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Molecularly Imprinted Templates for Solid-Phase Extraction (MISPE) Presented by: Janee’ Hardman Samantha Lawler.

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Presentation on theme: "Molecularly Imprinted Templates for Solid-Phase Extraction (MISPE) Presented by: Janee’ Hardman Samantha Lawler."— Presentation transcript:

1 Molecularly Imprinted Templates for Solid-Phase Extraction (MISPE) Presented by: Janee’ Hardman Samantha Lawler

2 Overview Overview Brief explanation of solid phase extraction Brief explanation of solid phase extraction What is MISPE? What is MISPE? Making MI polymers Making MI polymers –Polymerization –Reaction components –Covalent Imprinting –Non-covalent Imprinting Optimization of developing MIP’s Optimization of developing MIP’s –Trial and error –Computational approach Creating MISPE columns from MIP’s Creating MISPE columns from MIP’s Specific examples of MISPE used in industry. Specific examples of MISPE used in industry. Conclusions Conclusions References References

3 http://www.biotage.com/DynPage.aspx?id=35833 Solid Phase Extraction (SPE) Solid Phase Extraction (SPE) Used to selectively retain analytes for purification Used to selectively retain analytes for purification Use individual cartridges or 96-well plates. Use individual cartridges or 96-well plates. Retention can be based on ionic, polar, or non- polar interactions Retention can be based on ionic, polar, or non- polar interactions Sample added to column, impurities washed away, target analyte eluted Sample added to column, impurities washed away, target analyte eluted Can have problems with selectivity Can have problems with selectivity

4 Molecularly Imprinted Solid-Phase Extraction (MISPE) Technique introduced in early 1970’s Technique introduced in early 1970’s Similar theory to traditional SPE Similar theory to traditional SPE More selective, resulting in greater purification of final extracts More selective, resulting in greater purification of final extracts Sorbent composed of molecularly imprinted polymers (MIPs) that have a predetermined selectivity for a particular analyte, or group of structurally related compounds Sorbent composed of molecularly imprinted polymers (MIPs) that have a predetermined selectivity for a particular analyte, or group of structurally related compounds

5 MIPs Overview Creation of polymers based upon molecular recognition Creation of polymers based upon molecular recognition –Referred to as synthetic antibodies Polymer network is created around a template/imprint molecule Polymer network is created around a template/imprint molecule Removal of template/imprint molecule leaves cavity in polymer Removal of template/imprint molecule leaves cavity in polymer –Chemical affinity –Steric affinity

6 Polymerization Method Bulk Polymerization Bulk Polymerization –All components added to reaction vessel at once Template/imprint molecule Template/imprint molecule Monomers Monomers Initiator Initiator Cross-linker Cross-linker Porogen (Polymerization solvent) Porogen (Polymerization solvent) –Reaction initiated via heat or UV irradiation –Results in macroporous monolithic polymeric block Dried, manually ground, sieved Dried, manually ground, sieved

7 Additional Polymerization Methods Lee, Lim Lay. University Sains Malaysia, 2006, pp 1-52

8 Polymerization Reaction Most common type is free radical polymerization Most common type is free radical polymerization –Initiation I 2R* –Propagation where M = Monomers R* + M M* i M* i + M M* i+1,2,3…. –Termination M* i+n + M* i+n M n+n R* + R* I

9 Template/Imprint Molecule Target analyte or close structural analog Target analyte or close structural analog Must be chemically inert Must be chemically inert Stable under polymerization conditions Stable under polymerization conditions –No participation in free radical reaction –Thermally stable if polymerization initiated via heat –UV stable if polymerization initiated via UV irradiation Removal of template in MIP achieved via Soxhlet extraction Removal of template in MIP achieved via Soxhlet extraction

10 Functional Monomers Monomers chosen must be complementary in functionality to template/imprint molecule Monomers chosen must be complementary in functionality to template/imprint molecule Monomers may be Monomers may be –Acidic –Basic –Neutral Lee, Lim Lay. University Sains Malaysia, 2006, pp 1-52

11 Cross-linkers Fulfills three major functions Fulfills three major functions –Defines form and structure of polymer matrix –Makes imprint molecule insoluble in polymerization solvent (porogen) –Imparts mechanical stability to polymer matrix High degree of cross- linking required High degree of cross- linking required 70 – 90% 70 – 90% Lee, Lim Lay. University Sains Malaysia, 2006, pp 1-52

12 Initiators 2,2-Azobisisobutyronitrile (AIBN) Benzoyl peroxide http://polymer.w99of.com/tag/propagation / Function of initiator is to initiate free radical polymerization

