Molecularly Imprinted Templates for Solid-Phase Extraction MISPE

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

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

3. http://www.biotage.com/DynPage.aspx?id=35833 Solid Phase Extraction (SPE) Used to selectively retain analytes for purification Use individual cartridges or 96-well plates. Retention can be based on ionic, polar, or non-polar interactions Sample added to column, impurities washed away, target analyte eluted Can have problems with selectivity *with complex matrices, some impurities may also bind to column, and elute with target analyte Add examples of common SPE resins (C18, etc.)*with complex matrices, some impurities may also bind to column, and elute with target analyte Add examples of common SPE resins (C18, etc.)

4. Molecularly Imprinted Solid-Phase Extraction (MISPE) Technique introduced in early 1970’s Similar theory to traditional SPE 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 Wulff & Sarchan (Covalent), Mosbach (non-covalent)Wulff & Sarchan (Covalent), Mosbach (non-covalent)

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

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

7. Additional Polymerization Methods

8. Polymerization Reaction 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 Mn+n R* + R* I *Initiator – photochemical or thermal decomposition of initiator to form free radical *Can have homopolymerization (1 type monomer) or copolymerization (2 or more types of monomers)*Initiator – photochemical or thermal decomposition of initiator to form free radical *Can have homopolymerization (1 type monomer) or copolymerization (2 or more types of monomers)

9. Template/Imprint Molecule Target analyte or close structural analog Must be chemically inert 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

10. Functional Monomers Monomers chosen must be complementary in functionality to template/imprint molecule Monomers may be Acidic Basic Neutral Monomers are responsible for binding interactions in the imprinted binding site Ref from thesisMonomers are responsible for binding interactions in the imprinted binding site Ref from thesis

11. Cross-linkers 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 70 – 90%

12. Initiators

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

14. Covalent Imprinting Formation of reversible covalent bonds between template and monomers Polymerization occurs in presence of a cross-linker molecule 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

15. Non-Covalent Imprinting Most widely used production method Template molecule is non-covalently linked to monomers Polymerization occurs in presence of a cross-linker molecule Extraction of template molecule from polymer matrix *Cross-linker freezes the template molecule/monomer interactions (creates a non-pliable scaffold) *Non-covalent interactions can be H-bonding, ionic, dipole-dipole*Cross-linker freezes the template molecule/monomer interactions (creates a non-pliable scaffold) *Non-covalent interactions can be H-bonding, ionic, dipole-dipole

16. Comparison of Imprinting Techniques

17. Optimization 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 Standard formulations have been developed 1:4:20 template:monomer:cross-linker molar ratio More advanced techniques optimization techniques are being developed

18. Optimization 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 dried, manually crushed and sieved 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

20. MISPE Used in Industry 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 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 AMX and CFX are closely related in structure

21. Experimental Functional monomer: methacrylic acid (MAA) Cross-linker: ethylene glycol dimethacrylate (EGDMA) Two empty 6 mL polyethylene SPE cartridges were packed with ~500mg of the synthesized MIP Final extracts were analyzed using HPLC with UV detection

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

23. MISPE of Cholesterol

24. MISPE of Cholesterol

25. Conclusions

26. Conclusions Increased specificity from traditional SPE Binding of trace amounts of target analytes occurs from complex samples High % recovery Low quantification limits

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 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 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 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