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 MIPs
• Trial and error
• Computational approach
• Creating MISPE columns from MIPs
• Specific examples of MISPE used in industry.
• Conclusions
• References

3
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

4
Molecularly Imprinted Solid-Phase
Extraction (MISPE)

• Technique introduced in early 1970s
• 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

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
Lee, Lim Lay. University Sains Malaysia, 2006, pp
1-52
8
Polymerization Reaction

• Most common type is free radical polymerization
• Initiation
• I 2R
• Propagation where M Monomers
• R M Mi
• Mi M Mi1,2,3.
• Termination
• Min Min Mnn
• R R I

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

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

Lee, Lim Lay. University Sains Malaysia, 2006, pp
1-52
12
Initiators
Function of initiator is to initiate free radical
polymerization
2,2-Azobisisobutyronitrile (AIBN)
Benzoyl peroxide
http//polymer.w99of.com/tag/propagation/
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

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

http//www.imego.com/research/Molecularly-Imprinte
d-Polymers-(MIPs)/index.aspx
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

Möller, Kristina. Stockholm University, 2006,
p1-91, ISBN 91-7155-234-0
16
Comparison of Imprinting Techniques
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
17
Optimization

• Variables in producing MIPs 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
• 1420 templatemonomercross-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

http//www.biotage.com/DynPage.aspx?id35833
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 MIPs
• 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

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

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
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MISPE of Cholesterol
GC chromatogram of yolk sample after saponificatio
n
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
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
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Conclusions

• Increased specificity from traditional SPE
• 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
• 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