Molecularly imprinted polymers for selective separation of acetaminophen and aspirin by using supercritical fluid technology

Chemical Engineering Journal 226 (2013) 171–180

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Molecularly imprinted polymers for selective separation of acetaminophen and aspirin by using supercritical fluid technology Soon-Do Yoon, Hun-Soo Byun ⇑ Department of Chemical and Biomolecular Engineering, Chonnam National University, Yeosu, Jeonnam 550-749, South Korea

h i g h l i g h t s  AAP or AS imprinted polymers were prepared by supercritical polymerization in CO2.  Adsorption properties were evaluated with the adsorption isotherm and Scatchard analysis.  The recognition abilities were superior to that of the target molecules and others.  Selectivity of MIPs used via supercritical polymerization was better than other methods.

a r t i c l e

i n f o

Article history: Received 4 November 2012 Received in revised form 8 April 2013 Accepted 12 April 2013 Available online 21 April 2013 Keywords: Supercritical fluid technology Molecularly imprinted polymers Adsorption properties Acetaminophen Aspirin

a b s t r a c t In this study, we synthesize molecularly imprinted polymers (MIPs) by using supercritical fluid technology in carbon dioxide (CO2). To prepare MIPs, methyl methacrylate (MMA) is used as a third monomer, methacrylic acid (MAA) or 4-vinylpyridine (4-VP) as functional monomers, acetaminophen (AAP) and aspirin (AS) as templates, and ethylene glycol dimethacrylate (EGDMA) as a crosslinker. To evaluate the binding characteristics of MIPs for AAP and AS, equilibrium binding experiments are conducted. The results indicate that the adsorption equilibrium time is about 120 min, and the binding amount increases with the concentration of templates. The adsorption ability of the MIPs is also investigated by performing an HPLC analysis, measuring the adsorbed amounts for templates and their structural analogue, the selectivity factor (a), and the imprinting-induced promotion of binding (IPB). The results of the evaluation analysis indicate that the prepared MIPs have high separation abilities and selectivity. In addition, the molecular recognition properties according to the kind of functional monomers (MAA and 4-VP) and polymerization methods indicate that the use of 4-VP as a functional monomer is more efficient for binding yield and affinity, and the MIPs prepared by using supercritical fluid assisted polymerization are more efficient way to selectively separate and detect templates than bulk and emulsion polymerization process. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Supercritical fluids (SCFs) have specific properties which can be reinforced by many types of chemical process operations. An additional advantage of SCFs is that they can replace many environmentally harmful solvents currently used in industry. Especially, SCFs are an attractive alternative to organic solvents to be used as additives in polymer processing or preparation. That is, supercritical carbon dioxide (scCO2) is by far the most widely used SCF because its zero ozone-depletion potential makes it relatively cheap, nontoxic, nonflammable, eco-friendly, and acceptable. It has low critical constants of Tc = 31 °C and Pc = 74 bar [1,2]. Because CO2 is a gas under ambient conditions, it is very easily re-

⇑ Corresponding author. Tel.: +82 61 659 7296; fax: +82 61 653 3659. E-mail address: [email protected] (H.-S. Byun). 1385-8947/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cej.2013.04.052

moved from the polymeric product, avoiding the costly processes of drying or solvent removal, which is very important in processing and preparing of polymer-based materials. As a polymerization medium, scCO2 provides some advantages over the conventional solvents. High density of scCO2 enable easy adaptation to reactions of polymerization, thus composed polymer chains precipitate from the solution after reaching a specific molecular weight. Moreover, scCO2 can be used to extract unreacted monomers, initiators, catalysts, and some stabilizers from polymer products to achieve highly pure materials. Molecular imprinting is an applied technique to prepare a stable synthetic polymer matrix called molecularly imprinted polymers (MIPs) that contain highly specific sites having an affinity for a target or template molecule. The general preparation procedure of MIPs is illustrated in Fig. 1. MIPs are used as a selective support in liquid chromatography [3,4], capillary electrophoresis [5,6], and solid-phase extraction [7–9], and it is also used as biosensors

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Fig. 1. Schematic representation of molecularly imprinted polymers. (a) Template, (b) functional monomer, (c) print molecule, (d) crosslinker, (e) third vinyl monomer, (f) bulk polymer, and (g) template-imprinted polymer.

