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J. Ind. Eng. Chem., Vol. 13, No. 4, (2007) 552-557
Characteristics of a Monolithic Molecularly Imprinted Column and Its Application for Chromatographic Separation Hongyuan Yan and Kyung Ho Row
†
Center for Advanced Bioseparation Technology, Department of Chemical Engineering, Inha University, Incheon, 402-751, Korea Received September 26, 2006; Accepted March 15, 2007
Abstract: Monolithic molecularly imprinted columns were designed and prepared by in situ thermal-initiated copolymerization for rapid separation of the xanthine derivatives such as caffeine, theobromine, and theophylline. Molecular recognition was found to be dependent on the structures and arrangements of functional groups of the imprinted molecule and the cavities of the MIP. The morphological characteristics of the monolithic MIP were investigated using scanning electron microscopy, which showed that both mesopores and macropores were formed in the imprinted monolith. The effects of the chromatographic separation conditions on the molecular recognition were investigated; hydrogen bonding and hydrophobic interactions played an important role in the retention and separation. Thermodynamic data (△△H and △△S) obtained from Van’t Hoff plots revealed an enthalpy-controlled separation. The present method is very simple compared with the bulk MIP procedure; the macroporous structure has excellent separation properties. Keywords: monolithic column, molecularly imprinted polymer, in situ polymerization, xanthine derivatives
Introduction 1)
Molecular imprinting is a rapidly developing technique for the preparation of polymers having specific molecular recognition properties that can selectively recognize the template molecule in the presence of compounds with structures and functionality similar to that of the template [1-5]. The molecularly imprinted polymers (MIPs) possess several advantages over their biological counterparts, including low cost, simple and convenient preparation, storage stability, repeated operations without loss of activity, high mechanical strength, durability to heat and pressure, and applicability in harsh chemical media [6,7]. Due to such outstanding advantages, MIPs have drawn extensive attention and have been used successfully as affinity chromatographic stationary phases [8-10], artificial antibodies [11,12], and sensor components [13,14], for membrane separation [15], and as adsorbents for solid phase extraction [16-18]. The conventional approach is to synthesize the MIP in bulk, grind the resulting polymer block into particles, and †
To whom all correspondence should be addressed. (e-mail: [email protected])
sieve the particles into the desired size ranges. However, this tedious and time-consuming process often produces particles that are irregular in size and shape, resulting in minimal separation with a low column effect. In addition, some interaction sites are destroyed during grinding, which, thus, leads to a lower MIP loading capacity with respect to theoretical values. To overcome these problems, uniformly sized, and monodisperse particles have been made by suspension polymerization [19], multi-step swelling [20], precipitation polymerization [21], and surface imprinting polymerization [22]. These techniques offer their own merits, but often suffer from the need for special dispersing phases/surfactants or are too complicated. Monolithic molecularly imprinted technology, as a novel method for the preparation of chromatographic stationary phases, combines the advantage of monolithic column and MIP technology [23]. Monolithic MIPs are prepared by a one-step, in situ, free-radical polymerization “molding” process directly within a chromatographic column without the need for the tedious procedures of grinding, sieving, and column packing. Furthermore, preparation of these types of MIP is more cost-efficient, because the number of template molecules re-
Characteristics of a Monolithic Molecularly Imprinted Column and Its Application for Chromatographic Separation
553
Figure 2. Schematic principle of theophylline-imprinted polymers.
Figure 1. Molecular structures of caffeine, theophylline, and theobromine.
quired is much lower. The monolithic MIP technology has attracted significant interest because of the ease of preparation, high reproducibility, high selectivity and sensitivity, and rapid mass transport [24-26]. In this study, monolithic MIP columns were prepared using theophylline as the template, acrylamide as the functional monomer, and ethylene glycol dimethacrylate as the crosslinker by in situ thermal-initiated copolymerization. Chromatographic characteristics and molecular recognition of the monolithic MIP were investigated and evaluated using a series of xanthine derivatives: caffeine, theobromine and theophylline. Morphological characteristics and thermodynamic data (△H, △S', △△H, and △△S) are investigated and are discussed.
