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ANALYSIS OF AMPHETAMINE AND METHAMPHETAMINE
BY SURFACE-ENHANCED INFRARED SPECTROSCOPY

presented by

SRIVIDHYA KIDAMBI

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MS. degree in Criminal Justice

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ANALYSIS OF AMPHETAMINE AND METHAMPHETAMINE BY
SURFACE-ENHANCED INFRARED SPECTROSCOPY

By

Srividhya Kidambi

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Criminal Justice

2007

ABSTRACT

ANALYSIS OF AMPHETAMINE AND METHAMPHETAMINE BY SURFACE-
ENHANCED INFRARED SPECTROSCOPY

By

SRIVIDHYA KIDAMBI

In this thesis, an application of drug analysis using surface-enhanced infrared
spectroscopy (SEIRS) is presented. SEIRS can be considered as a more efficient form of
IR analysis, because of the signal enhancement effect observed. Amphetamine and
methamphetamine are used in this thesis as model systems to investigate and demonstrate
the surface enhanced infrared spectroscopic effect.

The presence of polyelectrolyte/nanoparticle films on the surface provided surface
enhancements as high as 33-fold in the absorbance of infrared peaks. It was verified that
the polymer does not contribute much to the surface infrared enhancement. The surface
modification with Ag nanoparticles resulted in more definitive peaks in the spectra, when
compared to Au nanoparticles. There was also an increase in the surface infrared
enhancement with the increase in the number of bilayers; however, there was no further
increase after 3 bilayers. This may have been caused by decreased surface area due to the
aggregation of the nanoparticle systems. This project is the first time where
nanoparticles-embedded polymer coatings are used as SEIRS-active substrate for drug
analysis. However, there is a lot of research to be done in future in order to employ this
technique in the actual crime cases as it currently shows only a preliminary study of the

potential of this technique in trace drug analysis.

TO MY FAMILY

iii

ACKNOWLEDGMENTS

I would like to express my deepest appreciation to my advisor Dr. Ruth Waddell,
for her guidance and invaluable assistance, without which this thesis would not have been
possible. I appreciate the freedom and encouragement Ruth gave me and her tolerance of
my independent streak in pursuing the thesis both during times when the research was in
its difficult stages and when it was sailing smoothly.

I would also like to thank the members of my M.S. committee, Dr. Merlin
Bruening and Dr. Vince Hoffman. I would have to thank Merlin in particular for his
insightful comments about my research and also critical reading of my thesis.

I am also thankful to my group members in the Waddell (forensic chemistry
program) and Bruening groups whom I learned a lot from. From the Waddell group:
Christin, Melissa, Heather, Elizabeth, Luther, Lisa, Lucas, Sarah, and Aggie; From the
Bruening group: Jamie, Fei, Maneesha, Lu, Lei, David, Parul, and Somnath; for making
my M.S. a pleasant experience. In particular, I thank Jamie for all the inspiring and fun
talks about the forensic program, and Fei for helping me with my TEM analysis during
her microscope time.

Thanks to Melissa Christle for her administrative support and answering all my
questions immediately.

It would be an understatement to say that my stay in MSU has been memorable
experience because of my friends. I would particularly like to thank Priya and Thara, for
the wonderful discussions we had about pretty much everything under the sun, and many

other things small and large, that contributed to my M.S., often in unpredictable ways.

iv

And to my family I owe the maximum gratitude, for years of their constant
support, patience and understanding. They provided me with the drive to finish with their
generous encouragement. To all of them, Amma, Appa, Srikanth (Annathey), Manni,
Srivats, Patti, Mamas, Mamis, all the kuttis and many many others I owe my heartfelt

thanks for always being there for me.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................ ix
LIST OF SCHEMES ............................................................................ x
LIST OF FIGURES ............................................................................. xi
LIST OF ACRONYMS ...................................................................... xiv
Chapter 1: Background Information ......................................................... 1
l . 1. Introduction .............................................................................. l
1.2. Amphetamines ........................................................................... 5
1.2.1. Amphetamine (alpha-methyl-phenethylamine) ................................ 6
1.2.1.a. Discovery of Amphetamine .................................................... 6
l.2.l.b. Synthesis of Amphetamine ..................................................... 6
1.2.1.0. Structure and Properties ......................................................... 7
1.2.1.d. Physiological and Psychological Effects ..................................... 7
l.2.l.e. Legislation ........................................................................ 8
l .2.2. Methamphetamine .................................................................. 9
l.2.2.a. Discovery of Methamphetamine ............................................... 9
1.2.2.b. Synthesis of Methamphetamine ................................................ 9
l.2.2.c. Structure and Properties ....................................................... 10
l.2.2.d. Physiological and Psychological Effects .................................... 10
1.2.2.e. Legislation ....................................................................... 11
1.3. Current Techniques for the Analysis of Amphetamine-type Drugs ............. 11
1.4. Surface Enhanced Techniques for Drug Analysis ................................. 12
1.5. Research Objectives .................................................................. 13
l .6. References .............................................................................. 15
Chapter 2: Instrumentation and Theory ............................................ ......22
2.1. Introduction ............................................................................ 22
2.2. Synthesis of Nanoparticles Embedded in Polymer Films for the Development
of SEIRS-Active Substrate ........................................................... 22
2.2.1. Layer-by—layer (LbL) Assembly of Polyelectrolytes ....................... .22
2.2.2. Incorporation of Nanoparticles in PEM Films as Polymer Composite
Materials ........................................................................... 24
2.3. Instrumentation Techniques Employed for Characterization of Nanoparticles
Embedded Polymer Films ............................................................ 27

vi

2.3.1. Transmission Electron Microscopy ............................................. 27

2.3.2. Energy Dispersive X-ray Spectroscopy (EDS) .............................. 30
2.3.3. UV-Visible Spectroscopy ........................................................ 31
2.4. Theory of Infrared Spectroscopy .................................................... 33
2.5. Surface-enhance Infrared Spectroscopy ........................................... 35
2.6. References .............................................................................. 38
Chapter 3: Materials and Methods .......................................................... 44
3. 1 . Introduction ............................................................................ 44
3.2. Materials ................................................................................ 45
3.3. Methods ................................................................................. 45
3.3.1. Preparation of Silver Nanoparticles ............................................ 45
3.3.2. Preparation of Gold Nanoparticles .............................................. 46
3.3.3. Preparation of Au Nanorods ..................................................... 46
3.3.4. Characterization of Nanoparticle-embedded Polymer Films ................ 47

3.3.4.a UV-Visible Spectroscopic Characterization of Layer-by-layer growth.47
3.3.4.b Transmission electron microscopy/ Energy dispersive X-ray

spectroscopy ..................................................................... 48
3.3.5. Film Deposition on Al wafers to S ynthesize SEIRS-active Substrates.48
3.3.6 Introduction of Anal yte ........................................................ 49
3.4. References .............................................................................. 50
Chapter 4: Results and Discussion ...................................................... ....51
4. 1 . Introduction ............................................................................ 5 1
4.2. Film Characterization ................................................................. 51
4.2.1. UV-Visible Spectroscopy ...................................................... 51
4.2.2. TEM/EDS ........................................................................ 54
4.2.3. TEM Analysis of Au Nanorods (AuNR) .................................... 57
4.3. SEIRS — Aminothiophenol as a Model System ................................... 58
4.3.1. Subtraction SEIR spectra of ATP ............................................. 58
4.3.2. SEIRS Analysis of ATP ....................................................... 59
4.4. Effect of Nanoparticle Shape on Infrared Enhancement .......................... 61
4.5. SEIRS of Amphetamine sulfate and Methamphetamine hydrochloride ....... 63
4.5.1. Subtraction SEIR Spectra of Amphetamine sulfate and
Methamphetamine hydrochloride Samples .................................. 64
4.5.2. Effect of Number of Bilayers on SEIRS Enhancement with Gold and
Silver Nanoparticles as Active Species ...................................... 67
4.5.2.a. Gold Nanoparticles ......................................................... 67
4.5.2.b. Silver Nanoparticles ....................................................... 69
4.6. Effect of Polymer Films on Surface Infrared Enhancement ..................... 72
4.7. Effect of Concentration of Analyte Solution on Surface Infrared
Enhancement ........................................................................... 74
4.8. Discussion of Mechanism for Surface Enhancement ............................. 75
4.9 Conclusions ............................................................................ 76
4. 10. References .............................................................................. 77

vii

Chapter 5: Conclusions and Future Work ................................................. 81

5.1. SEIRS Analysis of amphetamine sulfate and methamphetamine
hydrochloride .......................................................................... 81

5.2. Future Prospects ....................................................................... 81

5.3. Summary Statement .................................................................. 83

viii

LIST OF TABLES

Table 1.1 SWGDRUG classification for various analytical techniques .............. 3

ix

LIST OF SCHEMES

Scheme 1.1 Preparation of amphetamine by Leuckart reaction ............................ 7

Scheme 1.2 Preparation of methamphetamine from pseudoephedrine .................... 9

Figure 1.1.

Figure 1.2

Figure 2.1

Figure 2.2
Figure 2.3
Figure 2.4
Figure 2.5

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

LIST OF FIGURES

Number of dismantled amphetamine laboratories, reported to UNODC,
1985-2004 .......................................................................... 2

Structures of (a) amphetamine and (b) methamphetamine ................... 5

Layer-Layer assembly of polyelectrolytes for the formation of

polyelectrolyte multilayer films ................................................ 23
Block diagram of transmission electron microscopy ........................ 29
Schematic diagram of an EDS detector ....................................... 31
Schematic diagram of UV-Visible Spectrophotometer ..................... 32
Schematic diagram of interferometer in FTIR spectrometer ............... 35

UV-Visible spectra of (PAH/AgNP)n films on quartz with n=1 to n=6.
The increase in absorbance with the number of deposited layers
demonstrates layer-by-layer growth. ......................................... 52

UV-Visible spectra of (PAI-I/AuNP)n films on quartz with n=1 to n=5.
The increase in absorbance with the number of deposited layers
demonstrates layer-by-layer growth. ....................................... 53

TEM image of citrate-stabilized AuNPs deposited on a carbon-coated
copper grid by a ‘drop and dry’ method. ..................................... 55

EDS data of citrate-stabilized AuNPs deposited on a carbon-coated
copper grid by a ‘drop and dry’ method. ..................................... 56

TEM image of citrate-stabilized AgNPs deposited on a carbon-coated
copper grid by a ‘drop and dry’ method. ..................................... 57

TEM images of CTAB-stabilized AuNRs deposited on carbon-coated
copper grid by ‘drop and dry’method ........................................ 58

Reflectance infrared spectra of (a) [PAH/AuNPh on an Al-coated wafer
and (b) the same film after exposure to 50 mM ATP and rinsing. Both
the spectra were acquired using bare Al as a background. Spectrum c is
the difference between spectra b and a [(b)—(a)]. ............................ 59

xi

Figure 4.8

Figure 4.9

Figure 4.10

Figure 4.11

Figure 4.12

Figure 4.13

Figure 4.14

Figure 4.15

Figure 4.16

SEIR spectra of 4-aminothiophenol on different substrates — (a) Au-
coated wafer (Au background), (b) Al-coated wafer (Al background), (0)
subtraction spectrum of ATP on [PAH/AuNPh-coated Al, ((1) subtraction
spectrum of ATP on [PAH/AuNPh-coated Al. Prior to measuring each
spectrum, substrates were immersed in 50 mM ATP in ethanol for 30
min. Spectra c and d were obtained following the procedure mentioned
in Figure 4.7 ...................................................................... 60

Subtraction SEIR spectra of 4-aminothiophenol on Al-coated wafers
modified with polymer films containing gold nanostructures of different
shapes — (a) PAH/[PSS/AuNRb, (b) [PAH/AuNP]3on Al. ................ 63

IR spectra of a (PAH/Agh-coated Al wafer (a) before and (b) after
immersion in a 0.1 M amphetamine solution followed by a brief rinse
with water. Spectrum (0) gives the difference between spectra (a) and
(b). A bare Al surface was used as a background. .......................... 66

IR spectra of a (PAH/Ag)1-coated Al wafer (a) before and (b) after
immersed in 0.1 M methamphetamine solution. Spectrum (c) gives the
difference between spectra (a) and (b). A bare Al surface was used as a
background. ...................................................................... 66

Subtraction SEIR spectra of 0.1 M amphetamine on different coatings on
Al surfaces — (a) bare, (b) (PAH/Au)., (c) (PAH/Au)3, (d) (PAH/Au)5.

