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Biosensors and Bioelectronics 25 (2010) 1742–1747

Biosensors and Bioelectronics 25 (2010) 1742–1747 Contents lists available at ScienceDirect Biosensors and

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

journal homepage: www.elsevier.com/locate/bios Clean synthesis of molecular recognition polymeric materials

Clean synthesis of molecular recognition polymeric materials with chiral sensing capability using supercritical fluid technology. Application as HPLC stationary phases

Mara Soares da Silva a , Eva R. Vão a , Márcio Temtem a , Luís Mafra b , Jorge Caldeira a ,c , Ana Aguiar-Ricardo a , Teresa Casimiro a ,

a REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal

b Universidade de Aveiro, Departamento de Química, CICECO, Campus Santiago, P-3810-193 Aveiro, Portugal

c Centro de Investigac¸ ão Interdisciplinar Egas Moniz, Instituto Superior de Ciências da Saúde Egas Moniz, 2825-511 Caparica, Portugal

article info

Article history:

Received 31 August 2009 Received in revised form 10 November 2009 Accepted 18 December 2009 Available online 28 December 2009

Keywords:

Supercritical fluid technology Molecular imprinting MIP Tryptophan Enantiomeric separation HPLC

abstract

Molecularly imprinted polymers (MIPs) of poly(ethylene glycol dimethacrylate) and poly(N- isopropylacrylamide-co-ethylene glycol dimethacrylate) were synthesized for the first time in supercritical carbon dioxide (scCO 2 ), using Boc- l -tryptophan as template. Supercritical fluid technology provides a clean and one-step synthetic route for the preparation of affinity polymeric materials with sensing capability for specific molecules. The polymeric materials were tested as stationary HPLC phases for the enantiomeric separation of l - and d -tryptophan. HPLC results prove that the synthesized MIPs are able to recognize the template molecule towards its enantiomer which opens up potential applications in chromatographic chiral separation.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Molecular recognition technology is a promising alternative way to create highly specific sites for a molecule, within a polymer, via imprinting polymerization. Molecular imprinting technique uses the functionality of the target molecule (template), to assem- ble its own recognition site by forming specific interactions with the matrix (Alexander et al., 2006). In themolecular imprinting process, the functional group of the monomer interacts with the template molecule, in the presence of a porogen and a cross-linker agent that freezes the complex within a porous polymer matrix (Mosbach and Haupt, 1998). The imprinted sites formed, chemical and physically complementary to the template molecule are then made accessible by dissociation of the complex, by template removal. Regarding the interactions between monomer and template, three differentmolecular imprinting approaches can be considered, covalent (Wulff et al., 1986), non-covalent (Duarte et al., 2006; Wei and Mizaikoff, 2007; Zhang et al., 2006; Yin et al., 2005) and semi-

Corresponding author. Tel.: +351 212948385; fax: +351 212948385. E-mail address: teresa.casimiro@dq.fct.unl.pt (T. Casimiro).

URL: http://www.dq.fct.unl.pt/scf/ (T. Casimiro).

0956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.bios.2009.12.023

covalent (Sellergren and Andersson, 1990). The easy and cheap preparation, allied with a high specificity, turned out non-covalent imprinting as the most widely used approach. By means of the non-covalent route, the monomer–template complex is stabilized through numerous non-covalent interactions such as hydrogen bonds, ion-paring and dipole–dipole interactions. These robust materials have demonstrated high affinity and specificity, very good thermal and chemical stability, repeated operations without loss of activity, high mechanical strength, dura- bility to heat and pressure, and applicability in harsh chemical media. These properties have led to a significant increase in applica- tions in the last decade such as in synthesis and catalysis (Alexander et al., 2003), extraction (Baggiani et al., 2007), chromatography ( Wei andMizaikoff, 2007) and sensors (Guan et al., 2008). Their high selectivity, in particular, has propelled the development of MIPs as chromatographic stationary phases. MIPs have shown several advantages over other chiral matrixes (Maier and Lindner, 2007). They have proven to be more stable towards thermal, mechani- cal and chemical stress, which makes them perfect for analysis in aggressive environments (Svenson and Nicholls, 2001). A recent review details different synthesis approaches ( Alexander et al., 2006). The most used is bulk synthesis, which yields a hard block polymer, followed by grinding and sieving.