Experimental Materials Caffeine, theophylline, and theobromine, were obtained from Sigma (St Louis, MO, USA). The structures of these molecules are shown in Figure 1. Acrylamide (AM) from Duksan Pure Chemical Co., Inc. (Korea) was recrystallized prior to use. Ethylene glycol dimethacrylate (EDMA) was obtained from Tokyo Kasei Kogyo
Co., Ltd. (Tokyo, Japan) and was extracted with 2.0 mol/L NaOH solution and dried over anhydroxide magnesium sulfate. α, α'-Azobis (isobutyronitrile) (AIBN), the product of Junsei Chemical Co., Ltd. (Japan), was recrystallized prior to use. Cyclohexanol was obtained from Kanto Chemical Co. Inc. (Japan). Dodecyl alcohol, acetonitrile, and methanol were all of HPLC grade and obtained from Duksan Pure Chemical Co., Ltd. (Ansan, Korea). Acetic acid (analytical grade) was obtained from Oriental Chemical Industries (Incheon, Korea). Doubly distilled water was filtered through a 0.45-µm filter membrane before use. Preparation of Monolithic MIP Column The theophylline-imprinted polymers were directly prepared by in situ polymerization within the confines of a stainless-steel column tube of 150 × 3.9 mm I.D. The schematic principle of the imprinted polymers is shown in Figure 2. Theophylline (0.25 mmol), acrylamide (1.0 mmol), cross-linker (EDMA 5.0 mmol), and free-radical initiator (AIBN, 0.032 g) were dissolved in appropriate porogenic solvents (cyclohexanol and dodecanol) and sonicated for 10 min and sparged with helium for 5 min to remove oxygen. The stainless-steel tube sealed at the bottom was filled with the polymerization mixture and then sealed at the top. The polymerization was performed in a water bath with the temperature maintained o at 50 C for 16 h. After polymerization, the seals were removed, the column was provided with fittings and connected to an HPLC pump, and the system washed respectively with tetrahydrofuran, methanol, and acetic acid (80:20 v/v) to remove the porogenic solvents and template molecules. A blank monolithic column (in the absence of template) was prepared and treated in an
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Hongyuan Yan and Kyung Ho Row
(a)
(b)
(c)
Figure 3. Chromatograms of caffeine, theobromine, and theophylline on different columns. (a) Monolithic MIP column; (b) blank monolithic column; (c) bulk MIP column; mobile phase: acetonitrile; peak 1: caffeine; peak 2: theobromine; peak 3: theophylline.
identical manner. HPLC Analysis Separation characteristic of monolithic MIP columns were analyzed using a liquid chromatography system containing a Waters 600s Multisolvent Delivery System and a Waters 616 pump (Waters, Milford, MA, USA), a Waters 486 Tunable Absorbance Detector (Waters, Milford, MA, USA), and a Rheodyne injection valve (20-µL sample loop). Chromate software (V. 3.0, Interface Eng. Co. Ltd., Korea) was used as the data acquisition system. The UV wavelength was set at 270 nm. All the procedures were performed at room temperature. Morphological Characteristics of Imprinted Monolith After the chromatographic experiments had been completed, the column was washed with methanol/acetic acid (4:1, v/v) for 30 min. The bottom column fitting was removed and the monolith inside the column was pushed out of the tube using the pressure of the methanol mobile phase at a flow rate of 4.0 mL/min. The cylindrical mono olith was dried under vacuum at 40 C for 24 h and then cut into pieces using a razor blade. Microscopic analysis of the monolith was performed using an S-4200 Scanning Electron Microscope (Hitachi, Japan) at 15 kV.
