Molecular Imprinted Polymers: A Reviewshodhganga.inflibnet.ac.in/bitstream/10603/22423/... · MOLECULAR IMPRINTED POLYMERS: A REVIEW 2. 1. Introduction Molecular imprinting is a generic

Molecular Imprinted Polymers: A Review Chapter 2

MOLECULAR IMPRINTED POLYMERS: A REVIEW

2. 1. Introduction

Molecular imprinting is a generic technology that allows the introduction

of sites of specific molecular affinity into three-dimensional crosslinked

polymeric matrices. Specific molecular recognition is the fundamental process

governing control of biological form and function. In the past decade, great

interest in this new class of materials - molecular imprinted polymers (MIPs) - has

been expressed by specialists in various branches of chemistry1. This can be

explained by the presence of highly specific binding sites in MIPs, which are

complementary to various organic molecules in size, shape, structure, and

physicochemical properties. The potentially high selectivity of MIPs for organic

compounds, along with other useful properties opens up wide opportunities for

the use of these materials in analytical chemistry2-4. Molecular imprinted

polymers (MIPs) are a group of compounds synthesised by a process where

functional and crosslinking monomers are co-polymerised in the presence of a

target analyte, which acts as a molecular template. The functional monomers

initially form a complex with the imprint molecule, and following polymerisation,

their functional groups are held in position by the highly crosslinked polymeric

structure. Subsequent removal of the imprint molecule reveals binding sites that

are complementary in size and shape to the analyte, introducing a molecular

memory into the polymer, capable of rebinding the analyte (Scheme II. 1). The

MIPs possess two of the most important features of the biological receptors: the

ability to recognise and to bind specific target molecules. Because of the super

crosslinked nature, MIPs are stable to physical and chemical treatment including

12 Chapter 2

heating, organic solvents, acids and bases. Thus molecular imprinting is a

powerful method for preparing synthetic recognition sites with predetermined

selectivity. An important prerequisite for the preparation of these polymers is the

formation of a stable pre-polymerisation complex between the monomer and the

template molecules. The stability and low cost of molecular imprinted polymers

make them advantageous for use in analysis, industrial scale production and

application5-7.

Scheme II. 1. Principle of molecular imprinting

The properties of the polymer obtained by this technology are different

from the properties of the parent polymer because after the removal of the

template, its three-dimensional imprint remains in the MIP.

Molecular recognition between a molecular receptor (host) and a

substrate (guest) in matrix containing structurally related molecules requires

discrimination and binding. This can happen only if the binding sites of the

Molecular Imprinted Polymers: A Review 13

host and guest molecules complement 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 selection8. 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 complementary

array for the convergent interactions with the template. The relationship of the

template to the imprinted cavity corresponds to the lock and key principle

proposed by Emil Fisher for enzyme catalysis9.

2. 2. Approaches in molecular imprinting

MIPs are synthesised in the presence of specially introduced target

template molecules, which are intended for imprinting. Because of the formation

of a pre-polymerisation complex, the molecules of a functional monomer are

particularly arranged and fixed around the template molecule in the course of the

entire process of polymerisation. Generally molecular imprinting can be

approached in two ways: the self-assembly approach developed by Mosbach and

coworkers10 and the pre-organised approach developed by Wulff and

coworkers11. These two approaches, which differ with respect to the interaction

mechanism, follow common molecular recognition terminology12,13.

The pre-organised molecular imprinting approach (covalent imprinting)

involves formation of strong reversible, covalent arrangements of the monomers

with the print molecule before polymerisation. Thus the print molecule needs to

be derivatised with the monomers before the actual imprinting is performed.

After cleaving the covalent bonds that hold the print molecules to the

macroporous polymer matrix, recognition sites complementary to the analyte

14 Chapter 2

remain in the polymer matrix (Scheme II. 2a). The interaction between the pairs

like amine-aldehyde, diol-ketone and acid-amine are normally covalent in nature.

Thus imprinting with Schiff’s base14, boronic acid esters15 and metal

co-ordination bonds16 follow covalent method. Since the bond between the

functional monomer and the template is covalent, polar solvents can be used as

porogen during the polymerisation. However, slow binding kinetics restricts the

analytical application of covalently imprinted polymers. Furthermore, covalent

binding tends to be very limited since it is very specific for particular functional

groups and is generally directional.

In non-covalent imprinting a very important property of polymer

molecules, which forms the basis for the organisation of complex biological

structures is used. It is the capacity of macromolecules for self-organisation or

self-assembly. This method involves host-guest complexes produced from weak

inter molecular interactions such as ionic or hydrophobic interaction, hydrogen

bonding, π-π interactions, induction, dispersion and metal co-ordinations

between the analyte and the monomer precursors. These self-assembled

complexes are spontaneously established in the liquid phase and are then

sterically fixed by polymerisation with a high degree of crosslinking. After

removal of the print molecule from the resulting macroporous matrix, vacant

recognition sites that are specific to the print molecule are established (Scheme

II. 2b). The shape of the sites maintained by the polymer backbone and the

arrangement of the functional groups in the recognition sites results in affinity

for the analyte. In non-covalent imprinting the bonds are less specific,

non-directional and the binding sites are heterogeneous in nature. Although

hydrophobic forces are potentially more difficult to master because they are less

specific and have less direction, combinations of polar and hydrophobic

interactions may be used to generate strong and selective binding. The non-


covalent technique is more versatile in terms of the range of template molecules.

It is also easier to implement because simply mixing a template with a

functional monomer in an appropriate solvent performs complexation and the

template is removed by repeated washing of the polymer with a solvent or a

solvent mixture. The non-covalent approach was used in the synthesis of

polymers with the molecular imprints of dyes17-19, aminoacids and their

derivatives20-22, pharmaceuticals23-26, pesticides27-29 and other components30,20,3.

The great majority of currently known MIPs that are found in analytical

chemistry are synthesised by the method of non-covalent molecular imprinting.

(a) (b)

Scheme II. 2. Schematic representation of (a) covalent imprinting, and (b) non-covalent imprinting

Another technique is the intermediate semi-covalent technique, in

which a template is covalently bound to a functional monomer in the course of

polymerisation, whereas only non-covalent interactions are involved in the

secondary binding31,32.

16 Chapter 2

2. 3. Synthesis of imprinted polymers

Molecular imprinting provides a way to synthesise new materials

containing artificial receptors that can be employed in a variety of applications

such as in separation, sensors or catalysis. The latest developments in the

technique of molecular imprinting have made polymers that can be used in the

detection of drugs, toxins, peptides, food components and other molecules that

would be difficult to isolate otherwise. Research on MIPs is focused on two

main areas: at a molecular level on the improvement in the composition and

interactions of the polymer, and at a macromolecular level on the

improvement and development of new morphologies, processes of

polymerisation and their applications in sensors, assay technology and

separation. Different uses and potential applications of the MIP demand

different properties from the polymers. Factors such as specificity, capacity or

environment of the sample require particular characteristics. In response to

this demand, different methods to produce imprinted polymers have been

developed. So far MIPs have been prepared by bulk, suspension, two-step

swelling, precipitation and emulsion core-shell polymerisations. Other

methods employed are film synthesis33-38, aerosol polymerisation39 and

polymerisation on silica particles40-45.

The first polymerisation method employed to synthesise MIP was

based on “bulk” polymerisation11,13 and is widely used for imprinting because

of its simplicity and universality. It is used exclusively with organic solvents

and consists of mixing all the components (template, monomer, solvent and

initiator) and polymerising them, resulting in a polymeric block which is to be

crushed and ground to obtain particles of irregular shape and size between 20

and 50 µm. It has the disadvantage that a lot of the polymer produced is

wasted in the process of grinding. It may also produce areas of heterogeneity


in the polymeric matrix resulting from the lack of control of the process during

polymerisation, particularly when UV initiation is used.

A fast and reliable methodology that synthesises particles by UV

irradiation in less than 2 h is suspension polymerisation, first described by

Mayes and Mosbach46. The beads obtained have a diameter that can vary

between 5 and 50 µm depending on the stirring speed and the amount of

surfactant. It uses a perfluorocarbon solvent in the continuous phase, which

allows the establishment of the same interactions that occur in bulk

polymerisation. The fluorocarbon suspending medium can be easily recycled

by distillation.

