Research Article Synthesis and Characterization of ...

Research ArticleSynthesis and Characterization of Molecular ImprintingPolymer Microspheres of Piperine Extraction of Piperinefrom Spiked Urine

Rachel Marcella Roland and Showkat Ahmad Bhawani

Department of Chemistry Faculty of Resource Science and Technology Universiti Malaysia Sarawak (UNIMAS)94300 Kota Samarahan Sarawak Malaysia

Correspondence should be addressed to Showkat Ahmad Bhawani sabhawanigmailcom

Received 6 September 2016 Accepted 18 October 2016

Academic Editor Nuria Fontanals

Copyright copy 2016 R M Roland and S A Bhawani This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Molecularly imprinted polymer (MIP) microspheres for Piperine were synthesized by precipitation polymerization with anoncovalent approach In this research Piperine was used as a template acrylic acid as a functional monomer ethylene glycoldimethacrylate as a cross-linker and 221015840-azobisisobutyronitrile (AIBN) as an initiator and acetonitrile as a solvent The imprintedand nonimprinted polymer particles were characterized by using Fourier transform infrared spectroscopy (FT-IR) and ScanningElectron Microscopy (SEM) The synthesized polymer particles were further evaluated for their rebinding efficiency by batchbinding assay The highly selected imprinted polymer for Piperine was MIP 3 with a composition (molar ratio) of 05 3 8template monomer cross-linker respectively The MIP 3 exhibits highest binding capacity (8494) as compared to otherimprinted and nonimprinted polymers The extraction efficiency of highly selected imprinted polymer of Piperine from spikedurine was above 80

1 Introduction

Piperine a nitrogenous pungent substance is an alkaloidfound in important and oldest spices namely Piper nigrum(black peppers) and Piper longum (long peppers) [1] It is alsoknown as 1-piperoylpiperidine with the chemical formula ofC17H19NO3 Hamrapurkar et al [2] stated that Piperine isnaturally occurring organic compound that belongs to familyPiperaceae The fruits of Piperine possess antidepressanteffects hepatoprotective effects antioxidant activity antitu-mour effects antibacterial effects and anticonvulsant effects[2 3] Piperine also has the capability of reducing inflamma-tion relieving pain improving digestion and enhancing thebioavailability [4] Piperine is extensively used in medicinalfield for years due to various medicinal properties includingpainkiller antioxidant and bioavailability enhancer

Molecular Imprinting Technology (MIT) is used todesign molecular recognition materials because it is capableof mimicking natural recognition entities like antibodies and

biological receptors [5ndash14]The original concept ofmolecularimprinting is developed by Linus Pauling in 1940s but Wulffand Sarhan stimulated the interest in imprinting materials[6] According to Vlatakis et al [15] in the early 1980sthe molecular imprinting polymers (MIPs) were successfullyprepared by using noncovalent MIT

Molecular imprinting is a universal method to producepolymers with high affinity binding sites for organic inor-ganic biological and chemical molecules or ions MIPs allowthe functional and crosslinking monomers to copolymerizein the presence of the target compound or known as tem-plate [16] Molecular imprinting polymers [17] can be pre-pared by various methods such as bulk polymerization [18]electropolymerization [19] suspension polymerization [20]emulsion polymerization two-step polymerization [21] andprecipitation polymerization [22] Zhou et al [23] mentionedthat the controlledliving radical polymerization (CRP) isused to prepare MIP microspheres as it permits more precisecontrol over the molecular weight composition and end

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2016 Article ID 5671507 6 pageshttpdxdoiorg10115520165671507

2 Journal of Analytical Methods in Chemistry

group functionality of the obtained polymers [24ndash27] MIPsshow excellent thermal and chemical stability and can beused in aggressive media [15] According to Yan and Row[28] MIPs have many advantages over their biological coun-terparts including inexpensive simple preparation storagestability repeated operations without loss of activity highmechanical strength durability to heat and pressure andapplicability in harsh chemical media

Lai et al [29] stated that MIPs have been used inimportant application such as chemical sensors [30 31]capillary electrophoresis and electrochromatography [32]catalysis [33] HPLC stationary phases [34ndash39] and solid-phase extraction (SPE) [36]

In this research noncovalent imprinting or self-assemblyapproach is adopted during the course of polymerizationIn noncovalent imprinting the template and a functionalmonomer interact by noncovalent interactions in the pre-polymerization mixture According to Spivak [40] nonco-valent is simpler molecular imprinting method as comparedto covalent and semicovalent because it involves syntheticsteps toward the prepolymer complex In this way interactionbetween the monomer and template is achieved easily whenmixed in the solutionThis method has been used to produceimprinted polymers of cinnamic acid In this research as anapplication these imprinted polymers are used in extractionof Piperine from spiked urine sample