13 Porogens Polymerization solvent Polymerization solvent Functions to create pores in the macroporous polymer Functions to create pores in the macroporous polymer Porogen used is dependent on type of molecular imprinting Porogen used is dependent on type of molecular imprinting –Covalent Imprinting Wide range of porogens used Wide range of porogens used –Non-covalent Imprinting Aprotic, non-polar porogens used Aprotic, non-polar porogens used –Acetonitrile, toluene, or chloroform preferred

14 Covalent Imprinting Formation of reversible covalent bonds between template and monomers Formation of reversible covalent bonds between template and monomers Polymerization occurs in presence of a cross-linker molecule Polymerization occurs in presence of a cross-linker molecule Extraction of template molecule from polymer matrix Extraction of template molecule from polymer matrix Restrictive approach because under mild conditions it can be difficult to effectively induce reversible bond formation and cleavage Restrictive approach because under mild conditions it can be difficult to effectively induce reversible bond formation and cleavage http://www.imego.com/research/Molecularly-Imprinted-Polymers-(MIPs)/index.aspx

15 Non-Covalent Imprinting Most widely used production method Most widely used production method Template molecule is non-covalently linked to monomers Template molecule is non-covalently linked to monomers Polymerization occurs in presence of a cross-linker molecule Polymerization occurs in presence of a cross-linker molecule Extraction of template molecule from polymer matrix Extraction of template molecule from polymer matrix Möller, Kristina. Stockholm University, 2006, p1-91, ISBN 91-7155-234-0

16 Comparison of Imprinting Techniques FactorsCovalentNon-covalent Synthesis of monomer- template conjugate NecessaryUnnecessary Polymerization conditionsWide varietyRestricted Removal of template after polymerization DifficultEasy Target analyte binding and release SlowFast Target analyte selectivityBetter selectivity - Higher frequency of specific binding sites Less selectivity – mixture of specific & non-specific binding sites

17 Optimization Variables in producing MIP’s that affect capacity, and selectivity: Variables in producing MIP’s that affect capacity, and selectivity: –Amount of monomer –Type of monomer –Nature of cross-linker –Solvents Through trial and error optimization could take several weeks to complete Through trial and error optimization could take several weeks to complete Standard formulations have been developed Standard formulations have been developed – 1:4:20 template:monomer:cross-linker molar ratio More advanced techniques optimization techniques are being developed More advanced techniques optimization techniques are being developed

18 Optimization Advanced techniques: Computational approach Advanced techniques: Computational approach –Molecular modeling software used to screen monomers against the desired template. –Can calculate binding energies and estimate template-monomer interaction positions –Makes it possible to select the most efficient functional monomer to be used for the complex –Relatively new approach, so the polymers must still be prepared and evaluated prior to use

19 Creating MISPE Columns MIPs synthesized MIPs synthesized MIPs dried, manually crushed and sieved MIPs dried, manually crushed and sieved Prepared sorbent is placed between two frits in SPE cartridge Prepared sorbent is placed between two frits in SPE cartridge –25-500mg sorbent used –Reservoir volume of 1- 10mL Higher specificity for target analyte than SPE Higher specificity for target analyte than SPE http://www.biotage.com/DynPage.aspx?id=35833

20 MISPE Used in Industry 2009 study pertaining to the determination of cephalexin (CFX) in aqueous solutions (urine, and river water) 2009 study pertaining to the determination of cephalexin (CFX) in aqueous solutions (urine, and river water) Antibiotics are a commonly used family of pharmaceuticals, and are in many cases not fully eliminated during wastewater treatment Antibiotics are a commonly used family of pharmaceuticals, and are in many cases not fully eliminated during wastewater treatment Single target analyte at low concentration, and complex matrix make traditional SPE a poor choice for purification of CFX prior to quantification Single target analyte at low concentration, and complex matrix make traditional SPE a poor choice for purification of CFX prior to quantification Blank urine samples were spiked with CFX and amoxicillin (AMX) to determine cross-selectivity of the MIP’s Blank urine samples were spiked with CFX and amoxicillin (AMX) to determine cross-selectivity of the MIP’s –AMX and CFX are closely related in structure

21 Experimental Functional monomer: methacrylic acid (MAA) Functional monomer: methacrylic acid (MAA) Cross-linker: ethylene glycol dimethacrylate (EGDMA) Cross-linker: ethylene glycol dimethacrylate (EGDMA) Two empty 6 mL polyethylene SPE cartridges were packed with ~500mg of the synthesized MIP Two empty 6 mL polyethylene SPE cartridges were packed with ~500mg of the synthesized MIP Final extracts were analyzed using HPLC with UV detection Final extracts were analyzed using HPLC with UV detection Beltran, Antoni, et al. J. Sep. Sci. 2009, 32, 3319-3326

