Binding of IMP

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BINDING PROPERTIES OF A MOLECULARLY IMPRINTED POLYMER

WITH LEAD ACETATE FOR SEPARATION AND DETERMINATION OF
LEAD
A Thesis
Presented to
The Faculty of the Graduate School
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In Partial Fulfillment
of the
Requirements for the Degree
MASTER OF SCIENCE IN CHEMISTRY
LUCY AUSTRIA MAGLEO-DOMINGO

October 2008
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APPROVAL SHEET
In partial fulfillment of the requirements for the degree MASTER OF

SCIENCE IN CHEMISTRY, this thesis entitled “ BINDING PROPERTIES OF
MOLECULARLY IMPRINTED POLYMER WITH LEAD ACETATE FOR
THE SEPARATION AND DETERMINATION OF LEAD” has been prepared
and submitted by Lucy Austria Magleo – Domingo, who was recommended
for oral examination.
___________________________
MARIETTA A. ILAO, Ph.D.
Adviser
Approved by the Committee on Oral Examination with the grade of

______.
______________________________________
JOSE GENARO R. YAP – AIZON, Ph.D.
Chairman
__________________________ ____________________________
NORITA E. MANLY, Ph.D. DOROTEA T. OUANO, Prof.
Member Member
Comprehensive Examination Grade: 1.8

Date: October 2006
Accepted and approved in partial fulfillment of the requirements for the

degree MASTER OF SCIENCE IN CHEMISTRY.
_____________________________________
JOSE GENARO R. YAP–AIZON, Ph.D.
Dean, Graduate School
Date: ________________
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DEDICATION
I dedicate this masteral thesis to my One and True God.
To my family, my father 2nd Lt. Francisco Poyaoan Magleo and to my mother
Aldegunda Radam Austria-Magleo, thank you for giving me life and the
freedom to make decisions.
To my siblings, Kuya Boy, Ate Bheng, Kuya Rolly, Rolan, Es, Sato and Malen; to
my nephews and nieces at the same time my godchildren for their love and
inspiration.
To my dearly beloved true friends, in appreciation for the love and support.
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ACKNOWLEDGMENT
God is so good all the time and I would like to thank HIM for the
guidance, wisdom and strength. Without HIS overwhelming Love and
Provisions, this study would not have been possible. TO GOD BE THE GLORY
FOREVER!.
My sincerest thanks go to the following persons who have shared their
contributions to make this research successful:
Dr. Marietta A. Ilao, my adviser and consultant, for being patient,
helpful and understanding.
To the members of the defense panel, Dr. Jose Genaro R. Yap-Aizon , Dr.
Norita E. Manly and Prof. Dorotea T. Ouano, for their precious critical insights
and propositions geared towards the improvement of this research. Special
mention goes to Ms. Noreen Adonis-Gusi for her accommodation and support.
Dr. Cristina C. Salibay and Dr. Carmelita C. Cervillon, former and
concurrent Deans of DLSU-Dasma/College of Science and Engr. Imelda C.
Galera, chairperson of our Physical Sciences Department/College of Science,
DLSU-Dasmarinas for their permission to use the Chemistry Research
Laboratory.
Engr. Wilfredo Aguinaldo, previous Physics and Chemistry Research
Laboratory Coordinator and Engr. Jennifer Binas, current Chemistry/Physics
Laboratory Supervisor for their assistance in the CRL facility.
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Ms. Jane and Ms. Aimee, DLSU-Dasmariήas Chemistry Laboratory
Technicians, for their assistance during my experimentation.
Mr. Mike Ajero and Mr. Irving Chiong of the Analytical Services of the
Chemistry Department of De La Salle Manila, for his serenity during the
spectroscopic analyses of all my samples.
Mr. Rey Coria, technician of the DLSU-Manila Solid State Laboratory
and Ms. Prane Ong for their help in the surface and elemental analyses of all the
polymer samples.
Engr. Mildred Amador-Reyes and Atty. Myrna Villegas-Alcazar of the
USA and Ms. Noemi Ruth Quibote of EMB-DENR for sharing their other hand
in the acquisition of my much needed chemicals.
Engr. Rey Madelar of Petron and his wife, Lorna for providing me the
aviation gas sample and my other batchmates-friends from Mapua Institute of
Technology such as Bing Nerona-Caberte and Shirley Mercado-Bueno for their
inspiration.
Engr. Lani C. Antonio, Engr. Marina A. De Salit and Ms. Susan Sta Ana,
for lending me their thesis manuscript.
To all the other beautiful people of the College of Science -Physical
Sciences Department: Mam Jhaye, Mam Vernie, Mam Leng, Mam Betcha, Sir
Gidz, Sir Loy, Sir Peace, Sir Paul, Sir Mike, et al. for their help, encouragements
and heartfelt concerns.
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To my church family, especially Pastor Mike Clemeήa and Sis. Vangie
Baccar for their prayers and support.
To my niece Rona Jamaira Magleo, who had helped me in the internet
browsing and surfing for additional information related to this study.
And lastly to my parents who had instilled in me the virtue of patience,
perseverance and long-suffering.
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ABSTRACT
Title : BINDING PROPERTIES OF A MOLECULARLY

IMPRINTED POLYMER WITH LEAD ACETATE
FOR THE SEPARATION AND DETERMINATION
OF LEAD
Researcher : LUCY A. MAGLEO-DOMINGO
Adviser : DR. MARIETTA A. ILAO
Degree : Master of Science in Chemistry
Date Completed : October 2008
This research was carried out to prepare a molecularly imprinted
polymer potentially selective to lead acetate compound, lead nitrate solution
and tetraethyl lead from an aviation gasoline sample.
Characterization of the binding properties of these lead compounds with
the MIP and its use as an adsorbent/stationary phase for extraction and
separation processes were experimented. A simple sample extraction technique
(MEPS) microextraction in packed syringe was applied.
Free-radical polymerization was employed using methacrylic acid as the
monomer; N,N’-methylene bisacrylamide, the crosslinker; potassium
persulphate, the initiator; lead acetate, the template and distilled water as the
porogen. Polymerization time was 36 hours at 80oC temperature.
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Binding properties were investigated through a Scanning Electron
Microscope with Energy Dispersive X-rays capability. Binding sites were
characterized by IR Spectroscopy and Ultraviolet-Visible Spectrophotometry.
The correlation of the amount of lead adsorbed by the MIP coming from
the aviation gasoline sample and from lead nitrate solution was aided by the
Atomic Absorption Spectrometer.
Statistically, the decision that there is a significant difference in the
amount of lead present in the samples before and after extraction directs to a
conclusion that the type of a molecularly imprinted polymer prepared in this
research can be used as an adsorbent or a stationary phase in a column for the
selective quantification of lead present in lead compounds like lead acetate and
lead nitrate.
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TABLE OF CONTENTS
Title Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
Approval Sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii
Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv
Research Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
List of Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
CHAPTER PAGE
1 THE PROBLEM AND ITS SETTING . . . . . . . . . . . . . . . . 1
Background of the Study . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Statement of the Problem . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Significance of the Study . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Scope and Delimitation . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2 THEORETICAL FRAMEWORK . . . . . . . . . . . . . . . . . . . . 11
Review of Literature
a. Conceptual Literature . . . . . . . . . . . . . . . . . . . . . . . . . . 11
b. Related Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Conceptual Paradigm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Conceptual Framework . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Definition of Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3 METHODOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Research Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Research Locale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Research Reagents and Materials . . . . . . . . . . . . . . . . . . 40
Data Gathering Procedure . . . . . . . . . . . . . . . . . . . . . . . . 41
Data Analysis Procedure . . . . . . . . . . . . . . . . . . . . . . . . . 48
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4 RESULTS, ANALYSIS AND INTERPRETATION . . . . . 49
5 SUMMARY, CONCLUSIONS AND

RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
CURRICULUM VITAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
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LIST OF TABLES
1) Table 1. Solubility Tests on Monomer, Crosslinker, Initiator and

Template …………………………………………… . . . . . . . . . . 50
2) Table 2. Experiment on the amount of porogen used and results

on the observed physical properties of polymers
produced ……………………………………………………. . . . . 51
3) Table 3. Summary of IR Spectral Data for Undistilled MAA …. … . . . 53
4) Table 4. Summary of IR Spectral Data for Distilled MAA ………. . . . . 55
5) Table 5. Comparison between the use of EGDMA and N,N’-

Methylene bisacrylamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6) Table 6. Experiment on the best polymerization temperature and

time …. ……………………………………………………… . . . . 62
7) Table 7. Summary of IR Spectral Data of the PP ……………….. … . . . 67
8) Table 8. IR Spectral Data of the NIP ……………………………….. . . . . 67
9) Table 9. Outline of IR Spectral Data of the MIP . . . . . . . . . . . . . . . . . . . . 67
10) Table 10. Elemental Analysis of the NIP using EDX . . . . . . . . . . . . . . . . 69
11) Table 11. EDX analysis of the PP ……………………………………. . . . 71
12) Table 12. Elemental Analysis of the MIP …………………………… . . . 72
13) Table 13. Comparison of the % Element of experimented