Results and Discussion Molecular Recognition on the Monolithic MIP Columns To evaluate the molecular recognition ability of the monolithic MIP column, a homologous serious of xanthine derivatives, caffeine, theobromine, and theophylline, was applied to compare the retention and separation on the monolithic MIP column. Compared with our previous report [27] of a bulk MIP that showed broad and conjoint peaks of the three xanthine derivatives, due to the low mass transfer originating from irregularly shaped and sized particles of the bulk MIP column, baseline separation of caffeine, theophylline, and theobromine was
achieved on the monolithic MIP column with sharper and more-symmetrical peaks (Figure 3). Moreover, the blank monolithic column even provided partial separation due to the special macroproporous structure and large surface area of the monolith. When comparing the separation factor (k) of the three xanthine derivatives compounds, it can be seen that the k values increased in the order of caffeine, theobromine, and theophylline. Theophylline had been imprinted in the polymer, and the resulting MIP possessed microcavities with a three-dimensional structure complementary in both shape and chemical functionality to that of the template; thus, theophylline displayed the highest molecular recognition. From the structures of the three compounds, the amino group in theophylline could form a hydrogen bond with the monomer to produce specific sites in the polymer. Theobromine molecules also have an amino group. Caffeine displayed weak retention and recognition ability due to the lack of an amino group in its molecular structure and the methyl group providing steric encumbrance for the hydrogen bond. These results indicate that molecular recognition was dependent on the structure and arrangement of functional groups of the imprinted molecule and the cavities in the MIP. Effect of Chromatographic Conditions on Separation The influence of the mobile phase composition on the molecular recognition properties was investigated using methanol, acetonitrile, water, and 20 mol/L phosphate buffer as mobile phases. The results showed that the three xanthine derivatives could not be separated completely when using methanol, water, or the phosphate buffer as mobile phases. The best separation was obtained using acetonitrile as the mobile phase, presumably due to its weak hydrogen bonding capacity and, thus, limited ability to compete for hydrogen bonding sites on the template or at the binding sites. Furthermore, it solvates the polymer backbone well and is polar enough to dissolve a large number of compounds. The effects of polar additives in the mobile phase on molecular recognition were also evaluated using acetic acid as the polar
Characteristics of a Monolithic Molecularly Imprinted Column and Its Application for Chromatographic Separation
(a)
555
(b)
Figure 4. Effect of mobile phase composition on retention and separation (k1, k2, and k3 are the retention factors of caffeine, theobromine, and theophylline, respectively; a1 and a2 are the separation factors of theobromine and caffeine, and of theophylline and theobromine, respectively).
Figure 5. Effect of temperature on the separation and retention (k1, k2, and k3 are the retention factors of caffeine, theobromine, and theophylline, respectively; a1 and a2 are the separation factors of theobromine and caffeine, and of theophylline and theobromine, respectively).
additive in the range of 0 to 5.0 % (v/v); the results showed that the retention factors decreased with an increased proportion of acetic acid in the mobile phase (Figure 4). Moreover, the k value of caffeine changed slightly, while the k values of theophylline and theobromine change quickly with the change of the proportion of the acetic acid in the mobile phase. The apparent selectivity factor (a) decreased upon increasing the acetic acid proportion in the mobile phase, indicating that polar additives can interfere with the hydrogen bonding interactions between the matrix in the MIP and the functional groups of the analytes. To investigate the effect of temperature on the retention and separation, the temperature-dependence of the molecular recognition of the three analogues was investigated. Three replicate injections were made for each ana-
lyte at a flow rate of 0.5 mL/min with the temperature o changing from 25 to 45 C. The column was equilibrated with the mobile phase for ca. 30 min following each temperature change. We found that the retention factors of caffeine, theobromine, and theophylline all decreased upon increasing the temperature (Figure 5), presumably because the analytes have weaker adsorption to the substrate as the temperature increases and, therefore, migrate faster through the MIP stationary phase. This situation means that the hydrogen bonding and hydrophobic interactions between the template and polymer weakened with increasing temperature. Therefore, a relatively low temperature will help to provide better separation. Morphological Characteristics of the Imprinted Monolith Morphological analysis of the polymers was also investigated in this experiment. The SEM image in Figure 6 shows that many macropores and flow-through channels were inlaid in the network skeleton of the theophylline imprinted monolith. These macropores and channels allowed the mobile phase to flow through the monolith with a low flow resistance, and, thus, enabled fast mass transfer of the solutes. Moreover, the low backpressure allowed their operation at higher flow rates. The relationship between the backpressure and the flow rate on the monolithic MIP showed that even at a high flow rate of 2.0 mL/min, the backpressure was only 9.26 MPa. In contrast, the backpressure of the packed column was relatively higher over the whole range of flow rates, due to the irregular shape and non-uniform sizes of the packed particles.
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Hongyuan Yan and Kyung Ho Row
Table 1. Thermodynamic Parameters of Xanthine Derivative Separation on Monolithic MIP Column Analyte
△H (kJ.mol-1)
△S' (J.mol-1.K-1)
△△H (kJ.mol-1)
△△S (J.mol-1.K-1)
Caffeine
-5.52
-23.85
Theobromine
-9.21
-20.12
-9.42
-17.99
Theophylline
-9.42
-17.99
-5.52
-23.85
△H and △S' (△S' =△S + R lnφ) were obtained from linear regression of the Van’t Hoff plots by plotting ln k vs. 1/T; △△H and △△S were obtained from a plot of lnα vs. 1/T.
