SYNTHESIS AND CHARACTERIZATION OF MOLECULAR

IMPRINTING POLYMER MICROSPHERES FOR SELECTIVE

ADSORPTION OF SYRINGALDEHYDE

RB SRIPRASANTHI A/P BALAKRISHNAN

UNIVERSITI SAINS MALAYSIA

2015

SYNTHESIS AND CHARACTERIZATION OF MOLECULAR IMPRINTING

POLYMER MICROSPHERES FOR SELECTIVE ADSORPTION OF

SYRINGALDEHYDE

by

RB SRIPRASANTHI A/P BALAKRISHNAN

Thesis submitted in fulfilment of the requirements

for the degree of Master of Science

NOVEMBER 2015

ii

ACKNOWLEDGEMENT

First and foremost I would like to thank my supervisor Associate Profesor Dr.

Mohd. Nasir Mohd. Ibrahim for giving me an opportunity to carry out this research,

funding my work through his grants and his priceless guidance. Not forgetting Profesor

Farook Adam who is my co-supervisor who kept my work on track each time I slipped

away and for his indomitable wisdom and guidance.

Also, I would like to thank USM for giving financial support through the

Graduate Assistanship and PRGS-RU Grant which reduced the overwhelming financial

burden I was facing.

I would like to thank my lab buddies, Nor Liyana, Rashid, Nor Hidayah, Nor

Nadiah and Lim and the rest of the gang for their support and co-operation. Besides I

would like convey my sincere gratitude to Encik Ali and Encik Sobri for allowing me to

learn how to use the HPLC and centrifuge.

Then, I would like to offer my humblest gratitude to my sensei Professor

Takaomi Kobayashi for giving me a breakthrough idea and my lab mates in Nagaoka

University of Technology for dealing with me though I could have been an ordeal at

times. I would like to thank the JASSO association for providing the financial support

throughout my stay in NUT.

Before I could end, this thesis would not be here today if it is not for my parents’

faith, love and support. I would like to dedicate my thesis to my late father, without

whom I would not have come so far. Not forgetting each and everyone else who stood

by me through thick and thin and put up with my attitude so that this thesis could see the

light of the day. Lastly, I would like to offer my humblest gratitude to my thesis

examiners for examining and helping me to further improve my thesis. To all, I wish a

million thanks.

iii

CONTENTS

Halaman

ACKNOWLEDGEMENT ii

CONTENTS iii

LIST OF TABLES vi

LIST OF FIGURES viii

LIST OF ABBREVIATIONS xi

LIST OF SYMBOLS xii

ABSTRAK xii

ABSTRACT xv

CHAPTER I INTRODUCTION

1.1 An Overview 1

1.2 Problem Statement 3

1.3 Research Objectives 3

1.4 Research Scope 4

1.5 The outline of the thesis 5

CHAPTER II LITERATURE REVIEW

2.1 Molecular Recognition 6

2.2 Molecules Imprinting Polymer 7

2.2.1 Approaches to synthesis of MIP 8

2.2.2 A summary on MIP synthesis strategies 11

2.3 Syringaldehyde as target molecule 15

2.4 Properties of syringaldehyde 16

2.5 Polymerization of Molecular Imprinting Polymer 19

CHAPTER III MATERIALS AND METHODS

3.1 Introduction 25

3.2 Purification of Starting Materials 25

iv

3.2.1 Recrystallization of syringaldehyde, vanillin

and p-hydroxybenzaldehyde 25

3.2.2 Recrystallization of AIBN 26

3.2.3 Purification of cross-linkers 26

3.3 Synthesis of monomer (SYDM) 27

3.3.1 Recrystallization of monomer (SYDM) 27

3.3.2 FT-IR Analysis of SYDM 28

3.3.3 1H-NMR and

13C-NMR analysis of SYDM 29

3.4 Synthesis of polymer micro particles 29

3.4.1 Parameter I: Duration of Polymerization 31

3.4.2 Parameter II: Molar ratio of SYDM: EGDMA 32

3.4.3 Parameter III: Volume of acetonitrile 32

3.4.4 Parameter IV: Type of Cross-linker (DVB) 33

3.4.5 Control Polymer 33

3.5 Characterization of MIP particles 34

3.5.1 FT-IR analysis of MIP particles 34

3.5.2 SEM and particle size analysis of MIP particles 34

3.5.3 AFM analysis of MIP particles 34

3.6 Binding Experiments 36

3.6.1 Maximum binding capacity 37

3.6.2 Recovery of syringaldehyde 38

CHAPTER IV RESULTS AND DISCUSSION

4.1 Characteristics of monomer (SYDM) 39

4.1.1 FT-IR spectrum of SYDM and its structural analogues 40

4.1.2 1H-NMR characterization of SYDM 41

4.1.3 13

C-NMR characterization of SYDM 42

4.2 Template removal process 43

4.3 FTIR spectrum of semi-covalent imprinting polymer 50

4.4 Parameter I: Polymerization Duration 45

4.5 Growth Kinetics of MIPs during polymerization 51

4.6 Parameter II: Monomer to Cross-linker Ratio 53

4.7 Parameter III: Solvent Volume 59

v

4.8 Parameter IV: Type of Cross-linker (o-DVB) 62

4.9 Adsorption of SDM and SDV on MIP microspheres as

obtained from HPLC 68

4.10 Specific sorption and molecular recognition 70

4.11 Thermodynamic analysis 70

4.12 Adsorption isotherm models 73

4.13 Atomic Force Microscopy Analysis 79

4.13.1 Surface condition of Non-Imprinting Polymers 79

4.13.2 Surface condition of Molecularly Imprinting

Polymers (MIP) 79

4.14 HPLC Analysis of SDM-2 and SDV-3 89

CHAPTER V CONCLUSION & RECOMMENDATIONS 94

REFERENCES 96

LIST OF PUBLICATIONS 106

APPENDIX

A Calibration Curve & HPLC Chromatograms 107

vi

LIST OF TABLES

Table 2.1

Approaches to molecular imprinting

9

Table 2.2

A comparison on various imprinting polymer synthesis

13

Techniques

Table 2.3

Physical properties of syringaldehyde

15

(Ibrahim et al., 2012)

