Molecular Imprinted Polymers Review

Chapter 2
12
MOLECULAR IMPRINTED POLYMERS: A REVIEW
synthesis of polymeric materials with predetermined ligand selectivity8-11.

The MIPs possess two of the most important features of the biological
2.1. Introduction
The interactions between molecules that are important in processes
such as affinity separation and enzymatic catalysis relay on molecular
recognition. Through molecular imprinting, it is possible to develop tailormade polymers that are selective for different compounds. This technique
allows the formation of specific recognition and catalytic sites in
macromolecules by the use of templates1-3. Molecular imprinted polymers
(MIPs) are a group of compounds, where the functional and crosslinking
receptors the ability to recognize and bind specific target molecules.

Because of the super-crosslinked nature, MIPs are stable to physical and
chemical treatment, including heating, acids, bases and organic acids. The
imprinted polymers have been applied in an increasing number of
applications where molecular binding events are of interest. The stability
and low cost of molecular imprinted polymers make them advantageous for
use in analysis as well as in industrial scale production and application12-14.
monomers are copolymerised in the presence of a target analyte (imprint

molecule), which act as a molecular template. An important prerequisite for
the preparation of these polymers is the formation of a stable prepolymerisation complex between the template and the monomer4,5. The
molecules of the functional monomer are arranged and fixed around the
template molecule in the course of the entire process of polymerization. In
an imprinted polymer the chemically active moieties of the target
molecules are held in position by the highly crosslinked polymeric
networks (Scheme II.1). Subsequent removal of the imprint molecule
reveals binding sites that are complimentary in size and shape to the
imprinted analyte, comparable to that of antibodies6, 7 creating a molecular
memory in the polymer. Thus molecular imprinting is a technique for the
Scheme II. 1. Principle of molecular imprinting15

Molecular recognition between a molecular receptor (host) and a
substrate (guest) in a matrix containing structurally related molecules
requires discrimination and binding. This can happen only if the binding
Chapter 2
12
MOLECULAR IMPRINTED POLYMERS: A REVIEW
synthesis of polymeric materials with predetermined ligand selectivity8-11.

The MIPs possess two of the most important features of the biological
2.1. Introduction
The interactions between molecules that are important in processes
such as affinity separation and enzymatic catalysis relay on molecular
recognition. Through molecular imprinting, it is possible to develop tailormade polymers that are selective for different compounds. This technique
allows the formation of specific recognition and catalytic sites in
macromolecules by the use of templates1-3. Molecular imprinted polymers
(MIPs) are a group of compounds, where the functional and crosslinking
receptors the ability to recognize and bind specific target molecules.

Because of the super-crosslinked nature, MIPs are stable to physical and
chemical treatment, including heating, acids, bases and organic acids. The
imprinted polymers have been applied in an increasing number of
applications where molecular binding events are of interest. The stability
and low cost of molecular imprinted polymers make them advantageous for
use in analysis as well as in industrial scale production and application12-14.
monomers are copolymerised in the presence of a target analyte (imprint

molecule), which act as a molecular template. An important prerequisite for
the preparation of these polymers is the formation of a stable prepolymerisation complex between the template and the monomer4,5. The
molecules of the functional monomer are arranged and fixed around the
template molecule in the course of the entire process of polymerization. In
an imprinted polymer the chemically active moieties of the target
molecules are held in position by the highly crosslinked polymeric
networks (Scheme II.1). Subsequent removal of the imprint molecule
reveals binding sites that are complimentary in size and shape to the
imprinted analyte, comparable to that of antibodies6, 7 creating a molecular
memory in the polymer. Thus molecular imprinting is a technique for the
Scheme II. 1. Principle of molecular imprinting15

Molecular recognition between a molecular receptor (host) and a
substrate (guest) in a matrix containing structurally related molecules
requires discrimination and binding. This can happen only if the binding
Chapter 2
14
Review of Literature
13
sites of the host and guest molecules compliment each other in size, shape
and chemical functionality. Biological systems such as enzyme-substrate,
antigen-antibody and hormone-receptor systems demonstrate molecular
recognition properties originating from natural selection16. The working
hypothesis of the binding site structure in molecular imprinted polymers
is based on the idea that the pre-polymer complex is locked into place by
between the functional monomer and the template is covalent, polar

solvents can be used as porogen during the polymerization. However,
slow binding kinetics restricts the analytical application of covalently
imprinted MIPs. Furthermore, covalent binding tends to be very limited
since it is specific for particular functional groups and is generally
directional.
polymerisation. This assumption postulates the formation of a cavity with
The imprinting efficiency is very high in non-covalent imprinting,
functional groups in complimentary array for the convergent interactions
which has been pioneered by Mosbach and co-workers6, 22. This method
with the template. The relationship of template to the imprinted cavity
mimics the antigen-antibody or enzyme-substrate interactions in
corresponds to the lock and key principle proposed by Emil Fisher for
biomolecules. Noncovalent interactions involve hydrogen bonds, ionic,
enzyme catalysis17.
hydrophobic, - interactions, induction, dispersion and van der Waals

forces; the most important being electrostatic, ion pairing and hydrogen
2. 2. Approaches in molecular imprinting

Generally in molecular imprinting two approaches are employed
for the interaction between the functional monomer and the template
namely covalent and non-covalent. The process developed by Wulff and
co-workers18 is covalent where a pre-polymerisation derivative is formed
between
functional
monomer
and
template
followed
by
the
polymerization (Scheme II. 2). The interactions between the pairs:

amine - aldehyde, diol - ketone, acid - amine are normally covalent in
nature. Thus imprinting with Schiffs base19, boronic acid esters20 and
metal co-ordination bonds21 follow covalent method. Since the bond
bonding interactions. Here a pre-polymerisation complex is formed, the

stability of which depends on the affinity constants between imprint
molecule and the functional monomers (Scheme II. 2). The bonds are less
specific, non-directional and the binding sites are heterogeneous in nature.
Imprinting of a lot of compounds including pesticides like 2, 4-D, atrazine
and bentazone through non-covalent bonding are already reported23 - 25.
Chapter 2
14
13
sites of the host and guest molecules compliment each other in size, shape
and chemical functionality. Biological systems such as enzyme-substrate,
antigen-antibody and hormone-receptor systems demonstrate molecular
recognition properties originating from natural selection16. The working
hypothesis of the binding site structure in molecular imprinted polymers
is based on the idea that the pre-polymer complex is locked into place by
between the functional monomer and the template is covalent, polar

solvents can be used as porogen during the polymerization. However,
slow binding kinetics restricts the analytical application of covalently
imprinted MIPs. Furthermore, covalent binding tends to be very limited
since it is specific for particular functional groups and is generally
directional.
polymerisation. This assumption postulates the formation of a cavity with
The imprinting efficiency is very high in non-covalent imprinting,
functional groups in complimentary array for the convergent interactions
which has been pioneered by Mosbach and co-workers6, 22. This method
with the template. The relationship of template to the imprinted cavity
mimics the antigen-antibody or enzyme-substrate interactions in
corresponds to the lock and key principle proposed by Emil Fisher for
biomolecules. Noncovalent interactions involve hydrogen bonds, ionic,
enzyme catalysis17.
hydrophobic, - interactions, induction, dispersion and van der Waals

forces; the most important being electrostatic, ion pairing and hydrogen
2. 2. Approaches in molecular imprinting

Generally in molecular imprinting two approaches are employed
for the interaction between the functional monomer and the template
namely covalent and non-covalent. The process developed by Wulff and
co-workers18 is covalent where a pre-polymerisation derivative is formed
between
functional
monomer
and
template
followed
by
the
polymerization (Scheme II. 2). The interactions between the pairs:

amine - aldehyde, diol - ketone, acid - amine are normally covalent in
nature. Thus imprinting with Schiffs base19, boronic acid esters20 and
metal co-ordination bonds21 follow covalent method. Since the bond
bonding interactions. Here a pre-polymerisation complex is formed, the

stability of which depends on the affinity constants between imprint
molecule and the functional monomers (Scheme II. 2). The bonds are less
specific, non-directional and the binding sites are heterogeneous in nature.
Imprinting of a lot of compounds including pesticides like 2, 4-D, atrazine
and bentazone through non-covalent bonding are already reported23 - 25.
Chapter 2
16
15
graft polymerisation and precipitation polymerisation30,
31
. In suspension
polymerisation the beads obtained are of diameter 5-50 m depending on

the stirring speed and the amount of surfactant used. Liquid
perfluorocarbons be preferred to water since water may have detrimental
effect on the noncovalent complex formation between the template and the
functional monomer. Precipitation polymerisation is another method that
can provide particles in the submicron scale (0.3-10 m)15. In this case,
Scheme II. 2. Principle behind non-covalent and covalent imprinting
particles are prevented from coalescence by the rigidity obtained from the
Hybrid interactions are there in molecular imprinting where,
polymerisation through covalent and rebinding through non-covalent
interactions were observed26.
2.3. Synthesis of imprinted polymers
Majority of imprinted polymers are synthesised by radical
polymerisation from functional and crosslinking monomers having vinyl or
acrylic groups27. To suit the final application, molecular imprinted polymers
crosslinking of the polymer, and there is no need of any extra stabiliser.

