for chiral resolution of pharmaceutical intermediates, J. Chromatogr. A 1156 (2007) 220–227. [2] R.C. Williams, C.M. Riley, K.W. Sigvardson, J. Fortunak, P. Ma, E.C. Nicolas, S.E. Unger, D.F. Krahn, S.L. Bremner, Pharmaceutical development and specification of stereoisomers, J. Pharm. Biomed. Anal. 17 (1998) 917–924. [3] M. Yoshikawa, K. Murakoshi, T. Kogita, K. Hanaoka, M.D. Guiver, G.P. Robertson, Chiral separation membranes from modified polysulfone having myrtenalderived terpenoid side groups, Eur. Polym. J. 42 (2006) 2532–2539. [4] M. Lehmann, M. Dettling, H. Brunner, G.E.M. Tovar, Affinity parameters of amino acid derivative binding to molecularly imprinted nanospheres consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)], J. Chromatogr. B 808 (2004) 43–50. [5] T.Y. Guo, Y.Q. Xia, J. Wang, M.D. Song, B.H. Zhang, Chitosan beads as molecularly imprinted polymer matrix for selective separation of proteins, Biomaterials 26 (2005) 5737–5745. [6] Z.Y. Jiang, Y.X. Ying, H. Wu, Preparation and applications of molecularly imprinted polymer membranes, Membr. Sci. Technol. 26 (2006) 78–84 (in Chinese). [7] D. Silvestri, N. Barbani, C. Cristallini, P. Giusti, G. Ciardelli, Molecularly imprinted membranes for an improved recognition of biomolecules in aqueous medium, J. Membr. Sci. 282 (2006) 284–295. [8] Z.Y. Jiang, Y.X. Ying, H. Wu, Preparation of CS/GPTEMS hybrid molecularly imprinted membrane for efficient chiral resolution of phenylalanine isomers, J. Membr. Sci. 280 (2006) 876–882.
[9] R. Gupta, A. Kumar, Molecular imprinting in sol–gel matrix, Biotechnol. Adv. 26 (2008) 533–547. [10] M.E. Diaz-Garcia, R.B. Laino, Molecular imprinting in sol–gel materials: recent developments and applications, Microchim. Acta 149 (2005) 19–36. [11] Z.F. Cai, H.J. Dai, S.H. Si, F.L. Ren, Molecular imprinting and adsorption of metallothionein on nanocrystalline titania membranes, Appl. Surf. Sci. 254 (2008) 4457–4461. [12] J.H. Chen, Q.L. Liu, X.H. Zhang, Q.G. Zhang, Pervaporation and characterization of chitosan membranes cross-linked by 3-aminopropyltriethoxysilane, J. Membr. Sci. 292 (2007) 125–132. [13] R.L. Guo, H. Wu, Z.Y. Jiang, PVA–GPTMS/TEOS hybrid pervaporation membrane for dehydration of ethylene glycol aqueous solution, J. Membr. Sci. 281 (2006) 454–462. [14] C.K. Yeom, K.H. Lee, Vapor permeation of ethanol–water mixtures using sodium alginate membranes with crosslinking gradient structure, J. Membr. Sci. 135 (1997) 225–235. [15] S. Kalyani, B. Smitha, S. Sridhar, A. Krishnaiah, Pervaporation separation of ethanol–water mixtures through sodium alginate membranes, Desalination 229 (2008) 68–81. [16] H. Zimmermann, F. Wählisch, C. Baier, M. Westhoff, R. Reuss, D. Zimmermann, M. Behringer, F. Ehrhart, A. Katsen-Globa, C. Giese, U. Marx, V.L. Sukhorukov, J.A. Vásquez, P. Jakob, S.G. Shirley, U. Zimmermann, Physical and biological properties of barium cross-linked alginate membranes, Biomaterials 28 (2007) 1327–1345. [17] M.B. Patil, R.S. Veerapur, S.A. Patil, C.D. Madhusoodana, T.M. Aminabhavi, Preparation and characterization of filled matrix membranes of sodium alginate incorporated with aluminum-containing mesoporous silica for pervaporation dehydration of alcohols, Sep. Purif. Technol. 54 (2007) 34–43. [18] Y.Q. Dong, L. Zhang, J.N. Shen, M.Y. Song, H.L. Chen, Preparation of poly(vinyl alcohol)-sodium alginate hollow-fiber composite membranes and pervaporation dehydration characterization of aqueous alcohol mixtures, Desalination 193 (2006) 202–210. [19] W.S.W. Ngah, C.S. Endud, R. Mayanar, Removal of copper(II) ions from aqueous solution onto chitosan and cross-linked chitosan beads, React. Funct. Polym. 50 (2002) 181–190. [20] T. Jiang, L. Zhao, B. Chu, Q. Feng, W. Yan, J. Lin, Molecularly imprinted solidphase extraction for the selective determination of 17-estradiol in fishery samples with high performance liquid chromatography, Talanta 78 (2009) 442–447. [21] B. Okutucu, S. Onal, A. Telefoncu, Noncovalently galactose imprinted polymer for the recognition of different saccharides, Talanta 78 (2009) 1190–1193. [22] X. Li, S.M. Husson, Adsorption of dansylated amino acids on molecularly imprinted surfaces: a surface plasmon resonance study, Biosens. Bioelectron. 22 (2006) 336–348. [23] M. Ulbricht, Membrane separations using molecularly imprinted polymers, J. Chromatogr. B 804 (2004) 113–125.
