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Characteristic of theophylline imprinted monolithic column and its application for determination of xanthine derivatives caffeine and theophylline in green tea
Journal of Chromatography A, 1134 (2006) 194–200
Characteristic of theophylline imprinted monolithic column and its application for determination of xanthine derivatives caffeine and theophylline in green tea Han-wen Sun a,∗ , Feng-xia Qiao a,b , Guang-yu Liu a a
College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China b Department of Chemistry, Baoding Teachers College, Baoding 071000, China
Received 14 July 2006; received in revised form 28 August 2006; accepted 4 September 2006 Available online 16 October 2006
Abstract Theophylline imprinted monolithic columns were designed and prepared for rapid separation of a homologous series of xanthine derivatives, caffeine, and theophylline by an in situ thermal-initiated copolymerization technique. Caffeine and theophylline were fully separated both under isocratic and gradient elutions on this kind of monolithic molecularly imprinted polymers (MIP) column. The broad peak showed in isocratic elution could be improved in gradient elution. Some chromatographic conditions such as mobile phase composition, flow rate, and the temperature on the retention times were investigated. Hydrogen bonding interaction and hydrophobic interaction played an important role in the retention and separation. The binding capacity was evaluated by static adsorption and Scatchard analysis, which showed that the dissociation constant (KD ) and the maximum binding capacity (Qmax ) were 1.50 mol/L, and 236 mol/g for high affinity binding site, and 7.97 mol/L and 785 mol/g for lower affinity binding site, respectively. Thermodynamic data (H and S) obtained by Van’t Hoff plots revealed an enthalpy-controlled separation. The morphological characteristics of monolithic MIP were investigated by scanning electron microscope, which showed that both mesopores and macropores were formed in the monolith. The present monolithic MIP column was successfully applied for the quantitative determination of caffeine and theophylline in different kinds of green tea. © 2006 Elsevier B.V. All rights reserved. Keywords: Molecularly imprinted polymer; Monolithic column; Characteristic; Caffeine; Theophylline; Green tea
1. Introduction Molecularly imprinted polymers (MIP) that exhibit high selectivity and affinity to the predetermined molecule are a rapidly growing research focus [1–5]. The special binding sites are formed by the self-assembly of a template molecule with specific functional groups and the monomer, followed by a cross-linking co-polymerization. After the polymerization, the template is removed from the polymer, leaving microcavities and recognition sites that, in terms of size, shape, and chemical functionality, are complementary to that of the template. The MIP possess several advantages over their biological counterparts including low-cost, simple and convenient preparation, storage stability, repeated operations without loss of activity,
∗
Corresponding author. Tel.: +86 312 5079739; fax: +86 312 5079719. E-mail address: [email protected] (H.-w. Sun).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.09.004
high mechanical strength, durability to heat and pressure, and applicability in harsh chemical media [6]. Due to such outstanding advantages, MIP had been drawn extensive attention and been used successfully as affinity chromatographic stationary phases [7–10], artificial antibodies [11,12], sensor components [13,14], membrane separation [15], and adsorbents for solid phase extraction [16–18]. The conventional technology is to synthesize the MIP in bulk, grind the resulting polymer block into particles, and sieve the particles into the desired size ranges. Such ground and sieved particles can then be packed into conventional HPLC columns. Although the process of bulk polymerization is simple, the resulting polymer must be crushed, grinded and sieved to obtain the appropriate size, this tedious and timeconsuming process often produces particles that are irregular in size and shape, some interaction sites are destroyed during grinding, which lead to a negative impact on chromatographic performance and lower MIP loading capacity with respect
H.-w. Sun et al. / J. Chromatogr. A 1134 (2006) 194–200
to theoretical values. In order to overcome these problems, uniformly sized and monodispersed particles had been made by suspension polymerization [19], multistep swelling process [20], precipitation polymerization [21], and surface imprinting polymerization [22]. These techniques each offer its own merits, however, these methods often suffers from the use of special dispersing phases/surfactants or too complicated. Monolithic molecularly imprinted technology represents a novel method for preparation of stationary phases in recent years, which combined the advantage of monolithic column and molecular imprinted technology [23–25]. Monolithic MIP is prepared by a simple, one-step, in situ, free-radical polymerization “molding” process directly within a chromatographic column, thus avoiding the tedious procedures of grinding, sieving, and column packing. Compared with conventional particle columns, the monolithic column has attracted significant interest because of their ease of preparation, high reproducibility, and rapid mass transport. Furthermore, the preparation of this type of MIP is more cost-efficient, because it requires much smaller amount of template molecules. However, the prepared monolithic MIP often suffers from high backpressures and low efficiency, which results in their poor application and practical separation. Moreover, the most of monolithic MIP works were focused on preparation and recognition mechanisms, and application to real sample are very rarely. In this work, monolithic MIP columns were designed and prepared for rapid separation of a homologous series of xanthine derivatives, caffeine, and theophylline, in different kinds of green tea by in situ thermal-initiated copolymerization technique using theophylline as the template, acrylamide as the functional monomer and ethylene glycol dimethacrylate as crosslinker. Chromatographic conditions, such as separation mechanism, binding capacity, morphological characteristics and thermodynamic data of the monolithic column were also investigated and discussed. The present monolithic column was successfully applied for the quantitative determination of caffeine and theophylline in different kinds of green tea with satisfactory results. 