2 Materials and Methods

21 Materials Piperine (C17H19NO3) was purchased fromSigma-Aldrich Co Ltd (United States) acrylic acid (AA)was bought fromNippon Shokubai Co Ltd (Japan) ethyleneglycol dimethacrylate (EGDMA)was purchased from Sigma-Aldrich Co Ltd (United States) acetonitrile (ACN) wasobtained from Kunshan Yalong Trading Co Ltd (China) 221015840-azobisisobutyronitrile (AIBN) was obtained from Sigma-Aldrich Co Ltd (United States) methanol (MeOH) wasobtained fromNuasaKimia Sejati Co (Indonesia) acetic acid(CH3COOH) was purchased from Alpha Chemika (India)potassium bromide (KBr) was obtained from Powder PackChem Co (India) and hexane was obtained from SeidlerChemical Co (United States)

22 Equipment Branson 2510 ultrasonic cleaner was usedto disperse the mixtures Memmert W350T Water Bath-AAR 3060 was used to carry out the polymerization IRspectra of polymer particles were recorded with ThermoScientific Nicolet iS10 Scanning electron microscope (JEOLJSM-6390LA) was used to study the morphology of poly-mer particles Shaker (NB-101MT) was used to allow therebinding of polymer particleswith template EBA20-Hettichwas used to centrifuge and separate the polymer particlesfrom the solution Shimadzu LC-20A a reversed-phase highperformance liquid chromatography (RP-HPLC)was used toevaluate the batch binding of polymer particles

23 Synthesis of MIPs and NIP of Piperine The followingprocedure was followed during the preparation 05mmol

of template (Piperine) 2mmol of monomer (AA) 8mmolof cross-linker (EGDMA) 75mL of porogen (ACN) and0011 g of initiator (AIBN) were added into 150mL conicalflask respectively The mixture was sonicated for 10 minutesin order to remove bubbles and allow complete dissolutionThen the conical flask containing mixture was placed in abucket of ice cubes and the reaction mixture was purgedwith nitrogen gas for 15 minutes Ice cubes were used in thisexperiment to allow a suitable environment for noncovalentinteractions between Piperine and acrylic acid After that theconical flask was sealed and placed into a water bath Thepolymerization was conducted for 6 hours initially temper-ature was maintained at 60∘C for the first three hours andlater temperature was raised up to 80∘C and maintained foranother three hours in order to complete the polymerizationThe produced polymer particles were extracted out by usingthe centrifugation at 5000 rpm for 10min The template wasremoved by washing the MIPs successively in the mixture ofmethanol and acetic acid (9 1 vv) until the template was notdetected by RP-HPLC at 270 nm The HPLC was conductedby using the C18 column (250 times 4mm 5 120583m) with the mobilephase consisting of acetonitrile distilled water and aceticacid in the ratio of 60 395 05 vvv respectively The flowrate was set at 06mLmin with UV detection at 270 nm andinjection volume was set at 20 120583L

The nonimprinted polymeric particles (NIPs) were pre-pared in the same way without the addition of the templatemolecule The similar procedure was used for the synthesisof different molecular imprinted polymers of Piperine withvarying composition of AA and EGDMA (Table 1) by precip-itation polymerization namely MIP 2 MIP 3 and MIP 4 forMIPs as well as NIP

24 Batch BindingAssay A series of 150mLfive conical flaskscontaining 05 g of the MIP (MIP 1 MIP 2 MIP 3 and MIP4) and NIP beads were added with a 75mL of acetonitrilecontaining 05mmol of Piperine The conical flasks wereshaken on the shaker at 100 rpm and the samples werecollected at different time intervals (0 30 60 90 120 150 240and 360 minutes) The collected samples were centrifuged at5000 rpm for 10 minutes in order to remove any suspendedparticles and supernatant was used for further analysis Theconcentrations of Piperine after adsorption were recorded byusing RP-HPLC The binding capacity of MIPs and NIP ofPiperine was calculated [17] by using the following equation

Binding capacity () =119862119894 minus 119862119891119862119894times 100 (1)

where 119862119894 is the initial Piperine concentration in the solutionand 119862119891 is the final Piperine concentration in the solution

25 Competitive Binding Test Caffeine was used as a com-petitive template with the Piperine A 150mL conical flaskcontaining 05 g of the MIP 3 beads was added and a solu-tion of 75mL of acetonitrile containing equal concentration(05mmol) of Piperine and Caffeine Similarly for NIP sameprocedure was followed Both of the conical flasks wereshaken on the shaker at 100 rpm and the samples were

Journal of Analytical Methods in Chemistry 3

Table 1 Synthesis of molecularly imprinted polymers and nonimprinted polymer for Piperine by precipitation polymerization

Code MIP 1 MIP 2 MIP 3 MIP 4 NIP

Template (mmol) Piperine(05)

Piperine(05)

Piperine(05)

Piperine(05) mdash

Monomer (mmol) AA(2)

AA(2)

AA(3)

AA(3)

AA(3)

Cross-linker (mmol) EGDMA(8)

EGDMA(12)

EGDMA(8)

EGDMA(12)

EGDMA(8)

Porogen (mL) ACN(75)

ACN(75)

ACN(75)

ACN(75)

