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  1. 1. Nasrullah Shah, Mazhar UlIslam, Muhammad Haneef, Zahid Hussain, Muhammad Balal,Joong Kon Park / International Journal of Engineering Research and Applications (IJERA)ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 3, May-Jun 2013, pp.889-894889 | P a g ePhenylalanine Imprinted Microbeads Prepared by ShakingMethodNasrullah Shah*, Mazhar UlIslam**, Muhammad Haneef***, ZahidHussain*, Muhammad Balal*and Joong Kon Park***(Department of Chemistry, Abdul Wali Khan University, Mardan, Pakistan)**(Department of Chemical Engineering, Kyungpook National University, Daegu, Republic of Korea)***(Department of Physics, Hazara University, Mansehra, Pakistan)ABSTRACTMolecularly imprinted polymer (MIP)technology is an emerging tool for theseparation and isolation of structurally relatedcompounds. MIP beads are considered useful inHPLC, CC, SEC, TLC, drug delivery systems aswell as in chemical and biosensors. In this worka new shaking method using baffle flask asreactor for suspension polymerization for thepreparation of MIP microbeads has beendeveloped. This attempt proved to be simple,cheap and has greatly reduced the chances ofoxygen entrapment during polymerizationprocess. Characterization of the prepared non-imprinted beads (NIBs), D-Phe imprinted beads(DIBs) and L-Phe imprinted beads (LIBs) wasmade with SEM and FTIR analysis. Roundshape, porous structure beads with a sizedistribution of 1-12 µm have been synthesized.The prepared DIBs and LIBs were used for theseparation of D, L-Phe racemic mixture andshowed good adsorption capacity (0.109 and0.128 mg/g respectively) and adsorptionselectivity (1.271 and 1.303 respectively)compared to NIBs with adsorption capacity0.063 mg/g and adsorption selectivity 0.969.Keywords- D-Phe imprinted microbeads, L-Pheimprinted microbeads, shaking methodI. INTRODUCTIONMolecularly imprinted polymers (MIPs)often have affinity and selectivity comparable tothose exhibited by poly- or mono-clonal antibodies.MIPs are less expensive and quicker to preparethan biological receptors. Furthermore, they canstand at much harsher conditions than antibodies,such as high temperature, pressure, extreme pH,and organic solvents [1]. The promising areas ofapplication of imprinted polymers includechromatography, biosensors, catalysis, andimmunoassays [2, 3].MIPs prepared as bulk material requirecrushing or grinding and sieving. This process iseconomically not feasible. A lot of material iswasted and particles of irregular shapes areobtained which are not ideal for manychromatographic purposes [4, 5].To get maximum yield of MIPs with efficientseparation ability and useful for variety ofapplications the formation of microbeads is of greatimportance. The MIP microbeads can be useddirectly after synthesis and washing [5-8]. Incurrent time, much effort has been devoted indeveloping novel alternative methods to prepareimprinted stationary phases, which have highefficiency and better mass transfer properties.Spherical imprinted polymers (beads) of veryminute size have been prepared through severaltechniques, such as multistep swelling, suspensionpolymerization, dispersion, precipitation, emulsionand miniemulsion techniques etc [9-11].Suspension polymerization is much better andrelatively simple method used for the preparationof molecularly imprinted beads. This method doesnot require the common tedious mechanicalgrinding process. Sufficiently dilute system leads tothe formation of, uniform sized beads. MIP beadsusing different template molecules have beenprepared by the suspension polymerization in acontinuous aqueous or non aqueous phase in thepresence of suspension stabilizer [12-15].Suspension polymerization methods forproducing beads offer a striking alternative to bulkpolymerization [15-21]. The suspension methodscan produce a higher yield of particles withsuperior chromatographic characteristics [22].Moral and Mayes studied the performance ofimprinted polymer particles prepared by differentpolymerization methods. They concluded that theoverall performance of suspension polymerizationshowed better results for propranolol rebinding inaqueous solution as well as in organic solution[23]. Different techniques such as stirring,sonication, agitation method employ suspensionmethod for preparing imprinted microbeads.However, to further simplify the processes and toachieve good results other simpler techniques needto be developed.In the present study an effort is made toprepare D and L-Phe imprinted microbeads bysuspension polymerization employing a simple andeconomically feasible reactor and procedure. D andL-Phe imprinted microbeads were prepared byshaking method employing baffle flask as a reactor.