M. Soares da Silva et al. / Biosensors and Bioelectronics 25 (2010) 1742–1747

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Besides time-consuming, particles obtained are irregular and most interaction sites are destroyed reducing the recognition capacity ( Qiao et al., 2006). Also slow interaction kinetics and heteroge- neous nature of the binding sites with respect to geometry and accessibility have been reported (Alexander et al., 2003). Alter- native methods have been proposed, such as in situ, multi-step swelling and suspension polymerizations (Wei and Mizaikoff, 2007). However they still show limitations such as heterogeneity, complicated procedures, large use of template, phase partitioning and use of organic solvents. Much more has to be done so that these MIPs can be widely accepted in industry concerning specificity, reproducibility and sustainability. The increasing importance and world wide applications of these high affinity materials, the need to improve their efficiency and turn the processes “greener” eliminating or drastically reducing the use of volatile organic compounds (VOCs), are major driv- ing forces of this work. Conventional imprinting polymerizations involve excessive use of hazardous organic solvents. In most cases MIPs for enantiomers of amino acid derivatives have been prepared in organic solvents and used with organic mobile or in aqueous phases in HPLC (Haginaka and Kagawa, 2004). It is difficult to pre- pare MIPs in water since the high concentration of water molecules destroys the polar interactions between the functional monomer and the template, thus organic solvents are used (Yu and Mosbach, 1998). Due to environmental and human safety concerns, chemists and chemical engineers are currently seeking new and cleaner methods for polymer processing. Supercritical fluids are consid- ered interesting alternative to most traditional solvents because of their physical and chemical properties. Supercritical carbon dioxide (scCO 2 ), in particular, possesses innumerous properties that made it emerge as the most extensively studied supercritical fluid for polymerization reactions. It is inexpensive, has a low critical point, and is a gas under ambient conditions (Soares da Silva et al., 2007; Barroso et al., 2009). Recently we have synthesized for the first time a molecular recognition polymeric material using supercritical fluid technol- ogy. Micron-sized spherical particles of poly(diethylene glycol dimethacrylate), PDEGDMA MIP, were prepared in scCO 2 using sal- icylic acid and acetylsalicylic acids as model templates, for potential applications in controlled drug release devices (Duarte et al., 2006). Previously we had optimized the synthesis of this highly cross- linked material in scCO 2 ( Casimiro et al., 2005). This work was followed by the work of Ye et al. (2006) who synthesized MIPs of methacrylic acid and divinylbenzene using propanolol as template. This new clean synthetic approach for materials with molec- ular recognition capacity brings notorious advantages compared to conventional methods such as more controlled morphology, high porosity and specific area, being obtained as dry, pure, sterile flowing powders, without making use of organic solvents VOCs, or intensive purification and drying steps. The molecular recognition will be achieved by the self-assembly of the functional monomers and the template, for which the high affinity is wanted, in a highly cross-linked polymer using scCO 2 as porogenic agent. In the last years scCO 2 has already proved to be an excel- lent medium for the synthesis and processing of polymers due to its high density, high diffusivity and low viscosity (Cooper, 2000; Giles et al., 2001). It is well known that the polymerization conditions can highly influence the performance of the molec- ularly imprinted polymers (Piletska et al., 2009). Temperature, solvent polarity, pressure, monomer concentration and interac- tions solvent-monomers-template are known to have practical effects on selectivity, which are associated to the stability of the self-assembly complexes during polymerization, which in turn influences the recognition process. The high diffusivity and low vis- cosity of scCO 2 will decrease the mass transfer limitations found in conventional synthesis.