Polymers …………………………………………… . . . . . . . . . 73
14) Table 14. EDX Analysis of treated PP . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
15) Table 15. EDX Analysis of MIP rebinding with Lead Acetate . . . . . . . . 80
16) Table 16. Outline of IR Spectra of MIP rebinding with Lead Acetate . . 81
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17) Table 17. Elemental Analysis of MIP with Lead Nitrate ……………… 84
18) Table 18. Elemental Analysis of MIP with aviation gasoline…………. 89
19) Table 19. Concentration of Lead in samples …………………………….93
20) Table 20. Summary of Statistical Data ……………………………………95
21) Table 21. List of Matches for Aviation gasoline…………………………123
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LIST OF FIGURES
1) Figure 1. Molecularly Imprinted Polymer ………………………………. 14
2) Figure 2. Principles of Molecular Imprinting …………………………… 16
3) Figure 3. Paradigm of the Study …………………………………………. 35
4) Figure 4. UV-Visible Spectra of MAA …………………………………… 52
5) Figure 5. IR Spectra of Undistilled MAA ………………………………… 54
6) Figure 6. IR Spectra of Distilled MAA …………………………………… 56
7) Figure 7. UV-Visible Spectra of Lead Acetate and MAA ……………… 57
8) Figure 8. Scanning Electron Microscopy Results ………………………. 60
9) Figure 9. FT-IR Spectra of PP, NIP and MIP …………………………… 66
10) Figure 10. SEM Surface Structure of NIP ………………………………. . 68
11) Figure 11. Sample view of PP using 2 different magnification ………. 70
12) Figure 12. SEM of MIP before and after template removal …………… 74
13) Figure 13. UV-Vis spectra of collected filtrate ………………………….. 77
14) Figure 14. SEM of PP using a chelating agent and concentrated nitric
acid for removal of template …………………………………. 78
15) Figure 15. SEM of MIP binding with Lead acetate solution . . . . . . . . . . 79
16) Figure 16. IR Spectra of MIP binding with Lead acetate ……………… 82
17) Figure 17. SEM surface structure of MIP before and after binding with
Lead nitrate …………………………………………………… 83
18) Figure 18. IR Spectra of MIP binding with Lead nitrate ………………. 86
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19) Figure 19. SEM surface structure of MIP before and after the aviation
gasoline passed through ……………………………………… 88
20) Figure 20. IR Spectra of the Aviation gasoline sample ………………… 91
21) Figure 21. IR Spectra of MIP with the aviation gasoline sample ……… 92
LIST OF APPENDICES
1) Appendix A. Pictures of Reagents, PP and MIP ……………………….. 110
2) Appendix B. Pictures of DLSU Research Laboratories ……………….. 111
3) Appendix C. Sample computation of t-Test statistical tool …………… 112
4) Appendix D. Graphs of EDX Elemental Analysis …………………….. 114
5) Appendix E. Overlay Figures of IR Spectra ……………………………. 121
6) Appendix F. List of Matches for Aviation Gasoline…………………… 123
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Chapter 1
THE PROBLEM AND ITS SETTING
A technique called molecular imprinting has become popular in science
and technology most specifically in the fields of analytical chemistry and
biochemistry [Sellergen, 2001]. Molecular imprinting technology is the creation
of selective recognition sites in synthetic polymers utilizing suitable templates.
This imprinting method is quite simple and easy to perform which
involves the mixing of the imprint molecule with selected monomers, then
polymerization and finally, removal of the print molecule from the prepared
polymer. After washing, the resulting polymer now possesses a fingerprint of
the print molecule [Cada, et al, 2002].
From being a comprehensive concept, numerous strategies for still wider
and more versatile applications have been proposed and examined. Although
fundamental concepts have already been realized and several polymers are
now proceeding to practical application, this imprinting technique is still
growing [Schweitz, et al,2003].
Molecularly imprinted polymers (MIPs) are often compared to natural
antibodies although there are still wide gaps existing between them [Mosbach,
et al, 1996]. They are becoming more important for several application areas
such as, its use in the chiral and substrate-selective separations as well as
isolations of different components; as antibody and receptor mimics in
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competitive ligand binding assays[Vlatakis, et al, 1993] and diagnostic
applications ; as enzyme mimics in catalytic applications and in biosensor-like
devices [Kriz, et al, 1997].
This study explored on applying molecular imprinting technique in
preparing a stable synthetic polymer that possesses selective molecular
recognition properties. The molecularly imprinted polymer produced in this
research had a fingerprint of lead acetate on it. Investigation on the recognition
sites within the polymer matrix was accomplished by the aid of several
analytical instruments. The application of the MIP as a stationary phase or
adsorbent in any column for quantitative determination of lead present in
gasoline samples and/or other compounds containing this metal was also
experimented.
The hazardous contribution of lead in the environment and its adverse
effects on humans prompted the researcher to perform this thesis. Lead per se is
insoluble, but many lead compounds dissolve in water and act as neurotoxins
in the body. Insoluble lead compounds have been used in paints including lead
chromate for “yellow lines” and in petrol to improve the rating.
Tetraethyl lead(TEL) when put into petrol helps it to burn more slowly
and smoothly preventing knocking and giving higher octane ratings. TEL
improves the combustion characteristics of the petrol through its octane
number but when it burns in an engine, lead oxide is formed. Another additive
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to gasoline is ethylene dibromide and on burning, the resulting mixture forms
products that react with the lead oxide to form lead bromide, a toxic and
volatile compound that escapes from the engine with other exhausted products.
The use of tetraethyl lead in fuels, particularly in automotive gasolines has been
restricted due to health and environmental concerns [Seyferth, 2003]. However,
aviation gasolines have been allowed to contain tetraethyl lead since no suitable
substitute has been found with adequate knock resistance that will make
current fleet of aircraft engines to operate properly.
This process of lead contaminating the air brought environmentalists
and scientists to devise methods and suggest alternatives for the reduction and
even removal of this hazardous component.
Another disadvantage of lead is that it would poison the catalytic
converter (a standard equipment for cars) in the exhaust pipe. Catalytic
converters that serve to oxidize unburned hydrocarbons, carbon monoxide, and
nitrogen oxides contain platinum group metals (Ni, Pd & Pt) are inactivated by
lead [Lansdown & Yule, 1986].
Heavy metals like lead is a common effluent in industries such that it
has become the subject of many researches owing to its harmful effects on
living things [Ajero, 2002].
Particulate metals in air result from activities such as fossil fuel
combustion (including vehicles), metal processing industries and waste
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incineration. According to Hernberg, there are currently no EC standards for
metals other than lead, although several are under development. Lead is a
cumulative poison to the central nervous system, particularly detrimental to the
mental development of children [Singhal & Thomas, 1980].
Background of the Study
Although there had been extensive studies on molecularly imprinted
polymers for the last few decades, elucidation of reactions between these and
metal coordinated compounds specifically with heavy metal like Lead (Pb)
have not been entirely experimented [Steinke, et al]. Most of the scientific
researches used pure organic and bio-organic compounds as the template
[Komiyama, et al, 2003]. Thus, this study investigated the probability of lead
acetate’s binding properties with the imprinted polymer for selective
recognition and quantification of lead and other lead compounds like lead
nitrate.
An increased number of lead poisoning and other hazardous effects of
its compounds to an individual and the environment formed the basis for the
preparation of a molecularly imprinted polymer. An update by Ms. Amita
Legaspi of GMA News broadcasted on TV 08/04/2007 at 05:21 PM said that
“The Department of Trade and Industry (DTI) has ordered the Bureau of Food
and Drugs (BFAD) to test Fisher – Price toys made in China for high lead
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content”.
Another news gathered from the Philippines News.Net, Wednesday 5th
September, 2007 stressed that Mattel, the company who distributes dolls, has
apologized for the incident after having to take back 18 million China-made
toys because their paint was found to contain lead.
Toys made in China are not the only products laced with dangerous
heavy metals. Lipsticks manufactured in the United States of America also
contained surprisingly high levels of lead, according to new product tests
released October 11, 2007 by the Campaign for Safe Cosmetics. An independent
laboratory conducted the lead tests in September 2007 on red lipsticks bought in
Boston, Hartford, Connecticut, San Francisco and Minneapolis. Among the top
brands testing positive for lead were: L’Oreal Colour Riche “True Red” – 0.65
ppm; L’Oreal Colour Riche “Classic Wine” – 0.58 ppm; Cover Girl Incredifull
Lipcolor “Maximum Red” – 0.56 ppm; and Christian Dior Addict “Positive
Red” – 0.21 ppm. [Hartman, Brian, 2007]
Promoting green chemistry by using water as the solvent in preparing
the molecularly imprinted polymer was another consideration. Green
chemistry, also called sustainable chemistry, is a chemical philosophy
encouraging the design of products and processes that reduce or eliminate the
use and generation of hazardous substances. The usual solvents used in MIP
preparations are acetone, chloroform, acetonitrile, toluene, dodecanol and other
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organic solvents. Ionic liquids are also potential green solvents but are still
expensive as of todate. On-going studies for the preparation of these types of
liquids are on its way.
Detection and analysis of lead are mostly done inside an analytical
chemistry laboratory using sophisticated and automated instruments as well as
a technician. Molecularly imprinted polymers can be made portable and handy
to be used for field sampling and detection of unknown substances like lead
compounds. Although devising a standard method is required here and
method validation would entail another study, potential use of the MIP is
interesting and encouraging.
Statement of the Problem
The objective of the study was the preparation of a molecularly
imprinted polymer (MIP) using lead acetate as template for investigation on
whether this template undergoes molecular imprinting and will give rise to
isolated molecular binding sites. Whether these binding sites are able to bind
with compounds containing lead and rebind with the same template was also
experimented. The MIP prepared was employed for the quantitative
measurement of the presence of lead in samples like aviation gasoline and lead
nitrate solution.
Specifically, the researcher sought answers to the following questions:
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1. What are the optimum conditions in the preparation of a molecularly
imprinted polymer with lead acetate as template in terms of:
1.1 the porogen or solvent to be used in mixing
1.2 the concentration of monomer
1.3 the concentration of the template
1.4 the concentration of the crosslinker and initiator
1.5 the polymerization temperature and time?
2. What is the surface structure and the functional groups present in the
prepared polymer, non-imprinted polymer and molecularly
imprinted polymer as determined by:
2.1 Infrared Spectroscopy
2.2 Scanning Electron Microscopy?
3. How will the template be separated and removed from
the polymer matrix?
4. What is the difference in the IR spectra and the surface structure of the
MIP from the MIP binding again with lead acetate?
5. What is the binding capacity of the MIP with samples such as:
5.1 lead nitrate solution
5.2 aviation gasoline?
6. What is the concentration of lead from the samples before and after
passing through the MIP?
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7. Is there a significant difference in the concentration of lead present
in the samples before and after passing through the molecularly
imprinted polymer?
Hypothesis:
This study was guided by the null hypothesis:
There is no significant difference in the amount of lead from the samples
before and after passing through the molecularly imprinted polymer.
Significance of the Study
Toxic metals and its compounds from various sources have been found
to endanger human lives and other forms of biological life as well. Even the
intellectual capability of people living in a modern and polluted environment
has been linked to the presence of toxic lead released from cars exhausts and
dispersed into the environment. Studies indicated that children living near
motorways have lower IQs than those living in areas with less lead pollution,
suggesting that the lead has somehow lowered the brain function and
intelligence in children [Singhal & Thomas, 1980]. The high amount of toxic
lead in the surroundings has also been linked to a number of health problems.
Incorporation of the use of molecularly imprinted polymer MIP as an
alternative technique for trace residue analysis and as part of a properly
implemented quality check programs will surely give many advantages. One
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advantage is its use in a routine quality assurance monitoring of in-process
products and another as a collector of effluents in a certain manufacturing
plant.
An advantage of molecularly imprinted polymers is their long-term
stability and resistance to chemically harsh environments. This characteristic
can be used for pre-concentration of diluted lead components in a sample for
determination in an atomic absorption spectrophotometer.
Scope and Delimitation
The scope of this study started with the fabrication of a molecularly
imprinted polymer using methacryclic acid as the functional monomer, N,N’-
methylene-bisacrylamide as the crosslinker, water as the porogen , potassium
persulphate as the initiator and lead acetate as the template. Characterization
of the molecularly imprinted polymer was done by Ultraviolet Visible
Spectroscopy, Scanning Electron Microscopy, SEM-EDX and Fourier Transform
Infrared spectroscopy. Rebinding experiment was tested on lead acetate. The
probable use of the fabricated MIP and its binding properties for the
discrimination and detection of lead was restricted on the aviation gasoline
sample and lead nitrate solution quantitatively determined using Atomic
Absorption Spectroscopy. The possible binding of other metals that have the
same size as lead was not investigated. Correlation of data between SEM-EDX
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and IR spectra was made. Statistical tool used was the t-test for comparing
samples before and after passing through the MIP.
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Chapter 2
THEORETICAL FRAMEWORK
This chapter presents previous studies and researches, conceptual
paradigm and framework as well as definition of terms, all related to this study.
Conceptual Literature
The concept of molecular interactions is very old, having been used by
the Greek and Roman empires [Pauling, 1940]. However, the modern ideas of
these interactions began through the works of Van der Waals in his studies of
interactions between atoms in the gaseous state, and of Fisher who presented
his famous “lock-and-key” analogy of the interaction of a substrate with an
enzyme by the later half of the 19th century. The substrate fits like a key into
the lock of the enzyme's active site [Ramström et. al., 1996].
One approach of molecular interactions that scientists has worked on
and is now popular is molecular imprinting. Molecular imprinting, a fast
growing and powerful technique[Wulff,1995] with special relevance to
analytical problems is used for preparing synthetic macromolecules with the
tailor-made binding sites for the target molecule, achieved if the target molecule
is present as template or imprint molecule during the polymerization process.
In this technique, the functional monomer is very important since it is the
component principally involved in forming an effective chemical bond with the
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print molecule [Steinke et. al., 1995].
Ideal functional monomers, used in self-assembly imprinting and tested
in most researches are acrylic acid, methacrylic acid, trifluoro-methacrylic acid,
4-vinylbenzoic acid, itaconic acid, 1-vinylimidazole, 2-vinylpyridine, 4-
vinylpyridine, 4(5)-vinylimidazole, 4-vinylbenzyl-iminodiacetic acid and 2-
acrylamido-2-methyl-1-propane sulphonic acid.[MASAO DOI, 1996]
Solvents for polymerization have their trivial role of dissolving the
agents involved in the said process [Komiyama et. al., 2003]. But providing
porous structures to imprinted polymers and promote their rates of guest
binding is one of their crucial roles. In the process of polymerization, solvent
molecules are incorporated inside the polymers and are eradicated in the post-
treatment. The removal of the solvent tends to leave spaces that look like pores
in the polymers.
The choice of solvent is dependent on whether it is covalent imprinting
or non-covalent [Katz et. al., 1999]. In the covalent imprinting, a large number
of solvents are applicable as long as they satisfactorily dissolve the agents of
polymerization. In non-covalent imprinting, choosing a solvent is more decisive
on whether it would help in the promotion of the formation of non-covalent
reaction between the functional monomer and the template. Recent green
chemistry is being directed towards the use of non-organic and water based
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system. The relative amount of cross-linker reagent is of equal importance as
well since it affects the polymer structural rigidity and fixes the functional
monomer in specific orientation corresponding to the print molecule. They also
facilitate the practical applications of the imprinted polymers making them
insoluble in solvents. Cross-linkers commonly used are 4-divinylbenzene;
N,N’-methylene-bisacrylamide ; N,N’-phenylene-bisacrylamide ; 2,6-bisacryl
amidopyridine ; ethylene-glycol dimethacrylate and trimethylolpropane-
trimethacrylate. [Sherrington et. al., 1995]
For efficient imprinting, the reactivity of the crosslinkers should be the
same as that of the functional monomer, otherwise either the monomer or the
crosslinker polymerizes predominantly. [Takeuchi et. al., 2003]
Free-radical polymerization makes use of thermal decomposition of
initiators. The initiation radicals formed by decomposition attack the monomer
to produce the propagating radicals. These reactions are simple and
economical, but take note that the removal of molecular oxygen from the
polymerization mixtures is important if the initiator used is potassium
persulphate, since oxygen is produced which traps the radical and retards the
polymerization.
Other variables include polymerization time and polymerization
temperature . In some cases, non-covalent reaction between the monomer and
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template are very unstable to be used at higher temperatures. In this
condition, polymerization must be carried out at lower temperatures.
Under other conditions, initiators are decomposed with light irradiation, this is
photo-initiation that does not require high temperatures[Mukawa et. al., 2003].
A molecularly imprinted polymer (MIP) is generally described as a mold
or plastic cast of the analyte of interest, where the detection is based on the
shape, much like a lock and key. It is a polymer that was formed in the presence
of a molecule that is extracted afterwards, thus leaving complementary cavities
behind. These polymers show a certain chemical affinity for the original
molecule and can be used to fabricate sensors, catalysis or for separation
methods[Liu et. al., 2005]. The functional mechanism is similar to antibodies or
enzymes. In other words, MIPs are highly cross-linked polymer phases that
have pre-determined selectivity for a single analyte or a group of structurally
related analytes[Takeuchi, 1999]. See Figure 1 below.
Figure 1. Molecularly imprinted polymer
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Three steps are required to obtain molecularly imprinted polymers
(MIPs)[Vidyasankar et. al., 1995], see Figure 2. The first step (a) involves the
arrangement of monomers carrying certain functional groups around the
template through covalent or non- covalent interaction. During this step, pre-
organization of the binding sites is achieved. In the next (b), cross-linker is
added to the mixture and the monomers are frozen into position by
polymerization with high degree of cross-linking. The third step (c) is the
subsequent removal of the template from the macroporous polymer, yielding
complementary binding sites or cavities[Shea, K.J., 1994] that are
complementary to the template size, shape, and in the arrangement of
functional groups. This highly specific imprint sites are capable of rebinding
the imprint species in preference to other closely related structures.
Referring to Figure 2a, the self-assembly molecular imprinting approach
involves host-guest complexes produced from weak intermolecular interactions
such as ionic or hydrophobic interactions, hydrogen bonding, and metal
coordinations between the template and the functional monomer . These self-
assembled complexes are spontaneously established in the liquid phase and are
then fixed by polymerization with a high degree of crosslinking [Lanza et.
al.,1999]. After removal of the print molecules from the resulting macroporous
matrix, vacant recognition sites that are specific to the print molecule are
established.
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The interaction between a template and a functional monomer involved
in the imprinting and rebinding steps can occur in several ways some of which
have been mentioned previously ; (1) a non-covalent approach such as
hydrogen bond, ionic interaction, hydrophobic interactions; (2) covalent
approach, wherein a reversible covalent bond is formed between a print
molecule and functional monomers and (3) semi-covalent imprinting wherein a
reversible covalent bond formation during the imprinting step and non-
covalent interactions in the rebinding step.
(a) Self-assembly approach (b) Pre-organized approach.
Figure 2. Principles of molecular imprinting
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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 template [Percival, 2001]. The template in this study is lead acetate; lead
nitrate solution and the aviation gasoline served as the samples.
Lead(II) acetate is a chemical compound, a white crystalline substance
with a sweetish taste. It is made by treating litharge (lead(II) oxide, PbO) with
acetic acid. Like other lead compounds, it is very toxic. Lead acetate is soluble
in water and glycerin. With water it forms the trihydrate, Pb(CH3COO)2·3H2O,
a colorless or white efflorescent monoclinic crystalline substance. Lead(II)
acetate is also known as lead acetate, lead diacetate, plumbous acetate, sugar of
lead, lead sugar, salt of Saturn, and Goulard's powder (after Thomas Goulard).
The substance is used as a reagent to make other lead compounds and as
a fixative for some dyes. In low concentrations, it is the principal active
ingredient in progressive types of hair coloring dyes. Lead(II) acetate is also
used as a mordant in textile printing and dyeing, as a drier in paints and
varnishes, and in preparing other lead compounds.
Lead acetate has a sweet taste, which has led to its use as a sugar
substitute throughout history. The ancient Romans, who had few sweeteners
besides honey, would boil grape juice in lead pots to produce a reduced sugar
syrup called defrutum, concentrated again into sapa. This syrup was used to
sweeten wine, and to sweeten and preserve fruit. It is possible that lead acetate
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or other lead compounds leaching into the syrup might have caused lead
posoning in anyone consuming it [Singhal et. al., 1980].
Sugar of lead has also been used to treat poison ivy. Lead acetate is no
longer used as a sweetener in most of the world because of its recognized
toxicity. Lead acetate, as well as white lead, have been used in cosmetics
throughout history, though this practice has ceased in Western countries. It is
still used in men's hair coloring products like Grecian Formula. Lead acetate
paper is used to detect the poisonous gas hydrogen sulphide. The gas reacts
with lead(II) acetate on the moistened test paper to form a grey precipitate of
lead(II) sulphide.
Lead(II) acetate, among other lead salts, has been reported to cross the
placenta and to the embryo leading to fetal mortality. Lead salts also have
teratogenic effect in some animal species.
Other compound of lead that is another contributor of environmental
problems is tetraethyl lead. In 1922, Thomas Midgely, who also invented
CFCs(chlorofluorcarbons) found that if tetraethyl lead, Pb(CH2CH3)4 was put
into petrol, particles of lead and lead oxide PbO are formed on combustion.
This helps the petrol to burn more slowly and smoothly preventing knocking
and giving higher Octane ratings. 1,2-dibromoethane is also added to the petrol
to remove the lead from the cylinder as PbBr2, which is a vapour and removed
from the engine. This is how lead is released into the environment from leaded
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fuels. Using higher-Octane leaded petrol meant that more powerful high-
compression engines could be built [Seyferth, 2003].
The use of tetraethyl lead in fuels, particularly in automotive gasolines,
has been restricted for many years due, in part, to health and environmental
concerns as well as catalyst poisoning effects in automobile catalytic convertors.