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Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Molecularly imprinted organic–inorganic hybrid membranes for selective separation of phenylalanine isomers and its analogue Hong Wu a,b,∗ , Yanyan Zhao a , Mingcheng Nie a , Zhongyi Jiang a a b
Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072, China
a r t i c l e
i n f o
Article history: Received 6 February 2009 Received in revised form 3 April 2009 Accepted 10 April 2009 Keywords: Optical resolution Molecularly imprinted hybrid membrane Sodium alginate Silane d,l-Phenylalanine
a b s t r a c t A novel kind of molecularly imprinted organic–inorganic hybrid membrane, using sodium alginate (SA) and 3-aminopropyltriethoxysilane (APTES) as the organic and inorganic phase, respectively, was prepared and investigated for the optical resolution of chiral isomers. The hybrid membrane was prepared in an aqueous environment with the presence of d-phenylalanine (d-Phe) as imprinting templates, and the silica phase was introduced in the alginate matrix by sol–gel process of APTES. It was found by FTIR and SEM–EDX characterizations that strong covalent bonds were formed between the organic and inorganic phases, and silica was homogenously distributed in the matrix. These molecularly imprinted SA–APTES hybrid membranes showed chiral separation ability towards the d,l-Phe isomers with a maximum selectivity (˛D/L ) of 1.8 at 40% APTES content. The recognition mechanism was tentatively analyzed and discussed based on permeation and binding experiments. The separation ability of the above hybrid membranes toward d-Phe and its analogue, d-tyrosine (d-Tyr), was also investigated, and a separation factor of up to 2.9 was achieved. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Most important artificial pharmaceutical compounds produced by chemical reactions are chiral. The two enantiomers exist as either a right-handed or a left-handed molecule with almost identical physical and chemical properties but probably quite different physiological activities. One enantiomer may be effective as a drug, while the other is ineffective or even toxic [1]. Chiral separation has become an indispensible process for all the chiral drug productions and needs to be more precise and efficient to meet health and drug safety requirements. Due to their analogical structure and property, the optical resolution for enantiomers remains one of the most difficult separation tasks [2]. Current isomer separation methods mainly include recrystallization, chromatography, enzymatic resolution and membrane separation. Membrane separation process shows advantages over the other techniques in productivity, operational conditions and cost [3]. The invention of molecular imprinting technology further promotes the design and development of novel membranes specifically for efficient chiral separation. There has been an increasing interest in the synthesis and study of molecularly imprinted polymers (MIPs) used as syn-
∗ Corresponding author at: Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China. Tel.: +86 22 27890882; fax: +86 22 23500086. E-mail address: [email protected] (H. Wu). 1383-5866/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2009.04.014
thetic affinity materials [4]. Molecular imprinting is a technique to introduce molecular recognition sites for a specific analyte in a synthetic polymer for selective separation or concentration of target molecules. This technique involves the formation of complexes between imprinting molecules (templates) and functional monomers/polymers based on the following three interactions: covalent bonds, non-covalent interactions or metal ion coordinations [5]. The monomer/polymer–template complexes are then fixed by polymerization and/or crosslinking. Removal of the templates finally results in cavities with a shape, structure and functional groups complementary to the templates. MIPs have been widely studied in liquid chromatography, solid phase extraction and facilitated catalysis [6]. Employment of molecular imprinting technique in membrane preparation produces molecularly imprinted membranes (MIMs), highlighting the possibility of more precise separation