  2. 2. Nasrullah Shah, Mazhar UlIslam, Muhammad Haneef, Zahid Hussain, Muhammad Balal,Joong Kon Park / International Journal of Engineering Research and Applications (IJERA)ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 3, May-Jun 2013, pp.889-894890 | P a g eBoth D and L-Phe imprinted microbeads used forthe separation of respective enantiomers. Thecharacterization was conducted by FE-SEM andFTIR analyses.II. MATERIALS AND METHODS1. Chemicals and reagentsD-Phenylalanine (D-Phe), L-Phe, D,L-Phe, methacrylic acid (MAA),ethyleneglycoldimethacrylate (EGDMA), andtrifluoroacetic acid (TFA) were purchased fromSigma-Aldrich (St Louis, MO, USA); 2,2-azobisisobutyronitrile (AIBN) was obtained fromJunsei Chemical Co., Ltd. (Japan); toluene was theproduct of Duksun Pure Chemical Co., (Korea);SDS was from Fluka (Switzerland), polyvinylalcohol (PVA) and copper sulfate (CuSO4·5H2O)were obtained from Yakuri Pure Chemicals Co.,(Osaka, Japan). All reagents used were ofanalytical reagent grade.2. Polymerization process and beadspreparationThe aqueous and organic solutions were preparedby the same method as reported in our previousstudy [8, 24]. The prepared solutions were thenmixed in the baffle flask and shaked at 200 rpm at25˚C in shaking incubator for 10 mins. The mixedsolution was then sonicated under N2 supply forfive minutes to remove any oxygen present in thesolution. After N2 passing the baffle flaskscontaining the solutions were tightly sealed withaluminum foil and were putted in the shakingincubator at 200 rpm and 60˚C temperature for 24hours for complete polymerization. This methodwas followed for the preparation of both D-Phe andL-Phe imprinted microbeads (DIBs and LIBsrespectively). The diagrammatic representation isgiven Fig 1.Fig 1. Illustration of recognition sites formation in the Phe imprinted P(MMA-co-EGDMA) microbeads matrixafter removal of template molecules.3. Washing and removal of templateAfter polymerization the prepared DIBsand LIBs were washed with 5.0% ethanol solutionto remove the unreacted chemicals. Similarly, 5.0%aqueous solution of acetic acid was used for theextraction of template (D-Phe/L-Phe) molecules.This was followed by washing with distilled wateruntil all the acetic acid was removed from thebeads. The removal of acetic acid was checked bypH measurement.4. Adsorption experimentAn amount of 0.6 g of freeze dried beadswas taken and soaked in 5 mL of 100 ppm Pherecemate solution having pH 6 for 3 hours in ashaking incubator at 150 rpm and 25˚C.5. Characterization and analysisFE-SEM micrographs were taken withHITACHI (Japan) S-4300 Field Emission ElectronMicroscope to study surface morphology. The sizedistribution ratios were determined from FE-SEMmicrographs. The analysis of the racemic solutionafter adsorption experiment was performed withHPLC/UV system. FTIR ART spectrometer wasused for structural or functional group analysis ofthe prepared microbeads.6. HPLC AnalysisThe amounts of D-Phe and L-Phe in theobtained samples were determined with HPLCequipped with an M930 solvent delivery pump andM720 UV absorbance detector (Young-LinInstruments, Anyang, Korea). A TSK gel EnantioL2 column (Tosoh, Tokyo, Japan) with dimensionsof 4.6mm×250 mm was used. From HPLC resultsthe adsorbed amount of enantiomers and adsorptionselectivity of the prepared LIBs, DIBs and NIBswere calculated by the following equations [20].III. EQUATIONS1. Amount of Phe adsorbed WVCeCiQ / (1)Where Q is the amount of D- or L-Phe adsorbed on