[10,11] or artificial antibodies [12] due to physical–chemical stability and ability of high recognition. MIPs can be synthesized by the covalent [13] or noncovalent [14,15] method, which is now widely used in bulk polymerization [16], in situ polymerization [17], suspension polymerization [18], precipitation polymerization [19], and multi-step swelling polymerization [20]. The most common MIPs are prepared in a bulk form by using bulk polymerization. They are crushed and subsequently ground to gain appropriately sized particles. This method has the advantages of ease for preparing MIPs. However, heterogeneity of polymer matrix can occur during the polymerization process. In addition, the crushing and grinding process causes large loss and destruction of cavity in the template shape of the prepared MIPs. In order to complement the weakness of bulk polymerization, various methods have been attempted to prepare MIP particles such as suspension, emulsion, dispersion, solution, and precipitation polymerization. The MIP particles are easily gained by using these methods. Particle sizes of MIPs are also controlled for applications in various fields. However, these methods are flawed by the difficulty in selecting dispersion agent and mediums because the relation of combinations or solubility between components should be considered. Moreover, the process of polymerization for the formation of MIPs matrix is complex because the multi-step process required. Furthermore, in the case of dispersion, solution, and precipitation polymerization, MIP particles are prepared by using organic solvents. Use of organic solvents has an adverse effect not only on the environment, but also on the selective separation of template molecules. In order to overcome the drawbacks of these polymerization methods and to minimize the consumption of organic solvents, many studies have been carried out using supercritical fluid assisted polymerization in scCO2 as the reaction medium to prepare MIPs in a heterogeneous reaction system [1,21]. The preparation of MIPs using supercritical fluid assisted polymerization provides the following advantages: High purity products are easily obtained. They are synthesized using CO2 as an eco-friendly solvent. The preparation process of MIPs is relatively simple. In addition, the MIPs synthesized using supercritical fluid assisted polymerization are obtained as free-flowing powders with controlled morphology and porosity [22].

Acetaminophen (AAP) is commonly used for the relief of headaches and other minor aches and pains, and it is a major ingredient in numerous cold and flu remedies. However, its overdoses can cause potentially fatal liver damage [23]. The first drug in the non-steroidal anti-inflammatory (NSAID) class, Aspirin (AS) has been used as an anti-inflammatory agent or an antipyretic analgesic. More recently, AS is being evaluated for the prevention of two leading causes of death, i.e., cardiovascular disease and cancer [24,25]. Active pharmaceuticals such as antipyretic analgesics or antibiotics have become a major concern in the aquatic environment pollution [26]. In addition, it is necessary to detect these compounds for the prevention of their abuse. The environmental pollution could be attributed to excretion of pharmaceuticals and their metabolites in urine and feces, and inappropriate disposal of unused pharmaceuticals [27]. Detection of these pharmaceutical products in the blood of human or in the natural environment would not only make it possible to predict a particular disease or optimize the dosage but also protect the environment. In this study, AAP and AS imprinted polymers were synthesized by using supercritical fluid assisted polymerization in scCO2. The binding characteristics of prepared MIPs are investigated by adsorption kinetics, adsorption isotherms, and Scatchard plot analysis. The selective separation abilities of MIPs were analyzed by high performance liquid chromatography (HPLC) analysis, the adsorption of materials with structures similar to target molecules (AAP and AS), the selectivity factor (a), and the imprinting-induced promotion of binding (IPB). The effects of various polymerization methods and different functional monomers were also investigated.

