Article pubs.acs.org/IECR
Preparation and Characterization of Molecularly Imprinted Polymer for Selective Adsorption of 4‑Chlorophenol Molecules by Physical Selectivity Method Tenzin Pasang and Chikkakuntappa Ranganathaiah* Department of Studies in Physics, University of Mysore, Manasagangotri, Mysore-570006, India ABSTRACT: Molecular imprinted polymer (MIP) with methacrylate acid (MAA) as monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker in different molar ratios has been prepared. The free volume size and distribution have been derived from the measured positron lifetime spectra in MIP. The free volume size distribution observed clearly indicates the influence of the amount of cross-linker in the sample. Present results suggest that the optimum monomer cross-linker ratio is 1:5 for this MIP. Fourier tranform infrared (FTIR) results confirm complete extraction of the 4-chlorophenol template molecules (4CP) from the MIP with the absence of stretching frequencies at 648 and 825 cm−1 for the C−Cl group. The free volume distribution analysis indicates that the percentage of accessible cavities to 4-CP molecules in this MIP is only 85% and less than those values reported in earlier studies.
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INTRODUCTION Molecular imprinting techniques have been evolving as a practical tool for synthesizing several important materials for solid-phase extraction, sensors (chemical, biological), chromatographic stationary phases, etc. This has attracted a great deal of attention from material scientists and polymer chemists.1−3 The materials produced from this process are called molecular imprinted polymers (MIPs). MIPs have also been found to be potential candidates for drug delivery systems.4 Various methods of synthesis and the extensive uses of MIPs have been recently reviewed.5,6 The latest in this series is the molecular imprinted 3D cavity materials with high specific recognition sites in a synthetic polymer matrix.5 The bulk copolymerization of divinyl cross-linker, ethylene glycol dimethacrylate (EGDMA), with monovinyl monomer, methacrylate acid (MAA), is the most preferred combination in the design of MIPs.4,7 The template−monomer complexes are frozen with a high level of cross-linkers (>50%) to avoid strain in the polymer matrix which may affect the geometrical and morphological structure after the extraction of solvent and template molecules.7 The extraction of template molecules leaves behind cavities which are both physically and chemically complementary to the template molecules. The selectivity of MIPs to template molecules is the prime characteristic of the MIPs from the viewpoint of their numerous applications. However, in such designs, there is a probability for nonselective binding sites in the presence of foreign molecules with similar functional groups as the template molecule or native cavities of similar size of the template molecule in the polymer. Therefore, to achieve high selectivity of the MIPs for the particular template molecules for which it was designed, both functional selectivity (chemical) and pore-size selectivity (physical) must be thoroughly studied. Most of the previous studies were focused on finding an appropriate functional monomer since a direct relation between the specific binding site formation and the affinity of the functional monomer to template molecules exists. Second, in © 2013 American Chemical Society
such studies the characterization of MIPs from the point of view of pores (size and shape) had not been seriously attempted;7,8 nevertheless, the physical selectivity (pores) of MIPs is equally important in comparison to chemical selectivity, especially in the noncovalent imprinting polymers.4 MIPs with monomer (MAA) and cross-linker (EGDMA) with a wide range of template molecules were synthesized and characterized in the past.4,7 In these studies, the cavities left behind after extraction of the template molecules were present along with the native cavities of the same size as the template molecules and were not addressed which is a serious issue with regard to the percent selectivity. In this context, nonbinding cavities (native cavities) in the polymer matrix are to be addressed if the percent physical selectivity of the MIPs is to be measured precisely.9 Generally, in polymer science the micropores or the so-called free volume cavities do affect the macroscopic properties of polymers10 and, in the present case of MIPs, affect template transportation. Therefore, better understanding of the structure and porosity (micro) relation for transport properties of the polymers to imprint the template and then extract it from the polymer is very essential and fundamental. On the experimental front, the commonly employed methods to characterize the pores in MIPs are based on gas adsorption techniques like Brunauer−Emmett− Teller (BET) and Barrett−Joyner−Halenda (BJH).8 BET and BJH are based on the adsorption−desorption isotherms of nitrogen molecules at critical pressure and at liquefaction temperature (77 K). The limitation comes from the observed hysteresis between adsorption and desorption branches of isotherms and hence pore size calculated from these two branches of isotherms could differ considerably for the same kind of pores. These methods provide information on specific Received: Revised: Accepted: Published: 7445
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Table 1. o-Ps Lifetime Results for the Control Polymer (NIP) with Different Monomer to Cross-Linker Ratiosa sample
molar ratio (MAA:EGDMA)
o-Ps lifetime ±0.014 (ns)
o-Ps intensity ±0.18 (%)
average pore radius ±0.015 (Ǻ )
average pore volume ±0.8 (Ǻ 3)
Fv ±0.03 (%)
NIP0 NIP1 NIP2 NIP3 NIP4 NIP5 NIP7 NIP9
1:0 1:1 1:2 1:3 1:4 1:5 1:7 1:9
1.88 1.99 2.17 2.17 2.40 2.16 2.34 2.47
10.28 10.20 11.09 11.16 9.03 10.38 8.28 7.42
2.74 2.85 3.00 3.00 3.06 2.97 3.14 3.25
85.86 96.56 113.25 112.78 120.25 117.64 129.43 143.52
1.58 1.77 2.26 2.27 1.95 2.01 1.93 1.92
a
The word pore is used in the table for free volume cavities.