ACN(75)

collected at different time intervals (0 30 60 90 120 150240 and 360 minutes) After shaking at the appropriate timeintervals all the samples were centrifuged at 5000 rpm for 10minutesThe binding of both the templates wasmonitored byusing RP-HPLCThe distribution ratios (mL gminus1) of Piperinebetween the MIPs or NIP in the porogen (acetonitrile) weredetermined [41] by the following equation

Distribution ratio 119870119863 =(119862119894 minus 119862119891)119881119862119894119898

(2)

where 119862119894 is the initial Piperine concentration in the solution119862119891 is the final Piperine concentration in the solution 119881 isthe volume of porogen (ACN) used and 119898 is the mass ofMIPNIP used

The selectivity coefficients for Piperine relative to bindingcompetitor Caffeine forMIP 3 and NIP can be calculated [41]by

Selectivity coefficient 119870selPiperineCaffeine =119870Piperine119863

119870Caffeine119863

(3)

where 119870Piperine119863 is the batch binding assay of MIPNIP forPiperine and 119870Caffeine119863 is the batch binding assay of MIPNIPfor Caffeine

The relative selectivity coefficient (1198961015840) was determined[41] by the following equation

1198961015840 = 119896 (MIP 3)119896 (NIP)

(4)

26 Extraction of Piperine from Spiked Human Urine About100mL of fresh urine was collected from a drug free humanUrine sample was first centrifuged and filtered and thenspiked with a Piperine to get a concentration of 50 ppmAfter this 50mL of spiked urine sample was added in flaskcontaining 05 g of MIP 3 The samples were collected andanalysed the same as followed in batch bind assay The NIPwas treated in the same way

3 Results and Discussion

Synthesis of microsphere imprinted polymers is a verycrucial step in order to produce uniform shape and size ofparticles Previous studies revealed [42] that various prepa-ration methods have been carried out for the preparation of

polymer microspheres [43] such as the synthesis of polymermicrospheres by dispersion and emulsion polymerizationwhere the surfactants in aqueous solution [44] and stabilizersin organic solution [45] are crucial to stabilize the polymerphase and prevent the aggregation of particles Precipita-tion polymerization can form polymer microspheres withconstant size and shape that can lead to narrow dispersionwithout the need for any added surfactant or stabilizer[46ndash49] In this research we have successfully producedimprinted polymer microspheres by precipitation method inthe acetonitrile (porogen)

31 Fourier Transform Infrared Spectroscopy (FT-IR) IR anal-ysis is an important chemical characterization method todetect the functional groups present in a compoundThe FT-IR spectra of different MIPs and NIP are shown in Figure 1

Based on Figure 1 small peak in the range of 351929 cmminus1to 361306 cmminus1 attributed to the vibration mode of OndashHstretching was observed in both MIPs and NIP The bands inthe range of 292675 cmminus1 to 299602 cmminus1 and 285476 cmminus1to 295724 cmminus1 showed the vibrationmode ofCndashH stretchingof aliphatic compound as well as asymmetric and symmetricCH2 stretching inMIPs andNIP Strong peaks at 172611 cmminus1to 173642 cmminus1 indicated the presence of C=O of acrylicacid The vibration mode of C=C stretching of aromaticcompound can be foundwithin 163537 cmminus1 to 163684 cmminus1The CH2 bending at 145082 cm

minus1 to 145270 cmminus1 indicatedthe presence of alkane group in MIPs and NIP

The vibration mode of CndashN stretching of MIPs and NIPsafter leaching was detected at 138844 cmminus1 to 139006 cmminus1Peaks of MIPs and NIP at 125388 cmminus1 to 125961 cmminus1showed the vibration mode of ndashOndashCH2ndashOndash symmetricstretching Small band at 103588 cmminus1 to 105059 cmminus1 inMIPs and NIP explained the presence of symmetric stretch-ing of =CndashOndashC The vibration mode of CndashH stretchingof aliphatic compound can be observed at 295409 cmminus1 to298519 cmminus1

32 Scanning Electron Microscopy (SEM) SEM analysis is avery important morphological study for polymer particlesthat provides the idea about the shape and size Figure 2clearly indicates that spherical particles are produced withthe size in micrometresThis is because the polymer particleswere synthesized by precipitation polymerization According


0

minus50

minus100

minus150

minus250

minus300

minus200

minus350

minus400

minus450

minus500

minus550

minus600

minus650

T

Wavenumbers (cmminus1)

3500 3000 2500 2000 1500 1000 500

(a)

(b)

(c)

(e)

(d)

Figure 1 FT-IR spectra (a) MIP 1 (b) MIP 2 (c) MIP 3 (d) MIP 4and (e) NIP

0563120583m

Figure 2 SEM of imprinted polymer particles

Binding capacity versus time

MIP 1MIP 2MIP 3

MIP 4NIP

0102030405060708090

Bind

ing

capa

city

()

30 60 90 120 150 180 210 240 270 300 330 3600Time (min)