  3. 3. Nasrullah Shah, Mazhar UlIslam, Muhammad Haneef, Zahid Hussain, Muhammad Balal,Joong Kon Park / International Journal of Engineering Research and Applications (IJERA)ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 3, May-Jun 2013, pp.889-894891 | P a g ethe beads (mg g-1), while Ci and Ce are the initialconcentration (mg L-1) in feed solution andconcentration (mg L-1) after adsorptionrespectively. V is the volume in liter and W is thedry weight in grams.2. Adsorption selectivity        enantiomerCounterTemplateQQads/2/1 (2)Where α ads is the adsorption selectivity, (Q1) and(Q2) are the amounts (mg) of template and thecounter enantiomer adsorbed per 1 g of dry beads,respectively and [Template]/[Counter enantiomer]is the ratio of concentration (mg L-1) of templateand counter enantiomer.IV. RESULTS AND DISCUSSIONThe use of baffle flask as a reactor and theshaking method maintained uniform mixing ofchemicals for proper polymerization. This methodalso reduced the radical scavenging by theatmospheric oxygen which is a big problem inother techniques. The creation of the inert mediumat the start of polymerization and then sealing ofthe reactor was enough for complete and safepolymerization. Contrary to the stirring methodhere no separate heating system and special reactorwere required. This newly introduced shakingmethod may also be helpful for conducting otherpolymerization process involving the use of freeradical polymerization. In this method the priorsonication process caused micro size dropletswhich acted a micro reaction chambers and resultedin micron sized MIP beads.The SEM micrographs represented as Fig.2 indicate that the present method resulted in roundshape beads with porous surfaces. Beads sizedistribution ratio indicates that most of the beadsare in the range 0.10 to 12 µm. Fig. 3 shows thatthe ratio of the beads in the range of 3 to 6 µm isthe highest. Hence these beads are suitable for useas stationary phases in chromatographic techniquesand other useful applications. Contrary to theconventional stirring method where breaking downof beads occur as a result of mechanical stressapplied during stirring the beads remained safe inthe present study. The prepared DIBs and LIBs aresuitable for using in various chromatographictechniques and can also be applicable in preparingbiochemical sensors.From Table 1 it is clear that both DIBs and LIBsshowed selective adsorption for templatemolecules. The adsorption capacity of imprintedmicrobeads (DIBs and LIBs) is ≥1.3 times higherthan NIBs. Similarly, it is also evident that theadsorption capacity and the adsorption selectivityof the LIBs is relatively high compared to the DIBS[25, 26].FTIR spectra shown in Fig. 4 (a), (b) and (c)illustrate the functional groups present in theprepared Phe imprinted beads. The spectra revealedthat compared to non-imprinted beads the Pheimprinted beads exhibited broad and intense peaksfor -CO- and OH groups which are mainlyresponsible for template bonding. Thus the FTIRspectra act as a clue for active imprinting achievedin the imprinted polymers compared to non-imprinted polymers.The FT-IR spectra of the prepared beads are givenin Fig. 4 (a), (b) and (c) in which the peak at~3600 cm−1represents OH stretching of freecarboxylic group derived from MAA (functionalmonomer), while the peak at ~1,730 cm−1stand forC=O stretching. The peaks at ~1,240 and ~1,145cm−1were assigned to the CO bending of estergroup and CO stretching of COOH and estergroups. The small absorption bands at ~951, ~806,and ~753 cm−1were attributed to C-CH3 rocking,CH bending (vinyl out of plane), and CH2 rocking,respectively. Similar results were also obtained inpreviously reported studies [25, 27] related to theimprinted Poly(MAA-co-EGDMA) polymers.From these spectra, it is clear that the hydroxylpeak at ~3,600 cm−1for LIBs and DIBs is visiblethan that for NIBs. Similarly, the intensities ofother absorption peaks at ~1,730, ~1,240 and~1,145 cm−1which correspond to C=O and COgroups, respectively, were also found to be higherin case of LIBs and DIBs compared to NIBs. Thisindirectly confirms the formation of recognitionsites in the LIBs and DIBs because of the existenceof a large population of free carboxyl groups aftertemplate removal [25, 27].