Aviation gasolines have been allowed to contain tetraethyl lead since no
suitable substitute has been found with adequate knock resistance to allow the
current fleet of aircraft engines to operate properly. Current U.S. regulations set
a maximum amount of tetraethyl lead in aviation fuels at 4.0 mL/gallon. The
continued use of tetraethyl lead nonetheless remains an environmental and
health concern which has not been completely resolved. The possibility of
further restrictions , or a prohibition, on the use of tetraethyl lead in aviation
gasoline therefore exists.
Alternatives to the use of tetraethyl lead are known in the fuel art. For
example, methylcyclopentadienyl manganese tricarbonyl (MMT) has been used
as an antiknock agent in motor fuels since around 1975, first as a supplement to
leaded agents, and then as a replacement to produce lead-free gasoline.
However, questions have also been raised concerning the production of
undesirable emissions using MMT.
An aviation gasoline (Avgas) fuel composition possessing a high motor
octane number and which contains reduced amounts of tetraethyl lead is
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disclosed. The Aviation gas composition preferably comprises about 20 to about
80 vol % iso-octane, about 5 to about 18 vol % toluene, about 1 to about 20 vol %
C4 to C5 paraffins, about 0 to about 1 ml/gallon tetraethyl lead (TEL) and the
balance light alkylate. The Avgas composition may be economically produced
utilizing spare methyl tertiary butyl ether plant capacity to produce iso-octane
as one component of the composition.
The extraction and removal of the imprint molecules may be done with
methods from complex to simplest. One is solid phase extraction (SPE), it is a
sample preparation technique that concentrates the compound of interest
through some specific retention mechanisms, purifies them and are eluted into
a solvent appropriate for the final analysis. SPE is widely used for the
purification of small compound libraries synthesized in solution. It offers
several advantages when compared with the classic liquid-liquid extraction
using separatory funnel or preparative HPLC purification such as the reduction
of laboratory time, easy to manipulate, amount of solvent required will be
lesser, no disposal of large quantities of organic solvents, no problem with the
miscibility of solvent, higher concentration factor, and avoid problems like
incomplete phase separations, less-than-quantitative recoveries and emulsion
formation as encountered in liquid-liquid extractions. [Hosoya, K., et. al.,1996]
Chelation is another way of discriminating or isolating a target substance
from the polymer matrix. The word “chelator” refers to a substance consisting
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of molecules that bind tightly to metal atoms. Thus, metal atoms are forced to
go wherever the chelator goes. The bound pair, chelator plus metal atom, is
now called a “chelate”. One particular chelator utilized in this research is
EDTA, EthyleneDiamineTetraacetic Acid. Chelating agents can be classified
as bidentate and polydentate ligands. Ligands are molecules or ions that
surround the metal in a complex ion. The interactions between a metal atom
and the ligands can be thought of as Lewis acid-base reactions. EDTA is a
polydentate ligand used to treat metal poisoning, a controversial treatment
called “Chelation Therapy”. Six donor groups enable EDTA to form a very
stable complex ion with lead. EDTA is also used to clean up spills of radioactive
metals[Brown et. al. 1994].
The use of strong acids like concentrated nitric acid was used by Chemist
Richard Fish in his creation of imprinted polymers that would selectively trap
metal ions. After the complexes are cross-linked into a polymer, the metal ions
are washed away with a strong acid, leaving empty sites of the right size to fit
similar ions.
Metals and metalloids have long been extracted through mining. Their
numerous applications in the industry have increased production and the use
of these elements have resulted in their redistribution, including the
hazardous, impinging the upper layer of the earth’s crust[ Ajero, 2002].
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Heavy metals, a common effluent in industries have been the subject of
many researches because of its harmful effects on living things.
Particulate metals in air result from activities such as fossil fuel
combustion (including vehicles), metal processing industries and waste
incineration. There are currently no EC standards for metals other than lead,
although several are under development. Lead is a cumulative poison to the
central nervous system, particularly detrimental to the mental development of
children. Lead is the most widely used non-ferrous metal and has a large
number of industrial applications. Its single largest industrial use world-wide is
in the manufacture of batteries ( 60-70% of total consumption of some 4 million
tones ) and it is also used in paints, glazes, alloys, radiation shielding, tank
lining and piping [Lansdown & Yule,1986].
Pure lead is a soft, silvery-white or grayish metal in Group IVA of the
periodic table. It is malleable, ductile, dense, and a poor conductor of electricity.
Known in antiquity and believed by the alchemists to be the oldest of metals, it
is highly durable and resistant to corrosion, as is indicated by the continuing
use of lead water pipes installed by the ancient Romans.
It is rarely found in nature. It is present in several minerals, but all are of
minor significance except the sulfide, PbS (galena or lead glance) - major source
of lead production throughout the world. Lead may be extracted by roasting
the ore and then smelting it in a blast furnace or by direct smelting without
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roasting. Additional refining removes impurities present in the lead bullion
produced by either process. Almost half of all refined lead is recovered from
recycled scrap. When freshly cut, lead oxidizes quickly, forming a dull gray
coating, formerly thought to be lead suboxide, Pb2O, but now recognized as a
mixture of lead and lead monoxide, PbO, that protects the metal from further
corrosion.
Although lead is soluble in dilute nitric acid, it is only superficially
attacked by hydrochloric or sulfuric acid because the insoluble chloride or
sulfate coatings that are formed prevent continued reaction. Because of this
general chemical resistance, considerable amounts of lead are used in roofings,
as coverings for electric cables placed in the ground or underwater, and as
linings for water pipes and conduits and structures for the transportation and
processing of corrosive substances. They are also used as a protective shielding
around nuclear reactors, particle accelerators, x-ray equipment, and containers
used for transporting and storing radioactive materials because lead effectively
absorbs electromagnetic radiation of short wavelengths [Hernberg, 2000].
Caution about lead and its compounds are that they are toxic and are
retained by the body, accumulating over a long period of time-a phenomenon
known as cumulative poisoning-until a lethal quantity is reached. In children,
the accumulation of lead may result in cognitive deficits. In adults, it may
produce progressive renal disease [Thomas et. al.,1980].
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The most significant effects of lead on the body are on the brain and
nervous system. The effects of lead exposure in children include learning
disabilities, lower IQ; behavior problems, including inability to inhibit
misbehavior; distractibility, difficulty following directions, impulsiveness; sleep
disturbances and poor coordination. [Lansdown et. al., 1986]
Lead interferes with calcium's role in the neurons in the brain. Lead also
interferes with the incorporation of iron into hemoglobin, which carries oxygen
in the blood; damage kidneys, impair growth, interfere with hormones and
contribute to infertility.
For infants, a major source of lead can be milk formula prepared with
water from lead pipes, pipes soldered with lead, or brass or bronze fixtures
(which may contain lead). The amount of lead in water is higher for hot water
(which dissolves more lead) and for the first water drawn from the tap in the
morning ( which has absorbed more lead while in the pipes overnight ). For
toddlers and young children, major sources of lead include old paint (in houses
built before 1978), which may chip or form lead-containing dust (e.g. when
small particles of paint wear off as windows are opened and closed); soil
contaminated with lead from old paint or from leaded gasoline (which was
widely used in earlier decades).
Cigarette smoke contains lead, so one of the many health benefits of not
smoking is reduced lead exposure for yourself and your family.
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Increased iron and calcium in the diet can reduce uptake of lead. Food
sources for iron include red meat, liver, collard greens, spinach, tofu, baked
beans, fortified cereals, and raisins. Food sources for calcium include (low-fat)
milk, yogurt, cheese, and cooked greens.
One of the analytical instruments used in determining the lead content of
a sample is known as the atomic absorption spectrophotometer. It is capable of
determining traces (μg/mL) and ultratraces (sub- μg/mL) levels of elements or
metals in a wide variety of samples classified as biological, environmental,
geological, clinical, polymers and food[Antonio, 2005].
Although it has some limitations, atomic absorption spectrometry is one
of the predominant techniques in elemental analysis with good accuracy and
acceptable precision. This technique was discovered independently by Walsh
and Alkemade and Melatz in the early to mid 1950s. It involves the
impingement of light with a specific wavelength onto previously generated
ground-state atoms. This light is absorbed by atoms and a transition to a higher
energy level occurs. The intensity of this transition is related to the original
concentration of the ground state atoms [Sneddon, J., 1994].
In practice, the absorbance (A) used in AAS is related logarithmically to
the transmittance as follows: A = -log P/Po = log 1/T ; where P is the power of the
light source passing through the sample zone, Po is the power of the light source
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before it passes through the sample zone and T is the transmittance. AAS
involves the measurement of the drop in light intensity of Po to P.
Beer’s Law is fundamental in spectrophotometry being the basis for
estimating the concentration of a substance by measuring the light absorbed by
a solution of that substance. When a light of intensity Io is incident upon a
solution containing amounts as molar concentration (C) of an absorbing
substance, light will traverse the solution for a distance (b) and the intensity of
light is reduced to I. These quantities are related by Beer’s law where log I/Io = -
εbC, or I/Io = 10-εbC . The ratio I/Io is the transmittance, a dimensionless ratio; (ε)
is the molar absorptivity; (b) is the pathlength in centimeter and (C) is the
concentration with molar units [Sneddon, 1994].
Infrared Spectroscopy is the fastest and cheapest of the spectroscopic
techniques used by organic chemists. It is simply the measurement of the
absorption of the IR frequencies by organic compounds placed in the path of
the beam of light. Samples can be solids, liquids or gases and can be measured
in solution or as neat liquids mulled with KBr or mineral oil. Spectra can be
obtained in just a few minutes from partially purified materials giving an
indication that the reactions has proceeded as desired [Cooper, 1980].
IR spectroscopy is concerned only with IR frequencies of which only the
middle of the range is of interest for organic chemists. The reason why organic
chemists find IR spectroscopy of great interest is that most carbon-hydrogen,
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carbon-carbon and carbon-oxygen bonds stretch at frequencies in the mid-IR
region. The infrared region is divided into near, mid and far. These stretchings,
rockings and other vibrational modes are characteristic of the type of
compound and of its functional groups, resonance possibilities and shape so
that an organic chemist can identify rapidly a number of important structural
features from an IR spectrum. [Fawcett, A. H., 1996]
IR spectrometers consist of a number of functional blocks such as the
source of radiation, the sample, a grating, the detector and the output usually a
recorder.
Light absorption is also governed by Beer’s law as mentioned in AAS
A = log 1/T = log Io/I = Kcb
where I = transmitted radiation intensity
Io = intensity of incident radiation
A = absorbance
T = transmittance
c = concentration
b = pathlength
K = a proportionality constant for a given compound
This logarithmic relationship can be valuable in quantitative work, but in
most routine IR measurements, spectra are plotted as wave number against
percentage of transmittance (%T) which is not linearly related to concentration.
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IR spectra are divided into three more regions such as the functional
group region at 4000 cm-1 to 1300 cm-1 wavenumbers; the fingerprint region
(1300 – 910) cm-1 and the aromatic region (910 – 650) cm-1. Peaks in the
functional region are characteristics of particular group such as –OH, CH, NH,
SH, C=O, C=CH and C triple bond C-H as well as some overtones. The peaks in
the fingerprint region confirmed some of those present in the previous region
and the aromatic region signifies the presence of aromatic compounds.
Ultraviolet-Visible spectroscopy comprises electromagnetic radiation of
relatively high energy with the wavelength ranges of 200 to 400 nm, called
ultraviolet spectroscopy, and 400 to 800 nm, visible spectroscopy. These two are
useful for investigating the electronic structures of unsaturated molecules and
the extent of their conjugation. Spectroscopic techniques always complement
each other in elucidating the structure of investigated molecules in solid, liquid,
or gaseous form. This type of technique is one of the important t analytical tools
in the armory of a scientist for the characterization of polymers [Gurusamy,
1997].
The Scanning Electron Microscope (SEM) is patterned after reflecting
light microscopes and yield similar information. It gives the surface feature of
an object or “how it looks”, its texture, detectable features limited to a few
nanometers. It shows the shape, size and arrangement of the particles making
up the object that are lying on the surface of the sample or have been exposed
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by grinding or chemical etching. It also furnished information on the elements
the sample is composed of and their relative ratios, in area ~ 1 micrometer in
diameter.
SEM is a tool to observe an invisible tiny object in a stereographic
image with a magnified scale. It covers a wide range of magnification about
10 x - 1,000,000 x. The principle behind this equipment is when an electron
beam is irradiated on a specimen surface, interactions between the electron
beam and the atoms composing the specimen produce various kinds of
information [JEOL Datum Ltd.,1988].
Related Studies
Among the studies that involve separation of metals are the following:
Solid-phase extraction (SPE) columns packed with materials based on
molecularly imprinted polymers (MIPs) were used to develop selective
separation and preconcentration for Ni(II) ion from aqueous solutions. SPE is
more rapid, simple and economical method than the traditional liquid–liquid
extraction. The MIPs were used as column sorbent to increase the grade of
selectivity in SPE columns. In this study, a polymer obtained by imprinting
with Ni(II) ion as a ion-imprinted SPE sorbent has been developed by Ersöz, et.
al., using solid-phase extraction and preconcentration in aqueous solutions by
packed-bed columns. Another study made by Yavuz et. al. (2005), was Iron
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removal from human plasma based on molecular recognition using imprinted
beads. The aim of the study was to prepare ion-imprinted polymers used for
the selective removal of Fe3+ ions from Fe3+- overdosed human plasma. N-
Methacryloly-(l)-glutamic acid (MAGA) was chosen as the complexing
monomer.
Another study was Solid-phase extraction and preconcentration of
cadmium(II) in aqueous solution with Cd(II)-imprinted resin (poly-Cd(II)-
DAAB-VP) packed columns by Yongwen Liu et al. from China (2005) . A novel
chelating resin (poly-Cd(II)-DAAB-VP) was prepared by metal ion imprinted
polymer (MIIP) technique. The resin was obtained by one pot reaction of Cd(II)-
diazoaminobenzene-vinylpyridine with cross-linker ethyleneglycol
dimethacrylate (EGDMA). Comparing with non-imprinted resin, the poly-
Cd(II)-DAAB-VP has higher adsorption capacity and selectivity for Cd(II).
Based on poly-Cd(II)-DAAB-VP packed columns, a highly selective solid-phase
extraction (SPE) and preconcentration method for Cd(II) from aqueous solution
was developed.
The development of new enrichment techniques and selective separation
of closely related metals are two important frontier areas of research in
analytical chemistry[Pomogailo, 1988]. Solid-phase extraction (SPE) or solid-
liquid extraction (SLE) offers several advantages over other preconcentration or
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separation techniques. The introduction of ion-imprinted polymers (IIPs) in
trace and ultratrace analysis provide vital breakthroughs in preconcentration or
separation chemistry. The combination of the two approaches results in IIPs for
SPE ( IIP-SPE ) and has potential in trace and ultratrace analysis. With the
preparation of tailor-made materials, IIP-SPE offers an approach to the recovery
of valuable and rare metals from lean ores, minerals and dilute solutions and to
the removal of toxic uranium and heavy metal pollutants from industrial
wastes.
Highly selective determination of inorganic mercury(II) after
preconcentration with Hg(II)-imprinted diazoaminobenzene–vinylpyridine
copolymers is another study made by Yongwen Liu, et. al. The study involves
Hg(II) imprinted and non-imprinted copolymers prepared by metal ion
imprinted polymer (MIIP) technique. The copolymers were obtained by
copolymerizing mercury chloride (or without it), diazoaminobenzene (DAAB)
and vinylpyridine (VP) using ethyleneglycoldimethacrylate (EGDMA) as cross-
linker in the presence of 2,2′-azobisisobutryonitrile as initiator. The separation
and preconcentration characteristics of the copolymers for Hg(II) were
investigated by batch and column procedures.
A study by K. Tsukagoshi, et. al.(2004), includes metal-ion imprinted
microspheres prepared by the reorganization of coordinating groups on the
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surface imprinting by the use of seed emulsion polymerization; preparation
and physico–chemical characterization of the microsphores; metal adsorption
behaviour of the microspheres; FTIR and ESR studies on Cu (II) loaded
microspheres;metal-ion imprinted resins without emulsion polymerization;
surface imprinting by the use or w/o emulsion; potassium oleate and the
dioleyl hydrogenphosphate as functional monomers with y-rays irradiation.
Another research on metal-ion coordination in designing molecularly
imprinted polymeric receptors by P.K. Dhal covers binding site interactions in
molecularly imprinted polymers as well as advantages of the metal-ion
coordination interactions in molecular recognition; design of molecularly
imprinted polymers based on metal-coordination interaction and bulk
polymerized metal-coordinating polymeric receptors surface modified metal-
coordinated imprinted polymers. Alexander Katz, et. al. made an investigation
into the mechanisms of molecular recognition with imprinted polymers formed
by the self-assembly of binding monomer and imprint through non-covalent
interaction. [Davis et. al.,1999]
In another study by Prof. Dr. Gunter Wulff, involving molecular
imprinting polymers in cross-linked materials with the aid of molecular
templates, stated that binding sites can be produced in organic or inorganic
polymers-similar to those antibodies - which are able to recognize molecules
and which may have catalytic action. In this article a method analogous to a
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mechanism of antibody formation was proposed, by which in the presence of
interacting monomers a cross-linked polymer is formed around a molecule that
acts as a template. After, the removal of the template, an imprint containing
functional groups capable of chemical interaction remains in the polymer. The
shape of the imprint and the arrangements of the functional groups are
complementary to the structure of the template [Wulff, 1995].
Another advantage molecular imprinting offers is the possible feasibility
of constructing sensors for substances whose exact chemical composition is
unknown or one with too complex mixture [Mosbach, et. al., 1997].
Molecularly Imprinted Polymers (MIPs) have been recognized as a
valuable recognition element in chemical sensors [Ebarvia, B. S., 2004]. This
technique is suited for the determination of all kinds of analytes. The highly
specific imprint sites are capable of rebinding the imprint species in preference
to other closely related structures [Campbell et. al.,2000].
The original paper of Ebarvia, et. al. (2004), that studied the biomemetic
piezo-electric quartz sensor for caffeine based on a Molecularly Imprinted
Polymer prepared by copolymerizing methacrylic acid (MAA) and ethylene
glycol dimethacrylate (EGDMA) in the presence of azobis (isobutyronitile) as
initiator, caffeine as template molecule, and chloroform as solvent was
researched. The development of a caffeine chemical sensor was undertaken
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combining the recognition, capability of Molecularly Imprinted Polymer and
Piezoelectric Quartz Sensing.
Albaro, (2004) also conducted a study on Piezoelectric Quartz Crystal
Sensor for Surfactants based on Molecularly Imprinted Polypyrrole. In his
study, a piezoelectric quartz crystal sensor for sodium dodecyl sulfate was
developed based on Molecularly Imprinted Polypyrrole. The polypyrrole
membrane was deposited onto one side of the electrode of the quartz crystal by
galvanostic polymerization and molecular imprinting was achieved by
incorporating sodium dodecyl sulphate in the electrolysis reaction mixture.
Conceptual Paradigm
Molecular imprinting is quite a comprehensive, easy to apply and
perform concept which involves the mixing of the imprint molecule (template)
with selected monomers, crosslinker and initiator [Steinke et. al., 1995].
Polymerization results to the formation of an imprinted polymer. Removal of
the print molecule from the prepared polymer results to an MIP, which now
possesses a fingerprint of the print molecule [Komiyama et. al., 2003].
Characterization of the MIP and investigation on its binding capacity can be
performed by several analytical techniques [Mannivannan, 1997]. Various
reports from abroad has made use of MIP as a stationary phase in a simple
separation process intended for the determination of metals present in samples.
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Figure 3 shows the paradigm of this study.
Template Molecularly
(Lead acetate) Imprinted Polymer
Application
(MIP)
Monomer
Crosslinker
Initiator Quantitative
Solvent Measurement of
Characterization of Lead
the MIP
Investigation on
the binding
capacity
Figure 3. Paradigm of the study
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Conceptual Framework
Molecular imprinting concept was inspired by L. J. Pauling’s antibody
formation theory in which he used an antigen as the imprint molecule
[Ramstrom, et. al.,1997]. In his theory the template aids in the rearrangement of
antibody polypeptide chains so that this antibody has a configuration
complementing the antigen molecule. Although Pauling’s theory was proven
incorrect for antigen-antibody interactions, chemists used the idea in forming a
three dimensional structure around a template for preparing synthetic analogs
of antibodies that can recognize target molecules with selectivities similar to
their biological counterparts. It was in the early 1970’s when successful imprints
on synthetic organic polymers were achieved.
The formation of the molecularly imprinted polymer involves free-
radical polymerization composed of three steps: initiation, propagation and
termination. Initiation occurs by the use of an initiator, potassium persulphate
was used in this study. The initiator free radicals attack the vinylic monomer
such as methacrylic acid to form the monomer free-radical. These radicals
propagate to form a long chain. The crosslinker, N,N’ methylene bisacrylamide
facilitates linking of radicals to other radicals to form a crosslinked network.
The incorporation of the template depends on whether it is covalent or
non-covalent. Organic templates usually use covalent interactions while non-
covalent templates may use hydrogen bonding or ionic interactions [ Shea
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et.al., 1994 ]. In this study, lead acetate was used as the template and
investigation into the binding sites of this template with the polymeric matrix
was done. The binding capacity of the resulting polymer (MIP) with samples
containing lead (Pb) was performed using lead nitrate solution and aviation
gasoline. Quantitative determination of lead(Pb) present in samples like
aviation gasoline and lead nitrate solution was done by correlation.
Definition of Terms
For clarity and understanding the following terms are operationally
defined according to their use in this study.
Crosslinker A unit with two or more attachment possibilities
to the functional monomers and functions as a
crosslinking monomer.
Functional Monomer A polymerisable entity which interacts with the
print molecule.
Initiator A substance that induces polymerization such as
AIBN (azobis-isobutyronitrile) or potassium
persulphate.
Molecularly imprinted A polymer of three-dimensional networks that