The subtraction spectra were obtained following the procedure described
in Figure 4.10. ................................................................... 68

Subtraction SEIR spectra of 0.1 M methamphetamine hydrochloride on
different coatings on A1 surfaces — bare (a), (PAH/Aux (b), (PAH/Au)3
(c). The subtraction spectra were obtained following the procedure
described in Figure 4.10. ....................................................... 68

Subtraction SEIR spectra of 0.1 M amphetamine sulfate on different
coatings on Al surfaces - bare (a), (PAI-I/AgNP), (b), (PAH/AgNP)3 (c),
and (PAH/AgNP)5 (d). The subtraction spectra were obtained following
the procedure described in Figure 4.10. ...................................... 70

Subtraction SEIR spectra of 0.1 M methamphetamine hydrochloride on
different coating on Al wafer — bare (a), (PAH/Ag); (b), (PAH/Ag); (c).
The subtraction spectra were obtained following the procedure similar to
Figure 4.11. ...................................................................... 71

Subtraction SEIR spectra of 0.1 M amphetamine sulfate on different

coating on Al wafer - bare, (b) (PAH/PSS)1, (c) (PAH/Au)r. The
subtraction spectra were obtained following the procedure similar to

xii

Figure 4.17

Figure 4.18

Figure 4.10. ...................................................................... 73

Subtraction SEIR spectra of 0.1 M methamphetamine hydrochloride on

different coating on Al wafer - (a) bare, (b) (PAH/PSS)1, (c) (PAH/Au)..
The subtraction spectra were obtained following the procedure similar to
Figure 4.11. ...................................................................... 73

(a) IR spectrum of A1 wafer immersed in 0.1 M of amphetamine solution
in water. Subtraction SEIR spectra of (PAH/Ag)1-coated A1 wafer
immersed in amphetamine solutions in water of varying concentrations :
(b) 10 mM (c) 0.1 M and (d) 0.5 M. The subtraction spectra were
obtained following the procedure similar to Figure 4.10. ................. 75

xiii

LIST OF ACRONYMS

SWGDRUG: Scientific Working Group for the Analysis of Seized Drugs
UNODC: United Nations Office on Drugs and Crime
SEIRS: Surface-Enhanced Infrared Spectroscopy
SERS: Surface-Enhanced Raman Spectroscopy
CNS: Central Nervous System

GC: Gas Chromatography

MS: Mass Spectrometry

LbL: Layer-by-Layer

PAH: Poly(allylaminehydrochloride)

PSS: Poly(styrenesulfonate)

PEM: Polyelectrolyte Multilayers

TEM: Transmission Electron Microscopy

EDS: Energy dispersive X-ray Spectroscopy
UV-Vis: UV-Visible

FT IR: Fourier Transform Infrared Spectroscopy

NP: Nanoparticle

NR: Nanorod

ATP: Aminothiophenol

CTAB: Cetyltrimethylammonium bromide

xiv

Chapter 1. Background Information

1.1. Introduction

The extent of trafficking and the use of illicit drugs have been continuously
increasing throughout the world and may be expected to grow further in the future.1 For
instance, Figure 1.1 shows that the yearly number of amphetamine laboratories that were
dismantled from 1985 to 2004 has been rapidly increasing since 1996, as reported to the
United Nations Office on Drugs and Crime (UNODC). Furthermore, the situation could
be worsened by the development of new varieties of drugs that may be introduced to
exploit new markets. It is believed that the present ‘designer drugs’ (also referred to as
’club drugs') are related to techno-music events that are popular with today’s generation.
Therefore, there is a need for forensic scientists to be able to analyze these psychoactive
drugs, not only in bulk, but even at the trace levels present on drug paraphernalia that
may also be submitted to the forensic lab for analysis. Hence, it is crucial to develop

methods for rapid and sensitive detection of drugs.

600

 

 

 

 

 

n u mber of laboratories

 

mkDN O‘Ov—Nm LnkDNmOlOV—Nl‘lv
mmmmmmmmmmmmmmmooooo
wv—w—PF—F—wF—u—wu—NNNNN

I Comblned amphetamlne and other ATS
lAmphetamlne

Figure 1.1. Number of dismantled amphetamine laboratories, reported to UNODC,
1985-2004'. “Images in this thesis are presented in color”

The Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) is
an international working group, formed in 1997, that is dedicated to developing and
implementing minimum standards for the identification of drug exhibits in forensic
science laboratories.2 The primary objectives for the group are to promote professional
development in forensic drug analysis; provide guidelines for drug examinations and
reporting; specify requirements for analysts’ knowledge, skills, and abilities; establish
quality assurance guidelines; and to promote and gain international acceptance of
SWGDRUG standards. SWGDRUG requires the use of different uncorrelated techniques

to ensure the validity of an analysis, although it is recognized that the validated analytical

scheme and the competence of the analyst are crucial for the correct identification of a
drug.

Techniques for the analysis of drug samples were classified into three categories
by SWGDRUG based on the discriminating power of the technique. Table 1 provides

examples of these techniques listed in order of decreasing discriminating power from A

to C.

Table 1.1. SWGDRUG classification for various analytical techniques

 

 

 

 

Category A Category B ' Category C
(most discriminating) (least discriminating)
Infrared Spectroscopy Capillary Electrophoresis Color Tests
Mass Spectrometry Gas Chromatography Fluorescence Spectroscopy
Nuclear Magnetic Ion Mobility Spectrometry Immunoassay

Resonance Spectroscopy

 

Raman Spectroscopy Liquid Chromatography Melting point

 

Microcrystalline Tests Ultraviolet Spectroscopy

 

Thin Layer Chromatography

 

 

Cannabis only:
Macroscopic Examination

Microscopic Examination

 

 

 

 

Infrared (IR) spectroscopy, which is classified in category A, is a very useful tool

for the definitive identification of samples. The technique has been widely used in

forensic science for various applications such as analysis of paint samples”, drugs”,

"“2 and synthetic fibers.'0

explosives,

IR analysis has the ability to discriminate different drugs based on their specific
structures. The infrared portion of the electromagnetic spectrum is divided into three
regions; the near-, mid- and far- infrared, based on wavelengths and relative position with
respect to the visible light. The far-infrared (approx. 400-10 cm'l), lying adjacent to the
microwave region, has low energy and may be used for rotational spectroscopy. The
mid-infrared (approx. 4000-400 cm") may be used to study fundamental vibrations and
associated rotational-vibrational structure, whilst the higher energy near-IR (14000-4000
cm") can excite the vibrations of the bonds in a molecule. Mid-infrared spectra are
typically most useful in forensic analyses, with the 4000-1400 cm'1 region giving
functional group or structural information. The 1400-400 cm'l region is often referred to
as the molecular “fingerprint” region, and no two compounds have exactly the same
spectra. Hence, the fingerprint region of the spectrum can be used for definitive
identification of samples.

In this thesis, an application of drug analysis using surface-enhanced infrared
spectroscopy (SEIRS) is presented. SEIRS is based on the technique of infrared
spectroscopy; but in this case metal nanoparticles are incorporated on the surface of the
substrate in order to enhance the detection of compounds. SEIRS can be considered as a
more efficient form of IR analysis, because of the signal enhancement effect observed.”
'5 SEIRS has not yet been employed for illicit drug analysis, but has the potential to

identify drug samples at trace levels due to the signal enhancement afforded.

Amphetamine and methamphetamine were used in this thesis as model systems to

investigate and demonstrate the surface enhanced infrared spectroscopic effect.

1.2. Amphetamines

The term "amphetamines" refers to synthetic drugs that have the same basic
phenethylamine structure. "Amphetamines" broadly represent the class of drugs that
includes amphetamine, methamphetamine, and the designer drugs 3,4-
methylenedioxymethamphetamine (MDMA — also referred to as ecstasy) and 3,4-
methylendioxyamphetamine (MDA). These substances are similar in chemistry and also
have generally similar effects on users. Figure 1.2 shows the structures of amphetamine

and methamphetamine.

NH: NH-CHa

(a) (b)

Figure 1.2. Structures of (a) amphetamine and (b) methamphetamine

1.2.1. Amphetamine (alpha-methyl-phenethylamine)
1.2.1.a. Discovery of Amphetamine

Amphetamine was first synthesized in 1887 in Germany.'6 Even though it was
discovered in the 19th century, the drug did not have a significant impact until the early

20th century, when it was explored as a cure or treatment for a variety of illnesses. These

17-19 20-22 23, 24 25. 26

included epilepsy, schizophrenia, alcoholism, migraine, and irradiation
sickness, among many others. Later, amphetamine was found to raise blood pressure,
enlarge nasal and bronchial passages, and stimulate the central nervous system.
Amphetamine was marketed as Benzedrine in an over-the-counter inhaler to treat nasal
congestion for asthmatics, hay fever sufferers, and people with colds.27 In the 19603, all
amphetamines became illegal in the US. under the Controlled Substances Act, unless the
drug was required for medical use. Hence, the illicit use of amphetamine relied on

synthesis in clandestine laboratories.

I .2.1.b. Synthesis of Amphetamine

Over the years the Leuckart reaction has remained the most popular method for
synthesizing illicit amphetamine in clandestine labs in the US.28 Scheme 1.1 shows the
synthesis of amphetamine using the Leuckart route, in which the chemical handling is not

difficult and the necessary materials are easy to purchase.29

180-190°C
| + HCONH2—)

 

HZSOJHCI (dilute) ggf

90-125 °C‘)

Scheme 1.1. Preparation of amphetamine by Leuckart reaction

1.2.1.c. Structure and Properties

Amphetamine has a chiral center, where a carbon atom is bonded to four different
functional groups. Due to this chirality, it can exist in levo-(l) and dextro—(d) forms of
amphetamine, or as a racemic mixture that contains equal amounts of both the optically
isomeric forms. Previously the medical drug came in the racemic salt d, l-amphetamine
sulfate. However, since the d-amphetamine isomer is more active than the l-isomer,

- 3
abusers prefer the pure d-rsomer. 0

1.2. I.d. Physiological and Psychological Eflects

The physiological and psychological effects of amphetamine depend on several
factors such as the amount of drug consumed, the user's previous drug experience, the
route of administration of the drug, the chemical form of the drug, the user's

psychological and emotional stability, and the concurrent use of alcohol or other drugs.3|

33 34

Amphetamine limits appetite,32' increases plasma free fatty acid levels, and has
sympathetic effects on the cardiovascular and respiratory functions”. Chronic use can
result in "behavioral stereotypy", when the user gets caught up in the meaningless
repetition of a single activity such as repeatedly cleaning the same object.36 Other
general effects of amphetamines on the body include increased heart rate and increased
inability to sleep”, dry mouth, headache and dizziness, heat intolerance, and increased

kidney damage.38 Regular amphetamine use may lead to dependency, irritability,

fearfulness, paranoia hallucination, and even psychosis.

1.2.I.e. Legislation

In the United States, amphetamine is classified in Schedule H of the Controlled
Substances Act (CSA), as a Central Nervous System (CNS) stimulant. Schedule 11
substances have an accepted medical use although have a high potential for abuse and
potential for severe psychological and physiological dependence. Internationally,
amphetamine is a Schedule H drug under the Convention on Psychotropic Substances,
which is a United Nations treaty designed to control psychoactive drugs including

amphetamine.

 

1.2.2. Methamphetamine
1.2.2.a Discovery of Methamphetamine

Methamphetamine is a structural analog of amphetamine, the difference being the
presence of a methyl group on the a-carbon. Methamphetamine is more potent and easier
to synthesize than amphetamine, and was discovered from ephedrine in 1893.39 During
World War H, methamphetamine was widely used to increase the endurance and
performance of military personnel.40 In the United States in the 19503, legally
manufactured tablets of methamphetamine (Methedrine) became available, which were
used by students and athletes for non-therapeutic purposes. Methamphetamine is still
legally produced in the US. and used in the treatment of attention-deficit hyperactivity
disorder and narcolepsy.41 However, as with amphetamine, abuse of methamphetamine

relies mainly on clandestine production.

1.2.2.b. Synthesis of Methamphetamine

Illicit methamphetamine is primarily produced in the clandestine labs in the US.
by the reduction of pseudoephedrine with hydriodic acid and red phosphorus (HI/red P),
as shown in Scheme 1242’ 43 Psuedoephedrine can be easily obtained from several over-
the-counter preparations.

0H NH-CHa
NH-CH3
Red PIHI

\ ——>

/

(a) ('3)

Scheme 1.2. Preparation of methamphetamine from pseudoephedrine

Manufacture of methamphetamine from the reductive amination of phenyl-Z-
propanone and methylamine, which is another common method of synthesis, yields (1)-
methamphetamine, whereas the reduction of (—)-ephedrine or (+)-pseudoephedrine yields
(+)-methamphetamine. The (+)-form of methamphetamine is pharmacologically more
active than the (-)-form and hence, is more desirable. The HI/RedP synthesis produces

only (+)-methamphetamine, which may explain the popularity of this synthesis.

1.2.2. c. Structure and Chemical Properties

Methamphetamine has a chiral center like amphetamine, and thus can exist as
either the d- or l-isomer. The legal form of methamphetamine, Desoxyn (Abbott, USA),
contains d-methamphetamine hydrochloride. The l-isomer is less active; and thus a

racemic mixture of d/l-methamphetamine is not used medically.

1.2.2.a. Physical and Psychological Eflects

Methamphetamine causes euphoria and excitement; it is thus prone to abuse and
addiction. Methamphetamine rapidly enters the brain and triggers a dramatic release of
dopamine, norepinephrine and, to a lesser extent, serotonin. The desired acute effects
include well-being, increased alertness, increased activity, excitement, and decreased
appetite"0 Acute physical effects include elevation of blood pressure, pulse, respiration,
and body temperature. There are several physiological problems associated with
excessive dosages that include cerebral hemorrhage, stroke,"4 seizure,“ hyperthermia,45
arrhythmias, coma,46 and death."6 Psychological impairments associated with stimulant

use include insomnia, psychosis, paranoia, suicidal tendencies, and cognitive impairment.

10

1.2.2.e. Legislation

In the United States, methamphetamine is classified in Schedule II of the CSA as
a CNS stimulant, similar to amphetamine. Illicit methamphetamine has become a major
focus of the 'war on drugs' in the United States in recent years, due to the increase in the

clandestine synthesis and use of this drug"7

1.3. Current Techniques for the Analysis of Amphetamine-type Drugs
The goal of a forensic scientist is to identify the drug sample both qualitatively
(whether or not a controlled substance is present) and quantitatively (how much of the
controlled substance is present). In the case of amphetamines, qualitative analysis is
typically sufficient to determine the controlled substance present in the submitted exhibit.
The Marquis test is a useful presumptive test for amphetamines and other drugs,

48' 4° The presence of amphetamine and methamphetamine is

relying on a color reaction.
indicated by a yellow-orange coloration. Thin layer chromatography (TLC) can not only
determine the presence of the amphetamine drug class, but also the member of the class

5 l Confirmation of

(eg. amphetamine, methamphetamine, MDMA etc.) present.”
amphetamines is typically achieved using gas chromatography-mass spectrometry (GC-

MS). With suitable dcrivatization.53‘ 53

11

 

1.4. Surface Enhanced Techniques for Drug Analysis

In this thesis, the potential of SEIRS for amphetamine and methamphetamine
detection was investigated. Surface enhanced techniques such as SEIRS and surface
enhanced Raman spectroscopy (SERS) have been employed for analysis recently because
of their signal amplification. These techniques have been developed on various types of
surfaces which require a complicated production procedure, e.g., silver-island films,“
hydrosols}4 silver-films-over—nanospheres (AgFONs),55 and colloids.56 Of all these
methods that are developed, only some are suitable for a reliable, quick and, with respect
to the cost of preparation, reasonable method of analysis.

