A comparative study of the potential of acrylic and sol–gel polymers for molecular imprinting

Analytica Chimica Acta 542 (2005) 52–60

A comparative study of the potential of acrylic and sol–gel polymers for molecular imprinting Wayne Cummins∗ , Patrick Duggan, Peter McLoughlin Separation Science Research Group, Waterford Institute of Technology, Cork Road, Waterford, Ireland Received 15 October 2004; received in revised form 20 January 2005; accepted 20 January 2005 Available online 17 February 2005

Abstract The successful molecular imprinting of 2-aminopyridine (2-apy) in bulk polymerisations of acrylic and sol–gel based polymers has been achieved. Both polymeric systems reveal varying degrees of affinity in rebinding the original template as well as a number of structural analogues. Rebinding was conducted in chloroform, acetonitrile and methanol in order to assess the role of hydrogen bonding in imprinting. The acrylic imprinted polymer retained approximately 50% of the template in rebinding studies in chloroform compared to 100% for the sol–gel. However, this higher affinity for the sol–gel was accompanied by a higher degree of non-specific binding. While the acrylic polymer performed poorly in acetonitrile, the sol–gel maintained a high degree of discrimination. The acrylic polymer exhibited little discrimination between imprinted and reference polymers for 3-aminopyridine (3-apy) indicating the high selectivity of the MIP polymer for 2-apy relative to 3-apy. This selectivity was reduced in acetonitrile. Selectivity of the sol–gel for 2-apy in chloroform was poor as 3-apy was retained to a similar degree. Comparable results were obtained in acetonitrile. 4-Aminopyridine (4-apy) bound strongly to all polymers in all solvents and proved very difficult to remove due to the high degree of non-specific binding for both polymeric matrices. © 2005 Elsevier B.V. All rights reserved. Keywords: Molecular imprinting; Molecular recognition; 2-Aminopyridine; Sol–gel; Acrylic

1. Introduction Molecular imprinting can be envisaged as the selective manipulation of the shape, size and chemical functionality of a polymer matrix by a template molecule. The imprinting process consists of the polymerisation of functional monomers in the presence of a template species or a molecule closely related to the template species. The polymerisation is conducted in a solvent (porogen) which facilities the formation of template—monomer complexes by stabilisation of interactions. These complexes are then fixed into this spatial arrangement by the inclusion of a high proportion of cross-linking monomer, which imparts rigidity to the polymer network. Removal of the template species affords nano-cavites, which ∗

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are complementary in size, shape and chemical functionality to the templated species. These cavities have the ability to selectively rebind the template. The general principles, areas of applications and limitations of imprinted polymers have been extensively reviewed [1–5]. Unfortunately the imprinting process usually proceeds with a low degree of fidelity. An inherent characteristic of molecular imprinting is the formation of a distribution of binding sites possessing a range of binding affinities. This heterogeneity has implications for the applications of molecularly imprinted polymers (MIPs) and remains one of their most limiting characteristics. Heterogeneity has been cited as the main contributor to the broad asymmetric peaks obtained in chromatographic analysis [6]. An extensive and comprehensive body of work now exists detailing the use of molecularly imprinted polymers as selective media in a diversity of analytical applications ranging