Figure 7. Van’t Hoff plots obtained by plotting ln k vs. 1/T.
Fig. 6. Scanning electron microscopy (SEM) images of the monolithic and bulk MIP.
Thermodynamics of Separation on Monolithic MIP The retention behavior and thermodynamic parameters determined in this study were used to estimate the enthalpy, entropy, and Gibbs free energy of association between the xanthine derivatives and the monolithic MIP. Data obtained from retention and separation of the xanthine derivatives at temperatures ranging from 25 to 45 o C were processed using the Van’t Hoff equation [10,28] to estimate the thermodynamic properties of the separation (Figure 7):
In k' = - + In φ
In α= - where R, T, and φ are the gas constant, the absolute
temperature, and the phase volume ratio, respectively. The enthalpy (△H), entropy (△S'), enthalpy difference (△△H), and entropy difference (△△S) can be calculated from the slopes and intercepts of the linear portion of the above equations. The results are listed in Table 1. The values of △H and △S' indicated that theophylline has stronger affinity to the recognition sites, and could form a more-stable template-MIP complex than could caffeine or theobromine during their matching in the micro-cavities on the MIP. Moreover, the fact that |△△H| > T|△△S| indicates that the separation on the monolithic MIP column was an enthalpy-controlled process. In geno eral, for the temperature range 25∼45 C, the enthalpic contribution to the overall substrate’s transfer energy was more significant than the entropic one. In this case, the decrease in temperature led to an increase of the separation factor. This result is consistent with the results of the temperature-dependence experiment.
Conclusions A monolithic molecularly imprinted stationary phase was successfully prepared by in situ thermal-initiated copolymerization, and the effects of the specific molecular recognition ability for three xanthine derivatives were investigated. We found that recognition was dependent on the structure and arrangement of functional groups in
Characteristics of a Monolithic Molecularly Imprinted Column and Its Application for Chromatographic Separation
the cavities of the MIP. Moreover, hydrogen bonding interactions play an important role in the retention and separation. The morphological characteristics of the monolithic MIP showed that both mesopores and macropores were formed in the monolith. Thermodynamic data (△△H and △△S) obtained from Van’t Hoff plots revealed an enthalpy-controlled separation. The present method is very simple compared with the conventional MIP procedure; the macroporous structure has excellent separation properties.
Acknowledgment The authors gratefully acknowledge the financial support of the Center for Advanced Bioseparation Technology, Inha University.
References 1. M. J. Whitcombe, L Martin, and E. N. Vulfson, Chromatographia, 47, 457 (1998). 2. J. M. Hong, J. Ind. Eng. Chem., 4, 226 (2006). 3. H. Y. Yan and K. H. Row, Int. J. Mol. Sci., 7, 155 (2006). 4. S. Peter, L. Schweitz, and S. Nilsson, Electrophoresis, 24, 3892 (2003). 5. H. S. Wei, Y. L. Tsai, J. Y. Wu, and H. Chen, J. Chromatogr. B, 836, 57 (2006). 6. N. Lavignac, C. J. Allender, and K. R. Brain, Anal. Chim. Acta, 510, 139 (2004). 7. G. Vlatakis, L. I. Andersson, R. Miller, and K. Mosbach, Nature, 361, 645 (1993). 8. J. Xie, L. Zhu, H. Luo, L. Zhou, C. Li, and X. Xu, J. Chromatogr. A, 934, 1 (2001). 9. X. D. Huang, H. F. Zou, X. M. Chen, Q. Z. Luo, and L. Kong, J. Chromatogr. A, 984, 273 (2003). 10. J. Yin, G. Yang, and Y. Chen, J. Chromatogr. A,
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1090, 68 (2005). 11. L. Ye and K. Mosbach, React. Funct. Polym., 48, 149 (2001). 12. F. L. Dickert, P. Lieberzeit, and M. Tortschanoff, Sens. Actuators B, 65, 186 (2000). 13. O. Kriz, O. Ramstrom, and K. Mosbach, Anal. Chem., 69, 345A (1997). 14. K. Hirayama, Y. Sakai, K. Kameoka, K. Noda, and R. Naganawa, Sens. Actuator B, 86, 20 (2002). 15. M. Yoshikawa, T. Fujisawa, J. Izumi, T. Kitao, and S. Sakamoto, Anal. Chim. Acta., 365, 59 (1998). 16. A. Bereczki, A. Tolokan, G. Horvai, V. Horvath, F. Lanza, A. J. Hall, and B. Sellergren, J. Chromatogr. A, 930, 31 (2001). 17. P. D. Martin, G. R. Jones, F. Stringer, and I. D. Wilson, Analyst, 128, 345 (2003). 18. Y. Yin and K. H. Row, J. Ind. Eng. Chem., 12, 494 (2006). 19. L. Zhang, G. Cheng, and C. Fu, React. Func. Polym., 56, 167 (2003). 20. J. Haginaka and C. Kagawa, J. Chromatogr. A., 948, 77 (2002). 21. W. H. Li and D. H. Stover, Macromolecules, 33, 4354 (2000). 22. B. Sellergren, B. Ruckert, and A. J. Hall, Adv. Mater., 14, 1204 (2002). 23. H. Y. Liu, K. H. Row, and G. L. Yang, Chromatogr., 61, 429 (2005). 24. H. Li, Y. Liu, Z. Zhang, H. Liao, L. Nie, and S. Yao, J. Chromatogr. A, 1098, 66 (2005). 25. X. D. Huang, F. Qin, X. M. Chen, Y. Q. Liu, and H. F. Zou, J. Chromatogr. B, 804, 13 (2004). 26. H. Y. Yan, L. Jin, and K. H. Row, J. Liq. Chromatogr. Rel. Technol., 28, 3147 (2005). 27. D. X. Wang, S. P. Hong, and K. H. Row, Bull. Korean Chem. Soc., 25, 357 (2004). 28. H. Kim and G. Guiochon, Anal. Chem., 77, 1708 (2005).