These beads can be rendered magnetic through inclusion of iron oxide
particles32. In situ polymerisation can be done for certain applications
inside the chromatographic column33 or in a capillary34. In sensor
applications usually the imprinted polymer membranes were prepared as
thin crosslinked films by precipitation from solutions in the presence of
analyte35 or by casting polymer in the pores of an inert support
can be synthesised in a variety of physical forms. Chronologically, the first
membrane36. They can also be synthesised in situ at the electrode surface
polymerisation method employed to synthesise MIP was based on bulk
by electropolymerisation37 or at a non-conducting surface by chemical
polymerisation
28, 29
followed by mechanical grinding to obtain small
grafting38.
micrometer-sized particles suitable to use in chromatographic purposes.

One of the many attractive features of the molecular imprinting
The polymer beads of controlled diameter can be prepared directly by
method is that it can be applied to a diverse range of analytes, even though
alternate methods such as suspension polymerisation, seed polymerisation,
it is a problem for large molecules. A diverse range of substances including
Chapter 2
16
15
graft polymerisation and precipitation polymerisation30,
31
. In suspension
polymerisation the beads obtained are of diameter 5-50 m depending on

the stirring speed and the amount of surfactant used. Liquid
perfluorocarbons be preferred to water since water may have detrimental
effect on the noncovalent complex formation between the template and the
functional monomer. Precipitation polymerisation is another method that
can provide particles in the submicron scale (0.3-10 m)15. In this case,
Scheme II. 2. Principle behind non-covalent and covalent imprinting
particles are prevented from coalescence by the rigidity obtained from the
Hybrid interactions are there in molecular imprinting where,
polymerisation through covalent and rebinding through non-covalent
interactions were observed26.
2.3. Synthesis of imprinted polymers
Majority of imprinted polymers are synthesised by radical
polymerisation from functional and crosslinking monomers having vinyl or
acrylic groups27. To suit the final application, molecular imprinted polymers
crosslinking of the polymer, and there is no need of any extra stabiliser.

These beads can be rendered magnetic through inclusion of iron oxide
particles32. In situ polymerisation can be done for certain applications
inside the chromatographic column33 or in a capillary34. In sensor
applications usually the imprinted polymer membranes were prepared as
thin crosslinked films by precipitation from solutions in the presence of
analyte35 or by casting polymer in the pores of an inert support
can be synthesised in a variety of physical forms. Chronologically, the first
membrane36. They can also be synthesised in situ at the electrode surface
polymerisation method employed to synthesise MIP was based on bulk
by electropolymerisation37 or at a non-conducting surface by chemical
polymerisation
28, 29
followed by mechanical grinding to obtain small
grafting38.
micrometer-sized particles suitable to use in chromatographic purposes.

One of the many attractive features of the molecular imprinting
The polymer beads of controlled diameter can be prepared directly by
method is that it can be applied to a diverse range of analytes, even though
alternate methods such as suspension polymerisation, seed polymerisation,
it is a problem for large molecules. A diverse range of substances including
Chapter 2
18
17
sugars, polymers and amino acid derivatives are already imprinted. Novel
imaging strategies using fluorescently labelled template was also done39.
rebinding. The best results are obtained when the templates get attached to
more than one binding site.
Attempts to synthesise imprinted polymers, which can be operated in
The bond between the template and the binding group should be as
aqueous medium, are in the preliminary stage40. Metal ion imprinted
strong as possible during the polymerisation to enable the binding group to
polymeric beads are successfully applied in analytical applications41. One
be fixed by the template in a definite orientation on the polymer chain
of the first reports of imprinting of bacterial cells on the polymer surface
during crosslinking. The template should be able to be removed as
was also observed42. Computational design and synthesis of imprinted
completely as possible. The interaction of the binding site with the template
polymers, with high binding capacity for pharmaceutical applications is
should be very &ast and reversible.
reported as a model case. Special protocols have been proposed for the
2.4.
imprinting of proteins43.
Factors influencing the characteristics of the imprinted polymers

The structure of the polymer matrix is crucial in molecular
The templates are classified as convex and concave where metal ion
imprinting. The specific structure of the cavity is determined not only by
involving cyclisation is an example for convex template and enzyme is an
the low molecular weight template molecules but also by the fixed
example for concave. The substrate has to fit in the cavity created by the
arrangement of the polymer chains. The characteristics of the polymer
enzyme to undergo chemical transformation. Polymers are also categorised
matrix like the matrix structure, matrix configuration, matrix nature,
as reactive (catalytic) or non-reactive (non-catalytic) where the reaction rate
template used and the extent of crosslinking has a significant effect on
is accelerated or unaffected.
rebinding. Hence the optimisation of the polymer structure is extremely
The binding sites consist of a functional group attached, capable of

interacting with the template molecule and ideally the functional groups
exists on the surface of the cavity left by the template, readily available for
important44. Since random incorporation of the functional monomers in the

polymer matrix reduces its specificity, interactions between the monomer
and the template should be as strong as possible during polymerisation;
ideally they should be covalent. However, systems utilising reversible
covalent bonds exhibit slow kinetics during rebinding and often necessitate
Chapter 2
18
17
sugars, polymers and amino acid derivatives are already imprinted. Novel
imaging strategies using fluorescently labelled template was also done39.
rebinding. The best results are obtained when the templates get attached to
more than one binding site.
Attempts to synthesise imprinted polymers, which can be operated in
The bond between the template and the binding group should be as
aqueous medium, are in the preliminary stage40. Metal ion imprinted
strong as possible during the polymerisation to enable the binding group to
polymeric beads are successfully applied in analytical applications41. One
be fixed by the template in a definite orientation on the polymer chain
of the first reports of imprinting of bacterial cells on the polymer surface
during crosslinking. The template should be able to be removed as
was also observed42. Computational design and synthesis of imprinted
completely as possible. The interaction of the binding site with the template
polymers, with high binding capacity for pharmaceutical applications is
should be very &ast and reversible.
reported as a model case. Special protocols have been proposed for the
2.4.
imprinting of proteins43.
Factors influencing the characteristics of the imprinted polymers

The structure of the polymer matrix is crucial in molecular
The templates are classified as convex and concave where metal ion
imprinting. The specific structure of the cavity is determined not only by
involving cyclisation is an example for convex template and enzyme is an
the low molecular weight template molecules but also by the fixed
example for concave. The substrate has to fit in the cavity created by the
arrangement of the polymer chains. The characteristics of the polymer
enzyme to undergo chemical transformation. Polymers are also categorised
matrix like the matrix structure, matrix configuration, matrix nature,
as reactive (catalytic) or non-reactive (non-catalytic) where the reaction rate
template used and the extent of crosslinking has a significant effect on
is accelerated or unaffected.
rebinding. Hence the optimisation of the polymer structure is extremely
The binding sites consist of a functional group attached, capable of

interacting with the template molecule and ideally the functional groups
exists on the surface of the cavity left by the template, readily available for
important44. Since random incorporation of the functional monomers in the

polymer matrix reduces its specificity, interactions between the monomer
and the template should be as strong as possible during polymerisation;
ideally they should be covalent. However, systems utilising reversible
covalent bonds exhibit slow kinetics during rebinding and often necessitate
Chapter 2
20
19
severe conditions for desorption20. This renders them unsuitable for many
applications. Ideal imprinted polymers require mild conditions for
desorption and rebinding which can be attained only through non-covalent
crosslinked systems disappear below 10% crosslinking and at a higher

degree of crosslinking the selectivity increases45.
Some flexibility is necessary to allow a fast binding and splitting of
the template within the cavities. Cavities with accurate shape but without
macromolecule so that both the more favourable and less favourable
flexibility will show kinetic hindrance to reversible binding. The studies on
conformations will occur.
macroporous styrene-divinylbenzene46, 47 and styrene-diisopropylbenzene48, 49
method.
Functional
groups
are
statistically
distributed
over
(a) Rigidity/ Flexibility of the polymer support
co-polymer by Shea et al revealed that, relatively rigid, hydrophobic and
The stiffness of the polymer structure enables the cavities to retain
highly crosslinked materials provides chemical inertness needed to
their shape even after the removal of the template, thus giving higher
withstand subsequent chemical transformations. Thus the molecular
selectivity. This can be achieved by high degree of crosslinking. The
imprinted polymers should have optimum stiffness/flexibility to attain the
binding of the template into the polymer during polymerisation, directs the
rapid equilibrium with the template, imprinted cavities accessible for the
positioning of the imprinted substrate within the cavities of the crosslinked
template, mechanical stability to withstand stress in certain applications and
polymer and it is assumed that the polymers with optimum selectivity and
thermal stability to use at high temperature.
specificity characteristics were obtained by suitable selection of monomers
(b) Functional and crosslinking monomers
and crosslinking agents.