W. Cummins et al. / Analytica Chimica Acta 542 (2005) 52–60

from solid phase extraction [7], sorbant assays [8], capillary electrochromatography [9], micro extraction fibres [10], sensors [11] and catalysis [12]. Even the surface imprinting of bacterial cells has been realized [13]. In fact molecular imprinting has found application in virtually all areas of analytical chemistry where a selective substrate is a principal requirement. Imprinting may be achieved by three means, covalent, non-covalent and sacrificial spacer. Non-covalent imprinting relies on the ability of the template molecule to produce one or more strong intermolecular non-covalent interactions with the functional monomer, e.g. H-bonding, electrostatic or ␲–␲ interactions. In this thermodynamically stable orientation the functional monomer is incorporated into the polymer matrix. Removal of the template affords a cavity, which is complementary in size, shape and functionality to the template molecule. In covalent imprinting a pre-polymerisation template-monomer covalent bond is formed. Polymerisation is conducted in the usual manner but template removal and rebinding is achieved by a chemical rather than physical process. As only one type of interaction is involved (covalent) a reduced degree of heterogeneity is often realized. This form of imprinting, although resulting in a high degree of specificity, is limited by the amenability of the template to covalent binding with the functional monomer and the ease of its subsequent cleavage. Although not as widely used due to the relative complexity of the procedure, covalently imprinted media have found applications as specific recognition materials [14]. Sacrificial-spacer or semi-covalent imprinting involves covalent imprinting with non-covalent rebinding. This replaces some of the complexities of covalent imprinting with more practical characteristics of non-covalent imprinting. [15]. The vast majority of molecularly imprinted media are based on the use of organic acrylate or acrylic type polymers. A severe limitation of this type of imprinting is the requirement for an organic solvent in which all species are soluble. Template molecules that are only soluble in aqueous phases are generally not amenable to imprinting in such a manner. This presents obvious limitations for their use in environmental and biological applications. Although some promise has been shown for aqueous based rebinding procedures [16] such results are rare and considerable progress is required to overcome this limitation. Sol–gel imprinting may be one such progression. Sol–gel materials are inorganic (siloxane) based polymers formed by the acid or base catalysed hydrolysis and condensation of a series of silane monomers. Inclusion of a template species results in the formation of imprinted media. Sol–gels are extremely versatile materials and have a wide area of application in analytical chemistry [3]. Their optical transparency as well as chemical inertness, rigidity and porosity makes them ideally suited for use as optical sensing devices amongst other specialised applications. Their synthesis is straightforward and the availability of a range

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of pure functional monomers makes their use in molecular imprinting all the more attractive. The potential use of silica based gels as selective media was first demonstrated by Dickey [17]. After some initially positive work, the loss of performance of imprinted sol–gel materials with time proved a severe limitation. A resurgence of interest in the area has been fuelled by a more in depth understanding of the technical aspects of polymerisation and the availability of a wider range of starting monomers. The potential use of titanium butoxide as precursors to imprinted media have been illustrated by Kunitake and co-workers [18]. Marx and Liron have recently demonstrated the superior ability of a propanolol imprinted sol–gel material to rebind the template species over its acrylic based counterpart, with considerably lower non-specific binding [19]. In further work the ability of chirally imprinted sol–gels to selectively discriminate between enantiomers was demonstrated [20]. The kinetic uptake of the pesticide paraoxon was evaluated for a series of thin sol–gel films in aqueous systems [21]. Significantly much of this research was conducted using aqueous based polymerisations and rebinding environments. The possible application of sol–gel technology to electroanalytical sensing was also demonstrated through the formation of imprinted phases selective towards heavy metals [22]. Edmiston and coworkers employed both non-covalent and sacrificial spacer techniques in the sol–gel imprinting of dichlorodiphenyltrichloroethane (DDT) [23]. They found that while an imprinting effect was observed with the non-covalent approach a higher degree of specificity was obtained with the sacrificial spacer method. While the work conducted by Marx involved the use of thin films, the work described herein involved the formation of the sol–gels in bulk form. The utilisation of bulk polymerisation in sol–gel imprinted media has been demonstrated by Fernandez-Gonzalez et al. [24], who found that although bulk polymers could be produced they did exhibit higher degrees of non-specific binding (relative to film gels). The sensitivity of the sol–gel imprinting process was demonstrated by Maier and co-workers [25] who found that the rebinding of substrates was extremely sensitive to polymerisation conditions, particularly the amount of hydrochloric acid and water used in the hydrolysis stage. This study is the first, which compares the molecular imprinting of 2-aminopyridine in acrylic and sol based polymers. It is also the first to evaluate these systems by comparison of their performance in different solvents. Bulk polymerisation was chosen due to its simplicity and ease of subsequent analysis. 2-Aminopyridine (2-apy) presented some interesting considerations for imprinting as it has limited functionality only possessing a pyridyl nitrogen capable of acting as a hydrogen acceptor (i.e. base) and an amino function capable of acting as both a hydrogen donor and acceptor in a hydrogen bonding interactions. Previous work carried out by Zhou and He [26] describing the acrylic based molecular imprinting of 2-aminopyridine was replicated. An equivalent sol–gel polymer was also prepared and the binding characteristics of

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both polymeric materials were evaluated in different environments. Polymers were evaluated and compared for their ability to rebind the original template as well as a number of structural analogues in solvents of different polarity. The reproducibility, robustness and longevity of the polymers were also assessed.

