Available online at Journal of Chromatography A, 1187 (2008)

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1 Available online at Journal of Chromatography A, 1187 (2008) Molecularly imprinted polymer prepared with bonded -cyclodextrin and acrylamide on functionalized silica gel for selective recognition of tryptophan in aqueous media Lei Qin a, Xi-Wen He a, Wen-You Li a,, Yu-Kui Zhang a,b a Department of Chemistry, Nankai University, 94 Weijin Road, Tianjin , China b National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Acadamy of Sciences, Dalian , China Received 18 September 2007; received in revised form 4 February 2008; accepted 5 February 2008 Available online 8 February 2008 Abstract A novel molecularly imprinted polymer (MIP) selective for tryptophan (Trp) was described where polymerization was performed in aqueous media. Three kinds of molecularly imprinted polymers were prepared with surface molecular imprinting technique on functionalized silica gel (F-silica gel). MIPs prepared using bonded -cyclodextrin ( -CD) and acrylamide (AA), either separately or in combination have shown various recognition properties. The results of adsorption experiments indicated that the selectivity of MIP, which was synthesized with bonded -CD and AA [MIP(1)], was superior to those obtained with AA [MIP(2)] or bonded -CD [MIP(3)]. In addition, the high-performance liquid chromatography (HPLC) column packed with MIP(1) could not only separate Trp from other aromatic amino acids, but also separate the template from its enantiomer in aqueous mobile phase. This study developed a new method for chiral amino acid separation and purification Elsevier B.V. All rights reserved. Keywords: Surface molecular imprinting technique; Silica gel; -Cyclodextrin; Tryptophan; High-performance liquid chromatography; Enantiomer separation 1. Introduction The development of a synthetic approach that could be used to produce materials with comparable recognition properties is of obvious importance. By mimicking bio-system, molecular imprinting technology provides a promising alternative way to create highly specific recognition sites within a synthetic polymer network via the template polymerization process [1,2]. Recently, considerable attentions have been paid to molecular imprinting technology [3 5]. The remarkable advantages of MIP compared to bio-system like antibodies are their reusability and low cost. To date, MIPs have been widely used in many areas including chromatography stationary phase for separation purpose [6,7], solid-phase extraction [8 10], antibody and receptor mimics [11,12], various sensor strategies [13,14] and catalysis studies [15,16]. MIP has also been employed for the recognition of proteins [17,18]. Corresponding author. Tel.: ; fax: address: wyli@nankai.edu.cn (W.-Y. Li). Various attempts have been made in recent years to improve the preparation method of the imprinted polymers [19,20]. The preparation and application of MIP in aqueous media would be of great potential for the use of the imprinted polymer in the field of biomedical diagnostic [21 22]. However, preparing imprinted polymer in aqueous media has been proven to be a difficult task since water generally destroys the polar interaction between the functional monomer and the template molecule. In order to expand the molecular imprinting techniques, the study of preparing MIP in water gives a meaningful challenge. This unique polymer comprised a hydrophobic moietyselective recognition element (bonded -CD) and a hydrogen bond interaction functional monomer (AA). As shown in Fig. 1c, -CD moiety could interact with the indole ring of the template molecule (Trp) hydrophobicly [23 25] and AA could form hydrogen bond with the amino and carboxyl group of Trp [26 27]. This work used bonded -CD ( -CD and vinyl group were chemically bonded to silica gel), which was different from previous reports using acryloyl -CD [23,28,29]. This method provided more effective recognition sites using bonded -CD and AA on the surface of silica gel than the polymer of /$ see front matter 2008 Elsevier B.V. All rights reserved. doi: /j.chroma