polymer (MIP)
have a “memory” of the shape and functional
group positions of the template molecule. The
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system does not contain the template.
Non-imprinted polymer A polymer prepared without the presence of the

(NIP)
template.
Porogen The compatible solvent that is inducing porosity
in molecularly imprinted polymers.
Prepared polymer The imprinted polymer with the template.
Template molecule The imprint molecule or print molecule and the
selected target substance used in the imprinting
procedure.
Acronyms
AAS – Atomic Absorption Spectrophotometry
EDX – Energy Dispersive X-rays
FTIR – Fourier Transform Infrared Spectrophotometry
MAA – Methacrylic Acid
MIPs – Molecularly imprinted polymers
MEPS - Microextraction in packed syringe
UV-Vis spectroscopy- Ultraviolet Visible spectroscopy
SEM – Scanning Electron Microscopy
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Chapter 3
METHODOLOGY
This chapter discusses the research method used, research materials and
equipment, other methodologies and statistical tools employed in evaluating
experimental data.
Research Design
The experimental method of research was used in this study.
Experiments started with the distillation of the monomer methacrylic acid
(MAA) to remove the hydroquinone content [Elias, 1984]. Optimal methods for
studying various aspects of template–monomer-copolymer binding were
established in terms of the type of porogen (solvent) , temperature, monomer
concentration, effects of the amount of cross-linker and analysis by SEM –EDX,
Atomic absorption spectroscopy, FTIR and UV-Vis spectroscopy. Two methods
of removing the template molecule bound with the polymer were experimented
on. Quantitative analysis of the original percentage and the extracted lead was
carried out using the Atomic Absorption spectrophotometer and amount of
lead retained in the polymer was determined by Energy Dispersive X-ray
analysis of the Scanning Electron Microscope. Lead nitrate solution and
aviation gasoline samples were used in the batch-binding process.
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Research Locale
Experiments were performed inside the Chemistry Research Laboratory
of De La Salle University-Dasmarinas, Cavite and utilized the De La Salle
University - Manila laboratory services and facilities.
Reagents and Materials
Reagents used for this experimental research were all analytical grade
(AR). The functional monomer used was methacryclic acid (MAA) and the free-
radical initiator was potassium persulphate, both purchased from Chemline
Industries, N, N’ methylene bisacrylamide - the cross-linker was purchased
from Zibo Defeng Chemical Co.,Ltd, Tianzhuang Town, Huantai Country, Zibo
Shandong, China. Methacrylic acid was purified by simple distillation process
while potassium persulphate powder and the crosslinker were used as is.
Distilled water that served as the porogen and lead acetate purchased from
Yana Chemodities, were provided by DLSU-D Chemistry Laboratory. Aviation
fuel sample was availed from Petron Refining Plant, Batangas Station and was
used without further purification . The molecular structure of the reagents are:
Monomer: Methacrylic acid was distilled by simple distillation prior to use.
Molecular structure of MAA
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Initiator: Potassium persulphate was used as received.
Potassium persulphate
Crossllinker: Two kinds of crosslinker were tested
a) N,N’-methylene-bisacrylamide b) Ethylene glycol dimethacrylate
Template: Lead acetate powder was used as received.
Lead(II) acetate
Porogen: Distilled water was used.
Other solvents: Concentrated nitric acid, methanol, acetic acid were used as
received.
Data Gathering Procedure
Different methods of gathering data were performed using the above
mentioned materials, instruments and analytical techniques. Analytical balance
AND HR-200 was used in weighing the amounts of each reagent and materials.
Sonicating bath was used to agitate the particles in the mixture with the
application of sound energy thereby speeding the dissolution. Oven, Model:
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Memmert, was used in drying the polymer, pH determination was through the
use of Mettler Toledo pH meter. JASCO Model V-530 UV-Vis
Spectrophotometer Version 1.91 was used in ultraviolet and visible
spectroscopic technique. Atomic absorption spectrophotometer (Model: Varian
SpectrAA-200) was used in the analysis and quantification aspects of the study
such as the lead percentage. Infrared spectroscopy (FTIR) Model , and Energy
Dispersive X-ray (EDX) elemental analysis of the JSM-5310 JEOL scanning
electron microscope (SEM) were also used in the structure elucidation of the
complex formed before, during and after polymerization.
Likewise, the synthesized molecularly imprinted polymer was subjected
to surface characterization using the surface analysis technique of the Scanning
Electron Microscopy (SEM). Methodologies for acquiring data are as follows:
1.0 Procedure for Optimum Conditions:
1.1 Solubility tests
Solubility tests were done using a 1: 4 ratio and proportion (by volume)
of reagents to solvent.
1.2 Monomer Concentration:
Methacrylic acid concentration was tried out at different concentration of
0 to 0.8 M and the optimum baseline was determined by means of the UV-Vis
spectroscopic technique using a fixed amount of the template.
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Distillation of Methacrylic acid (MAA):
Since the purchased methacrylic acid (MAA) contains hydroquinone as
inhibitors or stabilizer for the purpose of preventing undesired polymerization
during storage, it was distilled before use. Simple distillation was used and
collection of MAA at a temperature of 81oC was performed.
1.3 Template (Lead Acetate) Concentration
Based on the solubility of the template to distilled water, its
concentration was fixed at 0.2 millimole per milliliter.
1.4 Amount of Crosslinker:
A typical water-soluble reagent such as N,N-methylenebisacrylamide was
chosen and utilized as the crosslinker. A constant amount of 0.300 grams of this
crosslinker was used all throughout the experiment after testing for efficiency.
Three (3) different amounts of this crosslinker such as 0.323 g, 0.7626 g and 1.00
g were actually tested for efficiency.
Amount of Initiator:
Potassium persulphate was chosen as the initiator using 0.05 grams for all
trials. It is non-combustible and dissolves in water at a 6.0% by weight
proportion in room temperature. Decomposition occurred upon heating at
80oC.
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1.5 Temperature and polymerization time:
Using potassium persulphate as the initiator, the mixture was
experimentally heated to different temperatures such as 60oC, 70oC and 80oC.
At the same time, polymerization time was monitored at time intervals of 12
hours, 24 hours and 36 hours.
2.0 Polymer Preparation and Characterization:
Molecularly Imprinted Polymers was synthesized wherein 2 millimole
MAA monomer (0.170 mL) , lead acetate (0.30 g) and 0.30 g of crosslinker were
dissolved first with 5 mL distilled water in test tubes to allow the molecules to
interact and form complexes. The initiator potassium persulphate (0.05 g) was
added. The mixture was degassed for 10 minutes in a sonicating bath under
vacuum and subsequently flushed with Nitrogen Gas for 5 minutes. It was
placed in a thermo-stated water bath kept at 80OC for 24 hours during which
polymerization had occurred. Drying in an oven for 12 hours followed at the
same temperature of 80oC.At the end of that period, the resulting polymer was
pulverized using a mortar and a pestle.
A non-imprinted polymer (NIP) was prepared using the same procedure
but omitting the addition of lead acetate.
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Template-monomer solutions for UV Spectroscopic Analysis
A series of template–monomer solutions were prepared by adding
different amounts of methacrylic acid to the 1 mmol lead acetate aqueous
solution. The concentrations of added methacrylic acid in the aqueous template
were set at 0 to 0.8 M. The solutions were prepared in 20 mL test tubes and put
in a sonicator for 10 minutes. Using distilled water as reference, the solutions
were scanned by a UV spectrophotometer at 200–400 nanometer (nm). A
separate scan of the pure methacrylic acid was also done to determine the effect
of distilling it first.
Characterization by FTIR Spectroscopy
For this kind of test, all spectra were recorded at room temperature on an
FTIR spectrometer with a 0.1 mm path length, using solid KBr window.
Scanning electron microscope (SEM) Analysis
A 0.01 g sample of polymer dried and kept in a sealed container tested for
surface structure were determined by using the scanning electron microscope
(JSM-5310 JEOL).
3.0 Removal of the template from the polymer matrix:
Two methods were experimented to remove the lead acetate template
from the prepared polymer. One method was using EDTA solution saturating
the polymer. The other method was achieved by saturating the prepared
polymer with nitric acid (1:1) mixed in distilled water then washing with
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methanol:acetic acid mixture (7:1) v/v. The remaining solid was dried at 60OC
over a water bath. These solids were packed and used as the stationary phase in
an MEPS system.
4.0 Binding Studies of the Molecularly Imprinted Polymer and its
Application:
Preparation of MIP as adsorbent/or stationary phase
Applying MEPS method for this study, approximately 0.1g of glass wool
was inserted into a syringe (2-mL) as a plug inside the syringe between the
barrel and needle.
The 0.500 g of the dried (MIP) molecularly imprinted polymer used as
the material in the selective microextraction of lead (Pb) from aviation gasoline
sample and lead nitrate was packed in the glass syringe.
AAS analysis of samples
A non-adulterated aviation gasoline sample was acquired from a
refinery. The initial concentration (in ppm) of lead(Pb) in the sample was
determined using Atomic Absorption Spectrophotometry. At least 5-mL of the
gasoline sample was passed through the MIP. Eluates coming out of the MIP
column were collected and tested for the ppm lead(Pb). The intial concentration
of lead in the aviation gasoline sample before binding minus the final eluted
ppm of lead in the same sample equals ppm of lead retained in the MIP. A
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sample using lead nitrate solution was likewise made to undergo the same
procedure.
Sample Preparation for Atomic Absorption Spectrophotometry
Samples for AAS must be in solution and the presence of organics must
be taken into consideration for it may affect the analysis. Since polymers
are of organic nature, it may complicate the sample matrix relative to that
of the standard and viscous solution may affect the uptake rate and
aerosol formation in the spray chamber. Digesting the sample with nitric acid
will correct this.
Samples for AAS analysis were digested in Erlenmeyer flasks by adding
5mL of nitric acid and heating to about 100 -110oC until 1 to 2 mL of clear
solution remained in the flask. Nitric acid and blank were prepared and
likewise digested. The digests were transferred to 250-mL volumetric flask and
diluted to volume with distilled water. Standard solutions containing lead were
prepared and their absorbance readings measured using the AAS. The
previously digested samples were also analyzed and their concentrations
determined by comparing the measured absorbance readings with those of the
standards.
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Data Analysis Procedure
Analysis which includes methods to compare observed data to expected
values and to compare sets of data to each other was used in this research.
t-Test : paired two sample for means statistical tool was employed.
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Chapter 4
RESULTS, ANALYSIS AND INTERPRETATION
This chapter presents the analysis and interpretation of all data gathered
from experiments performed in a chemistry laboratory.
1. Optimized Conditions in the Preparation of MIP
The following data shows the results of determined optimum conditions
in preparing a molecularly imprinted polymer with lead acetate as the
template such as the type and amount of solvent to be used in mixing, the
concentration of monomer, cross-linker, initiator and template. Data on the best
polymerization time and polymerization temperature are also presented.
1.1 Selection of Porogen or Suitable Solvent
The superficial role of solvents is to dissolve the agents for
polymerization but other than this they have more crucial roles. One is to
provide porous structure to imprinted polymers, and promote their rates of
guest binding. Porosity facilitates the release of the bound guest from the
polymer.
In the process of polymerization, solvent molecules are incorporated
inside the polymers and then removed in the post-treatment. The space
originally occupied by the solvent molecules is left as pores in the
polymers during the process.
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Dispersion of the heat of reaction generated on polymerization is another
role of solvents. Otherwise, undesired side-reactions occur and the temperature
of reaction mixture is locally elevated. Lastly, the formation of monomer-
template adduct, which is required for efficient non-covalent imprinting is
suppressed.
In the optimization process, a series of solubility test or miscibility test
using various solvents having different polarity and hydrogen bonding
properties was undertaken for each agent for polymerization, with the results
shown in Table 1.
Table 1
Solubility Tests on monomer, crosslinkers, initiator and template
Solvents
Reagents CH3CN CHCl3 (CH3)2CO CH3OH H2O
1. Monomer
Methacrylic m m m m m
acid
2. Crosslinkers
Ethylene m m m m n
Glycol
Dimethacrylate
(EGDMA)
N,N’- i i i ss s
methylene-
bisacrylamide
3. Initiator
Potassium s i i s s
Persulphate
4. Template
Lead acetate ss ss s s s
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Legend: m = miscible s = soluble i= insoluble