The high affinity of molecular recognition-based materials makes them a versatile tool in separation technology. MIPs can replace expensive chiral columns, avoid chiral mobile phase addi- tives, derivatisation with chiral reagents, etc. (Maier and Lindner,

2007).

The aim of this work was to address the possibility of developing such a high affinity material using this clean technology, able to distinguish very similar molecules such as enantiomeric species, which could have a real impact in chromatography. Here we report the preparation of a HPLC column in a two step process: first the benign synthesis of a MIP with specificity for Boc- l -tryptophan, using a clean technology, followed by its loading into a blank HPLC column in order to be tested as stationary phase. Poly- mers were characterized by scanning electron microscopy, SEM, adsorption of N 2 according to the BET method and by 13 C CPMAS NMR. Boc-l-tryptophan was used as a model enantiomer as a proof of concept. The separation of l- and d-enantiomers could have application for instance in the identification and quantification of amino acids in biological samples (Zhao and Liu, 2001). Herein we open up a new way of synthesizing chiral polymeric stationary phases for chromatography, with no use of organic sol- vents in the polymerization reaction. The molecular recognition capability of the developed materials was investigated concern- ing the influence of functional monomer addition, sample load and temperature.

2. Experimental

2.1. Materials

Ethylene glycol dimethacrylate (EGDMA, 98% purity) as cross- linker, N-isopropylacrylamide (NIPA, 97% purity) as functional monomer, Boc-l -tryptophan (Boc-l-trp, 99% purity) as template molecule, Boc-d -tryptophan (Boc-d-trp, 98% purity) and azo- bis(isobutyronitrile) (AIBN, 98% purity) were purchased from Sigma–Aldrich. HPLC grade acetonitrile from Scharlau was used. Carbon dioxide was obtained from Air Liquide with purity better than 99.998%. All chemicals were used without further purification.

2.2. MIP and NIP synthesis in scCO 2

Polymerization reactions in scCO 2 were carried out as described elsewhere (Casimiro et al., 2005) in a 33 ml stainless steel high- pressure cell equipped with two aligned sapphire windows and a Teflon coated magnetic stir bar inside. In a typical procedure to produce MIPs, cross-linker (3.43 g), monomer (12 wt% with respect to the amount of cross-linker, if included), initiator (2 wt%) and template (1 wt%) were loaded into the high-pressure cell. The cell was immersed in a thermostatted water bath with ±0.01 C of stability and temperature control was made with a RTD probe contacting the cell, connected to a Hart Scientific PID controller. Temperature was set to 65 C, the optimal AIBN initiation tem- perature, and after purging with nitrogen, CO 2 was introduced up to 21 MPa. This pressure was used since at these conditions an initial single homogeneous phase is assured, with all reactants com- pletely dissolved in the supercritical phase, as it can be observed through the sapphire windows. This is a crucial factor in order to have a completely homogeneous polymer initiation and forma- tion. The reaction proceeded for 24 h. At the end of the reaction, the resulting polymer was slowly washed with fresh high-pressure CO 2 in order to clean the remaining residues of template and unreacted monomer. For the production of NIPs the same proce- dure was followed except no template was added. Two MIPs were produced: MIP1 corresponding to imprinted PEGDMA and MIP2 corresponding to imprinted PEGDMA-co-PNIPA. NIP1 and NIP2 are the respective non-imprinted materials.