2. Experimental 2.1. Materials We purchased the following from Aldrich Chemical Company, Inc. (Milwaukee, WI, USA): acetaminophen (AAP), aspirin (AS), benzoic acid (BA), salicylic acid (SA), p-toluic acid (p-TA), 1-naphthoic acid (1NA), methyl methacrylate (MMA), ethylene glycol

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AAP–IP Control AS–IP Control AAP–IP Control AS–IP Control AAP–IP Control AS–IP Control AAP–IP Control AS–IP Control AAP–IP Control AS–IP Control AAP–IP Control AS–IP Control

Polymerization method

MMA (mmol)

MAA (mmol)

4-VP (mmol)

Supercritical Supercritical Supercritical Supercritical Supercritical Supercritical Supercritical Supercritical Bulk Bulk Bulk Bulk Bulk Bulk Bulk Bulk Emulsion Emulsion Emulsion Emulsion Emulsion Emulsion Emulsion Emulsion

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

25 25 25 25 – – – – 25 25 25 25 – – – – 25 25 25 25 – – – –

– – – – 25 25 25 25 – – – – 25 25 25 25 – – – – 25 25 25 25

dimethacrylate (EGDMA), methacrylic acid (MAA), 4-vinylpyridine (4-VP), potassium persulfate (K2S2O8, KPS), sodium dodecylsulfate (SDS), and 3, 3, 4, 4, 5, 5, 6, 6, 7, 7, 8, 8, 9, 9, 10, 10, 10-heptadecafluoro-decyl methacrylate (HDFDMA). The a,a-Azobis (isobutyronitrile) (AIBN) was purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). Tetrahydrofuran (THF, HPLC grade), toluene (HPLC grade), and n-hexane (HPLC grade) were obtained from Wako Pure Chemicals (Osaka, Japan). Ethanol was purchased from Duksan (Pharmaceutical Co., Ltd., Korea). Carbon dioxide (CO2, 99.8% minimum purity) was obtained from Daesung Industrial Gases Co. (Yeosu, Korea) and used as received. Monomers used in this work were distilled under vacuum to remove inhibitors before polymerization. 2.2. Preparation of molecularly imprinted polymers The molecularly imprinted polymers (MIPs) were synthesized by using dispersion polymerization in scCO2. AAP or AS was used

Templates (mmol) AAP

AS

10 – – – 10 – – – 10 – – – 10 – – – 10 – – – 10 – – –

– – 10 – – – 10 – – – 10 – – – 10 – – – 10 – – – 10 –

AIBN (wt.%)

Dispersion agent (wt.%)

2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 – – – – – – – – – – – – – – – –

as templates, MAA and 4-VP as functional monomers, MMA as a third vinyl monomer, EGDMA as a cross-linker, AIBN as an initiator, THF as a porogen solvent, and poly(heptadecafluorodecyl methacrylate) (PHDFDMA) as a dispersion agent for the supercritical fluid assisted polymerization. PHDFDMA was prepared by dispersion polymerization in scCO2 at 65 °C and 300 bar. Table 1 shows the composition of MIPs. 1.80 g AS (10 mmol) or 1.51 g AAP (10 mmol) was weighed into a 50 ml vial, respectively. Added to it were 2.15 g MAA (25 mmol) or 2.63 g 4-VP (25 mmol), 10.0 g MMA (100 mmol), 2.97 g EGDMA (15 mmol), 0.3 g AIBN, 0.65 g dispersion agent (PHDFDMA), and 1 mL THF were added, respectively. Dissolved oxygen was removed from the solutions by purging with dry nitrogen for 30 min. The prearranged solution was poured into a 100 mL stainless steel reactor equipped with mechanical stirrer, and it was pressurized at 70 bar by using a syringe pump (ISCO Series D, USA), which contains compressed CO2. The reaction mixture was heated up to 65 °C at 300 rpm, and the remaining CO2 was added into the system until the desired pressure of 300 bar and

Fig. 2. Supercritical fluid assisted polymerization apparatus for the preparation of molecularly imprinted polymers.