Table 2.4

A summary of bioactive properties of syringaldehyde

17

Table 2.5

A summary of other properties of syringaldehyde

18

Table 3.1

The amount of monomer (SYDM) and cross-linker

32

(EGDMA) used in the synthesis of each polymer

Table 3.2

The amount of monomer (SYDM) and cross-linker

33

(DVB) used in the synthesis of each polymer

Table 3.3

The parameters analyzed using AFM and their

36

definition

Table 4.1

The percent yield of polymerization reaction with

47

respect to time

Table 4.2

Adsorption concentrations at various time and

68

temperature

Table 4.3

Thermodynamic properties of MIP microspheres

73

Table 4.4

Adsorption isotherm models considered in this work

74

Table 4.5

Affinity constant, binding site density, heterogeneity

78

parameter and binding capacity of SDM-2 and SDV-3

Table 4.6

A summary of surface properties and 3D image of the

84

control polymer, NIP-SDM

vii

Table 4.7

A summary of surface properties and 3D image of the

85

imprinting polymer, SDM-2

Table 4.8

A summary of surface properties and 3D image of the

86

control polymer, NIP-SDV

Table 4.9

A summary of surface properties and 3D image of the

87

imprinting polymer, SDV-3

Table 4.10 Calibration data for p-hydroxybenzaldehyde, 90

syringaldehyde and vanillin in HPLC

Table 4.11 Concentration of syringaldehyde in solutions obtained 91

after binding study

viii

Figure 2.1

LIST OF FIGURES

Enzyme-substrate conformation in the catalysis of 6

amino acids to form peptides (Campbell et al., 2011)

Figure 2.2 Theoretical framework of semi-covalent type imprinting 11

Technique (Ibrahim et al., 2009)

Figure 2.3 Structure of syringaldehyde 15

Figure 2.4 The structures of syringaldehyde, Trimethoprim, 16

Bactrim and Biseptol (Ibrahim et al., 2012)

Figure 2.5 An illustration of molecules analogous to syringaldehyde 19

(Ibrahim et al., 2009)

Figure 2.6 An illustration depicting the formation of syringaldehyde 22

binding site

Figure 2.7 Synthesis and hydrolysis of SYDM-co-DVB co-polymer 24

Figure 2,8 Chemical structures of EGDMA and DVB 24 Figure 3.1 The picture above shows the apparatus set-up of 28

(a) synthesis of SYDM and (b) filtration of recrystallized

syringaldehyde methacrylate

Figure 3.2 The picture above shows the apparatus set-up for the 30

polymerization of imprinting polymer microspheres

Figure 3.3 Flowchart of SYDM-co-DVB polymer synthesis 31

Figure 3.4 An illustration of basic functioning methods of AFM 35

Figure 4.1 Synthesis of syringaldehyde methacrylate 39

Figure 4.2 FT-IR spectra of syringaldehyde methacrylate (SYDM) 40

Figure 4.3 The 1H-NMR spectra for SYDM 41

Figure 4.4 The 13C-NMR spectra for SYDM 43

ix

Figure 4.5 Base-catalyzed hydrolysis of SYDM-co-DVB polymer to 44

remove template molecule, syringaldehyde

Figure 4.6 FT-IR spectrum of molecules imprinting polymer synthesized 46

Figure 4.7 Micrographs of SEM images acquired at different hours into 49

polymerization (a)1 hour, (b) 4 hours, (c) 8 hours, (d) 12 hours, (e) 18 hours and (f) 24 hours

Figure 4.8 The growth rate of MIP microspheres during polymerization 50

Figure 4.9 The integral growth rate of MIP microspheres during 52

polymerization

Figure 4.10 SEM micrographs with particle size distribution for the 55-56

different monomer (SYDM) to cross-linker (EGDMA) ratio

used for polymerization with (a) 0.5:10, (b) 1.5:10,

(c) 2.5:10, (d) 3.5:10, (e) 4.5:10 and (f) 5.5:10

Figure 4.11 A cumulative frequency plot against particle size for 57

SDM-1 to SDM-6

Figure 4.12 SEM micrographs of polymerization system with volume of 60-61

acetonitrile used in (a) 250 mL, (b) 200 mL, (c) 150 mL

and (d) 100 mL and their respective distributions

Figure 4.13 A cumulative frequency plot against particle size for 62

SDM-2, SDM-7, SDM-8 and SDM-9

Figure 4.14 SEM micrographs and respective particle size distribution 64-65

for different SYDM : DVB ratio used (a) 0.5:10, (b) 1.0:10,

(c) 1.5:10 and (d) 2.0:10 and their respective particle size

distribution

Figure 4.15 A cumulative frequency plot against particle size for SDV-1 67

to SDV-4

Figure 4.16 A mean particle size plot for all the synthesized MIPs 67 Figure 4.17 Adsorption of SDM and SDV on the surface of templates 69