Another method that can provide particles in the submicron scale

(0.3-10 µm) is precipitation polymerisation47,48. It is based on the precipitation

of the polymeric chains out of the solvent in the form of particles, as they

grow more and more insoluble in an organic continuous medium. Here the

particles are prevented from coalescence by the rigidity obtained from the

crosslinking of the polymer and so it does not need any additional stabiliser.

Precipitation is one of the most simple and well suited methods to obtain

imprinted beads with the desired characteristics. The advantages of this

polymerisation strategy in terms of capacity and homogeneity associated to

binding sites have been demonstrated49, 50.

Monodisperse particles in the micron size range (2-50 µm) with good

control of the final size and number of the particles were synthesised by a

two-step polymerisation developed by Hosoya in 199451. It required several

swelling steps on the initial particles with the imprinting mixture before

polymerisation proceeds and the continuous phase of the polymerisation

medium was water.

18 Chapter 2

Core-shell particles are obtained by emulsion polymerisation52-54. They

have a structured morphology that allows the incorporation of any added

property into the core of the particle without interfering with the imprinted

shell. The continuous medium during polymerisation is water. Particles

obtained by this method are monodisperse and can be produced in colloidal

size with the range of 0.05 - 2 µm.

In situ polymerisation can be done for certain applications inside the

chromatographic column55 or in a capillary56. In sensor applications usually

imprinted polymer membranes were prepared as thin crosslinked films by

precipitation from solutions in the presence of analyte57 or by casting polymer

in the pores of an inert support membrane58. They can also be synthesised in

situ at the electrode surface by electropolymerisation37 or at a non-conducting

surface by chemical grafting59. Novel imaging strategies using fluorescent-

labelled template was also reported60. Metal ion imprinted polymeric beads are

successfully applied in analytical applications61. One of the first reports of

imprinting of bacterial cells on the polymer surface was also observed62.

Computational design and synthesis of imprinted polymers with high binding

capacity for pharmaceutical applications was demonstrated as the model case.

Special protocols have been proposed for the imprinting of proteins63.

Molecular imprinting in aqueous environments are in the preliminary

stage. It was noted that most imprints worked best in the solvent, which was

used for polymerisation13. Therefore the use of water as a solvent has been

studied for several targets with the aim of using the resultant imprinted

polymers in aqueous systems. Hydrophobic interactions, caused by the

exclusion of water, are frequently employed for imprinting in aqueous systems

and hydrophobically driven molecular imprints have proven successful with a

variety of small molecules in a range of polymer matrices.


Kugiyama relied on hydrophobic interactions between pyridine

monomers and the chosen template sialic acid to produce a MIP capable of

selective recognition in aqueous media64. Molecular imprinting of the

hydrophobic herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and

2,4,5 trichlorophenoxyacetic acid (2,4,5-T) in a mixture of methanol and water

was already reported65-70. This polar medium elicited hydrophobic binding site

within a 4-vinylpyridine (4-VP) functionalised imprinted polymer. Perez-

Moral produced core-shell particles with surface imprints that used shape-

specific hydrophobic interactions to recognise the hydrophobic molecule

cholesterol52,53. Similarly, Carter formed core-shell particles prepared in water

using emulsion polymerisation71,72. These particles were non-covalently

imprinted with caffeine and theophylline, using both hydrophobic and

electrostatic interactions. In a different approach to the same templates, Han

demonstrated a novel method of aqueous imprinting using an oil/water

emulsion73. The selected monomer and template sequestered into water

droplets within the oil and formed complexes. These complexes were then

transferred to a microporous membrane substrate and photopolymerised.

Dirion used a high-throughput synthesis and evaluation of MIP sorbents to

optimize a MIP for the local anaesthetic bupivacaine in aqueous systems74.

The resultant MIPs showed high imprinting factors in water, attributed to

reduced non-specific binding to the imprinted polymer. Batches of these

sorbents were successfully employed for the extraction of the template from

blood plasma samples.

The templates are classified as convex and concave. Metal ion

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

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

the enzyme to undergo chemical transformation. Polymers are also categorised

20 Chapter 2

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

accelerated or unaffected.

The binding site consists of a functional group attached, capable of

interacting with the template molecule and ideally the functional groups exist

on the surface of the cavity left by the template, readily available for

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

fast and reversible.

2. 4. Factors influencing the characteristics of imprinted polymers

A good success of imprinting method is predominantly dependent on

the polymer structure and on the type of binding site interaction. Binding site

interactions are necessary during the polymerisation procedure and are later

used for binding of the substrate to the polymer. The characteristics of the

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

nature of template and extent of crosslinking have a significant effect on

binding. Hence the optimization of the polymer structure is extremely

important75. Random incorporation of the functional monomer in the polymer

matrix reduces its specificity. So the interactions between the monomer and

the template should be as strong as possible during polymerisation and ideally

they should be covalent. However systems utilisng reversible covalent bonds

exhibit slow kinetics during rebinding and often necessitate severe conditions


for desorption15. 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 satisfactorily distributed over a macromolecule so that both the more

favourable and less favourable conformations will occur. The binding process

depends on the appropriate geometric organisation of functional groups,

including hydrophilic domains, within the host molecule that match or fit

reciprocal functionality on the guest76.

(i) Rigidity/Flexibility of the polymer support

An important prerequisite for the preparation of MIP is a high degree of

crosslinking. The rigidity of the highly crosslinked polymer maintains the

binding sites that feature the required functional groups in fixed three-

dimensional orientations necessary for rebinding of the template77. Yilmaz78

demonstrated that a molecular memory can be introduced by molecular

imprinting even in low crosslinked polymers (19%). The selectivity is mainly

influenced by the kind and amount of crosslinking agent used in the synthesis

of MIP79. Below a certain amount of crosslinking in the polymer (∼10%), no

selectivity can be observed because the cavities are not sufficiently

stabilised. Above 10% crosslinking selectivity increases steadily. Between

50 and 60% a surprisingly high increase in selectivity takes place, especially

in the case of EGDMA crosslinking. Crosslinking with DVB has the

advantage of less interaction with the functional groups80.

Some flexibility is also 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-divinylbenzene81,82, and styrene-diisopropyl benzene83,84

22 Chapter 2

copolymer by Shea revealed that relatively rigid, hydrophobic and highly

crosslinked materials provide chemical inertness required to withstand

subsequent chemical transformations. Thus the molecular imprinted polymers

should have optimum stiffness/flexibility to attain rapid equilibrium with the

template, to make imprinted cavities accessible for the template, to provide

mechanical stability to withstand stress in certain applications and thermal

stability to use at high temperature. Therefore crosslinking provides a

structural design that improves selective molecular recognition by MIPs85.

Thus there should be a compromise between rigidity and flexibility to ensure

favourable kinetics for substrate binding.

(ii) Functional and crosslinking monomers

Several 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, hydrogen bonding, hydrophobic and others (Fig. II. 1). Methacrylic acid

(MAA), acrylamide (AA) and 4-vinylpyridine (4-VP) were the most

frequently used functional monomers in non-covalent imprinting. Acrylamide

could be a promising functional monomer to form strong hydrogen bonds with

template molecule in polar solvents. In deciding a functional monomer, the

nature of the constituent donor atoms and the possibility of forming a stable

monomer-template associate are taken into consideration. Generally MAA is

used as a functional monomer in the synthesis of polymers with the molecular

imprints of organic compounds containing basic groups such as triazines86,87,


whereas 4-VP is used in the case of compounds containing acidic

groups88,89,65.

O

OH

O

OHF3C N

N N

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

N

O

OOH

O

NH2

(e) (f) (g)

(a) methacrylic acid, (b) trifluoromethacrylic acid, (c) N-vinylimidazole,

(d) 4-vinylpyridine, (e) 2-vinylpyridine, (f) hydroxyethyl methacrylate,

(g) acrylamide

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

While the amount of crosslinking agent affects the rigidity of MIPs, the

nature of this agent significantly affects the physicochemical properties of a

polymer matrix90. The choice of a crosslinking agent depends on the nature of the

functional monomer. For efficient imprinting, the reactivity of the crosslinking

agent should be similar to that of the functional monomer. DVB isomers are

most frequently used as crosslinking agents in the synthesis of MIPs based on

polystyrene, whereas EGDMA is used in the synthesis of MIPs based on acrylic

24 Chapter 2

or methacrylic acids3 in organic solvents. N,N’-methylene-bis-acrylamide

(NNMBA) is a typical water soluble crosslinking agent. The fundamental role of

these agents is to fix the guest binding sites firmly in the desired structure and

make the polymer insoluble in solvents. By the use of proper crosslinking agent,

random copolymerisation occurs successfully and the functional residues are

uniformly distributed in the polymer network.