Wulff and co-workers investigated the effect of the degree of
crosslinking on the selectivity of polymer (expressed as separation factor,
) for the resolution of phenyl-D, L-manopyranoside in batch processes as
a function of the amount of the crosslinking monomer using EGDMA as
crosslinking agent. It was found that the selectivity characteristics of
Majority of reports on molecular imprinting describe organic

polymers synthesised by radical polymerisation of functional and
crosslinking monomers having vinyl or acrylic groups. This can be
attributed to the fairly straightforward synthesis of these materials and to
the vast choice of available monomers. Other approaches are the
polystyrene-based and polysiloxane-based systems, but to a lesser extent.
The functional monomers can be basic, acidic, permanently charged,
Chapter 2
20
19
severe conditions for desorption20. This renders them unsuitable for many
applications. Ideal imprinted polymers require mild conditions for
desorption and rebinding which can be attained only through non-covalent
crosslinked systems disappear below 10% crosslinking and at a higher

degree of crosslinking the selectivity increases45.
Some flexibility is necessary to allow a fast binding and splitting of
the template within the cavities. Cavities with accurate shape but without
macromolecule so that both the more favourable and less favourable
flexibility will show kinetic hindrance to reversible binding. The studies on
conformations will occur.
macroporous styrene-divinylbenzene46, 47 and styrene-diisopropylbenzene48, 49
method.
Functional
groups
are
statistically
distributed
over
(a) Rigidity/ Flexibility of the polymer support
co-polymer by Shea et al revealed that, relatively rigid, hydrophobic and
The stiffness of the polymer structure enables the cavities to retain
highly crosslinked materials provides chemical inertness needed to
their shape even after the removal of the template, thus giving higher
withstand subsequent chemical transformations. Thus the molecular
selectivity. This can be achieved by high degree of crosslinking. The
imprinted polymers should have optimum stiffness/flexibility to attain the
binding of the template into the polymer during polymerisation, directs the
rapid equilibrium with the template, imprinted cavities accessible for the
positioning of the imprinted substrate within the cavities of the crosslinked
template, mechanical stability to withstand stress in certain applications and
polymer and it is assumed that the polymers with optimum selectivity and
thermal stability to use at high temperature.
specificity characteristics were obtained by suitable selection of monomers
(b) Functional and crosslinking monomers
and crosslinking agents.

Wulff and co-workers investigated the effect of the degree of
crosslinking on the selectivity of polymer (expressed as separation factor,
) for the resolution of phenyl-D, L-manopyranoside in batch processes as
a function of the amount of the crosslinking monomer using EGDMA as
crosslinking agent. It was found that the selectivity characteristics of
Majority of reports on molecular imprinting describe organic

polymers synthesised by radical polymerisation of functional and
crosslinking monomers having vinyl or acrylic groups. This can be
attributed to the fairly straightforward synthesis of these materials and to
the vast choice of available monomers. Other approaches are the
polystyrene-based and polysiloxane-based systems, but to a lesser extent.
The functional monomers can be basic, acidic, permanently charged,
Chapter 2
22
21
hydrogen bonding, hydrophobic and others (Fig. II.1). The imprinted

polymers prepared which are, rigid and insoluble have a lot of binding
sites, whereas the biological receptors are soluble, flexible, and have a few
or just one binding site. Generally, MIPs prepared in a relatively non-polar
solvent employ different functional monomers whereas acrylamide could
be a promising functional monomer to form strong hydrogen bonds with
crosslinking agent is NNMBA. The crosslinking agents control the

morphology of the polymer matrix by providing mechanical stability and
fix the guest-binding sites firmly in the desired structure. They also make
the imprinted polymers insoluble in solvents and facilitate their practical
applications. The DVB crosslinking imparts rigidity and hydrophobicity to
the polymer support which increases with increase in crosslinking. By
using different crosslinking agents we can control both the structure of the
template molecule in polar solvents.
guest-binding sites and the chemical environment around them.

For efficient imprinting, the reactivity of the crosslinking agent
should be similar to that of the functional monomer. The nature of the
(a)
(b)
(c)
crosslinking agent significantly affects the physicochemical properties of a

polymer matrix50. Divinylbenzene isomers are used as crosslinking agents
in the synthesis of MIPs based on polystyrene, whereas EGDMA is found
to be the most appropriate crosslinking agent for the synthesis of MIPs
(d)
(e)
(f)
(g)
(a) methacrylic acid (b) trifluoromethacrylic acid (c) N-vinylimidazole

(d) 4-vinyl pyridine (e) 2-vinyl pyridine (f) hydroxyethyl methacrylate
based on acrylic or methacrylic acid51. Not only the MIPs prepared with
the use of EGDMA as crosslinking agent found to be more specific, they
also exhibit good mechanical strength, thermal stability, porosity and
(g) acrylamide
wettability. In TEGDMA-crosslinked polymer the hydrophilic nature of the

Fig. II. 1. Functional monomers commonly used in molecular imprinting
crosslinking agent caused increased availability of binding sites for
For molecular imprinting in organic solvents, the crosslinking
agents EGDMA and DVB are often used. A typical water-soluble
template. By choosing the appropriate crosslinking agent, random
Chapter 2
22
21
hydrogen bonding, hydrophobic and others (Fig. II.1). The imprinted

polymers prepared which are, rigid and insoluble have a lot of binding
sites, whereas the biological receptors are soluble, flexible, and have a few
or just one binding site. Generally, MIPs prepared in a relatively non-polar
solvent employ different functional monomers whereas acrylamide could
be a promising functional monomer to form strong hydrogen bonds with
crosslinking agent is NNMBA. The crosslinking agents control the

morphology of the polymer matrix by providing mechanical stability and
fix the guest-binding sites firmly in the desired structure. They also make
the imprinted polymers insoluble in solvents and facilitate their practical
applications. The DVB crosslinking imparts rigidity and hydrophobicity to
the polymer support which increases with increase in crosslinking. By
using different crosslinking agents we can control both the structure of the
template molecule in polar solvents.
guest-binding sites and the chemical environment around them.

For efficient imprinting, the reactivity of the crosslinking agent
should be similar to that of the functional monomer. The nature of the
(a)
(b)
(c)
crosslinking agent significantly affects the physicochemical properties of a

polymer matrix50. Divinylbenzene isomers are used as crosslinking agents
in the synthesis of MIPs based on polystyrene, whereas EGDMA is found
to be the most appropriate crosslinking agent for the synthesis of MIPs
(d)
(e)
(f)
(g)
(a) methacrylic acid (b) trifluoromethacrylic acid (c) N-vinylimidazole

(d) 4-vinyl pyridine (e) 2-vinyl pyridine (f) hydroxyethyl methacrylate
based on acrylic or methacrylic acid51. Not only the MIPs prepared with
the use of EGDMA as crosslinking agent found to be more specific, they
also exhibit good mechanical strength, thermal stability, porosity and
(g) acrylamide
wettability. In TEGDMA-crosslinked polymer the hydrophilic nature of the

Fig. II. 1. Functional monomers commonly used in molecular imprinting
crosslinking agent caused increased availability of binding sites for
For molecular imprinting in organic solvents, the crosslinking
agents EGDMA and DVB are often used. A typical water-soluble
template. By choosing the appropriate crosslinking agent, random
Chapter 2
24
23
copolymerisation occurs successfully, and the polymer with uniform

distribution of monomers in the polymer network formed.
The mole ratios of crosslinking agent to functional monomer are
also important. If the ratios are too small, the guest binding sites are located
so closely to each other that they cannot work independently. In extreme
cases, the guest binding by one site completely inhibits the guest binding by
the neighbouring sites. At extremely high mole ratios, however, the
(a)
(b)
(c)
imprinting efficiency is damaged, especially when the crosslinking agents
(d)
O
O
show non-covalent interactions with functional monomers and/or
O
O
templates. Usually, a crosslinking agent is taken in 20-fold excess to the

functional monomer and its concentration in the reaction mixture is 70-
(e)
(f)
90%. At low degree of crosslinking, the selectivity is poor since the