Characteristics of a Monolithic Molecularly Imprinted Column and Its Application for Chromatographic Separation Hongyuan Yan and Kyung Ho Row
†
Center for Advanced Bioseparation Technology, Department of Chemical Engineering, Inha University, Incheon, 402-751, Korea Received September 26, 2006; Accepted March 15, 2007
Abstract: Monolithic molecularly imprinted columns were designed and prepared by in situ thermal-initiated copolymerization for rapid separation of the xanthine derivatives such as caffeine, theobromine, and theophylline. Molecular recognition was found to be dependent on the structures and arrangements of functional groups of the imprinted molecule and the cavities of the MIP. The morphological characteristics of the monolithic MIP were investigated using scanning electron microscopy, which showed that both mesopores and macropores were formed in the imprinted monolith. The effects of the chromatographic separation conditions on the molecular recognition were investigated; hydrogen bonding and hydrophobic interactions played an important role in the retention and separation. Thermodynamic data (△△H and △△S) obtained from Van’t Hoff plots revealed an enthalpy-controlled separation. The present method is very simple compared with the bulk MIP procedure; the macroporous structure has excellent separation properties. Keywords: monolithic column, molecularly imprinted polymer, in situ polymerization, xanthine derivatives
Introduction 1)
Molecular imprinting is a rapidly developing technique for the preparation of polymers having specific molecular recognition properties that can selectively recognize the template molecule in the presence of compounds with structures and functionality similar to that of the template [1-5]. The molecularly imprinted polymers (MIPs) possess several advantages over their biological counterparts, including low cost, simple and convenient preparation, storage stability, repeated operations without loss of activity, high mechanical strength, durability to heat and pressure, and applicability in harsh chemical media [6,7]. Due to such outstanding advantages, MIPs have drawn extensive attention and have been used successfully as affinity chromatographic stationary phases [8-10], artificial antibodies [11,12], and sensor components [13,14], for membrane separation [15], and as adsorbents for solid phase extraction [16-18]. The conventional approach is to synthesize the MIP in bulk, grind the resulting polymer block into particles, and †
To whom all correspondence should be addressed. (e-mail: [email protected])
sieve the particles into the desired size ranges. However, this tedious and time-consuming process often produces particles that are irregular in size and shape, resulting in minimal separation with a low column effect. In addition, some interaction sites are destroyed during grinding, which, thus, leads to a lower MIP loading capacity with respect to theoretical values. To overcome these problems, uniformly sized, and monodisperse particles have been made by suspension polymerization [19], multi-step swelling [20], precipitation polymerization [21], and surface imprinting polymerization [22]. These techniques offer their own merits, but often suffer from the need for special dispersing phases/surfactants or are too complicated. Monolithic molecularly imprinted technology, as a novel method for the preparation of chromatographic stationary phases, combines the advantage of monolithic column and MIP technology [23]. Monolithic MIPs are prepared by a one-step, in situ, free-radical polymerization “molding” process directly within a chromatographic column without the need for the tedious procedures of grinding, sieving, and column packing. Furthermore, preparation of these types of MIP is more cost-efficient, because the number of template molecules re-
Characteristics of a Monolithic Molecularly Imprinted Column and Its Application for Chromatographic Separation
553
Figure 2. Schematic principle of theophylline-imprinted polymers.