22 Cephalexin Results Chromatogram A: blank human urine sample Chromatogram A: blank human urine sample Chromatogram B: human urine spiked with CFX and AMX Chromatogram B: human urine spiked with CFX and AMX MIP showed good cross- selectivity for both analytes MIP showed good cross- selectivity for both analytes Recoveries of 78 and 60% for CFX & AMX, respectively Recoveries of 78 and 60% for CFX & AMX, respectively Some impurities were still present, but a clear chromatogram was obtained from MISPE extracts Some impurities were still present, but a clear chromatogram was obtained from MISPE extracts Beltran, Antoni, et al. J. Sep. Sci. 2009, 32, 3319-3326

23 MISPE 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

24 MISPE of Cholesterol GC chromatogram of yolk sample after saponification GC chromatogram of yolk sample after C18 SPE GC chromatogram of yolk sample after MISPE using MIP3 CG chromatogram of yolk sample after Shi, Yun et al., Journal of Pharmaceutical and Biomedical Analysis (2006) Vol. 42, p 549-555

25 Conclusions FactorTraditional SPEMISPE Type of SorbentUsually derivitized silica Tailored to target analyte SelectivityLowerHigher Binding CapacityLowerHigher % RecoveriesLowerHigher Limit of DetectionHigherLower CostLowerHigher

26 Conclusions Increased specificity from traditional SPE Increased specificity from traditional SPE Binding of trace amounts of target analytes occurs from complex samples Binding of trace amounts of target analytes occurs from complex samples –High % recovery –Low quantification limits x 20,000 electron scanning micrograph image of molecularly imprinted silica polymer Pilau, Eduardo J., et al. J. Braz. Chem. Soc. 2008, Vol. 19, No. 6, p 1136-1143

27 References Beltran, Antoni; Fontanals, Nuria; Marce, Rosa M.; Cormack, Peter A. G.; Borrull, Francesc. Molecularly imprinted solid-phase extraction of cephalexin from water-based matrices. J. Sep. Sci. 2009, Vol. 32, p 3319-3326 Beltran, Antoni; Fontanals, Nuria; Marce, Rosa M.; Cormack, Peter A. G.; Borrull, Francesc. Molecularly imprinted solid-phase extraction of cephalexin from water-based matrices. J. Sep. Sci. 2009, Vol. 32, p 3319-3326 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 Analysis. 2006, Vol. 42, p 549-555 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 Analysis. 2006, Vol. 42, p 549-555 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. J. Braz. Chem. Soc. 2008, Vol. 19, No. 6, p 1136-1143 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. J. Braz. Chem. Soc. 2008, Vol. 19, No. 6, p 1136-1143 Lee, Lim Lay. Synthesis and Application of Molecularly Imprinted Solid-Phase Extraction for the Determination of Terbutaline in Biological Matrices. Univeristy Sains Malaysia. 2006, p1-52 Lee, Lim Lay. Synthesis and Application of Molecularly Imprinted Solid-Phase Extraction for the Determination of Terbutaline in Biological Matrices. Univeristy Sains Malaysia. 2006, p1-52 Möller, Kristina. Molecularly Imprinted Solid-Phase Extraction and Liquid Chromatography/Mass Spectrometry for Biological Samples. Stockholm University. 2006, p 1-91, ISBN 91-7155-234-0 Möller, Kristina. Molecularly Imprinted Solid-Phase Extraction and Liquid Chromatography/Mass Spectrometry for Biological Samples. Stockholm University. 2006, p 1-91, ISBN 91-7155-234-0 Augusto, Fabio; Carasek, Eduardo; Silva, Raquel Gomes Costa; Rivellino, Sandra Regina; Batista, Alex Domingues; and Martendal, Edmar. New sorbents for extraction and microextraction techniques. Journal of Chromatography A, 2010, Vol. 1217, p 2533-2542 Augusto, Fabio; Carasek, Eduardo; Silva, Raquel Gomes Costa; Rivellino, Sandra Regina; Batista, Alex Domingues; and Martendal, Edmar. New sorbents for extraction and microextraction techniques. Journal of Chromatography A, 2010, Vol. 1217, p 2533-2542 Tamayo, F.G.; Turiel, E.; and Martin-Esteban, A. Molecularly imprinted polymers for solid- phase extraction and solid-phase microextraction: Recent developments and future trends. Journal of Chromatography A, 2007, Vol. 1152, p 32-40 Tamayo, F.G.; Turiel, E.; and Martin-Esteban, A. Molecularly imprinted polymers for solid- phase extraction and solid-phase microextraction: Recent developments and future trends. Journal of Chromatography A, 2007, Vol. 1152, p 32-40


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