Molecularly Imprinted Molecularly Imprinted Templates for Solid-Phase Templates for Solid-Phase

ExtractionExtraction(MISPE)(MISPE)

Presented by:Presented by:

Janee’ HardmanJanee’ Hardman

Samantha LawlerSamantha Lawler

OverviewOverview• Brief explanation of solid phase extractionBrief explanation of solid phase extraction• What is MISPE?What is MISPE?• Making MI polymers Making MI polymers

– Polymerization Polymerization – Reaction componentsReaction components– Covalent ImprintingCovalent Imprinting– Non-covalent ImprintingNon-covalent Imprinting

• Optimization of developing MIP’sOptimization of developing MIP’s– Trial and errorTrial and error– Computational approachComputational approach

• Creating MISPE columns from MIP’sCreating MISPE columns from MIP’s• Specific examples of MISPE used in industry.Specific examples of MISPE used in industry.• ConclusionsConclusions• ReferencesReferences

http://www.biotage.com/DynPage.aspx?http://www.biotage.com/DynPage.aspx?id=35833id=35833

Solid Phase Extraction Solid Phase Extraction (SPE) (SPE)

• Used to selectively Used to selectively retain analytes for retain analytes for purificationpurification

• Use individual Use individual cartridges or 96-well cartridges or 96-well plates. plates.

• Retention can be based Retention can be based on ionic, polar, or non-on ionic, polar, or non-polar interactionspolar interactions

• Sample added to Sample added to column, impurities column, impurities washed away, target washed away, target analyte eluted analyte eluted

• Can have problems with Can have problems with selectivityselectivity

Molecularly Imprinted Solid-Molecularly Imprinted Solid-Phase Extraction (MISPE)Phase Extraction (MISPE)

• Technique introduced in early 1970’sTechnique introduced in early 1970’s• Similar theory to traditional SPESimilar theory to traditional SPE• More selective, resulting in greater More selective, resulting in greater

purification of final extractspurification of final extracts• Sorbent composed of molecularly Sorbent composed of molecularly

imprinted polymers (MIPs) that have a imprinted polymers (MIPs) that have a predetermined selectivity for a predetermined selectivity for a particular analyte, or group of particular analyte, or group of structurally related compoundsstructurally related compounds

MIPs OverviewMIPs Overview

• Creation of polymers based upon Creation of polymers based upon molecular recognitionmolecular recognition– Referred to as synthetic antibodiesReferred to as synthetic antibodies

• Polymer network is created around a Polymer network is created around a template/imprint moleculetemplate/imprint molecule

• Removal of template/imprint molecule Removal of template/imprint molecule leaves cavity in polymerleaves cavity in polymer– Chemical affinityChemical affinity– Steric affinitySteric affinity

Polymerization MethodPolymerization Method• Bulk PolymerizationBulk Polymerization

– All components added to reaction vessel at All components added to reaction vessel at onceonce•Template/imprint moleculeTemplate/imprint molecule

•MonomersMonomers

• InitiatorInitiator

•Cross-linkerCross-linker

•Porogen (Polymerization solvent)Porogen (Polymerization solvent)

– Reaction initiated via heat or UV irradiationReaction initiated via heat or UV irradiation– Results in macroporous monolithic Results in macroporous monolithic

polymeric blockpolymeric block•Dried, manually ground, sievedDried, manually ground, sieved

Additional Polymerization MethodsAdditional Polymerization Methods

Lee, Lim Lay. University Sains Malaysia, 2006, pp 1-52

Polymerization ReactionPolymerization Reaction• Most common type is free radical Most common type is free radical

polymerizationpolymerization– InitiationInitiation

I 2R*I 2R*– Propagation where M = MonomersPropagation where M = Monomers

R* + M M*R* + M M*ii

M*M*ii + M M* + M M*i+1,2,3….i+1,2,3…. – TerminationTermination

M*M*i+ni+n ++ M*M*i+n i+n M Mn+nn+n

R* + R* IR* + R* I

Template/Imprint MoleculeTemplate/Imprint Molecule

• Target analyte or close structural Target analyte or close structural analog analog

• Must be chemically inertMust be chemically inert

• Stable under polymerization conditionsStable under polymerization conditions– No participation in free radical reactionNo participation in free radical reaction– Thermally stable if polymerization initiated Thermally stable if polymerization initiated

via heatvia heat– UV stable if polymerization initiated via UV UV stable if polymerization initiated via UV

irradiationirradiation

• Removal of template in MIP achieved Removal of template in MIP achieved via Soxhlet extractionvia Soxhlet extraction