and on blank surface at 30 0C

x

Figure 4.18 Adsorption of SDM and SDV on the surface of templates 69

and on blank surface at 40 0C

Figure 4.19 Variation of ln(1-Q•w/n0) with 1/T for SDM-2 72

Figure 4.20 Adsorption isotherm of syringaldehyde in (a) Freundlich, 75-76

(b) Langmuir, (c) Freundlich-Langmuir and

(d) Jovanovic models

Figure 4.21 Topographic 2D-AFM images of SDM-2 imprinting 80

polymers at (a) 2.5 µm and (b) 1.0 µm and phase images at

(c) 2.5 µm and (d) 1.0 µm

Figure 4.22 Topographic 2D-AFM images of SDV-3 imprinting 81

polymers at (a) 2.5 µm and (b) 1.0 µm and phase images at

(c) 2.5 µm and (d) 1.0 µm

Figure 4.23 Topographic 2D- AFM images of imprinting polymers at 5 82

µm for (a) SDM-2 and (b) SDV-3

Figure 4.24 Chromatogram obtained at (a) Initial Concentration, C0; 92

(b) After 2 hours of binding experiments; (c) After 14 hours or finalstage of binding experiments and (d) Concentration of syringaldehyde recovered from leaching solution

xi

LIST OF ABBREVIATIONS

MIP Molecules imprinting polymer

SYDM Syringaldehyde methacrylate

DVB Divinyl benzene

EGDMA Ethylene Glycol Dimethacrylate

AFM Atomic force microscopy

NMR Nuclear Magnetic Resonance Spectroscopy

FT-IR Fourier Transform Infrared Spectroscopy

HPLC High Performance Liquid Chromatography

SEM Scanning Electron Microscopy

Ra Average roughness

RMS Root Mean Square

Rq Roughness

Rp Maximum profile peak height

Rv Maximum profile valley depth

Rz Ten Point Height

xii

LIST OF SYMBOLS

C0

Molar concentration before binding

Ct

Molar concentrations after binding

V

Volume

W

Mass

μ0

Chemical potential in standard state

μ

Chemical potential in actual state

R

Universal gas constant, 8.314 J-1

mol-1

K-1

T

Absolute temperature in Kelvin

Q

Absorbance of MIP microspheres

n0

Number of moles

∆Hads

Adsorption enthalpy

∆Sads

Adsorption entropy

B

Quantity adsorbed by the column

k

Affinity constant

q

Binding site density

n

Heterogeneity parameter

S

Binding amount

C

Celcius

xiii

SINTESIS DAN PENCIRIAN POLIMER PERCETAKAN MOLEKUL

MIKROSFERA BAGI PENJERAPAN TERPILIH SIRINGALDEHID

ABSTRAK

Polimer percetakan (MIP) berasaskan molekul siringaldehid yang berbentuk mikrosfera

telah disintesis menggunakan teknik pempolimeran penyulingan-pemendakan serta ciri

fizikal dan kimia, pengecaman spesifik, penjerapan serta sifat pengikatan siringaldehid

terhadap MIP telah dikaji. Pada awalnya, monomer bagi polimer percetakan

menggunakan siringaldehid metakrilat telah disintesis dan dicirikan menggunakan

kaedah FT-IR, 13C-NMR dan 1H-NMR spektroskopi. Seterusnya, monomer tersebut

ditindakbalas dengan dua jenis polimer rangkai-silang iaitu etilena glikol dimetakrilat

(EGDMA) dan divinil benzena (DVB) dengan menggunakan asetonitril sebagai pelarut

serta azobisisobutironitril (AIBN) sebagai penggerak. Pembolehubah yang boleh

dikawal seperti tempoh pempolimeran, nisbah polimer rangkai-silang terhadap

monomer, jenis polimer rangkai-silang dan isipadu pelarut turut dikaji. Kaedah

penganalisaan FT-IR, SEM dan AFM telah dilakukan untuk mengkaji ciri fizikal, kimia

dan struktur mikrosfera MIP. Keupayaan pengikatan serta keafinan MIP yang telah

disintesis ditentukan menggunakan kaedah HPLC. Keupayaan kimia bagi penjerapan

dan keupayaan pengikatan MIP tersebut telah dikaji dengan menggunakan model

isoterma penjerapan Freundlich-Langmuir. MIP yang dihasilkan melalui kaedah ini

didapati berbentuk mikrosfera dengan saiz 1.4-1.7 µm selepas pempolimeran selama 24

jam. Hasil pempolimeran bagi SDM-2 ialah 80.1% dan bagi SDV-3 ialah 78.6%.

Polimer mikrosfera tersebut menunjukkan keafinan dan pemilihan terhadap

xiv

siringaldehid apabila diuji bersama dengan campuran analognya, vanilin dan p-

hidroksibenzaldehid. Kadar nisbah bagi polimer rantai-silang terhadap monomer serta

isipadu pelarut mempengaruhi saiz partikel serta kalakuan pengikatan. Penjerapan

polimer bukan percetakan (NIP) adalah kurang daripada 5 µmol/g. Sedangkan,

penjerapan bagi MIP ialah sebanyak 80% terhadap siringaldehid pada 40 ˚C. Keupayaan

kimia bagi penjerapan siringaldehid pada polimer SDM-2 adalah sebanyak -22.69

kJ/mol, yang mana adalah lebih tinggi, dibandingkan dengan vanilin dan p-

hidroksibenzaldehid.

xv

SYNTHESIS AND CHARACTERIZATION OF MOLECULAR IMPRINTING

POLYMER MICROSPHERES FOR SELECTIVE ADSORPTION OF

SYRINGALDEHYDE

ABSTRACT

The syringaldehyde based molecularly imprinted polymeric microspheres (MIP)

were synthesized using distillation-precipitation polymerization technique, and their

physico-chemical nature, specific recognition, adsorption and binding behaviours

were investigated in the present thesis. Initially, the monomer for the MIP

microspheres (syringaldehyde metacrylate) was synthesized and characterized using