Figure 1. Molecular structures of caffeine, theophylline, and theobromine.
quired is much lower. The monolithic MIP technology has attracted significant interest because of the ease of preparation, high reproducibility, high selectivity and sensitivity, and rapid mass transport [24-26]. In this study, monolithic MIP columns were prepared using theophylline as the template, acrylamide as the functional monomer, and ethylene glycol dimethacrylate as the crosslinker by in situ thermal-initiated copolymerization. Chromatographic characteristics and molecular recognition of the monolithic MIP were investigated and evaluated using a series of xanthine derivatives: caffeine, theobromine and theophylline. Morphological characteristics and thermodynamic data (△H, △S', △△H, and △△S) are investigated and are discussed.
Experimental Materials Caffeine, theophylline, and theobromine, were obtained from Sigma (St Louis, MO, USA). The structures of these molecules are shown in Figure 1. Acrylamide (AM) from Duksan Pure Chemical Co., Inc. (Korea) was recrystallized prior to use. Ethylene glycol dimethacrylate (EDMA) was obtained from Tokyo Kasei Kogyo
Co., Ltd. (Tokyo, Japan) and was extracted with 2.0 mol/L NaOH solution and dried over anhydroxide magnesium sulfate. α, α'-Azobis (isobutyronitrile) (AIBN), the product of Junsei Chemical Co., Ltd. (Japan), was recrystallized prior to use. Cyclohexanol was obtained from Kanto Chemical Co. Inc. (Japan). Dodecyl alcohol, acetonitrile, and methanol were all of HPLC grade and obtained from Duksan Pure Chemical Co., Ltd. (Ansan, Korea). Acetic acid (analytical grade) was obtained from Oriental Chemical Industries (Incheon, Korea). Doubly distilled water was filtered through a 0.45-µm filter membrane before use. Preparation of Monolithic MIP Column The theophylline-imprinted polymers were directly prepared by in situ polymerization within the confines of a stainless-steel column tube of 150 × 3.9 mm I.D. The schematic principle of the imprinted polymers is shown in Figure 2. Theophylline (0.25 mmol), acrylamide (1.0 mmol), cross-linker (EDMA 5.0 mmol), and free-radical initiator (AIBN, 0.032 g) were dissolved in appropriate porogenic solvents (cyclohexanol and dodecanol) and sonicated for 10 min and sparged with helium for 5 min to remove oxygen. The stainless-steel tube sealed at the bottom was filled with the polymerization mixture and then sealed at the top. The polymerization was performed in a water bath with the temperature maintained o at 50 C for 16 h. After polymerization, the seals were removed, the column was provided with fittings and connected to an HPLC pump, and the system washed respectively with tetrahydrofuran, methanol, and acetic acid (80:20 v/v) to remove the porogenic solvents and template molecules. A blank monolithic column (in the absence of template) was prepared and treated in an
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Hongyuan Yan and Kyung Ho Row
(a)
(b)
(c)
Figure 3. Chromatograms of caffeine, theobromine, and theophylline on different columns. (a) Monolithic MIP column; (b) blank monolithic column; (c) bulk MIP column; mobile phase: acetonitrile; peak 1: caffeine; peak 2: theobromine; peak 3: theophylline.