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pressure CO2 for 2 h in order to remove unreacted monomer. MIPs using bulk polymerization were synthesized by using the method previously reported [24]. AS (10 mmol) or AAP (10 mmol) as templates was weighed into a 15  150 mm borosilicate glass test tube, respectively. To it MAA or 4-VP (10 mmol), MMA (100 mmol), 30 mg AIBN, and 1 mL THF were added. Dissolved oxygen was removed from the solutions by purging with dry nitrogen for 10 min. The resulting solution was co-polymerized at 65 °C for 48 h. The resultant polymers were ground in a mortar and passed through a 65 mesh (212 lm) sieve. AAP and AS imprinted polymers using the method of emulsion polymerization were prepared as follows. Polymerization was performed in a 250 mL reactor at a constant stirring rate of 300 rpm in a nitrogen atmosphere. Distilled deionized water (180 mL), surfactant SDS (0.31 g), initiator KPS (0.28 g), and the prearranged solution were added into the reaction bottle. The reaction time and temperature for synthesizing MIP latex were 20 h and 65 °C. The resulting MIP latex was dialyzed in deionized water at room temperature for 3 days and dried under vacuum at 50 °C. Non-imprinted polymers (control polymers) were prepared by the same procedure without the addition of templates. All the MIP powders that had the template removed were dried in a vacuum oven at 50 °C. The templates were removed by the method of swelling process using Soxhlet equipment in ethanol. The degree of removal templates was verified by UV–vis. spectrophotometer (UV-160A, Shimadzu, Kyoto, Japan). The results showed that 97.0–99.0% of templates are removed repeatedly for four times. The size and shape of prepared MIPs were investigated with the scanning electron microscopy (SEM, S-4700, Hitachi, Japan), operating at an acceleration voltage of 5 kV. 2.3. HPLC analysis High performance liquid chromatography (HPLC) analysis was carried out using a HPLC system (YoungLin, Seoul, Korea), which consists of a pump (YoungLin M925), a UV–vis detector (YoungLin M720) and a 100 ll injector. First, each MIP was packed into a stainless steel column (150 mm  4.6 mm i.d.). Then, the column was washed with ethanol until a stable base line was obtained to ensure the removal of templates. Subsequent chromatographic analysis was executed with the selected mixed mobile phase (toluene:n-hexane = 20:80, v/v) at a flow rate of 1.0 mL/min and the eluant was detected at its maximum absorption wavelength on the UV detector. Acetone was used as a void marker. Each substrate was injected independently and the substrate volume and the concentration injected were 100 lL and 0.1 mmol/L, respectively. 2.4. Evaluation of adsorption for MIPs

Fig. 3. UV absorption spectrum of the extraction of AAP with the extraction times (a), integrated ratio (%) of the AAP extraction with the extraction times (b), and integrated ratio (%) of the AS extraction with the extraction times (c).

the temperature of 65 °C were reached. Polymerization was then carried out at 300 bar for 20 h. The polymerization apparatus for preparing MIPs is represented in Fig. 2. At the end of the polymerization process, the polymer was slowly washed with fresh high-

In order to evaluate their recognition properties, we investigated the MIP imprinted templates for their adsorption kinetics, the binding isotherms, and the adsorption of materials with structures similar to templates. The mixed solvents (toluene:n-hexane (v/v)) were used as the adsorption solution. Kinetic studies were carried out with an initial concentration of 0.8 mmol/L and 20 mg of dried MIPs. After shaking with an isothermal shaker at 200 rpm and 25 °C, the MIP samples were withdrawn at intervals of 30 min, filtered through a 0.45 lm membrane filter (Millipore Corp., Bedford, Massachusetts, USA) and then analyzed for AAP and AS concentrations with a UV–vis. spectrophotometer. The binding isotherms were calculated by adding a fixed amount of 20 mg of the dried MIPs into 20 mL conical flasks containing 10 mL of different initial concentrations (0.1–1.5 mmol/L). The flasks were agitated in an isothermal shaker at 200 rpm and 25 °C for 5 h until the equilibrium was reached. Then, aqueous samples were taken from the solutions and the concentrations

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175

Fig. 4. SEM images of AAP–IP and AS–IP prepared using supercritical fluid assisted polymerization (SP) before/after removal templates, bulk polymerization (Bulk), and emulsion polymerization (Emu). (a) AAP–IP with 4-VP as the functional monomer (SP), (b) AAP-4VPIP removed AAP as the template, (c) AS–IP with 4-VP as the functional monomer (SP), (d) AS–IP removed AS as the template, (e) AAP–IP with MAA as the functional monomer (SP), (f) AS–IP with MAA as the functional monomer (SP), and (g) AAP– IP prepared using bulk, and AAP–IP prepared using Emu.