Fourier-Transform Infrared (FT-IR), 13

C and 1H Nuclear Magnetic Resonance

(NMR) technique for compound structural verification and its purity. Subsequently,

the monomer was reacted with two different types of cross-linkers: ethylene glycol

dimethyacrylate (EGDMA) and divinyl benzene (DVB) using acetonitrile as the

solvent and azobisisobutyronitrile (AIBN) as initiator. The controlable variables such

as duration of polymerization, ratio of cross-linker to monomer, types of cross-

linkers and solvent volume were also investigated. FT-IR, Scanning Electronic

Microscope (SEM) and Atomic Force Microscope (AFM) analysis were carried out

to study the physico-chemical and structural properties of the MIP spheres. The

binding capacity and the affinity of the synthesized MIPs were determined using the

HPLC method. The chemical potential for adsorption and binding capacity of the

MIP spheres were also investigated using Freundlich-Langmuir adsorption

isotherms. The resultant MIP were found to be spherical with the size of 1.4-1.7 µm

xvi

after 24 hours of polymerization. The polymer yield obtained for SDM-2 was 80.1%

whereas for SDV-3 was 78.6%. These polymeric microspheres exhibited affinity and

selectivity towards syringaldehyde when tested with a mixture of its analogues,

vanillin and p-hydroxybenzaldehyde. The ratio of the cross linkers against monomer

as well as volume of solvent greatly influenced the particle size and binding

behaviour. The adsorption of non-imprinting polymers was found to be less than 5

µmol/g while the imprinting polymers showed about 80% adsorption at 40 ˚C for

syringaldehyde. The chemical potential for adsorption of syringaldehyde is -22.69

kJ/mol, which is very high, in comparison to vanillin and p-hydroxybenzaldehyde for

the SDM-2 imprinting polymer.

1

CHAPTER 1

INTRODUCTION

1.1 An overview

The science of shape and size recognition has become a treasured skill, for

appearance is an important aspect that identifies an object of its given name such as a

car, a banana plant or a tower. However, we need additional tools to recognize a

bacteria or a molecule which is too small to be identified by our naked eyes. A

widely known fact is that no two molecules are alike. Therefore, many efforts have

been carried out in order to selectively recognize and separate a molecule from its

environment which consists of a mixture of other molecules typically those

synonymous in structure. Interestingly, this process can be attributed to the

fabrication of dentures.

Firstly, an impression of a patient’s dental layout is created by placing a

viscous liquid material on the gums and remaining teeth. This material then gives “a

copy” of the patient’s gums and teeth once it sets into an elastic “mold”. This “mold”

is later used to make a denture that perfectly fits the patient’s mouth cavity. In a way,

this process can be considered as a “macro” imprinting technique. Hence, in order to

develop a recognition method for a molecule, we can employ molecular imprinting

technique which is rather similar to a certain extent, conceptually.

Molecular imprinting technique was defined by (Alexander et al., 2006) as

follows:

The construction of selective ligand in synthetic polymers where a template

(atom, ion, molecule, complex or a macromolecular assembly including micro-

2

organisms) is employed in order to facilitate recognition site formation during the

covalent assembly of the bulk phase by a polymerization or polycondensation

process with subsequent removal of some or all of the template being necessary for

recognition to occur in the spaces vacated by the templating species.

Literally, it means a polymer is shaped and fabricated to meet a target

molecule (template) and when the template is removed, a cavity is left in the polymer

which corresponds to the template shape and size and bonding properties. If this

vacant polymer comes across a molecule exactly like the template molecule, it

recognizes this molecule and binds to it momentarily. These synthetic smart

materials have become a fascination in the field of chemistry due to its recognition

capabilities being at par with those of biological receptors (Vlatakis et al., 1993).

Among the numerous advantages of the molecular imprinting systems are their

robustness over a wide range of pH and temperature while still being able to retain

the recognitive attributes over long term usages (Svenson and Nicholls, 2001).

Another exquisite characteristic of MIPs are their advantage over biological

receptors whereby they can be used in organic solvents without being denatured.

Nevertheless, the main motivation for the research and development of MIPs are

definitely the time and cost that can be saved or an environment-safe procedure when

compared to current common practices in separation like crystallization, solvent

extraction and distillation. The needs for procedures like MIPs is widely justified by

the exponential increase in research publications pertaining to this field (Alexander et

al., 2006).

3

1.2 Problem Statement

Syringaldehyde is a precious compound with a myriad of applications

ranging from pharmaceuticals to biological control in the ecosystem and it naturally

occurs in minute amount. Conventional extraction and purification methods may no

longer be relevant for selective separation at µmol level. Therefore, one of the best

modern tools developed for selective separation or and quantification would be

molecular imprinting especially for substances like syringaldehyde which naturally

coexist with several structural analogues. Furthermore, previous study done by

Ibrahim et. al, 2008 on the purification on vanillin using molecules imprinting

polymer which were fabricated using bulk polymerization. This research serves as a

platform for a more uniform and microspheric imprinting polymers and to

understand adsorption and selectivity properties of these advanced materials.

Syringaldehyde was chosen for this research due to its availability in abundance in

the pulping waste of oil palm empty fruit bunch. This research is expected to

contribute to the conversion of waste into a value-added compound.

1.3 Research Objectives

The objectives of this research are:

i. To synthesize specific molecular imprinting polymer microspheres for

syringaldehyde

ii. To investigate the characteristics of the syringaldehyde imprinting polymer

microspheres

iii. To determine the specificity and adsorptivity of syringaldehyde imprinted

microparticles towards syringaldehyde in a mixture of phenolic aldehydes.

iv. To determine the capacity of the molecular imprinting polymer microspheres.