H
N
polymer matrix is not crosslinked enough to retain the shape of the cavity.
2
In deciding a crosslinking agent its good solubility in a pre-polymerisation
H
N
O
(g)
(h)
mixture is also taken into account. For large templates such as proteins, the
crosslinking agents having optimal length such as TTEGDMA or PEG 400
(a) trimethylpropane trimethacrylate (TRIM),
(b) divinylbenzene (DVB),
(c) 2, 6-bisacryloylamidopyridine, (d) N, O - bisacryloyl-L- phenylalaninol,
DMA are more suitable52. However, these findings suggest that the
(e) ethylene glycol dimethacrylate (EGDMA), (f) triethylene glycol
crosslinking monomers are an active part of imprinting, rather than merely
dimethacrylate (TEGDMA), (g) butanediol dimethacrylate (BDDMA) and

(h) N, N'- methylene bis acrylamide (NNMBA)
inert scaffolding for functional monomers. The most frequently used

Fig. II. 2. Frequently used crosslinking monomers in molecular imprinting
crosslinking agents53 are exhibited (Fig. II. 2).
Chapter 2
24
23
copolymerisation occurs successfully, and the polymer with uniform

distribution of monomers in the polymer network formed.
The mole ratios of crosslinking agent to functional monomer are
also important. If the ratios are too small, the guest binding sites are located
so closely to each other that they cannot work independently. In extreme
cases, the guest binding by one site completely inhibits the guest binding by
the neighbouring sites. At extremely high mole ratios, however, the
(a)
(b)
(c)
imprinting efficiency is damaged, especially when the crosslinking agents
(d)
O
O
show non-covalent interactions with functional monomers and/or
O
O
templates. Usually, a crosslinking agent is taken in 20-fold excess to the

functional monomer and its concentration in the reaction mixture is 70-
(e)
(f)
90%. At low degree of crosslinking, the selectivity is poor since the

H
N
polymer matrix is not crosslinked enough to retain the shape of the cavity.
2
In deciding a crosslinking agent its good solubility in a pre-polymerisation
H
N
O
(g)
(h)
mixture is also taken into account. For large templates such as proteins, the
crosslinking agents having optimal length such as TTEGDMA or PEG 400
(a) trimethylpropane trimethacrylate (TRIM),
(b) divinylbenzene (DVB),
(c) 2, 6-bisacryloylamidopyridine, (d) N, O - bisacryloyl-L- phenylalaninol,
DMA are more suitable52. However, these findings suggest that the
(e) ethylene glycol dimethacrylate (EGDMA), (f) triethylene glycol
crosslinking monomers are an active part of imprinting, rather than merely
dimethacrylate (TEGDMA), (g) butanediol dimethacrylate (BDDMA) and

(h) N, N'- methylene bis acrylamide (NNMBA)
inert scaffolding for functional monomers. The most frequently used

Fig. II. 2. Frequently used crosslinking monomers in molecular imprinting
crosslinking agents53 are exhibited (Fig. II. 2).
26
Chapter 2
25
(c) Monomertemplate ratio

The molar relationship between the functional monomer and
template (M/T) has been found to be important with respect to the number
and quality of MIP recognition sites54-57. Low M/T ratios result in less than
optimal complexation on account of insufficient functional monomers.
Most of the functional monomer will be complexed and only a limited
number of functional monomer will be located outside the imprinted
cavities scattered on the surface of the polymer particles. During rebinding,
the imprinted binding sites may compete successfully with the residual
monomers for a limited number of guests, thus increasing selectivity. But
when the ratio (M/T) is high, only a limited number of functional monomer
is complexed with the template while those located outside the cavities,
remain scattered in the bulk of the polymer particles. Thus too high ratios
Fig. II. 3. Hypothetical model of the anticipated methacrylic acidnicotine

complex states in the pre-polymerisation complex55.
yield non-selective binding the extreme case being the non-imprinted
An excess of monomer on the other hand would favour the opposite
58
polymer . The parameter imprinting effect gives an estimation of how
situation where, the complexes formed will be of the type IV, but again
59
well the polymer was imprinted by the template . This cannot be
excess monomer would yield high number of non-complexed randomly
considered as a property of the stationary phase, but only a measure of the
distributed monomer which contribute to non-specific binding60. As
net molecular recognition effect due to the imprinting process in well-
selectivity is considered to arise from the pre-polymerisation of monomer
defined experimental conditions. The excess of template during
by the template prior to polymerisation, we should expect complex state IV
polymerisation is unfavourable towards selectivity. Thus excess nicotine
to be the one that is responsible for the production of higher affinity, more
favours the formation of complex states II and III over ideal IV (Fig. II. 3).
selective, binding sites. States I to III would lead to the formation of site
26
Chapter 2
25
(c) Monomertemplate ratio

The molar relationship between the functional monomer and
template (M/T) has been found to be important with respect to the number
and quality of MIP recognition sites54-57. Low M/T ratios result in less than
optimal complexation on account of insufficient functional monomers.
Most of the functional monomer will be complexed and only a limited
number of functional monomer will be located outside the imprinted
cavities scattered on the surface of the polymer particles. During rebinding,
the imprinted binding sites may compete successfully with the residual
monomers for a limited number of guests, thus increasing selectivity. But
when the ratio (M/T) is high, only a limited number of functional monomer
is complexed with the template while those located outside the cavities,
remain scattered in the bulk of the polymer particles. Thus too high ratios
Fig. II. 3. Hypothetical model of the anticipated methacrylic acidnicotine

complex states in the pre-polymerisation complex55.
yield non-selective binding the extreme case being the non-imprinted
An excess of monomer on the other hand would favour the opposite
58
polymer . The parameter imprinting effect gives an estimation of how
situation where, the complexes formed will be of the type IV, but again
59
well the polymer was imprinted by the template . This cannot be
excess monomer would yield high number of non-complexed randomly
considered as a property of the stationary phase, but only a measure of the
distributed monomer which contribute to non-specific binding60. As
net molecular recognition effect due to the imprinting process in well-
selectivity is considered to arise from the pre-polymerisation of monomer
defined experimental conditions. The excess of template during
by the template prior to polymerisation, we should expect complex state IV
polymerisation is unfavourable towards selectivity. Thus excess nicotine
to be the one that is responsible for the production of higher affinity, more
favours the formation of complex states II and III over ideal IV (Fig. II. 3).
selective, binding sites. States I to III would lead to the formation of site
Chapter 2
28
27
population of low selectivity, although weaker additional interactions to the

crosslinker may partly compensate for the absence of MAA. There is the
chance of self association of template at concentrations comparable to those
reactions occur. The volume of the porogen used is generally half of the
total volume of the mixture, and creates pores by phase separating into
channels during polymerisation. The porogen parameters such as polarity
and hydrogen bonding are important in determining the final morphology
used in imprinting studies.
of the network structure and porosity.

The rate of radical polymerisation depends on the nature and
It is believed that solvents with low permittivities (toluene,
concentration of the initiator61, 25. Polymerisation in early applications was

initiated by thermal decomposition of AIBN62,
63
. AIBN is the most
effective initiator; which undergoes homolytic cleavage to form two

isobutyronitrile radicals with the release of a nitrogen molecule upon
heating the reaction mixture to 60oC.
dichloromethane, and chloroform) are best suited for molecular

imprinting64. Chloroform is one of the most widely used solvents, since it
satisfactorily dissolves many monomers and templates and hardly
suppresses hydrogen bonding. In these solvents, the monomer-template
non-covalent interactions are stronger than in polar solvents.
(d)
Porogen / Rebinding solvent

Solvent is used in the imprinting studies both as the porogen during
polymerisation and as rebinding medium. The trivial role of solvents is to

dissolve the agents for polymerisation. However they have more crucial
roles. It provides porosity within the network, which facilitates mass
transfer of the analyte to and from the binding sites. In the polymerisation,
solvent molecules are incorporated inside the polymers and are removed in
the post-treatment. Then the space originally occupied by the solvent
molecules is left as pores in the polymers. Another role of solvents is to
disperse the heat of reaction generated on polymerisation. Otherwise, the
temperature of reaction mixture is locally elevated, and undesired side-
(e)
Polymerisation temperature
The position of equilibrium between free template - monomer(s)
and their corresponding complexes is a product of both temperature and

pressure65. Sellergren group showed that high pressure (1000 bar)
polymerisation could be used to enhance the selectivity of the resultant
imprinted polymers66. Previous studies found that lower temperature of
polymerisation is favourable for the preparation of MIPs based on
electrostatic interaction due to the greater strength of electrostatic
interaction
at
lower
temperature67,
68
At
low
temperature
the
polymerisation process is slow and the chain formation does not interfere
Chapter 2
28
27
population of low selectivity, although weaker additional interactions to the

crosslinker may partly compensate for the absence of MAA. There is the
chance of self association of template at concentrations comparable to those
reactions occur. The volume of the porogen used is generally half of the
total volume of the mixture, and creates pores by phase separating into
channels during polymerisation. The porogen parameters such as polarity
and hydrogen bonding are important in determining the final morphology
used in imprinting studies.
of the network structure and porosity.