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2.3. HPLC analysis

The synthesized polymers were slurry packed into a blank col- umn (Supelco; 150 mm × 4.6 mm) using a mixture of Millipore water and acetonitrile (8%, v/v). Chromatographic analyses were carried out using a Merck L-7100 HPLC pump equipped with an L-7400 UV detector, D-7000 computer interface and a XT Mara- ton autosampler. UV detection was made at 275 nm. Prior to the experiments column was thoroughly washed with acetonitrile with 1% (v/v) of acetic acid in order to further eliminate any unreacted material, until a stable baseline was achieved. Experiments were carried out at 25 and 65 C using a column oven (XT Maraton). The mobile phase used was hydro-organic with 8% acetonitrile by vol- ume at a flow rate of 0.5 ml min 1 . 0.25–4 mM sample were injected for analysis using loop volume of 5 l. Acetone was used as void marker, to determine the column dead time. MIP2 was packed in a longer column (Supelco; 250 mm × 4.6 mm) in order to increase the enantiomeric resolution. This column was also tested in a Knauer equipment, with IR detection Smartline 2300 and pump Smartline

1000.

2.4. Polymer characterization

Polymers were morphologically characterized using scanning electron microscopy (SEM) in a Hitachi S-2400 instrument, with an accelerating voltage set to 15 kV. Samples were mounted on aluminium stubs using carbon tape and were gold coated. Specific surface area and pore diameter of the polymeric par- ticles were determined by adsorption of N 2 according to the BET method. An accelerated surface area and porosimetry system (ASAP 2010 Micromeritics) was used under nitrogen flow. 13 C CPMAS NMR spectra were recorded on a Bruker AVANCE 400 (DSX)WB spectrometer (9.4 T), using a double-resonance 4 mm VTN probe, with a Larmor frequency of 100.62 MHz. The sam- ples were packed into a 4 mm diameter ZrO 2 cylindrical rotor and spun at MAS rates of 7 and 10 kHz. The 13 C CPMAS spectrum was recorded using a RAMP-CP shape (100–50% amplitude); rf field strengths between 50 and 70 kHz for Hartman-Hahn condition cal- ibration for 13 C and 1 H channels; number of scans = 2–4 K; recycle delay of 5 s; contact time = 1.5 ms; 1 H decoupling was employed using the Small Phase INcremental ALternation (SPINAL64) mul- tiple pulse scheme with 64 steps (Fung et al., 2000), during the 13 C acquisition. A pulse length of 4.5 s ( ca. 165 pulses) was used for the basic unit of the SPINAL64 scheme, while the 1 H radio- frequency field strength (ω 1 /2 ) employed was 105 kHz. Chemical shifts were quoted in parts per million (ppm) from solid glycine.

3. Results and discussion

3.1. Characterization of the synthesized polymers

The molecular imprinted and non-imprinted polymers were obtained as dry, free-flowing powders in high yields (99%, deter- mined gravimetrically). The synthesized polymeric material is obtained ready to be slurry packed which can be considered one major advantage over traditional MIPs formation where materials have to be grounded and sieved prior to use, leading to prod- uct losses that sometimes reach more than 50% (Baggiani et al., 2005). The pressure-temperature conditions of the reaction assure an initial homogeneous system, as it can be seen through the sapphire windows. SEM images revealed that the imprinted and non-imprinted polymers show similar morphology, aggregates of smooth surfaced discrete nanoparticles (see Fig. S3, supplementary information). It seems that by introducing a small amount of template during the polymerization step, the morphology of the polymers is not greatly affected.

Table 1 Physical characteristics (surface area, average pore diameter and specific pore vol- ume) of both MIPs and NIPs, obtained by multipoint BET method (type II).

Polymer

BET surface area (m 2 /g)

Pore volume (cm 3 /g)

Pore diameter

(nm)