There has been some research undertaken to identify illicit drug samples by
means of SERS on a roughened silver surface.S7 More advanced techniques involve the
use of a fiber-optic probe for detection.58 Furthermore, immunoassays were also utilized
for the identification of drugs with enzymatic detection using colloids as the substrates9
Recently, a microtest plate in combination with silver or gold hydrosols was introduced
by Bell and Spence for amphetamine analysis."0 There has been a lot of research
involving trace level identification of drugs using SERS, where comparatively low
concentrations in the range of 10" to 10'5 M concentrations can be detected.”‘ 58’ 6" (’2
This can be achieved by analyzing characteristic vibrational bands of the analytes, thus
overcoming the major challenge of conventional Raman spectroscopy of low sensitivity.

SEIRS is a vibrational spectroscopy technique that can identify analyte substances
uniquely based on their vibrational bands and thus it is most appropriate when unique

identification of the analytes is warranted. For SEIRS, the incorporation of metal

nanoparticles onto the substrate serves to enhance the signal in a manner similar to that

12

 

described for SERS. Hence, this research combines the advantages of IR (structural
information and definitive sample identification) with the advantages of surface enhanced
spectroscopic techniques (enhanced signals).

There are several advantages of using SEIRS as an analytical tool for detecting
trace levels of illicit substances, compared to the IR techniques currently used. Firstly,
because of the enhancement attained from the presence of nanoparticles, high sensitivity
can be achieved. Secondly, IR produces unique data for a particular compound, and it is
impossible to have two compounds with exactly the same IR spectra. As previously
mentioned, there have been several designer drugs that have been synthesized to create
new market potentials. In this case, it would not be possible to have standards to
compare available for the analysis, which is a requirement for not only the color tests, but
also for comparison analysis using established techniques such as gas chromatography.63
In the case of IR analysis, it is possible to identify the functional groups, and possibly
identify the drug class of the designer samples. Thus, SEIRS is an ideal candidate for a
novel analytical tool, which has a potential to cope with the growing illegal drug market

throughout the world.

1.5. Research Objectives

In this work, the amplification of IR signals is demonstrated with surface-
enhanced infrared spectra of amphetamine compounds, and is then compared with the
infrared technique without any surface enhancement. Surface-enhanced substrates were
synthesized by a simple procedure of layer by layer assembly of charged polymers, which

was first developed by Decher in early 19903.64' 65 The method was used for depositing

13

stabilized silver and gold nanoparticles, which act as enhancers, on a substrate that was
subsequently employed for SEIRS. The SEIRS analysis of amphetamine and
methamphetamine is presented in this thesis for the first time. With further development,

SEIRS may provide a novel and sensitive method for trace drug analyses in crime labs.

14

1.6. References

1. UN World Drug Report.
http://www.(mot/c.org/pdf/ll’DR 2006/mlr2006 volunrcIJnM (last accessed 05/01/07)

2006.

2. Bono, J. P., Report of the scientific working group for the analysis of seized drugs
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3. Bell, S. E. J.; Fido, L. A.; Speers, S. J.; Armstrong, W. J.; Spratt, S., Forensic
analysis of architectural finishes using Fourier transform infrared and Raman
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4. Flynn, K.; O'Leary, R.; Lennard, C.; Roux, C.; Reedy, B. J ., Forensic applications
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5. Zieba-Palus, J .; Borusiewicz, R., Examination of multilayer paint coats by the use
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6. Lundquist, P., Analysis of g-hydroxybutyrate (GHB) and g-butyrolactone (GBL)
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Zagadnien Nauk Sadowych 2001, 47, 345-357.

7. Olsen, B. A.; Kiehl, D. E., Authentication and fingerprinting of suspect
counterfeit drugs. American Pharmaceutical Review 2006, 9, (1), 115-118.

8. Praisler, M.; Dirinck, 1.; Van Bocxlaer, J. F.; De Leenheer, A. P.; Massart, D. L.,
Computer-aided screening for hallucinogenic and stimulant amphetamines with gas
chromatography-Fourier transform infrared spectroscopy (GC-FTIR). Journal of
Analytical Toxicology 2001, 25, (1), 45-56.

9. Roux, C.; Bull, 8.; Goulding, J .; Lennard, C., Tracing the source of illicit drugs
through plastic packaging-a database. Journal of Forensic Sciences 2000, 45, (1), 99-114.

10. Bartick, E. G.; Tungol, M. W.; Reffner, J. A., A new approach to forensic analysis
with infrared microscopy: internal reflection spectroscopy. Analytica Chimica Acta 1994,
288, (1-2), 35-42.

15

 

11. Primera-Pedrozo, O. M.; Soto-Feliciano, Y.; Pacheco-Londono, L.; De La Torre-
Quintana, L. F.; Hemandez-Rivera, S. P., Detection of explosive mixtures on surfaces
using grazing angle probe - FTIR: model for classification. Proceedings of SPIE-The
International Society for Optical Engineering 2006, 6201, (Sensors, and Command,
Control, Communications, and Intelligence (C31) Technologies for Homeland Security
and Homeland Defense V), 62012A/1-62012A/8.

12. Zitrin, 8., Analysis of explosives by infrared spectrometry and mass spectrometry.
Forensic Investigation of Explosions 1998, 267-314.

13. Huo, S.-J.; Li, Q.-X.; Yan, Y.-G.; Chen, Y.; Cai, W.-B.; Xu, Q.-J.; Osawa, M.,
Tunable surface-enhanced infrared absorption on au nanofilms on si fabricated by self-
assembly and growth of colloidal particles. The journal of physical chemistry. B,
Condensed matter, materials, surfaces, interfaces & biophysical 2005, 109, (33), 15985-
91.

14. Leyton, P.; Domingo, C.; Sanchez-Cortes, S.; Campos-Vallette, M.; Diaz, G. F.;

Garcia-Ramos, J. V., Reflection-absorption IR and surface-enhanced IR spectrOSCOpy of
tetracarboethoxy t-butyl-calix[4]arene, as a host molecule with potential applications in

sensor devices. Vibrational Spectroscopy 2007, 43, (2), 358-365.

15. Sanchez-Cortes, S.; J ancura, D.; Miskovsky, P.; Bertoluzza, A., Near infrared
surface-enhanced raman spectroscopic study of antiretroviral y drugs hypericin and
emodin in aqueous silver colloids. Spectrochimica Acta, Part A: Molecular and
Biomolecular Spectroscopy 1997, 53A, (5), 769-779.

16. Brent Q. Hafen; Soulier, D., Amphetamines: Facts, Figures, and Information.
Hazelden PES 1990.

17. Borowski, T. B.; Kirkby, R. D.; Kokkinidis, L., Amphetamine and antidepressant
drug effects on GABA- and NMDA-related seizures. Brain Research Bulletin 1993, 30,
(5-6), 607-10.

18. Culic, M.; Saponjic, J .; Jankovic, B.; Rakic, L., Amphetamine and haloperidol
modulatory effects on Purkinje cell activity and on EEG power spectra in the acute rat
model of epilepsy. Neuroscience Letters 1994, 182, (2), 259-62.

19. Storm Van Leeuwen, W.; Elink Sterk, C.; Zierfuss, E., Therapeutic effect of
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16

I’m—Fl

20. Casey, J. F.; Hollister, L. E.; Klett, C. J .; Lasky, J. J .; Caffey, E. M., Jr.,
Combined drug therapy of chronic schizophrenics. Controlled evaluation of placebo,
dextro-amphetamine, imipiramine, isocarboxazid and trifluoperazine added to
maintenance doses of chlorpromazine. The American journal of psychiatry 1961, 117,
997-1003.

21. Huston, P. E.; Senf, R., Psychopathology of schizophrenia and depression. I.
Effect of amytal and amphetamine sulfate on level and maintenance of attention. The
American journal of psychiatry 1952, 109, (2), 131-8.

22. van der Elst Martino, C. J .; Wunderink Yvette, S.; Ellenbroek Bart, A.; Cools
Alexander, R., Differences in the cellular mechanism underlying the effects of
amphetamine on prepulse inhibition in apomorphine-susceptible and apomorphine-
unsusceptible rats. Psychopharrnacology 2007, 190, (1), 93-102.

23. Bennett, A. E., Compulsive, Addictive Personality Problems. Medical times 1964,
92, 433-42.

24. Little, W. G., Alcoholism. II. Etiology and therapy. The Journal of osteopathy
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25. Gupta Vinod, K., Amphetamine, migraine, and brain noradrenergic activation:
contradictions in headache research. Headache 2006, 46, (1), 180-1; author reply 181-2.

26. Radke, R. A., Treatment of migraine. Military medicine 1956, 118, (3), 205-8.

27. Bradley, C., Benzedrine and dexedrine in the treatment of children's behavior
disorders. Pediatrics 1950, 5, (1), 24-37.

28. Frank, R. S., The clandestine drug laboratory situation in the United States.
Journal of forensic sciences 1983, 28, (1), 18-31.

29. Palhol, F.; Boyer, S.; Naulet, N.; Chabrillat, M., Impurity profiling of seized
MDMA tablets by capillary gas chromatography. Analytical and Bioanalytical Chemistry
2002, 374, (2), 274-281.

30. Rebec, G. V.; Groves, P. M., Differential effects of the optical isomers of
amphetamine on neuronal activity in the reticular formation and caudate nucleus of the
rat. Brain research 1975, 83, (2), 301-18.

17

 

31. Hall, W.; Hando, J .; Darke, 8.; Ross, J ., Psychological morbidity and route of
administration among amphetamine users in Sydney, Australia. Addiction 1996, 91, (1),
81-7.

32. Hsieh, Y.-S.; Yang, S.-F.; Kuo, D.-Y., Amphetamine, an appetite suppressant,
decreases neuropeptide Y immunoreactivity in rat hypothalamic paraventriculum.
Regulatory Peptides 2005, 127, (1-3), 169-176.

33. Hollister, L. E., Hunger and appetite after single doses of marihuana, alcohol, and
dextroamphetamine. Clinical Pharmacology & Therapeutics 1971, 12, (1), 44-9.

34. Hajos, G. T.; Garattini, 8., Effect of (+)- and (-)-amphetamine on lipid
metabolism. Journal of Pharmacy and Pharmacology 1973, 25, (5), 418-19.

35. Phillis, B. D.; Ong, J .; White, J. M.; Bonnielle, C., Modification of d-
amphetamine-induced responses by baclofen in rats. Psychopharmacology 2001, 153,
(3), 277-284.

36. Chandra, G.; Gangopadhyay, P. K.; Kumar, K. S. S.; Mohanakumar, K. P., Acute
intranigral homocysteine administration produces stereotypic behavioral changes and
striatal dopamine depletion in Sprague-Dawley rats. Brain Research 2006, 1075, (l), 81-
92.

37. Lucas, E. A.; Rogers, J .; Sterman, M. B., Effect of amphetamine and
pentobarbital on sleep-wake patterns of cats with basal forebrain lesions.
Psychopharmacology 1980, 68, (2), 179-84.

38. Dunnick, J. K.; Elwell, M. R., Toxicity studies of amphetamine sulfate, ampicillin
trihydrate, codeine, 8-methoxypsoralen, a-methyldopa, penicillin VK and propantheline
bromide in rats and mice. Toxicology 1989, 56, (2), 123-36.

39. Nordeste, B., The potential expansion of methamphetamine production and
distribution in Canada. http://ii'ww.car/emu.ca/c'ifp/docs/nordcstcmcthrcportpdf, 2004.

(last accessed 05/02/07)

40. Freese Thomas, E.; Miotto, K.; Reback Cathy, J ., The effects and consequences of
selected club drugs. Journal of substance abuse treatment 2002, 23, (2), 151-6.

18

 

 

41. Fry, J. M., Treatment modalities for narcolepsy. Neurology 1998, 50, (2, Suppl.
1), $43-$48.

42. Skinner, H. F., Methamphetamine synthesis via hydriodic acid/red phosphorus
reduction of ephedrine. Forensic Science International 1990, 48, (2), 123-34.

43. Cantrell, T. S.; John, B.; Johnson, L.; Allen, A. C., A study of impurities found in
methamphetamine synthesized from ephedrine. Forensic Science International 1988, 39,
(1), 39-53.

44. Yen, D. J.; Wang, S. J.; Ju, T. H.; Chen, C. C.; Liao, K. K.; Fuh, J. L.; Hu, H. H.,
Stroke associated with methamphetamine inhalation. European neurology 1994, 34, (1),
16-22.

45. Kiyatkin, E. A., Drug-induced brain hyperthermia: mechanisms and functional
implications. International Journal of Neuroprotection and Neuroregeneration 2006, 2,
(3), 168-174.

46. Ago, M.; Ago, K.; Hara, K.; Kashimura, S.; Ogata, M., Toxicological and
histopathological analysis of a patient who died nine days after a single intravenous dose
of methamphetamine: A case report. Legal Medicine 2006, 8, (4), 235-239.

47. Werrnuth, L., Methamphetamine use: hazards and social influences. J Drug Educ
FIELD Full Journal Title.°Journal of drug education 2000, 30, (4), 423-33.

48. Agarwal, S. P.; Chandrasekhara, N.; Madu, A. U., A colorimetric method for the
determination of amphetamines. Acta Pharmaceutica Technologica 1981, 27, (3), 181-4.

49. Rendle, D. F., Advances in chemistry applied to forensic science. Chemical
Society Reviews 2005, 34, (12), 1021-1030.

50. Kalasz, H., Spot and mobile-phase front anomalies in planar (thin-layer)
chromatography. Chromatographia 2005, 62, (Suppl.), $57-$62.

51. Decker, W. J .; Thompson, J. D., Rapid detection of amphetamine in urine by
micro thin-layer chromatography and fluorescence. Clinical toxicology 1978, 13, (5),
545-9.

19

52. Yahata, M.; Namera, A.; N ishida, M.; Yashiki, M.; Kuramoto, T.; Kimura, K., In-
matrix deri vatization and automated headspace solid-phase microextraction for GC-MS
determination of amphetamine-related drugs in human hair. Forensic Toxicology 2006,
24, (2), 51-57.

53. Dasgupta, A.; Spies, J ., A rapid novel derivatization of amphetamine and
methamphetamine using 2,2,2-trichloroethyl chloroforrnate for gas chromatography
electron ionization and chemical ionization mass spectrometric analysis. American
Journal of Clinical Pathology 1998, 109, (5), 527-532.