The rate of radical polymerisation depends on the nature and
It is believed that solvents with low permittivities (toluene,
concentration of the initiator61, 25. Polymerisation in early applications was

initiated by thermal decomposition of AIBN62,
63
. AIBN is the most
effective initiator; which undergoes homolytic cleavage to form two

isobutyronitrile radicals with the release of a nitrogen molecule upon
heating the reaction mixture to 60oC.
dichloromethane, and chloroform) are best suited for molecular

imprinting64. Chloroform is one of the most widely used solvents, since it
satisfactorily dissolves many monomers and templates and hardly
suppresses hydrogen bonding. In these solvents, the monomer-template
non-covalent interactions are stronger than in polar solvents.
(d)
Porogen / Rebinding solvent

Solvent is used in the imprinting studies both as the porogen during
polymerisation and as rebinding medium. The trivial role of solvents is to

dissolve the agents for polymerisation. However they have more crucial
roles. It provides porosity within the network, which facilitates mass
transfer of the analyte to and from the binding sites. In the polymerisation,
solvent molecules are incorporated inside the polymers and are removed in
the post-treatment. Then the space originally occupied by the solvent
molecules is left as pores in the polymers. Another role of solvents is to
disperse the heat of reaction generated on polymerisation. Otherwise, the
temperature of reaction mixture is locally elevated, and undesired side-
(e)
Polymerisation temperature
The position of equilibrium between free template - monomer(s)
and their corresponding complexes is a product of both temperature and

pressure65. Sellergren group showed that high pressure (1000 bar)
polymerisation could be used to enhance the selectivity of the resultant
imprinted polymers66. Previous studies found that lower temperature of
polymerisation is favourable for the preparation of MIPs based on
electrostatic interaction due to the greater strength of electrostatic
interaction
at
lower
temperature67,
68
At
low
temperature
the
polymerisation process is slow and the chain formation does not interfere
Chapter 2
30
29
with the template - monomer interactions. An optimal condition of

temperature should be found for each combination of template and
monomer55. pH of the rebinding medium also induces a significant role in
molecular imprinting, especially in covalent imprinting69.
2.5.
Characterisation of molecular imprinted polymers

In the non-covalent approach the stability of the template/
functional monomer complex formed in the pre-polymerisation mixture

will govern the resulting binding site distribution and the distribution
properties of the imprinted polymer matrix. Close analysis of the prepolymerisation solution can provide fundamental insights to the various
interactions occurring during imprinting. Consequently, spectroscopic
studies of the pre-polymerisation mixtures provide prevalent information
on the imprinting process. Since reorganisation of functional groups at the
binding sites is required during rebinding the spectral studies before and
interacting structures that engage in complexation. Thus evaluating the shift

of a proton signal due to participation in hydrogen bond was used as the
selection criterion for complex formation, M/T ratio and interacting
forces70, 71.
(b)
FT-IR Spectra
In addition to 1H NMR, FT-IR spectra provide the fundamental
analytical basis for rationalising the mechanisms of recognition during the

imprinting process probing the governing interactions for selective binding
site formation at the molecular level72. The interaction between the
functional monomer and template during pre-polymerisation complex
formation and the template incorporation into the imprinted polymer during
rebinding can be confirmed by the characteristic FT-IR absorption
analysis27, 73.
(c) 13C-CP-MAS-NMR
Being rigid solids, neither usual NMR spectroscopy nor X-ray
after rebinding can also put light into rebinding.

diffraction methods can be applied successfully to follow rebinding with
(a) 1H NMR Spectra

Proton NMR titration experiments facilitate observation of
hydrogen bond formation between bases and carboxylic acid through
hydrogen bonding. These studies has been introduced in molecular
imprinting for investigating the extent of complex formation in prepolymerisation solutions and as a means of identifying the specific sites in
imprinted polymers74. Therefore it is not possible to obtain reliable

structure information of the interactions occurring in the cavities between
binding site and template. The polymer analysis using 13C-CP-MAS-NMR
technique can give information on the polymer backbone.
Chapter 2
30
29
with the template - monomer interactions. An optimal condition of

temperature should be found for each combination of template and
monomer55. pH of the rebinding medium also induces a significant role in
molecular imprinting, especially in covalent imprinting69.
2.5.
Characterisation of molecular imprinted polymers

In the non-covalent approach the stability of the template/
functional monomer complex formed in the pre-polymerisation mixture

will govern the resulting binding site distribution and the distribution
properties of the imprinted polymer matrix. Close analysis of the prepolymerisation solution can provide fundamental insights to the various
interactions occurring during imprinting. Consequently, spectroscopic
studies of the pre-polymerisation mixtures provide prevalent information
on the imprinting process. Since reorganisation of functional groups at the
binding sites is required during rebinding the spectral studies before and
interacting structures that engage in complexation. Thus evaluating the shift

of a proton signal due to participation in hydrogen bond was used as the
selection criterion for complex formation, M/T ratio and interacting
forces70, 71.
(b)
FT-IR Spectra
In addition to 1H NMR, FT-IR spectra provide the fundamental
analytical basis for rationalising the mechanisms of recognition during the

imprinting process probing the governing interactions for selective binding
site formation at the molecular level72. The interaction between the
functional monomer and template during pre-polymerisation complex
formation and the template incorporation into the imprinted polymer during
rebinding can be confirmed by the characteristic FT-IR absorption
analysis27, 73.
(c) 13C-CP-MAS-NMR
Being rigid solids, neither usual NMR spectroscopy nor X-ray
after rebinding can also put light into rebinding.

diffraction methods can be applied successfully to follow rebinding with
(a) 1H NMR Spectra

Proton NMR titration experiments facilitate observation of
hydrogen bond formation between bases and carboxylic acid through
hydrogen bonding. These studies has been introduced in molecular
imprinting for investigating the extent of complex formation in prepolymerisation solutions and as a means of identifying the specific sites in
imprinted polymers74. Therefore it is not possible to obtain reliable

structure information of the interactions occurring in the cavities between
binding site and template. The polymer analysis using 13C-CP-MAS-NMR
technique can give information on the polymer backbone.
Chapter 2
32
(d)
31
Scanning electron microscopy (SEM)
rebinding reaction very effectively. The extent of swelling can be
SEM can be used in a variety of distinct ways to probe imprinted
determined in terms of change in weight80, 81. Alte2native,y, by packing a
polymers on a variety of length scales. Scanning electron microscopy is the
definite weight of the polymer in a capillary tube and measuring the
most widely used technique to study the shape, size, morphology and
volume before and after incubation in the solvent, the swelling ratio can be
porosity of polymers75, 76.
determined in terms of change in volume 82.
(e) Pore analysis
2.7. Selectivity parameters of molecular imprinted polymers
It is possible to probe the morphology of imprinted polymers in
The binding parameters of the MIPs are usually estimated from
much the same way as one is able to do with most porous solids.
adsorption isotherms74,
Depending on the method of analysis, useful information may be gleaned
perform binding performance is based on saturation experiments and
on the specific pore volumes, pore sizes, pore size distributions and specific
subsequent Scatchard analysis85-88. The obtained binding data were
surface area of materials. Pore and surface area analysis was performed by
transformed into linear form and analysed to create Scatchard plots based
N2 adsorption. The surface area was determined using the BET model, the
on Scatchard equation
83, 84
using mathematical models. One strategy to
t-plot using Harkin-Jura average thickness equation and the pore volume
[S]b / [S]f = (Smax [S]b) / KD
and pore size distribution using the BJH model77.
where, KD is an equilibrium dissociation constant, Smax an apparent
2.6.
Swelling studies
The efficiency of a functional polymer is governed by the
aCcessibility of the reactive functIonal groups anchored on it, which in turn