2 L. Qin et al. / J. Chromatogr. A 1187 (2008) Fig. 1. The first step (a) and the second step (b) for the synthesis of F-silica gel, and the procedure of the immobilization of imprinted polymer on F-silica gel (c). acryloyl -CD which was just simply random grafted on the matrix. Using bonded -CD also avoided using virulent acryloyl chloride when synthesizing the acryloyl -CD. Comparing to previous work, this method is much easier to get better MIP. 2. Experimental 2.1. Apparatus UV-2450 UV vis spectrophotometer was from Shimadzu (Kyoto, Japan). HPLC system consisted of two Shimadzu LC- 20AD pumps and a Shimadzu SPD-M20A photodiode array detector. Fourier transform infrared (FT-IR) spectra were performed on the AVATAR 360 FT-IR spectrophotometer (Nicolet, Waltham, MA, USA). Scanning electron microscopy (SEM) images were performed on Quanta 200 (FEI, Eindhoven, The Netherlands). Nitrogen adsorption desorption analysis was done at 77 K on a Micromeritics TriStar 3000 porosimeter (Norcross, GA, USA) Materials Silica gel of ultra pure (40 60 m, 150 Å, Acros Organics, Geel, Belgium) was activated with acid before being silanized. -Glycidoxypropyltrimethoxysilane (GOTMS) and 3-methylacryloxypropyltrimethoxysilane (MATMS) were purchased from Chemical Factory of Wuhan University (Wuhan, China). -Cyclodextrin ( -CD) (Institute of Tianjin JingKe Fine Chemicals, Tianjin, China) was recrystallized three times and dried under vacuum at 110 C for 24 h. Acrylamide (AA), N,N -methylenebiacrylamide (MBAA) and N,N,N,N,- tetramethylethlenediamine (TEMED) were purchased from Chemistry Reagent Factory of Chinese QianJin (Tianjin, China). d-tryptophan and l-tryptophan (d-trp and l-trp, 99%) were

3 96 L. Qin et al. / J. Chromatogr. A 1187 (2008) purchased from Acros Organics. Ammonium persulfate (APS), l-phenylalanine (l-phe, 99%, BC) and l-tyrosine (l-tyr, 99%, BC) were obtained from Institute of Tianjin GuangFu Fine Chemicals (Tianjin, China). N,N-Dimethylformamide (DMF) was dried over 3-Å molecular sieves before being used for modifying silica gel. All chemical reagents were of analytical or HPLC grade. Phosphate buffer solution (PBS, 0.01 mol/l Na 2 HPO 4 and 0.01 mol/l NaH 2 PO 4, ph 6.2) was used as working medium Synthesis of F-silica gel GOTMS and MATMS as silane coupling agents were used to synthesize functionalized silica gel (F-silica gel). Surface modification of silica gel needed two steps. The first step was to bond -CD with the epoxy group of GOTMS (Fig. 1a), and the following step was that GOTMS bonded -CD and MATMS were chemically bonded onto the surface of silica gel (Fig. 1b). In the first step, -CD (2.29 g) was dissolved in 50.0 ml of anhydrous DMF to which 0.25 g of NaH was added. The mixture was stirred at room temperature until no gas was emitted. Excessive NaH was removed by filtration. 1.0 ml of GOTMS was added to the filtrate, which was allowed to react at 90 C under nitrogen protection for 5 h. After this process, -CD moiety was bonded to the epoxy group of GOTMS. The following step was that 8.00 g of activated silica gel, 50.0 ml of DMF and 1.0 ml of MATMS were dispersed to the first step s product. After the mixture was stirred at C for 24 h under nitrogen protection, the F-silica gel was collected and washed several times successively with anhydrous DMF, methanol, distilled water and acetone. Finally, the F-silica gel was dried under vacuum at 110 C. After the above two steps, -CD and vinyl group were bonded on the surface of silica gel (F-silica gel A). The silica gel only modified with vinyl group (F-silica gel B) was prepared according to the second step (no GOTMS and - CD added). The ratio of bonded -CD towards the initial added amount was 12.5% through elemental analysis [30] Polymer preparation A series of molecularly imprinted and non-imprinted polymers were prepared in aqueous media according to the amounts presented in Table 1. F-silica gel A was used as matrix for MIP(1) and MIP(3). F-silica gel B was used as matrix for MIP(2). In a typical experiment procedure (Fig. 1c), Trp was dissolved in 8.0 ml of PBS (0.01 mmol/l, ph 6.2), and then 1.00 g of F-silica gel A was dispersed in the solution. Under stirring, appropriate amounts of AA and MBAA were added. The polymerization was started by