4

1.4 Research Scope

In order to fulfill the objectives mentioned, the research is divided into

smaller scopes:-

i. The semi-covalent synthesis process for syringaldehyde specific molecular

imprinting polymer is studied in this research in terms of cross-linker to

monomer ratio, duration of polymerization and volume of solvent used.

ii. The two common cross-linkers ethylene glycol dimethacrylate, EGDMA and

o-divinyl benzene, o-DVB are used in this synthesis

iii. The characterization of the monomer is done by Fourier Transform Infra-red,

FT-IR and Nuclear Magnetic Resonance, NMR non-destructive spectroscopic

methods

iv. The characterization of synthesized syringaldehyde imprinting polymers are

carried out using Fourier Transform Infra-red spectroscopy, FT-IR, Scanning

Electron Microscopy, SEM and Atomic Force Microscopy, AFM

v. The adsorption concentration of syringaldehyde for the imprinting and non-

imprinting polymers are measured using High Performance Liquid

Chromatography, HPLC.

5

1.5 The outline of the thesis

This thesis focuses on synthesizing and characterizing molecules imprinting

polymer which are more uniformly shaped and with the capability to adsorb the

intended target molecule i.e, syringaldehyde. In order to fabricate these imprinting

microspheres, common polymerization method called precipitation-distillation is

utilized. To complement the synthesis method, a lesser known semi-covalent

imprinted polymers approach is explored to achieve the objectives of this study. The

properties of the imprinted polymers are studied from the aspect of morphology,

adsorption ability and selectivity. A concise literature review is discussed in the

following chapter pertaining to this study, followed by materials used and methods of

experimentation and the results obtained from the study are analyzed and discussed

in Chapter 4. To conclude the research, a brief summary on this research findings

and recommendations for future are suggested.

6

CHAPTER 2

LITERATURE REVIEW

2.1 Molecular Recognition

Molecular recognition has been and is presently receiving a great deal of

scientific attention due to its vast array of applications. Molecular recognition in

biological contexts can be exemplified by protein-protein interactions, inter-lipid

interactions in membranes and mechanism of protein-based catalysis. Taking the

protein-based catalysis as an example, Figure 2.1 illustrates how the host molecule or

the enzyme recognizes its substrate by shape and conformation. This crucial

phenomenon in biological systems has driven modern chemical scientists to mimic

such systems to lead the development of new technologies.

Figure 2.1 Enzyme-substrate conformation in the catalysis of amino acids to form peptides (Campbell et al., 2011).

7

In this illustration, the amino acids were recognized by the enzyme due to its

morphology. The substrate and only this substrate will fit perfectly in this enzyme for

further reaction or transformation. This idea became the key enabling technology for

modern day separation and sensing materials. Its application at the micro scale has

catapulted molecular recognition to a much higher level. Selectivity and molecular

recognition using molecularly imprinted materials was discovered by Soviet

scientist, M.V. Polyakov in the 1930s who worked on the impression and the effects

of the presence of benzene, toluene and xylene during its preparation using silica

pore structure (Polyakov et al., 1931). However, it was not until 1972 when Wulff

and Klotz reported the preparation of polymers with predetermined ligand selectivity

which marked the technology as it is known today (Andersson and Nicholls, 2000).

They later coined the term molecularly imprinted polymers (MIP) for their functional

polymer. In the past four decades, MIP has become a vast and fast developing field

in molecular recognition.

2.2 Molecular Imprinting Polymers

MIPs are smart materials which have a “memory” of the functional groups

and precise cavity mimicking the template molecule. The embedded key molecules

or templates are then extracted to leave a complementary morphology of the original

template molecules. MIPs are designed in such a way to recognize selectively the

molecules of interest specifically in the presence of structurally and functionally

similar compounds.

Using the enzyme-substrate catalysis as a model representing Fischer’s

“Lock-and-Key” concept, the molecular imprinting technology can be explained as

follows. The intended molecule for imprinting is classified as “target molecules” or

“template molecules” similar to the substrate in the catalysis model. The role of the

8

enzyme is denoted by the functional monomers in MIPs. The “molecular glues” that

bind the substrate to the enzyme in the exemplified model referring to Figure 2.1 are

unanimously called cross-linkers in the MIP system. The cavities formed in the

polymerization arrangement after the removal of target molecules are called “binding

sites”, “receptor sites” or “recognition sites”.

2.2.1 Approaches to synthesis of MIP

Knowing the basic concept of what MIPs consists of; the next step would be

in understanding the “how’s” of developing an imprinting polymer using

syringaldehyde as target molecules. Before that a brief history on the imprinting

methods or approaches are detailed as follows. In general, MIPs can be synthesized

using the covalent, non-covalent or the semi-covalent approaches.

Wulff G. and co-researchers introduced the covalent imprinting methods for

the first time. These researchers successfully synthesized phenyl-R-D-

mannopyranoside imprinted molecules. The polymerization method included 4-

vinylphenylboronic acid as the functional monomer and ethylene glycol

dimethacrylate (EDMA) as a cross-linker (Wulff and Sarhan, 1972). The template

was covalently bonded to the polymer and it had to be cleaved in order for re-binding

processes. During the re-binding process, a reformation of the covalent bond between

the template and the polymer chain was observed. However, to design an appropriate

template-monomer molecule with readily reversible covalent bonds under mild

pressures and temperatures can be a rather arduous and un-economical. At present,

there are three imprinting approaches: covalent, non-covalent and semi-covalent as

shown in Table 2.1.