2. Experimental 2.1. Chemicals Caffeine and theophylline were obtained from Sigma (ST Louis, MO, USA). Acrylamide (AM) from Tianjin No. 1 Chemical Reagent Factory (Tianjin, China) was recrystallized prior to use. Ethylene glycol dimethacrylate (EDMA) from SigmaAldrich (Shanghai) Trading Co., Ltd. (Shanghai, China) was extracted with 2 mol/L NaOH solution and dried over anhydroxide magnesium sulfate. 2,2 -Azobisisobutyronitrile (AIBN) from Beijing Chemical Reagent company (Beijing, China) recrystallized prior to use. Toluene was purchased from Beijing Chemical Industries (Beijing China). Dodecyl alcohol, acetonitrile, chloroform and methanol were all of HPLC grade and purchased from Tianjing Kermel Chemical Reagents Development Centre (Tianjin, China). Acetic acid (analytical grade) was purchased from Jinli Industries Co. (Tianjin, China). All of the
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solutions were filtered through a 0.45 m membrane filter (Millipore) before use. 2.2. Preparation of monolithic MIP column The monolithic MIP stationary phase was directly prepared by in situ thermal-initiated polymerization within a chromatographic column tube (150 mm × 4.0 mm I.D.). The polymerization mixture composed of 0.047 g template molecule, 0.212 g acrylamide, 1.134 g ethylene glycol dimethacrylate (EDMA), and 0.010 g 2,2 -Azobis (isobutyronitrile) (AIBN) was dissolved in the appropriate porogenic solvents (toluene and dodecanol). The solution was ultasonicated for 15 min and purged with nitrogen gas for 10 min at room temperature to remove oxygen. The ends of the stainless-steel tube were connected with plastics tubes, and then sealed at the bottom, filled with the above polymerization mixture and then sealed at the top. Subsequently, the polymerization was performed in water bath at 50 ◦ C for 24 h. After the polymerization, the seals and plastics tube were removed. The resulted column was provided with fittings, and connected to an HPLC pump and washed with tetrahydrofuran, methanol:acetic acid (4:1, v/v), respectively, to remove the templates and porogenic solvents. At last, it was washed with methanol until no residue of template was found in the rinses. A non-imprinted blank monolithic column was prepared in the absence of template, and treated in an identical manner. 2.3. HPLC analysis Separation characteristic of monolithic MIP column was performed with a Shimadzu HPLC system (Shimadzu, Kyoto, Japan). This HPLC system is equipped with a photodiode array detector (SPD-M10Avp), a gradient controller (SCL-10Avp), a workstation (CLASS-vp), a degasser (DGU-12A) and a column thermostat (CTO-10Avp). A Shimadzu UV-265 UV–vis spectrophotometer (Shimadzu, Japan) was used to detect UV absorption. The UV detection wavelength was set at 271 nm. All the procedures were carried out at the room temperature. The separation factor (α) was determined by the following equation, α = k2 /k1 , where k1 and k2 are the retention factor of the caffeine and the theophylline, respectively. The retention factors were determined by k = (tM − t0 )/t0 , where tM is the retention time of the solute and t0 is the void time of the column. 2.4. Characterization of monolithic stationary phases After all chromatographic experiments had been completed, the column was washed with methanol/acetic acid (10:1, v/v) for 40 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 monolith was then dried under vacuum at 35 ◦ C for 2 days and cut into pieces with a razor blade. The pore properties and microscopic analysis of the monolith was carried out in a KYKY-2800B scanning electron microscopy (Beijing, China) at 25 kV.
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2.5. Binding capacity and adsorption isotherms Static method was performed by placing 20 mg polymer particles, 3.00 mL acetonitrile and different concentration of the template solution into 10 mL flasks, then the flasks were oscillated in a constant temperature bath oscillator at 25 ◦ C for 12 h. The mixture was transferred into a centrifuge tube and centrifuged at 4000 rpm for 5 min. The concentrations of free compounds in the solutions were determined by an UV-Spectrometer at 271 nm. The absorption quantity (Q) was calculated by subtracting the free concentrations from the initial concentrations. 2.6. Solvent extraction of green tea Five grams of green tea was extracted by 150 mL doubly distilled water with continuously stirring under 50 ◦ C for 8 h. The obtained extraction was filtered with 0.2 mm, 25 mm syringe filter, then it was stored in 4 ◦ C for further work. 3. Results and discussion 3.1. Molecular recognition on the monolithic MIP columns To evaluate the molecular recognition ability of the monolithic MIP columns, two homologous series of xanthine derivatives, caffeine, and theophylline were applied to compare the retention and separation on the monolithic MIP column and blank column. Caffeine and theophylline were baseline separated on the monolithic MIP column, but no separation was observed on blank monolithic column (Fig. 1). It indicates that molecular recognition was dependent on the stereo structures and the arrangement of functional groups of the imprinted molecule and the cavities on MIP. The structures of the two compounds are shown in Fig. 2. There is an active amino group in theophylline molecule, which could form hydrogen bond with the monomer, on the other hand, because H in the amino group has smaller volume, contiguous O and N could also form hydrogen bond with the monomer, however, the other O to both methyl (CH3 ) could not form hydrogen bond with the monomer because of bigger position encumbrance from both CH3 . Therefore, it is possible that theophylline molecule has three active sites. Comparison with theophylline molecule, caffeine molecule has no hydrogen atom which forms hydrogen bond with the monomer, moreover, because there is bigger position encumbrance from contiguous CH3 , the contiguous O and N could not form or could only form very weak hydrogen bond with the monomer. It is hard to produce effectively specific sites, appearing weakly recognition and retention ability. Based above descriptions, molecular recognition process on the monolithic MIP columns can be shown in Fig. 3.