determine the deviation from the normal or Gaussian distribution indicating the presence of undesired native cavities in the polymer matrix. FTIR scans have been used to identify the formation of MIPs and to detect the presence and absence of template molecules. The SEM pictures are used for morphological information like grain size of the MIPs synthesized in the present work.
surface area from which pore sizes are derived in the range of meso-/macropores (2−50 nm). Notably, these methods do not explicitly reveal the presence of micropores (2 nm) or bigger size pores, and hence, the surface area measured is small. Therefore, from this point of view, PLS is the most attractive and accurate method of measuring micropores which are important for small molecule imprinting. 3.4. Free Volume Distribution Results from CONTIN Program. Pores associated with the template molecules in the MIPs are of primary interest if the MIP is designed for sensing a particular template molecule or for some such application. However, we learn from the earlier investigation32 that the inherent native free volume cavities in the base polymer of MIP have not been addressed but discussed only the pores corresponding to template molecules. These native cavities acquire significance when the template molecule imprinted is a small molecule like 4-CP. To address this issue, the abovediscussed average free volume cavity size evaluation is not adequate since evolution of free volume cavities in polymers is due to chain folding and molecular architecture and hence cavities of varying sizes are evolved which exhibit a distribution of cavity sizes. To get free volume distribution in NIP5, NIP5 + template, MIP5, and the RMIP5, we have used the same measured positron lifetime spectra (those used in PATFIT-88 analysis) and employed the CONTIN-PALS226 program. The program deconvolutes the spectra and generates positron annihilation rate probabilities using Laplace inversion technique.26 The details of the program and interpretations can be found in the original work26 and a host of other publications. The fraction of positrons annihilating with rates between λ and λ + dλ is given by α(λ) dλ with
(2)
where Vf = (4/3)πR3 is average size of free volume cavity and C is a constant whose value is taken as 0.0018 Å−3.16,28 The positron lifetime results so derived from the measured lifetime spectra for NIP samples with different molar ratio of monomer to cross-linker are tabulated in Table 1. From the data in Table 1, it is clear that different molar ratio brings in clearly observable microstructural changes in the NIPs. As the concentration of cross-linker in the sample is increased, the oPs lifetime, cavity radius, and, hence, the cavity size (volume) exhibits more or less increasing tendency. However, the fractional free volume does not show this systematic like other positron parameters. To select a set of samples from the nine NIP samples for the development of MIPs, we used free volume radius and volume as the basis parameters. The criterion for physical selectivity is that the NIPs shall not have native cavities (average radius) bigger than that of the template molecule size (2.99 Å radius for 4-CP).31 However, the polymers used in the synthesis of MIPs generally come with native cavities of varying sizes which cannot be eliminated. Therefore, with this premise, we selected NIP2, NIP5, and NIP9 out of nine samples shown in Table 1 for further development of MIPs. Although NIP1 has small size cavity, this cannot be selected due to less cross-linkers. This basis seems to be lot easier, cost-effective, and time saving compared to what is generally practiced in the synthesis of MIPs as reported in literature. If we follow the general or normal procedure, for each of the nine samples, MIPs are to be made and then rebind the temples from which evaluate the binding efficiency of the NIP structure which is certainly time-consuming, cumbersome, and expensive. Sufficient cross-linkers in the NIP structure are required to give structure stability so that the cavities shall have stable configuration when imprinted. Positron results of the selected three samples of NIPs with template, after extraction (MIP) and after rebinding (RMIP) are tabulated in Table 2. From these three, we need to qualify the best MIP for the 4-CP template molecule