MOLECULAR IMPRINTED POLYMERS: A REVIEW

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 tailor-

made 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

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 pre-

polymerisation 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

12 Chapter 2

synthesis of polymeric materials with predetermined ligand selectivity8-11.

The MIPs possess two of the most important features of the biological

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.

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

MOLECULAR IMPRINTED POLYMERS: A REVIEW

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 tailor-

made 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

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 pre-

polymerisation 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

12 Chapter 2

synthesis of polymeric materials with predetermined ligand selectivity8-11.

The MIPs possess two of the most important features of the biological

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.

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

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

polymerisation. This assumption postulates the formation of a cavity with

functional groups in complimentary array for the convergent interactions

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

corresponds to the lock and key principle proposed by Emil Fisher for

enzyme catalysis17.

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

14 Chapter 2

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.

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

which has been pioneered by Mosbach and co-workers6, 22. This method

mimics the antigen-antibody or enzyme-substrate interactions in

biomolecules. Noncovalent interactions involve hydrogen bonds, ionic,

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

forces; the most important being electrostatic, ion pairing and hydrogen

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.

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

polymerisation. This assumption postulates the formation of a cavity with

functional groups in complimentary array for the convergent interactions

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

corresponds to the lock and key principle proposed by Emil Fisher for

enzyme catalysis17.

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

14 Chapter 2

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.

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

which has been pioneered by Mosbach and co-workers6, 22. This method

mimics the antigen-antibody or enzyme-substrate interactions in

biomolecules. Noncovalent interactions involve hydrogen bonds, ionic,

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

forces; the most important being electrostatic, ion pairing and hydrogen

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.

Review of Literature 15

Scheme II. 2. Principle behind non-covalent and covalent imprinting

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

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

polymerisation method employed to synthesise MIP was based on bulk

polymerisation28, 29 followed by mechanical grinding to obtain small

micrometer-sized particles suitable to use in chromatographic purposes.

The polymer beads of controlled diameter can be prepared directly by

alternate methods such as suspension polymerisation, seed polymerisation,

16 Chapter 2

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,

particles are prevented from coalescence by the rigidity obtained from the

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

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

by electropolymerisation37 or at a non-conducting surface by chemical

grafting38.

One of the many attractive features of the molecular imprinting

method is that it can be applied to a diverse range of analytes, even though

it is a problem for large molecules. A diverse range of substances including

Review of Literature 15

Scheme II. 2. Principle behind non-covalent and covalent imprinting

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

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

polymerisation method employed to synthesise MIP was based on bulk

polymerisation28, 29 followed by mechanical grinding to obtain small

micrometer-sized particles suitable to use in chromatographic purposes.

The polymer beads of controlled diameter can be prepared directly by

alternate methods such as suspension polymerisation, seed polymerisation,

16 Chapter 2

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,

particles are prevented from coalescence by the rigidity obtained from the

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

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

by electropolymerisation37 or at a non-conducting surface by chemical

grafting38.

One of the many attractive features of the molecular imprinting

method is that it can be applied to a diverse range of analytes, even though

it is a problem for large molecules. A diverse range of substances including

Review of Literature 17

sugars, polymers and amino acid derivatives are already imprinted. Novel

imaging strategies using fluorescently labelled template was also done39.

Attempts to synthesise imprinted polymers, which can be operated in

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

polymeric beads are successfully applied in analytical applications41. One

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

was also observed42. Computational design and synthesis of imprinted

polymers, with high binding capacity for pharmaceutical applications is

reported as a model case. Special protocols have been proposed for the

imprinting of proteins43.

The templates are classified as convex and concave where metal ion

involving cyclisation is an example for convex template and enzyme is an

example for concave. The substrate has to fit in the cavity created by the

enzyme to undergo chemical transformation. Polymers are also categorised

as reactive (catalytic) or non-reactive (non-catalytic) where the reaction rate

is accelerated or unaffected.

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

18 Chapter 2

rebinding. The best results are obtained when the templates get attached to

more than one binding site.

The bond between the template and the binding group should be as

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

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

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

completely as possible. The interaction of the binding site with the template

should be very &ast and reversible.