adding APS and TEMED under N 2 protection at 37 C, and then it was stirred for 1 h. The polymer was collected and washed with double distilled water and methanol subsequently in order to sufficiently remove the template. As a control, molecularly non-imprinted polymer (NIP) was immobilized on the surface of F-silica gel in the similar manner described as above, except for the absence of the template. Finally, the prepared MIP (or NIP) was packed into the HPLC column or used for adsorption experiments. In the present study, l-trp or d-trp was used as template, respectively Guest adsorption experiments The rebinding properties of MIP for l-trp were investigated. In a centrifuge tube, 20.0 mg of MIPL(1) or NIP(01) was suspended in 5.0 ml of an initial l-trp concentration of 0.2 mmol/l. The tube was incubated at room temperature with shaking. Ten samples were taken at defined time intervals (at 1, 2, 3, 6, 9, 12, 15, 18, 21 and 24 h, respectively). The amount of l-trp adsorbed by MIPL(1) or NIP(01) particles was determined by UV vis spectrophotometer. In binding isotherm experiments, 20.0 mg of MIP was equilibrated with adsorbate of varied initial concentrations in each centrifuge tube. After 24 h, the saturated polymer was separated by centrifugation, and the residual concentration of the adsorbate was measured by UV vis spectrophotometer. The adsorption capacity (Q ( g/mg)) of the template or analogue bound to the polymer are calculated by Q = (C 0 C t ) VM (1) W where C 0 and C t (mmol/l) are the initial concentration and the residual concentration of the template or analogue, respectively, V (ml) the volume of the initial solution, M (g/mol) the molar mass of the template or analogue, and W (mg) is the weight of the polymer. Table 1 The preparative composition of different polymers Polymer l-(or d-) Trp ( mol) AA ( mol) -CD a ( mol) MBAA ( mol) APS (mg) TEMED ( l) MIPL(1) MIPD(1) MIPL(2) MIPD(2) MIPL(3) MIPD(3) NIP(01) NIP(02) NIP(03) a Represents the amount of -CD bonded on F-silica gel.

4 The specific recognition property of MIP is evaluated by imprinting factor (α), which is defined as α = Q MIP Q NIP (2) where Q MIP and Q NIP are the adsorption capacity of the template or analogue on MIP and the corresponding NIP, respectively. The selectivity factor (β) is defined as β = α tem α ana (3) where α tem is the imprinting factor towards the template molecule and α ana is the imprinting factor towards the analogue. L. Qin et al. / J. Chromatogr. A 1187 (2008) High-performance liquid chromatography experiments MIPL(1), MIPD(1), MIPL(2), MIPD(2), MIPL(3), MIPD(3) and NIP(01), were packed into stainless steel columns (100 mm 4.6 mm i.d.), respectively. 20 l of 2.0 mmol/l of analytes in PBS (0.01 mmol/l, ph 6.2) were injected for analysis. PBS (0.01 mmol/l, ph 6.2) with a flow rate of 0.25 ml/min was used as mobile phase in order to simulate the interaction existing prior to and during the polymerization [7]. Retention behavior of the analyte was estimated by capacity factor (k), which is calculated according to k = t 1 t 0 t 0 (4) where t 1 and t 0 are the retention times of the analyte and acetone (as void marker), respectively. The chromatographic imprinting factor (CIF) for analyte is calculated from the capacity factors obtained on MIP and NIP column (CIF = k MIP /k NIP ). Effective separation factor (γ) is calculated as γ = k tem k ana (5) where k tem and k ana are the capacity factors of the template and analogue, respectively. Enantioseparation factor (γ ) is calculated from γ = k ( D or k ) L (6) k L k D where k D and k L are the capacity factors of d-trp and l-trp, respectively. The resolution (R) is calculated by R = 2(t tem t ana ) W tem + W ana (7) where t tem and t ana are the retention times of the template and analogue, respectively, and W tem and W ana are the peak widths of the template and analogue, respectively. Fig. 2. FT-IR spectra of activated silica-gel (a), MIPL(1) (b) and F-silica gel (c). Peak identification: ν OH : cm 1, cm 1 ; ν Si O Si : cm 1 ; ν Si O : cm 1, cm Results and discussion 3.1. Characteristics of molecular imprinting polymers FT-IR spectra of activated silica gel, F-silica gel and MIPL(1) were shown in