2. Experimental

2.3.2. Condensation After 2 h 16 mL of this pre-polymer solution were added to 188 mg (2 mmol) of 2-apy dissolved in 20 mL of distilled water. This solution was agitated for a further 5 min to ensure thorough mixing, after which it was placed into an oven at 80 ◦ C for 12 h. Note: Reference non-imprinted polymers (NIPs) were prepared for both acrylic and sol–gel polymers. These were prepared in an identical manner to that of the MIP but in the absence of the template.

2.1. Materials 2.4. Template removal 2-Aminopyridine (2-apy), 3-aminopyridine (3-apy), and 4-aminopyridine (4-apy) were purchased from Sigma–Aldrich (IRL). Methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), 2,2 -azo-bis-isobutyronitrile (AIBN), tetraethoxysilane (TEOS), phenyltrimethoxysilane (PTMOS), and 3-aminopropyltriethoxysilane (APTES) were also purchased from Sigma–Aldrich (IRL) and used without further purification. Chloroform, acetonitrile and methanol were of HPLC grade minimum. All other reagents used were of analytical grade. Chloroform and acetonitrile were stored under molecular sieves to ensure the absence of moisture. 2.2. Preparation of the 2-aminopyridine imprinted acrylic polymer The 2-apy imprinted polymers were formed according to the method previously described by Zhou and He [26]. Ninety-four milligrams of 2-apy (1 mmol) and 389 mg of MAA (4.5 mmol) were dissolved in 12.5 mL of chloroform in a 30 mL glass ampoule. Six grams (30 mmol) of EGDMA together with 65 mg of AIBN (0.4 mmol) were added to this solution. Solutions were crimp sealed and agitated by bubbling with nitrogen for 5 min. This was followed by a 15 min sonication to remove dissolved gases. Solutions were placed in a stationary water bath at 60 ◦ C for 24 h and allowed to polymerise. 2.3. Preparation of sol–gel imprinted polymers A typical sol–gel preparation consisted of two stages. Firstly the hydrolysis of monomers in the absence of the template, followed by the condensation step in the presence of the template. 2.3.1. Hydrolysis Twenty-four milliliters (0.11 mol) of TEOS together with 24 mL (0.25 mol) of ethoxyethanol were agitated in a 150 mL beaker. To this solution 800 ␮L (4.3 mmol) of PTMOS, 1200 ␮L (5.16 mmol) of APTES and 800 ␮L of concentrated HCl were added. Four milliliters of distilled water were added dropwise to the agitating solution, which became warm due to the exothermic hydrolysis reaction. Hydrolysis was continued over a 2-hour period.

The same procedure was employed for the removal of the template from both sol–gel and acrylic polymers. The resulting bulk polymers were ground and wet sieved (acetone). Particles ranging from 25 to 75 ␮m were collected. These particles were agitated in a hot solution of acetic acid acidified methanol (10%, v/v), for 15 min. Particles were gravity filtered and washed with approximately 50 mL of hot acidified methanol. Continuous washing with hot methanol was used to achieve the removal of the template. This also achieved the removal of the acetic acid. UV analysis of the filtrate stream allowed for the removal of both the template and acetic acid to be monitored. Polymers were re-suspended in hot methanol and Buchner filtered followed by washing with a further 50 mL of hot methanol, to effect the final removal of residual acetic acid. Polymers were then oven dried at 80 ◦ C to constant weight. 2.5. Binding experiments Three hundred and fifty milligrams of polymer were slurried in 5 mL of acetonitrile and packed into Varian SPE 3 mL cartridges, fitted with Varian, 20 ␮m frits. Columns were preconditioned with the solvent chosen for the analysis. A 3 mL aliquot of a 2 mM solution was loaded onto both a NIP and MIP column. Solutions were eluted through the column under vacuum. The quantity of reloaded substrate removed was determined via UV spectrophotometery. Successive 3 mL aliquots of solvent were used to elute the remaining bound substrate. The quantity removed by each 3 mL washing was quantified and elution profiles produced. In all cases extracts 1–20 inclusive were collected by washing with the reloading solvent. Extracts 21+ were collected by washing with methanol. Regeneration of the column was achieved by re-conditioning of the column with the solvent of choice post analysis. Rebinding experiments were conducted in both chloroform and acetonitrile to investigate the effect of solvent polarity on the retention of the substrate materials. The binding characteristics of the structural analogues, 3-aminopyridine (3-apy) and 4-aminopyridine (4apy) were tested in chloroform and acetonitrile to investigate the selectivity of imprinted sites. Triplicate analyses were preformed to evaluate the reproducibility of the analysis system.