  4. 4. Nasrullah Shah, Mazhar UlIslam, Muhammad Haneef, Zahid Hussain, Muhammad Balal,Joong Kon Park / International Journal of Engineering Research and Applications (IJERA)ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 3, May-Jun 2013, pp.889-894892 | P a g eFig. 2. (a) to (d). FE-SEM images for the size and morphological study of the beads prepared by shaking baffleflask method.Fig. 3. Size distribution of MIP microbeads prepared by shaking method of suspension polymerization where (a)DIBs (b) LIBs(a)Wave number (cm-1)1000200030004000%T9698100102104 (b)Wave number (cm-1)1000200030004000%T9698100102104D-Phe Beads Size DistributionBeads Size0.1~3µm 3~6µm 6~9µm 9~12µmPercentageinNumber[%]010203040L-Phe Beads Size DistributionBeads Size0.1~3µm 3~6µm 6~9µm 9~12µmPercentageinNumber[%]01020304050
  5. 5. Nasrullah Shah, Mazhar UlIslam, Muhammad Haneef, Zahid Hussain, Muhammad Balal,Joong Kon Park / International Journal of Engineering Research and Applications (IJERA)ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 3, May-Jun 2013, pp.889-894893 | P a g e(c)Wave number (cm-1)1000200030004000%T92949698100102104Fig. 4. FTIR spectra of (a) NIBs (b) DIBs and (c) LIBs prepared by shaking flask method.Table 1. Adsorption capacity and selectivity of NIBs, DIBs and LIBs prepared by suspension polymerizationemploying shaking method at 200 rpm, 60 oC for 24 h.Microbeads Phe enantiomerAdsorption capacity Q(mg/gdry wt)Adsorption selectivity (α)NIBsD-Phe 0.0310.969L-Phe 0.032DIBsD-Phe 0.0611.271L-Phe 0.048LIBsD-Phe 0.0551.303L-Phe 0.073V. CONCLUSIONAn economically feasible and simpleshaking method for suspension polymerization wassuccessfully employed for the preparation of DIBsand LIBs. The wastage of product was small andsize distribution ratio was favorable. Both DIBsand LIBs showed selective adsorption for theirrespective template molecules. The adsorptioncapacity and the adsorption selectivity of L-Pheimprinted beads was relatively high compared tothe D-Phe imprinted beads. FTIR analysissupported the formation of specific functionalgroups in the prepared imprinted polymermicrobeads. Besides synthesizing imprintedpolymers this technique will also be feasible forother polymerization techniques involving risk ofradical scavenging.REFERENCES1. A. Strikovsky, J. Hradil, and G. Wulff ,Catalytically active, molecularly imprintedpolymers in bead form, Reactive &Functional Polymers, 54, 2003, 49–61.2. L. Ye, and K. Mosbach , Molecularlyimprinted microspheres as antibodybinding mimics, Reactive & FunctionalPolymers 48, 2001, 149–157.3. N. Shah, M. Ul-Islam, M. Haneef and J.K.Park, A Brief Overview of MolecularlyImprinted Polymers: From Basics toApplications, Journal of PharmacyResearch 5(6), 2012, 3309-3317.4. O. Bruggemann, K. Haupt, L. Ye, E.Yilmaz, and K. Mosbach, Newconfigurations and applications ofmolecularly imprinted Polymers, Journalof Chromatography A, 889, 2000, 15–24.5. V.B. Kandimalla and H. Ju, Molecularimprinting: a dyanamictechnique fordiverse application in analyticalchemistry, Anal. Bioanal. Chem., 380,2004, 587-605.6. J.D. Lei, and A.J. Tong, Preparation of Z-l-Phe-OH-NBD imprinted microchanneland its molecular recognition study,Spectrochimica Acta Part A, 61, 2005,1029-1033.7. H. Khan and J.K. Park, The preparation ofd-phenylalanine imprinted microbeads bya novel method of modified suspension