MIP1

10.30

0.0122

4.7

NIP1

10.14

0.0133

5.2

MIP2

8.41

0.0102

4.8

NIP2

8.53

0.0120

5.6

The physical characteristics of both MIPs and NIPs concern- ing surface area, average pore diameter and specific pore volume, obtained by multipoint BET method (type II) are shown in Table 1. The surface areas obtained were very similar with those obtained for PNIPA synthesized in scCO 2 ( Temtem et al., 2007), mainly due to the known porogenic ability of CO 2 . MIP1 and NIP1 show a slight increase on the surface area compared to MIP2 and NIP2 which could be explained by the fact that the primary parti- cles in this case are smaller, thus, providing a material with higher porosity comprising micron-sized agglomerates of nano-primary particles, as it can be observed on SEM images. This morphology is consistent with other reported precipitation polymerizations in scCO 2 without using stabilizers (Cooper et al., 1999; Casimiro et al., 2005). The small differences observed, on the pore volume and diameter for the MIPs and NIPs could be explained by the interfer- ence of the template molecule in the nucleation process and/or the presence of template residues within the final polymer. Fig. 1 depicts the 13 C CPMAS NMR spectra of imprinted and non- imprinted polymers. The strong similarities in the 13 C chemical shifts and relative intensities between MIP2 and NIP2 co-polymers,

and relative intensities between MIP2 and NIP2 co-polymers, Fig. 1. 1 3 C CPMAS NMR spectra

Fig. 1. 13 C CPMAS NMR spectra and peak assignment. (a) MIP1, (b) MIP2, (c) NIP1 and (d) NIP2. Asterisks depict spinning sidebands.

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and MIP1 and NIP1 polymers, suggest that imprinted and non- imprinted materials are chemically equivalent. Being aware of the insensitive character of NMR to detect very diluted species, the NMR data indicates that the Boc-l-trp template is not visible on the 13 C spectra after washing-out the drug template from MIP1 and MIP2 polymers. This observation suggests a successful poly- mer cleaning upon washing (if not completely removed, at least only a very low template concentration remains). Structural differences between the co-polymer (MIP2/NIP2) and the polymer (MIP1/NIP1) may be observed by the presence of a faint resonance at ca. 42 ppm on MIP2/NIP2 due to the additional contri- bution of the CH 2 groups on NIPA’s ligands, which have slightly more shielded chemical shifts.Moreover, the additional peak inten- sity at ca. 22 ppm, observed on MIP2/NIP2 (Fig. 1b and d), with respect to same chemical shift region on the MIP1/NIP1 polymers (compare Fig. 1a and c), is originated from NIPA’s terminal methylic carbons, which are only present on MIP2/NIP2 polymers. A detailed peak assignment of the relevant 13 C resonances is also shown in Fig. 1. The presence of weak resonances appearing at ca. 125, 137 ppm ( unsaturated carbons from NIPA and EGDMA) and ca. 167 ppm (carbonyl groups from NIPA and EGDMA) shows that a relatively small amount of unreacted double bonds exists on the poly- mers, which is in accordance with other works found in literature (Skogsberg et al., 2007).

3.2. Retention factor and enantioselectivity

The molecular recognition and affinity of the polymers for the template enantiomer was gauged by their ability to selectively retain this molecule. Retention or capacity factors, k , of the pre- pared MIPs were evaluated according to Eq. (1) .

k = t r t 0

(1)

t 0

where t r is the retention time of the amino acid enantiomer and t 0 is the retention time of the void marker on the column, acetone. The enantioseparation factor was calculated using the following equation:

˛

=

k L

k D

(2)

Resolution of the racemic mixture was calculated from Eq. (3), where W D and W L are the baseline widths at 4.4% of peak height.

R

= 2(t L t D )

W D + W L

(3)

In order to evaluate the cross-linker capability of creating specific bonds with the imprinted molecule, a PEGDMA and PEGDMA-co-PNIPAMIPs were synthesized and tested as chromato- graphic stationary phases. MIP1 stationary phase was injected with 1 mM amino acid solu- tions of Boc-l-trp and Boc-d-trp. The HPLC peaks obtained are broad and show some asymmetry, which could be explained by the het- erogeneity of the binding sites within the matrix (chromatogram showed on Fig. S4, supplementary information). It is reported in literature that this heterogeneity is the major contributor to broad and asymmetric peaks in chromatography (Sellergren and Shea, 1995). MIP1 shows some molecular recognition for Boc-d- trp, although it is evident that higher retention factors are obtained for the template molecule (t D = 19 min compared to t L = 25 min). This means that the imprinted stationary phase has non-specific sites (recognizes both enantiomers) but shows higher specificity for Boc-l-trp (better recognition of the template enantiomer). Fig. 2 shows the chromatograms of 0.25 mM amino acid solu- tions of Boc-l -trp and Boc-d-trp injected separately on MIP2 and