2.4. Factors influencing the characteristics of the imprinted polymers

The structure of the polymer matrix is crucial in molecular

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

the low molecular weight template molecules but also by the fixed

arrangement of the polymer chains. The characteristics of the polymer

matrix like the matrix structure, matrix configuration, matrix nature,

template used and the extent of crosslinking has a significant effect on

rebinding. Hence the optimisation of the polymer structure is extremely

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

Review of Literature 17

sugars, polymers and amino acid derivatives are already imprinted. Novel

imaging strategies using fluorescently labelled template was also done39.

Attempts to synthesise imprinted polymers, which can be operated in

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

polymeric beads are successfully applied in analytical applications41. One

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

was also observed42. Computational design and synthesis of imprinted

polymers, with high binding capacity for pharmaceutical applications is

reported as a model case. Special protocols have been proposed for the

imprinting of proteins43.

The templates are classified as convex and concave where metal ion

involving cyclisation is an example for convex template and enzyme is an

example for concave. The substrate has to fit in the cavity created by the

enzyme to undergo chemical transformation. Polymers are also categorised

as reactive (catalytic) or non-reactive (non-catalytic) where the reaction rate

is accelerated or unaffected.

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

18 Chapter 2

rebinding. The best results are obtained when the templates get attached to

more than one binding site.

The bond between the template and the binding group should be as

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

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

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

completely as possible. The interaction of the binding site with the template

should be very &ast and reversible.

2.4. Factors influencing the characteristics of the imprinted polymers

The structure of the polymer matrix is crucial in molecular

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

the low molecular weight template molecules but also by the fixed

arrangement of the polymer chains. The characteristics of the polymer

matrix like the matrix structure, matrix configuration, matrix nature,

template used and the extent of crosslinking has a significant effect on

rebinding. Hence the optimisation of the polymer structure is extremely

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

Review of Literature 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

method. Functional groups are statistically distributed over a

macromolecule so that both the more favourable and less favourable

conformations will occur.

(a) Rigidity/ Flexibility of the polymer support The stiffness of the polymer structure enables the cavities to retain

their shape even after the removal of the template, thus giving higher

selectivity. This can be achieved by high degree of crosslinking. The

binding of the template into the polymer during polymerisation, directs the

positioning of the imprinted substrate within the cavities of the crosslinked

polymer and it is assumed that the polymers with optimum selectivity and

specificity characteristics were obtained by suitable selection of 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

20 Chapter 2

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

flexibility will show kinetic hindrance to reversible binding. The studies on

macroporous styrene-divinylbenzene46, 47 and styrene-diisopropylbenzene48, 49

co-polymer by Shea et al revealed that, relatively rigid, hydrophobic and

highly crosslinked materials provides chemical inertness needed to

withstand subsequent chemical transformations. Thus the molecular

imprinted polymers should have optimum stiffness/flexibility to attain the

rapid equilibrium with the template, imprinted cavities accessible for the

template, mechanical stability to withstand stress in certain applications and

thermal stability to use at high temperature.

(b) Functional and crosslinking monomers 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,

Review of Literature 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

method. Functional groups are statistically distributed over a

macromolecule so that both the more favourable and less favourable

conformations will occur.

(a) Rigidity/ Flexibility of the polymer support The stiffness of the polymer structure enables the cavities to retain

their shape even after the removal of the template, thus giving higher

selectivity. This can be achieved by high degree of crosslinking. The

binding of the template into the polymer during polymerisation, directs the

positioning of the imprinted substrate within the cavities of the crosslinked

polymer and it is assumed that the polymers with optimum selectivity and

specificity characteristics were obtained by suitable selection of 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

20 Chapter 2

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

flexibility will show kinetic hindrance to reversible binding. The studies on

macroporous styrene-divinylbenzene46, 47 and styrene-diisopropylbenzene48, 49

co-polymer by Shea et al revealed that, relatively rigid, hydrophobic and

highly crosslinked materials provides chemical inertness needed to

withstand subsequent chemical transformations. Thus the molecular

imprinted polymers should have optimum stiffness/flexibility to attain the

rapid equilibrium with the template, imprinted cavities accessible for the

template, mechanical stability to withstand stress in certain applications and

thermal stability to use at high temperature.