A non-covalent synthesis method was first experimented by Mosbach K. and

team by polymerizing L-phenylalanine anilide (template) and methacrylic acid as the

9

functional polymer (Mosbach and Kempe, 1995). This pre-arrangement produced a

complex which contains hydrogen bonds and electrostatic bonds between the

monomer and complex. This complex is copolymerized with ethylene glycol

dimethacrylate, a cross-linking agent. The template molecule was extracted from the

polymer matrix which leaves behind active binding sites for molecules with exact

space and bonding characteristics as the template molecule to selectively rebind. This

reversible binding and interactions occurs mainly through hydrogen bonds and even

through ionic interactions or other weak intermolecular forces like Van der Waals

interactions, π-π interactions and others.

Table 2.1 Approaches to molecular imprinting

Imprinting

Approach

Nature of the Bonds

Template and

Monomer

Analytes and

Polymer

Covalent Covalent Covalent Non-covalent Non-covalent Non-covalent Semi-covalent Covalent Non-covalent

As you can see, this method is relatively simpler than the covalent synthesis

method since lesser synthetic steps are involved in the template preparation. The

versatility of the process which permits a wider range of templates to react with

monomers to form the imprinting polymer makes it a better option than covalent

synthetic approach. However, the downside of the non-covalent approach is the

generation of non-specific or heterogeneous reception sites distributed randomly

around the binding sites. As a consequence, undesirable molecules could be attracted

towards these sites leading to low purification yield. The low yield of functional

high-affinity receptor sites (relative) to amount of template present in the pre-

polymerization mixture might have been contributed by the change in morphology

after template extraction process.

10

Therefore, a semi-covalent approach was developed by Whitcombe and co-

researchers. The semi-covalent approach comprises of two main methodologies. The

first method is where the template and monomer is linked directly and another which

uses a spacer or a cross-linker to connect them. In the idea proposed by (Whitcombe

and Vulfson, 2001), a carbonate ester was used to covalently bound the template to

the monomer which when hydrolyzed will release CO2. A ester (COO) link functions

as a sacrificial spacer to link the cholesterol template to a phenolic residue in the

polymer matrix. The theoretical framework for semi-covalent polymerization

technique is given in Figure 2.2.

Semi-covalent imprinting method has surpassed the impediments of

homogenous binding sites, reduced non-specific sites generation and possible

reduced redundant monomers in the imprinting polymer synthesized using the

covalent and non-covalent method. However, there still exist certain limitations to

this approach which is the complete extraction of templates from the polymer matrix.

11

Figure 2.2. Theoretical framework of semi-covalent-type imprinting technique

(Ibrahim et al., 2009)

2.2.2 A summary on MIP synthesis strategies

Using the covalent, non-covalent and semi-covalent imprinting approaches,

the morphology and shape of the MIP can be controlled. The outcome of the MIP

polymerization process such as beads, films and gels largely depends on the

polymerization methods chosen. Choosing the right method and fine-tuning the

procedures can yield a product of functional capability. A summary of MIP synthesis

strategies are summarized in Table 2.2.

Traditionally, MIPs were polymerized by using cross-linkers which produced

monolithic bulk polymers which had to be grinded (Ibrahim et al., 2009). The

12

grinded polymer particles had very irregular shapes and sizes. Once grounded into

powder, there is a high likeliness for the active binding sites to be cleaved and lost.

13

Table 2.2 A comparison on various imprinting polymer synthesis techniques Polymerization

Methods Procedure & Mechanism Product Advantages Disadvantages References

Bulk Monomer, cross-linker, initiator and solvent are polymerized and ground into fine powder.

Irregular particles in shape and size

Easy. Free from surfactant.

Low surface area. Poor purity. Poor yield of active sites.

(Che Ku et al., 2008) (Takeda and Kobayashi, 2005a)

Emulsion Surfactants are added to stabilize monomer micelles in a non-solvent media. Once polymerization is complete, evenly dispersed polymer beads are formed.

Spherical beads obtained, typically< 1µm

Uniform, controllable size, micro to nano scale beads.

Interference by surfactants. Poor purity. Poor yield.

(Dvorakova et al., 2010)

Precipitation Monomers and other components are dissolved in a desired media. The polymer precipitates to form spherical particulate.

Spherical beads 1µm<id<10 µm

Fairly simple. No surfactants. High purity.

The polymer beads obtained are not necessarily uniform. Poor yield.

(Wang et al., 2011)

Suspension/ Dispersion

Droplets of monomer, initiator and solvent are dispersed in a continuous phase is maintained by continuous mechanical stirring whereby usage of surfactant is optional. The polymer beads are formed from these droplets once polymerization is completed.

Spherical beads 20µm<id<200µm

Fairly simple. Good yield. High purity.

Interferences by surfactants (if used). Particle size is in a wide range.

(Son et al., 2011)

14

Table 2.2 A comparison on various imprinting polymer synthesis techniques Polymerization Methods

Procedure & Mechanism Product Advantages Disadvantages References

Multi-step swelling

Seeds are produced using emulsion polymerization technique and made to swell in an activating solvent. The monomers, cross-linkers and other components are dispersed in the solvent. The resultant polymers are droplet-like beads absorbed on the swollen seeds surface.

Spherical beads 1µm<id<20µm

Excellent packing for HPLC column. Good yield. High purity. Very effective.

Complex process and expensive.

(Hoshina et al., 2011, Yang et al., 2010)

Imprinting on pre-formed beads

MIP layer is adsorbed on the surface of pre-formed particles by modifying or grafting method to produce composite beads.

Depends on the pre-formed particles size

High separation capacity. Special functions. Good yield. High purity. Very effective.

Complex. High Cost.