54. Creighton, J. A.; Blatchford, C. G.; Albrecht, M. G., Plasma resonance
enhancement of Raman scattering by pyridine adsorbed on silver or gold sol particles of
size comparable to the excitation wavelength. Journal of the Chemical Society, Faraday
Transactions 2: Molecular and Chemical Physics 1979, 75, (5), 790-8.

55. Van Duyne, R. P.; Hulteen, j. C.; Treichel, D. A., Atomic force microscopy and
surface-enhanced Raman spectroscopy. 1. Silver island films and silver film over polymer
nanosphere surfaces supported on glass. Journal of Chemical Physics 1993, 99, (3),
2101-15.

56. Lee, P. C.; Meisel, D., Adsorption and surface-enhanced Raman of dyes on silver
and gold sols. Journal of Physical Chemistry 1982, 86, (17), 3391-5.

57. Sulk, R. A.; Corcoran, R. C.; Carron, K. T., Surface-enhanced Raman scattering
detection of amphetamine and methamphetamine by modification with 2-
mercaptonicotinic acid. Applied Spectroscopy 1999, 53, (8), 954-959.

58. Carter, J. C.; Brewer, W. E.; Angel, S. M., Raman spectroscopy for the in situ
identification of cocaine and selected adulterants. Applied Spectroscopy 2000, 54, (12),
1876-1881.

59. Don, X.; Takama, T.; Yamaguchi, Y.; Yamamoto, H.; Ozaki, Y., Enzyme
immunoassay utilizing surface-enhanced raman scattering of the enzyme reaction
product. Analytical Chemistry 1997, 69, (8), 1492-1495.

60. Bell, S. E.; Spence, S. J ., Disposable, stable media for reproducible surface-
enhanced Raman spectroscopy. The Analyst 2001, 126, (1), 1-3.

20

61. Faulds, K.; Smith, W. B.; Graham, D.; Lacey, R. J ., Assessment of silver and gold
substrates for the detection of amphetamine sulfate by surface enhanced Raman
scattering (SERS). The Analyst 2002, 127, (2), 282-6.

62. Sagmuller, B.; Schwarze, B.; Brehm, G.; Trachta, G.; Schneider, 8., Identification
of illicit drugs by a combination of liquid chromatography and surface-enhanced Raman
scattering spectroscopy. Journal of Molecular Structure 2003, 661-662, 279-290.

63. Laks, S.; Pelander, A.; Vuori, E.; Ali-Tolppa, E.; Sippola, E.; Ojanperae, 1.,
Analysis of Street Drugs in Seized Material without Primary Reference Standards.
Analytical Chemistry 2004, 76, (24), 7375-7379.

64. Decher, 6., Fuzzy nanoassemblies: toward layered polymeric multicomposites.
Science 1997, 277, (5330), 1232-1237.

65. Lvov, Y.; Decher, G.; Mohwald, H., Assembly, structural characterization, and
thermal behavior of layer-by-layer deposited ultrathin films of poly(vinyl sulfate) and
poly(allylamine). Langmuir 1993, 9, (2), 481-6.

21

 

 

Chapter 2. Instrumentation and Theory

2.1. Introduction

The goal of this project is to develop an analytical tool that exploits recent
advances in nanotechnology for the detection of illicit drugs, specifically amphetamine
and methamphetamine, using SEIRS. This can be achieved by incorporating
nanoparticles on a surface, which in this project was an Al wafer, to make a substrate
SEIRS-active. The technique of layer by layer (LbL) assembly of polyelectrolytes
(charged polymers) is used as the basis for preparing the active nanoparticle-coated
substrates. This chapter discusses the theory and background of the LbL technique, and
also the instrumentation that will be employed for both characterization of modified
substrates and analysis of amphetamine and methamphetamine using these modified

surfaces.

2.2. Synthesis of N anoparticles Embedded in Polymer Films for the

Development of SEIRS-Active Substrates
2.2.1. Layer by layer (LbL) Assembly of Polyelectrolytes

Probably the most prominent area of recent research in the formation of ultrathin
coatings is electrostatic layer by layer assembly, which was first introduced by Decher in
1991." 2 This technique, which is based on the alternating deposition of multiply charged
cationic and anionic species, has experienced an explosion of growth in both applications

3

and theoretical and experimental advances. The success of the method is due to its

ability to form polymer thin films with an almost unlimited range of functional groups.

22

 

This flexibility is achieved through the use of a simple, yet convenient deposition process

that is inexpensive and reproducible (Figure 2.1).

     

Polyanion Polycation
Solution Solution

<2 <:

   

i) Polyanion i) Polycation .
ii) Rinse Q ii) Rinse 1 bllayer

Substrate

   

Figure 2.1. Layer-Layer assembly of polyelectrolytes for the formation of
polyelectrolyte multilayer films

In this method, a charged substrate (e.g. positively charged alumina) is immersed
into a solution of oppositely charged polyelectrolyte (e.g. a negatively charged polyanion
such as poly(styrene sulfonate), poly(acrylic acid), or hyaluronic acid). A layer of
polyion is formed on the surface due to electrostatic interactions between the substrate
and the polyelectrolyte. This process is driven by the increase in entropy due to the
displacement of counter—ions from the surface as well as the deposited polyelectrolyte

chain. This step is followed by rinsing with water, to remove excess unadsorbed

23

polyelectrolyte solution, and subsequent immersion of the substrate into an oppositely
charged polyelectrolyte (e.g. positively charged polycations such as poly(ethyleneimine),
poly(allylaminehydrochloride), or chitosan), to form another adsorbed layer. This
sequence of steps results in one bilayer of polyelectrolyte film and can be repeated as
many times as necessary, depending on the application, to form polyelectrolyte
multilayer films (PEMs). The thickness of such films is dependent on the polymer used
and can be tuned by controlling the pH and ionic strength of the deposition solutions and
the number of bilayers deposited.

The LbL technique, which was initially developed with polyelectrolyte molecules,
has now branched out to include incorporation of literally any charged species onto a
substrate including electro-active polymers”, quantum dotss, DNA", inorganic sheets",
and a wide variety of proteins,7 and other biosystemss. One of the most advantageous
and interesting aspects of the LbL-technique is that active species can be introduced into
the polyelectrolyte multilayer films without significantly altering their electrical,
chemical, or biological properties. This fact has led to an expansion in the applications of
the LbL technique to areas ranging from sensing9 and electronics to tissue engineering'0

and biotechnology”.

2.2.2. Incorporation of Nanoparticles in PEM Films as Polymer Composite Materials
Nanotechnology is expected to be the basis of many of the main scientific and

industrial innovations of the 21‘“ century because of it’s application in research areas

from electronics to biotechnology.” There is tremendous growth in the research and

development in this field throughout the world. A major outcome of this global interest

24

 

is the development of new materials at the nanometer scale, including nanoparticles.
Metal nanoparticles are an interesting class of materials because they often exhibit
properties different from those of the corresponding bulk metals?“3 For example, bulk
Au is not catalytically active, but recent studies have shown that Au nanoparticles can
serve as catalysts for oxidation and hydrogenation reactions.“ 45 Additionally,

42, 44, 45

nanoparticle properties can be tuned by varying their sizes and environments.'3

Because of these unique characteristics, metal nanoparticles are being intensively studied

42. 47 5

for applications in catalysis, optoelectronics”, preservatives’ , cosmetics” and

sensing”. In forensic science, nanoparticles have found applications in the analyses of
drugs,'8 explosives,’9 and DNA,20 as well as in imaging fingerprints?" 22

Recent research using nanoparticles was employed in the detection of cocaine in
serum where lateral flow devices, similar to pregnancy tests, were used as a platform to
separate aptamer-assembled nanoparticle (NP) aggregates. The difference in color
between isolated and aggregated nanoparticles was used as the colorimetric indicator for
the presence of cocaine, while the use of DNA aptamers yielded a highly specific
device.23 The DNA aptamers were bound to gold nanoparticles and, in the absence of
cocaine, bonding between complementary strands of DNA resulted in the formation of
aggregates that have a distinct blue-purple color. In the presence of cocaine, which was
the specific analyte for the chosen aptamer, DNA-cocaine interactions resulted in
dissociation of the nanoparticles. Individual Au nanoparticles are red in color and hence,

the observation of a color change from purple to red was indicative of the presence of

cocaine.

25

 

Based on the intensity of the red bands, the concentration of cocaine in buffer was
determined. The detection limit for this device was ~10 uM. When similar experiments
were conducted using cocaine samples in blood serum, the presence of cocaine was
observed at levels as low as 0.2 mM. The decrease in sensitivity in blood was attributed
to the differences in the untreated blood serum and buffer conditions, and degradation of
cocaine in serum. Luong et al. also recently used metal nanoparticles (Pt, Au, or Cu)
together with multi-walled and single-walled carbon nanotubes solubilized in nafion to
form nanocomposites for electrochemical detection of trinitrotoluene (TNT) and several
other nitroaromatics.19

One challenge in working with metal nanoparticles is that these materials may
aggregate, reducing surface area and eventually altering the electronic, optical, and
catalytic properties of the particles. To overcome this problem, metallic nanoparticles
have been immobilized on solid supports such as metal oxides,24 or stabilized by
introducing capping ligands that range from small organic molecules to large polymers.25
In this work, the metal nanoparticles were stabilized by depositing them in multilayer
films using the LbL procedure. The resulting FEM/nanoparticle systems are
characterized by numerous analytical techniques to demonstrate polymer layer growth

and nanoparticle introduction.

26

2.3. Instrumentation Techniques Employed for Characterization of

N anoparticles Embedded in Polymer Films
2.3.1. Transmission Electron Microscopy

The most widely used technique for characterizing nanostructures is transmission
electron microscopy (TEM),26 which provides direct visual information on the size,
shape, dispersity, structure, and morphology of nanostructures. TEM is capable of
routine magnifications of 400,000, which can typically provide a resolution of 2 A. The
first practical transmission electron microscope was built by Albert Prebus and James
Hillier at the University of Toronto in 1938 using concepts developed earlier by Max
Knoll and Ernst Ruska.27 The TEM operates on the same basic principles as the light
microscope but uses electrons instead of light to magnify and visualize the image.

Figure 2.2 shows a block diagram of TEM with the major components.26
Electrons are generated by a process known as thermionic discharge in the gun chamber,
where the filament tip is exposed to high voltage supply in the order of 200,000 V to
1,000,000 V. A higher accelerating voltage leads to better resolution. The electron beam
thus formed passes through the condenser-lens system (a combination of two condenser
lenses), which determines the electron illumination on the specimen. Thus, the
condenser-lens system helps in controlling the beam brightness during imaging. Some
electrons are scattered by the specimen, while others are transmitted through a series of
lenses, becoming magnified and eventually being detected using photographic film, a
fluorescent screen, or a CCD camera. The less dense areas of the specimen allow more
electrons to be transmitted, thus appearing bright on the image, while the more dense

areas of the sample appear as dark spots of the image.

27

 

In this work, TEM was used to characterize the nanoparticles embedded in the
polymer films. Particle shape, size, and size dispersity, were determined from the TEM

image of the FEM/nanoparticle system.

28

High-voltage cable

 

 

 

 

Electron Source

 

Gun chamber

 

 

Condenser lens 1

 

 

 

Condenser System

 

 

  
 

4—Condenser lens 2

................. Specimen plane

+— Objective lens

................. First image plane

 

Diffraction lens

 

 

 

Projector System Intermediate lens

 

Projector lens

 

 

 

    

 

[ 1— Screen

 

 

 

Figure 2.2. Block diagram of transmission electron microscopy

29

2.3.2. Energy Dispersive X-ray Spectroscopy (EDS)

EDS is an analytical tool predominantly used for characterizing chemical
composition by analyzing the interactions between electrons and a sample.26 X-rays are
produced in all electron microscopes because of the inelastic scattering during specimen
interactions with the electron beam. When an electron beam interacts with the sample, an
atom within the sample is excited from the ground state to a higher energy level, resulting
in the formation of an electron hole within the atom’s electronic structure. The vacancy
in an inner orbital shell is then filled by an electron from a higher-energy shell, and the
energy difference between the core and outer level is released in the form of an X-ray.
The energy and the wavelength of the X-rays created are characteristic of the elements
present in the sample. The X-rays produced are named based on the name of the shell
(K, L, M, N) in which the vacancy was created, and also on the number of the orbital
shells that the electron jumped to fill the vacancy. Hence, a one-shell transfer is
represented as a, a two-shell transfer as [3, a three-shell transfer as 7, etc.

An EDS detector is attached to a TEM (or any other electron microscope) to
detect the emitted X-rays. The EDS consists of collimator, window, detector crystal, and
field—effect transistor as shown in Figure 2.3. The collimator has a carbon lining that
helps prevent the entrance of stray radiation from the sample onto the crystal. The crystal
(e.g. silicon doped with lithium) is a semiconductor material which converts the X-rays
produced into a brief voltage pulse. The field-effect transistor amplifies the voltage pulse
from the detector crystal. An X-ray spectrum is generated of X-ray intensity versus
energy. The energy of the X-ray is characteristic of elements present in the sample, while

the intensity of the X-ray is related to element concentration. Thus, the detection of X-

30

 

rays allows the determination of the presence, amount, and distribution of the elements in
the sample. The advantages of X-ray analysis are that it does not destroy the sample, and
it does allow spatial analysis. EDS was used in this work to confirm the presence of the

nanoparticles on the substrate.

 

_ _ Stray
quUId X-ray -

nitrogen

l:

  
 

 

 

 

Electron beam

 

Sample

 

 

X-ray from sample

 

 

 

Collimator

Field-effect transistor Crystal

Figure 2.3. Schematic diagram of an EDS detector

2.3.3. U V- Visible Spectroscopy

Ultraviolet-visible spectroscopy (UV-Vis) is based on the absorption of visible
and ultraviolet (UV) radiation by a sample. This technique has been commonly used in

forensic analysis for sample characterization, along with other analytical techniques such

31

as gas chromatography”, liquid chromatography/mass spectrometry,29 Fourier transform

infrared spectroscopy,30 thin-layer chromatography and mass spectrometry“.