by membranes. A number of researchers have carried out systematic investigations on the molecularly imprinted membranes prepared in non-aqueous solutions (organic solvents) through covalent-bond or noncovalent-bond approaches followed by phase inversion process [7]. However, in case of biomolecular imprinting, an aqueous imprinting process is obviously much preferred owing to the much less solubility of the templates and to minimize the risk of losing bioactivities in organic solvents. In aqueous environment, the nonspecific interactions (mainly hydrogen bonding) between the templates and the functional monomers/polymers may become much weaker due to the presence of water. How to realizing and
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maintaining the imprinting of biomolecules in the membrane is a challenge for MIM development [8]. Molecularly imprinted inorganic materials, usually prepared by sol–gel process, have advantages in keeping the structure of the imprinted cavities unchanged after removal of the templates owing to their rigid nature compared to flexible organic polymers [9–11]. However, the fact that it is rather difficult or even impossible to remove templates inside the inorganic matrix greatly confines its use only to surface imprinting. Moreover, the frangible nature of inorganic materials which makes it hard to prepare large-area membrane pieces also limits their practical application. Developing organic–inorganic hybrid membranes may provide a solution to satisfy the requirements from the two aspects: unchanged imprinted cavity and good film-forming property. Hybrid membranes have shown extraordinary properties in pervaporative separation area with improved physical and mechanical properties arising form the synergism of the two kinds of building blocks [12,13]. Alginate, a polysaccharide extracted from seaweeds, is a biodegradable, biocompatible and non-toxic polymer with good film-forming property. Besides, its natural chiral environment and the large number of carboxyl groups which can assemble with such template molecules as amino acids through forming static interactions makes it an attractive material for the preparation of optical resolution membranes. But alginate alone cannot fulfill this task successfully since imprinted cavities would not be well retained due to its severe swelling and eventual dissolution in aqueous solutions. One way to overcome this drawback is by chemical crosslinking. The commonly used crosslinkers for alginate include glutaraldehyde [14], phosphoric acid [15] and divalent cations (e.g. Ba2+ , Ca2+ ) [16]. Herein, 3-aminopropyltriethoxysilane, a silane-coupling agent with a 3-aminopropyl and three ethoxysilane groups, was used as an efficient crosslinking agent for alginate modification as well as to introduce inorganic silica phases. A novel molecularly imprinted hybrid membrane, denoted as SA–APTES for simplicity, was prepared by using sodium alginate (SA) as polymer material and APTES as not only a precursor for introducing inorganic component into organic polymer matrix through sol–gel process but also a crosslinking agent to strengthen the mechanical strength of SA membrane and thus reduce its swelling degree in aqueous solutions. One of the target enantiomers for separation, d-phenylalanine (d-Phe), was used as the template molecule. The binding and permeation property and mechanism of the membranes for chiral resolution of the d,l-Phe aqueous mixtures as well as d-Phe and d-Tyr mixture was investigated and analyzed.
The hot solution was filtered and a known amount of APTES was added into the filtrate followed by addition of a small amount of 1 mol/L ammonia solution. The mixture was vigorously stirred for 12 h before 80 mg of d-Phe template was added. The solution was stirred for another 12 h. The resulting homogeneous solution was cast onto a clean glass plate with the aid of a casting knife. The membrane was allowed to dry at room temperature for 48 h first and then annealed at 60 ◦ C for 2 h. The dried membrane was peeled off from the glass plate and immersed in a crosslinking bath containing 10 vol.% of GA, 0.05 vol.