Functional MonomersFunctional Monomers

• Monomers chosen must Monomers chosen must be complementary in be complementary in functionality to functionality to template/imprint template/imprint moleculemolecule

• Monomers may beMonomers may be– AcidicAcidic– BasicBasic– NeutralNeutral

Lee, Lim Lay. University Sains Malaysia, 2006, pp 1-52

Cross-linkersCross-linkers• Fulfills three major Fulfills three major

functionsfunctions– Defines form and structure Defines form and structure

of polymer matrixof polymer matrix– Makes imprint molecule Makes imprint molecule

insoluble in polymerization insoluble in polymerization solvent (porogen)solvent (porogen)

– Imparts mechanical Imparts mechanical stability to polymer matrixstability to polymer matrix

• High degree of cross-High degree of cross-linking requiredlinking required

•70 – 90%70 – 90%

Lee, Lim Lay. University Sains Malaysia, 2006, pp 1-52

InitiatorsInitiators

2,2-Azobisisobutyronitrile

(AIBN)

Benzoyl peroxide

http://polymer.w99of.com/tag/

propagation/

Function of initiator is to initiate free radical polymerization

PorogensPorogens

• Polymerization solventPolymerization solvent

• Functions to create pores in the Functions to create pores in the macroporous polymermacroporous polymer

• Porogen used is dependent on type Porogen used is dependent on type of molecular imprintingof molecular imprinting– Covalent ImprintingCovalent Imprinting

•Wide range of porogens usedWide range of porogens used

– Non-covalent ImprintingNon-covalent Imprinting•Aprotic, non-polar porogens usedAprotic, non-polar porogens used

– Acetonitrile, toluene, or chloroform preferredAcetonitrile, toluene, or chloroform preferred

Covalent ImprintingCovalent Imprinting

• Formation of reversible covalent bonds between template Formation of reversible covalent bonds between template and monomersand monomers

• Polymerization occurs in presence of a cross-linker moleculePolymerization occurs in presence of a cross-linker molecule

• Extraction of template molecule from polymer matrixExtraction of template molecule from polymer matrix

• Restrictive approach because under mild conditions it can Restrictive approach because under mild conditions it can be difficult to effectively induce reversible bond formation be difficult to effectively induce reversible bond formation and cleavage and cleavage

http://www.imego.com/research/Molecularly-Imprinted-Polymers-(MIPs)/index.aspx

Non-Covalent ImprintingNon-Covalent Imprinting

• Most widely used Most widely used production methodproduction method

• Template molecule Template molecule is non-covalently is non-covalently linked to monomerslinked to monomers

• Polymerization Polymerization occurs in presence occurs in presence of a cross-linker of a cross-linker moleculemolecule

• Extraction of Extraction of template molecule template molecule from polymer from polymer matrixmatrix

Möller, Kristina. Stockholm University, 2006, p1-91, ISBN 91-7155-234-0

Comparison of Imprinting Comparison of Imprinting TechniquesTechniques

Factors Covalent Non-covalent

Synthesis of monomer-template conjugate

Necessary Unnecessary

Polymerization conditions

Wide variety Restricted

Removal of template after polymerization

Difficult Easy

Target analyte binding and release

Slow Fast

Target analyte selectivity

Better selectivity - Higher frequency of specific binding sites

Less selectivity – mixture of specific & non-specific binding sites

OptimizationOptimization

• Variables in producing MIP’s that affect Variables in producing MIP’s that affect capacity, and selectivity:capacity, and selectivity:– Amount of monomerAmount of monomer– Type of monomerType of monomer– Nature of cross-linkerNature of cross-linker– SolventsSolvents

• Through trial and error optimization could Through trial and error optimization could take several weeks to completetake several weeks to complete

• Standard formulations have been developed Standard formulations have been developed – 1:4:20 template:monomer:cross-linker molar 1:4:20 template:monomer:cross-linker molar

ratio ratio • More advanced techniques optimization techniques More advanced techniques optimization techniques

are being developedare being developed

OptimizationOptimization

• Advanced techniques: Computational Advanced techniques: Computational approachapproach– Molecular modeling software used to screen Molecular modeling software used to screen

monomers against the desired template. monomers against the desired template. – Can calculate binding energies and estimate Can calculate binding energies and estimate

template-monomer interaction positions template-monomer interaction positions – Makes it possible to select the most efficient Makes it possible to select the most efficient

functional monomer to be used for the functional monomer to be used for the complexcomplex