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

neighbouring sites. At extremely large ratios 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%91. At low crosslinking, the

selectivity is poor since the polymer matrix is not stable enough to retain the

shape of the cavity. In deciding on crosslinking agents, their 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 PEG400 DMA are more suitable92. Thus the crosslinking

monomers are an active part of imprinting, rather than merely ‘inert’

scaffolding for functional monomers. The most frequently used crosslinking

agents30 are depicted in Fig. II. 2.


ONH

NHO

O

O

O

O

N

HNO

HNO

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

NHO

HNO

OO

O

O

O O

(e) (f)

(a) Divinylbenzene (DVB), (b) N,N’-Methylene-bis-acrylamide (NNMBA), (c) Ethyleneglycol dimethacrylate (EGDMA), (d) 2,6-Bisacrylamidopyridine (e) N,N’-Phenylene-bis-acrylamide (NNPBA), (f) Trimethylolpropane trimetha- crylate (TRIM)

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

A simple method of molecular imprinting using a single crosslinking

monomer N,O-bis-methacryloyl ethanolamine (NOBE) along with template,

initiator and solvent was recently reported93. This formulation eliminates the

26 Chapter 2

need for additional functional monomers and empirical optimization of

relative ratio of functional monomers, crosslinking monomer and template.

Utilisation of NOBE alone often provides MIPs with better performance than

MIPs incorporating functional monomer.

(iii) Monomer-template ratio

The molar relationship between the functional monomer and template

had been found to be important with respect to the number and quality of MIP

recognition sites94. Low M/T ratios afford less than optimal complexation on

account of insufficient functional monomer. Too high an M/T ratio, the

extreme case being a non-imprinted polymer, yields non-selective binding95.

Studies70 on the column capacity, selectivity factor and imprinting effect of

2,4,5-T imprinted polymer with different molar ratios between template and

functional monomer revealed that the retention of 2,4,5-T was proportional to

the molar ratio between the functional monomer and template. When the

molar ratio is low, the pyridine rings are mostly included in high affinity

binding sites generated by the imprinting mechanism, where the imprinting

effect should be very significant. When the molar ratio is high, the pyridine

rings are mostly located outside the imprinted cavities, scattered in regions of

the polymer where the imprinting effect is absent or almost negligible. A well

designed imprinted polymer shows marked specific molecular recognition

effects and negligible non-specific partition effects, while a non-imprinted

polymer shows only partition effects without any sort of molecular recognition

effects. Thus a parameter called “imprinting effect” can be considered as a

good estimate of how much and how well the polymer was imprinted by a

template95. Thus imprinting effect is only a measure of the net molecular

recognition effect due to the imprinting process in defined experimental

conditions. The molar ratio between template and monomer could be


approximately set from 1+2 to 1+4 for many of the polymers described in the

literature96-98. It has been demonstrated that for equilibrium constants between

10 and 1000 M-1, a significant fraction of the template is complexed, provided

that functional monomers will be in considerable excess99,100. Anyway it may

not be considered as a general criterion, since imprinting effects with a 1+1

molar ratio between template and functional monomer was reported101-103. One

problem with increasing the functional monomer is that the amount of

crosslinker becomes too low, and the MIP loses its recognition properties from

random motion of non-crosslinked polymer domains104.

(iv) Porogen/Rebinding solvent

Molecular imprinted polymers utilise a solvent both as a porogen in

polymerisation and as a medium for rebinding studies. The trivial role of

solvents is to dissolve the agents for polymerisation and also to disperse the heat

of reaction generated during polymerisation. The solvent also serves to facilitate

mass transfer of the analytes to and from the binding sites and to create pores by

phase separating into channels during polymerisation105. It also affects

adsorbent characteristics such as specific surface area and pore size106. Thus

porogen parameters such as polarity and hydrogen bonding have an important

impact on the final morphology of the network structure, porosity and

recognition properties of MIPs.

It is believed that solvents with low permitivities (toluene,

dichloromethane, chloroform) are best suited for molecular imprinting107,108

because the monomer-template non-covalent interactions are stronger than in

polar solvents. Chloroform is one of the most widely used solvents, since it

satisfactorily dissolves many monomers and templates and hardly suppresses

hydrogen bonding.

28 Chapter 2

Choice of solvent depends on the kind of imprinting. In covalent

imprinting many kinds of solvents are employable as long as they

satisfactorily dissolve all the components. In non-covalent imprinting the

choice of solvent is more critical to the promotion of the formation of non-

covalent adducts between the functional monomer and the template enhancing

imprinting efficiency. Polar solvents will interfere with the hydrogen bonding

interaction between substrate and MIP and the substrate will tend to remain in

solution rather than binding to the polymer due to strong solvation of the

substrate.

The solvent specific behavior of the imprinted polymers has been

explained in terms of the solvating properties of the solvents109. The solvating

ability of the porogen for polymer chains during polymerisation has been

suggested to adjust the shape of the binding sites and the distance between the

functional groups in the binding site. Thus the ability of the binding medium

to recreate the binding site dimensions determines the binding performance of

the polymer. Generally best recognition of imprinted polymers occurs when

the rebinding medium and the porogen are the same110-112.

(v) Polymerisation temperature

The position of equilibrium between free template-monomer and their

corresponding complex is a product of both temperature and pressure113.

Sellergren group showed that high pressure (1000 bar) polymerisation could

be used to enhance the selectivity of the resultant imprinted polymers114.

Previous studies have shown that lower temperature is favourable for the

preparation of MIPs based on electrostatic interaction due to the greater

strength of electrostatic interaction at lower temperature115,116. At low

temperature, the polymerisation process is slow and the chain formation does


not interfere with the template-monomer interactions. An optimum

temperature should be found for each combination of template and

monomer117. Synthesis of MIPs could also be performed at reduced

temperatures (15 to 20oC), while initiating the polymerisation reaction by

UV- irradiation118-120. In this case MIPs with a greater capacity for molecular

recognition were obtained because the monomer-template complex in the

polymerisation mixture is more stable at low temperatures.

The radical polymerisation of corresponding functional monomers is

the main method for preparing MIPs. The rate of radical polymerisation

depends on the nature and concentration of the initiator121. Polymerisation in

early applications was initiated by thermal decomposition of AIBN122,123, an

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.

2. 5 Characterisation of molecular imprinted polymers

In the non-covalent approach the stability of the template-monomer

pre-polymerisation complex will govern the resulting binding site

distribution and the binding properties of the imprinted polymer matrix.

Close analysis of the pre-polymerisation solution can provide fundamental

insights into 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, spectral studies before and after rebinding can also put light into

the binding process.

30 Chapter 2

(i) FT-IR

FT-IR spectra provide the fundamental analytical basis for

rationalising the mechanism of interactions for selective binding site formation

at the molecular level124. 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 analysis125.

(ii) 1H NMR

Proton NMR titration experiments facilitate observation of hydrogen

bond formation between bases and carboxylic acid through hydrogen bonding.

These studies have been introduced in molecular imprinting for investigating

the extent of complex formation in pre-polymerisation solutions and to

identify the specific sites in interacting structures that engage in complexation.

Thus evaluating the shift of proton signals due to participation in hydrogen

bonding and other interactions were used as the criteria for complex

formation, M/T ratio and interacting forces126,81.

(iii) 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 polymers103. Therefore it is not possible to obtain reliable structural

information of the interactions occurring in the cavities between binding site

and template. The polymer analysis using 13C CP-MAS-NMR techniques can

give information about the polymer backbone.


(iv) Scanning electron microscopy (SEM)

SEM can be used in 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 polymers127, 128.

2. 6. Swelling studies

The efficiency of a functional monomer is governed by the

accessibility of the reactive functional groups anchored on it, which, in turn,

depends upon the extent of swelling and solvation129. The rate of diffusion of a

reagent into the polymer matrix mainly depends on the extent of swelling130.

Thus swelling is an important parameter, which controls the success of

rebinding. The most effective solvent can carry out the rebinding reaction very

effectively. The extent of swelling can be determined in terms of change in

weight131,132. Alternatively, 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 volume133.