depends upon the extent of swelLing and solvation78. The raTe of diffusion
of a reagent into the polymer matrix mainly dePends on the extent of
swelling79. Thus swelling is an important parameter, which controls the
success of rebinding. The most effective solvent can carry out th%
maximum number of binding sites and [S]b is the amount of template

bound to MIP at equilibrium. In this plot, the X-axis is bonded
concentration and the Y-axis is the ratio between bonded and free ligand
concentration. It is possible to estimate the Smax and KD from a Scatchard
plot where Smax is the X intercept; KD is the negative reciprocal of the
slope. For non-covalently synthesised MIPs the Scatchard plots result in a
Chapter 2
32
(d)
31
Scanning electron microscopy (SEM)
rebinding reaction very effectively. The extent of swelling can be
SEM can be used in a variety of distinct ways to probe imprinted
determined in terms of change in weight80, 81. Alte2native,y, by packing a
polymers on a variety of length scales. Scanning electron microscopy is the
definite weight of the polymer in a capillary tube and measuring the
most widely used technique to study the shape, size, morphology and
volume before and after incubation in the solvent, the swelling ratio can be
porosity of polymers75, 76.
determined in terms of change in volume 82.
(e) Pore analysis
2.7. Selectivity parameters of molecular imprinted polymers
It is possible to probe the morphology of imprinted polymers in
The binding parameters of the MIPs are usually estimated from
much the same way as one is able to do with most porous solids.
adsorption isotherms74,
Depending on the method of analysis, useful information may be gleaned
perform binding performance is based on saturation experiments and
on the specific pore volumes, pore sizes, pore size distributions and specific
subsequent Scatchard analysis85-88. The obtained binding data were
surface area of materials. Pore and surface area analysis was performed by
transformed into linear form and analysed to create Scatchard plots based
N2 adsorption. The surface area was determined using the BET model, the
on Scatchard equation
83, 84
using mathematical models. One strategy to
t-plot using Harkin-Jura average thickness equation and the pore volume
[S]b / [S]f = (Smax [S]b) / KD
and pore size distribution using the BJH model77.
where, KD is an equilibrium dissociation constant, Smax an apparent
2.6.
Swelling studies
The efficiency of a functional polymer is governed by the
aCcessibility of the reactive functIonal groups anchored on it, which in turn

depends upon the extent of swelLing and solvation78. The raTe of diffusion
of a reagent into the polymer matrix mainly dePends on the extent of
swelling79. Thus swelling is an important parameter, which controls the
success of rebinding. The most effective solvent can carry out th%
maximum number of binding sites and [S]b is the amount of template

bound to MIP at equilibrium. In this plot, the X-axis is bonded
concentration and the Y-axis is the ratio between bonded and free ligand
concentration. It is possible to estimate the Smax and KD from a Scatchard
plot where Smax is the X intercept; KD is the negative reciprocal of the
slope. For non-covalently synthesised MIPs the Scatchard plots result in a
Chapter 2
34
33
curve with the degree of curvature containing the information on the

heterogeneity of the binding sites within the MIP matrix. The random
arrangement of the templates at the binding sites and the incomplete
complexation between the template and the functional monomer led to the
heterogeneity in the binding sites typical for the non-covalent imprinting.
The effectiveness of imprinting was verified by comparison of the
binding of template versus molecules with similar structure89-92. A
complete secondary screen for binding and selectivity was performed for
all the polymers in terms of separation factor93
It provides a good indication for the molecular recognition of the

comparative molecule to the template.
2.8. Applications of molecular imprinted polymers

The great potential of molecular imprinting for the separation and
purification of wide range of molecules is quite apparent and make this
technique the ultimate in those areas.
(a) Enantioseparation of racemic mixtures

The first and the most important application of molecular imprinted
polymers is in the enantioseparation of racemic mixtures of chiral
compounds. This is because it provides a convenient method for
Separation factor ( template) = Kmip /Knip

MIPs possessing high separation factors should be capable of completely
recovering the target molecule by the simple process of stirring the
imprinted polymers with template solution. In general, the values of
separation factor are high when ionic interactions are utilised between the
template and the functional monomers. However, when non-covalent
interactions are employed, comparatively less values in the range one to
two is obtained. The selectivity of the imprinted polymer can also be
expressed as selectivity factor56, 94.
quantitative assessment of the quality of imprints produced by a particular

recipe or strategy. Most work has concentrated on the resolution of chiral
compounds. This reflects both the importance of this procedure in
analytical and synthetic chemistry and also the ease of distinguishing
between specific and non-specific recognition in this case. If the polymer is
imprinted with L-enantiomer of an amino acid, the MIP will retain the
L-enantiomer more than D-enantiomer and vice-versa, whereas the column
containing non-imprinted polymer will not be able to separate the
enantiomers. The typical values of enantioseparation factor () are between
Selectivity factor = template / comp
1.5 and 5, although in some cases much higher values are obtained.
Cinchona alkaloids cinchonidine and cinchonine showed selectivity with
Chapter 2
34
33
curve with the degree of curvature containing the information on the

heterogeneity of the binding sites within the MIP matrix. The random
arrangement of the templates at the binding sites and the incomplete
complexation between the template and the functional monomer led to the
heterogeneity in the binding sites typical for the non-covalent imprinting.
The effectiveness of imprinting was verified by comparison of the
binding of template versus molecules with similar structure89-92. A
complete secondary screen for binding and selectivity was performed for
all the polymers in terms of separation factor93
It provides a good indication for the molecular recognition of the

comparative molecule to the template.
2.8. Applications of molecular imprinted polymers

The great potential of molecular imprinting for the separation and
purification of wide range of molecules is quite apparent and make this
technique the ultimate in those areas.
(a) Enantioseparation of racemic mixtures

The first and the most important application of molecular imprinted
polymers is in the enantioseparation of racemic mixtures of chiral
compounds. This is because it provides a convenient method for
Separation factor ( template) = Kmip /Knip

MIPs possessing high separation factors should be capable of completely
recovering the target molecule by the simple process of stirring the
imprinted polymers with template solution. In general, the values of
separation factor are high when ionic interactions are utilised between the
template and the functional monomers. However, when non-covalent
interactions are employed, comparatively less values in the range one to
two is obtained. The selectivity of the imprinted polymer can also be
expressed as selectivity factor56, 94.
quantitative assessment of the quality of imprints produced by a particular

recipe or strategy. Most work has concentrated on the resolution of chiral
compounds. This reflects both the importance of this procedure in
analytical and synthetic chemistry and also the ease of distinguishing
between specific and non-specific recognition in this case. If the polymer is
imprinted with L-enantiomer of an amino acid, the MIP will retain the
L-enantiomer more than D-enantiomer and vice-versa, whereas the column
containing non-imprinted polymer will not be able to separate the
enantiomers. The typical values of enantioseparation factor () are between
Selectivity factor = template / comp
1.5 and 5, although in some cases much higher values are obtained.
Cinchona alkaloids cinchonidine and cinchonine showed selectivity with
Chapter 2
36
35
values upto 3195. By adopting capillary electrochromatographic technique,

in combination with MIPs appreciable resolution and separation factors can
and also for pre-concentration of dilute samples.
(c)
be achieved96.
(b)
product recovery from fermentation broths or production waste streams,
Binding assays
Since MIPs share with antibodies one of their most important
Solid phase extraction