(b) Functional and crosslinking monomers 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,

Review of Literature 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

template molecule in polar solvents.

(a) (b) (c)

(d) (e) (f) (g)

(a) methacrylic acid (b) trifluoromethacrylic acid (c) N-vinylimidazole (d) 4-vinyl pyridine (e) 2-vinyl pyridine (f) hydroxyethyl methacrylate (g) acrylamide

Fig. II. 1. Functional monomers commonly used in molecular imprinting

For molecular imprinting in organic solvents, the crosslinking

agents EGDMA and DVB are often used. A typical water-soluble

22 Chapter 2

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

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

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

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

wettability. In TEGDMA-crosslinked polymer the hydrophilic nature of the

crosslinking agent caused increased availability of binding sites for

template. By choosing the appropriate crosslinking agent, random

Review of Literature 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

template molecule in polar solvents.

(a) (b) (c)

(d) (e) (f) (g)

(a) methacrylic acid (b) trifluoromethacrylic acid (c) N-vinylimidazole (d) 4-vinyl pyridine (e) 2-vinyl pyridine (f) hydroxyethyl methacrylate (g) acrylamide

Fig. II. 1. Functional monomers commonly used in molecular imprinting

For molecular imprinting in organic solvents, the crosslinking

agents EGDMA and DVB are often used. A typical water-soluble

22 Chapter 2

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

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

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

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

wettability. In TEGDMA-crosslinked polymer the hydrophilic nature of the

crosslinking agent caused increased availability of binding sites for

template. By choosing the appropriate crosslinking agent, random

Review of Literature 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

imprinting efficiency is damaged, especially when the crosslinking agents

show non-covalent interactions with functional monomers and/or

templates. Usually, a crosslinking agent is taken in 20-fold excess to the

functional monomer and its concentration in the reaction mixture is 70-

90%. At low degree of crosslinking, the selectivity is poor since the

polymer matrix is not crosslinked enough to retain the shape of the cavity.

In deciding a crosslinking agent its good solubility in a pre-polymerisation

mixture is also taken into account. For large templates such as proteins, the

crosslinking agents having optimal length such as TTEGDMA or PEG 400

DMA are more suitable52. However, these findings suggest that the

crosslinking monomers are an active part of imprinting, rather than merely

inert scaffolding for functional monomers. The most frequently used

crosslinking agents53 are exhibited (Fig. II. 2).

24 Chapter 2

(a) (b) (c) (d)

O

OO

O3

(e) (f)

2

O

HN

O

NH

(g) (h)

(a) trimethylpropane trimethacrylate (TRIM), (b) divinylbenzene (DVB), (c) 2, 6-bisacryloylamidopyridine, (d) N, O - bisacryloyl-L- phenylalaninol, (e) ethylene glycol dimethacrylate (EGDMA), (f) triethylene glycol dimethacrylate (TEGDMA), (g) butanediol dimethacrylate (BDDMA) and (h) N, N'- methylene bis acrylamide (NNMBA)

Fig. II. 2. Frequently used crosslinking monomers in molecular imprinting

Review of Literature 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

imprinting efficiency is damaged, especially when the crosslinking agents

show non-covalent interactions with functional monomers and/or

templates. Usually, a crosslinking agent is taken in 20-fold excess to the

functional monomer and its concentration in the reaction mixture is 70-

90%. At low degree of crosslinking, the selectivity is poor since the

polymer matrix is not crosslinked enough to retain the shape of the cavity.

In deciding a crosslinking agent its good solubility in a pre-polymerisation

mixture is also taken into account. For large templates such as proteins, the

crosslinking agents having optimal length such as TTEGDMA or PEG 400

DMA are more suitable52. However, these findings suggest that the

crosslinking monomers are an active part of imprinting, rather than merely

inert scaffolding for functional monomers. The most frequently used

crosslinking agents53 are exhibited (Fig. II. 2).