W. Cummins et al. / Analytica Chimica Acta 542 (2005) 52–60

3. Results and discussion 3.1. Molecular imprinting of acrylic polymers The molecular imprinting of 2-aminopyridine in acrylic based polymer materials was achieved according to the method described by Zhou and He [26] without further optimisation. Fig. 1(a) illustrates the elution profile obtained for a reloading of 3 mL of 2 mM 2-apy (6 ␮mol) in chloroform. The quantity removed in each extraction is expressed as the percentage of the total quantity loaded. Elution profiles are useful as a visual aid to compare the performance of molecularly imprinted polymers. It is evident from this profile that the quantity of material loaded was insufficient to overload either NIP (non-imprinted polymer) or MIP. After an initial period of retention the substrate was quickly removed from the NIP in one relatively sharp band with 94% being removed by extraction 10 (Table 1). The MIP demonstrates a much higher affinity with only 9% removed by extraction 10. This was followed by a more gradual release with consecutive washings of chloroform. Prior to switching

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the solvent to methanol at extraction 21, 52% of the 2-apy had been removed from the MIP while 98% was removed from the NIP. Elution with methanol easily displaced the remaining 48% of 2-apy from the MIP polymer. These results demonstrate that under these conditions of loading the MIP was capable of binding the substrate (2apy) with a much higher affinity than the NIP, i.e. a specific response was produced. This binding was however heterogeneous in nature resulting in the bleed observed from the MIP. Nevertheless these profiles indicate the successful imprinting of the acrylic polymer with 2-apy. 3.2. Effect of solvent polarity on rebinding in acrylic polymers Zhou and He [26] have suggested that the mechanism of interaction of the 2-aminopyridine template and methacrylic acid precursor involves hydrogen bonding with the amino group acting as a proton donator and the pyridyl nitrogen acting as a proton acceptor in a co-operative hydrogen bonding interaction. Chloroform was chosen as the porogen as its non-polar character would not be expected to interfere with hydrogen bonding (dielectric constant ε = 5). However, a more polar solvent (e.g. acetonitrile ε = 36) would be expected to interfere with this cooperative interaction and hence reduce the degree of binding and lessen any discrimination between MIP and NIP polymers. In order to investigate this hypothesis, reloading in acetonitrile was performed (Fig. 1(b) and Table 1). The NIP profiles showed a negligible difference with acetonitrile being slightly more efficient in removing the reloaded template compared to chloroform. However, the MIP produced a dramatic loss of specificity for 2-apy in acetonitrile relative to chloroform. Although some degree of discrimination still exists for analysis in acetonitrile, with 84% being removed from the NIP compared to 8% for the MIP by extraction 5, a substantial loss of specificity was demonstrated, as the total quantity removed up to extraction 10 is 64% compared to 9% for the chloroform MIP analysis (Fig. 1(a)). 3.3. Selectivity of the 2-aminopyridine imprinted acrylic polymer

Fig. 1. (a) Elution profiles for 2-apy from imprinted (MIP, ) and nonimprinted (NIP, 䊉) acrylic polymers in chloroform. (b) Elution profiles for 2-apy from imprinted (MIP, ) and non-imprinted (NIP, 䊉) acrylic polymers in acetonitrile. Errors bars are based on ±1S.D. from triplicate analysis.