2. 7. Selectivity parameters of molecular imprinted polymers

The binding parameters of the MIPs are usually estimated from

adsorption isotherms103,109 using mathematical models. One strategy to

perform binding performance is based on saturation experiments and

subsequent Scatchard analysis134-137. The 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 the equilibrium dissociation constant, Smax an apparent

maximum number of binding sites and [S]b is the amount of template bound to

32 Chapter 2

MIP at equilibrium. From the plot of bound concentration ([S]b), against the

ratio between bound and free template concentration ([Sb]/[S]f), it is possible

to estimate the Smax and KD where Smax is the X intercept and KD is the

negative reciprocal of the slope. For non-covalently synthesised MIPs the

Scatchard plots result in a 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 functional monomer led to

the heterogeneity in the binding sites typical of the non-covalent imprinting.

The effectiveness of imprinting was verified by comparison of the

binding of template with structural analogues138-141. A complete secondary

screen for binding and selectivity was performed for all the polymers in terms

of separation factor142.

Separation factor (α 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 value of separation

factor depends on the type of interactions between the template and the

functional monomers. However, when non-covalent interactions are

employed, comparatively less values in the range 1-2 is obtained. The

selectivity of the imprinted polymer can also be expressed as selectivity

factor143,70.

Selectivity factor = α template /α analogue

It provides a good indication for the molecular recognition of the

comparative molecule to the template.


2. 8. Applications of molecular imprinted polymers

The initial imprinting structures of Wulff in the 1970s using sugar and

amino acid derivatives inspired the application of molecular imprinted polymers

to a wide range of substrates and functions. Investigations have involved the

development of MIPs for separating mixtures of racemates of sugars144 and

amino acid126 derivatives. But within the past few years the variety of substrates

to which MIPs has been developed has increased to include pesticides145, metal

ions146-148, steroids149, drugs150, peptides151 and antibiotics152.

(i) Enantioseparation of racemic mixtures

The application of MIPs that has received significant attention is that

of molecular imprinted chromatography (MIC). Numerous templates had been

examined in imprinting protocols for separation science, ranging from small

molecules such as amino acids to much larger species such as proteins153.

Since most commercially available chiral drugs are administered as racemate

mixtures, the chiral resolution of drugs is a major potential application of

MIPs154. Molecular imprinted polymers had been prepared against chiral

compounds and applied to enantiomeric separation24. One major advantage of

the use of MIPs in enantiomeric separations is that this technique allows the

preparation of ‘custom-made’ supports155. Optimized MIPs exhibit predictable

elution orders with the imprinted enantiomer always retaining in the column

for a long time156.

In MIC, template selectivity is dependent upon the differences in

specific and non–specific interactions between the analytes and the template in

question157. MIPs possessing separation factors of greater than 10 should be

capable of completely resolving compounds by the simple process of stirring

the racemate with the imprinted polymer and filtering158. In general, the values

34 Chapter 2

exhibited by MIP stationary phases range between 4-8157, when ionic

interactions are utilised between the template and the functional monomers.

Yu and Mosbach159 have carried out extensive optimization of the

chromatographic resolution of racemic mixtures of α-amino acids using amide

groups as the key recognition element within imprinted polymer networks.

A very pronounced stereo selectivity has been observed with an MIP

specific for the cinchona alkaloids cinchonidine and cinchonine, resulting in

chromatographic values up to 31160. It is even possible to obtain chromatographic

supports selective for compounds containing several chiral centers151. If the

molecule of interest contains more than two chiral centers, as is the case with

carbohydrates, these properties of molecular imprinted materials become even

more relevant. When polymers were imprinted against a glucose derivative,

very high selectivity between the various stereoisomers and anomers were

recorded161.

Capillary electrochromatography (CEC) might be one of the more

promising chromatographic techniques to be used in combination with MIPs,

in particular for chiral separations162,163. Enantioseparation of the β–blockers

propranolol and metoprolol was achieved with MIP-CEC. The polymer was

cast in situ in the capillary in the form of a microporous monolith attached to

the inner wall, and the capillary could be prepared and conditioned within a

few hours56. The racemate of propranolol was resolved within only 120 s and

when non-racemic samples were injected containing mainly the R-enantiomer,

very small amounts (1%) of the S-enantiomer could be distinguished. Other

possibilities of using MIPs in combination with capillary electrophoresis is in

the form of continuous polymer rods164, particles included in a gel matrix165

and small particles suspended in the carrier electrolyte166.


(ii) Solid phase extraction

The separation technique that has been most intensively studied in the

past few years with respect to the possible use of imprinted materials is solid

phase extraction (SPE)167-170. MIPs are not only more selective than common

sample treatment methods using C18 or ion exchange materials, but are at the

same time more stable than immuno extraction171 matrices. Since MIPs are

compatible with organic solvents, MIP-SPE can be applied directly after a

solvent pre-extraction step. As SPE works in the adsorption-desorption mode,

the low-resolution factors are not an issue. Thus SPE seems to be one of the

most promising application niches for MIPs today. MIP-SPE has been used to

extract the target analyte from blood plasma and serum172, urine173, bile168,

liver extract86, chewing gum174, environmental water and sediment119 and

plant tissue170. The SPE procedures were also applied to the clean up of phenyl

urea herbicides in carrot, potato, corn and pea sample extracts149. The

quantification of the herbicide atrazine in beef liver is a good demonstrative

example of the utility of imprinted polymers in SPE86.

When MIPs are to be used as SPE materials, one of their more

troublesome features is template leakage leading to inaccuracies in the

quantitative measurement in the levels of the analyte of interest in the sample

and it is a source of concern in the application of MIPs in the pharmaceutical

industry. A possible method of circumventing the bleeding problem entirely is

to use a template analogue during the imprinting step rather than the template

itself. This approach was described by several researchers175,176.

Barzana and co-workers177 have utilised MIPs for the selective

adsorption of organosulfur compounds from fuel, with the ultimate aim of

desulfurising diesel. The cross-reactivity of MIPs was exploited by Hui and

36 Chapter 2

Qin in the determination of the degradation products of nerve agents in human

serum via MIP-SPE178. MIPs for the solid phase extraction of propranolol168,

pentamidine167, sameridine148, tamoxifen179 and atenolol180 were also reported.

MIPS have also been used in other separation techniques such as thin layer

chromatography181-183, membrane based separations184-187 and adsorptive

bubble flotation fractionation188.

(iii) Binding assays For analytes lacking physicochemical properties that allow selective

and sensitive quantitation in a given matrix, ligand-binding assays can be

used. The wide variety of techniques developed for the determination of

analytes by immunoassay includes radio immunoassays (RIA) and enzyme

immunoassays189-191(ELISA). Since MIPs can bind a target molecule

selectively like antibodies, they could conceivably be employed in

immunoassay type binding assays in place of antibodies. This was first

demonstrated by Mosbach’s group, who developed MIP based assays for the

bronchodilator theophylline and the tranquilizer diazepam25, analogous to the

first solid-phase immunoassay for human growth hormone192. This showed

not only a very good correlation with an antibody-based enzyme

immunoassay but even yielded a cross reactivity profile very similar to that

of the natural antibodies. In MIA the most commonly used label is a

radioactive isotope25, but also detection systems based on fluorescence have

been suggested68. An innovative technical improvement is the use of

magnetic MIP beads to facilitate separation of free and bound radiolabelled

marker193. Also sub-micron beads, more resistant to precipitation and

aggregation, requiring less agitation during incubation may simplify the

assay procedure194. Use of MIA for analysis of drug compounds in blood-

derived biofluids has already been presented195. Some analyte MIP systems


can be employed equally well for organic solvent or aqueous buffer based

assays196,146,. An MIA method by which plasma samples could be assayed

directly was also demonstrated145. 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

hydrolysis by 103-104 fold197. The enhancement is due to the preferred

binding of the transition state of the reaction.