24 Chapter 2

(a) (b) (c) (d)

O

OO

O3

(e) (f)

2

O

HN

O

NH

(g) (h)

(a) trimethylpropane trimethacrylate (TRIM), (b) divinylbenzene (DVB), (c) 2, 6-bisacryloylamidopyridine, (d) N, O - bisacryloyl-L- phenylalaninol, (e) ethylene glycol dimethacrylate (EGDMA), (f) triethylene glycol dimethacrylate (TEGDMA), (g) butanediol dimethacrylate (BDDMA) and (h) N, N'- methylene bis acrylamide (NNMBA)

Fig. II. 2. Frequently used crosslinking monomers in molecular imprinting

Review of Literature 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

yield non-selective binding the extreme case being the non-imprinted

polymer58. The parameter imprinting effect gives an estimation of how

well the polymer was imprinted by the template59. This cannot be

considered as a property of the stationary phase, but only a measure of the

net molecular recognition effect due to the imprinting process in well-

defined experimental conditions. The excess of template during

polymerisation is unfavourable towards selectivity. Thus excess nicotine

favours the formation of complex states II and III over ideal IV (Fig. II. 3).

26 Chapter 2

Fig. II. 3. Hypothetical model of the anticipated methacrylic acidnicotine complex states in the pre-polymerisation complex55.

An excess of monomer on the other hand would favour the opposite

situation where, the complexes formed will be of the type IV, but again

excess monomer would yield high number of non-complexed randomly

distributed monomer which contribute to non-specific binding60. As

selectivity is considered to arise from the pre-polymerisation of monomer

by the template prior to polymerisation, we should expect complex state IV

to be the one that is responsible for the production of higher affinity, more

selective, binding sites. States I to III would lead to the formation of site

Review of Literature 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

yield non-selective binding the extreme case being the non-imprinted

polymer58. The parameter imprinting effect gives an estimation of how

well the polymer was imprinted by the template59. This cannot be

considered as a property of the stationary phase, but only a measure of the

net molecular recognition effect due to the imprinting process in well-

defined experimental conditions. The excess of template during

polymerisation is unfavourable towards selectivity. Thus excess nicotine

favours the formation of complex states II and III over ideal IV (Fig. II. 3).

26 Chapter 2

Fig. II. 3. Hypothetical model of the anticipated methacrylic acidnicotine complex states in the pre-polymerisation complex55.

An excess of monomer on the other hand would favour the opposite

situation where, the complexes formed will be of the type IV, but again

excess monomer would yield high number of non-complexed randomly

distributed monomer which contribute to non-specific binding60. As

selectivity is considered to arise from the pre-polymerisation of monomer

by the template prior to polymerisation, we should expect complex state IV

to be the one that is responsible for the production of higher affinity, more

selective, binding sites. States I to III would lead to the formation of site

Review of Literature 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

used in imprinting studies.

The rate of radical polymerisation depends on the nature and

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.

(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-

28 Chapter 2

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

of the network structure and porosity.

It is believed that solvents with low permittivities (toluene,

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.

(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

Review of Literature 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

used in imprinting studies.

The rate of radical polymerisation depends on the nature and

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.

(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-

28 Chapter 2

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

of the network structure and porosity.

It is believed that solvents with low permittivities (toluene,

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.

(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

Review of Literature 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 pre-

polymerisation 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

after rebinding can also put light into rebinding.

(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 pre-

polymerisation solutions and as a means of identifying the specific sites in

30 Chapter 2

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

diffraction methods can be applied successfully to follow rebinding with

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.

Review of Literature 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 pre-

polymerisation 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

after rebinding can also put light into rebinding.

(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 pre-

polymerisation solutions and as a means of identifying the specific sites in

30 Chapter 2

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

diffraction methods can be applied successfully to follow rebinding with

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.

Review of Literature 31

(d) Scanning electron microscopy (SEM) SEM can be used in a variety of distinct ways to probe imprinted

polymers on a variety of length scales. Scanning electron microscopy is the

most widely used technique to study the shape, size, morphology and

porosity of polymers75, 76.

(e) Pore analysis It is possible to probe the morphology of imprinted polymers in

much the same way as one is able to do with most porous solids.