whose physical selectivity shall be good. The bigger size micropores in the matrix of the MIPs without compromising the structural strength is important as far as template transport in the MIP is concerned.7 Using the abovementioned aspects, we can say that the best binding MIP sample is certainly MIP5 as seen from Table 2. This decision is made by defining the binding efficiency as follows: If the cavity radius in NIP + templalate sample and that of the reloaded sample with template (RMIP) is the same, the MIP has the best binding efficiency. From Table 2, if we use the above definition of binding efficiency, we find for MIP2, MIP5, and MIP9 samples, the percentage binding is found to be 94.58%, 98.36%, and 95.6%, respectively. Therefore, we can conclude that MIP5 with monomer to cross-linker ratio 1:5 is the good candidate based on average free volume cavity size from PATFIT analysis. These results are in good agreement with the early studies as well.29 To test the efficacy of the above method of deciding the best MIP sample, we provide in Table 2 for NIP 5, the total pore volume measured by BET method13 which in excellent agreement with the present value. For MIP5 we have given the total pore volume based on free volume data,16 this is very much different from that reported in ref 13 because the
∫0
∞
α(λ ) d λ = 1
(3)
A typical annihilation rate probability distribution so obtained for the present samples is shown in Figure 3. The three peaks correspond to three lifetimes resolved from PAFFIT analysis. From the annihilation rate probability distribution function (PDF), free volume radius PDF and free volume size PDF (spherical shape) were transformed by the method of Gregory.26 Accordingly, free volume radius PDF:f(R) and free volume size PDF:g(V) are expressed as f (R ) = 2ΔR {cos[2πR /(R + ΔR )] − 1}α(λ)/(R + ΔR )2 (4)
and g (V ) = f (R )/4πR2
(5)
where ΔR = 1.656 Å is the same as in eq 1. The average values of lifetimes and intensities obtained from the PATFIT and CONTIN-PALS2 programs were compared and found to agree very well. We found that the free volume radius and volume distributions of NIP5, NIP5 + template, MIP5, and of the rebind sample RMIP5 are distinct with respect to peak width, peak position, and skewedness. None of 7449
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that not all pores in the MIPs are accessible to template molecules when the same is reloaded. To show that what percentage of pores really is accessible to the template molecules, we used the free volume radius PDF curve and evaluated the area under this radius PDF curve corresponding to template molecule radius and beyond. This is done by first fitting a Gaussian to the radius PDF curve. For the NIP5 sample (Figure 5), we have computed the percentage area
Figure 3. Typical annihilation rate probability distribution curves in MIP sample.
the distributions exhibited normal distribution; rather all them are skewed to the left from the center reflecting that the distributions are more dense with cavities of lesser radius (than the template molecule radius) as reflected in Figure 4 for NIP5, NIP5 + template, MIP5, and RMIP5. The shift in the peaks indicates the process of addition and removal of template molecules from the polymer matrix. When the MIP5 is rebind with 4-CP molecules, the shift of the curve to the left clearly demonstrates that 4-CP molecules certainly occupy the native free volume cavities. This cannot be seen from the PATFIT results. We see from Figure 4 that the free volume cavities come with a distribution and the maximum probability corresponds to the peak of this distribution. What is important from such an analysis is the following: if one uses the physical selectivity (pore size and shape) as the prime criterion for the design of a MIP, what is the efficiency of this method as for as sensing the molecules which are imprinted in the polymer? Physical selectivity depends on chemical selectivity too. Several investigations in the past used BET and other allied techniques for pores estimation and claim the selectivity to be close to 95− 98%. This depends on the binding ability of the MIP structure. We propose in this work based on the PDF analysis of pore size
Figure 5. Template reloaded NIP5 with pores radius PDF. Gaussian fitting shows the deviation from the normal distribution.