(iv) Polymeric sensors

An area in which MIPs have considerable potential is as the

recognition element in biosensors198-200. In sensor systems, the MIPs replace

the naturally occurring sensing element, and therefore provide mechanical

stability to the system. Many sensors for environmental monitoring,

biomedical and food analyses rely on biomolecules such as antibodies or

enzymes as the specific recognition elements. Because of the poor chemical

and physical stability of biomolecules, artificial receptors are therefore gaining

increasing interest. The first reported integrated sensor based on an MIP was a

capacitance sensor201. Recently capacitive detection was employed in

conjunction with imprinted electropolymerised polyphenol layers on gold

electrodes34. In another report, thin films of TiO2 were imprinted with

chloroaromatic acids such as 2,4-D and used as recognition layers in sensors

based on ion-sensitive field-effect transistors202. During the last few years

there has been a big boost in the use of mass-sensitive acoustic transducers

such as the surface acoustic wave (SAW) oscillator203,204, the Love-wave

oscillator205 (LWO) and the quartz crystal microbalance206-210 (QCM) for the

design of MIP based sensors. If the target analyte exhibits a special property

such as fluorescence211, 212 or electrochemical activity213 that can be exploited

38 Chapter 2

for the design of MIP based sensors. A very sensitive sensor for the detection

of the hydrolysis product of a chemical warfare agent Soman has been

described based on a polymer-coated fiber optic probe and a luminescent

europium complex214. The selectivity of a sensor is dependent on differences

in interaction between analytes and sorbent to the same extent as in ligand

binding assays. The sensitivity of a sensor is dependent on the affinity of the

analyte or of the labelled ligand and on the detection principle. In conclusion

due to their stability molecular imprints can be readily used as the sensing

surface of a chemical sensor.

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.

(v) Herbicide selective polymers

Maximising the world’s agricultural efficiency depends largely on

controlling a variety of diseases and pests, especially weeds. Herbicides are

the chemicals that are widely used in agricultural field for controlling the

growth of herbs, weeds and bushes. The herbicide industry was built on the

success of 2,4-D and 2,4,5-T and weed control research over the last 50 years

had been focused almost exclusively on synthetic herbicides215. The use of

pesticides is accompanied by a variety of undesirable environmental effects.

Current concerns about potential health hazards connected with pesticide use

have focused on 2,4-D and 2,4,5-T as suspected cancer-causing agents. 2,4-D

is extensively used as a broad leaf weed killer on field crops, turf, and non-

crop lands. The wide spread use of 2,4-D and associated health concerns have

made monitoring of environmental samples for the presence of 2,4-D. 2,4,5-T

also is a broad range herbicide whose use has been banned in Europe and the


USA owing to the danger of dioxin contamination connected with the

commercial product216,217. Thus it may represent an interesting environmental

contaminant of anthropic origin. These compounds are polar chlorinated

herbicides with a carboxylic group attached to the benzene ring by means of

an oxygen atom.

O COOH O COOH

Cl

Cl

O COOH

Cl

Cl

Cl

COOH

OH POA 2,4-D 2,4,5-T p-HB

With human’s increasing concern of environmental protection, the

requirement of a sensitive and convenient method for environmental assay is

strongly required. The classical analytical methods for herbicide analysis

include HPLC, GC, GC-MS, ELISA and spectrophotometric methods218,219.

These conventional methods are still tedious and time consuming and are

mostly laboratory-bound. An alternative modern method that eliminates some

of these difficulties of conventional methods is the use of molecular imprinted

polymers.

Attempts were made previously to imprint herbicides in polymers

and to use them in various applications. The 2,4-D imprinted polymer

coated bulk acoustic wave (BAW) sensor was fabricated in liquid phases29,

polymer layers on the surface of zinc selenide attenuated total reflection

elements38, immunosensing methods220, differential-pulse voltammetry

method66, competitive fluoroimmunoassays68, radioligand binding assay65,

biosensors 221, and solid phase extraction by means of polymeric resins222 were

reported for the selective binding of the herbicide 2,4-D.

40 Chapter 2

Chromatographic characterisation of 2,4,5-T imprinted polymers was

reported69. HPLC columns packed with 2,4,5-T imprinted polymers in

hydrophilic solvent show column capacity, selectivity and imprinting effects

controlled by ion-pair and hydrophobic interactions between the analyte and

the stationary phase70. Molecular imprinting of phenoxyacetic acid is not yet

reported to our knowledge.

Many phenolic compounds are toxic for living beings, easily

penetrating through natural membranes, causing a broad spectrum of

genotoxic, mutagenic and hepatotoxic effects, and also modulating bio-

catalysed reaction in respiration and photosynthesis223,224. Because of their

toxicity, as well as their unpleasant organoleptic properties, phenols have been

included in the priority pollutant list of the EEC and US-EPA225-229. Molecular

imprinting of such a phenolic compound p-hydroxybenzoic acid (p-HB) was

reported230, but the study was based on HPLC.

Most of the reported techniques especially that employed for the

studies of 2,4-D, 2,4,5-T and p-HB imprinted polymers were complex, costly,

time consuming, require sophisticated instruments and skilled technicians.

Thus there is the need of developing faster screening methods that should be

technically simple and useful for routine analysis of a large number of

samples. Here an attempt is made to design imprinted polymers of the selected

templates and to tailor the conditions for maximum specificity and selectivity

employing the simple UV-spectrophotometric technique.