Fig. 1. Chromatograms of caffeine and theophylline on non-imprinted column (a) and monolithic MIP column (b, c). (a) and (b), Isocratic elution; mobile phase, acetonitrile; flow rate, 0.6 mL/min; (c), gradient elution: mobile phase, dichloromethane, and methanol–acetic acid (20:1); detection wavelength, 271 nm; peak 1, caffeine; peak 2, theophylline.
of polar additives in the mobile phase were also evaluated with mixtures of acetonitrile–acetic acid as the mobile phase. The experiment showed that the polarity of the organic solvents had significant effect on the retention behavior of the template (Fig. 4). With the increasing of the solvent polarity in the mobile phase, the retention factors of caffeine and theo-
3.2. Effect of the mobile phase composition The effect of the mobile phase on the retention and separation was investigated using methanol, dichloromethane and acetonitrile as mobile phase, respectively. The best separation was obtained by using acetonitrile as mobile phase. The effects
Fig. 2. Molecular structure of caffeine (1) and theophylline (2).
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Fig. 3. The schematic process of synthesized monolithic MIP.
Fig. 4. Effect of mobile phase composition on the retention and separation. Mobile phase, acetonitrile and acetic acid; flow rate, 0.6 mL/min; detection wavelength, 271 nm; k1 , the retention factor of caffeine; k2 , the retention factor of theophylline; a, separation factor of caffeine and theophylline.
phylline all decreased. It is suggested that polar additives can interfere the hydrogen-bonding interactions between the MIP matrix and the functional groups of the analytes. The more polar the mobile phase, the more the affinity of caffeine and theophylline to it, suggesting that the hydrogen-bonding interactions were destroyed much, so the retention factors decreased. Though using acetonitrile as mobile phase could get pleasant separation, the peak of theophylline is very broad and asymmetric. In order to get more sharper and symmetrical peak, gradient elutions with dichloromethane, methanol and acetic acid (20:1) were investigated and optimized. By grade elution (procedure is listed in Table 1), the narrower and higher elution peak could Table 1 Parameters of gradient elution Time (min)
Percent pump B (%)
3.00 8.00 37.00 45.00 45.01
0 0 100 100 0
Note. Pump B, methanol and acetic acid (20:1); total flow rate, 0.6 mL/min.
obtain. Furthermore, from Fig. 1c higher apparent separation factor was obtained than that acetonitrile as the mobile phase. The dichloromethane reagent has the ability for eluting the nonidentify component out, but hardly eluting theophylline out. With the increasing of methanol and acetic acid in mobile phase, the hydrogen-bonding interactions were destroyed quickly, so the eluted speed was quick, which result to more sharper and symmetrical peak. 3.3. Effect of temperature on the separation The effects of temperature over the range from 25 to 45 ◦ C on the separation were also investigated (Fig. 5). The results showed that the retention time of theophylline changed very faster than that of caffeine under higher temperature. The retention factor of theophylline decreased with increasing temperature, because the adsorption of the analytes to the substrate weakened with increasing temperature, allowing the analytes to migrate faster through the monolithic column. It means the hydrogen-bonding interaction and hydrophobic interaction between the template and polymer weakened with increasing temperature. Therefore, a lower temperature will lead to a higher separation.
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Fig. 5. Effect of different temperatures on the retention and separation.
3.4. Effect of the flow rate on the separation The SEM image (Fig. 6) of theophylline monolithic column showed that there were many macropores and flowthrough channels inlaid in the network skeleton of theophylline imprinted monolith. Through-pores provide flow paths through the column, and the size and density of the macropore network gives the monolithic columns a high external porosity and, consequently, a large permeability and low column hydraulic resistance. At the same time, the network of mesopores is responsible for the large specific surface area of the monolith. For these reasons, the monolithic MIP columns are efficient at high flowrates and allow the achievement of very high efficiencies. In this experiment, the flow rate of the mobile phase was investigated over the range of 0.2–2.0 mL/min (Fig. 7), and the result showed that although the migration times of the caffeine and theophylline decreased with increasing of the flow rate, merely a slight decrease of the separation factor was found with increasing flow rate. This is the typical characteristic of monolithic column. 3.5. Thermodynamics of separation on monolithic MIP
and Gibbs free energy of association between the xanthine derivatives and the monolithic MIP. Data obtained from retention and separation at temperature ranging from 25 to 45 ◦ C were processed using the Van’t Hoff equation to estimate the thermodynamic properties of the separation (Fig. 5b). ln k =
−H S + + ln ϕ RT R
ln α =
−H S + RT R
Where R, T, and ϕ are the gas constant, the absolute temperature and the phase volume ratio, respectively. Enthalpy (H) and entropy (S ) were obtained from linear regression of the Van’t Hoff plots by plotting ln k versus 1/T. Enthalpy difference (H) and entropy difference (S) can be calculated form the slopes and intercepts of linear portion of plot of ln α versus 1/T. The results are listed in Table 2. The more H indicated strong affinity between template molecules and MIP, and more easily formed stable compound, which indicates that theophylline has stronger affinity to the recognition sites, and could form a more stable template-MIP
The retention behavior and thermodynamic parameters determined in this study were used to estimate the enthalpy, entropy,
Fig. 6. Scanning electron microscope (SEM) of the monolithic MIP column.
Fig. 7. The relationship of separation factor or backpressure and flow rates on monolithic MIP column. Column: 150 mm × 4.00 mm I.D., mobile phase: acetonitrile.