Figure 3 A graph of binding capacity of MIPs 1 2 3 and 4 and NIPat different time intervals

Table 2 The distribution ratio selectivity coefficients and relativeselectivity coefficient of MIP 3 and NIP

119870119863 (MIP 3)(mL gminus1)

119870119863 (NIP)(mL gminus1) 119896sel 1198961015840

Piperine 7672 2679 534 mdashCaffeine 1437 770 347 154

to Tamayo et al [50] uniform size of imprinted polymers canbe formed by using a noncovalent imprinting approach byprecipitation polymerization

Arabzadeh and Abdouss [41] stated that interactionbetween monomer and template could be another factorthat contributed to uniform size distribution with cleansurfaces Research conducted by Park et al [51] mentionedthat there are various factors that affect the production ofuniform polymer microspheres including volume of solventreaction of solvent and presence of template ion Excesssolvent or porogen used in the synthesis of polymer particleswill produce highly uniform polymer microspheres withimprinted binding sites

33 Batch Binding Assay of MIPs and NIP RP-HPLC wasused to evaluate the binding efficiency of MIPs and NIP ofPiperine Figure 3 depicts the binding capacity of differentMIPs and NIP at different time intervals

MIP 3 showed the highest binding capacity (8494)followed by MIP 2 (7586) MIP 1 (6940) and MIP 4(6080) MIP 3 contains a higher amount of monomer ratioas compared toMIP 1 andMIP 2 but MIP 4 contains a higheramount of cross-linker In this study increasing amount ofmonomer would produce specific interaction sites with thePiperine and hence rebinding efficiency was also increasedBut the increase in amount of cross-linker has produced areverse effect as can be seen inMIP 4 If we compare theMIPswith NIP it is clear from Figure 3 the binding capacity is lowThis can be conferred that NIP does not contain any bindingsite complimentary with the Piperine

34 Competitive Binding Assay In order to evaluate theproperties of MIP of Piperine as a sensing material theselectivity test was conducted In this test two compounds(Piperine and Caffeine) were tested using both MIP 3 andNIP The selectivity of Piperine and Caffeine was calculatedby using RP-HPLC measurements The distribution ratioof Piperine in both MIP 3 and NIP was higher than thedistribution ratio of Caffeine in bothMIP 3 andNIP resultingin higher selectivity coefficient of Piperine than that ofCaffeine in bothMIP 3 andNIP (Table 2)The results indicatethat the imprinted polymer has got complimentary bindingsites or cavities with the Piperine as compared to Caffeine

35 Extraction of Piperine from Spiked Human Urine Theextensive use of Piperine in medicine and spices has gener-ated this idea to first use the selected MIP 3 in the extractionof Piperine from urine This will provide us a way forward toexpand the application of these imprinted polymer particles


From this study it was found that about 8118 of Piperinewas successfully extracted from the spiked urine sample

4 Conclusion

Molecularly imprinted polymeric microspheres of Piperinewere synthesized by using precipitation polymerization Thebinding efficiencies of MIPs and NIP of Piperine wereevaluated by batch binding assay MIP 3 exhibited the highestbinding capacity (8494) as compared to NIP (40) Theseimprinted polymer particles successfully extracted (8118)Piperine from spiked urine

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

Financial support from SGS UNIMAS Grant no F07(S168)12432015(05) is highly appreciated

References

[1] K P Umesh S Amrit and K C Anup ldquoRole of piperineas an bioavailability enhancerrdquo Journal of Recent Advances inPharmaceutical Research vol 4 pp 16ndash23 2011

[2] PDHamrapurkar K Jadhav and S Zine ldquoQuantitative estima-tion of piperine in Piper nigrum and Piper longum using highperformance thin layer chromatographyrdquo Journal of AppliedPharmaceutical Science vol 1 no 3 pp 117ndash120 2011

[3] A Khajuria NThusa U Zusthi and K L Bedi ldquoEstimation ofpiperine in commercial Ayurvedic formulationsrdquo Indian Drugsvol 34 no 10 pp 557ndash563 1997

[4] A MMujumdar J N Dhuley V K Deshmukh and S R NaikldquoEffect of piperine on bioavailability of oxyphenylbutazone inratsrdquo Indian Drugs vol 36 no 2 pp 123ndash126 1999

[5] T Takagishi and I M Klotz ldquoMacromolecule-small moleculeinteractions introduction of additional binding sites inpolyethyleneimine by disulfide cross-linkagesrdquo Biopolymers-Peptide Science Section vol 11 no 2 pp 483ndash491 1972

[6] G Wulff and A Sarhan ldquoUse of polymers with enzyme-analogous structures for the resolution of racematesrdquo Ange-wandte Chemie International Edition vol 11 pp 341ndash342 1972

[7] K Mosbach and O Ramstrom ldquoThe emerging technique ofmolecular imprinting and its future impact on biotechnologyrdquoNature Biotechnology vol 14 no 2 pp 163ndash170 1996