References

1. Piletsky, S. A.; Alocock, S.; Turner, A. P. F. Trends Biotechnol. 2001, 19, 9

2. Anderson, L. I. J. Chromatogr. B: Biomed. Appl. 2000, 745, 3

3. Sellergren, B. Molecularly Imprinted Polymers: Man-Made Mimics of

Antibodies and their Application in Analytical Chemistry, Elsevier, 2001

4. Haupt, K. Analyst 2001, 126, 747

5. Mosbach, K.; Haupt, K. J. Mol. Recognit. 1998, 11, 62

6. Haupt, K. Chem. Commun. 2003, 2, 171

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

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

9. Fisher, E. Ber. Dtsch. Chem. Ges. 1994, 27, 2985

10. Lindsey, J. S. New J. Chem. 1991, 15, 153

11. Mosbach, K.; Ramstrom, O. Biotechnology 1996, 14, 163

12. Cram, D. J. Angew. Chem., Int. Ed. Engl. 1988, 27, 1009

13. Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812

14. Wulff, G.; Vietmeier, J. Makromol. Chem. 1989, 190, 1717.

15. Wulff, G.; Vesper, W.; Grobe, E. R.; Sarhan, A. Macromol. Chem. 1977,

178, 2799

16. Takeuchi, T.; Mukawa, T.; Matsui, J.; Higashi, M.; Shimizu, K. D. Anal.

Chem. 2001, 73, 3869

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

18. Norrlew, O.; Glad, M.; Mosbach, K. J. Chromatogr. A 1984, 299, 29

19. Glad, M.; Norrlew, O.; Sellergren, B. J. Chromatogr. A 1985, 347, 11

42 Chapter 2

20. Takeuchi, T.; Haginaka, J. J. Chromatogr. B: Biomed. Appl. 1999, 728, 1

21. Kempe, M.; Mosbach, K. Anal. Lett. 1991, 24, 1137

22. Andersson, L. I.; Mosbach, K. J. Chromatogr. 1990, 516, 313

23. Andersson, L. I. J. Chromatogr. B: Biomed. Appl. 2000, 739, 163

24. Fisher, L.; Muller, R.; Erberg, B.; Mosbach, K. J. Am. Chem. Soc. 1991, 113, 9358

25. Vlatakis, G.; Anderson, L. I.; Muller, R.; Mosbach, K. Nature 1993, 361, 645.

26. Hagginaka, J.; Sakai, Y.; Narimatsu, S. Anal. Sci. 1998, 14, 823

27. Tamayo, F. G.; Casillas, J.; Martin-Esteban, A. Anal. Bioanal. Chem. 2005,

381, 1234

28. Marx, S.; Zaltsman, A.; Turyan, I.; Mandler, D. Anal. Chem. 2004, 76, 120

29. Liang, C.; Peng, H.; Nie, L.; Yao, S. Fresenius J. Anal. Chem. 2000, 367, 551

30. Kempe, M.; Mosbach, K. J. Chromatogr. A 1995, 694, 3

31. Sellergren, B.; Andersson, L. I. J. Org. Chem. 1990, 55, 3381

32. Whitcombe, M. J.; Rodriquez, M. E.; Villar, P.; Vulfson, E. N. J. Am. Chem.

Soc. 1995, 117, 7105

33. Haupt, K.; Noworyta, K.; Mosbach, K. Anal. Commun. 1999, 36, 391

34. Panasyuk, T. L.; Mirsky, V. M.; Piletsky, S. A.; Wolfbeis, O. S. Anal. Chem.

1999, 71, 4609

35. Piletsky, S. A.; Piletska, E. V.; Chem, B.; Karim, K.; Weston, D.; Barret, G.;

Lowe, P.; Turner, A. P. F. Anal. Chem. 2000, 72, 4381

36. Malitesta, C.; Losito, I.; Zambonin, P. G. Anal. Chem. 1999, 71, 1366

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

2000, 423, 221


38. Jakusch, M.; Janotta, M.; Mizaikoff, B.; Mosbach, K.; Haupt, K. Anal. Chem.

1999, 71, 4786

39. Vonderbruggen, M. A.; Wu, K.; Breneman, C. M. Chem. Mater. 1996, 8, 1106

40. Makote, R.; Collinson, M. M. Chem. Commun. 1998, 3, 425

41. Makote, R.; Collinson, M. M. Chem. Mater. 1998, 10, 2440

42. Markowitz, M. A.; Kust, P. R.; Deng, G.; Schoen, P. E.; Dordick, J. S.; Clark,

D. S.; Gaber, B. P. Langmuir 2000, 16, 1759

43. Katz, A.; Davis, M. E. Nature 2000, 403, 286

44. Sasaki, D. Y.; Alam, T. M. Chem. Mater. 2000, 12, 1400

45. Markowitz, M. A.; Deng, G.; Gaber, B. P. Langmuir 2000, 16, 6148

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

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

48. Wang, J.; Cormack P. A. G.; Sherrington, D. C.; Khoshdel, E. Angew. Chem.

2003, 115, 5494

49. Tamayo, F. G.; Casillas, J. L.; Martin-Esteban, A. Anal. Chim. Acta 2002, 482, 165

50. Tamayo, F. G.; Casillas, J. L.; Martin-Esteban, A. J. Chromatogr. A 2005,

1069, 173

51. Hosoya, K.; Yoshizako, K.; Tanaka, N.; Kimata, K.; Araki, T.; Haginaka, J.

Chem. Lett. 1994, 1437

52. Perez, N.; Whitcombe, M. J.; Vulfson, E. N. J. Appl. Polym. Sci. 2000, 77, 1851

53. Perez, N.; Whitcombe, M. J.; Vulfson, E . N. Macromolecules 2001, 34, 830

54. Perez-Moral, N.; Mayes, A. G. MRS Symp. Proc. 2002, 723, 61

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

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

44 Chapter 2

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

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

59. Piletsky, S. A.; Piletska, E. V.; Matuschewski, H.; Schedler, U.; Wilpert, A.;

Thiele, T. A.; Ulbricht, M. Macromolecules 2000, 33, 3092

60. Hawkins, D. M.; Trache, A.; Ellis, E. A.; Stevenson, D.; Holzenburg, A.;

Meininger, G. A. Biomacromolecules 2006, 7, 2560

61. Saatcilar, O.; Satiraglu, N.; Say, R.; Bettas, S.; Denizli, A. J. Appl. Polym.

Sci. 2006, 101, 3520

62. Aherne, A.; Alexander, C.; Payne, M. J.; Perez, N.; Vulfson, E. N. J. Am.

Chem. Soc. 1996, 118, 8771

63. Guo, T. Y.; Xia, Y. Q.; Hao, G. J.; Song, M. D.; Zhang, B. H. Biomaterials

2005, 26, 5737

64. Kugimiya, A.; Matsui, J.; Takeuchi, T. Mater. Sci. Eng., C 1997, 4, 263

65. Haupt, K.; Dzgoev, A.; Mosbach, K. Anal. Chem. 1998, 70, 628

66. Kroger, S.; Turner, A. P. F.; Mosbach, K.; Haupt, K. Anal. Chem. 1999, 71, 3698

67. Wen, Y. C.; Chin, S. C.; Fu, Y. L. J. Chromatogr. A 2001, 923, 1

68. Haupt, K.; Mayes, A. G.; Mosbach, K. Anal. Chem. 1998, 70, 3936

69. Baggiani, C.; Giraudi, G.; Giovannoli, C.; Trotta, F.; Vanni, A. J.

Chromatogr. A 2000, 883, 119

70. Baggiani, C.; Anfossi, L.; Giavannoli, C.; Tozzi, C. Talanta 2004, 62, 1029

71. Carter, S. R.; Rimmer, S. Abstr. Pap. Am. Chem. Soc. 2002, 224, 363

72. Carter, S. R.; Rimmer, S. Adv. Mater. 2002, 14, 667

73. Han, M.; Kane, R.; Goto, M.; Belfort, G. Macromolecules 2003, 36, 4472


74. Dirion, B.; Cobb, Z.; Schillinger, E.; Andersson, L. I.; Sellergren. B. J. Am.

Chem. Soc. 2003, 125, 15101

75. Wulff, G.; Sarhan, A. Angew. Chem., Int. Ed. Engl. 1972, 11, 341

76. Turner, N. W.; Jeans, C. W.; Brain, K. R.; Allender, C. J.; Hlady, V.; Britt, D.

W. Biotechnol. Prog. 2006, 22, 1474

77. Davidson, L.; Hayes, W. Curr. Org. Chem. 2002, 6, 265

78. Yilmaz, E.; Mosbach, K.; Haupt, K. Anal. Commun. 1999, 35, 167

79. Wulff, G.; Kemmerer, R.; Vietmeier, J.; Poll, H. G. Nouv. J. Chim. 1982, 6, 681

80. Wulff, G.; Vietmeier, J.; Poll, H. G. Makromol. Chem. 1987, 188, 731

81. Shea, K. J.; Sasaki, D. Y. J. Am. Chem. Soc. 1991, 113, 4103

82. Shea, K. J.; Thompson, E. A. J. Org. Chem. 1978, 43, 4253

83. Shea, K. J.; Dougherty, T. K. J. Am. Chem. Soc. 1986, 108, 1091

84. Shea, K. J.; Stoddard, G. T.; Shavelle, D. M.; Wakui, F.; Choate, M.

Macromolecules 1990, 23, 4497

85. Martha, S. V.; David, A. S. J. Org. Chem. 2003, 68, 9604

86. Muldoon, M. T.; Stanker, L. H. Anal. Chem. 1997, 69, 803

87. Dauwe, C.; Sellergren, B. J. Chromatogr. A 1996, 753, 191

88. Masque, N.; Marce, R. M.; Borrul, F. Anal. Chem. 2000, 72, 4122

89. Zhy, L.; Chen, L.; Xu, X. Anal. Chem. 2003, 75, 6381

90. Zhy, Q. Z.; Haupt, K.; Knopp, D.; Niessner, R. Anal. Chim. Acta 2002, 468, 217

91. Masque, N.; Marce, R. M.; Borrul, F. Trends Anal. Chem. 2001, 20, 477

92. Lin, H. Y.; Hsu, C. Y.; Thomas, J. L.; Wang, S. E.; Chen, H. C.; Chou, T. C.

Biosens. Bioelectron. 2006, 22, 534

93. Martha, S. V.; David, A. S. J. Am. Chem. Soc. 2004, 126, 7827

46 Chapter 2

94. Andersson, H. S.; Koch-Schmidt, A.; Ohlson, S.; Mosbach, K. J. Mol.

Recognit. 1996, 9, 675

95. Nicholls, I. A. Chem. Lett. 1995, 1035

96. Yano, K.; Nakagiri, T.; Takeuchi, T.; Matsui, J.; Ikcbukuro, K.; Karube, I.

Anal. Chim. Acta 1997, 357, 91

97. Cheong, S. H.; Rachkov, A. E.; Park, J. K.; Yano, K.; Karube, I. J. Polym.

Sci., A: Polym. Chem. 1998, 36, 1725

98. Andersson, H. S.; Karlsson, J. G.; Piletsky, S. A.; Koch-Schmidt, A.;

Mosbach, K.; Nicholls, I. A. J. Chromatogr. A 1999, 848, 39

99. Andersson, H. S.; Nicholls, I. A. Bioorg. Chem. 1997, 25, 203

100. Whitcombe, H. S.; Martin, L.; Vulfson, E. N. Chromatographia 1998, 47, 457

101. Wulff, G.; Schonfeld, R. Adv. Mater. 1998, 10, 957

102. Lubke, C.; Lubke, M.; Whitcombe, M. J.; Vulfson, E. N. Macromolecules

2000, 33, 5098

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

104. Sellergren, B. Makromol. Chem. 1989, 190, 2703

105. Guyot, A. Syntheses and Separations Using Functional Polymers, Sherrington,

D. C.; Hodge, P. (Eds.), J. Wiley & Sons , New York, 1989, p.1

106. Sellergren, B.; Shea, K. J. J. Chromatogr. A 1993, 31, 635

107. Martin-Esteban, A. Fresenius J. Anal. Chem. 2001, 370, 795

108. Lanza, F.; Hall, A. J.; Sellergren, B. Anal. Chim. Acta 2001, 435, 91

109. David, S.; Gilmore, M. A.; Shea, K. J. J. Am. Chem. Soc. 1997, 119, 4388

110. Yu, C.; Mosbach, K. J. Chromatogr. A 2000, 888, 63


111. Umpleby, R. J.; Rushton, G. T.; Shah, R. N.; Rampey, A. M.; Bradshaw, J.

C.; Berch, J. K.; Shimizu, K. D. Macromolecules 2001, 34, 8446

112. Sharma, A. C.; Borovik, A. S. J. Am. Chem. Soc. 2000, 122, 8946

113. Nicholls, I. A.; Adbo, K.; Andersson, H. S.; Ankarloo, J.; Hedin-Dahlstrom,

J.; Jokela, P.; Karlsson, J. G.; Olofsson, L.; Rosengren, J.; Shorvi, S.;