Boc- l -trp and Boc- d -trp injected separately on MIP2 and Fig. 2. Chromatograms of

Fig. 2. Chromatograms of MIP2 and NIP2 injected with 0.25 mM solutions of each tryptophan enantiomer at 25 C. Boc-l-trp (dotted line) and Boc-d-trp (solid line).

NIP2 columns. The ability of a MIP stationary phase to retain the

template molecule compared to the retention at the correspond- ing non-imprinted column is a commonly accepted indicator for the selectivity of the produced MIP (O‘Mahony et al., 2006). As it can be seen the injected amino acid derivatives do not show any retention on the non-imprinted stationary phase NIP2. In opposi- tion MIP2 show affinity for both molecules, although presenting

a much higher retention factor for the Boc-l -trp, the template

molecule present during polymerization, than for its enantiomer. Also its peak is broader meaning that there are more interactions

of this molecule with the stationary phase, retarding its elution time. Chromatographic experiments were performed at two different column temperatures 25 and 65 C.

Fig. 3 shows the capacity factors of Boc-l -trp and Boc-d-trp for both imprinted polymers, MIP1 and MIP2, for the range of sample loads studied, as calculated by Eq. (1). As it can be seen higher capac- ity factors can be obtained at 25 C than at 65 C, for both matrixes and both enantiomers and for all the range of compositions. We suggest that this could be due to the increasing solvent power of the mobile phase with temperature, destabilizing the hydrogen bonds between the eluted species and the stationary phase material and leading to lower retention times at 65 C. This temperature depen- dency of the capacity factor is also typically observed in literature (Haginaka and Kagawa, 2004). Also the difference between k L and k D is more pronounced for MIP2 than for MIP1 as it can be seen comparing Fig. 3c and d, to Fig. 3a and b, meaning that MIP2 is more selective for the template enantiomer. MIP2 has less affinity to Boc-d-trp than MIP1, while it shows higher retention times for Boc-l-trp. This can be explained by the introduction of NIPA as functional monomer. The introduc- tion of an amide functional group increases the available hydrogen bonds with the template, which establishes stronger interaction with the stationary phase in both polar and apolar solvents. This is

in accordance with literature (Cong and Mosbach, 1997). For exam-

ple, Boc-l-trp showed a higher retention factor on MIP2 at 25 C and sample load of 0.5 mM (k = 5.03), while the corresponding MIP without amide functional monomer showed a much lower capacity factor (k = 3.06) at the same conditions. For MIP1 at 25 C, k L and k D increase for loads up to 1 mM, remaining constant for higher sample loads. For MIP2 at the same temperature, both capacity factors are almost constant, only a slight increasing trend is observed up to 4 mM. At 65 C the capacity factors of MIP and MIP2 for both enantiomers have a notorious increase for injection loadings up to 1 mM, followed by a less pronounced increase. Around 3 mM the retention factors tend to

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al. / Biosensors and Bioelectronics 25 (2010) 1742–1747 Fig. 3. Effect of sample load and temperature

Fig. 3. Effect of sample load and temperature on the capacity factors of Boc-l -trp ( ) and Boc-d -trp ( ). MIP1: (a) 25 C, (b) 65 C and MIP2: (c) 25 C and (d) 65 C.