[8] M J Whitcombe M E Rodriguez P Villar and E NVulfson ldquoA new method for the introduction of recognitionsite functionality into polymers prepared bymolecular imprint-ing synthesis and characterization of polymeric receptors forcholesterolrdquo Journal of the American Chemical Society vol 117no 27 pp 7105ndash7111 1995

[9] CAlexanderH S Andersson L I Andersson et al ldquoMolecularimprinting science and technology a survey of the literaturefor the years up to and including 2003rdquo Journal of MolecularRecognition vol 19 no 2 pp 106ndash180 2006

[10] S Yan Y Fang and Z Gao ldquoQuartz crystal microbalance forthe determination of daminozide using molecularly imprinted

polymers as recognition elementrdquo Biosensors and Bioelectronicsvol 22 no 6 pp 1087ndash1091 2007

[11] L Ye and K Mosbach ldquoMolecular imprinting synthetic mate-rials as substitutes for biological antibodies and receptorsrdquoChemistry of Materials vol 20 no 3 pp 859ndash868 2008

[12] E V Piletska A R Guerreiro M J Whitcombe and S APiletsky ldquoInfluence of the polymerization conditions on the per-formance of molecularly imprinted polymersrdquoMacromoleculesvol 42 no 14 pp 4921ndash4928 2009

[13] A Poma A P F Turner and S A Piletsky ldquoAdvances in themanufacture of MIP nanoparticlesrdquo Trends in Biotechnologyvol 28 no 12 pp 629ndash637 2010

[14] G Vasapollo R D Sole L Mergola et al ldquoMolecularlyimprinted polymers present and future prospectiverdquo Interna-tional Journal ofMolecular Sciences vol 12 no 9 pp 5908ndash59452011

[15] G Vlatakis L I Andersson R Muller and K Mosbach ldquoDrugassay using antibody mimics made by molecular imprintingrdquoNature vol 361 no 6413 pp 645ndash647 1993

[16] Q-Z Feng L-X Zhao B-L Chu W Yan and J-M Lin ldquoSyn-thesis and binding site characteristics of 246-trichlorophenol-imprinted polymersrdquo Analytical and Bioanalytical Chemistryvol 392 no 7-8 pp 1419ndash1429 2008

[17] N A Yusof A Beyan J Haron and N A Ibrahim ldquoSyn-thesis and characterization of a molecularly imprinted poly-mer for Pb2+ uptake using 2-vinylpyridine as the complexingmonomerrdquo Sains Malaysiana vol 39 no 5 pp 829ndash835 2010

[18] S S Milojkovic D Kostoski J J Comor and J M NedeljkovicldquoRadiation induced synthesis of molecularly imprinted poly-mersrdquo Polymer vol 38 no 11 pp 2853ndash2855 1997

[19] M C Blanco-Lopez M J Lobo-Castanon A J Miranda-Ordieres and P Tunon-Blanco ldquoVoltammetric sensor forvanillylmandelic acid based onmolecularly imprinted polymer-modified electrodesrdquoBiosensors and Bioelectronics vol 18 no 4pp 353ndash362 2003

[20] L Zhang G Cheng and C Fu ldquoSynthesis and characteristicsof tyrosine imprinted beads via suspension polymerizationrdquoReactive and Functional Polymers vol 56 no 3 pp 167ndash1732003

[21] N Perez-Moral and A G Mayes ldquoComparative study ofimprinted polymer particles prepared by different polymerisa-tionmethodsrdquoAnalytica Chimica Acta vol 504 no 1 pp 15ndash212004

[22] M Andac S Mirel S Senel R Say A Ersoz and A DenizlildquoIon-imprinted beads for molecular recognition basedmercuryremoval from human serumrdquo International Journal of BiologicalMacromolecules vol 40 no 2 pp 159ndash166 2007

[23] T Zhou L Joslashrgensen M A Mattebjerg I S Chronakisand L Ye ldquoMolecularly imprinted polymer beads for nicotinerecognition prepared by RAFT precipitation polymerization astep forward towards multi-functionalitiesrdquo RSC Advances vol4 no 57 pp 30292ndash30299 2014

[24] S Boonpangrak M J Whitcombe V Prachayasittikul KMosbach and L Ye ldquoPreparation of molecularly imprintedpolymers using nitroxide-mediated living radical polymeriza-tionrdquo Biosensors and Bioelectronics vol 22 no 3 pp 349ndash3542006

[25] G Pan Y Zhang X Guo C Li and H Zhang ldquoAn efficientapproach to obtainingwater-compatible and stimuli-responsivemolecularly imprinted polymers by the facile surface-graftingof functional polymer brushes via RAFT polymerizationrdquoBiosensors and Bioelectronics vol 26 no 3 pp 976ndash982 2010


[26] C Gonzato M Courty P Pasetto and K Haupt ldquoMagneticmolecularly imprinted polymer nanocomposites via surface-initiated RAFT polymerizationrdquo Advanced Functional Materi-als vol 21 no 20 pp 3947ndash3953 2011