% of concentrated HCl in water–acetone (30:70) mixture at 40 ◦ C for 24 h. After crosslinking, the membrane was washed repeatedly with deionized water to remove any possible residual of HCl and GA. For the extraction of templates, the membrane was washed by or immersed in deionized water until no d-Phe molecules could be detected by HPLC (Agilent 1100). Finally, the membrane was either dried at room temperature or in a hot air oven at 65 ◦ C. The relative mass content of APTES to SA was varied by 30, 40, 50, and 60%, and the resulting membranes were designated correspondingly as SA–APTES-30, SA–APTES-40, SA–APTES-50, SA–APTES-60. The resulting membrane was transparent in appearance and self-standing with a thickness of 30–40 m. Non-imprinted control membrane was prepared in the same way without adding any template molecules during preparation. 2.3. Membrane characterizations FT-IR spectra of the plain SA and SA/APTES were scanned in the range of 4000–600 cm−1 on a Nicolet 5DX instrument equipped with horizontal attenuated total reflectance accessories. The surface and cross-section morphologies of the hybrid membranes were observed by scanning electron microscopy (SEM) and the distribution of silica in the SA polymer domains was observed by EDX Si-mapping analysis attached to the SEM (Philips XL30E SEM). Membranes were frozen in liquid nitrogen, broken, and sputtered with gold before observation. 2.4. Binding experiments Static binding experiments were conducted to evaluate the recognition property of the membranes toward the target molecule. The imprinted and control membrane samples were, respectively, immersed in the isomer mixture of d,l-Phe with a concentration of 0.5 mmol/L for each isomer at room temperature for 12 h. The concentration of d-Phe and l-Phe in the solution was monitored
2. Experimental 2.1. Materials SA was purchased from Shanghai Tianlian Fine Chemical Corporation (Shanghai, China). APTES was purchased from Fluka (USA). d,l-Phe (>98%) and d-tyrosine (d-Tyr, ≥99%) from Sigma–Aldrich Co.(USA) were used as purchased. Glutaraldehyde (GA) (50% content in water), ammonia (25% content), hydrochloric acid (HCl) (35% content) and acetone were supplied by Tianjin Jiangtian Chemical Corporation (Tianjin, China). Acetic acid and methanol of HPLC grade were purchased from Tianjin Guangfu Chemical Corporation. Deionized water was used throughout this study. 2.2. Membrane preparation The SA solution with a concentration of 1.6 wt% was prepared by dissolving 0.8 g of SA in water at 60 ◦ C under stirring for 6 h.
Fig. 1. FT-IR spectra of SA and SA–APTES membranes.
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by analyzing with an HPLC (Agilent 1100) equipped with a UV detector and a Regis ChirosilSCA(−) column (150 mm × 4.6 mm), using methanol–water–acetic acid solution as mobile phase. The uptake (Q, mol/g dry membrane) of d,l-Phe and the adsorption selectivity (˛S ) were calculated according to the following two equations: Qi =
(Cfi − Cei )V , m
˛S =
(CfD − CeD )/(CfL − CeL ) CeD /CeL
i = D or L
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was calculated as follows: ˛D =
˛D/L ˛S
where ˛D/L represents the separation factor obtained by separation experiments described in the following section. 2.5. Separation experiments
where Cfi and Cei are the initial and equilibrium concentrations of d,l-Phe, respectively, m is the weight of dry hybrid membrane sample and V is the volume of solution. According to the solution–diffusion mechanism, commonly used to describe the transportation behavior of molecules across dense membrane, the diffusion selectivity (˛D ) of the membrane
To evaluate the separation performance of the imprinted membrane towards two isomers, permeation tests were conducted. The membrane sample was mounted tightly between two chambers (20 mL for each chamber) in a diffusion cell. A mixture of d,l-Phe with a total concentration of 1 mmol/L (0.5 mmol/L for each isomer) was added into the feeding chamber while water was added into the receiving chamber. The concentration change of d,l-Phe in the receiving chamber was monitored and determined by HPLC. The permeability coefficient P (cm2 /s) and the separation factor (˛D/L )
Fig. 2. Schematic depiction of the formation of d-Phe imprinted SA–APTES hybrid membrane.