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Table 2 The thermodynamic parameters of the separation on theophylline monolithic column Analyte
H (KJ mol−1 )
S (KJ mol−1 K)
H (KJ mol−1 )
S (KJ mol−1 K)
Caffein Theophylline
−4.09 −11.8
−0.0297 −0.0315
−7.73
−0.00173
complex than caffeine during their matching in the microcavities on the MIP. Moreover, the result of |H| > T|S| indicates that the separation on the monolithic MIP column was an enthalpy-controlled process. In general, for the temperature range of 25–45 ◦ C, the enthalpic contribution to the overall substrates transfer energy was found to be more significant than the entropic one. In this case, the decrease of temperature led to an increase of separation factors. This result is consistent with the results of the temperature-dependence experiment. 3.6. Binding capacity and adsorption isotherms Fig. 9. Chromatograms of caffeine (1) and theophylline (2) in green tea.
The equilibrium binding experiments for the imprinted and non-imprinted polymers were carried out by varying the initial concentration of theophylline in the range of 0–2.6 mmol/L. The adsorption isotherms were shown in Fig. 8a. For imprinted polymer, the amount of theophylline bound to the imprinted polymer increased with increasing the initial concentration of theophylline, and reached to saturation at higher concentration of theophylline. But for non-imprinted polymer, there was no a typical saturation profile. In order to estimate the binding parameters of MIP, the obtained binding data in Fig. 8a were plotted according to the Scatchard equation: Q/Cfree = (Qmax − Q)/KD , where Q is the amount of theophylline bound to MIP at quilibrium, Qmax is the apparent maximum number of binding sites, Cfree is the free concentration of theophylline and KD is the dissociation constant. The scatchard plot is shown in Fig. 8b. The scatchard plot is not linear indicating that the binding sites in MIP are heterogeneous in respect to the afinity for theophylline. The linear regression equations for the two linear regions are Q/Cfree = 157.66 − 0.667Q (r = 0.996) and
Q/Cfree = 98.49 − 0.125Q (r = 0.998). This suggests that there are two heterogeneous hydrogen bonds (H· · ·O and N· · ·H) in the MIP with specific binding properties and the affinity. From the slope (KD ) and the intercept (Qmax ) of the scatchard plot for the higher affinity binding sites can be calculated to be 1.50 mol/L and 236 mol/g dry polymer, respectively. Similarly, the KD and Qmax for the lower affinity binding sites were found to be 7.97 mmol/L and 785 mol/g dry polymer, respectively. 3.7. Analysis of green tea A calibration curve was obtained by loading 5.0 L standard solutions of theophylline over the range of (0–0.80 mg/mL). Good linearity was obtained in this concentration range (r = 0.9992). The quantification limits for this compound was 80 pg. All the samples were determined by standard addition method (Fig. 9). The recoveries were calculated from the corresponding calibration curve and the results are shown in Table 3.
Fig. 8. Adsorption isotherms of caffeine and theophylline (a) and Scatchard analysis (b) of theophylline.
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Table 3 The content of caffeine and theophylline in green teas Sample
Caffeine (g/100 g)
Theophylline (g/100 g)
Recovery (%) Caffeine
1 2 3 4
4.38 10.3 11.8 11.2
0.357 0.486 0.265 0.328
4. Conclusion Theophylline imprinted monolithic columns were successfully designed and applied for quantitative determination of caffeine and theophylline in different kinds of green tea. Caffeine and theophylline were fully separated both under isocratic and gradient elution on this kind of monolithic MIP column and hydrogen bonding interaction and hydrophobic interaction played an important role in the retention and separation. Thermodynamic data obtained by Van’t Hoff plots reveals an enthalpy-controlled separation. The morphological characteristics of the monolithic MIP showed that both mesopores and macropores were formed in the monolith. The study results presented here have substantiated the significant research interest in monolithic MIP columns due to their ease of preparation, high separation efficiency, and rapid mass transport. Acknowledgments The authors gratefully appreciate the financial support by the Specialized Research Funds of China Education Ministry (No. 20050075003). References [1] P.K. Owens, L. Karlsson, E.S.M. Lutz, L.I. Andersson, Trends Anal. Chem. 18 (1999) 146. [2] N. Perez-Moral, A.G. Mayes, Anal. Chim. Acta 504 (2004) 15.