[27] G Pan Y Zhang Y Ma C X Li and H Q Zhang ldquoEfficientone-pot synthesis of water-compatible molecularly imprintedpolymer microspheres by facile RAFT precipitation polymer-izationrdquo Angewandte Chemie International Edition vol 50 no49 pp 11731ndash11734 2011

[28] H Yan and K H Row ldquoCharacteristic and synthetic approachof molecularly imprinted polymerrdquo International Journal ofMolecular Sciences vol 7 no 5-6 pp 155ndash178 2006

[29] J-P Lai M-L Yang R Niessner and D Knopp ldquoMolecularlyimprinted microspheres and nanospheres for di(2-ethylhexyl)phthalate prepared by precipitation polymerizationrdquo Analyticaland Bioanalytical Chemistry vol 389 no 2 pp 405ndash412 2007

[30] S TWei AMolinelli and BMizaikoff ldquoMolecularly imprintedmicro and nanospheres for the selective recognition of 17120573-estradiolrdquo Biosensors and Bioelectronics vol 21 no 10 pp 1943ndash1951 2006

[31] D Vaihinger K Landfester I Krauter H Brunner and GE M Tovar ldquoMolecularly imprinted polymer nanospheres assynthetic affinity receptors obtained by miniemulsion poly-merisationrdquo Macromolecular Chemistry and Physics vol 203no 13 pp 1965ndash1973 2002

[32] L I Andersson and K Mosbach ldquoEnantiomeric resolutionon molecularly imprinted polymers prepared with only non-covalent and non-ionic interactionsrdquo Journal of Chromatogra-phy A vol 516 no 2 pp 313ndash322 1990

[33] O Ramstrom and K Mosbach ldquoSynthesis and catalysis bymolecularly imprinted materialsrdquo Current Opinion in ChemicalBiology vol 3 no 6 pp 759ndash764 1999

[34] K Yano and I Karube ldquoMolecularly imprinted polymers forbiosensor applicationsrdquo TrACmdashTrends in Analytical Chemistryvol 18 no 3 pp 199ndash204 1999

[35] E Caro R M Marce F Borrull P A G Cormack and D CSherrington ldquoApplication of molecularly imprinted polymersfor the analysis of pesticide residues in Food a highly selectiveand innovative approachrdquo Trends in Analytical Chemistry vol25 no 2 pp 143ndash154 2006

[36] J-P Lai X-Y Lu C-Y Lu H-F Ju and X-W He ldquoPreparationand evaluation of molecularly imprinted polymeric micro-spheres by aqueous suspension polymerization for use as ahigh-performance liquid chromatography stationary phaserdquoAnalytica Chimica Acta vol 442 no 1 pp 105ndash111 2001

[37] J-P Lai X-F Cao X-L Wang and X-W He ldquoChro-matographic characterization of molecularly imprinted micro-spheres for the separation and determination of trimethoprimin aqueous buffersrdquoAnalytical and Bioanalytical Chemistry vol372 no 2 pp 391ndash396 2002

[38] E Turiel J L Tadeo P A G Cormack and A Martin-EstebanldquoHPLC imprinted-stationary phase prepared by precipitationpolymerisation for the determination of thiabendazole in fruitrdquoAnalyst vol 130 no 12 pp 1601ndash1607 2005

[39] J Wang P A G Cormack D C Sherrington and E KhoshdelldquoMonodisperse molecularly imprinted polymer microspheresprepared by precipitation polymerization for affinity separationapplicationsrdquoAngewandte Chemie International Edition vol 42no 43 pp 5336ndash5338 2003

[40] D A Spivak ldquoOptimization evaluation and characterizationof molecularly imprinted polymersrdquo Advanced Drug DeliveryReviews vol 57 no 12 pp 1779ndash1794 2005

[41] N Arabzadeh andM Abdouss ldquoSynthesis and characterizationof molecularly imprinted polymers for selective solid-phaseextraction of pseudoephedrinerdquo Colloid Journal vol 72 no 4pp 446ndash455 2010

[42] F Bai X Yang R Li B Huang and W Huang ldquoMonodis-perse hydrophilic polymermicrospheres having carboxylic acidgroups prepared by distillation precipitation polymerizationrdquoPolymer vol 47 no 16 pp 5775ndash5784 2006

[43] M Okubo and T Nakagawa ldquoFormation of multihollow struc-tures in crosslinked composite polymer particlesrdquo Colloid andPolymer Science vol 272 no 5 pp 530ndash535 1994

[44] M R Ferrick J Murtagh and J K Thomas ldquoSynthesis andcharacterization of polystyrene latex particlesrdquoMacromoleculesvol 22 no 4 pp 1515ndash1517 1989

[45] A J Paine W Luymes and J McNulty ldquoDispersion polymer-ization of styrene in polar solvents 6 Influence of reactionparameters on particle size and molecular weight in poly(N-vinylpyrrolidone)-stabilized reactionsrdquoMacromolecules vol 23no 12 pp 3104ndash3109 1990