Figure 2.4 shows the major components of an UV/Vis spectrophotometer32’ 33

which include a stable light source (UV and visible light), a wavelength selector
(diffraction grating), sample (and/or reference), and a detector (e.g. a photomultiplier

tube) that converts the radiant energy to a detectable electrical signal.

 

 

 

 

  
 

 

Slit 1 UV light
Diffraction grating I source
Visible Mirror 1
$1112 __ SOUI’CC
Filter W S I
am e
Mirror 3 Lens 1
Sam le beam ‘ A 355
w p U 2?? (I)
’ - Detector 1
Half mirror’.
,.’ Mirror 2
.’. Reference Lens 2
I
.’ Reference beam
% ............................ _ ............ (10)
Mirror 4 Detector 2

Figure 2.4. Schematic diagram of UV-Visible Spectrophotometer

When a beam of light passes through the sample (or reference), some of the light

is absorbed by the sample solution leading to a decrease in the intensity of light. The

32

 

extent of the incident light absorbed depends on the nature and concentration of the

solution, which can be determined by Beer’s law as shown in the following equation:

A = -log(Io/l) = ebc
where A is the absorbance, 10 and I correspond to intensity of light passing through the
reference and sample respectively, a is the molar absorptivity (L mol’l cm"), b is the path
length (cm), and c is the concentration of the absorbing species (mol L"). The intensity
of light passing through the sample (I) is measured and compared to the intensity of light
passing through the reference (10). A UV-visible spectrum is essentially a graph of light
absorbance versus wavelength in the range of ultraviolet or visible regions. The
wavelength with maximum absorbance (Amax) is a characteristic of the sample. This
technique has been typically employed in analyzing the layer by layer growth of active
species such as metal ions or nanoparticles since the nanoparticles have specific
wavelengths where maximum absorption can be observed in the spectrum.“ In this

work, layer by layer growth was confirmed using UV/visible spectroscopy.

2.4. Theory of Infrared Spectroscopy

Infrared (IR) spectroscopy is a very useful tool for the definitive identification of
samples. The technique has been widely used in forensic science for various applications
such as characterization of the molecular structure of paint samples35'37, drugs,38'4'
explosives,4244 synthetic fibers”.

The infrared spectrum is formed as a consequence of the absorption of

electromagnetic radiation at frequencies that correlate to the vibration of specific sets of

chemical bonds within a molecule. A molecule absorbs IR of the frequency

33

corresponding to it’s natural vibrational frequency. This allows the characterization and
identification of a molecule from its IR spectrum. The vibrational motion is quantized
i.e. it must follow the rules of quantum mechanics, and the only transitions which are
allowed fit the following formula:
E = (n + 1/2) be

where u is the frequency of the vibration and n is the quantum number (0, 1, 2, 3, . .).
One important selection rule that influences the intensity of infrared absorptions is that a
change in dipole moment should occur for a vibration to absorb infrared energy.

IR spectroscopy has undergone significant developments over the years. With the
technology of Fourier Transform Infrared spectroscopy (FTIR), all wavelengths are
scanned simultaneously, resulting in rapid scan times compared to conventional
dispersive spectrometers. In addition, with integrated computer databases of known IR
spectra, comparison and definitive identification of samples is simpler.

In FTIR, the IR light is guided through an interferometer (Figure 2.5), which
consists of a moving mirror, a fixed mirror, and a beam splitter. The IR radiation from the
source is divided at a beam splitter; half the beam passes to the fixed mirror and half is
reflected to the moving mirror. After reflection, the two beams recombine at the beam
splitter and, for any particular wavelength, undergo constructive or destructive
interference. With a constant mirror velocity, the intensity of the emerging radiation at
any particular wavelength modulates in a regular sinusoidal manner. After passing
through the sample, the emergent beam is focused onto the detector. The resultant

measured signal from the detector is an interferogram, which is measured in the time-

34

domain. A mathematical process known as Fourier transformation, is used to convert the

interferogram into a spectrum in the frequency domain.

_ Fixed mirror

    

Beam splitter

   
 

From IR source

 

:] Moving mirror

<—>

Sample

To detector

Figure 2.5. Schematic diagram of interferometer in FTIR spectrometer

2.5. Surface-Enhanced Infrared Spectroscopy

Since its discovery in 1977, surface enhanced Raman scattering (SERS) has
become a well-developed analytical technique as discussed in Chapter 1. In 1977, Van
Duyne and Jeanmaire46 and, independently, Albrecht and Creighton47 observed enhanced
Raman signals from pyridine on a rough silver electrode due to the enhancement of the
Raman scattering efficiency itself. An early report by Hartstein et al."8 showed that silver
films could also be employed in surface-enhanced infrared spectroscopy (SEIRS) to
enhance the absorption of IR by molecules adsorbed onto an active substrate. However,
most of the research involving SEIRS is quite recent."9 Due to the vast number of IR

spectra that have been collected for gases, liquids, and solids, any improvement in the

35

 

sensitivity offered by SEIRS will have a major impact in the field of vibrational
spectroscopy of molecules on surfaces.50

The intensification of infrared-active vibrational modes in SEIRS occurs when
molecules are in close proximity to nanometer-thick metal. The theory of this effect is
rather complicated and not yet completely understood. It is believed that, according to
the electromagnetic mechanism, the surface plasmons (resulting from the excitation of
conduction electrons) can be excited in metals such as gold or silver by an incident
electric field which leads to an enhancement of the local field at the surface. Since
absorbance is directly related to the square of electric field, this can result in dramatic
enhancements in infrared absorbance.5| In addition to the electromagnetic mechanism,”’
53 several studies have suggested an additional enhancement caused by the chemical
interaction between the adsorbed molecule and the metal island surface.54 Osawa and
IkedaS3 have suggested that both electromagnetic and chemical contributions can cause
the total enhancement effect.55

Although the SEIRS effect was originally explored with the attenuated total
reflection (ATR) sampling technique,56 it was later reported that SEIRS spectra were
successfully observed with both transmission52 and external reflection57 techniques. Due
to its remarkable absorption enhancement and the availability of versatile sampling
methods, SEIRS spectroscopy has become a novel surface-sensitive technique for
detecting a minute quantity of sample with high sensitivity. In particular, the SEIRS
technique has been extensively applied to micro- and trace analyses of molecules and/or

monolayers on a variety of metal surfaces.” 58 Even though, SEIRS has been used for

several applications, it has never been employed in trace drug analysis.

36

 

In this work, the potential high sensitivity offered by SEIRS in the analysis of

amphetamine and methamphetamine will be investigated.

37

 

2.6. References

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9. Zhang, S.; Yang, W.; Niu, Y.; Li, Y.; Zhang, M.; Sun, C., Construction of glucose
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736-741.

38

10. Zhu, H.; Ji, J .; Shen, J ., Biomacromolecules Electrostatic self-assembly on 3-
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11. Caruso, F.; Mohwald, H., Protein multilayer formation on colloids through a
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12. Lee, L. L.; Chan, C. K.; N giam, M.; Ramakrishna, S., Nanotechnology patent
landscape 2006. Nano 2006, 1, (2), 101-113.

13. Li, Y.; El-Sayed, M. A., The effect of stabilizers on the catalytic activity and
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14. Park, J. H.; Lim, Y. T.; Park, 0. 0.; Kim, J. K.; Yu, J.-W.; Kim, Y. C.,
Polymer/gold nanoparticle nanocomposite light-emitting diodes: enhancement of
electroluminescence stability and quantum efficiency of blue-1i ght-emitting polymers.
Chemistry of Materials 2004, 16, (4), 688-692.

15. Lee, J.-E.; Kim, J.-W.; Jun, J.-B.; Ryu, J.-H.; Kang, H.-H.; Oh, S.-G.; Suh, K.-D.,
Polymer/Ag composite microspheres produced by water-in-oil-in-water emulsion
polymerization and their application for a preservative. Colloid and Polymer Science
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16. Katz, L. M., Nanotechnology and applications in cosmetics: general overview.
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17. Ye, J.-S.; Ottova, A.; Tien, H. T.; Sheu, F.-S., Nanostructured platinum-lipid
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18. Loane, C. J ., Device for the detection of explosive residues. in application: AU
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19. Hrapovic, S.; Majid, E.; Liu, Y.; Male, K.; Luong, J. H. T., Metallic nanoparticle-
carbon nanotube composites for electrochemical determination of explosive nitroaromatic
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39

20. Zhao, X.; Tapec-Dytioco, R.; Wang, K.; Tan, W., Collection of trace amounts of
DNA/mRNA molecules using genomagnetic nanocapturers. Analytical Chemistry 2003,
75, (14), 3476-3483.

21. Bouldin, K. K.; Menzel, E. R.; Takatsu, M.; Murdock, R. H., Diimide-enhanced
fingerprint detection with photoluminescent CdS/dendrimer nanocomposites. Journal of
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22. Rowell, F. J .; Theaker, B. J. Nanoparticles as agents for imaging fingerprints.
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23. Liu, J .; Mazumdar, D.; Lu, Y., A simple and sensitive "dipstick" test in serum
based on lateral flow separation of aptamer-linked nanostructures. Angewandte Chemie,
International Edition 2006, 45, (47), 7955-9.

24. Mallick, K.; Scurrell, M. 8., CO oxidation over gold nanoparticles supported on
TiOz and T iOz-ZnO: catalytic activity effects due to surface modification of TiOz with
ZnO. Applied Catalysis, A: General 2003, 253, (2), 527-536.

25. Park, M. J.; Park, J .; Hyeon, T.; Char, K., Effect of interacting nanoparticles on
the ordered morphology of block copolymer/nanoparticle mixtures. Journal of Polymer
Science, Part B: Polymer Physics 2006, 44, (24), 3571-3579.

26. Flegler, S. L.; John W. Heckman, J .; Klomparens., K. L., Scanning and
transmission electron microscopy: an introduction. 1993.

27. Zhang, X.-F.; Zhang, Z.; Editors, Progress in Transmission Electron Microscopy
1. Concepts and Techniques, 2001; 38, p 365 pp.

28. Xu, Y.; Wang, J .; Yao, L., Dating the writing age of black roller and gel inks by
gas chromatography and UV-vis spectrophotometer. Forensic Science Intemational
2006, 162, (1-3), 140-143.

29. Asea, P. E.; MacNeil, J. D.; Boison, J. 0., An analytical method to screen for 6
thyreostatic drug residues in the thyroid gland and muscle tissues of food producing
animals by liquid chromatography with ultraviolet absorption detection and liquid
chromatography/mass spectrometry. Journal of AOAC International 2006, 89, (2), 567-
575.

40

 

30. Aalberg, L.; Andersson, K.; Bertler, C.; Boren, H.; Cole, M. D.; Dahlen, J.;
Finnon, Y.; Huizer, H.; Jalava, K.; Kaa, E.; Lock, E.; Lopes, A.; Poortman-van der Meer,
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31. Kulikowska, J .; Celinski, R.; Soja, A.; Sybirska, H., Identification study of tablets
of unknown composition originating from illicit drug sale. Z Zagadnien Nauk Sadowych
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32. Skoog, D. A., Principles of Instrumental Analysis, 3rd Ed. 1985.

33. Skoog, D. A.; West, D. M.; Holler., F. J .; Crouch., S. R., Analytical Chemistry:
An Introduction. 7th Ed. 2000; p 773 pp.

34. Kidambi, S.; Bruening, M. L., Multilayered polyelectrolyte films containing
palladium nanoparticles: synthesis, characterization, and application in selective
hydrogenation. Chemistry of Materials 2005, 17, (2), 301-307.

35. Bell, S. E. J .; Fido, L. A.; Speers, S. J .; Armstrong, W. J .; Spratt, S., Forensic
analysis of architectural finishes using Fourier transform infrared and Raman
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36. Flynn, K.; O'Leary, R.; Lennard, C.; Roux, C.; Reedy, B. J ., Forensic applications
of infrared chemical imaging: Multi-layered paint chips. Journal of Forensic Sciences
2005, 50, (4), 832-841.

37. Zieba-Palus, J .; Borusiewicz, R., Examination of multilayer paint coats by the use
of infrared, Raman and XRF spectroscopy for forensic purposes. Journal of Molecular
Structure 2006, 792-793, 286-292.

38. Lundquist, P., Analysis of g-hydroxybutyrate (GHB) and g-butyrolactone (GBL)
in liquids performed at national laboratory of forensic science (SKL), Sweden. Z
Zagadnien Nauk Sadowych 2001, 47, 345-357.

39. Olsen, B. A.; Kiehl, D. E., Authentication and fingerprinting of suspect
counterfeit drugs. American Pharmaceutical Review 2006, 9, (1), 115-118.

41

40. Praisler, M.; Dirinck, 1.; Van Bocxlaer, J. F.; De Leenheer, A. P.; Massart, D. L.,
Compater-aided screening for hallucinogenic and stimulant amphetamines with gas
chromatography-Fourier transform infrared spectroscopy (GC-FI‘ IR). Journal of
Analytical Toxicology 2001, 25, (1), 45-56.

41. Roux, C.; Bull, S.; Goulding, J .; Lennard, C., Tracing the source of illicit drugs
through plastic packaging-a database. Journal of Forensic Sciences 2000, 45, (1), 99-114.

42. Beveridge, A. D.; Payton, S. F.; Audette, R. J .; Lambertus, A. J .; Shaddick, R. C.,
Systematic analysis of explosive residues. Journal of Forensic Sciences 1975, 20, (3),
431-54.

43. Primera-Pedrozo, O. M.; Soto-Feliciano, Y.; Pacheco-Londono, L.; De La Torre-
Quintana, L. F .; Hemandez-Rivera, S. P., Detection of explosive mixtures on surfaces
using grazing angle probe - FT IR: model for classification. Proceedings of SPIE-The
International Society for Optical Engineering 2006, 6201, (Sensors, and Command,
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44. Zitrin, 8., Analysis of explosives by infrared spectrometry and mass spectrometry.
Forensic Investigation of Explosions 1998, 267-314.

45. Bartick, E. G.; Tungol, M. W.; Reffner, J. A., A new approach to forensic analysis
with infrared microscopy: internal reflection spectroscopy. Analytica Chimica Acta 1994,
288, (1-2), 35-42.