Depending on the method of analysis, useful information may be gleaned

on the specific pore volumes, pore sizes, pore size distributions and specific

surface area of materials. Pore and surface area analysis was performed by

N2 adsorption. The surface area was determined using the BET model, the

t-plot using Harkin-Jura average thickness equation and the pore volume

and pore size distribution using the BJH model77.

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%

32 Chapter 2

rebinding reaction very effectively. The extent of swelling can be

determined in terms of change in weight80, 81. Alte2native,y, by packing a

definite weight of the polymer in a capillary tube and measuring the

volume before and after incubation in the solvent, the swelling ratio can be

determined in terms of change in volume 82.

2.7. Selectivity parameters of molecular imprinted polymers

The binding parameters of the MIPs are usually estimated from

adsorption isotherms74, 83, 84 using mathematical models. One strategy to

perform binding performance is based on saturation experiments and

subsequent Scatchard analysis85-88. The obtained binding data were

transformed into linear form and analysed to create Scatchard plots based

on Scatchard equation

[S]b / [S]f = (Smax [S]b) / KD

where, KD is an equilibrium dissociation constant, Smax an apparent

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

Review of Literature 31

(d) Scanning electron microscopy (SEM) SEM can be used in a variety of distinct ways to probe imprinted

polymers on a variety of length scales. Scanning electron microscopy is the

most widely used technique to study the shape, size, morphology and

porosity of polymers75, 76.

(e) Pore analysis It is possible to probe the morphology of imprinted polymers in

much the same way as one is able to do with most porous solids.

Depending on the method of analysis, useful information may be gleaned

on the specific pore volumes, pore sizes, pore size distributions and specific

surface area of materials. Pore and surface area analysis was performed by

N2 adsorption. The surface area was determined using the BET model, the

t-plot using Harkin-Jura average thickness equation and the pore volume

and pore size distribution using the BJH model77.

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%

32 Chapter 2

rebinding reaction very effectively. The extent of swelling can be

determined in terms of change in weight80, 81. Alte2native,y, by packing a

definite weight of the polymer in a capillary tube and measuring the

volume before and after incubation in the solvent, the swelling ratio can be

determined in terms of change in volume 82.

2.7. Selectivity parameters of molecular imprinted polymers

The binding parameters of the MIPs are usually estimated from

adsorption isotherms74, 83, 84 using mathematical models. One strategy to

perform binding performance is based on saturation experiments and

subsequent Scatchard analysis85-88. The obtained binding data were

transformed into linear form and analysed to create Scatchard plots based

on Scatchard equation

[S]b / [S]f = (Smax [S]b) / KD

where, KD is an equilibrium dissociation constant, Smax an apparent

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

Review of Literature 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

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.

Selectivity factor = template / comp

34 Chapter 2

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

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

1.5 and 5, although in some cases much higher values are obtained.

Cinchona alkaloids cinchonidine and cinchonine showed selectivity with

Review of Literature 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

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.

Selectivity factor = template / comp

34 Chapter 2

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

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

1.5 and 5, although in some cases much higher values are obtained.

Cinchona alkaloids cinchonidine and cinchonine showed selectivity with

Review of Literature 35

values upto 3195. By adopting capillary electrochromatographic technique,

in combination with MIPs appreciable resolution and separation factors can

be achieved96.

(b) 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 liquid-

liquid 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

MIPs are also used in other separation techniques such as molecularly

imprinted solid phase extraction-differential pulse elution106 (MISPE-DPE),

membrane based separations107 and adsorptive bubble floatation

fractionation108. Alternatively, finely ground imprinted polymer mixed with

binders are spread on a support has been investigated to use in thin layer

chromatography109.

MIPs could also be used as adsorbents, stirred with a large volume

of liquid before collection by filtration. This approach might be suitable for

36 Chapter 2

product recovery from fermentation broths or production waste streams,

and also for pre-concentration of dilute samples.

(c) Binding assays

Since MIPs share with antibodies one of their most important

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.

Molecular imprinting based on non-covalent interactions can also

be used to produce catalytic polymers. Polymers that mimic the

hydrolytication of proteases on amino acid esters have been prepared from

imidazole monomers113. Recently other approaches have been explored to

improve enzyme mimicking polymers. Thus antibodies prepared by

imprinting the transition state analogue, p-nitrophenyl methyl phosphonate

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.