approach meaning that the stationary phase looses selectivity due to the saturation of the column. The capacity factor cannot be directly related to enantiomeric separation since besides the specific interactions between the tem- plate and MIP, there is also the binding site competition of the enantiomeric species when injecting the racemic solution. The racemic mixture of Boc-trp was injected on the 150 mm × 4.6 mm columns loaded with MIP1 and MIP2, but no resolution of the peaks were obtained despite the different capacity factors obtained for each enantiomer. This can be explained by the competition for the specific and non-specific sites within the matrix. By increas- ing the length of the column we were expecting to increase the enantiomeric resolution. Gilar et al. (2004) described an increase of the column efficiency with the increase of the column length. Fig. 4(a) and (b) shows the enantioseparation chromatograms for MIP2 by injecting the racemate at 65 and 25 C respectively using a longer column (250 mm × 4.6 mm). As it can be seen there was a significant improvement in the resolution of the enantiomers by increasing the length of the column, since both species can be now clearly distinguished. Table 2 presents the retention, enantios- electivity and resolution for Boc-trp enantiomers on MIP2 at dif- ferent temperatures, when injecting the racemic mixture. As it can be seen promising enantiomeric separation factors and resolutions were obtained for both temperatures. By decreasing the column temperature the stationary phase increased its affinity for the tem- plate enantiomer, with an evident increase of the capacity factor. These results would be quite suitable for instance, for moving- bed chromatography, where full separation of pure enantiomers is

Table 2 Retention, enantioselectivity and resolution factors of Boc-trp enantiomers on MIP2 at different temperatures.

 

k

k

D

 

R

L

˛

25 C 65 C

 

0.98

0.43

2.27

0.55

0.71

0.40

1.76

0.41

0.98 0.43 2.27 0.55 0.71 0.40 1.76 0.41 Fig. 4. Enantioseparation on MIP2 of a 1

Fig. 4. Enantioseparation on MIP2 of a 1 mM tryptophan racemic mixture at: (a) 65 C and (b) 25 C.

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obtained even when the resolution of the two peaks is not excellent ( Juza et al., 2000). Typically MIPs show better selectivity/rebinding results when analysed in the same solvent used in their preparation. It has been reported that changing solvents can affect the integrity of the bind- ing sites weakening the molecular recognition of the template (Yu and Mosbach, 1998). Since the polymers were synthesized in scCO 2 current work is being developed in order to increase resolution by testing these materials in supercritical fluid chromatography.

4. Conclusions

Herein we were able to prepare polymeric materials with chi- ral recognition capability by molecular imprinting in scCO 2 . This technology showed to be a promising “greener” alternative to conventional techniques in the preparation of chromatographic columns for enantioseparation. We proved that the synthesized polymers demonstrate high affinity to the template molecule intro- duced in the polymerization step, being able to recognize and differentiate molecules as similar as enantiomeric species by HPLC. We foresee that a wide range of areas such as sensors, separation, catalysis, etc., could benefit from the clean development of these high affinity materials, especially when purity and morphology of the molecular sensing materials is a key issue.

Acknowledgements

The authors would like to thank Fundac¸ ão para a Ciência e Tecnologia (FCT-Lisbon) for financial support through projects PTDC/QUI/66086/2006 and PTDC/CTM/70513/2006, and doctoral grant SFRH/BD/31085/2006 (M.S.S.), FEDER, FSE and POCTI. The Portuguese NMR Network is acknowledged for granting access to the NMR equipment.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2009.12.023.

References

Alexander, C., Davidson, L., Hayes, W., 2003. Tetrahedron 59, 2025–2057. Alexander, C., Andersson, H.S., Andersson, L.I., Ansell, R.J., Kirsch, N., Nicholls, I.A., O’Mahony, J., Whitcombe, M.J., 2006. J. Mol. Recogn. 19, 106–180.

Baggiani, C., Anfossi, L., Baravalle, P., Anfossi, L., Tozzi, C., 2005. Anal. Chim. Acta 542,

125–134.

Baggiani, C., Anfossi, L., Giovannoli, C., 2007. Anal. Chim. Acta 59, 29–39.