Svenson, J.; Wikman, S. Anal. Chim. Acta 2001, 435, 9

114. Sellergren, B.; Dauwe, C.; Schneider, T. Macromolecules 1997, 30, 2454

115. O’ Shannessy, D. J.; Ekberg, B.; Mosbach, K. Anal. Biochem. 1989, 177, 144

116. Sellergren, B. Macromol. Chem. 1989, 190, 2703

117. Andersson, H. S.; Karlsson, J. G.; Piletsky, S. A.; Schmidt, K.; Mosbach, K.;

Nicholls, I. A. J. Chromatogr. A 1999, 848, 39

118. Bjarnason, B.; Chimuka, L.; Ramstrom, O. Anal. Chem. 1999, 36, 263

119. Ferrer, I.; Lanza, F.; Tolokan, A. Anal. Chem. 2000, 72, 3934

120. Hwang, C. C.; Lee, W. C. J. Chromatogr. B 2001, 765, 45

121. Baggiani, C.; Trotta, F.; Giraudi, G.; Giovannoli, C.; Vanni, A Anal.

Commun. 1999, 36, 263

122. Andersson, L. I.; Sellergren, B.; Mosbach, K. Tetrahedron Lett. 1984, 25, 5211

123. Andersson, L. I.; Ekberg, B.; Mosbach, K. Tetrahedron Lett. 1985, 26, 3623

124. Molinelli, A.; O’Mahony, J.; Nolan, K.; Smyth, M. R.; Jakusch, M.;

Mizaikoff, B. Anal. Chem. 2005, 77, 5196

125. Cormack, P. A. G.; Elorza, A. Z. J. Chromatogr. B 2004, 804, 173

126. Sellergren, B.; Lepisto, M.; Mosbach, K. J. Am. Chem. Soc. 1988, 110, 5853

127. Tomoi, M.; Oda, H.; Kaikuchi, H. Makromol. Chem. Rapid. Commun. 1987, 8, 339

128. Buyot, A. Pure Appl. Chem. 1988, 60, 365

48 Chapter 2

129. Manzer, M.; Trommsdroff, E. Polymerisation Processes, Schildkencht, C. E.

Skeist, I. (Eds.), Wiley Interscience, New York, 1977, ch. 5

130. Dawkins, J. V. Comprehensive Polymer Science, Eartmond, G. C.; Ledweth,

A.; Segwalt, R. P. (Eds.), Pergamon Press, New York, 1989, 4, 231

131. Piletsky, S. A.; Piletska, E. V.; Karim, K.; Freebairn, K. W.; Legge, C. H.;

Turner, A. P. F. Macromolecules 2002, 35, 7499

132. Davaran, S.; Rashidi, M. R.; Hashemi, M. AAPS Pharmsci. Tech. 2001, 2, 9

133. Liotta, R.; Rose, K.; Hippo, E. J. Org. Chem. 1981, 46, 277

134. Piletsky, S. A.; Piletska, E. V.; Panayuk, T. L.; El’skaya, A. V.; Levi,

R.; Karube, I.; Wulff, G. Macromolecules 1998, 31, 2137

135. Luka, M. F.; Chambers, J. P.; Valdes, E. R.; Thompson, R. G.; Valdes, J.

Anal. Lett. 1997, 30, 2301

136. Takeuchi, T.; Ugata, S.; Tasuda, S.; Matsui, J.; Takase, M. Org. Biomol.

Chem. 2004, 2, 2563

137. Scatchard, G. Ann N. Y. Acad. Sci. 1949, 51, 660

138. Segel, I. H. Enzyme Kinetics: Behaviour and Analysis of Rapid Equilibrium

and Steady state Enzyme Systems, J. Wiley, New York, 1975

139. Joshi, V. P.; Kulkarni, M. G.; Mashelkar, R. A. J. Chromatogr. A 1999, 849, 319

140 Andersson, L. I.; O’Shannessy, D. J.; Mosbach, K. J. Chromatogr. 1990, 513, 617

141. O’Dela Cruz, E.; Mugarurna, H.; Jose, W. I.; Pederson, H. Anal. Lett. 1999, 32, 841

142. O’Brien, T. P.; Snow, N. H.; Grinberg, N.; Crocker, L. J. Liq. Chrom. & Rel.

Technol. 1999, 22, 183

143. Lanza, F.; Sellergren, B. Anal. Chem. 1999, 71, 2092

144. Wulff, G.; Sarhan, A.; Zabrocki, K. Tetrahedron Lett. 1973, 44, 4329


145. Bengtsson, H.; Roos, U.; Anderson, L. I. Anal. Commum. 1997, 34, 233

146. Anderson, L. I.; Muller, R.; Vlatakis, G.; Mosbach, K. Proc. Natl. Acad. Sci. USA

1995, 92, 4788

147. Marcos, M. P.; Gee, S.; Hammock, B. D. Trends Anal. Chem. 1995, 14, 341

148. Hock, B.; Dankwardt, A.; Kramer, K.; Marx, A. Anal. Chim. Acta 1995, 311, 393

149. Anderson, L. I.; Paprica, A.; Arvidsson, T. Chromatographia 1997, 46, 57

150. Kempe, M.; Mosbach, K. J. Chromatagr. A 1994, 664, 276

151. Ramstrom, O.; Nicholls, I. A.; Mosbach, K. Tetrahedron: Asymmetry 1994, 5, 649

152. Siemann, M.; Andersson, L. I.; Mobach, K. J. Antibiot. 1997, 50, 89

153. Kempe, M.; Mosbach, K. J. Chromatogr. A 1995, 691, 317

154. Ramstorm, O.; Anderson, L. I.; Mosbach, K. J. Org. Chem. 1993, 58, 7562

155. Ramstorm, O.; Ansell, R. J. Chirality 1998, 10, 195

156. Ansell, R. J.; Kriz, D.; Mosbach, K. Current Opin. Biotech. 1996, 7, 89

157. Ensing, K.; de Boer, T. Trends Anal. Chem. 1999, 18, 138

158. Adbo, K.; Nicholls, I. A. Anal. Chim. Acta 2001, 435, 115

159. Yu, C.; Mosbach, K. J. Org. Chem. 1997, 62, 4057

160. Matsui, J.; Nicholls, I. A.; Takeuchi, T. Tetrahedron: Asymmetry 1996, 2, 1357

161. Mayes, A. G.; Anderson, L. I.; Mosbach, K. Anal. Biochem. 1994, 222, 483

162. Nilsson, K. G. I.; Lindell, J.; Norrlew, O.; Sellergren, B. J. Chromatogr. A

1994, 680, 57

163. Vallano, P. T.; Remcho, V. T. J. Chromatogr. A 2000, 887, 125

164. Lin, J. M.; Nakagama, T.; Uchiyama, K.; Hobo, T. J. Pharm. Biomed. Anal.

1997, 15, 1351

50 Chapter 2

165. Lin, J. M.; Nakagama, T.; Uchiyama, K.; Hobo, T. Chromatographia 1996, 43, 585

166. Schweitz, L.; Spegel, P.; Nilsson, S. Analyst 2000, 125, 189.

167. Sellergren, B. Anal. Chem. 1994, 66, 1578

168. Martin, P.; Wilson, I. D.; Morgan, D. E.; Jones, G. R.; Jones, K. Anal. Commun.

1997, 34, 45

169. Matsui, J.; Okada, M.; Tsuruoka, M.; Takeuchi, T. Anal. Commun. 1997, 34, 85

170. Mullet, W. M.; Lai, E. P. C.; Sellergren, B. Anal. Commun. 1999, 36, 217

171. Pichon, V.; Bouzige, M.; Miege, C.; Hennion, M. C. Trends Anal. Chem.

1999, 18, 219

172. Mullet, W. M.; Lai, E. P. C. Anal. Chem. 1998, 70, 3636

173. Berggren, C.; Bayoudh, S.; Sherrington, D.; Ensing, K. J. Chromatogr. A

2000, 889, 105

174. Zander, A.; Frindlay, P.; Renner, T.; Sellergren, B.; Swietlow, A. Anal.

Chem. 1998, 70, 3304

175. Matsui, J.; Fujiwara, K.; Takeuchi, T. Anal. Chem. 2000, 72, 1810.

176. Quaglia, M.; Chenon, K.; Hall, A.J.; Lorenzi, E. D.; Sellergren, B. J. Am.

Chem. Soc. 2001, 123, 2146.