Polymers were evaluated for their ability to rebind two structural analogues of 2-apy. Although in theory 2-apy, 3-apy and 4-apy all possess the ability to interact via the same functional interactions described previously (Section 3.2) with the carboxylate groups embedded in the polymer matrix, varying degrees of affinity were observed for both specific and non-specific interactions of the analogues tested. The results of 3-apy loading in chloroform are shown in Fig. 2(a) and Table 2. The NIP extraction profiles were similar to that of the equivalent 2-apy run, with a slightly lower degree of non-specific binding observed for 3-apy relative to 2-apy (Fig. 1(a)). The MIP profiles however exhibited substantial

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Table 1 Cumulative extraction quantities from the loading of 2-apy onto NIP and MIP acrylic and sol–gel polymers Polymer

Analogue

Solvent

Cumulative percentage quantity extracted 1

Acrylic

Sol–gel

2-apy

Chloroform

2-apy

Acetonitrile

2-apy

Chloroform

2-apy

Acetonitrile

5

10

15

20

21+

NIP MIP NIP MIP

0.1 0.0 0.3 0.4

53.4 0.0 83.6 8.0

94.1 8.7 96.9 64.5

97.4 36.7 97.9 78.3

98.2 52.5 98.5 84.4

100.0 100.0 100.0 100.0

NIP MIP NIP MIP

23.5 0.0 72.9 4.0

30.5 0.1 91.8 95.2

31.8 0.2 94.3 99.0

32.5 0.3 95.4 99.1

32.8 0.3 96.3 99.2

100.0 100.0 100.0 100.0

differences. By extraction 5, 50% of the reloaded 3-apy was displaced from the MIP by chloroform while, for the equivalent 2-apy extraction, template removal was negligible. This had increased to 80% by extraction 10 with only 9% of 2-apy being removed by the equivalent 2-apy reloading (Table 2). These results illustrate that the imprinted acrylic polymer is more selective for the template 2-apy than the structural 3apy analogue in chloroform. A reloading in acetonitrile was also preformed (Fig. 2 (b) and Table 2), in order to evaluate selectivity in a more polar environment. The performances of both 2-apy and 3-apy in acetonitrile were similar. While some degree of specificity was maintained for both analogues with 8% displaced form the MIP compared to 71.8 for 3-apy by extraction 5, similarities in specificities indicate an overall loss of selectivity for 2-apy relative to 3-apy in acetonitrile, compared to chloroform. The ability of the 2-apy imprinted polymer to selectively bind 4-apy was also evaluated in both solvents (Table 2). 4apy bound to both the NIP and MIP polymers to such an extent that it was very difficult to remove regardless of the solvent system used. Similar results have been reported by He and co-workers [27]. This phenomenon was attributed to the higher basicity of the 4-apy, pKa 9.3 compared to 6.7 for

2-apy. Both 2-apy and 4-apy have the ability of resonance stabilisation by delocalising the charge on the nitrogen of the pyridyl group. This stabilisation is not possible for 3-apy. The interaction between a target molecule and the carboxylic acid group in the polymer becomes stronger with increasing analyte basicity. This resulted in a higher degree of both specific and non-specific binding of 4-apy to the polymers. There is however some important information which can be interpreted from this data. Firstly it appeared that chloroform was a more efficient solvent in displacing the analyte from both NIP and MIP polymers, 59 and 3.6%, respectively compared to acetonitrile with 47 and 0.8%, respectively (Table 2, extract 20). This would suggest that although less polar than acetonitrile, chloroform (porogen) provides the optimal conditions for swelling the polymer allowing for easier access to and from the imprinted sites. Secondly, neither chloroform nor acetonitrile exhibited a capability to displace any significant quantity of 4-apy from the MIP. Both solvents displaced a considerable amount from the equivalent NIP extractions with 59 and 47% being displaced, respectively. Therefore, although not entirely quantifiable both imprinted polymers exhibit a specific retention of 4-apy relative to their nonimprinted counterparts.