Fig. 2. The typical feature of -CD was the peak around cm 1. The carbonyl groups of MATMS were confirmed by the absorption peak at cm 1 (Fig. 2c). These new bands were attributed to the fact that GOTMS bonded -CD and MATMS were modified on the surface of silica gel. Successful preparation of MIPL(1) was judged from the strong peak of cm 1 which was attributed to the carbonyl groups of polyacrylamide (Fig. 2b). As shown in Fig. 3, the surface coated with molecularly imprinted material was multiporous while the surface of activated silica gel and F-silica gel were homogeneous (Fig. 4). From nitrogen sorption data of Table 2, three kinds of polymers had different pore structure (total pore volume and average pore diameter), which were consistent with SEM images. The surface area decreased in the order of MIPL(1) > MIPL(3) > MIPL(2). Large surface area was convenient for the template to diffuse and be adsorbed. Therefore, recognition properties of MIPL(1) were better than that of MIPL(3) and MIPL(2), which can been seen in Sections 3.2 and 3.3. It also can be seen from the nitrogen sorption data that the pore structure (total pore volume and average pore diameter) of MIPL(3) was not better than that of MIPL(2), but the surface area of MIPL(3) was larger than that Table 2 Physical properties of imprinted polymers prepared on F-silica gel Surface area a (m 2 /g) Total pore volume b (ml/g) MIPL(1) MIPL(2) MIPL(3) Average pore diameter c (nm) a Determined using multipoint BET method. b BJH cumulative desorption pore volume of pores between 1.7 and 300 nm. c BJH desorption average pore diameter of pores between 1.7 and 300 nm.

5 98 L. Qin et al. / J. Chromatogr. A 1187 (2008) Fig. 3. The SEM microphotographs of MIPL(1) (a), MIPL(2) (b) and MIPL(3) (c). of MIPL(2). So the adsorption properties of MIPL(3) performed better than MIPL(2), which can been seen in Sections 3.2 and Adsorption properties of molecularly imprinted polymers Binding kinetics Fig. 5 showed the kinetic adsorption processes of l-trp to MIPL(1) and NIP(01). The adsorption reached equilibrium on MIPL(1) after 21 h, which indicated that the imprinted sites were saturated with l-trp. Compared with NIP(01), it was evidential that a much higher adsorption capacity was achieved on MIPL(1). The imprinting factor (α) was 8.24 after adsorption equilibrium. These data indicated that molecular imprinting process had resulted in the formation and preservation of specific recognition cavities of the template on the surface of MIPL(1), which benefited for l-trp to diffuse into the inner cavities of the polymer. For NIP(01), however, the non-specific adsorption had dominant effect due to the lack of imprinting process and there Fig. 4. The SEM microphotographs of activated silica gel (a) and F-silica gel (b). Fig. 5. Adsorption kinetic curves of MIPL(1) ( ) and NIP(01) ( ). Experimental conditions: 5.0 ml of 0.2 mmol/l l-trp with 20.0 mg of MIPL(1) or NIP(01) for certain hours, respectively. The error bar represents the standard deviation. The measurements were repeated five times.

6 L. Qin et al. / J. Chromatogr. A 1187 (2008) Fig. 6. The adsorption isotherms of l-trp on MIPL(1) ( ), MIPL(3) ( ) and MIPL(2) ( ). Experimental conditions: 5.0 ml of mmol/l l-trp with 20.0 mg of the polymers for 24 h. The error bar represents the standard deviation. The measurements were repeated five times. was no suitable cavities on the surface for l-trp to diffuse into the inner of polymer Binding isotherms A binding isotherm is a measure of the concentrationdependent recognition behavior of a system. Binding isotherms were plotted by the adsorption capacity (Q) of adsorbate bound to the polymer versus initial concentration of the adsorbate (Fig. 6). The adsorption capacity to l-trp increased with the increase of initial concentration of l-trp. When the polymer was saturated and all the sites were filled, the adsorption capacity levelled off and remain constant. It can be seen from Fig. 6 that the adsorption efficiencies of these three MIPs were in the order of MIPL(1) > MIPL(3) > MIPL(2). MIPL(1) had the highest adsorption efficiency due to the combination of bonded -CD and AA. The adsorption capacity of MIPL(3) was