46. Jeanmaire, D. L.; Van Duyne, R. P., Surface Raman spectroelectrochemistry. Part
I. Heterocyclic, aromatic, and aliphatic amines adsorbed on the anodized silver electrode.
Journal of Electroanalytical Chemistry and Interfacial Electrochemistry 1977, 84, (l), 1-
20.

47. Albrecht, M. G.; Creighton, J. A., Anomalously intense Raman spectra of
pyridine at a silver electrode. Journal of the American Chemical Society 1977, 99, (15),
5215-17.

48. Hartstein, A.; Kirtley, J. R.; Tsang, J. C., Enhancement of the infrared absorption
from molecular monolayers with thin metal overlayers. Physical Review Letters 1980, 45,
(3), 201-4.

42

49. Osawa, M., Dynamic processes in electrochemical reactions studied by surface-
enhanced infrared absorption spectroscopy (SEIRAS). Bulletin of the Chemical Society of
Japan 1997, 70, (12), 2861-2880.

50. Aroca, R.; Price, B., A new surface for surface-enhanced infrared spectroscopy:
tin island films. Journal of Physical Chemistry B 1997, 101, (33), 6537-6540.

51. Osawa, M., Surface-enhanced infrared absorption. Topics in Applied Physics
2001, 81, (Near-Field Optics and Surface Plasmon Polaritons), 163-187.

52. Osawa, M.; Ataka, K.; Yoshii, K.; Nishikawa, Y., Surface-enhanced infrared

spectroscopy: the origin of the absorption enhancement and band selection rule in the
infrared spectra of molecules adsorbed on fine metal particles. Applied Spectroscopy

1993, 47, (9), 1497-502.

53. Osawa, M.; Ikeda, M., Surface-enhanced infrared absorption of p-nitrobenzoic
acid deposited on silver island films: contributions of electromagnetic and chemical
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54. Wadayama, T.; Sakurai, T .; Ichikawa, S.; Suetaka, W., Charge-transfer
enhancement in infrared absorption of thiocyanate ions adsorbed on a gold electrode in
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55. Otto, A., Surface-enhanced Raman scattering of adsorbates. Journal of Raman
Spectroscopy 1991, 22, (12), 743-52.

56. Hatta, A.; Suzuki, N.; Suzuki, Y.; Suetaka, W., Infrared absorption of
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57. Nishikawa, Y.; Fujiwara, K.; Ataka, K.; Osawa, M., Surface-enhanced infrared
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58. Zhang, Z.; Imae, T.; Sato, H.; Watanabe, A.; Ozaki, Y., Surface-enhanced Raman
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metalloporphyrin monolayer film formed on pyridine self-assembled monolayer-
modified gold. Langmuir 2001, 17, (15), 4564-4568.

43

Chapter 3. Materials and Methods

3.1. Introduction

In this project, substrates for SEIRS were prepared by incorporating nanoparticles
on Al substrates using layer by layer assembly. The layer by layer (LbL) growth of a
film comprised of positively charged pol yallylamine hydrochloride (PAH) and negatively
charged citrate-stabilized metal nanocolloids (gold and silver) was performed on an Al
wafer to make the substrate active for SEIRS technique.

Since it is necessary to verify the layer by layer growth of the system, UV-visible
spectroscopic analysis of the multilayer system was performed on a quartz slide. The
properties of the nanocolloids such as size, shape, size dispersity, structure, and
morphology must also be characterized to understand the nanoparticle system. This was
achieved by employing transmission electron microscopy (TEM) coupled with energy
dispersive X-ray spectroscopy (EDS).

Finally, the surface enhancement in infrared spectroscopy was analyzed by
building the nanoparticle-containing multilayers on an Al wafer, and introducing this
active substrate to solutions of amphetamine sulfate and methamphetamine hydrochloride
to study the enhancement in IR absorbances due to adsorption of these molecules onto the
active substrate. The active substrate was compared to Al wafers without any
nanoparticle incorporation to verify that the nanoparticles were responsible for the
enhancement effect. The detailed procedure for film preparation on each of the substrates

is described in the following sections.

3.2. Materials

Poly(allylamine hydrochloride) (PAH) (Mw = 25,000 Da), poly(styrene sulfonate)
(PSS) (sodium salt, Mw = 70,000), sodium citrate dihydrate, sodium borohydride, 4-
aminothiophenol (ATP), cetyltrimethylammonium bromide (CTAB), and gold(III)
chloride trihydrate were purchased from Aldrich (St. Louis, Missouri). Silver nitrate was
obtained from EM Science (Gibbstown, New Jersey). All reagents were used as received
and solutions were prepared with deionized water (Milli-Q, 18.2 MO). The aluminum
wafers were obtained by sputtering 200 nm of Al (by Lance Goddard Associates, Foster
City, CA) on Si wafers (Silicon Quest International, Santa Clara, CA). d-Amphetamine
sulfate (Lot # 64H0032) and d-methamphetamine hydrochloride (Lot # 35H0193) were

obtained from Aldrich (St. Louis, Missouri).

3.3. Methods
3.3.1. Preparation of Silver Nanoparticles

Aqueous suspensions of silver nanoparticles (Ag hydrosols) were prepared using
the procedure described by Lee and Meisel.1 Briefly, 9 mg of silver nitrate was added to
50 mL of ultrapure water in an Erlenmeyer flask and the mixture was heated to 100 °C.
A 1 mL aliquot of 1% by weight aqueous sodium citrate was added drop-wise to the
boiling water with vigorous stirring. A gradual change of the solution color from
transparent to greenish gray was indicative of the progress of the silver particle
formation. The Erlenmeyer flask containing the solution was loosely capped with a

ceramic crucible to avoid significant solvent loss due to evaporation. After 40 minutes of

45

boiling, the hydrosol was left to cool to room temperature, after which it was stored in a

dark bottle at 4°C in a refrigerator.

3.3.2. Preparation of Gold Nanoparticles

Monodisperse gold nanoparticles were prepared according to a modified literature
procedurez‘ 3 All glassware was cleaned thoroughly with aqua regia (3 parts HCl, 1 part
HNO3) and rinsed with deionized water. In an Erlenmeyer flask, 50 mL of aqueous 1
mM HAuCl4-3 H20 was heated to a rolling boil with stirring. 5 mL of 38.8 mM sodium
citrate dihydrate was also heated to a rolling boil and then added rapidly to the gold
solution. After about 20 seconds, the mixture became dark and then burgundy, and was
subsequently heated with stirring for 10 minutes and stirred without heating for an
additional 15 minutes. The aqueous citrate-stabilized Au nanoparticles were stored in a

dark bottle and kept refrigerated at 4°C until use.

3.3.3. Preparation of Au Nanorods

The preparation of CT AB-stabilized gold nanorods was based on the procedure
reported by Murphy’s group." 5 A 20 mL aqueous solution containing 0.25 mM HAuCl4
and 0.25 mM sodium citrate was prepared in a conical flask. This was followed by the
addition of 0.6 mL of ice cold 0.1 M NaBI-L, solution to the conical flask containing the
gold solution with constant stirring. The solution turned burgundy immediately after
adding NaBI-I4, indicating particle formation. This acts as the seed solution for the

formation of gold nanorods.

46

In a clean test tube, 10 mL of growth solution, containing 0.25 mM HAuCl4 and
0.1 M CT AB, was mixed with 0.05 mL of 0.1 M freshly prepared ascorbic acid solution.
Then, 0.025 mL of the seed solution was added resulting in the formation of Au

nanorods.

3.3.4. Characterization of Nanoparticle-embedded Polymer Films

The nanoparticle-containing polymer films were characterized prior to their use
for the analysis of amphetamine and methamphetamine. The LbL growth was first
verified using UV-visible spectroscopy. The composition, size, and shape of the
nanoparticles were also characterized using TEM/EDS as these properties play an

important role in determining the enhancement factor by SEIRS.6

3.3.4.a. U V- Visible Spectroscopic Characterization of Layer by layer growth

UV-visible spectroscopy is an absorption technique where the light beam is
transmitted through the substrate and thus a transparent substrate, quartz, was employed.
To form films on transparent quartz, slides were alternatively immersed into 10 mL of 20
mM PAH (concentration given with respect to polymer repeating unit) and 10 mL of
citrate-stabilized gold colloidal solutions (as prepared in section 3.3.2) for 10 min, with a
1-min water rinse after each deposition. Using a Perkin-Elmer UV/Visible Lambda 40
spectrophotometer, a background spectrum was first taken using an uncoated quartz slide
and subsequent UV-Visible absorption spectra of polyelectrolyte multilayer films on
quartz slides were then obtained. UV-Visible spectra were obtained after deposition of

each bilayer. Since Au nanoparticles have maximum absorbance at 517 nm, layer by

47

layer growth was monitored by the increase in absorbance at 517 nm. For Ag
nanoparticles, layer by layer growth was monitored by the increase in absorbance at 370

nm, corresponding to the wavelength of maximum absorbance for Ag.

3.3.4. b. Transmission electron microscopy/ Energy dispersive X-ray spectroscopy

Since a very thin sample is required for TEM/EDS analysis, an ultra-thin carbon-
coated copper grid was used as the substrate. The grids were cleaned in a UV/ozone
cleaner for 1 min. prior to introducing the nanoparticles.

Samples of the citrate-stabilized Au nanocolloids were prepared by introducing a
drop of solution of Au nanoparticles (as prepared by following the procedure mentioned
in section 3.3.2) onto the carbon-coated copper grid and letting it dry ('drop and dry'
preparation). TEM was performed on a JEOL 100CX microscope using an accelerating
voltage of 100 kV. The digital images were taken with a Mega View III Soft Imaging

System.

3.3.5. Film Deposition on Al wafers to Synthesize SEIRS-active Substrates.

Al-coated Si wafers (24 mm by 11 mm) were UV/ozone-cleaned for 15 min
before film deposition. PAH/Nanoparticle (NP) deposition on Al substrate began with a
10-min immersion of the substrate in an aqueous solution containing 20 mM of PAH.
The Al substrate coated with a PAH layer, was then rinsed with deionized water for 1
min before exposure to Au or Ag NPs for 10 min, and this was followed by another water
rinse for 1 min. This process was repeated until the desired number of bilayers (1, 3 or 5)

was deposited. Reflectance FTIR spectra of the coated Al wafers were collected using a

48

Nicolet Magna 560 spectrophotometer with a grazing angle (80°) accessory. The
following parameters were employed for the FF IR analysis: number of scans of 128,

resolution of 4 cm", and a wavenumber range of 4000-1000 cm".

3.3. 6. Introduction of Analyte

Initially, ATP was used as a model system to verify the surface enhanced effect of
NP-coated substrate. The NP-coated Al wafer was immersed into a solution of 0.05 M
ATP in ethanol for 30 min. The substrate was then rinsed briefly in ethanol for 15 s,
followed by IR analysis using the same parameters as before. The adsorption of the ATP
anal yte on the active substrate was studied by subtracting the spectra of the substrate with
and without the introduction of ATP.

The analysis of ATP was followed by the analyses of amphetamine sulfate and
methamphetamine hydrochloride on the active substrates. An NP-coated substrate was
immersed into a solution of 0.1 M amphetamine or methamphetamine in water for 30
min. The substrate was then rinsed briefly in water for 15 s, followed by IR analysis.
The adsorption of the drug on the active substrate was studied by subtracting the spectra
of the substrate with and without the drug immersion, similar to ATP analysis.

To verify that the NP-system was responsible for the enhancement effect, and not
the polymer film, the analyses were performed using the same procedure as above, except
that NP was replaced by a negatively charged polymer, PSS. The PS8 was deposited
using a 10 min immersion in a 0.02 M solution of PSS, and a subsequent l-min water
rinse. In this case, there was no incorporation of nanoparticles on the surface of the

substrate.

49

3.4. References

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and gold sols. Journal of Physical Chemistry 1982, 86, (17), 3391-5.

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3. Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J ., Preparation and
characterization of Au colloid monolayers. Analytical Chemistry 1995, 67, (4), 735-43.

4. Gole, A.; Murphy, C. J., Seed-mediated synthesis of gold nanorods: role of the
size and nature of the seed. Chemistry of Materials 2004, 16, (19), 3633-3640.

5. Jana, N. R.; Gearheart, L.; Murphy, C. J ., Wet chemical synthesis of high aspect
ratio cylindrical gold nanorods. Journal of Physical Chemistry B 2001, 105, (19), 4065-
4067.

6. Hu, X.; Cheng, W.; Wang, T.; Wang, Y.; Wang, E.; Dong, S., Fabrication,
characterization, and application in SERS of self-assembled polyelectrolyte-gold nanorod
multilayered films. Journal of Physical Chemistry B 2005, 109, (41), 19385-19389.

50

Chapter 4: Results and Discussion

4.1. Introduction

This chapter describes the formation and characterization of polymer/nanoparticle
films and the subsequent investigation of these systems as substrates for SEIRS. As a
prelude to possible forensics applications, Al substrates coated with these
FEM/nanoparticle (NP) films were introduced into solutions containing amphetamine
sulfate or methamphetamine hydrochloride to investigate the SEIRS activity achievable
with these controlled substances. The enhancement effect of both Ag and Au NPs was
investigated. Surface enhancement of infrared signals occurred, but only with relatively

high concentrations of the amphetamines.

4.2. Film Characterization
4.2. I. U V- Visible Spectroscopy

UV/vis spectroscopy of [PAI-I/AgNP]n and [PAH/AuNP]n (n represents number of
bilayers) films on quartz was performed to demonstrate that layer by layer deposition
occurs. As shown in Figure 4.1, the spectrum of a [PAI-I/AgNP], film exhibited a peak
with maximum absorbance (Amax) at 370 nm," 2 which stems from an absorbance of the
silver nanoparticles. The absorbance of this peak increased linearly with the number of
PAI-I/AgNP bilayers because the number of immobilized silver nanoparticles increased
regularly each time a PAH/AgNP bilayer was added. It is also interesting to note that
after the deposition of the first 3 bilayers, a second Am, appeared around 410 nm. This
peak likely results from aggregation of particles, as has been reported by Rast and

Stanishevsky.3 The aggregation of nanoparticles may be mediated by diffusion in the

51

polymeric matrix during the deposition process and the state of aggregation should
depend on the number of nanoparticles per unit area. The aggregation of nanoparticles is
probably not desirable in SEIRS because high local electrical fields around single

particles cause the greatest enhancement.4

0.2

O
'_t
01

Absorbance
P

0.05

 

190 290 390 490 590

Wavelength (nm)

Figure 4.1. UV-Visible spectra of (PAH/AgNP)n films on quartz with n=1 to n=6.
The increase in absorbance with the number of deposited layers demonstrates layer

by layer growth.