were analyzed. The adsorbed amount (Q) of AAP or AS bound to the imprinted polymer was calculated by the following equation:

Q ðlmol=gÞ ¼

ð Ci  Ce Þ  V W

ð1Þ

where Ci and Ce are the initially measured and equilibrium concentrations (mmol/L). V(L) is the volume of the solution and W(g) is the mass of the dried MIPs used. To estimate the binding affinity of the MIPs for templates, a saturation binding experiment and Scatchard analysis were carried out. The Scatchard equation was calculated by the following equation:

Q =½Template ¼

ðQ max  Q Þ KD

ð2Þ

where Q is the amount of templates bound to MIPs at equilibrium, Qmax is the apparent maximum number of binding sites, [Template]

is the free AAP and AS concentration at equilibrium and KD is the equilibrium dissociation constant of binding sites. The selectivity factor (a) of the imprinted polymer is the relative adsorbed amount of AAP, AS, BA, SA, p-TA, or 1NA bound to the imprinted polymer compared with that of the AAP or AS as templates. The a value was calculated by the following formula:

a¼

Q ðAAP;AS;BA;SA;pTA;or1NAÞ Q ðAAPorASÞ

ð3Þ

where Q(AAP, AS, BA, SA, p-TA, or 1NA) is the binding amount of AAP, AS, BA, SA, p-TA, or 1NA for AAP–IP and AS–IP and Q(AAP or AS) is the binding amount of AAP and AS for AAP–IP and AS–IP. The effect of molecular imprinting was also investigated in terms of the imprinting-induced promotion of binding (IPB). This value is defined by the following equation:

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Fig. 5. The adsorbed amount (Q) of AAP–IP with MAA and 4-VP as functional monomers in terms of mixing ratio toluene:n-hexane (v/v).

Fig. 7. Binding isotherm of (a) AAP–IP with MAA and 4-VP as functional monomers, and (b) AS–IP with MAA and 4-VP as functional monomers.

Fig. 6. Adsorbed equilibrium profiles of AAP–IP and AS–IP extracted templates with the adsorption time.

IPB ¼

ðQ IP  Q Control Þ Q Control

ð4Þ

where QIP is the amount of the guest molecule that was bound by the imprinted polymer under the conditions described above and QControl is the corresponding value for the control polymer. 3. Results and discussion 3.1. Extraction of the template on MIPs Molecularly imprinted polymers (MIPs) are functional polymers that have been synthesized using the molecular imprinting technique which forms cavities in the polymer matrix with affinity to a target molecule (template). The preparation of MIPs generally involves initiating the polymerization of monomers with a crosslinker, third vinyl monomer, and functional monomer in the presence of a target molecule that is extracted after polymerization, thus leaving complementary cavities behind. The function of MIPs is determined by the types of functional monomers, the degree of crosslinkage or polymerization, the methods of polymerization process, and the degree of removal template. Of these

conditions of MIP performance, the extraction of the removal template plays an important role in improving the ability of MIPs since it related to the adsorbed amount and selectivity of the MIPs. In our previous study [24], the effects of extraction were reported with soxhlet extraction and swelling process as the methods of removal template. The method of swelling process was confirmed to be more effective than that of soxhlet extraction. The method of swelling process is closely related with the type of third vinyl monomers. In order to use this process, MIPs were prepared using MMA as the third vinyl monomer, and the templates were extracted. Fig. 3 shows the removal ratio (%) of each MIP. The removal template was calculated from the removal ratio (%) of AAP and AS imprinted polymers (2.0 g) including AAP and AS. The degree of removal template was verified in terms of UV–vis spectrophotometer (Fig. 3a). Fig. 3b and c represent the removal ratio (%) with extraction times (h) for prepared MIPs. The templates were removed above 98.0% in about 36 h. The degree of template removal was relatively fast due to the use of the swelling process. 3.2. Scanning electron microscope (SEM) images of MIPs Fig. 4 shows the SEM images of MIPs synthesized by various methods of polymerization such as supercritical fluid assisted polymerization (SP), bulk polymerization (Bulk), and emulsion polymerization (Emu). The images show that MIPs particles are

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177

Fig. 8. Scatchard plot of binding for (a) AAP–IP with MAA and 4-VP as functional monomers, and (b) AS–IP with MAA and 4-VP as functional monomers.