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were calculated by the following equations: Pi =
Ki Vd A(CFi − CRi )
˛D/L =
i = L, D
PD PL
where Ki is the concentration change rate of the receiving solution obtained by calculating the slope value of the diffused analyte concentration–time curve, V is the volume of solution, A is the effective membrane area, d is the membrane thickness and (CFi − CRi ) is the concentration difference between feeding and receiving chambers. The separation ability of the hybrid membranes for separating d-Phe and its analogue, d-Tyr, was also investigated by permeation tests. The experimental procedure was the same as that described above except that l-Phe was replaced by d-Tyr. 3. Results and discussion 3.1. Formation of SA/APTES hybrid molecularly imprinted membranes The chemical structure of the SA–APTES hybrid membranes was analyzed by FT-IR spectra in the range of 4000–650 cm−1 as shown in Fig. 1. The absorption peaks appeared at 1602 cm−1 and 1412 cm−1 were assigned to the asymmetric and symmetric stretching of carboxylate salt groups on sodium alginate [17,18]. The absorption bands of the amino groups (–NH2 ) attributed to the addition of APTES, which should have appeared at 1596 cm−1 , was overlapped with the peak of carboxylate groups. The overlapped peak of –COOH and –NH2 was weakened with the increase of relative mass content of APTES added to the membrane. This was due to the chemical reaction happened between the –COOH group of SA and the –NH2 group of APTES, resulting in a new peak appeared at 1486 cm−1 which was attributed to the amide covalent bonds (–CO–NH–). The absorption peaks at 1000–1100 cm−1
were attributed to both the C–O groups on SA and the formation of Si–O–Si bonds (1095 cm−1 ) as a result of condensation of the silanol groups (Si–OH) after hydrolysis of APTES. The formation of –CO–NH– bonds was expected to remarkably improve the compatibility between the organic and the inorganic components and thus benefit the maintenance of the structure of the imprinted cavities. A possible process for the formation of the imprinted hybrid membrane was presented and illustrated schematically in Fig. 2. The difficulty in imprinting under aqueous condition lies in the weakening effect of water on the formation of noncovalent interactions between the template and the polymer since water would compete with the templates for functional groups of the polymer to form hydrogen bonds. The abundance of carboxyl groups and hydroxyl groups on SA was supposed to enhance the interactions between template and functional polymer to form pre-assemble complex. Besides, the amino groups that remained after hydrolysis of APTES might also interact with the carboxyl groups on the amino acid templates, thus making additional favorable contribution to keeping the structure of the imprinting sites after silica condensation. A portion of the residual carboxyl groups as well as hydroxyl groups of SA would be crosslinked by forming covalent bonds with the amino groups and the silanol groups of the hydrolyzed APTES during sol–gel process. A dense organic–inorganic network was thus fabricated. In addition, the propyl chain connecting the amino group and the silicon was thought to produce a transition area between the inorganic silica phase and the organic SA chains. After removal of templates, the imprinted cavities with distinct shape, size and chemical functionality remained in the hybrid matrix and the specific recognition sites were formed. 3.2. Membrane morphology and silica distribution The SEM images of the membrane surface of the SA–APTES hybrid membrane were shown in Fig. 3. The SEM images (Fig. 3(a)–(c)) confirmed the dense structure of the hybrid membranes prepared. The transparent appearance of the hybrid membranes was a macroscopic indication of the good compatibil-
Fig. 3. SEM images of imprinted SA–APTES hybrid membrane surface: (a) SA–APTES-40; (b) SA–APTES-50; (c) SA–APTES-60; (d) SA–APTES-40 with a SA/d-Phe mass ratio of 6. The SA/d-Phe mass ratio of (a)–(c) membrane was 10.
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ity between the polymer and the silica phases. When the APTES content reached 50%, some silica nanoparticles could be observed on the membrane surface by SEM, and when this content further increased to 60%, the particles became larger, revealing an increasing aggregation of the silica under higher concentrations. In this study, the APTES content was kept lower than 60%. The amount of templates used for imprinting is a key factor that influences the amount of imprinting cavities and final selectivity. The effect of template content on the separation factor was investigated by decreasing the polymer/template ratio from 20, 10 to 6 (data not shown). It had been found that the separation selectivity increased first notably with the increase of the template amount (ratio decreasing from 20 to 10), and then level off, little change in selectivity was found when the ratio further decreased from 10 to 6. However, the quality of the membrane at higher template content was rather poor. Fig. 3(d) showed the SEM image of the imprinted SA–APTES-40 membrane surface with a higher SA/d-Phe mass ratio of 6. Some diamond- or rectangle-shaped delves could be clearly observed on the surface. These defects resulted from the crystallization of the amino acids during evaporation of solvent due to the high concentration of templates. The higher the template content was, the bigger the crystal particles became, leading to big defects after template removal. Taking both selectivity and membrane quality into consideration, a ratio of 10 was applied for membrane preparation. Fig. 4 showed the EDX Si-mapping image of the hybrid membrane. A uniform dispersion of silica domains (the bright spots) inside the SA matrix was observed, revealing a successful introduction of inorganic phase in organic polymer at molecular level by APTES sol–gel process. 3.3. Effect of APTES content on the enantioselectivity The separation factors (enantioselectivity) of SA–APTES hybrid membranes prepared with different contents of APTES and dried at room temperature were shown in Fig. 5. Both the imprinted and control membranes under study showed a preferential permeation for d-Phe over l-Phe. Pre-experiments showed that when APTES content was low (<30%), almost no selective ability was found due to the loose and too flexible structure. With the increase of APTES content from 30 to 60%, the selectivity of the imprinted hybrid membrane increased first and then decreased. For the imprinted membrane, besides the hybrid structure, the imprinting effect and the maintenance of the imprinted cavity were another
101
Fig. 5. Effect of APTES content on the enantioselectivity of imprinted and control SA–APTES hybrid membranes dried at room temperature (d,l-Phe mixture with a concentration of 0.5 mmol/L for each isomer).