Review of Literature 35

values upto 3195. By adopting capillary electrochromatographic technique,

in combination with MIPs appreciable resolution and separation factors can

be achieved96.

(b) 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 liquid-

liquid 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

MIPs are also used in other separation techniques such as molecularly

imprinted solid phase extraction-differential pulse elution106 (MISPE-DPE),

membrane based separations107 and adsorptive bubble floatation

fractionation108. Alternatively, finely ground imprinted polymer mixed with

binders are spread on a support has been investigated to use in thin layer

chromatography109.

MIPs could also be used as adsorbents, stirred with a large volume

of liquid before collection by filtration. This approach might be suitable for

36 Chapter 2

product recovery from fermentation broths or production waste streams,

and also for pre-concentration of dilute samples.

(c) Binding assays

Since MIPs share with antibodies one of their most important

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.

Molecular imprinting based on non-covalent interactions can also

be used to produce catalytic polymers. Polymers that mimic the

hydrolytication of proteases on amino acid esters have been prepared from

imidazole monomers113. Recently other approaches have been explored to

improve enzyme mimicking polymers. Thus antibodies prepared by

imprinting the transition state analogue, p-nitrophenyl methyl phosphonate

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.

Review of Literature 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

enzymes as the specific recognition elements. Due to the poor chemical and

physical stability of biomolecules, artificial receptors are gaining increasing

interest. Molecular imprinted polymers are having the advantage that the

recognition sites are tailor made, and at the same time incorporated into a

solid polymeric support. Taking into account the very high specificity that

can be obtained as well as the chemical and physical stability of imprinted

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

of MIPs in such devices is to develop a transducing mechanism to translate

the binding event into a measurable signal. Several MIP based sensing

systems have been proposed, including sensors utilising field effect

devices119, conductometric measurements120, amperometric measurements121,

fluorescence measurements 122, 1123.

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

38 Chapter 2

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

Any compound that can cause toxicity to living species can be

considered as toxicant. In that sense nicotine, theophylline and caffeine are

toxicants. The toxicity differ from one compound to another. Like most

organic pesticides, nicotine break down rapidly meaning that the highest

hazard is to the applicator, birds and other wildlife present at the time of

application.

N

N

HN

N

O

O

Theophylline

N

N

Nicotine

N

N

O

O N

N

Caffeine

A drop of pure nicotine will kill a person the lethal dose being one mg /

kg body weight. In fact nicotine can be used as a pesticide on crops124.

Theophylline is used as a bronchodilator but its toxicity continues to be a

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

Review of Literature 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

enzymes as the specific recognition elements. Due to the poor chemical and

physical stability of biomolecules, artificial receptors are gaining increasing

interest. Molecular imprinted polymers are having the advantage that the

recognition sites are tailor made, and at the same time incorporated into a

solid polymeric support. Taking into account the very high specificity that

can be obtained as well as the chemical and physical stability of imprinted

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

of MIPs in such devices is to develop a transducing mechanism to translate

the binding event into a measurable signal. Several MIP based sensing

systems have been proposed, including sensors utilising field effect

devices119, conductometric measurements120, amperometric measurements121,

fluorescence measurements 122, 1123.

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

38 Chapter 2

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

Any compound that can cause toxicity to living species can be

considered as toxicant. In that sense nicotine, theophylline and caffeine are

toxicants. The toxicity differ from one compound to another. Like most

organic pesticides, nicotine break down rapidly meaning that the highest

hazard is to the applicator, birds and other wildlife present at the time of

application.

N

N

HN

N

O

O

Theophylline

N

N

Nicotine

N

N

O

O N

N

Caffeine

A drop of pure nicotine will kill a person the lethal dose being one mg /

kg body weight. In fact nicotine can be used as a pesticide on crops124.

Theophylline is used as a bronchodilator but its toxicity continues to be a

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

Review of Literature 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

mental disorder with symptoms such as restlessness, nervousness or

excitement. It also leads to reduction in sperm movement as well as birth

defects129. Chronic toxicity may affect functional aspects of every organ130.