Table 2 Qmax and Kd values to be calculated from the slope and intercept of the Scatchard plot. MIPs

Qmax1 (lmol/L)

Qmax2 (lmol/L)

KD1 (lmol/g)

KD2 (lmol/g)

AAP–IP (MAA) AAP–IP (4-VP) AS–IP (MAA) AS–IP (4-VP)

104.06 108.15 111.76 109.18

315.73 333.02 349.93 590.81

1.61  10 2.30  10 1.40  10 4.72  102

6.67  102 8.33  102 6.25  102 2.00  103

successfully synthesized through the SP process. In addition, use of the solvent for the removal of templates does not cause any large structural changes. The average size of MIPs particles prepared by using SP was about 250–300 nm. Generally, MIPs are prepared by using Bulk. Fig. 4g shows the SEM image of AAP-imprinted polymer prepared Bulk. We found that particles of irregular shape and size are gained due to the process of crushing and grinding for the resulting polymer. Fig. 4h represents the SEM image of AAP-imprinted polymer prepared by Emu. The surface of the resulting polymer particles is smooth and has a regular spherical shape. 3.3. Binding characterization of MIPs We evaluated the binding characteristics of MIPs prepared using SP through the adsorption kinetics, the binding isotherm,

Fig. 9. Selective adsorption of (a) AAP–IP and control polymer with MAA and 4-VP as functional monomers for materials with structures similar to templates, and (b) AS–IP and control polymer with MAA and 4-VP as functional monomers for materials with structures similar to templates.

and Scatchard analysis. In order to determine the adsorption of the mixed solvent, we investigated the adsorbed amount (Q) for imprinted polymer with respect to mixed solvent ratios (toluene:n-hexane (v/v)) of 10:90, 20:80, 25:75, 50:50, 75:25, 80:20, and 90:10. As shown in Fig. 5, Q values increased as the n-hexane content increased in toluene because the proper binding sites for the adsorption of specific target molecules was formed by using the mixed solvent which exerts a swelling and shrinking action on prepared MIPs. This result indicates that the optimum mixed solvent ratio was 20:80 as toluene:n-hexane ratio (v/v). Fig. 6 shows the adsorption kinetics of AAP and AS imprinted polymers prepared by using different functional monomers MAA and 4-VP. The results indicated that the adsorption rate of each MIP reached the equilibrium after 120 min although maximum Q values were slightly different in terms of the sort of functional monomers. The binding abilities such as the binding isotherm and Scatchard plot analysis of imprinted templates for prepared MIPs are significant factors in determining the specific binding amount of templates from solution on each MIP. Fig. 7 shows the results of binding isotherms for templates on the AAP and AS imprinted polymers and on the control polymers, respectively. The binding

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amount slowly increased with the concentration of templates in the initial solution, but the binding amount of templates on the imprinted polymers was greater than that of control polymers. This difference of Q values is ascribed to the effect of molecular imprinting for target molecules. The binding Q can reach a stable value due to some non-specific adsorption. In addition, regarding the functionality of monomers, Q values of MIPs with MAA as functional monomer were higher than that of MIPs with 4-VP as shown in Fig. 7. Fig. 8 shows the result of Scatchard analysis for prepared MIPs. The method of Scatchard analysis is an important factor in determining the binding site for template on prepared MIPs. The calculated binding isotherm data was plotted by using Scatchard Eq. (2). The results showed two distinct sections within the plot which can be regarded as straight lines. The two lines indicate that there are two classes of binding sites in the prepared MIPs (AAP–IP and AS– IP). The steep line and flat line are related to the high affinity sites (specific binding sites) and the low affinity sites (non-specific binding sites), respectively. Thus, the Eq. (2) can be rewritten as follows:

Q ðQ  QÞ ðQ max2  Q Þ ¼ max1 þ ½Template K D1 K D2

ð5Þ

For each MIP, Fig. 9a and b show the adsorbed amount (Q) of AAP or AS as templates and of the materials that are structurally similar to templates. These results show that all of the MIPs are selectively separated. This allowed us to determine the abilities of the MIPs because the Q values of the MIPs that use the templates were higher than those materials with structures similar to imprinted templates. In addition, we found that Q values of MIPs that used MAA as functional monomer were higher than that of MIPs with 4-VP. However, when Q values of adsorption materials of the prepared MIPs and the control polymer were compared, the degree of adsorption of MIPs with 4-VP was relatively higher than that of using MAA. The results indicate that the selective separation is carried out by using 4-VP as functional monomer. It was also verified by the analysis of the selectivity factor (a) and the imprinting-induced promotion of binding (IPB) (Table 3). As shown in Table 3, the prepared MIPs were found to have good recognition capabilities. The a and IPB values for each MIP were calculated by Eqs. (2) and (3). Comparison of a values indicated that the selective separation of prepared MIPs particles is carried out. The IPB values directly reflect the imprinting efficiency more correctly than the QIP dose, because the difference in the intrinsic binding activities of various guests molecules is normalized toward functional monomer residues [30]. The larger the IPB value of the MIP for var-

where Q is the adsorbed amount of templates bound to MIPs at equilibrium, Qmax is the apparent maximum number of binding sites, [Template] is the free AAP or AS concentration at equilibrium and KD is the equilibrium dissociation constant of binding sites. In addition, Q1, Qmax1, and KD1 describe the high affinity sites and Q2, Qmax2, and KD2 explain the low affinity sites [28]. The KD value, equilibrium dissociation constant at imprinted sites, and Qmax, the apparent maximum number of binding sites are calculated from the slope and intercept of the plot (see Table 2). The results indicate the values of high and low affinity sites have similar Qmax and KD values except for AS–IP (4-VP) prepared using 4-VP as the functional monomer. However, the total Qmax and KD values of MIPs with 4-VP were higher than that of MIPs with MAA. A possible explanation of this result is that when 4-VP as the basic functional monomer was used on prepared MIPs, the formation of cavities for the separation and detection of the specific molecules was greater than that of MAA as the acidic functional monomer [29]. 3.4. Evaluation of MIPs The prepared MIPs are evaluated and characterized by comparing the adsorption of rebinding templates (AAP and AS) and the molecular structures similar to templates, HPLC analysis, selectivity factor (a), and the imprinting-induced promotion of binding (IPB). Table 3 Selectivity factor (a) and imprinting-induced promotion of binding (IPB) as templates and various adsorption materials for molecularly imprinted polymers. Molecularly imprinted polymers

Template AAP

AS

BA

SA

p-TA

1NA

Selectivity factor (a) AAP–IP (MAA) AAP–IP (4-VP) AS–IP (MAA) AS–IP (4-VP)

1 1 0.297 0.243

0.392 0.272 1 1

0.326 0.236 0.235 0.212

0.352 0.240 0.232 0.172

0.273 0.251 0.204 0.200

0.395 0.223 0.227 0.210

IPB AAP–IP (MAA) AAP–IP (4-VP) AS–IP (MAA) AS–IP (4-VP)

2.961 2.840 0.275 0.192

0.775 0.278 2.622 2.754

0.395 0.165 0.010 0.108

0.389 0.212 0.177 0.063

0.275 0.123 0.140 0.073

1.181 0.130 0.158 0.018

Fig. 10. HPLC analysis for the selective separation of acetaminophen (AAP), aspirin (AS), benzoic acid (BA), salicylic acid (SA), p-toluic acid (p-TA), and 1-naphthoic acid (1NA) on each MIP. (a) HPLC analysis for the selective separation of AAP, AS, BA, SA, p-TA, and 1NA on AAP–IP, and (b) HPLC analysis for the selective separation of AAP, AS, BA, SA, p-TA, and 1NA on AS–IP.