The layer by layer growth of PAH/AuNP films on quartz slides was also analyzed
using UV-vis spectroscopy. As seen in Figure 4.2, there was an absorbance maximum at
517 nm for thin films, which is due to the AuNPs, and this absorbance increased linearly
with the number of PAH/AuNP bilayers deposited. After deposition of 2 bilayers, a new
band appeared with a Am“ of 620 nm. This band is attributed to the strong
electromagnetic coupling between neighboring AuNPs, which has been described

previouslyf"9 With more layers and an increasing amount of NP deposition, the coupling

52

between particles becomes more extensive, resulting in an increase in the intensity of the

peak at 620 nm relative to the peak at 517 nm.

 

.0
on

0.2 J

Absorbance

.0
_L

 

 

 

 

0 T l l l I
200 300 400 500 600 700 800

Wavelength (nm)

Figure 4.2. UV-Visible spectra of (PAH/AuNP)n films on quartz with n=1 to n=5.
The increase in absorbance with the number of deposited layers demonstrates layer

by layer growth.

In summary, UV/Vis spectra confirmed the layer by layer growth of both
[PAH/AgNP]n and [PAH/AuNP]n films. However, as more layers were deposited there
was more interaction between particles, resulting in a red shift in nanoparticle

absorbance, indicating the aggregation of nanoparticles with increase in the number of

bilayers.

53

4.2.2. T EM/EDS

To investigate the morphological and chemical properties of nanoparticles, both
TEM and EDS were performed on AuNPs and AgNPs. Since TEM requires an
extremely thin transparent sample, carbon-coated copper grids were used as the substrate
for analyzing the citrate-stabilized Au and Ag nanoparticles. Typically, two different
samples were prepared by the same procedure and at least three different positions on the
grid were analyzed on each sample to determine the reproducibility of the nanoparticle
size distribution.

The TEM image in Figure 4.3 shows that AuNPs have diameters of 15-20 nm.
There is minimal aggregation of the AuNPs, presumably because of electrostatic
repulsion due to the stabilizing, negatively charged citrate molecules on the particle
surfaces. Most of the particles had an approximately spherical structure, thus possessing
an aspect ratio (length/width ratio, n) of about 1. Several images of the samples showed
similar size and shape distributions of the nanoparticles, confirming reproducibility in the

nanOparticle size distribution.

54

    

20 nm

 

Figure 4.3. TEM image of citrate-stabilized AuNPs deposited on a
carbon-coated copper grid by a ‘drop and dry’method.

Figure 4.4 shows the EDS spectrum of these nanoparticles, which clearly reveals
the presence of Au in the system. The signals due to Cu and C resulted from the
supporting TEM grid. In this experiment, spectra were also obtained using EDS from at
least two different nanoparticles in a sample, and all spectra were similar to that

represented in Figure 4.4.

55

 

 

 

 

 

 

Cu
.C
,Au
Cu
[Au
Cu J r A“ A

u
1' - A
0 2 4 6 8 10 12 14 keV

 

Figure 4.4. EDS of citrate-stabilized AuNPs deposited on a carbon-coated
copper grid by ‘drop and dry’method.

TEM analysis of citrate-stabilized AgNPs was also performed to determine the
characteristics of these nanomaterials. Figure 4.5 indicates that the diameter of the
AgNPs was about 40 nm, which is in the size range reported in the literature.10 Also, the
nanoparticles were mostly spherical in shape, and aggregation of these particles was not
extensive. EDS confirmed the presence of Ag in these samples. The size of citrate-

stabilized AgNPs was however about double the size of citrate-stabilized AuNPs.

56

 

Figure 4.5. TEM image of citrate-stabilized AgNPs deposited on a
carbon-coated copper grid by a ‘drop and dry’method.

4.2.3. TEM Analysis of Au Nanorods (AuNR)

Citrate-stabilized gold nanorods (AuNRs) were also prepared to analyze the effect
of shape of nanoparticle shape on the SEIRS enhancement. Figure 4.6 shows a TEM
image of AuNRs produced following the procedure reported by Murphy and co-
workers.” '2 Two types of particles were formed—one type that was approximately
spherical and a second type that was rod shaped, the spherical type being more
predominant. The diameter of the nanospheres was about 20 nm. Figure 4.6b shows an
enlarged image of a typical sized nanorod, which was about 60 nm in width and 200 nm

in length, thus possessing an aspect ratio (1]) of about 3.

57

 

Figure 4.6. TEM images of CTAB—stabilized AuNRs deposited on carbon-coated
copper grid by ‘drop and dry’method.

4.3. SEIRS — Aminothiophenol as a Model System

The SEIRS activity of the FEM/nanoparticle systems was first verified using 4—
aminothiophenol (ATP), a molecule that has been extensively examined in surface
5.13-16

enhanced spectroscopy.

4.3.1. Subtraction SEIR spectra of A TP

Figure 4.7a shows the spectrum obtained for a (PAH/AuNP)1 film deposited on an
Al—coated substrate. Bare Al was employed as a background. Small absorbances due to
PAH were apparent at 1600 cm" due to the presence of the NH; group. After introducing
the ATP analyte by immersing the substrate in an ATP solution and rinsing, the IR
spectrum was acquired again (Figure 4.7b). New peaks at 1591 and 1488 cm'l are due to
adsorbed ATP, and the difference of spectra 4.7b and 4.7a, shown in spectrum 4.7c, was
simply the SEIR spectrum of adsorbed ATP. Most of the spectra that follow are

subtracted spectra that were obtained in a similar manner.

58

 

 

 

 

 

 

0.005
-_._\ c
8
C
6
e
o
0)
a .
<
b
a
2200 2000 1800 1600 1400 1200
Wavenumber tom")

Figure 4.7. Reflectance infrared spectra of (a) [PAH/AuNP]. on an Al-coated wafer
and (b) the same film after exposure to 50 mM ATP and rinsing. Both spectra were
acquired using bare Al as a background. Spectrum 0 is the difference between

spectra b and a [(b)-(a)].

4.3.2. SEIRS Analysis of A TP

Figure 4.8 shows the SEIR spectrum of ATP on both nanoparticle and bare metal
surfaces. Spectrum (a) corresponds to a layer of ATP molecules on a Au wafer (bare Au
was used as the background) and reveals low-intensity peaks at 1591 and 1488 cm",
which result from C-C stretching of the phenyl ring.'7 Au substrates have been typically
used for the formation of monolayers or multilayers of molecules containing groups such
as thiols (-SH)'8'2| or amines (-NH2)22'24 because of the strong interaction between Au
and the functional groups. In the case of ATP, it is primarily the —SH group that interacts

with the Au surface since it has a higher affinity towards gold. Thus, spectrum (a) was

59

 

used as a reference for comparison with spectra on other substrates. Spectrum (b) was
obtained from an Al-coated wafer that was exposed to a 50 mM ATP solution. In this
case, an Al-coated wafer was used as the background. This spectrum did not show
appreciable absorbances at 1591 and 1488 cm'1 because ATP does not strongly adsorb to
Al. Thus, Al may be an ideal candidate for the analysis of SEIRS enhancement by metal

nanoparticles because there is no background ATP signal.

 

 

 

 

 

 

 

 

0.01
c .
10 d
'9
O
m
n
<
C
_ __ b
——: .— - .‘M 1 "__a _
2200 2000 1800 1600 1400 1200

Wavenumber (cm")

Figure 4.8. SEIR spectra of 4-aminothiophenol on different substrates — (a) Au-
coated wafer (Au background), (b) Al-coated wafer (Al background), (c) subtraction
spectrum of ATP on [PAH/AuNPL-coated Al, (d) subtraction spectrum of ATP on
[PAH/AuNPh-coated Al. Prior to measuring each spectrum, substrates were
immersed in 50 mM ATP in ethanol for 30 min. Spectra c and d were obtained
following the procedure mentioned in Figure 4.7

Although the bare Al wafer did not show a significant signal due to ATP

molecules (Figure 4.8, spectrum b), the SEIRS-active substrate obtained by deposition of

60

 

PAH/AuNP bilayers on the Al. wafer demonstrated significant surface infrared
enhancement (Figure 4.8, spectra c and d).

The signal due to adsorbed ATP obtained using an Al substrate modified with one
bilayer of (PAH/AuNP) was about 17-fold greater than that on an unmodified Al surface
(Figure 4.8c — [PAH/AuNPL). The enhancement factor is determined based on the
increase in the absorbance of the most intense peaks at 1591 cm", which is attributed to
C-C stretching.l7 The peak at 1623 cm'1 in the enhanced spectrum corresponds to the
NH; deformation. As reported previously, the presence of metal nanoparticles induces a
strong SEIRS enhancement.4 There was a further increase (~30 compared to the
unmodified Al wafer substrate) in the intensity of ATP absorbances when the number of
bilayers was increased to 3 (Figure 4.8d — [PAH/AuNPh). This further enhancement
could be attributed to the presence of a higher number of Au nanoparticles on the surface,
which leads to increased numbers of active sites on the SEIRS substrate. A similar trend
of increase in the enhancement factor with increases in the number of bilayers of active
species was observed by Hu et al. in the SERS analysis of aminothiophenol with
cetyltrimethylammonium bromide (CTAB)-stabilized gold nanorods and

poly(styrenesulfonate) (PSS) films.5

4.4. Effect of Nanoparticle Shape 0n Infrared Enhancement

Osawa has reported that the shape of the nanoparticles plays an important role in
the magnitude of the SEIRS enhancement.4 Since nanorods possess an aspect ratio
higher than spherical particles (11 = 1), the nanorods are typically expected to have a

better enhancement than nanospheres due to high local electric fields provided by the

61

 

nanorod substrate.25 Thus, SEIRS using polyelectrolyte/Au nanorod (NR) films was
examined and was then compared to similar films with AuNPs. The AuNRs employed
were stabilized by a positively charged surfactant, CTAB, according to the method of
Murphy.” '2 Thus, in order to build the multilayers, negatively charged poly(styrene
sulfonate) was employed as the counter polyelectrolyte, after depositing a first layer of
PAH on Al.

Figure 4.9 shows that the surface enhancement of the infrared spectrum of ATP
with a PAH/(PSS/AuNR)3 film on Al was about 4 times lower than that of a substrate
coated with 3 bilayers of (PAH/AuNP) (spheres). The decrease in the enhancement with
AuNRs could indicate the nanorods do not exist as discrete island structures, but have
aggregated on the Al wafer during deposition of the bilayers. Aggregation can greatly
decrease surface infrared enhancement by reducing local electromagnetic fields.4
However, it was not determined whether the mass of nanorods deposited was equivalent
to the mass of nanospheres. Also, the films and nanoparticle stabilizers were different, so
a direct comparison between the two was difficult. However, since optimum signal
enhancement was observed with nanospheres, the analyses of amphetamines were

performed using this system.

62

 

 

0.005

(b)

(a) W

Absorbance

 

 

 

 

2800 2400 2000 1600 1200
Wavenumber (cm-1)

Figure 4.9. Subtraction SEIR spectra of 4-aminothiophenol on Al—coated wafers
modified with polymer films containing gold nanostructures of different shapes —
(a) PAH/[PSS/AuNR]3, (b) [PAH/AuNP]3 on A1.

4.5. SEIRS of Amphetamine sulfate and Methamphetamine hydrochloride

As shown above, AuNPs provided better enhancement than the AuNR system in
SEIRS analysis of ATP. Hence, SEIRS of amphetamine sulfate and methamphetamine
hydrochloride was examined using spherical nanoparticles. The extent of surface
infrared enhancement depends not only on the shape of the nanostructures, but also on
the composition of the metal nanoparticles and the nature of the analyte molecules." 2“"

To examine compositional effects, SEIRS analysis was performed on both gold and silver

nanoparticles that were citrate-stabilized.

63

4.5.1. Subtraction SEIR Spectra of Amphetamine sulfate and Methamphetamine
hydrochloride

The surface infrared enhancement effect on the spectra of adsorbed amphetamine
sulfate and methamphetamine hydrochloride molecules was analyzed by the following
sequence of steps. Firstly, Al-coated Si was coated with the surface active species
(multilayers containing AuNPs or AgNPs), and an IR spectrum of the polyelectrolyte/NP
film was taken using a bare Al surface as the background. Then, the surface-active
substrate was immersed into a solution of analyte molecules for 30 min, followed by brief
rinsing of the substrate for 15 5. After drying, another IR spectrum was obtained, with
bare Al as the background. The instrument software was then used to subtract the
spectrum obtained before exposure to analyte from that measured after immersion in the
anal yte solution.

Figure 4.10, shows a sample set of spectra for amphetamine sulfate. Spectrum (a)
corresponds to a bilayer of (PAI-I/AgNP), on the Al wafer, and there were no definitive
absorbance peaks observed. After immersing this substrate into a solution of 0.1 M
amphetamine sulfate and rinsing, spectrum (b) was acquired, and new absorbance peaks
were observed. Spectrum (c) was then obtained by subtracting the spectrum (b) from (a),
to give the SEIR spectrum of amphetamine sulfate on a [PAH/AgNPh-coated Al wafer.
In this case, peaks at 1142, 1497, and 1554 cm”I are representative of amphetamine
sulfate as reported in the UN Recommended Methods for Testing Amphetamine and
Methamphetamine Manual.29 There are other peaks at 619, 697, and 737 cm’l that are
also recommended; however the Mercury-Cadmium Telluride IR detector that was

employed in this research cannot detect these low-energy peaks.