79.5 86.3 117 107
Theophylline 127 138 81.6 90.8
[3] H.Y. Yan, K.H. Row, Int. J. Mol. Sci. 7 (2006) 155. [4] E. Busi, R. Basosi, F. Ponticelli, M. Olivucci, J. Mol. Catal. A: Chem. 217 (2004) 31. [5] H.S. Wei, Y.L. Tsai, J.Y. Wu, H. Chen, J. Chromatogr. B 836 (2006) 57. [6] N. Lavignac, C.J. Allender, K.R. Brain, Anal. Chim. Acta 510 (2004) 139. [7] B. Sellergren, K.J. Shea, J. Chromatogr. A. 690 (1995) 29. [8] J. Xie, L. Zhu, H. Luo, L. Zhou, C. Li, X. Xu, J. Chromatogr. A 934 (2001) 1. [9] X.D. Huang, H.F. Zou, X.M. Chen, Q.Z. Luo, L. Kong, J. Chromatogr. A 984 (2003) 273. [10] J. Yin, G. Yang, Y. Chen, J. Chromatogr. A 1090 (2005) 68. [11] L. Ye, K. Mosbach, React. Funct. Plym. 48 (2001) 149. [12] F.L. Dickert, P. Lieberzeit, M. Tortschanoff, Sens. Actuators B 65 (2000) 186. [13] O. Kriz, O. Ramstrom, K. Mosbach, Anal. Chem. 69 (1997) 345A. [14] K. Hirayama, Y. Sakai, K. Kameoka, K. Noda, R. Naganawa, Sens. Actuator B 86 (2002) 20. [15] M. Yoshikawa, T. Fujisawa, J. Izumi, T. Kitao, S. Sakamoto, Anal. Chim. Acta 365 (1998) 59. [16] C. Baggiani, C. Giovannoli, L. Anfossi, C. Tozzi, J. Chromatogr. A 938 (2001) 35. [17] P.D. Martin, G.R. Jones, F. Stringer, I.D. Wilson, Analyst 128 (2003) 345. [18] O. Br¨uggemann, A. Visnjevski, R. Burch, P. Patel, Anal. Chim. Acta 504 (2004) 81. [19] A.G. Mayes, K. Mosbach, Anal. Chem. 68 (1996) 3769. [20] J. Haginaka, C. Kagawa, J. Chromatogr. A 948 (2002) 77. [21] W.H. Li, D.H. Stover, Macromolecules 33 (2000) 4354. [22] B. Sellergren, B. Ruckert, A.J. Hall, Adv. Mater. 14 (2002) 1204. [23] H.Y. Liu, K.H. Row, G.L. Yang, J. Chromatogr. 61 (2005) 429. [24] H. Li, Y. Liu, Z. Zhang, H. Liao, L. Nie, S. Yao, J. Chromatogr. A 1098 (2005) 66. [25] X.D. Huang, F. Qin, X.M. Chen, Y.Q. Liu, H.F. Zou, J. Chromatogr. B 804 (2004) 13.
Characteristic of theophylline imprinted monolithic column and its application for determination of xanthine derivatives caffeine and theophylline in green tea Han-wen Sun a,∗ , Feng-xia Qiao a,b , Guang-yu Liu a a
College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China b Department of Chemistry, Baoding Teachers College, Baoding 071000, China
Received 14 July 2006; received in revised form 28 August 2006; accepted 4 September 2006 Available online 16 October 2006
Abstract Theophylline imprinted monolithic columns were designed and prepared for rapid separation of a homologous series of xanthine derivatives, caffeine, and theophylline by an in situ thermal-initiated copolymerization technique. Caffeine and theophylline were fully separated both under isocratic and gradient elutions on this kind of monolithic molecularly imprinted polymers (MIP) column. The broad peak showed in isocratic elution could be improved in gradient elution. Some chromatographic conditions such as mobile phase composition, flow rate, and the temperature on the retention times were investigated. Hydrogen bonding interaction and hydrophobic interaction played an important role in the retention and separation. The binding capacity was evaluated by static adsorption and Scatchard analysis, which showed that the dissociation constant (KD ) and the maximum binding capacity (Qmax ) were 1.50 mol/L, and 236 mol/g for high affinity binding site, and 7.97 mol/L and 785 mol/g for lower affinity binding site, respectively. Thermodynamic data (H and S) obtained by Van’t Hoff plots revealed an enthalpy-controlled separation. The morphological characteristics of monolithic MIP were investigated by scanning electron microscope, which showed that both mesopores and macropores were formed in the monolith. The present monolithic MIP column was successfully applied for the quantitative determination of caffeine and theophylline in different kinds of green tea. © 2006 Elsevier B.V. All rights reserved. Keywords: Molecularly imprinted polymer; Monolithic column; Characteristic; Caffeine; Theophylline; Green tea
1. Introduction Molecularly imprinted polymers (MIP) that exhibit high selectivity and affinity to the predetermined molecule are a rapidly growing research focus [1–5]. The special binding sites are formed by the self-assembly of a template molecule with specific functional groups and the monomer, followed by a cross-linking co-polymerization. After the polymerization, the template is removed from the polymer, leaving microcavities and recognition sites that, in terms of size, shape, and chemical functionality, are complementary to that of the template. The MIP possess several advantages over their biological counterparts including low-cost, simple and convenient preparation, storage stability, repeated operations without loss of activity,
∗
Corresponding author. Tel.: +86 312 5079739; fax: +86 312 5079719. E-mail address: [email protected] (H.-w. Sun).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.09.004
high mechanical strength, durability to heat and pressure, and applicability in harsh chemical media [6]. Due to such outstanding advantages, MIP had been drawn extensive attention and been used successfully as affinity chromatographic stationary phases [7–10], artificial antibodies [11,12], sensor components [13,14], membrane separation [15], and adsorbents for solid phase extraction [16–18]. The conventional technology is to synthesize the MIP in bulk, grind the resulting polymer block into particles, and sieve the particles into the desired size ranges. Such ground and sieved particles can then be packed into conventional HPLC columns. Although the process of bulk polymerization is simple, the resulting polymer must be crushed, grinded and sieved to obtain the appropriate size, this tedious and timeconsuming process often produces particles that are irregular in size and shape, some interaction sites are destroyed during grinding, which lead to a negative impact on chromatographic performance and lower MIP loading capacity with respect
H.-w. Sun et al. / J. Chromatogr. A 1134 (2006) 194–200