The SPE technique has been most intensively studied from 1998
onwards with respect to the possible use of imprinted materials for the
extraction of significant materials97-99. The advantages of SPE over liquidliquid extraction (LLE) are that it is faster and more reproducible. The
MIP-SPE has been used to extract the target analyte from blood plasma and
serum100, urine101, bile102, chewing gum99, sediment103, diesel104 and plant
tissue105. The quantification of the herbicide atrazine in beef liver is a good
demonstrative example of the utility of imprinted polymers in SPE98. The
features, the ability to bind a target molecule selectively, they could

conceivably be employed in immunoassay type binding assays in place of
antibodies. This was first demonstrated by Mosbachs group, who
developed MIP based assay for the bronchodilator theophylline and the
tranquilizer diazepam6. The assay yielded a cross reactivity profile very
similar to that of the natural monoclonal antibodies. Later imprints against
morphine110, glycosides111, and propanolol112 have all shown very high
affinity and selectivity for the template.
MIPs are also used in other separation techniques such as molecularly
Molecular imprinting based on non-covalent interactions can also
imprinted solid phase extraction-differential pulse elution106 (MISPE-DPE),
be used to produce catalytic polymers. Polymers that mimic the
floatation
hydrolytication of proteases on amino acid esters have been prepared from
fractionation108. Alternatively, finely ground imprinted polymer mixed with
imidazole monomers113. Recently other approaches have been explored to
binders are spread on a support has been investigated to use in thin layer
improve enzyme mimicking polymers. Thus antibodies prepared by
chromatography109.
imprinting the transition state analogue, p-nitrophenyl methyl phosphonate
membrane
based
separations107 and
adsorptive
bubble
MIPs could also be used as adsorbents, stirred with a large volume

of liquid before collection by filtration. This approach might be suitable for
against a phosphonic ester for alkaline ester hydrolysis, enhanced the rate
of ester hydrolysis by 103-104 fold114. The enhancement is due to the
preferred binding of the transition state of the reaction.
Chapter 2
36
35
values upto 3195. By adopting capillary electrochromatographic technique,

in combination with MIPs appreciable resolution and separation factors can
and also for pre-concentration of dilute samples.
(c)
be achieved96.
(b)
product recovery from fermentation broths or production waste streams,
Binding assays
Since MIPs share with antibodies one of their most important
Solid phase extraction

The SPE technique has been most intensively studied from 1998
onwards with respect to the possible use of imprinted materials for the
extraction of significant materials97-99. The advantages of SPE over liquidliquid extraction (LLE) are that it is faster and more reproducible. The
MIP-SPE has been used to extract the target analyte from blood plasma and
serum100, urine101, bile102, chewing gum99, sediment103, diesel104 and plant
tissue105. The quantification of the herbicide atrazine in beef liver is a good
demonstrative example of the utility of imprinted polymers in SPE98. The
features, the ability to bind a target molecule selectively, they could

conceivably be employed in immunoassay type binding assays in place of
antibodies. This was first demonstrated by Mosbachs group, who
developed MIP based assay for the bronchodilator theophylline and the
tranquilizer diazepam6. The assay yielded a cross reactivity profile very
similar to that of the natural monoclonal antibodies. Later imprints against
morphine110, glycosides111, and propanolol112 have all shown very high
affinity and selectivity for the template.
MIPs are also used in other separation techniques such as molecularly
Molecular imprinting based on non-covalent interactions can also
imprinted solid phase extraction-differential pulse elution106 (MISPE-DPE),
be used to produce catalytic polymers. Polymers that mimic the
floatation
hydrolytication of proteases on amino acid esters have been prepared from
fractionation108. Alternatively, finely ground imprinted polymer mixed with
imidazole monomers113. Recently other approaches have been explored to
binders are spread on a support has been investigated to use in thin layer
improve enzyme mimicking polymers. Thus antibodies prepared by
chromatography109.
imprinting the transition state analogue, p-nitrophenyl methyl phosphonate
membrane
based
separations107 and
adsorptive
bubble
MIPs could also be used as adsorbents, stirred with a large volume

of liquid before collection by filtration. This approach might be suitable for
against a phosphonic ester for alkaline ester hydrolysis, enhanced the rate
of ester hydrolysis by 103-104 fold114. The enhancement is due to the
preferred binding of the transition state of the reaction.
Chapter 2
38
37
(d) Polymeric sensors

One of the areas where specific recognition phenomena play a key
role is in sensor technology. Many sensors for environmental monitoring,
biomedical and food analysis relay on biomolecules such as antibodies or
view of the toxic effects of chloramphenicol, a broad spectrum antibiotic,

the molecular imprinted polymers of even this compound was developed
which serves as a model system for investigations.
(e)
Toxicant selective polymers
enzymes as the specific recognition elements. Due to the poor chemical and
Any compound that can cause toxicity to living species can be
physical stability of biomolecules, artificial receptors are gaining increasing
considered as toxicant. In that sense nicotine, theophylline and caffeine are
interest. Molecular imprinted polymers are having the advantage that the
toxicants. The toxicity differ from one compound to another. Like most
recognition sites are tailor made, and at the same time incorporated into a
organic pesticides, nicotine break down rapidly meaning that the highest
solid polymeric support. Taking into account the very high specificity that
hazard is to the applicator, birds and other wildlife present at the time of
can be obtained as well as the chemical and physical stability of imprinted
application.
polymers; there have been a number of attempts to construct chemical
sensors based on these materials as the recognition elements115-118. The
challenge currently facing those wishing to exploit the recognition properties
O
H
N
N
Nicotine
Theophylline
N
O
of MIPs in such devices is to develop a transducing mechanism to translate

the binding event into a measurable signal. Several MIP based sensing
Caffeine
systems have been proposed, including sensors utilising field effect
A drop of pure nicotine will kill a person the lethal dose being one mg /
devices119, conductometric measurements120, amperometric measurements121,
kg body weight. In fact nicotine can be used as a pesticide on crops124.
fluorescence measurements 122, 1123.
Theophylline is used as a bronchodilator but its toxicity continues to be a
Usually the template selected for molecular imprinting studies were

of either biological or environmental significance. This is the reason for the
release of enormous imprinted polymers of various types mentioned. In
commonly encountered clinical problem. Theophylline is iatro-genic and can

cause seizures and convulsions either by acute overdose or chronic use125, 126.
However major toxicity in children has been documented at lower
Chapter 2
38
37
(d) Polymeric sensors

One of the areas where specific recognition phenomena play a key
role is in sensor technology. Many sensors for environmental monitoring,
biomedical and food analysis relay on biomolecules such as antibodies or
view of the toxic effects of chloramphenicol, a broad spectrum antibiotic,

the molecular imprinted polymers of even this compound was developed
which serves as a model system for investigations.
(e)
Toxicant selective polymers
enzymes as the specific recognition elements. Due to the poor chemical and
Any compound that can cause toxicity to living species can be
physical stability of biomolecules, artificial receptors are gaining increasing
considered as toxicant. In that sense nicotine, theophylline and caffeine are
interest. Molecular imprinted polymers are having the advantage that the
toxicants. The toxicity differ from one compound to another. Like most
recognition sites are tailor made, and at the same time incorporated into a
organic pesticides, nicotine break down rapidly meaning that the highest
solid polymeric support. Taking into account the very high specificity that
hazard is to the applicator, birds and other wildlife present at the time of
can be obtained as well as the chemical and physical stability of imprinted
application.
polymers; there have been a number of attempts to construct chemical
sensors based on these materials as the recognition elements115-118. The
challenge currently facing those wishing to exploit the recognition properties
O
H
N
N
Nicotine
Theophylline
N
O
of MIPs in such devices is to develop a transducing mechanism to translate

the binding event into a measurable signal. Several MIP based sensing
Caffeine
systems have been proposed, including sensors utilising field effect
A drop of pure nicotine will kill a person the lethal dose being one mg /
devices119, conductometric measurements120, amperometric measurements121,
kg body weight. In fact nicotine can be used as a pesticide on crops124.
fluorescence measurements 122, 1123.
Theophylline is used as a bronchodilator but its toxicity continues to be a
Usually the template selected for molecular imprinting studies were

of either biological or environmental significance. This is the reason for the
release of enormous imprinted polymers of various types mentioned. In
commonly encountered clinical problem. Theophylline is iatro-genic and can

cause seizures and convulsions either by acute overdose or chronic use125, 126.
However major toxicity in children has been documented at lower
Chapter 2
40
39
concentrations127. The lethal dose of caffeine in adults is 150mg/kg weight128.

