Molecularly Imprinted Templates
for Solid-Phase Extraction
(MISPE)
Presented by:
Janee’ Hardman
Samantha Lawler
Overview
• Brief explanation of solid phase extraction
• What is MISPE?
• Making MI polymers
–
–
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–
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
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 nonpolar interactions
Sample added to
column, impurities
washed away, target
analyte eluted
Can have problems with
selectivity
http://www.biotage.com/DynPage.aspx?id=35833
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
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
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
Additional Polymerization Methods
Lee, Lim Lay. University Sains Malaysia, 2006, pp 1-52
Polymerization Reaction
• Most common type is free radical
polymerization
– Initiation
I
2R*
– Propagation where M = Monomers
R* + M
M*i + M
M*i
M*i+1,2,3….
– Termination
M*i+n + M*i+n
R* + R*
I
Mn+n
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
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
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 crosslinking required
• 70 – 90%
Lee, Lim Lay. University Sains Malaysia, 2006, pp 1-52
Initiators
Function of initiator is to initiate free radical
polymerization
2,2-Azobisisobutyronitrile
(AIBN)
Benzoyl peroxide
http://polymer.w99of.com/tag/propagation/
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
Covalent Imprinting
• Formation of reversible covalent bonds between template
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•
•
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-Imprinted-Polymers-(MIPs)/index.aspx
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
Comparison of Imprinting
Techniques
Factors
Covalent
Non-covalent
Synthesis of monomertemplate 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 Less selectivity – mixture
frequency of specific
of specific & non-specific
binding sites
binding sites
Optimization
• Variables in producing MIP’s that affect capacity,
and selectivity:
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–
–
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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
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
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 110mL
• Higher specificity for
target analyte than SPE
http://www.biotage.com/DynPage.aspx?id=35833
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
Experimental
• Functional monomer:
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•
•
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
Cephalexin Results
• Chromatogram A: blank
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•
•
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human urine sample
Chromatogram B: human
urine spiked with CFX and
AMX
MIP showed good crossselectivity 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
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
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
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
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
References
• Beltran, Antoni; Fontanals, Nuria; Marce, Rosa M.; Cormack, Peter A. G.; Borrull, Francesc.
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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 solidphase extraction and solid-phase microextraction: Recent developments and future trends.
Journal of Chromatography A, 2007, Vol. 1152, p 32-40