to theoretical values. In order to overcome these problems, uniformly sized and monodispersed particles had been made by suspension polymerization [19], multistep swelling process [20], precipitation polymerization [21], and surface imprinting polymerization [22]. These techniques each offer its own merits, however, these methods often suffers from the use of special dispersing phases/surfactants or too complicated. Monolithic molecularly imprinted technology represents a novel method for preparation of stationary phases in recent years, which combined the advantage of monolithic column and molecular imprinted technology [23–25]. Monolithic MIP is prepared by a simple, one-step, in situ, free-radical polymerization “molding” process directly within a chromatographic column, thus avoiding the tedious procedures of grinding, sieving, and column packing. Compared with conventional particle columns, the monolithic column has attracted significant interest because of their ease of preparation, high reproducibility, and rapid mass transport. Furthermore, the preparation of this type of MIP is more cost-efficient, because it requires much smaller amount of template molecules. However, the prepared monolithic MIP often suffers from high backpressures and low efficiency, which results in their poor application and practical separation. Moreover, the most of monolithic MIP works were focused on preparation and recognition mechanisms, and application to real sample are very rarely. In this work, monolithic MIP columns were designed and prepared for rapid separation of a homologous series of xanthine derivatives, caffeine, and theophylline, in different kinds of green tea by in situ thermal-initiated copolymerization technique using theophylline as the template, acrylamide as the functional monomer and ethylene glycol dimethacrylate as crosslinker. Chromatographic conditions, such as separation mechanism, binding capacity, morphological characteristics and thermodynamic data of the monolithic column were also investigated and discussed. The present monolithic column was successfully applied for the quantitative determination of caffeine and theophylline in different kinds of green tea with satisfactory results. 2. Experimental 2.1. Chemicals Caffeine and theophylline were obtained from Sigma (ST Louis, MO, USA). Acrylamide (AM) from Tianjin No. 1 Chemical Reagent Factory (Tianjin, China) was recrystallized prior to use. Ethylene glycol dimethacrylate (EDMA) from SigmaAldrich (Shanghai) Trading Co., Ltd. (Shanghai, China) was extracted with 2 mol/L NaOH solution and dried over anhydroxide magnesium sulfate. 2,2 -Azobisisobutyronitrile (AIBN) from Beijing Chemical Reagent company (Beijing, China) recrystallized prior to use. Toluene was purchased from Beijing Chemical Industries (Beijing China). Dodecyl alcohol, acetonitrile, chloroform and methanol were all of HPLC grade and purchased from Tianjing Kermel Chemical Reagents Development Centre (Tianjin, China). Acetic acid (analytical grade) was purchased from Jinli Industries Co. (Tianjin, China). All of the
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solutions were filtered through a 0.45 m membrane filter (Millipore) before use. 2.2. Preparation of monolithic MIP column The monolithic MIP stationary phase was directly prepared by in situ thermal-initiated polymerization within a chromatographic column tube (150 mm × 4.0 mm I.D.). The polymerization mixture composed of 0.047 g template molecule, 0.212 g acrylamide, 1.134 g ethylene glycol dimethacrylate (EDMA), and 0.010 g 2,2 -Azobis (isobutyronitrile) (AIBN) was dissolved in the appropriate porogenic solvents (toluene and dodecanol). The solution was ultasonicated for 15 min and purged with nitrogen gas for 10 min at room temperature to remove oxygen. The ends of the stainless-steel tube were connected with plastics tubes, and then sealed at the bottom, filled with the above polymerization mixture and then sealed at the top. Subsequently, the polymerization was performed in water bath at 50 ◦ C for 24 h. After the polymerization, the seals and plastics tube were removed. The resulted column was provided with fittings, and connected to an HPLC pump and washed with tetrahydrofuran, methanol:acetic acid (4:1, v/v), respectively, to remove the templates and porogenic solvents. At last, it was washed with methanol until no residue of template was found in the rinses. A non-imprinted blank monolithic column was prepared in the absence of template, and treated in an identical manner. 2.3. HPLC analysis Separation characteristic of monolithic MIP column was performed with a Shimadzu HPLC system (Shimadzu, Kyoto, Japan). This HPLC system is equipped with a photodiode array detector (SPD-M10Avp), a gradient controller (SCL-10Avp), a workstation (CLASS-vp), a degasser (DGU-12A) and a column thermostat (CTO-10Avp). A Shimadzu UV-265 UV–vis spectrophotometer (Shimadzu, Japan) was used to detect UV absorption. The UV detection wavelength was set at 271 nm. All the procedures were carried out at the room temperature. The separation factor (α) was determined by the following equation, α = k2 /k1 , where k1 and k2 are the retention factor of the caffeine and the theophylline, respectively. The retention factors were determined by k = (tM − t0 )/t0 , where tM is the retention time of the solute and t0 is the void time of the column. 2.4. Characterization of monolithic stationary phases After all chromatographic experiments had been completed, the column was washed with methanol/acetic acid (10:1, v/v) for 40 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 monolith was then dried under vacuum at 35 ◦ C for 2 days and cut into pieces with a razor blade. The pore properties and microscopic analysis of the monolith was carried out in a KYKY-2800B scanning electron microscopy (Beijing, China) at 25 kV.