Table 2 Cumulative extraction quantities from the loading of 3-apy and 4-apy onto NIP and MIP acrylic and sol–gel polymers Polymer

Analogue

Solvent

Acrylic

3-apy

Chloroform

3-apy

Acetonitrile

4-apy

Chloroform

4-apy

Acetonitrile

3-apy

Chloroform

3-apy

Acetonitrile

4-apy

Chloroform

4-apy

Acetonitrile

Cumulative percentage quantity extracted 1

Sol–gel

NIP MIP NIP MIP NIP MIP NIP MIP NIP MIP NIP MIP NIP MIP NIP MIP

0.08 0.05 0.2 0.1 0.0 0.0 0.0 0.0 41.1 0.1 71.0 0.6 9.2 0.1 34.1 0.1

5

10

15

20

21+

92.2 50.1 94.4 71.8 0.3 0.0 0.1 0.1

97.01 79.11 97.6 86.9 33.2 0.0 5.4 0.2

98.39 86.94 98.7 91.8 52.8 0.0 29.9 0.3

99.07 90.95 99.3 94.5 59.4 3.6 47.0 0.8

100 100 100.0 100.0 61.2 56.2 88.0 78.6

71.5 0.2 90.8 89.2 12.1 0.1 45.6 0.5

80.7 2.6 94.3 98.4 12.1 0.1 48.3 1.8

85.5 18.4 95.9 98.7 12.1 0.1 49.8 4.9

90.0 47.5 96.8 98.8 12.1 0.1 50.6 10.7

100.0 100.0 100.0 100.0 41.9 44.0 55.7 33.4

W. Cummins et al. / Analytica Chimica Acta 542 (2005) 52–60

Fig. 2. (a) Elution profiles for 3-apy from imprinted (MIP, ) and nonimprinted (NIP, 䊉) acrylic polymers in chloroform. (b) Elution profiles for 3-apy from imprinted (MIP, ) and non-imprinted (NIP, 䊉) acrylic polymers in acetonitrile. Errors bars are based on ±1S.D. from triplicate analysis.

3.4. Molecular imprinting of sol–gel polymers To allow for direct comparisons between the polymer systems the binding characteristics of the sol–gel imprinted polymers were evaluated in exactly the same manner as with the acrylic polymers. The sol–gel functional monomer system was chosen to include two functional monomers, phenyltrimethoxysilane (PTMOS) containing an aromatic ring facilitating the formation of possible ␲–␲ interactions with the aromaticity of the pyridine ring, and 3aminopropyltriethoxysilane (APTES), capable of hydrogen bonding with both the amine and pyridyl nitrogen of the aminopyridine. The data obtained from the reloading of the sol–gel polymers with 2-apy are shown in Fig. 3(a) and Table 1. Immediately differences in binding characteristics relative to acrylic polymers are apparent. Firstly from the reloading conducted in chloroform it can be seen that 23.5% of the reloaded template fails to bind to the NIP. This suggests the capacity of the NIP polymer was exceeded at the reloaded concentration. The MIP retains the entire quantity of reloaded template showing no displacement in chloroform (0.3% by

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Fig. 3. (a) Elution profiles for 2-apy from imprinted (MIP, ) and nonimprinted (NIP, 䊉) sol–gel polymers in chloroform. (b) Elution profiles for 2-apy from imprinted (MIP, ) and non-imprinted (NIP, 䊉) sol–gel polymers in acetonitrile. Errors bars are based on ±1S.D. from triplicate analysis.

extraction 20 compared to 32.8% by the NIP). Switching the elution solvent to methanol immediately displaces the entire quantities from both the NIP and MIP. Analysis in chloroform affords limited insight into the true specificity of sol–gel for the template as 2-apy binds completely in chloroform and is immediately displaced in methanol. However, it would appear that the capacity of the MIP under the conditions of this analysis is significantly higher than that of the NIP. 3.5. Effect of solvent polarity on rebinding in sol–gels As with the acrylic polymers, sol–gel polymers were loaded with 2-apy in acetonitrile (Fig. 3(b) and Table 1). Although there is a dramatic loss of affinity for the MIP compared to that observed in chloroform, there is also a corresponding loss of affinity for the NIP. There is little or no retention of the 2-apy by the NIP signifying non-specific binding for the NIP is negligible, with only 27% of the reloaded template binding to the stationary phase. The MIP however binds virtually all of the analyte (96%), indicating a high degree of