– Relatively new approach, so the polymers Relatively new approach, so the polymers must still be prepared and evaluated prior to must still be prepared and evaluated prior to useuse

Creating MISPE Columns Creating MISPE Columns

• MIPs synthesizedMIPs synthesized

• MIPs dried, manually MIPs dried, manually crushed and sievedcrushed and sieved

• Prepared sorbent is Prepared sorbent is placed between two placed between two frits in SPE cartridgefrits in SPE cartridge– 25-500mg sorbent used25-500mg sorbent used– Reservoir volume of 1-Reservoir volume of 1-

10mL10mL

• Higher specificity for Higher specificity for target analyte than target analyte than SPESPE

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MISPE Used in IndustryMISPE Used in Industry• 2009 study pertaining to the determination of 2009 study pertaining to the determination of

cephalexin (CFX) in aqueous solutions (urine, cephalexin (CFX) in aqueous solutions (urine, and river water) and river water)

• Antibiotics are a commonly used family of Antibiotics are a commonly used family of pharmaceuticals, and are in many cases not pharmaceuticals, and are in many cases not fully eliminated during wastewater treatmentfully eliminated during wastewater treatment

• Single target analyte at low concentration, and Single target analyte at low concentration, and complex matrix make traditional SPE a poor complex matrix make traditional SPE a poor choice for purification of CFX prior to choice for purification of CFX prior to quantificationquantification

• Blank urine samples were spiked with CFX and Blank urine samples were spiked with CFX and amoxicillin (AMX) to determine cross-selectivity amoxicillin (AMX) to determine cross-selectivity of the MIP’sof the MIP’s– AMX and CFX are closely related in structure AMX and CFX are closely related in structure

ExperimentaExperimentall

• Functional monomer: Functional monomer: methacrylic acid (MAA)methacrylic acid (MAA)

• Cross-linker: ethylene Cross-linker: ethylene glycol dimethacrylate glycol dimethacrylate (EGDMA)(EGDMA)

• Two empty 6 mL Two empty 6 mL polyethylene SPE cartridges polyethylene SPE cartridges were packed with ~500mg were packed with ~500mg of the synthesized MIPof the synthesized MIP

• Final extracts were Final extracts were analyzed using HPLC with analyzed using HPLC with UV detectionUV detection

Beltran, Antoni, et al. J. Sep. Sci. 2009, 32, 3319-3326

Cephalexin ResultsCephalexin Results• Chromatogram A: Chromatogram A:

blank human urine blank human urine sample sample

• Chromatogram B: Chromatogram B: human urine spiked human urine spiked with CFX and AMXwith CFX and AMX

• MIP showed good MIP showed good cross-selectivity for cross-selectivity for both analytesboth analytes

• Recoveries of 78 and Recoveries of 78 and 60% for CFX & AMX, 60% for CFX & AMX, respectivelyrespectively

• Some impurities were Some impurities were still present, but a clear still present, but a clear chromatogram was chromatogram was obtained from MISPE obtained from MISPE extractsextracts

Beltran, Antoni, et al. J. Sep. Sci. 2009, 32, 3319-3326

MISPE of CholesterolMISPE of Cholesterol

Shi, Yun, et al. extracted cholesterol from biological samples using four MIPs created under different optimization conditions and compared % recoveries against traditional SPE

Shi, Yun et al., Journal of Pharmaceutical and Biomedical Analysis (2006) Vol. 42, p 549-555

MISPE of CholesterolMISPE of Cholesterol

GC chromatogramof yolk sample after

saponification

GC chromatogramof yolk sample after

C18 SPE

GC chromatogramof yolk sample afterMISPE using MIP3

CG chromatogramof yolk sample after

Shi, Yun et al., Journal of Pharmaceutical and Biomedical Analysis (2006) Vol. 42, p 549-555

ConclusionsConclusions

Factor Traditional SPE

MISPE

Type of Sorbent Usually derivitized silica

Tailored to target analyte

Selectivity Lower Higher

Binding Capacity Lower Higher

% Recoveries Lower Higher

Limit of Detection Higher Lower

Cost Lower Higher

ConclusionsConclusions

• Increased specificity Increased specificity from traditional SPEfrom traditional SPE