(Yang et al., 2010)

15

2.3 Syringaldehyde as target molecule

Over the years, however, a meager number of articles dedicated to

syringaldehyde have been published. This can probably be attributed to its simple

synthesizability and the lack of understanding of its true capacities. The molecular

structure of syringaldehyde is given in Figure 2.3 and its physical properties are

provided in Table 2.3.

O

OH

O

O

Figure 2.3 Structure of syringaldehyde

Table 2.3 Physical Properties of Syringaldehyde (Ibrahim et al., 2012) Property Data

Molecular Formula C9H10O4 CAS number 134-96-3 Molecular Weight 182.18 Physical State Solid (yellow-brown) Melting Point 110-113 ˚C Boiling Point 192-193 ˚C Water Solubility Insoluble

[

16

2.4 Properties of syringaldehyde

Advancements in analytical instruments coupled with breakthroughs in

chemistry and pharmacology have allowed for the identification, quantification, and

isolation of phenolic aldehydes for the diverse applications such as antioxidants,

antifungal or antimicrobial, and anti-tumorigenesis agents in pharmaceuticals.

Syringaldehyde is vastly used as a precursor for pharmaceutical antifungal products

like Trimethoprim and Bactrim (Tripathi et. al). The structures of Trimethoprim and

Bactrim are given in Figure 2.4. In the food industry there is also a tendency to

utilize naturally occurring flavor compounds that exhibit antioxidant and

antimicrobial properties, hence providing a potential source of non-synthetic

preservatives and additives. Only preliminary in vitro tests have been reported in

most cases, but a new potential research area and application of syringaldehyde has

been identified. Keeping this in mind, some of the reported properties of

syringaldehyde are exemplified in Tables 2.4 and 2.5.

Figure 2.4 The structures of syringaldehyde, as well as Trimethoprim, Bactrim, or Biseptol (Ibrahim et al., 2012)

17

Table 2.4 A summary of bioactive properties of syringaldehye Bioactive

Properties Description References

1.

Antioxidant capacity

Wines aged in oak barrels exhibits increased levels of antioxidants due to the presence of phenolic aldehydes like syringaldehyde.

Phenolic extracts containing syringaldehyde from wild rice hulls and olive tree pruning.

Syringaldehyde shows high antioxidant capacity when tested using Trolox C and DPPH method.

The dimethoxy substitution in syringaldehyde as well as its syringol moiety was acknowledged for exhibiting enhanced antioxidant properties. Test conducted using Trolox and CB assays.

Syringaldehyde-based dendrimers exerted strong antioxidant character-istics but reduced pro-oxidant (mutagenicity and carcinogenicity on healthy cells) effects.

(Canas et al., 2008) (Matejícek et al., 2005) (Asamarai et al., 1996), (Bortolomeazzi et al., 2007) (Boundagidou et al., 2010) (Cao et al., 1997) (Lee et al., 2009)

2.

Antimicrobial/antifungal activity

Promising effect against Candida guilliermondii Tested against strains of Plasmodium falsiparum and showed a mild anti-malarial

activity with an IC50 value of 21.4 ± 8.2 µg mL-1 Reduced the population of Staphylococcus aureus (Gram +), Klebsiella pneumonia

(Gram -), and Pseudomonas aeruginosa (Gram -) by 70% using the ASTM Standard Test Method E2149-01 on paper and pulp.

(Gurpilhares et al., 2006, Kelly et al., 2008, Cheplogoi et al., 2008, Fillat et al., 2012)

3.

Anti-oncogenic property

Syringaldehyde was noted to show potency in inhibiting the formation of OPBA and NNAL (lung oncogenesis metabolites) in hepatic microsomes.

Syringaldehyde synergetically with seven other compounds showed a high potential in inhibiting the proliferation of HCT-116 cancer cell lines.

Morse et al. (1995) (González-Sarrías et al., 2012)

18

Table 2.5 A summary of other properties of syringaldehye Other

Properties Description References

1. Mediator First natural mediators in the degradation of indigo carmine by bacterial laccase

A redox mediator for recalcirant dye de-colorization Syringaldehyde shows high antioxidant capacity when tested using

Trolox C and DPPH method. Bio bleaching and delignification of pulp and paper. A mediator with both oxidoreductive enzymes and inorganic catalysts for

the conversion of cyprodinil

(Kawai et al., 1989), (Singh et al., 2007) (Satar and Husain, 2009) Camarero et al. (2005), (Camarero et al., 2007) (Murugesan et al., 2009) (Moldes et al., 2008) (Kang et al., 2002), Dec et al., 2004

2. Inhibitors for hydrolysis

Inhibition of xylose conversion into xylitol by Gluconobacter oxydans Tested against strains of Plasmodium falsiparum and showed a mild anti-

malarial activity with an IC50 value of 21.4 ± 8.2 µg mL-1 Reduced the population of Staphylococcus aureus (Gram +), Klebsiella

pneumonia (Gram -), and Pseudomonas aeruginosa (Gram -) by 70% using the ASTM Standard Test Method E2149-01 on paper and pulp.

Selective inhibition of urease instead of α-chymotrypsin that prevents peptic ulcers.

(Zaldivar et al., 1999) (Buchert and Niemela, 1990), (Delgenes et al., 1996, Taherzadeh et al., 1999, Klinke et al., 2004, Klinke et al., 2001)

3. Organic markers in wood smoke

Used as a molecular marker for biomass smoke to monitor global climate Syringaldehyde synergetically with seven other compounds showed a

high potential in inhibiting the proliferation of HCT-116 cancer cell lines.

(Simoneit et al., 1995), (Robinson et al., 2006), (Simoneit, 2002)

4. Biological activity

Agrobacterium tumefaciens virulence gene inducer that causes death of Acanthoscelides obtectus beetles that destroys crops.