n = not miscible ss = slightly soluble
Based on these solubility tests, distilled water was chosen as the best
solvent for the preparation of the molecularly imprinted polymer where all
the reagents for polymerization have dissolved and shown compatibility.
Amount of Chosen Porogen:
The optimum amount of chosen solvent was also experimented on the
basis of the amount of product produced and physical properties such as
porosity/texture and density (Table 2).
Too firm and dense polymers result from preparation involving the
absence of a solvent. These types of polymers hardly bind guests.
Table 2
Experiment on the amount of porogen used and results on the observed
physical properties of polymers produced.
Volume H2O, Polymer Weight, Texture Density of polymer,

(mL) (g) (g/mL)
10 2.1106 Too soft 0.11
7 0.7958 Slightly porous 0.22
5 0.3334 Porous /rough 0.63
1 0.2035 Too firm 1.34
The ideal volume of water added as the porogen in the preparation of the
polymer was 5 mL because of the texture quality and density factors. Density of
the prepared polymer was determined by mass per unit volume method.
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1.2 Concentration of Monomer
Results on the Distillation of Monomer (MAA)
Methacrylic acid (MAA) was used as the functional monomer as it has
dominated the field of molecular imprinting for its broad applicability as
related to the fact that the carboxylic acid group serves well as a hydrogen bond
and proton donor as well as a hydrogen bond acceptor. Absorbance property
was revealed by the UV-Visible spectra (Figure 4). The difference in the
character of an undistilled and distilled MAA is shown by the IR spectra
(Figures 5 and 6).
Ultraviolet-Visible Spectra of MAA
2.5
2
Absorbance
1.5
1
0.5
0
0 50 100 150 200 250 300 350 400 450
Wavelength
Figure 4. UV-Visible spectra of methacrylic acid (MAA)
The ultraviolet-visible spectra of methacrylic acid in Figure 4 showed a
λmax at 260 nanometers which indicates the presence of conjugated double
bonds.
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Distillation of monomer was accomplished to remove the hydroquinone
content of the purchased MAA. Figure 5- IR spectra demonstrate the presence
of such compound on the fingerprint region when absorption peaks were still
very high. An abrupt change in the spectra profile along the functional group
region also showed the elimination of the inhibitor.
Table 3
Summary of IR Spectral Data for Methacrylic acid, undistilled
Frequency, cm-1 Assignment Intensity

2950-3100 O-H stretching broad, strong
2989.80 asymmetric C-H stretch weak
2620. 48 symmetric C-H stretch weak
1696.62 C=O stretch strong
1634.46 C=C stretch much weaker
1429.96-1454.04 C-H bend; doublet strong
1375 C-O bending medium
1088.66-1300.11 C-O bend weak
The IR spectra, Figures 5 and 6 clearly showed the difference in the
profile of MAA most specifically in the double-bond region 1634.46 cm-1 to
1696.62 cm-1. The strong peak and medium peak at this region indicated a C=C
stretch due to alkenes and C=O stretch due to the presence of a ketone
functional group.
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Figure 5. IR Spectra of Undistilled Methacrylic acid

54
Table 4
Summary of IR Spectral Data for Methacrylic acid, distilled

3112.53 O-H stretch strong
2925.00 Symmetric C-H stretch weak
1634.97 C=C stretch medium
The IR spectra of distilled methacrylic acid, Figure 6 have strong peaks at
3111.53 cm-1 due to the O-H stretch and 2925 cm-1 are ascribed to the symmetric
C-H stretch of a saturated system. Medium peaks at 1634.97 cm-1 to strong
peak at 1696.58 cm-1 are stretching vibration peak of C=C and C=O stretch.
Peaks of the distilled MAA were sharp while peaks of the undistilled
MAA are somewhat broader. See Appendix E for overlay figures of Distilled
MAA and undistilled MAA.
Methacrylic acid interacting with the lead acetate template prior to
polymerization is shown by Figure 7. These monomers that represent an equal
percentage in the composition of the molecularly imprinted polymers (MIPs)
with the other components gave a large influence on the MIPs properties. The
solution structure of the resulting assembly defines the subsequently formed
binding site. Stabilizing the monomer template assemblies increases the
number of imprinted sites and minimized number of non-specific binding sites.
The monomer having the carboxylate functionality binds the lead acetate
template utilizing non-covalent interactions forming the pre-polymer complex.
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Figure 6. IR Spectra of Distilled Methacrylic acid

56
Uv-Vis spectra of template with monomer
6
5
absorbance 4 1 mmol
2 mmol
3
3 mmol
2
4 mmol
1 blank
0
-1 0 100 200 300 400 500
wavelength
Figure 7. Uv-Vis spectra of lead acetate and MAA
1.3 Concentration of the Template:
The functional groups present in the template provide points for
electrostatic and hydrogen-bonding interactions for the complex formation
prior to polymerization.
According to their structure, the interactions between the template and
monomer could be due to hydrogen bonds produced by their self-assembling.
When the amount of methacrylic acid was increased from 1 mmol to 4.0 mmol,
the differences between the spectra were not apparent ( see Figure 4 ) ; this
means that interactions between the functional monomer and the template
occurred at any ratio, as evident in the absorbance readings ranging from 0.075
to 0.336 for all concentrations. Regardless of the concentration, lambda max
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remains unchanged. Thus, the chosen ratio of template to monomer used in
preparing the polymer was 1 mmol : 2 mmol.
The Ultraviolet-Visible spectrophotometer was useful in investigating
the interactions between lead acetate template and the functional monomer
methacrylic acid (Figure 7). It was used to study the binding mechanism of MIP
and decide on the optimum ratio of the template to the functional monomer.
The amount of lead acetate was fixed and the amounts of methacrylic acid
varied from 0 to 4.0 mmol. A series of the absorption spectra of the solution of
lead acetate and methacrylic acid in Figure 7 was then obtained. The changes in
the spectra relative to Figure 4 indicated that interactions had taken place
between the template and the monomer.
1.4 Concentration of Crosslinker and Initiator:
This research compared the efficiency of two crosslinkers namely
ethylene glycol dimethacrylate (EGDMA) and N,N’-methylene-bisacrylamide.
Mole ratio of crosslinker to monomer established in this research was 5 mmol: 2
mmol , experimented for both EGDMA and N,N’-methylene-bisacrylamide.
The basic role of the crosslinker is to fix the guest-binding sites firmly in
the desired structure and make the imprinted polymers insoluble in solvents to
facilitate their practical applications. By choosing an appropriate crosslinking
agent, random copolymerization occurs successfully and the functional
residues-derived from the functional monomers , are uniformly distributed in
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the polymer network. The surface structure of the resulting polymer and
solubility of the crosslinker to monomer, template and the common solvent was
the basis of efficiency.
The mole ratios of crosslinking agent to functional monomer are also
important. If the mole ratios are too small, the guest-binding sites are located so
closely to each other that they can not work independently. In extreme cases,
the guest binding by one site completely inhibits the guest binding by the
neighboring sites. An extremely large mole ratio, however, means the
imprinting efficiency is damaged, especially when the crosslinking agents show
non-covalent interactions with functional monomers and/or templates.
Surface structure of prepared polymers was tested using the scanning electron
microscope to distinguish the difference in using the two types of crosslinkers
mentioned. Refer to figures 8a to 8d. All these tests used potassium persulphate
as the initiator.
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Figure 8a to 8d. Scanning Electron Microscopy Results
a) SEM image of Polymer without template using EGDMA crosslinker
b)SEM image of Polymer with template using EGDMA crosslinker
c) SEM image of Polymer without template using N,N’- methylene-bisacrylamide

crosslinker
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d) SEM image of Polymer with template using N,N’- methylene-bisacrylamide

crosslinker
Table 5
Comparison between the use of EGDMA and N,N methylene
bisacrylamide based on SEM results.
Polymer Ethyleneglycol N,N’-methylene- bisacrylamide

dimethacrylate
EGDMA
PP (with template) Non-uniform binding Uniform distribution and binding
NIP(without template) Slight uniform binding Uniform cavities
On the basis of these results, where there is uniform distribution and
binding as well as uniformcavities formed, N, N’- methylene-bisacrylamide was
chosen as the crosslinker , while potassium persulphate was selected as the
initiator for the preparation of a molecularly imprinted polymer selective to
lead acetate.
The crosslinker, N,N’-methylene-bisacrylamide and initiator, potassium
persulphate were chosen on the basis of their solubility and compatibility with
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the other agents of polymerization as well as their availability in the local
market.
1.5 Optimum Temperature and Polymerization Time
The experiment on the determination of the best polymerization
temperature and polymerization time was summarized on Table 6. It showed
that the optimum time was 36 hours and the best polymerization temperature
was 80oC producing acceptable texture and porosity of polymer.
Table 6
Experiment on the best polymerization temperature and time.
Temperature Polymerization time Results