identical manner. HPLC Analysis Separation characteristic of monolithic MIP columns were analyzed using a liquid chromatography system containing a Waters 600s Multisolvent Delivery System and a Waters 616 pump (Waters, Milford, MA, USA), a Waters 486 Tunable Absorbance Detector (Waters, Milford, MA, USA), and a Rheodyne injection valve (20-µL sample loop). Chromate software (V. 3.0, Interface Eng. Co. Ltd., Korea) was used as the data acquisition system. The UV wavelength was set at 270 nm. All the procedures were performed at room temperature. Morphological Characteristics of Imprinted Monolith After the chromatographic experiments had been completed, the column was washed with methanol/acetic acid (4:1, v/v) for 30 min. The bottom column fitting was removed and the monolith inside the column was pushed out of the tube using the pressure of the methanol mobile phase at a flow rate of 4.0 mL/min. The cylindrical mono olith was dried under vacuum at 40 C for 24 h and then cut into pieces using a razor blade. Microscopic analysis of the monolith was performed using an S-4200 Scanning Electron Microscope (Hitachi, Japan) at 15 kV.
Results and Discussion Molecular Recognition on the Monolithic MIP Columns To evaluate the molecular recognition ability of the monolithic MIP column, a homologous serious of xanthine derivatives, caffeine, theobromine, and theophylline, was applied to compare the retention and separation on the monolithic MIP column. Compared with our previous report [27] of a bulk MIP that showed broad and conjoint peaks of the three xanthine derivatives, due to the low mass transfer originating from irregularly shaped and sized particles of the bulk MIP column, baseline separation of caffeine, theophylline, and theobromine was
achieved on the monolithic MIP column with sharper and more-symmetrical peaks (Figure 3). Moreover, the blank monolithic column even provided partial separation due to the special macroproporous structure and large surface area of the monolith. When comparing the separation factor (k) of the three xanthine derivatives compounds, it can be seen that the k values increased in the order of caffeine, theobromine, and theophylline. Theophylline had been imprinted in the polymer, and the resulting MIP possessed microcavities with a three-dimensional structure complementary in both shape and chemical functionality to that of the template; thus, theophylline displayed the highest molecular recognition. From the structures of the three compounds, the amino group in theophylline could form a hydrogen bond with the monomer to produce specific sites in the polymer. Theobromine molecules also have an amino group. Caffeine displayed weak retention and recognition ability due to the lack of an amino group in its molecular structure and the methyl group providing steric encumbrance for the hydrogen bond. These results indicate that molecular recognition was dependent on the structure and arrangement of functional groups of the imprinted molecule and the cavities in the MIP. Effect of Chromatographic Conditions on Separation The influence of the mobile phase composition on the molecular recognition properties was investigated using methanol, acetonitrile, water, and 20 mol/L phosphate buffer as mobile phases. The results showed that the three xanthine derivatives could not be separated completely when using methanol, water, or the phosphate buffer as mobile phases. The best separation was obtained using acetonitrile as the mobile phase, presumably due to its weak hydrogen bonding capacity and, thus, limited ability to compete for hydrogen bonding sites on the template or at the binding sites. Furthermore, it solvates the polymer backbone well and is polar enough to dissolve a large number of compounds. The effects of polar additives in the mobile phase on molecular recognition were also evaluated using acetic acid as the polar
Characteristics of a Monolithic Molecularly Imprinted Column and Its Application for Chromatographic Separation
(a)
555
(b)
Figure 4. Effect of mobile phase composition on retention and separation (k1, k2, and k3 are the retention factors of caffeine, theobromine, and theophylline, respectively; a1 and a2 are the separation factors of theobromine and caffeine, and of theophylline and theobromine, respectively).
Figure 5. Effect of temperature on the separation and retention (k1, k2, and k3 are the retention factors of caffeine, theobromine, and theophylline, respectively; a1 and a2 are the separation factors of theobromine and caffeine, and of theophylline and theobromine, respectively).