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ious guests molecules, the better the selectivity of the MIP. Compared with the IPB values for each MIP, IPB value on MIPs prepared by using AAP and AS as templates was higher than the other values. As a results, AAP and AS is completely selectively bound by the imprinted polymers. In addition, IPB values of MIPs that used 4-VP as functional monomer were higher than that of MIPs that used using MAA. As mentioned above, 4-VP which is a widely used basic functional monomer forms a strong interaction with electron-deficient aromatic rings, as well as acid–base interactions and H-bond acceptance. Thus, it can easily interact with the template through acid–base interactions and H-bond interaction. For these reasons, the imprinting efficiency, high affinity, and selectivity of the 4-VP used was superior to that of MAA. Therefore, the use of 4-VP as the functional monomer is more efficient for receptor yield and affinity. Fig. 10 shows the HPLC analysis of AAP–IP and AS–IP using 4-VP as functional monomer. The recognition ability of the prepared MIPs was investigated by comparing the retention time of templates (AAP and AS) and its structural analogue on columns packed

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with MIPs. As shown in Fig. 10, the retention time of AAP and AS used as templates on the prepared MIPs was longer than that of their structural analogue. The separation abilities of prepared MIPs are confirmed when compared to retention time of templates with similar molecular structure. 3.5. The efficiency of imprinting with polymerization methods MIPs have been prepared by using various methods such as bulk, suspension, emulsion, precipitation, and supercritical fluid assisted polymerization, and so on. This work evaluated the efficiency of imprinting in terms of polymerization methods for the preparation MIPs was evaluated by comparing Q and IPB values. Fig. 11 shows the results of Q, IPB values, HPLC analysis of MIPs synthesized by SP, Bulk, and Emu. The results indicated that the Q values of MIPs that used SP were higher than that of MIPs that used Bulk, and Emu. The IPB value of MIPs that used SP to provide the imprinting efficiency was also superior to that of MIPs that used Bulk and Emu. In addition, the retention time of AAP as the template on the MIPs prepared by using SP was longer than that of MIPs that used other methods. These results verify that the MIPs prepared via SP is more efficient in selectively separating and detecting the target molecules than other methods. 4. Conclusions MIPs that selectively separate acetaminophen (AAP) and aspirin (AS) were successfully synthesized by using supercritical fluid assisted polymerization in scCO2. MIPs were prepared using MAA or 4-VP as functional monomers, MMA as the third vinyl monomer, and EGDMA as the crosslinker. The fine MIP particles with average particle size of about 250–300 nm were obtained by applying supercritical fluid technology that uses scCO2 as an eco-friendly solvent. The adsorption properties of the prepared MIPs particles were identified by using the adsorption kinetics, the binding isotherm, Scatchard analysis. In addition, the separation abilities were investigated by using HPLC analysis, the adsorption of templates (AAP and AS) and of its structural analogue, the selectivity factor (a), and the imprinting-induced promotion of binding (IPB). The results of the analysis revealed that the prepared MIPs have high separation abilities and selectivity. In addition, the results of comparative analysis of the adsorption properties of MIPs with MAA and 4-VP showed that use of 4-VP as the functional monomer was more efficient in binding yield and affinity to have a low a value and high IPB value. The separation properties obtained by polymerization methods demonstrated that the MIPs by prepared using SP are more efficient in selectively separating and detecting the templates than other methods because they have large amounts of adsorbed imprinted templates and high IPB values. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant No. 2011-0022371). References

Fig. 11. Adsorbed amount (Q), IPB values and HPLC analysis for MIPs prepared by using Supercritical fluid assisted polymerization (SP), bulk polymerization (Bulk), and emulsion polymerization (Emu). (a) Q and IPB values of AAP for AAP–IP with polymerization methods, (b) Q and IPB values of AS for AS–IP with polymerization methods, and (c) HPLC analysis of AAP for AAP–IP and control polymers synthesized by using SP, Bulk, and Emu.

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