[46] K Li and H D H Stover ldquoSynthesis of monodispersepoly(divinylbenzene) microspheresrdquo Journal of Polymer Sci-ence Part A Polymer Chemistry vol 31 no 13 pp 3257ndash32631993

[47] T J Romack E E Maury and J M DeSimone ldquoPrecipitationpolymerization of acrylic acid in supercritical carbon dioxiderdquoMacromolecules vol 28 no 4 pp 912ndash915 1995

[48] S Sosnowski M Gadzinowski and S Slomkowski ldquoPoly(ll-lactide) microspheres by ring-opening polymerizationrdquoMacro-molecules vol 29 no 13 pp 4556ndash4564 1996

[49] F Bai X L Yang Y Z Zhao and W Q Huang ldquoSynthesisof narrow ormonodisperse poly(divinylbenzene) microspheresby distillation-precipitation polymerizationrdquo Polymer Interna-tional vol 54 no 26 pp 168ndash174 2004

[50] F G Tamayo J L Casillas and A Martin-Esteban ldquoEvaluationof new selective molecularly imprinted polymers prepared byprecipitation polymerisation for the extraction of phenylureaherbicidesrdquo Journal of Chromatography A vol 1069 no 2 pp173ndash181 2005

[51] J Park H A Dam and D Kim ldquoSelective sorption behav-ior of metal(II) ion-imprinted polymethacrylate microspheressynthesized via precipitation polymerization methodrdquo KoreanJournal of Chemical Engineering vol 32 no 5 pp 967ndash973 2015

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group functionality of the obtained polymers [24ndash27] MIPsshow excellent thermal and chemical stability and can beused in aggressive media [15] According to Yan and Row[28] MIPs have many advantages over their biological coun-terparts including inexpensive simple preparation storagestability repeated operations without loss of activity highmechanical strength durability to heat and pressure andapplicability in harsh chemical media

Lai et al [29] stated that MIPs have been used inimportant application such as chemical sensors [30 31]capillary electrophoresis and electrochromatography [32]catalysis [33] HPLC stationary phases [34ndash39] and solid-phase extraction (SPE) [36]

In this research noncovalent imprinting or self-assemblyapproach is adopted during the course of polymerizationIn noncovalent imprinting the template and a functionalmonomer interact by noncovalent interactions in the pre-polymerization mixture According to Spivak [40] nonco-valent is simpler molecular imprinting method as comparedto covalent and semicovalent because it involves syntheticsteps toward the prepolymer complex In this way interactionbetween the monomer and template is achieved easily whenmixed in the solutionThis method has been used to produceimprinted polymers of cinnamic acid In this research as anapplication these imprinted polymers are used in extractionof Piperine from spiked urine sample

2 Materials and Methods

21 Materials Piperine (C17H19NO3) was purchased fromSigma-Aldrich Co Ltd (United States) acrylic acid (AA)was bought fromNippon Shokubai Co Ltd (Japan) ethyleneglycol dimethacrylate (EGDMA)was purchased from Sigma-Aldrich Co Ltd (United States) acetonitrile (ACN) wasobtained from Kunshan Yalong Trading Co Ltd (China) 221015840-azobisisobutyronitrile (AIBN) was obtained from Sigma-Aldrich Co Ltd (United States) methanol (MeOH) wasobtained fromNuasaKimia Sejati Co (Indonesia) acetic acid(CH3COOH) was purchased from Alpha Chemika (India)potassium bromide (KBr) was obtained from Powder PackChem Co (India) and hexane was obtained from SeidlerChemical Co (United States)

22 Equipment Branson 2510 ultrasonic cleaner was usedto disperse the mixtures Memmert W350T Water Bath-AAR 3060 was used to carry out the polymerization IRspectra of polymer particles were recorded with ThermoScientific Nicolet iS10 Scanning electron microscope (JEOLJSM-6390LA) was used to study the morphology of poly-mer particles Shaker (NB-101MT) was used to allow therebinding of polymer particleswith template EBA20-Hettichwas used to centrifuge and separate the polymer particlesfrom the solution Shimadzu LC-20A a reversed-phase highperformance liquid chromatography (RP-HPLC)was used toevaluate the batch binding of polymer particles

23 Synthesis of MIPs and NIP of Piperine The followingprocedure was followed during the preparation 05mmol

of template (Piperine) 2mmol of monomer (AA) 8mmolof cross-linker (EGDMA) 75mL of porogen (ACN) and0011 g of initiator (AIBN) were added into 150mL conicalflask respectively The mixture was sonicated for 10 minutesin order to remove bubbles and allow complete dissolutionThen the conical flask containing mixture was placed in abucket of ice cubes and the reaction mixture was purgedwith nitrogen gas for 15 minutes Ice cubes were used in thisexperiment to allow a suitable environment for noncovalentinteractions between Piperine and acrylic acid After that theconical flask was sealed and placed into a water bath Thepolymerization was conducted for 6 hours initially temper-ature was maintained at 60∘C for the first three hours andlater temperature was raised up to 80∘C and maintained foranother three hours in order to complete the polymerizationThe produced polymer particles were extracted out by usingthe centrifugation at 5000 rpm for 10min The template wasremoved by washing the MIPs successively in the mixture ofmethanol and acetic acid (9 1 vv) until the template was notdetected by RP-HPLC at 270 nm The HPLC was conductedby using the C18 column (250 times 4mm 5 120583m) with the mobilephase consisting of acetonitrile distilled water and aceticacid in the ratio of 60 395 05 vvv respectively The flowrate was set at 06mLmin with UV detection at 270 nm andinjection volume was set at 20 120583L