better than that of MIPL(2). MIPL(3) recognized the template mainly based on hydrophobic effect, while MIPL(2) recognized the template mainly depending on hydrogen-bonding interaction which was easily destroyed in aqueous media. MIPL(1) and MIPL(3) all employed bonded -CD, so it was concluded that in aqueous media the adsorption properties of the polymers were governed mainly by hydrophobic effect. The binding properties of isotherms could be extracted by application of specified binding models. In this work, the isotherms mathematically adapted the model of Bi Langmuir. The Scatchard analysis was applied by replotting the binding isotherm in the format of Q/[l-Trp] versus Q (Fig. 7) [31]. Linear regression analysis yielded the slope and Y-intercept from which the equilibrium dissociation constant (K) and the apparent maximum number of binding sites (Q max ) were obtained. Scatchard Plot yielded two sets of binding parameters (K 1, Q max1 and K 2, Q max2 ) corresponding to the high- and low-affinity binding sites (Table 3). The Q max calculated was reasonably compared with the experimental data (Fig. 6). Fig. 7. Scatchard plot of MIPL (1) selective for l-trp. The error bar represents the standard deviation. The measurements were repeated five times. Table 3 Fitted affinity parameters using Bi Langmuir model Model Bi Langmuir (Scatchard) Parameters High affinity site K 1 = 0.20 mmol/l Q max1 = mg/g Low affinity site K 2 = 1.02 mmol/l Q max2 = mg/g The imprinting effects of three kinds of polymers towards l-trp were also shown in Table 4. The imprinting factor (α) was in the order of α 1 > α 3 > α 2. Thus, it was confirmed that an enhanced affinity was generated between l-trp and MIPL(1) by using bonded -CD and AA. Then, l-tyr and l-phe were used as analogues to evaluated the selectivity of MIPL(1). When l-tyr was used as the analogue, the selectivity factor (β) of MIPL(1) was When l-phe was used as the analogue, the β was In a word, MIPL(1) demonstrated significant binding specificity toward l-trp comparing with the analogues. By using bonded -CD and AA, MIPL(1) exhibited good adsorption capacity and imprinting effect Chromatographic analysis Imprinted effect of MIPL(1) The imprinting effect of MIPL(1) was also investigated by HPLC. The results were shown in Fig. 8. The column packed with NIP(01) was analyzed at the same condition as MIPL(1) column. Fig. 8 indicated that MIPL(1) (t R = min) could adsorb l-trp much stronger than NIP(01) (t R = min). NIP(01) did not have the imprinting cavities complementary to the template, so that functional groups were distributed ran- Table 4 Imprinting factors (α) of the three kinds of polymers towards l-trp l-trp MIPL(1), α MIPL(2), α MIPL(3), α Conditions: 5.0 ml of 2.0 mmol/l l-trp with 20.0 mg of the polymers for 24 h.

7 100 L. Qin et al. / J. Chromatogr. A 1187 (2008) Fig. 8. Chromatograms of l-trp on MIPL(1) ( ) and NIP(01) ( ). Sample amount: 20 l (2.0 mmol/l); detection wavelength: 278 nm; mobile phase: 0.01 mmol/l PBS (ph 6.2); flow rate: 0.25 ml/min. Peak identification: 1, l-trp. domly throughout the polymer matrix, leading to weak binding sites. The chromatographic imprinting factor (CIF) for l-trp was figured out to be Column separation properties It can be seen from Fig. 9 that the baseline separation of l- Trp and l-tyr (or l-trp and l-phe) was obtained on MIPL(1) column. The R was 2.16 for the mixture of l-trp and l-tyr, and 2.19 for the mixture of l-trp and l-phe on MIPL(1) column (Table 5). The properties of the column packed with NIP(01) was analyzed at the same condition. Though there were also separated peaks, the separation factors were not as good as that of MIPL(1), and the retention capability was weaker than MIPL(1). A baseline separation of the template from its analogues was only achieved on the imprinted polymer due to the imprinting effect. It also can be seen that l-tyr and l-phe first flowed out on both MIPL(1) column and NIP(01) column. These results may be explained by the imprinting effect and the molecular volume of the analytes. The imprinting cavities were suitable for the template rather than l-tyr and l-phe, so the retention time of l-tyr or l-phe on MIP(1) was shorter