additive in the range of 0 to 5.0 % (v/v); the results showed that the retention factors decreased with an increased proportion of acetic acid in the mobile phase (Figure 4). Moreover, the k value of caffeine changed slightly, while the k values of theophylline and theobromine change quickly with the change of the proportion of the acetic acid in the mobile phase. The apparent selectivity factor (a) decreased upon increasing the acetic acid proportion in the mobile phase, indicating that polar additives can interfere with the hydrogen bonding interactions between the matrix in the MIP and the functional groups of the analytes. To investigate the effect of temperature on the retention and separation, the temperature-dependence of the molecular recognition of the three analogues was investigated. Three replicate injections were made for each ana-
lyte at a flow rate of 0.5 mL/min with the temperature o changing from 25 to 45 C. The column was equilibrated with the mobile phase for ca. 30 min following each temperature change. We found that the retention factors of caffeine, theobromine, and theophylline all decreased upon increasing the temperature (Figure 5), presumably because the analytes have weaker adsorption to the substrate as the temperature increases and, therefore, migrate faster through the MIP stationary phase. This situation means that the hydrogen bonding and hydrophobic interactions between the template and polymer weakened with increasing temperature. Therefore, a relatively low temperature will help to provide better separation. Morphological Characteristics of the Imprinted Monolith Morphological analysis of the polymers was also investigated in this experiment. The SEM image in Figure 6 shows that many macropores and flow-through channels were inlaid in the network skeleton of the theophylline imprinted monolith. These macropores and channels allowed the mobile phase to flow through the monolith with a low flow resistance, and, thus, enabled fast mass transfer of the solutes. Moreover, the low backpressure allowed their operation at higher flow rates. The relationship between the backpressure and the flow rate on the monolithic MIP showed that even at a high flow rate of 2.0 mL/min, the backpressure was only 9.26 MPa. In contrast, the backpressure of the packed column was relatively higher over the whole range of flow rates, due to the irregular shape and non-uniform sizes of the packed particles.
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Table 1. Thermodynamic Parameters of Xanthine Derivative Separation on Monolithic MIP Column Analyte
△H (kJ.mol-1)
△S' (J.mol-1.K-1)
△△H (kJ.mol-1)
△△S (J.mol-1.K-1)
Caffeine
-5.52
-23.85
Theobromine
-9.21
-20.12
-9.42
-17.99
Theophylline
-9.42
-17.99
-5.52
-23.85
△H and △S' (△S' =△S + R lnφ) were obtained from linear regression of the Van’t Hoff plots by plotting ln k vs. 1/T; △△H and △△S were obtained from a plot of lnα vs. 1/T.
Figure 7. Van’t Hoff plots obtained by plotting ln k vs. 1/T.
Fig. 6. Scanning electron microscopy (SEM) images of the monolithic and bulk MIP.
Thermodynamics of Separation on Monolithic MIP The retention behavior and thermodynamic parameters determined in this study were used to estimate the enthalpy, entropy, and Gibbs free energy of association between the xanthine derivatives and the monolithic MIP. Data obtained from retention and separation of the xanthine derivatives at temperatures ranging from 25 to 45 o C were processed using the Van’t Hoff equation [10,28] to estimate the thermodynamic properties of the separation (Figure 7):
In k' = - + In φ
In α= - where R, T, and φ are the gas constant, the absolute
temperature, and the phase volume ratio, respectively. The enthalpy (△H), entropy (△S'), enthalpy difference (△△H), and entropy difference (△△S) can be calculated from the slopes and intercepts of the linear portion of the above equations. The results are listed in Table 1. The values of △H and △S' indicated that theophylline has stronger affinity to the recognition sites, and could form a more-stable template-MIP complex than could caffeine or theobromine during their matching in the micro-cavities on the MIP. Moreover, the fact that |△△H| > T|△△S| indicates that the separation on the monolithic MIP column was an enthalpy-controlled process. In geno eral, for the temperature range 25∼45 C, the enthalpic contribution to the overall substrate’s transfer energy was more significant than the entropic one. In this case, the decrease in temperature led to an increase of the separation factor. This result is consistent with the results of the temperature-dependence experiment.
Conclusions A monolithic molecularly imprinted stationary phase was successfully prepared by in situ thermal-initiated copolymerization, and the effects of the specific molecular recognition ability for three xanthine derivatives were investigated. We found that recognition was dependent on the structure and arrangement of functional groups in
Characteristics of a Monolithic Molecularly Imprinted Column and Its Application for Chromatographic Separation
the cavities of the MIP. Moreover, hydrogen bonding interactions play an important role in the retention and separation. The morphological characteristics of the monolithic MIP showed that both mesopores and macropores were formed in the monolith. Thermodynamic data (△△H and △△S) obtained from Van’t Hoff plots revealed an enthalpy-controlled separation. The present method is very simple compared with the conventional MIP procedure; the macroporous structure has excellent separation properties.
Acknowledgment The authors gratefully acknowledge the financial support of the Center for Advanced Bioseparation Technology, Inha University.
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