Barroso, T., Temtem, M., Casimiro, T., Aguiar-Ricardo, A., 2009. J. Supercrit. Fluids 51,

57–66.

Casimiro, T., Banet-Osuna, A.M., Nunes da Ponte, M., Aguiar-Ricardo, A., 2005. Eur. Polym. J. 41, 1947–1953. Cong, Y., Mosbach, K., 1997. J. Org. Chem 62, 4057–4064.

Cooper, A.I., Hems, W.P., Holmes, A.B., 1999. Macromolecules 32, 2156–2166. Cooper, A.I., 2000. J. Mater. Chem. 10, 207–234.

Duarte, A.R.C., Casimiro, T., Aguiar-Ricardo, A., Simplício, A.L., Duarte, C.M.M., 2006.

J. Supercrit. Fluids 39, 102–106.

Fung, B.M., Khitrin, A.K., Ermolaev, K., 2000. J. Magn. Reson. 142, 97–101.

Gilar, M., Daly, A.E., Kele, M., Neue, U.D., Gebler, J.C., 2004. J. Chromatogr. A 1061,

183–192.

Giles, M.R., Griffiths, R.M.T., Aguiar-Ricardo, A., Silva, M.R.C.G., Howdle, S.M., 2001. Macromolecules 34, 20–25. Guan, G., Liu, B., Wang, Z., 2008. Sensors 8, 8291–8320. Haginaka, J., Kagawa, C., 2004. Anal. Bioanal. Chem. 378, 1907–1912. Juza, M., Mazzotti, M., Morbidelli, M., 2000. Trends Biotechnol. 18, 108–118. Maier, N.M., Lindner, W., 2007. Anal. Bioanal. Chem. 389, 377–397. Mosbach, K., Haupt, K., 1998. J. Mol. Recogn. 11, 62–68. O‘Mahony, J., Molinelli, A., Nolan, K., Smyth, M.R., Mizaikoff, B., 2006. Biosens. Bio- electron. 21, 1383–1392. Piletska, E.V., Guerreiro, A.R., Whitcombe, M.J., Piletsky, S.A., 2009. Macromolecules 42, 4921–4928. Qiao, F., Sun, H., Yan, H., Row, K.H., 2006. Chromatographia 64, 625–634. Sellergren, B., Andersson, L., 1990. J. Org. Chem. 55, 3381–3383. Sellergren, B., Shea, H.J., 1995. J. Chromatogr. A 690, 29–39.

Skogsberg, U., Meyer, C., Rehbein, J., Fischer, G., Schauff, S., Welsch, N., Albert, K., Hall, A.J., Sellergren, B., 2007. Polymer 48, 229–238. Soares da Silva, M., Temtem, M., Henriques, S., Casimiro, T., Aguiar-Ricardo, A., 2007.

J. Chem. Eng. Data 52, 1970–1974.

Svenson, J., Nicholls, I.A, 2001. Anal. Chim. Acta 435, 19–24. Temtem, M., Casimiro, T., Mano, J.F., Aguiar-Ricardo, A., 2007. Green Chem. 9, 75–

79.

Wei, S., Mizaikoff, B., 2007. J. Sep. Sci. 30, 1794–1805. Wulff, G., Heide, B., Helfmeier, G., 1986. J. Am. Chem. Soc. 108, 1089–1091. Ye, L., Yoshimatsu, K., Kolodziej, D., Francisco, J.D.C., Dey, E.S., 2006. J. Appl. Polym. Sci. 102, 2863–2867. Yin, J., Yang, G., Chen, Y., 2005. J. Chromatogr. A 1090, 68–75. Yu, C., Mosbach, K., 1998. J. Mol. Recogn. 11, 69–74. Zhang, H., Ye, L., Mosbach, K., 2006. J. Mol. Recogn. 19, 248–259. Zhao, S., Liu, Y., 2001. Electrophoresis 22, 2769–2774.