(Delmotte et al., 1991, Lee et al., 1996), (Regnault-Roger et al., 2004)

19

2.5. Polymerization process of the MIPs

The molecular imprinting system reported in the present thesis is a semi-

covalent system which is less explored. In general, the semi-covalent polymerization

process comprises of a monomer, cross-linker (o-DVB and EGDMA), template

molecule (syringaldehyde), initiator (AIBN) and a porogenic solvent. The template

molecule is similar in geometry and analogous in structure to vanillin and p-

hydroxybenzaldehyde. Figure 2.5 illustrates the structural similarity of p-

hydroxybenzaldehyde, vaniline and syringaldehyde respectively. The MIPs exhibits

high affinity and selectivity towards the family of related compounds due to similar

functional group and structural make-up. Thus, both MIPs and NIPs (non-imprinting

polymer) are synthesized to determine the selectivity of the MIP towards

syringaldehyde as well as its analogues. In order to assess the selectivity of MIPs

towards syringaldehyde, reference polymers or blanks are synthesized without

involving any template molecules which are called NIPs.

Figure 2.5 An illustration of the molecules analogous to syringaldehyde (Ibrahim et al., 2009)

Optimum processing parameters for the synthesis of MIP for syringaldehyde

were investigated with different factors which are most likely to impact the

20

selectivity and reusability of the MIP towards syringaldehyde. For this purpose, the

purity of all starting materials are ensured through recrystallization and only HPLC

grade acetonitrile is utilized for synthesis and rinsing. Deoxygenated nitrogen gas

was used to purge off oxygen from the pre-polymerization solution. Besides factors

like polymerization technique, duration of polymerization, ratio of monomer to

cross-linker and volume of solvent used are examined as well. Two types of cross-

linkers were used in order to understand the influence of bonding types on the batch-

binding properties of imprinted polymers.

In the semi-covalent polymers, o-DVB and EGDMA were used as cross-

linker and their fraction was high in the polymer. In many other protocols, both o-

DVB and EGDMA had been successfully employed in non-covalent synthesis

methods (Takeda and Kobayashi, 2005b). The cross-linker, o-DVB is styrenic and

hydrophobic in nature (Cormack and Elorza, 2004) which is copolymerized with

syringaldehyde methacrylate to produce a narrow disperse microspheres with active

carboxylic acid groups. In order to synthesize this semi-covalent polymer,

syringaldehyde was reacted with methacryloyl chloride to form a methacrylate in the

first stage. Subsequently, the monomer, SYDM was co-polymerized with o-DVB to

form the MIP-template network. Syringaldehyde methacrylate is more likely to

encourage the formation of inter-chain hydrogen bonding between the carboxylic

acid groups to reduce the polymer-solvent interactions during the distillation

precipitation copolymerization (Wang et al., 2007). Upon hydrolysis, the template

molecule is released and an imprinting cavity with active sites is available for the

entrapment of target syringaldehyde molecules. Such method is exclusive for the

semi-covalent imprinting technique.

21

From previous studies (Quesada-Molina et al., 2012, Valero-Navarro et al.,

2009) the most common method of synthesizing MIP is by adding the template

molecule to an independent monomer, cross-linker and an initiator in a solvent

media. The shortcome of this method is that the mechanism by which all these inputs

actually form the MIP of interest remain a little vague. However, this shortcome is

addressed by the semi-covalent imprinting method by synthesizing a monomer which

contains the template molecule. In addition, the template molecule can be easily

removed through hydrolysis for the semi-covalent method (Takeda and Kobayashi,

2005b, Poma et al., 2010) The reaction outline of copolymerization of SYDM-co-

DVB and its hydrolysis process is illustrated in Figure 2.6 and Figure 2.7.

22

Figure 2.6 An illustration depicting the formation of syringaldehyde binding site

23

Acetonitrile was chosen as the porogenic solvent because it solubilizes the

template even in high concentration. Furthermore, using neat acetonitrile aids the

formation of less distorted and more spherically shaped microspheres which will

produce uniformed distribution regardless of a styrenic or vinylic cross-linker usage

(Son et al., 2011). It exhibited a rather good thermal stability during batch-binding

reactions. Nevertheless, the solvent chosen should soluble the templet but it should

not dissolve the resulting polymer. Therefore, acetonitrile is the best solvent for this

purpose.

A mono-vinyl cross-linker, EGDMA was also selected to synthesize

imprinting polymer microspheres. The relatively low viscosity of EGDMA enables it

to homogenize the monomer mixture prior to polymerization (Muhammad et al.,

2012). Meanwhile, the methacrylate functional group was used in an attempt to allow

formation of H-bonds between the template molecule (syringaldehyde) and polymer

matrix. The SYDM (monomers) are hydrophilic in nature thus expected to promote

inter-chain hydrogen bond formation among the carboxylic acid groups. These inter-

chain H-bonding in return reduced the solvent-monomer interactions and increased

the interactions of the monomer with EGDMA or o-DVB during distillation process.

A similar observation was reported by (Bai et al., 2006). The structures of o-DVB

and EGDMA are given in Figure 2.8.

24

Figure 2.7 Synthesis and hydrolysis of SYDM-co-DVB co-polymer

Figure 2.8 Chemical structures of EGDMA and o-DVB

O

O

O

O O

O

OO

OO

OO

O O

O

OH

O

OHO

+

Hydrolysis

DVBSYDM

OH

O O

O

+

SYDM-co-DVB copolymer

SYDM-co-DVB imprinting polymer

syringaldehyde

OO

O

O

Ethylene glycol dimethacrylate (EGDMA)

o-DVB p-divinylbenzene (DVB)