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)

acrylic acid as the functional monomer the selectivity was found to be high94.

Attempts were done even to remove nicotine from cigarette smoke using

molecular imprinted polymers as substitute for filter tip132.

Theophylline recognition copolymer membranes were prepared by

phase-inversion method in molecular imprinting with acrylic acid segments as

functional sites133 as well as by acrylonitrile-acrylic acid copolymerisation134.

High Performance Liquid Chromatography (HPLC) method has been applied

commonly to determine theophylline and caffeine simultaneously135, 136. The

40 Chapter 2

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

polyacrylonitrile, pyridine and styrene employing QCM recognition assay,

imprinting by one step, in-situ free-radical polymerisation within the

chromatographic column138 and imprinting on cellulose/silica composite139

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.

Selectivity is determined by steric exclusion, the trend that appears

to be in accord with other cross-reactivity studies on MIP in the

literature145-147.

Most of the reported techniques especially that employed for the

imprinting of nicotine, theophylline and caffeine were complex and costly,

which required sophisticated instruments, skilled technicians and large

laboratory based instrumentation. An attempt is made here to optimise the

rebinding conditions and to design polymers with the optimum specificity

and selectivity employing the simple UV-spectroscopic technique.

Review of Literature 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

mental disorder with symptoms such as restlessness, nervousness or

excitement. It also leads to reduction in sperm movement as well as birth

defects129. Chronic toxicity may affect functional aspects of every organ130.

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)

acrylic acid as the functional monomer the selectivity was found to be high94.

Attempts were done even to remove nicotine from cigarette smoke using

molecular imprinted polymers as substitute for filter tip132.

Theophylline recognition copolymer membranes were prepared by

phase-inversion method in molecular imprinting with acrylic acid segments as

functional sites133 as well as by acrylonitrile-acrylic acid copolymerisation134.

High Performance Liquid Chromatography (HPLC) method has been applied

commonly to determine theophylline and caffeine simultaneously135, 136. The

40 Chapter 2

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

polyacrylonitrile, pyridine and styrene employing QCM recognition assay,

imprinting by one step, in-situ free-radical polymerisation within the

chromatographic column138 and imprinting on cellulose/silica composite139

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.

Selectivity is determined by steric exclusion, the trend that appears

to be in accord with other cross-reactivity studies on MIP in the

literature145-147.

Most of the reported techniques especially that employed for the

imprinting of nicotine, theophylline and caffeine were complex and costly,

which required sophisticated instruments, skilled technicians and large

laboratory based instrumentation. An attempt is made here to optimise the

rebinding conditions and to design polymers with the optimum specificity

and selectivity employing the simple UV-spectroscopic technique.

Review of Literature 41

References

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2. Alexander, C.; Davidson, L.; Hayes, W. Tetrahedron 2003, 59, 2025

3. Haginaka, J. Ana.l Bioanal. Chem. 2004, 379, 332

4. Sellergren, B. Trends Anal. Chem. 1997, 16, 310

5. Mayers, A. G.; Mosbach, K. Trends Anal.Chem. 1997, 16, 321.

6. Vlatakis, G.; Andersson, L. I.; Muller, R.; Mosbach, K. Nature 1993, 361, 645.

7. Ramstrom, O.; Ye, L.; Mosbach, K. Chem .Biol. 1996, 3, 471

8. Wulff, G. Chem. Rev. 2002, 102, 2

9. Wulff, G.; Knorr, K. Bioseparation 2001, 10, 257

10. Nicholls, I. A.; Rosengren, J. P. Bioseparation 2001, 10, 301

11. Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495

12. Mosbach, K; Haupt, K. J. Mol. Recogn. 1998, 11, 62

13. Haupt, K. Chem. Commun. (Camb.) 2003, 2, 171

14. Kandimalla,V. B.; Ju, H. Anal. Bioanal. Chem. 2004, 380, 587.

15 Ye, L.; Mosbach, K. React. Funct. Polym. 2001, 48, 149

16 Takeuchi, T.; Matsui, J. Acta Polym. 1996, 47, 471

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, 2799

42 Chapter 2

21 Takeuchi, T.; Mukawa, T.; Matsui, J.; Higashi, M.; Shimizu, K.D. Anal. Chem. 2001, 73, 3869

22 Arshady, R.; Mosbach, K. Macromol. Chem. 1981, 182, 687

23 Jakusch, M.; Janota, M.; Mizaikoff, B.; Mosbach, K.; Haupt, K. Anal. Chem. 1999, 71, 4786

24 Sergeyeva, T. A.; Piletsky, S. A.; Panasyuk, T. L.; Elskaya, A. V.; Brovko, A. A.; Slinchenko, E. A.; Sergeeva, L. M. Analyst 1999, 124, 331