(h)
60oC 12 Incomplete polymerization, still soft
24 Incomplete polymerization, still soft
36 Incomplete polymerization, still soft
70oC 12 Incomplete polymerization
24 Still soft
36 Slightly soft
80oC 12 Incomplete polymerization
24 Slightly soft
36 Complete polymerization, porous
2. Polymerization and Characterization Results
Successful polymerization and characterization of the prepared polymer
(PP), non-imprinted polymer(NIP) and the molecularly imprinted polymer
(MIP) were undertaken through the aid of several analytical equipment such as
the Fourier Transform Infrared Spectroscope ( FTIR ), Scanning Electron
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Microscope (SEM) and SEM-EDX (Energy Dispersive X-rays). In this study, the
binding characteristic of the prepared polymer that would be useful for the next
stage of producing an MIP column was investigated and compared with the
non-imprinted polymer. The binding properties of the molecularly imprinted
polymer with lead acetate, lead nitrate and tetraethyl lead from an aviation
gasoline sample were likewise studied.
A. Infrared (IR) Analysis of Prepared Polymer, NIP and MIP
A polymer generally consists of assemblages of bonds, each bond having
vibrational modes. FT-IR spectroscopy provides information about the
structure and local environment of the polymer chains as well as the
extent of incorporation of the monomers into the polymeric network. By using
this technique, some aspects of the macromolecular contributions by the
polymeric network to the molecular recognition process can be evaluated.
Figure 9a to 9c shows the FT-IR spectra of the prepared polymer (PP), the non-
imprinted polymer (NIP) and the molecularly imprinted polymer (MIP).These
spectra were used as basis to clarify the functionality and composition of the
polymers. The Infrared (IR) spectra of the polymers showed absorptions
associated with the complex interacting vibrating systems and a pattern of
vibration which is uniquely characteristic of a particular molecule.
Comparing the IR spectra of the distilled methacrylic acid with that of
the prepared polymer, it was evident that the C-OH stretch at 1000 cm-1 to 1400
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cm-1 was shown. The proof of polymerization existed when a bathochromic
shift to higher wavelength occurred from 1004.3 cm-1 to 1118.12 cm-1. The 1634
cm-1 peak also proved that the C=C bond are still present which suggests that
the interaction of the methacrylic acid and the crosslinker were at the C=C bond
not with the N-H bonds. Another confirmation that polymerization was
successful was the non-existence of the OH peak attributed to MAA. This was
mainly due to the linking of the electron withdrawing group like Pb+2 of lead
acetate with the two Oxygen of MAA. The less broad N-H stretch of PP as
compared to that of MAA was evident. The presence of a doublet at 1430 - 1464
cm-1 in the MAA due to a primary N-H became a very weak singlet in the PP
due to a secondary N-H. This may be attributed to the interaction of the MAA
with the crosslinker at the double bonds (C=C).
The prepared polymer as represented by Figure 9a illustrated the broad,
strong peak around 3434 cm-1 attributed to the presence of N-H stretching
present in the crosslinker’s structure. At 1648 cm-1, strong peak signified that
after polymerization, the alkenes functional group of the methacrylic acid in the
polymer is still present. The presence of lead acetate is depicted by a weak peak
at 2000.35 cm-1where the interaction of lead with the carbonyl oxygen was
implied. The peak at 1647.51 cm-1 was due to C=O bonds stretching. Still the
C-O peak is there but with a bathochromic shift at 1124.86 cm-1 while the C=C
peak disappeared.
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The non-imprinted polymer (NIP) having no template in its system also
produced different vibrational modes. Figure 9b showed the IR spectrum of the
non-imprinted polymer (NIP) where the strong peak at 1640 cm-1 shows the
stretching vibration peak of the C=O bond. The weak peak at 2086 for C=C
stretch indicated the presence of the cross-linker. The C-OH stretch was
stronger and the 1634 cm-1 of the C=C peak was not present. The broad peak of
N-H or O-H is obvious and N-H bending can be seen at 1537 cm-1. The presence
of a stronger C=C peak at 2000 cm-1 indicate that termination mechanism may
be by disproportionation. The presence of the 1391 cm-1 was not as intense as in
the MIP, because one C-O bond is there and the other is still C=O bond.
The IR spectra of the molecularly imprinted polymer (Figure 9c)
described the functionality of the bonds present in the said long-chain
molecule. The presence of an amide was shown by a strong peak at 3406.42 cm-1
that signifies N-H stretching. This peak is narrower compared to that of the PP
which means that the C-OH peak became weak. Weak peaks at 1455.65 cm-1
indicated C-H bending while strong peak at 1658.23 showed C=O stretching.
Since methacrylic acid is a carboxylic acid, the small peak at 2945.95 cm-1
absorption region maybe attributed to the C-H stretch. The 2000 cm-1 (C=C)
peak disappeared indicative that template was removed and termination
mechanism may be due to symmetry, combination type of termination
mechanism or dipole moment reason.
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80
Figure 9a
60
40
20
80
%Transmittance
60 Figure 9b
40
20
80
Figure 9c
60
40
4000 3000 2000 1000
wave numbers (cm-1)
Figure 9. IR Spectra (9a:)the Prepared Polymer; (9b:)Non-imprinted Polymer

and (9c:) Molecularly Imprinted Polymer.
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See Appendix E for overlay figures. The IR spectra showing the
assignment of the functional groups present in the prepared polymer (PP), non-
imprinted polymer (NIP) and molecularly imprinted polymer (MIP) with
corresponding frequency and intensity is given in the following tables:
Table 7
Summary of IR Spectral Data of PP
Frequency,cm-1 Assignment Intensity
3434 N-H stretch strong
2000 Metal carbonyl weak
1648 C=O stretch strong
1544 N-H bending strong
1444 C-O bending medium
1211 C-N bending weak
Table 8
IR Spectral Data of Non-imprinted Polymer
Frequency,cm-1 Assignment Intensity

3473 O-H and N-H stretch Broad, strong
2634 Symmetric C-H weak
2086 C=C stretch weak
1640 C=O stretch strong
1537 N-H bend medium
Table 9
Outline of the IR Spectral Data of the MIP

3406.42 N-H stretch strong
2945.95 C-H stretch weak
1455.65 C-H bend weak
1384.54 C-O stretch strong
1117.57 C-O stretch weak
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B. SEM and EDX Analysis
The structure and morphology of the polymers were characterized by
scanning electron microscopy. Elemental analysis of the polymeric samples was
provided by the Energy Dispersive X-rays technique of the SEM machine.
Figure 10. SEM Surface Structure of the Non-imprinted polymer
a) SEM image of sample NIP x 10,000 magnification
b) SEM image of sample NIP x 20,000 magnification
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Magnification ability of the SEM machine was determined and found out
that the 10,000 magnification gave a more distinct profile while that of the
20,000 gave blurred figures as evidently seen from Figure 10a and 10b.
Therefore, all SEM tests were undertaken under the said magnification.
As shown in the figures, all particles have a similar spheric configuration
and the size of the polymers was homogeneous.
Table 10
Elemental Analysis of the NIP using EDX technique
Element Element % Atomic %

Carbon 29.51 35.44
Nitrogen 30.94 31.86
Oxygen 33.63 30.32
Sulfur 2.31 1.04
Potassium 3.62 1.34
Lead 0.00 0.00
Table 10 clearly states that the non-imprinted polymer does not contain
the template. Most of the component elements were carbon, nitrogen and
oxygen, present due to the interactions of monomer and crosslinker. The
presence of the potassium persulphate initiator is indicated by the percentage of
potassium and sulfur as seen from the elemental analysis of the EDX technique.
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Figure 11. Sample view of the prepared polymer (PP) using two different
magnifications.
a) SEM image of PP x 20,000 magnification
b) SEM image of PP x 10,000 magnification
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Table 11
EDX Analysis of the prepared polymer (PP) as shown in Figure 11b.

Carbon 32.10 39.13
Oxygen 22.51 20.60
Potassium 0.42 0.16
Lead 7.07 0.50
The percentage of lead bound with other molecules was read to be 7.07%
and as an atom the microscope scanned it as 0.50% present. The analysis
showed that most of the elements present that are binding with one another are
carbon with nitrogen and/or oxygen.
Structural Characterization of the Molecularly Imprinted Polymer (MIP)
In order to understand molecularly imprinted polymers such as that
illustrated by Figure 12c and its properties, it was necessary to characterize the
structure and morphology of these materials. Knowledge about processes such
as polymer chain mobility or side chain mobility and transport of small
molecules within the pores, is important to understand the behavior of these
materials. Some of these characteristics were visually revealed by the use of the
Scanning Electron Microscope (see figure below). Particle size ranges from 10
microns to 13 microns.
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SEM image of the MIP also shown as Figure 12c
Table 12
Elemental Analysis of the MIP

Carbon 34.92 39.88
Oxygen 26.11 22.38
Sulfur 0.06 0.03
Potassium 0.03 0.01
Lead 0.41 0.03
An assessment of the EDX analysis in terms of % element of the prepared
polymer, non-imprinted polymer and the molecularly imprinted polymer is
presented below as Table 13 and evaluated.
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Table 13
Comparison of the % Element of experimented Polymers
Element PP NIP MIP

Carbon 32.10 29.51 34.92
Nitrogen 37.90 30.94 38.47
Oxygen 22.51 33.63 26.11
Sulfur 0.00 2.31 0.06
Potassium 0.42 3.62 0.03
Lead 7.07 0.00 0.41
The MIP left without the template has a carbon percentage higher than
NIP and PP which may mean that linkages of bonds between this element and
others like nitrogen and oxygen has remained strong. The actual configuration
of the assemblages can be determined by x-ray crystallography.
The crosslinker containing nitrogen in its molecular arrangement has its
configuration changed together with the template during the lead acetate
removal process. This caused the difference in the nitrogen percentage between
the PP and MIP.
3. Removal of Template from the Polymer Matrix
Removal of the template was done by saturating the prepared polymer
PP with 1:1 (v/v) nitric acid solution overnight and washing with 7:1 (v/v)
methanol :acetic acid solution to produce a molecularly imprinted polymer
(MIP).
The different changes in the structure and character of the molecule were
monitored using the Scanning Electron Microscope as it undergoes treatment
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with the agents for separation. Further confirmation on several observations
was provided by the succeeding analytical tests.
A. SEM Results:
Figure 12. Scanning electron microscopy image of the molecularly

imprinted polymer at 10,000 X magnification before and after removal.
a) SEM Surface structure image of original prepared polymer (PP)
b) SEM image of Prepared Polymer soaked in 1:1 HNO3
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c) SEM image of Polymer washed with methanol:acetic acid
Figures 12a to 12c showed the transformation of the prepared polymer to
a moleculary imprinted polymer by undergoing treatment with nitric acid and
methanol:acetic acid. From the figure, the surface structure of the polymer after
soaking was very much different from the surface profile of the original
prepared polymer. Figure 12a showed a prominent shape of the surface
structure that looked like an encircled boundary of the crosslinker, linking the
template and the polymerized monomer. Observed in Figure 12b was the
disappearance of that unusual shape and structure.
Figure 12c showed that methanol:acetic acid mixture has created cavities
that is characteristic of a molecularly imprinted polymer. This has resulted from
the washing off molecules that are not selectively bound to the polymer and
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destroyed selective interaction between the template and the polymer.
The MIP obtained and characterized by scanning electron microscope as
shown in Figure 12 signified that all particles possess a similar morphology in
the form of rounded mass. The sizes of the particles are 1-15 microns. The IR
spectra of the MIP, from which the template had been extracted with 1:1 nitric
acid and washed with methanol:acetic acid, implied that the structures of the
particles were not changed or destroyed but the composition has varied.
B. EDX Technique gave an elemental analysis on the different stages the
prepared polymer has undergone. Table 14 shows percentages of the major
elements present in the molecule after treatment with 1:1 nitric acid.
Table 14
EDX analysis of treated PP as shown in Fig 12b

Carbon 35.81 40.84
Oxygen 29.57 25.32
Potassium 0.03 0.01
Lead 0.00 0.00
From the surface profile, EDX analysis, and UV-Vis spectra, it can be
deduced that the removal of the template from the polymer matrix using nitric
acid (1:1) and methanol: acetic acid mixture was effectual.
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C. UV-Visible Spectroscopy Result:
UV - Vis spectra of NIP and PP with 1:1 HNO3