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2.5. Binding capacity and adsorption isotherms Static method was performed by placing 20 mg polymer particles, 3.00 mL acetonitrile and different concentration of the template solution into 10 mL flasks, then the flasks were oscillated in a constant temperature bath oscillator at 25 ◦ C for 12 h. The mixture was transferred into a centrifuge tube and centrifuged at 4000 rpm for 5 min. The concentrations of free compounds in the solutions were determined by an UV-Spectrometer at 271 nm. The absorption quantity (Q) was calculated by subtracting the free concentrations from the initial concentrations. 2.6. Solvent extraction of green tea Five grams of green tea was extracted by 150 mL doubly distilled water with continuously stirring under 50 ◦ C for 8 h. The obtained extraction was filtered with 0.2 mm, 25 mm syringe filter, then it was stored in 4 ◦ C for further work. 3. Results and discussion 3.1. Molecular recognition on the monolithic MIP columns To evaluate the molecular recognition ability of the monolithic MIP columns, two homologous series of xanthine derivatives, caffeine, and theophylline were applied to compare the retention and separation on the monolithic MIP column and blank column. Caffeine and theophylline were baseline separated on the monolithic MIP column, but no separation was observed on blank monolithic column (Fig. 1). It indicates that molecular recognition was dependent on the stereo structures and the arrangement of functional groups of the imprinted molecule and the cavities on MIP. The structures of the two compounds are shown in Fig. 2. There is an active amino group in theophylline molecule, which could form hydrogen bond with the monomer, on the other hand, because H in the amino group has smaller volume, contiguous O and N could also form hydrogen bond with the monomer, however, the other O to both methyl (CH3 ) could not form hydrogen bond with the monomer because of bigger position encumbrance from both CH3 . Therefore, it is possible that theophylline molecule has three active sites. Comparison with theophylline molecule, caffeine molecule has no hydrogen atom which forms hydrogen bond with the monomer, moreover, because there is bigger position encumbrance from contiguous CH3 , the contiguous O and N could not form or could only form very weak hydrogen bond with the monomer. It is hard to produce effectively specific sites, appearing weakly recognition and retention ability. Based above descriptions, molecular recognition process on the monolithic MIP columns can be shown in Fig. 3.
Fig. 1. Chromatograms of caffeine and theophylline on non-imprinted column (a) and monolithic MIP column (b, c). (a) and (b), Isocratic elution; mobile phase, acetonitrile; flow rate, 0.6 mL/min; (c), gradient elution: mobile phase, dichloromethane, and methanol–acetic acid (20:1); detection wavelength, 271 nm; peak 1, caffeine; peak 2, theophylline.
of polar additives in the mobile phase were also evaluated with mixtures of acetonitrile–acetic acid as the mobile phase. The experiment showed that the polarity of the organic solvents had significant effect on the retention behavior of the template (Fig. 4). With the increasing of the solvent polarity in the mobile phase, the retention factors of caffeine and theo-
3.2. Effect of the mobile phase composition The effect of the mobile phase on the retention and separation was investigated using methanol, dichloromethane and acetonitrile as mobile phase, respectively. The best separation was obtained by using acetonitrile as mobile phase. The effects
Fig. 2. Molecular structure of caffeine (1) and theophylline (2).
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Fig. 3. The schematic process of synthesized monolithic MIP.
Fig. 4. Effect of mobile phase composition on the retention and separation. Mobile phase, acetonitrile and acetic acid; flow rate, 0.6 mL/min; detection wavelength, 271 nm; k1 , the retention factor of caffeine; k2 , the retention factor of theophylline; a, separation factor of caffeine and theophylline.
phylline all decreased. It is suggested that polar additives can interfere the hydrogen-bonding interactions between the MIP matrix and the functional groups of the analytes. The more polar the mobile phase, the more the affinity of caffeine and theophylline to it, suggesting that the hydrogen-bonding interactions were destroyed much, so the retention factors decreased. Though using acetonitrile as mobile phase could get pleasant separation, the peak of theophylline is very broad and asymmetric. In order to get more sharper and symmetrical peak, gradient elutions with dichloromethane, methanol and acetic acid (20:1) were investigated and optimized. By grade elution (procedure is listed in Table 1), the narrower and higher elution peak could Table 1 Parameters of gradient elution Time (min)
Percent pump B (%)
3.00 8.00 37.00 45.00 45.01
0 0 100 100 0
Note. Pump B, methanol and acetic acid (20:1); total flow rate, 0.6 mL/min.
obtain. Furthermore, from Fig. 1c higher apparent separation factor was obtained than that acetonitrile as the mobile phase. The dichloromethane reagent has the ability for eluting the nonidentify component out, but hardly eluting theophylline out. With the increasing of methanol and acetic acid in mobile phase, the hydrogen-bonding interactions were destroyed quickly, so the eluted speed was quick, which result to more sharper and symmetrical peak. 3.3. Effect of temperature on the separation The effects of temperature over the range from 25 to 45 ◦ C on the separation were also investigated (Fig. 5). The results showed that the retention time of theophylline changed very faster than that of caffeine under higher temperature. The retention factor of theophylline decreased with increasing temperature, because the adsorption of the analytes to the substrate weakened with increasing temperature, allowing the analytes to migrate faster through the monolithic column. It means the hydrogen-bonding interaction and hydrophobic interaction between the template and polymer weakened with increasing temperature. Therefore, a lower temperature will lead to a higher separation.
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Fig. 5. Effect of different temperatures on the retention and separation.