two determining factors for efficient chiral recognition. This chiral recognition ability was enhanced by increasing the APTES content since the inorganic phase helped in maintaining the structure of the cavities. However, when the APTES content was higher than 40%, too much more carboxyl groups on SA would be consumed by crosslinking with APTES, resulting in a weaker ability to form template–polymer complexes and thus leading to a poorer imprinting effect. Therefore, there was an optimal content of inorganic phase in the hybrid membrane to achieve a balance between structure maintenance and pre-assembly formation. Moreover, the compatibility between the organic and the inorganic phases became poorer as the APTES content increased up to 60% (Fig. 3(c)). Regarding the imprinted SA–APTES hybrid membranes prepared and dried at room temperature, the highest selectivity of 1.8 was achieved at an APTES content of 40%. 3.4. Binding isotherms Equilibrium adsorption experiments were conducted to investigate the binding behavior of the imprinted SA–APTES(D)-40 membrane for d-Phe. The equilibrium adsorption data were fitted, respectively, according to the Langmuir model and Freundlich model as shown by the following two equations: Langmuir model :
Ce 1 Ce + = Q Qmax bQmax
where Ce is the equilibrium of final concentration of d-Phe in solution, Q is the adsorption capacity of d-Phe adsorbed on the membrane at equilibrium concentration, Qmax is the maximum adsorption capacity, and b is the Langmuir constant. Freundlich model :
1/n
Q = Qf Ce
where Qf and n are the two Freumdlich constants. The linearized plots of Ce /Q versus Ce and ln Q versus ln Ce , plotted based on the above two equations respectively, were shown in Fig. 6. The calculated results were listed in Table 1. It can be Table 1 Langmuir and Freundlich isotherm constants of imprinted SA–APTES-40 membrane. Langmuir
Fig. 4. EDX Si-mapping image of SA–APTES-40 membrane.
Freundlich
Qmax (mmol/g)
b (L/mmol)
RL
Qf (mmol/g)
n
RF
0.0073
4.71
0.992
0.0064
2.90
0.972
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Fig. 6. Adsorption isotherms of imprinted SA–APTES-40 membrane for d-Phe fitted by (a) Langmuir model and (b) Freundlich model.
seen from the data in Table 1 that both the Langmuir equation and the Freundlich equation fitted well for d-Phe adsorption on the imprinted hybrid membrane within the experimental concentration range with a Langmuir correlation coefficient rL = 0.992 and a Freundlich correlation coefficient rF = 0.972. The Langmuir dimensionless constant separation factor, RL , was also calculated according to the dimensionless expression, RL = 1/(1 + bC0 ) where C0 is the initial concentration of d-Phe. RL is commonly used as an indicator in analyzing the adsorption isotherms which can be classified into four types, unfavorable, linear, favorable and irreversible, respectively corresponding to the following four different cases, RL > 1, RL = 1, 0 < RL < 1 and RL = 0 [19]. All the RL values, decreasing from 0.462 to 0.240 while the initial d-Phe concentration increased from 0.25 to 0.67 mmol/L, were lower than 1, revealing a favorable adsorption of d-Phe on the imprinted hybrid membrane. Regarding the Freundlich model, the parameter Qf and 1/n indicates the adsorption capacity and adsorption intensity, respectively. The value n = 2.9 (>1) again confirmed the favorable adsorption behavior of the imprinted hybrid membrane. In addition, Scatchard analysis was also carried out to estimate the binding performance of the imprinted membrane using the saturation binding data: Scatchard equation :
Qmax − Q Q = C0 Kd
where Kd is the equilibrium dissociation constant. The adsorption data were plotted according to the Scatchard equation as shown in Fig. 7. A straight line with a slope and intercept of −1/Kd and Qmax /Kd , respectively, was obtained, indicating that only one kind of binding sites existed in the imprinted hybrid membrane and they were homogeneous in respect to the affinity for d-Phe. A similar observation of this one kind of binding sites could be found in some other literatures [20,21], while most imprinted materials were found to possess multiple kinds of binding sites. This strong affinity was supposed to originate from the template effect in the imprinting process and the better structural maintenance attributed to the hybridizing effect via the introduction of inorganic phase. The above Scatchard analysis agreed with the results of Langmuir and Freundlich fitting where the Langmuir model fitted relatively better than the Freundlich model (rL = 0.992 > rF = 0.972). Langmuir model assumes a homogeneous binding site distribution with equal energy while the Freundlich model assumes a heterogeneous surface binding energy [22]. The non-specific adsorption not in the imprinted cavities could be ignored in the concentration range studied. The Kd and Qmax values can be calculated to be 0.226 mmol/L and 7.465 mol/g from the slope and the intercept of the Scatchard regression, respectively.