177. Castro, B.; Whitcombe, M. J.; Vulfson, E. N.; Vazquez-Duhalt, R.; Barzana,

E. Anal. Chim. Acta 2001, 435, 83

178. Zi-Hu, M.; Qin, L. Anal. Chim. Acta 2001, 435, 121.

179. Rashid, B. A.; Briggs, R. J.; Hay, J. N.; Stevenson, D. Anal. Commun. 1997 ,

34, 303

180. Stevenson, D. Trends Anal. Chem. 1999, 18, 154.

181. Kriz, D.; Berggren-kriz, C.; Anderson, L. I.; Mosbach, K. Anal. Chem.

1994, 66, 2636.


182. Suedee, R.; Songkram, C.; Petmoreekul, A.; Sangkunakup, S.; Sankasa, S.;

Kongyarit, N. J. Planar Chromatogr. 1998, 11, 272.

183. Suedee, R.; Songkram, C.; Petmoreekul, A.; Sangkunakup, S.; Sankasa, S.;

Kongyarit, N. J. Pharm. Biomed. Anal. 1999, 19, 519.

184. Yoshikawa, M.; Izumi, J.; Kitao, T.; Sakamoto, S. Macromolecules 1996,

29, 8197.

185. Yoshikawa, M.; Izumi, J.; Kitao, T. Chem. Lett. 1996, 8, 611.

186. Kobayashi. T.; Wang, H. Y.; Fujii, N. Anal. Chim. Acta 1998, 365, 81.

187. Dzgoev, A.; Haupt, K. Chirality 1999, 11, 465.

188. Armstrong, D. W.; Schneiderheinze, J. M.; Hwang, Y. S.; Sellergren, B.

Anal. Chem. 1998, 70, 3717

189. Porstmann, T.; Kiessig, S. T. J. Immunol. Methods 1992, 150, 5

190. Yarmush, M. L.; Weiss, A. M.; Antonsen, K. P.; Odde, D. J.; Yarmush, D.

M. Biotechnol. Adv. 1992, 10, 413

191. Borrebaeck, C. A. K. Immunol. Today 2000, 21, 379

192. Catt, K.; Niall, H. D.; Tregear, G. W. Nature 1967, 25, 825

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

194. Ye, L.; Cormack, P. A. G.; Mosbach, K. Anal. Commun. 1999, 36, 35

195. Senholdt, M.; Siemann, M.; Mosbach, K.; Anderson, L. I. Anal. Lett. 1997,

30, 1809

196. Anderson, L. I. Anal. Chem. 1996, 68, 111

197. Robinson, D. K.; Mosbach, K. J. Chem. Soc., Chem. Commun. 1989, 14, 969

198 Kriz, D.; Ramstrom, O.; Mosbach, K. Analytical Chemistry News and

Features 1997, 345

52 Chapter 2

199. Dickert, F. L.; Hayden, O. Trends Anal. Chem. 1999, 18, 192

200. Yano, K.; Karube, I. Trends Anal. Chem. 1999, 18, 199

201 Hedborg, E.; Winquist, F.; Lundstrom, I.; Anderson, L. I.; Mosbach, K. Sens.

Actuators A 1993, 796, 36

202 Lahav, M.; Kharitonov, A. B.; Katz, O.; Kunitake, T.; Willner, I. Anal.

Chem. 2001, 73, 720

203. Dickert, F. L.; Tortschanoff, M.; Bulst, W. E.; Fischerauer, G. Anal. Chem.

1999, 71, 4559

204. Dickert, F. L.; Forth, P.; Lieberzeit, P.; Tortschanoff, M. Fresenius J. Anal.

Chem. 1998, 360, 759

205. Jakoby, B.; Ismail, G. M.; Byfield, M. P.; Vellekoop, M. J. Sens. Actuators, A

1999, 76, 93

206. Dickert, F. L.; Thierer, S. Adv. Mater. 1996, 8, 987

207. Haupt, K.; Noworyta, K.; Kutner, W. Anal. Commun. 1999, 36, 391

208. Ji, H. S.; McNiven, S.; Ikebukuro, K.; Karube, I. Anal. Chim. Acta 1999, 390, 93

209. Ji, H. S.; McNiven, S.; Lee, K. H.; Saito, T.; Ikebukuro, K.; Karube, I.

Biosens. Bioelectron. 2000, 15, 403

210. Liang, C.; Peng, H.; Bao, X.; Nie, L.; Yao, S. Analyst 1999, 124, 1781

211. Kriz, D.; Ramstrom, O.; Svensson, A.; Mosbach, K. Anal. Chem. 1995, 67, 2142

212. Suarez-Rodriguez, J. L.; Diaz-Garcia, M. E. Anal. Chim. Acta 2000, 405, 67

213. Kriz, D.; Mosbach, K. Anal. Chim. Acta 1995, 300, 71

214. Jenkins, A. L.; Uy, O. M.; Murray, G. M. Anal. Chem. 1999, 71, 373

215. Kropff, M. J.; Walter, H. Weed Res. 2000, 40, 7


216. Pesticide Manual, Worthing, C. R.; Hance, R. J. (Eds.), British Crop Protection

Council, Farnham, Surrey, UK, IX ed., 1991, 779

217. The Merck Index, XII ed., 1996

218. Muldoon, M. T.; Stanker, L. H. J. Agric. Food Chem. 1995, 43, 1424

219. Siemann, M.; Andersson, L. I.; Mosbach, K. J. Agric. Food Chem. 1996, 44, 141

220. Wittmann, C.; Bier, F. F.; Eremin, S. A.; Schmid, R. D. J. Agric. Food

Chem. 1996, 44, 343

221. Verma, N.; Dhillon, S. S. Intern. J. Environ. Studies 2003, 60, 29

222. Menor-Higueruelo, S.; Perez-Arribas, L. V.; Leon-Gonzalez.; Polo-Diez, L.

M. J. Liq. Chrom. & Rel. Technol. 2002, 25, 445

223. Brodfuehrer, J. I.; Chapman, D. E.; Wilke, T. J.; Pawis, W. Drug Metab.

Dispos. Biol. Fate Chem. 1990, 18, 20

224. Yager, J. W.; Eastmond, D. A.; Robertson, M. L.; Paradisin, W. M.; Smith,

M. T. Cancer Res. 1990, 15, 393

225. Vincent, G. Organic Micro-Pollutants in the Aquatic Environment, Kluwer

Academic Publishers, Dordrecht, 1991

226. Hennion, M. C.; Pishon, V.; Barcelo, D. Trends Anal. Chem. 1994, 13, 361

227. EPA Method 604, Phenols, Environmental Protection Agency, Part VIII, 40,

CFR Part 136, Washington, D.C., Fed. Regist., 26 October 1984, p. 58

228. EPA Method 625, Base/Neutrals and Acids, Environmental Protection

Agency, Part VIII, 40, CFR Part 136, Washington, D.C., Fed. Regist., 26

October 1984, p. 153

229. EPA Method 8041, Phenols by Gas Chromatography: Capillary Column

Technique, Environmental Protection Agency, Washington, D.C., 1995, p.1

230. Bao Wei, S.; Yuan Zong, L.; Wen Bao, C. J. Mol. Recognit. 2001, 14, 388

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