3.4. Effect of the flow rate on the separation The SEM image (Fig. 6) of theophylline monolithic column showed that there were many macropores and flowthrough channels inlaid in the network skeleton of theophylline imprinted monolith. Through-pores provide flow paths through the column, and the size and density of the macropore network gives the monolithic columns a high external porosity and, consequently, a large permeability and low column hydraulic resistance. At the same time, the network of mesopores is responsible for the large specific surface area of the monolith. For these reasons, the monolithic MIP columns are efficient at high flowrates and allow the achievement of very high efficiencies. In this experiment, the flow rate of the mobile phase was investigated over the range of 0.2–2.0 mL/min (Fig. 7), and the result showed that although the migration times of the caffeine and theophylline decreased with increasing of the flow rate, merely a slight decrease of the separation factor was found with increasing flow rate. This is the typical characteristic of monolithic column. 3.5. Thermodynamics of separation on monolithic MIP
and Gibbs free energy of association between the xanthine derivatives and the monolithic MIP. Data obtained from retention and separation at temperature ranging from 25 to 45 ◦ C were processed using the Van’t Hoff equation to estimate the thermodynamic properties of the separation (Fig. 5b). ln k =
−H S + + ln ϕ RT R
ln α =
−H S + RT R
Where R, T, and ϕ are the gas constant, the absolute temperature and the phase volume ratio, respectively. Enthalpy (H) and entropy (S ) were obtained from linear regression of the Van’t Hoff plots by plotting ln k versus 1/T. Enthalpy difference (H) and entropy difference (S) can be calculated form the slopes and intercepts of linear portion of plot of ln α versus 1/T. The results are listed in Table 2. The more H indicated strong affinity between template molecules and MIP, and more easily formed stable compound, which indicates that theophylline has stronger affinity to the recognition sites, and could form a more stable template-MIP
The retention behavior and thermodynamic parameters determined in this study were used to estimate the enthalpy, entropy,
Fig. 6. Scanning electron microscope (SEM) of the monolithic MIP column.
Fig. 7. The relationship of separation factor or backpressure and flow rates on monolithic MIP column. Column: 150 mm × 4.00 mm I.D., mobile phase: acetonitrile.
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Table 2 The thermodynamic parameters of the separation on theophylline monolithic column Analyte
H (KJ mol−1 )
S (KJ mol−1 K)
H (KJ mol−1 )
S (KJ mol−1 K)
Caffein Theophylline
−4.09 −11.8
−0.0297 −0.0315
−7.73
−0.00173
complex than caffeine during their matching in the microcavities on the MIP. Moreover, the result of |H| > T|S| indicates that the separation on the monolithic MIP column was an enthalpy-controlled process. In general, for the temperature range of 25–45 ◦ C, the enthalpic contribution to the overall substrates transfer energy was found to be more significant than the entropic one. In this case, the decrease of temperature led to an increase of separation factors. This result is consistent with the results of the temperature-dependence experiment. 3.6. Binding capacity and adsorption isotherms Fig. 9. Chromatograms of caffeine (1) and theophylline (2) in green tea.
The equilibrium binding experiments for the imprinted and non-imprinted polymers were carried out by varying the initial concentration of theophylline in the range of 0–2.6 mmol/L. The adsorption isotherms were shown in Fig. 8a. For imprinted polymer, the amount of theophylline bound to the imprinted polymer increased with increasing the initial concentration of theophylline, and reached to saturation at higher concentration of theophylline. But for non-imprinted polymer, there was no a typical saturation profile. In order to estimate the binding parameters of MIP, the obtained binding data in Fig. 8a were plotted according to the Scatchard equation: Q/Cfree = (Qmax − Q)/KD , where Q is the amount of theophylline bound to MIP at quilibrium, Qmax is the apparent maximum number of binding sites, Cfree is the free concentration of theophylline and KD is the dissociation constant. The scatchard plot is shown in Fig. 8b. The scatchard plot is not linear indicating that the binding sites in MIP are heterogeneous in respect to the afinity for theophylline. The linear regression equations for the two linear regions are Q/Cfree = 157.66 − 0.667Q (r = 0.996) and
Q/Cfree = 98.49 − 0.125Q (r = 0.998). This suggests that there are two heterogeneous hydrogen bonds (H· · ·O and N· · ·H) in the MIP with specific binding properties and the affinity. From the slope (KD ) and the intercept (Qmax ) of the scatchard plot for the higher affinity binding sites can be calculated to be 1.50 mol/L and 236 mol/g dry polymer, respectively. Similarly, the KD and Qmax for the lower affinity binding sites were found to be 7.97 mmol/L and 785 mol/g dry polymer, respectively. 3.7. Analysis of green tea A calibration curve was obtained by loading 5.0 L standard solutions of theophylline over the range of (0–0.80 mg/mL). Good linearity was obtained in this concentration range (r = 0.9992). The quantification limits for this compound was 80 pg. All the samples were determined by standard addition method (Fig. 9). The recoveries were calculated from the corresponding calibration curve and the results are shown in Table 3.
Fig. 8. Adsorption isotherms of caffeine and theophylline (a) and Scatchard analysis (b) of theophylline.
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Table 3 The content of caffeine and theophylline in green teas Sample
Caffeine (g/100 g)
Theophylline (g/100 g)
Recovery (%) Caffeine
1 2 3 4
4.38 10.3 11.8 11.2
0.357 0.486 0.265 0.328
4. Conclusion Theophylline imprinted monolithic columns were successfully designed and applied for quantitative determination of caffeine and theophylline in different kinds of green tea. Caffeine and theophylline were fully separated both under isocratic and gradient elution on this kind of monolithic MIP column and hydrogen bonding interaction and hydrophobic interaction played an important role in the retention and separation. Thermodynamic data obtained by Van’t Hoff plots reveals an enthalpy-controlled separation. The morphological characteristics of the monolithic MIP showed that both mesopores and macropores were formed in the monolith. The study results presented here have substantiated the significant research interest in monolithic MIP columns due to their ease of preparation, high separation efficiency, and rapid mass transport. Acknowledgments The authors gratefully appreciate the financial support by the Specialized Research Funds of China Education Ministry (No. 20050075003). References [1] P.K. Owens, L. Karlsson, E.S.M. Lutz, L.I. Andersson, Trends Anal. Chem. 18 (1999) 146. [2] N. Perez-Moral, A.G. Mayes, Anal. Chim. Acta 504 (2004) 15.
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