3.5. Selective binding ability towards d-Phe Binding experiments were conducted by immersing the d-Phe imprinted hybrid membranes in a d,l-Phe mixture to investigate the chiral recognition ability. The adsorption amounts for d- and l-Phe were shown in Fig. 8. Both the imprinted and control membranes showed a preferential adsorption for d-Phe over l-Phe, corresponding to the previous permeation experimental results. Compared with control membranes, d-Phe imprinted hybrid membranes showed both remarkably stronger binding ability and higher adsorption selectivity (˛S ) for d-Phe relative to l-Phe. Imprinting led to an increase in the adsorption ability for d-Phe over l-Phe by more than five times (from 1.11 to 6.14 mol/g) for SA–APTES-40 membrane and more than four times (from 1.08 to 4.63 mol/g) for SA–APTES-50 membrane compared to their corresponding control ones. Meanwhile, the adsorption selectivity of d-Phe to l-Phe for the imprinted membranes was also significantly increased by nearly two times. These adsorption data proved the efficient imprinting effect in the hybrid membranes. The improved enantioselectivity of imprinted membranes was attributed to the selective recognition and binding ability of the imprinting sites with “memory” created after the removal of templates. 3.6. Separation mechanism analysis The following two mechanisms are commonly applied in analyzing the selective transport of molecules in imprinted membranes:
Fig. 7. Scatchard plot estimate for binding nature of imprinted SA–APTES-40 membrane.
H. Wu et al. / Separation and Purification Technology 68 (2009) 97–104
103
Fig. 8. Comparison of binding properties of imprinted and control SA–APTES membranes for each isomer (d,l-Phe mixture with a concentration of 0.5 mmol/L for each isomer).
(i) facilitated permeation driven by preferential affinity and transport of the target molecules while the transport of other molecules are relatively much slower; (ii) retarded permeation due to too strong affinity binding of the target molecules while the transport of other molecules are relatively much faster until a saturation of MIP sites with target molecules is reached [23]. For SA–APTES-40 and SA–APTES-50 membranes dried at 65 ◦ C, diffusion selectivity (˛D ) was calculated according to the solution–diffusion mechanism model based on the separation factor (˛D/L ) data obtained by permeation experiments and the adsorption selectivity (˛S ) data obtained by binding experiments, and was listed in Table 2. It could be seen that both the ˛D and ˛S were higher than one, revealing that the diffusion and adsorption behavior of the hybrid membranes were both preferential toward d-isomer. The transport of the target molecules, d-Phe, across the hybrid membrane occur via a fixed carrier-mediated (“facilitated”) process by adsorbing on and desorbing from the imprinted cavities. Therefore, the transport mechanism of SA–APTES hybrid membranes for d,l-Phe isomer separation was in accordance with the first mechanism (facilitated permeation) mentioned above. This transport-selectivity mechanism was a good indication for practical application of this kind of imprinted membrane in continuous separation rather than only as a membrane adsorber for batch adsorption. Fabrication of composite membranes with a porous support and a thin imprinted skin layer might provide a possibility for continuous enantioseparation. 3.7. Separation of d-Phe and its analogue d-Tyr The separation performance of the imprinted SA–APTES hybrid membrane for d-Phe and its analogue d-Tyr was investigated. The separation factor (ˇ) was defined as the ratio of the permeability coefficients of the two molecules, ˇ = Pd-Phe /Pd-Tyr . Fig. 9 showed the separation ability of the imprinted SA–APTES membranes with various APTES contents for d-Phe and its analogue, d-Tyr. The separation factor of the control hybrid membrane for these two analogues was around 1.0 and changed little with APTES content, Table 2 The separation factor (˛D/L ), adsorption selectivity (␣S ) and diffusion selectivity (˛D ) of the imprinted and control membranes dried at 65 ◦ C. SA–APTES-40
˛D/L ˛S ˛D
SA–APTES-50
Imprinted
Control
Imprinted
Control
2.87 1.46 1.97
1.48 1.31 1.13
2.52 1.70 1.68
1.34 1.04 1.29
Fig. 9. Separation performance of imprinted SA–APTES hybrid membranes for d-Phe and its analogue d-Tyr (d-Phe and d-Tyr mixture with a concentration of 0.5 mmol/L for each amino acid).