beyond the line corresponding to size of the template molecule. The area to the right of this line is also computed and tabulated for sample series NIP5 samples in Table 3. From Figure 5 we see that the percentage area accessible to 4-CP template molecules is marked in the hatched region for RMIP5 sample which includes native free volume cavities as well, which are inherent to the structure. From Table 3, we see that the area under radius distribution curves (column 2) decreases when template is incorporated (by 0.98%) and increases when the template is removed (by 1.33%). But interestingly when we rebind the template molecules into MIP5, the area decreases (by 2.30%) and is noticeably large. The possible reason for this may be that the
Figure 4. (a) Radius PDF and (b) volume PDF of pores in NIP5, NIP5 + template, MIP5, and RMIP5 samples. 7450
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Table 3. Area under the Radius PDF Curves and Percentage of Pores Accessible to the Template Molecules
sample NIP5 NIP5 + template MIP5 RMIP5
percentage of total area under the radius distribution
percentage area of pores accessible to the template molecule (4-CP)
percentage area of pores with size less than template molecule (4-CP)
9.10 8.12
4.79(100) 4.43(92.48)
4.31 3.69
9.45 7.15
5.28(110.23) 4.07(84.97)
4.17 3.08
Article
AUTHOR INFORMATION
Corresponding Author
*E-mail: crang1@rediffmail.com. Tel.: 91 821 2361128. Fax: 91 821 2419361. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Authors thank Prof. Lokanath Rai K.M., Chemistry Department, and Prof. K. Byrappa, Material Science Department, for the valuable help and discussion. The authors also thank the University of Mysore for providing the facility to carry out this research, and one of the authors (T.P.) thanks the Tibetan Children’s Village, Dharamsala, India, for providing financial help.
native pores also host the template molecules during the rebinding process. In Table 3 we have also given the percentage of pores accessible to template molecules (column 3) and those not accessible to template molecule rebinding (column 4). From the third column of the Table 3, we can understand the use of present analysis of free volume cavity distribution in the following way. If we consider the available native free volume cavities in NIP5 accessible to 4-CP template molecules as 100%, then with the imprinting of the template molecules, this comes down to 92.48% that is a decrease of 7.52%. This decrease refers to template molecules going into native cavities as well as to those binding outside these cavities. When the template molecules are extracted (MIP5 is produced), the available accessible cavities increases to 110.23% that is 10.23% more than in NIP5. If we take the difference between MIP5 and NIP5 + template, the accessible pores turn out to be 17.75%. Now the MIP5 is rebound with the template molecules as is done by adopting the standard procedure by many investigators, and the available accessible pores decrease to 84.97%. This is less by 15.03% as compared to NIP5. From this simple calculation, we understand that the difference of 2.72% pores could not rebind the template molecules or these pores are nonbinding sites for the template. Therefore, the present MIP5 is characterized to have (15.03/17.75) × 100 = 84.68% ∼ 85% physical selectivity for 4-CP template molecules.
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4. CONCLUSION A commonly used monomer MAA with the cross-linker EGDMA has been used to synthesize molecularly imprinted polymer (MIP) with 4-CP as the template molecules. Different stages of the molecular imprinting process have been studied using positron annihilation lifetime spectroscopy. From the results of PATFIT analysis, the MIP with monomer-cross-linker ratio of 1:5 has been qualified as the suitable candidate for imprinting 4-CP molecules and the results of CONTIN analysis proved to be very useful to quantify the binding sites. The MIP5 synthesized in this study has been found to have 85% physical selectivity (pore size and of spherical shape) for 4-CP template molecules. This particular type of quantification of rebinding sites in an MIP is reported for the first time from positron lifetime measurements. It is also evident from the present results that the physical selectivity of MIPs relies on the size and shape of the pores in the microrange and can be easily evaluated from PLS. Also, it is a simple technique for measuring the surface area of the binding sites if proper care is taken with regard to the Positronium formation probability and its influence on o-Ps intensity in the MIP under investigation. Further work in this direction is needed to consolidate the results of this investigation. 7451
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