A
b 6
s 4 Blank
o
r 2 PP 1
b
a 0 PP 2 (M)
n -2 0 100 200 300 400 500
c
NIP
e -4
Wavelength (nm)
Figure 13. Ultraviolet-visible spectra of the collected filtrate from the

prepared polymers soaked in 1:1nitric acid
The absorbance readings and shifting of lambda max shown in Figure 13
indicated that removal of lead acetate template with 1:1 nitric acid:water
solution (v/v) has taken place. The blank used was the 1:1 HNO3 in water
solution (v/v) . Polymer PP1 and PP2 represent the two trials made resulting to
absorbance readings being of the same amount. The high absorbance readings
of the PP ranging from 3.144 to 4.348 mean that a certain amount of the
template has reacted with the nitric acid.
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Figure 14. SEM image of the PP in which a chelating agent and concentrated
nitric acid were used for the removal of template.
(a) (b)
Figure 14a is the picture of a trial using concentrated nitric acid to
remove the lead acetate template from the prepared polymer. Another trial,
figure 14b was made using EDTA solution (10%) being passed through the
prepared polymer to test for removal of lead. By the looks of both, structure of
particles were changed and destroyed.
Although EDTA, a polydentate ligand (or a chelating agent) has the
ability to hold a metal atom like a claw , the resulting reaction with the
prepared polymer was unexpectedly negative.
4. Test on the Rebinding Capacity of the MIP with Lead Acetate
The molecularly imprinted polymer (MIP) was subjected to a rebinding
experiment. A 0.2-0.4 g of this polymer was placed in a vial and added with 2
mL of the prepared known concentration of lead acetate. It was soaked and
equilibrated overnight then filtered with glass wool. Rebinding capacity of the
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MIP with lead acetate was also tested by SEM technique and compared with the
SEM of prepared polymer.
A. Scanning Electron Microscopy Result
For the rebinding capacity, a known concentration of lead acetate
solution ( 0.10 %) was used as the analyte and SEM test was compared with the
SEM results of the prepared polymer (PP).
The following figures showed the difference in the surface structure of
the prepared polymer with the MIP binding again with lead acetate.
Figure 15. SEM images at 10,000 X magnification, of MIP showing

rebinding with lead acetate solution.
a) SEM image of MIP
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b) SEM image of MIP rebinding with lead acetate
Interpretation:
The cavities of the MIP in Figure 15a has been filled up after a known
concentration of lead acetate passed through the MIP. Figure 15b illustrates the
effectiveness of the MIP as a stationary phase or adsorbent for chromatographic
purposes.
Elemental Analysis of the MIP
Table 15
EDX analysis of MIP rebinding with lead acetate
Element Element(%) Atomic(%)

Carbon 32.40 37.46
Oxygen 24.36 21.14
Sulfur 0.22 0.09
Potassium 0.01 0.00
Lead 1.43 0.10
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Based on Table 15 showing the elemental analysis of the molecularly
imprinted polymer, the 1.43% indicated that lead(Pb) has rebinded to the
polymer. This showed that the MIP has retained in its memory the fingerprint
of the template thus has the capacity to rebind.
B. FT-IR Results
An outline of the characterization using Infrared spectroscopy is on Table
16 below:
Table 16
Outline of the IR Spectral Data of the MIP rebinding with template

3430.66 N-H stretch strong
2946 C-H stretch weak
1529.02 N-H bending strong
1384 C-C stretch strong
1208.92 C-N bending medium
Referring to Figure 16 – the spectra of the MIP rebinding with lead
acetate; the strong peak at 3430.66 cm-1 with 32% Transmittance maybe
attributed to absorption due to the N-H stretch bond. The presence of an amide
methacrylic acid were the sources of such absorption. A strong absorption at
1655.57 cm-1 confirmed the C=O stretching of acetates or carboxylic acid groups.
N-H bend was depicted by the spectra at 1529.02 cm-1. The C-C stretch bonds
are present as represented by peak 1384.23 cm-1. C-N bending was
characterized by the absorption peak of 1208.92 cm-1.
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Figure 16. IR spectra of MIP showing rebinding of lead acetate

82
5. Experiment on the Binding Capacity
This part of the research determined the selectivity of the
molecularly imprinted polymer with other lead compounds.
5.1 Binding with Lead nitrate
The analytical tests applied here to determine if there will occur
binding between the imprinted polymer and lead nitrate were the following:
A. SEM technique
Surface analysis of the MIP was given by the scanning electron
microscope at 10,000 magnification. Figure 17 shows,
Figure 17. SEM image before and after binding with lead nitrate.
a) SEM image of MIP
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b) SEM image of MIP with lead nitrate
Images from Figure 17a and 17b are evidences that the binding of lead
nitrate with the molecularly imprinted polymer were not manifested clearly.
The two SEM results are almost similar in structure and looks like nothing
happened.
B. EDX Analysis of the Polymer Binding with Lead Nitrate
Table 17
Elemental Analysis of the MIP with lead nitrate
Element Element (%) Atomic (%)

Carbon 34.28 39.25
Oxygen 28.99 24.93
Sulfur 0.11 0.05
Potassium 0 0
Lead 0.20 0.01
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From the elemental analysis of Table 17, noticed that potassium has
become zero since it might have reacted to form potassium nitrate. Percentages
of the elements as determined by SEM-EDX have actually different readings as
it is dependent on the surface area of the sample being striked by irradiation.
The percentage lead (Pb) was very minimal but might go higher if
further interactions, by increasing the time of reaction to reach equilibrium
condition, was made to allow the substrates to interact more. Overnight
standing was not enough for the lead nitrate to penetrate the molecular
structure of the MIP.
C. FT-IR Spectroscopy
Characterization of the MIP after its binding with lead nitrate was
supported by Infrared spectroscopy. The IR spectra is shown in Figure 18.
The spectra is very much different from that of the original MIP as seen
in the shape of the profile. A strong peak at 3426.38 cm-1 maybe attributed to a
the presence of an amide group due to N-H stretch; while N-H bend was
shown at peak 1632.37 cm-1 and C=O stretch at peak 1653.92 cm-1.
See Appendix E for overlay figures of the MIP binding with lead acetate
compared with the graph of MIP binding with lead nitrate.
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Figure 18. A spectra of the MIP binding with lead nitrate

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5.2 Aviation Gasoline
The composition of the aviation gasoline (Avgas) fuel comprises about
20-80 vol % iso-octane, 5-18 vol % toluene, 1-20 vol % C4 to C5 paraffins, about
0-1 ml/gallon tetraethyl lead and the balance light alkylate.
As per specifications given by the source of the gasoline, the amount of
lead (Pb) present and analyzed was 0.5 gram per liter. Retesting the sample
using atomic absorption spectrophotometer resulted to a 28. 7565 parts per
million value.
The MIP with 10-15mm particle size that were packed and evaluated as
selective stationary phase for lead in lead nitrate sample was also applied to
direct analysis of lead from aviation gasoline samples.
A. SEM Results
Visual comparison of the surface profile on the effect of extracting lead
from a sample of aviation gasoline after letting it pass through the MIP was
undertaken. Filled up cavities means binding took place.
Judgment on the difference of the surface character of the MIP before
and after the sample passed through it was based mainly on the images or
actual pictures developed by the scanning electron microscope. Figure 19 gave
the profile and comparison was made between the before and after conditions.
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Figure 19. SEM comparison of surface structure of the MIP before and
after an aviation gasoline passed through it.
a) SEM image of MIP
b) SEM image of the MIP with aviation gasoline sample
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From the SEM images, the efficiency of the MIP as a stationary
phase/adsorbent was verified judging from the density and particle size of the
magnified surface structure before and after the aviation gasoline sample was
purged into the MIP. Figure 19b demonstrated a denser organization of greater
particle size of the MIP after an aviation gasoline sample had passed through it
as compared with the size and density of the original MIP, Figure 19a.
B. EDX Result
The elemental composition of the MIP was determined by EDX
Technique of the Scanning electron microscope. Table 18 below summarized it.
Table 18
Elemental Analysis of the MIP with aviation gasoline
% Element % Atomic
Carbon 30.59 35.30
Oxygen 30.82 26.889
Sulfur 0.04 0.02
Potassium 0.02 0.01
Lead 0.15 0.01
Comparing the percentages of carbon, nitrogen and oxygen elements of
the MIP used as stationary phase for an aviation gasoline sample with the
original MIP, it can be inferred that the organic components of the gasoline
sample has greatly reacted with the MIP as shown by the increase in the
percentages. The table of data reveals that some lead components were trapped
by the MIP.
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C. FT-IR Spectroscopy
Figure 20 gives the IR spectra of the aviation gasoline fuel demonstrating
the functionality of the different components that can react with the MIP. It was
evident that the strong peak at 3026.76 cm-1 signified the sp-sp2 C-H stretch and
peak 2873.12 cm-1 indicated C-H stretching due to alkanes and confirmed by
1365.87 cm-1 to 1488.78 cm-1 due to C-H bend. Peaks showing monosubstituted
components at the combinatory band were at regions 1800 -2000 cm-1. A list of
matches for the Aviation fuel using correlation as the search algorithm at
regions 3495.26 to 667.16 frequency (cm-1) was tabulated in Appendix D.
Figure 21 shows the spectra of the molecularly imprinted polymer after
an aviation gasoline sample was allowed to pass through it. At least three
prominent peaks were depicted by spectra. One is the strong peak at 3414.22
cm-1 which maybe attributed to the presence of an amide group due to N-H
stretch; while N-H bend was shown at peak 1633.33 cm-1. A strong peak at
1384.53 cm-1 indicates that C-O stretch present in the original MIP remained
untouched by the presence of an aviation gasoline.
The covalent interaction of the tetraethyl lead present in small amount in
the aviation gasoline sample maybe depicted by the small weak peaks along the
1540.85 cm-1 region caused by the C-H bend of alkane group.
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Figure 20. IR Spectra of Aviation Fuel
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Figure 21. IR Spectra of MIP binding with the aviation fuel
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6. MIP as stationary phase for the correlation of the concentration of lead in
samples.
The application part of this study limits the use of the MIP for two
samples such as the aviation gasoline fuel and lead nitrate. The MIP was
utilized as the stationary phase for the MEPS (microextraction in packed
syringe) technique. One (1) mL of the eluate was collected and digested with
concentrated nitric acid then analyzed for lead content using the atomic
absorption spectrophotometer. The difference in the lead (Pb) concentration
(ppm) before and after the extraction is related to the binding capacity of the
polymer.
A. Atomic Absorption Spectrophotometry Results
Detection of lead from the two samples’ eluate was accomplished using
the atomic absorption spectrophotometer. Result of examination is tabulated as
follows:
Table 19
Concentration of Lead (ppm) in samples
Samples Trial 1 Trial 2 Mean Difference % Diff

Aviation gasoline
sample
Before 29.4602 28.0527 28.7565
After 27.8305 27.2564 27. 54345 1.21305 4.2
Lead nitrate sample
Before 198.7840 198.7840 198.7840
After 65.3331 62.6754 64.0043 134.7797 67.8
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From these tabulated data, the amount of lead retained by the MIP can
be computed by subtracting the mean-after passing the MIP, from the mean
before passing the same column.
The amount of lead (Pb) in the aviation gasoline sample considered
imprinted into the MIP is equal to 1.2038 ppm (4.2%), a minimal value which
means that the MIP produced is not that effective for use in gasoline samples.
But for other lead compounds like lead nitrate, the amount of lead retained in
the MIP column was 134.7797 ppm which is 67.8% of the initial composition.
This percentage can be increased by increasing the amount of MIP to be used as
the adsorbent or the stationary phase in the extraction process. A higher
proportion of the MIP with the amount of sample that passed through it will
give a higher yield.
7. Statistical Analysis
Statistical investigations and analyses of data involved the inferential
category which concerned developing and using mathematical tools to make
forecasts and inferences.
The t-test is the most commonly used method to evaluate the differences
in means between two groups. In this research, the t-test was used to infer the
difference between the concentration (in ppm) of two samples before it passed
the MIP column and after passing through the same MIP column.
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This statistical study limits the test for two samples, lead nitrate and the
aviation gasoline with two trials each.
Since the difference is in the predicted direction, only one half (one "tail")
of the probability distribution was considered.
Table 20
Summary of Statistical Data
Aviation Gas Lead Nitrate