than that of Trp. The molecular volume of l-tyr and l-phe are and Å 3, respectively. However, the molecular volume of l-trp is Å 3 [32]. Small Table 5 Separation properties of the columns of MIPL(1), MIPD(1) and NIP(01) Analyte MIPL(1) MIPD(1) NIP(01) γ R γ R γ R l-trp + l-tyr a l-trp + l-phe b D-Trp + l-tyr a D-Trp + l-phe b a Conditions: sample amount: 20 l (2.0 mmol/l); detection wavelength: 278 nm; mobile phase: 0.01 mmol/l PBS (ph 6.2); flow rate: 0.25 ml/min. b Conditions: sample amount: 20 l (2.0 mmol/l); detection wavelength: 257 nm; mobile phase: 0.01 mmol/l PBS (ph 6.2); flow rate: 0.25 ml/min. Fig. 9. Chromatograms of the mixture of l-trp and l-tyr (a) and the mixture of l-trp and l-phe (b) on MIPL(1) ( ) and NIP(01) ( ). Sample amount: 20 l (2.0 mmol/l); detection wavelength: 278 nm (a) and 257 nm (b); mobile phase: 0.01 mmol/l PBS (ph 6.2); flow rate: 0.25 ml/min. Peak identification: 1, l-trp; 2, l-tyr; 3, l-phe. molecules first eluted, which accorded with the principle of the chromatograph outflow order. The separation properties of MIPD(1) was also evaluated (Table 5). It can be seen from Table 5 that MIPs prepared with bonded -CD and AA could selectively recognize the template, which was coincidental with the adsorption experimental data Column enantioselective properties The difficulty in achieving selectivity for enantiomer molecules follows that the enantiomer molecules differ only in the three-dimensional geometry of the constituent atoms in space, while the remaining properties of both molecules are identical. Further efforts were directed toward studying the enantioselectivity of these polymers when employed as HPLC stationary phases. MIPL(1) imprinted with l-trp yielded the γ and R of 1.60 and 0.98, respectively, whereas MIPD(1) produced the γ and R of 1.84 and 1.19, respectively (Table 6). As shown in Fig. 10, MIPL (1) and MIPD(1) demonstrated selectivity for its target template, respectively. However, the capacity factors of l-trp and d-trp were almost the same on NIP(01) column.

8 L. Qin et al. / J. Chromatogr. A 1187 (2008) Table 6 Separation properties of the enantiomers of Trp on the three kinds of molecularly imprinted polymer stationary phases MIPL(1) MIPD(1) MIPL(2) MIPD(2) MIPL(3) MIPD(3) NIP(01) k L k D γ R Conditions: sample amount: 20 l (2.0 mmol/l); detection wavelength: 278 nm; mobile phase: 0.01 mmol/l PBS (ph 6.2); flow rate: 0.25 ml/min. Fig. 10. Retention behaviors of the enantiomers of Trp on MIPL(1) (a), MIPD(1) (b) and NIP(01) (c). Sample amount: 20 l (2.0 mmol/l); detection wavelength: 278 nm; mobile phase: 0.01 mmol/l PBS (ph 6.2); flow rate: 0.25 ml/min. Peak identification: 1, l-trp; 2, d-trp. The non-imprinted polymer was unable to separate the enantiomer of Trp. In the case of MIPL(2) and MIPD(2), which was imprinted using only acrylamide as functional monomer, the weak hydrogen-bonding interaction contributed by the acrylamide did not allow for sufficient enantioselective capability. The MIPL(3) and MIPD(3) have chiral separation properties. However, the values of the R of MIPL(3) and MIPD(3) were 0.53 and 0.50, respectively. Meanwhile, it can be seen from Table 6 that l-trp exhibited higher capacity factor than d-trp on both MIPL(3) and MIPD(3) columns. So the absence of the acrylamide did not allow reversing the inherent selectivity of the -CD moiety for the l-form. All above results indicated that MIPs prepared with bonded -CD and AA owned better chiral recognition property than MIPs prepared with bonded -CD or AA. In the case of the polymers prepared with bonded -CD and AA, the imprinting effect was sufficient to reverse the natural selectivity of the - CD moiety. The good resolution of MIPL(1) and MIPD(1) for the enantiomer of Trp was mainly attributed to the combination interactions of hydrophobic effect between bonded -CD and Trp [23 25] and hydrogen bond between AA and Trp [26,27]. 4. Conclusion In the present work, MIPs were successfully prepared and applied in aqueous media. By using surface imprinting polymerization method, the amount of the template used was reduced. Bonded -CD and AA cooperated together could improve the

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