Caffeine serves as a form of pest control for many plants where, it causes the
insects to collapse from over-stimulation. Intake of higher doses at once causes
recognition sites moulded in the polymer mimic the binding sites of

natural antibodies in their interaction with the target antigen137.
Caffeine
imprinting
by
phase
inversion
technique
using
mental disorder with symptoms such as restlessness, nervousness or
polyacrylonitrile, pyridine and styrene employing QCM recognition assay,
excitement. It also leads to reduction in sperm movement as well as birth
imprinting by one step, in-situ free-radical polymerisation within the
defects129. Chronic toxicity may affect functional aspects of every organ130.
chromatographic column138 and imprinting on cellulose/silica composite139
Attempts were done previously to imprint nicotine in polymers and to

use them in various purposes where molecular imprinted polymers are of
interest. Analysis by solid-phase extraction using chromatographic HPLC
technique74 and quartz crystalshear mode TSM bio-mimetic sensor131 was
developed using molecular imprinted polymers. By using 2-(trifluoromethyl)
were reported. Selective solid phase extraction sorbent
140-142
as well as
biomimetic piezoelectric quartz sensor143 was also made for caffeine by

molecular imprinting. Surface plasmon resonance (SPR) technique using
molecular imprinted polymers was used to detect theophylline as well as
caffeine simultaneously144.
acrylic acid as the functional monomer the selectivity was found to be high94.
Selectivity is determined by steric exclusion, the trend that appears
Attempts were done even to remove nicotine from cigarette smoke using
to be in accord with other cross-reactivity studies on MIP in the
molecular imprinted polymers as substitute for filter tip132.
literature145-147.
Theophylline recognition copolymer membranes were prepared by
Most of the reported techniques especially that employed for the
phase-inversion method in molecular imprinting with acrylic acid segments as
imprinting of nicotine, theophylline and caffeine were complex and costly,
functional sites133 as well as by acrylonitrile-acrylic acid copolymerisation134.
which required sophisticated instruments, skilled technicians and large
High Performance Liquid Chromatography (HPLC) method has been applied
laboratory based instrumentation. An attempt is made here to optimise the
commonly to determine theophylline and caffeine simultaneously135, 136. The
rebinding conditions and to design polymers with the optimum specificity

and selectivity employing the simple UV-spectroscopic technique.
Chapter 2
40
39
concentrations127. The lethal dose of caffeine in adults is 150mg/kg weight128.

Caffeine serves as a form of pest control for many plants where, it causes the
insects to collapse from over-stimulation. Intake of higher doses at once causes
recognition sites moulded in the polymer mimic the binding sites of

natural antibodies in their interaction with the target antigen137.
Caffeine
imprinting
by
phase
inversion
technique
using
mental disorder with symptoms such as restlessness, nervousness or
polyacrylonitrile, pyridine and styrene employing QCM recognition assay,
excitement. It also leads to reduction in sperm movement as well as birth
imprinting by one step, in-situ free-radical polymerisation within the
defects129. Chronic toxicity may affect functional aspects of every organ130.
chromatographic column138 and imprinting on cellulose/silica composite139
Attempts were done previously to imprint nicotine in polymers and to

use them in various purposes where molecular imprinted polymers are of
interest. Analysis by solid-phase extraction using chromatographic HPLC
technique74 and quartz crystalshear mode TSM bio-mimetic sensor131 was
developed using molecular imprinted polymers. By using 2-(trifluoromethyl)
were reported. Selective solid phase extraction sorbent
140-142
as well as
biomimetic piezoelectric quartz sensor143 was also made for caffeine by

molecular imprinting. Surface plasmon resonance (SPR) technique using
molecular imprinted polymers was used to detect theophylline as well as
caffeine simultaneously144.
acrylic acid as the functional monomer the selectivity was found to be high94.
Selectivity is determined by steric exclusion, the trend that appears
Attempts were done even to remove nicotine from cigarette smoke using
to be in accord with other cross-reactivity studies on MIP in the
molecular imprinted polymers as substitute for filter tip132.
literature145-147.
Theophylline recognition copolymer membranes were prepared by
Most of the reported techniques especially that employed for the
phase-inversion method in molecular imprinting with acrylic acid segments as
imprinting of nicotine, theophylline and caffeine were complex and costly,
functional sites133 as well as by acrylonitrile-acrylic acid copolymerisation134.
which required sophisticated instruments, skilled technicians and large
High Performance Liquid Chromatography (HPLC) method has been applied
laboratory based instrumentation. An attempt is made here to optimise the
commonly to determine theophylline and caffeine simultaneously135, 136. The
rebinding conditions and to design polymers with the optimum specificity

and selectivity employing the simple UV-spectroscopic technique.
Chapter 2
42
41
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645.
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Nicholls, I. A.; Rosengren, J. P. Bioseparation 2001, 10, 301
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Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495
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17
Fisher,E. Ber. Dtsch. Chem. Ges. 1894, 27, 2985.
18
Wulff, G. Angew. Chem., Int. Ed. Eng. 1995, 34, 1812.
19
Wulff, G.; Vietmeier, J. Makromol. Chem. 1989, 190, 1717
20
Wulff, G.; Vesper, W.; Grobe E. R.; Sarhan, A. Macromol. Chem. 1977, 178,
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Cormack, P. A. G.; Elorza A. Z. (Rev.) J. Chromatogr. B 2004, 804, 173
28
Mosbach, K.; Ramson, O. Biotechnology 1996, 14, 163
29
Wulff. G. Angew. Chem. Int. Ed. Engl. 1995, 34, 1812
30
2799
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32
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34
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43
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54
Anderson, H. S.; Koch Schmidt, A. C.; Ohison, S.; Mosbach, K. J. Mol.

Recogn. 1996, 9, 675
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42
53
55
Andersson, H. S.; Karlsson, J. G.; Piletsky, S. A.; Schmidt, K.; Mosbach, K.;
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56
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58
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62
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Molecular Imprinted Polymers Review

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Molecular Imprinted Polymers Review

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Chapter 2

MOLECULAR IMPRINTED POLYMERS: A REVIEW

synthesis of polymeric materials with predetermined ligand selectivity8-11.

receptors the ability to recognize and bind specific target molecules.

monomers are copolymerised in the presence of a target analyte (imprint

Scheme II. 1. Principle of molecular imprinting15

MOLECULAR IMPRINTED POLYMERS: A REVIEW

synthesis of polymeric materials with predetermined ligand selectivity8-11.

receptors the ability to recognize and bind specific target molecules.

monomers are copolymerised in the presence of a target analyte (imprint

Scheme II. 1. Principle of molecular imprinting15

between the functional monomer and the template is covalent, polar

polymerisation. This assumption postulates the formation of a cavity with

The imprinting efficiency is very high in non-covalent imprinting,

functional groups in complimentary array for the convergent interactions

with the template. The relationship of template to the imprinted cavity

mimics the antigen-antibody or enzyme-substrate interactions in

biomolecules. Noncovalent interactions involve hydrogen bonds, ionic,

hydrophobic, - interactions, induction, dispersion and van der Waals

2. 2. Approaches in molecular imprinting

polymerization (Scheme II. 2). The interactions between the pairs:

bonding interactions. Here a pre-polymerisation complex is formed, the

between the functional monomer and the template is covalent, polar

polymerisation. This assumption postulates the formation of a cavity with

The imprinting efficiency is very high in non-covalent imprinting,

functional groups in complimentary array for the convergent interactions

with the template. The relationship of template to the imprinted cavity

mimics the antigen-antibody or enzyme-substrate interactions in

biomolecules. Noncovalent interactions involve hydrogen bonds, ionic,

hydrophobic, - interactions, induction, dispersion and van der Waals

2. 2. Approaches in molecular imprinting

polymerization (Scheme II. 2). The interactions between the pairs:

bonding interactions. Here a pre-polymerisation complex is formed, the

graft polymerisation and precipitation polymerisation30,

polymerisation the beads obtained are of diameter 5-50 m depending on

crosslinking of the polymer, and there is no need of any extra stabiliser.

can be synthesised in a variety of physical forms. Chronologically, the first

membrane36. They can also be synthesised in situ at the electrode surface

polymerisation method employed to synthesise MIP was based on bulk

by electropolymerisation37 or at a non-conducting surface by chemical

followed by mechanical grinding to obtain small

micrometer-sized particles suitable to use in chromatographic purposes.

graft polymerisation and precipitation polymerisation30,

polymerisation the beads obtained are of diameter 5-50 m depending on

crosslinking of the polymer, and there is no need of any extra stabiliser.

can be synthesised in a variety of physical forms. Chronologically, the first

membrane36. They can also be synthesised in situ at the electrode surface

polymerisation method employed to synthesise MIP was based on bulk

by electropolymerisation37 or at a non-conducting surface by chemical

followed by mechanical grinding to obtain small

micrometer-sized particles suitable to use in chromatographic purposes.

Attempts to synthesise imprinted polymers, which can be operated in

aqueous medium, are in the preliminary stage40. Metal ion imprinted

strong as possible during the polymerisation to enable the binding group to

polymeric beads are successfully applied in analytical applications41. One

be fixed by the template in a definite orientation on the polymer chain

of the first reports of imprinting of bacterial cells on the polymer surface

during crosslinking. The template should be able to be removed as

was also observed42. Computational design and synthesis of imprinted

polymers, with high binding capacity for pharmaceutical applications is

should be very &ast and reversible.

Factors influencing the characteristics of the imprinted polymers

imprinting. The specific structure of the cavity is determined not only by