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affinity relative to the NIP in acetonitrile. This is a promising situation as in molecular imprinting a low degree of nonspecific binding is as desirable as a high degree of specific binding. 3.6. Selectivity of the sol–gel imprinted polymers The selectivity of the 2-apy imprinted sol–gel polymer was determined by comparing the binding characteristics of 3-apy and 4-apy to that of 2-apy (Table 2). The rebinding characteristics of 3-apy in chloroform were first evaluated (Fig. 4(a) and Table 2). It can be seen that the 2-apy and 3-apy behaved differently in binding to the NIP. 41.1% of the 3-apy was not retained to any degree, compared to 23.5% of 2-apy. 80.7% and 31.8%, respectively were removed by extraction 10. This suggests a lower capacity of 3-apy relative to that of 2-apy. MIP profiles for both 2-apy and 3-apy in chloroform indicate a high degree of binding with the former being bound to a greater extent (100% of 2-apy bound by extraction 20 compared to 52.5% for 3-apy).

These results show a specific response of the sol–gel for both 2-apy and 3-apy. The MIP profiles also demonstrate a greater degree of binding of 2-apy over 3-apy (with 32% and 90%, respectively eluted by extraction 20). This shows the selectivity of the imprinted polymer for 2-apy relative 3-apy. 3-apy was also loaded in acetonitrile to evaluate the selectivity of the sol–gel in a more polar environment (Fig. 4(b) and Table 2). As with the equivalent analysis for 2-apy little or no non-specific binding was observed, with 71.0% (72.9% for 2-apy) of the reloaded 3-apy failing to bind to the sol–gel. Similarly little or no difference between MIP profiles was observable with 99.9% of the reloaded 3-apy binding in acetonitrile (96% for 2-apy). As both analogues exhibit similar specificities for the sol–gel polymers it can be concluded that the selectivity of the sol–gel for 2-apy in acetonitrile is low. As with the acrylic polymers the sol–gel polymers were loaded with 4-apy. A large proportion of the 4-apy bound to both NIP and MIP. Chloroform displaced 12% from the NIP while it was incapable of displacing any from the MIP. Acetonitrile was more effective, removing 51% from the MIP and 11% from the NIP (Table 2). Elution with methanol proved to be of little benefit only removing approximately up to 50% in all cases. The quantity that was removed however did show evidence of greater retention by the MIP in both solvents. 3.7. Acrylic versus sol–gel specificity versus selectivity In this discussion, the binding characteristics of acrylic and sol–gel polymers will be discussed in terms of the difference in specificity, i.e. the success of the imprinting process and the selectivity of each polymer system in relation to which analytes it can bind and how efficiently.

Fig. 4. (a) Elution profiles for 3-apy from imprinted (MIP, ) and nonimprinted (NIP, 䊉) sol–gel polymers in chloroform. (b) Elution profiles for 3-apy from imprinted (MIP, ) and non-imprinted (NIP, 䊉) sol–gel polymers in acetonitrile. Errors bars are based on ±1S.D. from triplicate analysis.

3.7.1. Specificity comparison of acrylic and sol–gel polymers Results suggest that both polymers were successfully imprinted with 2-apy (Figs. 1(a), 3(a) and Table 1). The binding characteristics of each polymer system were in certain cases quite different. Both NIP and MIP acrylic polymers completely bound a fixed quantity of reloaded 2-apy substrate. The MIP exhibited much higher affinity than the NIP, whose entire quantity was promptly displaced by chloroform. A large proportion was also removed from the MIP by washing with chloroform although not as efficiently. The sol–gel produced significantly different profiles, particularly for the NIP, which only succeeded in binding approximately 75% of the reloaded template. However, the quantity it did bind was bound to a very high degree, similar to that of the acrylic MIP polymer, i.e. was only displaced by extraction with methanol. The sol–gel MIP polymer bound the entire quantity to the highest degree, with chloroform incapable of displacing any amount. Therefore, although the sol–gel demonstrated the highest degree of affinity for rebinding the template molecule it was accompanied by a relatively high degree of non-specific binding. The acrylic MIP