revealing that non-imprinted SA–APTES membrane was unable to selectively separate the two D-type structural analogues only by its own chiral nature. In comparison, the separation ability was significantly enhanced by imprinting. The effect of APTES content on the separation factor, ˇ, showed a similar trend to that in separating enantiomers, i.e., ˇ increased first and then decreased with the increase of APTES content, a maximum of 2.9 appearing at 40% APTES content. There did exist an optimal organic–inorganic phase ratio where the crosslinking structure and the formation and maintenance of the imprinted cavities reached a proper balance to get the best recognition and separation performance. 4. Conclusions Molecularly imprinted SA–APTES hybrid membranes with enantioselectivity were prepared by sol–gel process in aqueous solution and successfully applied in chiral resolution of underivatized d,lPhe racemic mixture. The addition of APTES by hydrolysis and condensation significantly improved the compatibility between the organic and inorganic phase via covalent interactions, creating a dense and uniform hybrid network and thus reducing the degree of swelling of the materials in aqueous system, and finally ensuring the formation and maintenance of imprinting sites. The target molecules’ transport was facilitated by strengthening the binding
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ability of the imprinted membrane through specific interactions between the imprinting cavities and the templates, resulting in an enhanced chiral resolution ability. The adsorption behavior of the imprinted hybrid membrane fitted the Langmuir model well, suggesting only one kind of binding site existed in the membrane. In addition, these imprinted hybrid membranes also exhibited a promising separation performance for separation of D-type structural analogues. Acknowledgements We gratefully acknowledge financial supports from National Natural Science Foundation of China (No. 20306021) and the Programme of Introducing Talents of Discipline to Universities (No. B06006). References [1] T.Q. Yan, C. Orihuela, Rapid and high throughput separation technologies— steady state recycling and supercritical fluid chromatography for chiral resolution of pharmaceutical intermediates, J. Chromatogr. A 1156 (2007) 220–227. [2] R.C. Williams, C.M. Riley, K.W. Sigvardson, J. Fortunak, P. Ma, E.C. Nicolas, S.E. Unger, D.F. Krahn, S.L. Bremner, Pharmaceutical development and specification of stereoisomers, J. Pharm. Biomed. Anal. 17 (1998) 917–924. [3] M. Yoshikawa, K. Murakoshi, T. Kogita, K. Hanaoka, M.D. Guiver, G.P. Robertson, Chiral separation membranes from modified polysulfone having myrtenalderived terpenoid side groups, Eur. Polym. J. 42 (2006) 2532–2539. [4] M. Lehmann, M. Dettling, H. Brunner, G.E.M. Tovar, Affinity parameters of amino acid derivative binding to molecularly imprinted nanospheres consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)], J. Chromatogr. B 808 (2004) 43–50. [5] T.Y. Guo, Y.Q. Xia, J. Wang, M.D. Song, B.H. Zhang, Chitosan beads as molecularly imprinted polymer matrix for selective separation of proteins, Biomaterials 26 (2005) 5737–5745. [6] Z.Y. Jiang, Y.X. Ying, H. Wu, Preparation and applications of molecularly imprinted polymer membranes, Membr. Sci. Technol. 26 (2006) 78–84 (in Chinese). [7] D. Silvestri, N. Barbani, C. Cristallini, P. Giusti, G. Ciardelli, Molecularly imprinted membranes for an improved recognition of biomolecules in aqueous medium, J. Membr. Sci. 282 (2006) 284–295. [8] Z.Y. Jiang, Y.X. Ying, H. Wu, Preparation of CS/GPTEMS hybrid molecularly imprinted membrane for efficient chiral resolution of phenylalanine isomers, J. Membr. Sci. 280 (2006) 876–882.
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