• Binding of trace Binding of trace amounts of target amounts of target analytes occurs analytes occurs from complex from complex samplessamples– High % recoveryHigh % recovery– Low quantification Low quantification

limitslimits

x 20,000 electron scanningmicrograph image of molecularly imprinted silica polymer

Pilau, Eduardo J., et al. J. Braz. Chem. Soc. 2008, Vol. 19, No. 6, p 1136-1143

ReferencesReferences• Beltran, Antoni; Fontanals, Nuria; Marce, Rosa M.; Cormack, Peter A. G.; Borrull, Beltran, Antoni; Fontanals, Nuria; Marce, Rosa M.; Cormack, Peter A. G.; Borrull,

Francesc. Molecularly imprinted solid-phase extraction of cephalexin from water-Francesc. Molecularly imprinted solid-phase extraction of cephalexin from water-based matrices. based matrices. J. Sep. Sci. J. Sep. Sci. 2009, Vol. 32, p 3319-33262009, Vol. 32, p 3319-3326

• Shi, Yun; Zhang, Jiang-Hua; Shi, Dan; Jiang, Ming; Zhu, Ye-Xiang; Mei, Su-Rong; Shi, Yun; Zhang, Jiang-Hua; Shi, Dan; Jiang, Ming; Zhu, Ye-Xiang; Mei, Su-Rong; Zhou, Yi-Kai; Dai, Kang; and Lu, Bin. Journal of Pharmaceutical and Biomedical Zhou, Yi-Kai; Dai, Kang; and Lu, Bin. Journal of Pharmaceutical and Biomedical Analysis. 2006, Vol. 42, p 549-555Analysis. 2006, Vol. 42, p 549-555

• Pilau, Eduardo J.; Silva, Raquel G. C.; Jardim, Isabel C. F. S.; and Augusto, Fabio. Pilau, Eduardo J.; Silva, Raquel G. C.; Jardim, Isabel C. F. S.; and Augusto, Fabio. Molecularly Imprinted Sol-Gel for Solid Phase Extraction of Phenobarbital. Molecularly Imprinted Sol-Gel for Solid Phase Extraction of Phenobarbital. J. Braz. J. Braz. Chem. Soc. Chem. Soc. 2008, Vol. 19, No. 6, p 1136-11432008, Vol. 19, No. 6, p 1136-1143

• Lee, Lim Lay. Synthesis and Application of Molecularly Imprinted Solid-Phase Lee, Lim Lay. Synthesis and Application of Molecularly Imprinted Solid-Phase Extraction for the Determination of Terbutaline in Biological Matrices. Extraction for the Determination of Terbutaline in Biological Matrices. Univeristy Univeristy Sains Malaysia.Sains Malaysia. 2006, p1-52 2006, p1-52

• Möller, Kristina. Molecularly Imprinted Solid-Phase Extraction and Liquid Möller, Kristina. Molecularly Imprinted Solid-Phase Extraction and Liquid Chromatography/Mass Spectrometry for Biological Samples. Chromatography/Mass Spectrometry for Biological Samples. Stockholm University.Stockholm University. 2006, p 1-91, ISBN 91-7155-234-02006, p 1-91, ISBN 91-7155-234-0

• Augusto, Fabio; Carasek, Eduardo; Silva, Raquel Gomes Costa; Rivellino, Sandra Augusto, Fabio; Carasek, Eduardo; Silva, Raquel Gomes Costa; Rivellino, Sandra Regina; Batista, Alex Domingues; and Martendal, Edmar. New sorbents for Regina; Batista, Alex Domingues; and Martendal, Edmar. New sorbents for extraction and microextraction techniques. extraction and microextraction techniques. Journal of Chromatography AJournal of Chromatography A, 2010, Vol. , 2010, Vol. 1217, p 2533-25421217, p 2533-2542

• Tamayo, F.G.; Turiel, E.; and Martin-Esteban, A. Molecularly imprinted polymers for Tamayo, F.G.; Turiel, E.; and Martin-Esteban, A. Molecularly imprinted polymers for solid-phase extraction and solid-phase microextraction: Recent developments and solid-phase extraction and solid-phase microextraction: Recent developments and future trends. future trends. Journal of Chromatography AJournal of Chromatography A, 2007, Vol. 1152, p 32-40, 2007, Vol. 1152, p 32-40