Before After Before After
Trial 1 29.4602 27.8305 200.0000 65.3331
Trial 2 28.0527 27.2564 198.7840 62.6754
Mean 28.7565 27.54345 199.3920 64.0043
Variance 0.99053 0.1648 0 3.5317
df 1 1
Level of significance 0.05 0.05
t-critical value from table 6.3138 6.3138
t-test calculated 2.9110 101.4259
Statistical Decision Accept null hypothesis Reject null
hypothesis
The selected level of significance was 0.05 usually employed for
analytical chemistry experimental researches.
t-Test : paired two sample for means statistical tool was employed.
From Table 20, for the aviation gasoline sample the calculated value of
test statistic is less than the t-critical taken from the distribution table.
Therefore, the null hypothesis was accepted meaning that there is no significant
difference between the concentration of lead that has passed through an MIP
before and after. But for the lead nitrate sample, the null hypothesis was
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rejected which means that there is a significant difference between the amounts
of lead before and after it had passed through the MIP, since the calculated test
statistic is greater than the t-critical from the distribution table.
This decision directs to a conclusion that the type of a molecularly
imprinted polymer prepared in this research can be used as a stationary phase
in a column or a separation media for the selective quantification of lead
compounds that can be directly correlated with lead.
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Chapter 5
SUMMARY , CONCLUSIONS AND RECOMMENDATIONS
This chapter presents the summary of findings, conclusions drawn based
on the findings and the recommendations offered by the researcher.
Summary of Findings
The first aim of this research is to prepare molecularly imprinted
polymers with selectivity on lead acetate template for its use on the separation,
and quantification of a health hazard substance like heavy metal lead (Pb).
Secondly, to determine whether this template undergoes molecular
imprinting by self-assembly in the imprint molecule system giving rise to
isolated molecular binding sites. Third, to test whether these binding sites are
able to bind with compounds containing lead and rebind with another solution
of the template. Lastly, to utilize this MIP as an adsorbent or a stationary phase
in the quantitative measurement of lead in the aviation gasoline sample and
lead nitrate solution.
The experimental method of research was used in this study. The
molecularly imprinted polymer was prepared by dissolving 0.3 g of lead acetate
in a 5.0 mL distilled water then adding 0.3 g N,N’ methylene bisacrylamide,
followed by the addition of 0.20 mL of MAA and 0.05 g of potassium
persulphate. Degassing and sonication of the solution was done at 80oC and at
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polymerization time of 36 hours. Drying; crushing and grounding; removal of
template by soaking in 1:1 nitric acid solution and washing with methanol
:acetic acid completed the preparation of the MIP.
The effects of the kind and amount of cross-linkers such as EGDMA and
N,N’-methylene-bisacrylamide were differentiated. Between the two, it was
proven that N,N’-methylene-bisacrylamide gave the best effect.
Characterization of the prepared polymer , NIP and the MIP was done by
several analytical techniques like SEM –EDX, FTIR and UV-Visible
spectroscopy. The suitable method of removing the imprint molecule from the
prepared polymer was determined by saturating the polymer with 1:1 nitric
acid solution and then washing it with methanol-acetic acid mixture and water.
The resulting dried MIP packed in a 2-mL glass syringe using glass wool as
support was applied as a stationary phase and/or an adsorbent using the MEPS
process.
Quantitative analysis on the concentration of lead (in ppm) before
extraction as well as the extracted lead in the eluate was carried out using the
Atomic Absorption spectrophotometer. The concentration of lead retained in
the polymer was determined by subtracting the ppm after extraction from the
ppm of the original sample.
A series of the absorption spectra of the solution of lead acetate and
methacrylic acid in Figure 4 was obtained. The changes in the spectra relative to
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the original ones indicated that interactions had taken place between the
template and the monomer. The functional groups present in the template
provide points for electrostatic and hydrogen-bonding interactions for the
complex formation prior to polymerization.
The IR spectra of imprinted polymers (Figures 6a to 6c) showed that
most of the monomers and the cross-linker were polymerized. Spectra also gave
an idea that after the polymerization there are still functional groups in the
molecularly imprinted polymer that can interact with the template molecule.
By choosing N,N’-methylene-bisacrylamide crosslinker, random
copolymerization occured successfully and the functional residues-derived
from the functional monomers, are uniformly distributed in the polymer
network as seen through SEM (Figures 5 a-d) and FT-IR. The surface structure
of the resulting polymer and solubility of the crosslinker to monomer, template
and the common solvent was the basis of efficiency. The crosslinker and
initiator, potassium persulphate were chosen to study their application in
molecular imprinting on the basis of their solubility and compatibility with the
other agents of polymerization as well as their availability in the local market.
Molecular recognition and characterization of the prepared polymer (PP)
were undertaken through the aid of several analytical instruments such as the
Scanning Electron Microscope (SEM), Fourier Transform Infrared Spectrometer
(FTIR) and Ultraviolet-Visible Spectrophotometer.
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The structure and morphology of the polymers were characterized by
scanning electron microscopy. Elemental analysis of the polymeric samples was
provided by the Energy Dispersive X-rays technique of the SEM machine.
Magnification ability of the SEM machine was determined and found out
that the 10,000 magnification gave a more distinct profile. All SEM tests were
undertaken under the said magnification. As shown in the figures, all particles
have a similar spheric configuration and the size of the polymers is
homogeneous.
SEM figures that showed the structure of the polymer after the removal
of the template produced cavities, characteristic of a molecularly imprinted
polymer MIP.
The MIP was subjected to a binding experiment. The difference in the
concentration of lead (Pb) in the eluate before and after the extraction expressed
in terms of parts per million (ppm) gave the binding capacity of the polymer.
For the rebinding capacity, a known concentration of lead acetate
solution was used as the analyte and underwent the same treatment as lead
nitrate. SEM profile was compared with the previous SEM results of the
prepared polymer (PP) that clearly showed the rebinding capacity of the MIP
selective to lead acetate. The selectivity of the MIP with lead compounds was
proven by the binding experiment with a known concentration of lead acetate
and lead nitrate, respectively.
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For the application part, the aviation gasoline sample that contained
tetraethyl lead has a slight binding ability with the MIP as illustrated by SEM
results judging from the density and particle size of the magnified surface
structure before and after an aviation gasoline sample was purged into it.
Conclusions
The interactions between lead acetate template and the pre-polymer
complex were investigated, characterized and elucidated in this research. Based
on the aforementioned findings, the synthesized molecularly imprinted
polymer showed specific recognition capability for the template lead acetate
and sample lead nitrate solution but not with the aviation gasoline.
Statistically, the t-test decision leads to a conclusion that the type of a
molecularly imprinted polymer prepared in this research can be used as a
stationary phase in a column for the selective separation of lead. Stating it other
way, such MIP is a capable stationary adsorbent for chromatographic
techniques.
Recommendations
Based on the findings of the study and the conclusions drawn, the
researcher recommends the following:
In a future work, the plan of packing the MIP materials in a glass column
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or to fabricate them on a conductive substrate in order to resolute the racemic
mixture directly using electrochemical method is recommended. However, the
type of MIP produced in this study is not highly recommended for the
stationary phase of High Pressure Liquid Chromatography (HPLC) since the
obtained polymers are not stiff enough.
Having proven that the MIP of this study is a good separation media,
other compounds containing heavy and toxic metals like mercury can be
experimented on using the same monomer, crosslinker, porogen and initiator.
The making of a portable adsorbent containing this molecularly
imprinted polymer may be tested on a polluted environment where emissions
of gasoline smoke are very high. The binding properties of the molecularly
imprinted polymer prepared in this study could also be exploited for its
possible use in the pre-concentration and extraction of other heavy metals from
natural waters.
Further studies or in-depth investigation on the molecular
configuration of the molecularly imprinted polymer produced in this study
using computational chemistry is also recommended, for the purpose of
product development and broad application in the field of environmental
science.
Incorporation of the function of this type of molecularly imprinted
polymer (MIP) as an alternative technique for trace residue analysis and as part
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of a properly implemented quality check programs will surely give some
advantages. One advantage is its use in a routine quality assurance monitoring
of in-process products and another as a collector of effluents in a certain
manufacturing plant or of pollution instruments.
An advantage of molecularly imprinted polymers is their long-term
stability and resistance to chemically harsh environments. This characteristic
can be used for pre-concentration of diluted lead components in a sample for
determination in an atomic absorption spectrophotometer.
The simple and selective sample extraction technique, MEPS used in this
study for the determination of lead in the aviation gasoline and lead nitrate
samples was reliable. This method that reduced sample preparation time and
organic solvent consumption can be recommended for use in other extraction
procedures.
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Appendix C
Sample Computation for T-Test of Significance of Difference Between Paired

Two Sample for Means
Aviation Gas Lead Nitrate

Before After Before After
Trial 1 29.4602 27.8305 200.0000 65.3331
Trial 2 28.0527 27.2564 198.7840 62.6754
Mean 28.7565 27.54345 199.3920 64.0043
Variance 0.99053 0.1648 0 3.5317
df 1 1
Level of significance 0.05 0.05
t-critical value 6.3138 6.3138
t-test 2.9110 101.4259
Statistical Decision Accept null hypothesis Reject null hypothesis
Let μ1 = mean concentration in ppm of lead before passing the MIP column
μ2 = mean concentration in ppm of lead after passing the MIP column
Step 1: A decrease in mean shows μ1 > μ2
Null Hypothesis (Ho): There is no significant difference in the
concentration of lead before and after passing through the MIP column.
Ho : μ1 = μ2
Alternative Hypothesis (Ha) : There is a significant difference in the
concentration of lead before and after passing through the MIP column.
Ho : μ1 > μ2
Step 2: Level of significance, α = 0.05
Step 3: Degrees of freedom (df) = n – 1 = 2 – 1 = 1
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The critical value of t for df = 1 and 0.05 area in the right tail of the t-
distribution, from table = 6.3138
Step 3: Decision rule = Reject Ho if computed value of t < 6.3138 otherwise
accept.
Step 4: Calculate the value of t-test statistic
d= ppm before – ppm after = 29.4602 – 27.8305 = 1.6297
_
d = Σd/n = 2.426/2 = 1.213
_
Σd = d-d = 1.6297 – 1.213 = 0.4167
___________________
Sd =√ [Σd 2 – (Σd)2/n]/n-1 = 0.5893
_
d __ 1.213 _
t = --------- √n = ------------ √2 = 2.9110
Sd 0.5893
Step 5: Statistical Decision
Since computed t = 2.9110 which is less than the t-critical value of 6.3138,
therefore the null hypothesis is rejected meaning that there is a significant
difference between the before and after of the sample. In relation to the
research, there is a significant difference on the concentration of lead from lead
nitrate solution before and after the sample had passed through the molecularly
imprinted polymer column but no significant difference for the aviation
gasoline sample.
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Appendix E
Overlay Spectra 1: MIP binding with lead acetate (blue graph); the prepared
polymer (red graph).
Overlay Spectra 2: Molecularly imprinted polymer(red graph); Lead acetate

rebinding(light blue graph) and Lead nitrate binding (dark blue graph)
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Overlay Spectra 3: Prepared polymer (light blue); Non-imprinted

polymer(dark blue) and Molecularly imprinted polymer (red)
Overlay Spectra 4: Undistilled MAA(red graph) and Distilled MAA(light

blue graph)
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Appendix F
List of Matches for Aviation Gasoline
Search results list of matches for the Aviation fuel using correlation as
the search algorithm at regions 3495.26 to 667.16 frequency (cm-1) are tabulated
as follows:
Table 21
List of Matches for the Aviation gasoline sample
Index Match Compound Name Library Name

1 1147 93.14 Hydrocarbons, aliphatic Hummel Polymer and
additives
2 1146 92.89 Hydrocarbons, aliphatic same as above
3 1520 91.89 2,2,4-Trimethylhexane same as above
4 31 91.59 2-Methylpentane,99+% Aldrich Hydrocarbons
5 30 90.48 2-methylbutane,99+% same
6 38 90.18 2,2-Dimethylpentane same
7 36 89.89 2,2-Dimethylbutane same
8 41 89.87 2,4-Dimethylpentane,99% same
9 44 89.46 2,2,4-Trimethylpentane,99% same
10 28 88.65 Mineral spirits same
More or less, the presence of the aliphatic hydrocarbons shown on Table
19 gave a hint on the composition of the aviation gasoline sample.
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CURRICULUM VITAE
PERSONAL DATA:
Name : Lucy Austria Magleo – Domingo
Date of Birth : March 23, 1959
Place of Birth : Quezon City
Sex : Female
Citizenship : Filipino
Height : 162.56 centimeters
Weight : 59.09 kilograms
Permanent Address : No. 13 Jacob-Stolt Nielsen St. Seamen’s Village,

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June 1975 – April 1980
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ADAMSON UNIVERSITY

BINDING PROPERTIES OF A MOLECULARLY IMPRINTED POLYMER

LUCY AUSTRIA MAGLEO-DOMINGO

In partial fulfillment of the requirements for the degree MASTER OF

Approved by the Committee on Oral Examination with the grade of

Comprehensive Examination Grade: 1.8

Accepted and approved in partial fulfillment of the requirements for the

I dedicate this masteral thesis to my One and True God.

To my family, my father 2nd Lt. Francisco Poyaoan Magleo and to my mother

freedom to make decisions.

guidance, wisdom and strength. Without HIS overwhelming Love and

My sincerest thanks go to the following persons who have shared their

contributions to make this research successful:

Dr. Marietta A. Ilao, my adviser and consultant, for being patient,

helpful and understanding.

and propositions geared towards the improvement of this research. Special

Dr. Cristina C. Salibay and Dr. Carmelita C. Cervillon, former and

concurrent Deans of DLSU-Dasma/College of Science and Engr. Imelda C.

Galera, chairperson of our Physical Sciences Department/College of Science,

DLSU-Dasmarinas for their permission to use the Chemistry Research

Engr. Wilfredo Aguinaldo, previous Physics and Chemistry Research

Laboratory Coordinator and Engr. Jennifer Binas, current Chemistry/Physics

Laboratory Supervisor for their assistance in the CRL facility.

Ms. Jane and Ms. Aimee, DLSU-Dasmariήas Chemistry Laboratory

Technicians, for their assistance during my experimentation.

Chemistry Department of De La Salle Manila, for his serenity during the

spectroscopic analyses of all my samples.

Mr. Rey Coria, technician of the DLSU-Manila Solid State Laboratory

Engr. Mildred Amador-Reyes and Atty. Myrna Villegas-Alcazar of the

in the acquisition of my much needed chemicals.

aviation gas sample and my other batchmates-friends from Mapua Institute of

Technology such as Bing Nerona-Caberte and Shirley Mercado-Bueno for their

for lending me their thesis manuscript.

To all the other beautiful people of the College of Science -Physical

and heartfelt concerns.

To my church family, especially Pastor Mike Clemeήa and Sis. Vangie

Baccar for their prayers and support.

To my niece Rona Jamaira Magleo, who had helped me in the internet

browsing and surfing for additional information related to this study.

And lastly to my parents who had instilled in me the virtue of patience,

perseverance and long-suffering.

Title : BINDING PROPERTIES OF A MOLECULARLY

Researcher : LUCY A. MAGLEO-DOMINGO

Adviser : DR. MARIETTA A. ILAO

Degree : Master of Science in Chemistry

Date Completed : October 2008

This research was carried out to prepare a molecularly imprinted

polymer potentially selective to lead acetate compound, lead nitrate solution

and tetraethyl lead from an aviation gasoline sample.

Characterization of the binding properties of these lead compounds with

separation processes were experimented. A simple sample extraction technique

(MEPS) microextraction in packed syringe was applied.

Free-radical polymerization was employed using methacrylic acid as the

monomer; N,N’-methylene bisacrylamide, the crosslinker; potassium

porogen. Polymerization time was 36 hours at 80oC temperature.

Binding properties were investigated through a Scanning Electron

Microscope with Energy Dispersive X-rays capability. Binding sites were

characterized by IR Spectroscopy and Ultraviolet-Visible Spectrophotometry.

Atomic Absorption Spectrometer.

Statistically, the decision that there is a significant difference in the

conclusion that the type of a molecularly imprinted polymer prepared in this

research can be used as an adsorbent or a stationary phase in a column for the

1 THE PROBLEM AND ITS SETTING . . . . . . . . . . . . . . . . 1

Background of the Study . . . . . . . . . . . . . . . . . . . . . . . . . . 4