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while not binding 2-apy to the same degree as the sol–gel MIP was accompanied by a lower degree of non-specific binding. The ability of both polymers to bind 2-apy in acetonitrile was similar (Figs. 1(b), 3(b) and Table 1). Both sol and acrylic MIPs showed a dramatically reduced ability to rebind 2-apy in acetonitrile relative to chloroform, however both did initially bind the entire quantity. A significant difference was observed for the NIP profiles. While the acrylic NIP also initially bound the entire quantity of reloaded 2-apy the sol–gel NIP did not, only binding approximately 10%, hence exhibiting an extremely low degree of non-specific binding. 3.7.2. Selectivity of acrylic and sol–gel polymers The binding characteristics of 3-apy to the NIP acrylic polymer in chloroform was very similar to that observed for 2-apy (Figs. 1(a), 2(a) and Tables 1 and 2), indicating a similar degree of non-specific binding for both. However, the binding of 3-apy to the MIP polymer, although higher than that of the NIP, was dramatically lower than that of 2-apy. This indicates the 2-apy imprinted acrylic polymer is far more selective for 2-apy than 3-apy in chloroform. Binding characteristics of 3-apy to the sol–gel in chloroform were similar to those of 2-apy (Figs. 3(a), 4(a) and Tables 1 and 2). This implies that although the 2-apy imprinted sol–gel exhibits a high specificity for the substrates tested, it is equally selective for both substrates indicating little or no selectivity for 2-apy relative to 3-apy for the sol–gel in chloroform. Only slight specificity is produced for either 2-apy or 3-apy with the acrylic polymers in acetonitrile. In both cases this specificity is similar, indicating little or no selectivity in acetonitrile. A high degree of specificity is produced by the sol–gels in acetonitrile, due mainly to the very low degree of non-specific binding demonstrated by the NIP studies. However, similar specificities are produced for both 2-apy and 3-apy resulting in a low selectivity for 2-apy. The ability of each polymer to rebind 4-apy was also examined. It was found that regardless of the solvent used, chloroform, acetonitrile or methanol, extraction proved very difficult and in all cases complete removal was not achieved. It should be noted that of the quantities recovered the MIPs displayed higher retention for both sol and acrylic polymers in both solvent systems tested. Although 4-apy was most strongly bound by both polymeric systems, a high degree of non-specific binding was observed in all cases. Binding was sufficiently strong especially for MIP polymers that displacement with the solvents used was not achieved and hence a complete evaluation of the binding characteristics of 4-apy was not possible. It is clear however that the difference in the extraction profiles from the NIP and MIP indicate a difference in specificity.

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Table 3 Evaluation of batch-batch variability Extract 10

Extract 20

Polymer 1 NIP MIP

93 13

97 56

Polymer 2 NIP MIP

97 14

98 56

Data produced from two separate batches prepared and evaluated in an identical manner. Values correspond to cumulative percentage extractions.

ner. For all data triplicate analyses were performed. Statistical errors in the elution profiles were produced based on ±1 standard deviation. Error bars were produced for all figures displayed and illustrate the highly reproducible response of the system. Several samples of each polymer were prepared in order to investigate the batch–batch variability of the polymers. Reloading in chloroform was used to examine reproducibility. Negligible deviation in results was observed (Table 3). In order to study the longevity and robustness of the polymers a change in performance over several months after treatment with different analytes and solvents was examined. In a typical comparison two acrylic polymers prepared at different times were examined (Table 3), to evaluate the batch–batch variability. 4. Conclusions The successful molecular imprinting of 2-aminopyridine in bulk polymerisations of two chemically different matrices has been achieved. While the use of acrylic based polymers in numerous analytical applications is well established, the application of sol–gels to such applications is not yet as substantial, with a relatively small but well presented body of material available. Analysis of the binding properties reveals that hydrogen bonding does indeed play a key role in the molecular recognition observed in acrylic based polymers, and to a lesser extent in sol–gel polymers. While the acrylic polymers only exhibited negligible performance of MIP over NIP in acetonitrile, the sol–gel polymers produced a significant difference demonstrating their greater ability to maintain recognition in a more polar environment. While a higher degree of selectivity was observed for the acrylic polymers a more refined sol–gel polymerisation system might see higher selectivity being produced in future work. The ability to prepare materials capable of molecular recognition has great implications for a host of chemical applications. This coupled with the versatility of sol–gel technology should prove invaluable in the further development of molecular recognition media.

3.8. Reproducibility and ruggedness

Acknowledgements

An essential prerequisite for any analytical procedure is the ability of the system to respond in a reproducible man-

Funding was provided by the Technological Sector Research Initiative (2001) (Strand III). The authors gratefully

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