The nonimprinted polymeric particles (NIPs) were pre-pared in the same way without the addition of the templatemolecule The similar procedure was used for the synthesisof different molecular imprinted polymers of Piperine withvarying composition of AA and EGDMA (Table 1) by precip-itation polymerization namely MIP 2 MIP 3 and MIP 4 forMIPs as well as NIP

24 Batch BindingAssay A series of 150mLfive conical flaskscontaining 05 g of the MIP (MIP 1 MIP 2 MIP 3 and MIP4) and NIP beads were added with a 75mL of acetonitrilecontaining 05mmol of Piperine The conical flasks wereshaken on the shaker at 100 rpm and the samples werecollected at different time intervals (0 30 60 90 120 150 240and 360 minutes) The collected samples were centrifuged at5000 rpm for 10 minutes in order to remove any suspendedparticles and supernatant was used for further analysis Theconcentrations of Piperine after adsorption were recorded byusing RP-HPLC The binding capacity of MIPs and NIP ofPiperine was calculated [17] by using the following equation

Binding capacity () =119862119894 minus 119862119891119862119894times 100 (1)

where 119862119894 is the initial Piperine concentration in the solutionand 119862119891 is the final Piperine concentration in the solution

25 Competitive Binding Test Caffeine was used as a com-petitive template with the Piperine A 150mL conical flaskcontaining 05 g of the MIP 3 beads was added and a solu-tion of 75mL of acetonitrile containing equal concentration(05mmol) of Piperine and Caffeine Similarly for NIP sameprocedure was followed Both of the conical flasks wereshaken on the shaker at 100 rpm and the samples were





Piperine(05)

Piperine(05)

Piperine(05) mdash


AA(2)

AA(3)

AA(3)

AA(3)


EGDMA(12)

EGDMA(8)

EGDMA(12)

EGDMA(8)


ACN(75)

ACN(75)

ACN(75)

ACN(75)



(2)





(3)



1198961015840 = 119896 (MIP 3)119896 (NIP)

(4)











0

minus50

minus100

minus150

minus250

minus300

minus200

minus350

minus400

minus450

minus500

minus550

minus600

minus650

T


3500 3000 2500 2000 1500 1000 500

(a)

(b)

(c)

(e)

(d)


0563120583m



MIP 1MIP 2MIP 3

MIP 4NIP

0102030405060708090

Bind

ing

capa

city

()

30 60 90 120 150 180 210 240 270 300 330 3600Time (min)



119870119863 (MIP 3)(mL gminus1)

119870119863 (NIP)(mL gminus1) 119896sel 1198961015840










4 Conclusion


Competing Interests


Acknowledgments


References































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





Piperine(05)

Piperine(05)

Piperine(05) mdash


AA(2)

AA(3)

AA(3)

AA(3)


EGDMA(12)

EGDMA(8)

EGDMA(12)

EGDMA(8)


ACN(75)

ACN(75)

ACN(75)

ACN(75)



(2)





(3)



1198961015840 = 119896 (MIP 3)119896 (NIP)

(4)











0

minus50

minus100

minus150

minus250

minus300

minus200

minus350

minus400

minus450

minus500

minus550

minus600

minus650

T


3500 3000 2500 2000 1500 1000 500

(a)

(b)

(c)

(e)

(d)


0563120583m



MIP 1MIP 2MIP 3

MIP 4NIP

0102030405060708090

Bind

ing

capa

city

()

30 60 90 120 150 180 210 240 270 300 330 3600Time (min)



119870119863 (MIP 3)(mL gminus1)

119870119863 (NIP)(mL gminus1) 119896sel 1198961015840










4 Conclusion


Competing Interests


Acknowledgments


References































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0

minus50

minus100

minus150

minus250

minus300

minus200

minus350

minus400

minus450

minus500

minus550

minus600

minus650

T


3500 3000 2500 2000 1500 1000 500

(a)

(b)

(c)

(e)

(d)


0563120583m



MIP 1MIP 2MIP 3

MIP 4NIP

0102030405060708090

Bind

ing

capa

city

()

30 60 90 120 150 180 210 240 270 300 330 3600Time (min)



119870119863 (MIP 3)(mL gminus1)

119870119863 (NIP)(mL gminus1) 119896sel 1198961015840










4 Conclusion


Competing Interests


Acknowledgments


References































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



4 Conclusion


Competing Interests


Acknowledgments


References































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Research Article Synthesis and Characterization of ...

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