  6. 6. Nasrullah Shah, Mazhar UlIslam, Muhammad Haneef, Zahid Hussain, Muhammad Balal,Joong Kon Park / International Journal of Engineering Research and Applications (IJERA)ISSN: 2248-9622 www.ijera.com Vol. 3, Issue 3, May-Jun 2013, pp.889-894894 | P a g epolymerization, Biotechnol. Bioprocess.Eng. 11, 2006, 503–509.8. N. Shah, J.H. Ha, M. Ul-Islam and J.K.Park, Highly improved adsorptionselectivity of L-Phenylalanine imprintedpolymeric submicron/nanoscale beads bymodified uspension polymerization,Korean Journal of Chemical Engineering,28(9), 2011, 1936-1944.9. F.G. Tamayo, M.M. Titirici, A.M. Estebanand B. Sellergren, Synthesis andevaluation of new propazine-imprintedpolymer formats for use as stationaryphases in liquid chromatography, Anal.Chim. Acta, 542, 2005, 38-46.10. K. Yang, Z. Liu, M. Mao, X. Zhang, C.Zhao and N. Nishi, Molecularly imprintedpolyethersulfone microspheres for thebinding and recognition of bisphenol A,Anal. Chim. Acta, 546, 2005, 30-36.11. A.G. Mayes and K. Mosbach, Molecularlyimprinted polymer beads: suspensionpolymerization using a liquidperfluorocarbon as the dispersing phase,Anal. Chem., 68, 1996, 3769-3774.12. E. Birlik, A. Ersoz, A. Denizli and R. Say,Preconcentration of coper using double-imprinted polymer via solid phaseextraction, Anal. Chim. Acta, 565, 2006,145-151.13. M. Andac, R. Say, and A. Denizli,Molecular recognition based cadmiumremoval from human plasma. J.Chromatography. B, 811, 2004, 119-126.14. O. Saatcilar, N. Satiroglu, R. Say, S.Bektas and A denzili, Binding behaviourof Fe3+on ion imprinted polymeric beadsfor analytical applications, J. Appl.Polym.Sci., 101, 2006, 3520-3528.15. F.F. Svec, and J. M. Chet, Continuousrods of macroporous polymer as high-performance liquid chromatographyseparation media, Anal. Chem., 64, 1992,820-822.16. D. C. Sherrington, and H. P. Guyot, Insynthesis and separations using functionalpolymers; Eds., John Wiley and Sons:London, 1988, 1-42.17. M. Munger, E. Trommsdroff, C. E.Schildknecht, and I. Skeist, InPolymerization Processes; Eds. Wiley-Interscience, New York, 1977, 106-142.18. H. Nilsson, R. Mosbach, and K. Mosbach,The use of bead polymerization of acrylicmonomers for immobilization ofenzymes, Biochem. Biophys. Acta, 268,1972, 253-256.19. P. Reinholdsson, T. Hargitai, R. Isaksson,and B. Tornell, Preparation and propertiesof porous particles fromtrimethylolpropane trimethacrylate,Angew. Macromol. Chem., 192, 1991,113-132.20. K. E. J, Barrett, DispersionPolymerization in Organic Media, Ed.;John Wiley and Sons: London, 1975, 13.21. B. Williamson, R.Lukas, M. A. Winnik,and M. D. J Croucher, The preparation ofmicron-size polymer particles in nonpolarmedia, Colloid Interface Sci., 119, 1987,559-564.22. S. Lu, G. Cheng and X. pang, Preparationof molecularly imprinted Fe3O4/P(St-DVB) composite beads with magneticsusceptibility and their characteristics ofmolecular recognition for amino-acid, J.Appl. Polym. Sci., 89, 2003, 3790-3796.23. N. Perez-Moral and A. G. Mayes,Comparative study of imprinted polymerparticles prepared by differentpolymerisation methods, AnalyticaChimica Acta, 504, 2004, 15-21.24. N. Shah, M. Ulislam, J.H. Ha, and J.K.Park, Chiral separation of phenylalanineusing D-phenylalanine imprintedmembrane containing L-phenylalanineimprinted submicron/nanoscale beads,Journal of Pharmacy Research, 5(8),2012, 4425-4433.25. N. Shah, J. H. Ha, M. Ul-Islam and J. K.Park, Highly improved adsorptionselectivity of L-Phenylalanine imprintedsubmicron/nanoscale beads prepared bymodified suspension polymerizationmethod, Korean Journal of ChemicalEngineering, 28(9), 2011, 1936-1944.26. N. Ul-Haq, T. Khan and J.K.Park,Enantioseparation with D-Phe and L-Pheimprinted PAN-based membranes byultrafiltration, J Chem Technol Biotechnol,83, 2008, 524–533.27. G. Socrates, Infrared characteristic groupfrequencies, second edition, John Wiley &Sons Ltd., ISBN 0471942308 (1994).