25 Baggiani, C.; Trotta, F.; Giraudi, G.; Giovannoli, C.; Vanni,A. Anal. Commun. 1999, 36, 263

26 Klein, J. U.; Whitcombe, M. J.; Mulholland F.; Vulson, E. N. Angew. Chem., Int. Ed. Engl.1999, 38, 2057.

27 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 Hosoya, K.; Yoshihako, K.; Shirasu,Y.; Kimata, K.; Araki,T.; Tanaka, N.; Haginaka, J. J. Chromatogr. A 1996, 728, 139.

31 Mayes, A. G.; Mosbach, K. Anal. Chem. 1996, 68, 3769

32 Ansell, R. J.; Mosbach, K. Analyst 1998, 123, 1611

33 Sellergren, B. J. Chromatogr. A 1994, 673, 133.

34 Schweitz, L.; Andersson, L. I.; Nilsson, S. Anal. Chem. 1997, 69, 1179

35 Mathew K. J.; Shea, K. J. J. Am. Chem. Soc. 1996, 118, 8154.

36 Hong, J. M.; Andersson, P. E.; Qian, J.; Martin, C. E. Chem. Mater. 1998, 10, 1029

37 Peng, H.; Liang, C.; Zhou, A.; Zhang, Y.; Xie, Q.; Yao, S. Anal. Chim. Acta 2000, 423, 221

Review of Literature 41

References

1. Haupt, K. Anal. Chem. 2003, 75, 376A.

2. Alexander, C.; Davidson, L.; Hayes, W. Tetrahedron 2003, 59, 2025

3. Haginaka, J. Ana.l Bioanal. Chem. 2004, 379, 332

4. Sellergren, B. Trends Anal. Chem. 1997, 16, 310

5. Mayers, A. G.; Mosbach, K. Trends Anal.Chem. 1997, 16, 321.

6. Vlatakis, G.; Andersson, L. I.; Muller, R.; Mosbach, K. Nature 1993, 361, 645.

7. Ramstrom, O.; Ye, L.; Mosbach, K. Chem .Biol. 1996, 3, 471

8. Wulff, G. Chem. Rev. 2002, 102, 2

9. Wulff, G.; Knorr, K. Bioseparation 2001, 10, 257

10. Nicholls, I. A.; Rosengren, J. P. Bioseparation 2001, 10, 301

11. Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495

12. Mosbach, K; Haupt, K. J. Mol. Recogn. 1998, 11, 62

13. Haupt, K. Chem. Commun. (Camb.) 2003, 2, 171

14. Kandimalla,V. B.; Ju, H. Anal. Bioanal. Chem. 2004, 380, 587.

15 Ye, L.; Mosbach, K. React. Funct. Polym. 2001, 48, 149

16 Takeuchi, T.; Matsui, J. Acta Polym. 1996, 47, 471

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, 2799

42 Chapter 2

21 Takeuchi, T.; Mukawa, T.; Matsui, J.; Higashi, M.; Shimizu, K.D. Anal. Chem. 2001, 73, 3869

22 Arshady, R.; Mosbach, K. Macromol. Chem. 1981, 182, 687

23 Jakusch, M.; Janota, M.; Mizaikoff, B.; Mosbach, K.; Haupt, K. Anal. Chem. 1999, 71, 4786

24 Sergeyeva, T. A.; Piletsky, S. A.; Panasyuk, T. L.; Elskaya, A. V.; Brovko, A. A.; Slinchenko, E. A.; Sergeeva, L. M. Analyst 1999, 124, 331