64

Similar analysis was performed for methamphetamine hydrochloride, and the
results are shown in Figure 4.11. In spectrum 4.11c, peaks at 1061, 1488, and 1604 cm'1
are representative of methamphetamine hydrochloride as reported in the UN
Recommended Methods for Testing Amphetamine and Methamphetamine Manual.29

The peaks at less than 1000 cm'1 wavenumbers that are also recommended were not

detected since the IR detector was not sensitive at these low energy levels.

65

 

0.005

 

 

 

M
03
0
C
N
.D
b
O
3
< (b)
(a)
3500 3000 2500 2000 1500

Wavenumber (cm-‘)
Figure 4.10. IR spectra of a (PAH/Ag)l-coated Al wafer (a) before and (b) after
immersion in a 0.1 M amphetamine solution followed by a brief rinse with water.
Spectrum (c) gives the difference between spectra (a) and (b). A bare Al surface
was used as a background.

 

0.005

(c)

Absorbance

 

 

 

3500 3000 2500 2000 1 500

Wavenumber (cm-1)

Figure 4.11. IR spectra of a (PAI-I/Agh-coated Al wafer (a) before and (b) after
immersed in 0.1 M methamphetamine solution. Spectrum (c) gives the difference
between spectra (a) and (b). A bare Al surface was used as a background.

66

4.5.2. Effect of Number of Bilayers on SEIRS Enhancement with Gold and Silver
Nanoparticles as Active Species
4.5.2.a. Gold Nanoparticles

When gold nanoparticles ([PAH/AuNP] films) were incorporated onto the surface
of Al-coated Si, surface infrared enhancement effects were observed for amphetamine
sulfate detection; however, the bare Al wafer (Figure 4.12a) had very low absorbance
values when immersed into the same solution containing 0.1 M amphetamine sulfate. As
the number of bilayers of (PAH/AuNP) increased, there was an increase in the intensity
of the peaks at 1142, 1497, 1554 cm", A (PAH/AuNP).-coated Al wafer showed an
enhancement factor of ~15 (Figure 4.12b) compared to the unmodified substrate (Figure
4.123) while, with a further increase in the number of bilayers to 3 (Figure 4.12c) and 5
(Figure 4.12d), the enhancement factor increased to 23 and 28, respectively.

When similar experiments were performed with 0.1 M methamphetamine
hydrochloride solution, there were no identifiable peaks when using bare A1 (Figure
4.13a). Chappell has reported that some of the predominant peaks obtained for the IR
spectrum of methamphetamine hydrochloride occur at 1060, 1488, 1604, 2461, 2730 and
2968 cm".30 Figure 4.13 shows that these peaks (except for 1060 cm") are consistent
with UN recommendations as mentioned earlier, and were observed during the analysis

with AuNP-coated substrates.

67

 

 

 

 

 

 

 

 

 

l 0.02
0
0
5
e d
O
W
.0
< w - L

w c

f fi— %
”A
a
3500 3000 2500 2000 1500 1000

Wavenumber (cm")

Figure 4.12. Subtraction SEIR spectra of 0.1 M amphetamine on different
coatings on Al surfaces — (a) bare, (b) (PAH/Au)., (c) (PAH/Au)3, (d) (PAH/Au)5.
The subtraction spectra were obtained following the procedure described in Figure

4.10.

 

0.02

 

Absorbance

 

 

 

 

3000 2500 2000 1 500 1 000

Wavenumber (cm-1)

Figure 4.13. Subtraction SEIR spectra of 0.1 M methamphetamine
hydrochloride on different coatings on Al surfaces - bare (a), (PAH/Au), (b),
(PAI-I/Au)3 (c). The subtraction spectra were obtained following the procedure

described in Figure 4.10.

68

When a (PAH/Au),-coated Al wafer was used as a substrate, the peaks
corresponding to methamphetamine hydrochloride were observed as shown in Figure
4.13b. However, the peaks were not as definitive as observed for amphetamine sulfate
samples. The resolution improved when (PAH/Au)3-coated Al was employed (Figure
4.13c). Interestingly, when the number of bilayers was further increased, very broad
peaks were noticed from which not much useful information could be obtained due to
poor resolution. This could be due to extensive aggregation of the nanoparticle system,

initiated by their interaction with analyte molecules.

4.5.2.b. Silver Nanoparticles

As mentioned earlier, the nature of the nanoparticles plays an important role in
determining the extent of surface infrared enhancement of a specific analyte. The surface
infrared enhancement effect obtained using gold nanoparticles was compared with that of
silver nanoparticles for amphetamine sulfate and methamphetamine hydrochloride
detection.

Figure 4.14 shows that the peaks obtained by employing silver nanoparticles were
more definitive than those obtained by using gold nanoparticles for amphetamine sulfate
analysis. Also, there were more peaks detected with the AgNP-coated surface, again
indicating that the sensitivity of AgNP-coated substrate was better than that of the AuNP-
coated substrate. The enhancement factor of the peaks using the substrate coated with 3
PAH/AgNP bilayers (Figure 4.14c) was 33, whereas a 1 bilayer film showed an
enhancement factor of ~17 (Figure 4.14b); however, there was not an appreciable

increase in the intensity of the amphetamine sulfate bands on going from substrate coated

69

 

with 3 PAH/AgNP bilayers to 5 PAH/AgNP bilayers (Figure 4.14d). This might be
because of the aggregation of nanoparticles, which could potentially cause a decrease in

the surface area of the active species.

 

 

 

 

0.005
0
O
E
.9 d
0
1'0
.0
<
C
b
M
3500 3000 2500 2000 1 500 1 000

Wavenumber tom")
Figure 4.14. Subtraction SEIR spectra of 0.1 M amphetamine sulfate on different
coatings on Al surfaces — bare (a), (PAH/AgNP); (b), (PAH/AgNP); (c), and
(PAH/AgNP)5 (d). The subtraction spectra were obtained following the procedure
described in Figure 4.10.

70

Similarly, when 0.1 M methamphetamine hydrochloride was analyzed using
AgNP-coated Al substrate, the peaks were more definitive, when compared to AuNP-
coated substrates (compare Figures 4.15 and Figure 4.13). Also, peaks at 2740 and 2970
cm‘1 were more intense than on the AuNP-coated substrate, indicating a better sensitivity

of the AgNP system.

 

Absorbance

 

 

 

 

3500 3000 2500 2000 1500 1000
Wavenumber (cm-1)

Figure 4.15. Subtraction SEIR spectra of 0.1 M methamphetamine hydrochloride
on different coating on Al wafer — bare (a), (PAH/Ag), (b), (PAH/Ag)3 (c). The
subtraction spectra were obtained following the procedure similar to Figure 4.11.

The differences in the surface enhancement effects of the analyte molecules on
gold and silver-modified substrates may arise due to the differences in the adsorption
properties of the analyte molecules on gold and silver.” 32 Also, with the increase in the

number of bilayers, it is highly probable that the nanoparticles aggregate, as suggested by

71

the UV-visible data, causing a decrease in the surface area. Thus, there was no further
increase in the surface infrared enhancement after increasing the number of bilayers to 3.

(Figure 4.14)

4.6. Effect of Polymer Films on Surface Infrared Enhancement

To verify that the increase in the intensity of the peaks was not due to absorption
or adsorption of molecules in the polymer films, an Al-coated wafer was modified with a
bilayer of PAH/PSS, and then immersed into analyte solutions. In this case, since there
were no active nanoparticles present, the spectrum did not show a large enhancement of
infrared absorbance (Figure 4.16b) when compared to nanoparticle-containing films
(Figure 4.16c). Moreover, the spectrum looked quite different than that obtained with the
nanoparticle films since the predominant peaks of amphetamine at 1554 and 1142 cm'1
were missing in spectrum 4.16b, indicating that the polymer films in the multilayers do
not contribute to infrared surface enhancement.

Figure 4.17 confirms a similar trend in that the polymer-coated wafer did not
exhibit any enhancement compared to nanoparticle-coated substrate for 0.1 M

methamphetamine hydrochloride.

72

 

0.01

Absorbance

 

 

 

 

3500 3000 2500 2000 1500 1000

Wavenumber (cm-‘1
Figure 4.16. Subtraction SEIR spectra of 0.1 M amphetamine sulfate on different
coating on Al wafer - bare, (b) (PAH/PSS)., (c) (PAH/Au)1, The subtraction spectra

were obtained following the procedure similar to Figure 4.10.

 

 

 

 

 

 

0.02
0)
O
C
N
'9
O C
(I)
.D
<
—_ — — A “h ‘a A “h
3000 2500 2000 1500 1000

Wavenumber (cm-1)

Figure 4.17. Subtraction SEIR spectra of 0.1 M methamphetamine hydrochloride
on different coating on Al wafer — (a) bare, (b) (PAH/PSS)1, (c) (PAI-I/Au)l. The

subtraction spectra were obtained following the procedure similar to Figure 4.11.

73

4.7. Effect of Concentration of Analyte Solution 0n Surface Infrared

Enhancement.

The effect of the concentrations of the analyte solutions on SEIRS activity was
then investigated. When the concentration of amphetamine sulfate was 10 mM, there
was no SEIRS effect detected when using a AgNP-coated substrate (Figure 4.18 b), but
when the concentration was increased to 0.1 M, the SEIRS effect was very apparent as
seen in Figure 4.180. There was an enhancement factor of ~15 when (PAI-I/Ag)1-Coated
A1 wafer was employed as the substrate, when compared to unmodified Al substrate for
analyzing 0.1 M of amphetamine sulfate (Figure 4.18, spectrum c compared to spectrum
a). When the concentration of amphetamine sulfate was further increased to 0.5 M (500
mM), the intensity of the characteristic peaks increased further; and the surface
enhancement calculated for (PAH/AgNP)1-coated Al wafer was ~60, when compared to

bare Al immersed in 0.5 M amphetamine solution.

74

 

Absorbance

 

I 0.02

 

(d) .,
(c) A ,, A.

 

 

(b)

 

 

(a)

 

 

 

3500 3000 2500 2000 1 500 1 000

Wavenumber (cm-1)

Figure 4.18. (a) IR spectrum of Al wafer immersed in 0.1 M of amphetamine
solution in water. Subtraction SEIR spectra of (PAH/Ag).-coated Al wafer
immersed in amphetamine solutions in water of varying concentrations : (b) 10 mM
(c) 0.1 M and (d) 0.5 M. The subtraction spectra were obtained following the
procedure similar to Figure 4.10.

4.8. Discussion of Mechanism for Surface Enhancement

The enhancement of infrared intensities is attributed to two separate contributions:
the electromagnetic enhancement mechanism33'35, which is based on the increase in the
local dipole moment at the nanoparticle surface, and the chemical mechanism, which is
attributed to the changes in the optical parameters of the adsorbateg’é'39 Also, it is likely
that the increased surface area attributed by the presence of nanoparticles could
potentially enhance the concentration due to adsorption on the surface.25 In this work,

since the enhancement effect is saturated after specific number of bilayers, it is highly

75

 

probable that the actual enhancement arises from the increase in the surface area of the

nanoparticles on the substrate.

4.9. Conclusions

In this chapter, it has been established that nanoparticles incorporated in polymer
films act as surface-active substrates for SEIRS analysis. The presence of
PAH/nanoparticle films on the surface provided surface enhancements as high as 33-fold
in the absorbance of infrared peaks. It was verified that the polymer does not contribute
to surface infrared enhancement. The surface modification with AgNPs resulted in more
definitive peaks in the spectra, when compared to AuNPs. There was also an increase in
the surface infrared enhancement with the increase in the number of bilayers; however,
there was no further increase after 3 bilayers. This may have been caused by decreased

surface area due to the aggregation of the nanoparticle systems.

76

4.10. References

1. Lee, P. C.; Meisel, D., Adsorption and surface-enhanced Raman of dyes on silver
and gold sols. Journal of Physical Chemistry 1982, 86, (17), 3391-5.

2. Magdassi, S.; Bassa, A.; Vinetsky, Y.; Kamyshny, A., Silver nanoparticles as
pigments for water-based ink-jet inks. Chemistry of Materials 2003, 15, (11), 2208-2217.

3. Rast, L.; Stanishevsky, A., Aggregated nanoparticle structures prepared by
thermal decomposition of poly(vinyl)-N-pyrrolidone/Ag nanoparticle composite films.
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4. Osawa, M., Surface-enhanced infrared absorption. Topics in Applied Physics
2001, 81, (Near-Field Optics and Surface Plasmon Polaritons), 163-187.

5. Ha, X.; Cheng, W.; Wang, T.; Wang, Y.; Wang, E.; Dong, 8., Fabrication,
characterization, and application in SERS of self-assembled polyelectrolyte-gold nanorod
multilayered films. Journal of Physical Chemistry B 2005, 109, (41), 19385-19389.

6. Cheng, W.; Dong, S.; Wang, E., Two- and three-dimensional Au
nanoparticle/CoTMPyP self-assembled nanotructured materials: film structure, tunable
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108, (50), 19146-19154.

7. Malikova, N.; Pastoriza-Santos, I.; Schierhom, M.; Kotov, N. A.; Liz-Marzan, L.
M., Layer-by-layer assembled mixed spherical and planar gold nanoparticles: control of
interparticle interactions. Langmuir 2002, 18, (9), 3694-3697.

8. Ung, T.; Liz-Marzan, L. M.; Mulvaney, P., Optical properties of thin films of
Au@SiOz particles. Journal of Physical Chemistry B 2001, 105, (17), 3441-3452.

9. Schmitt, J .; Maechtle, P.; Eek, D.; Mohwald, H.; Helm, C. A., Preparation and
optical properties of colloidal gold monolayers. Langmuir 1999, 15, (9), 3256-3266.

10. Taurozzi, J. S.; Tarabara, V. V., Silver nanoparticle arrays on track etch
membrane support as flow-through optical sensors for water quality control.
Environmental Engineering Science 2007, 24, (1), 122-137.

77

 

 

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Chapter 5. Conclusions and Future Work
5.1. SEIRS Analysis of Amphetamine sulfate and Methamphetamine

hydrochloride

IR analysis has the ability to discriminate different drugs based on their specific
functional groups and spectra. In this thesis, SEIRS has been demonstrated as a more
efficient form of IR analysis because of the signal enhancement effect observed due to
the incorporation of nanoparticles on a surface. Nanoparticles encapsulated in polymer
films act as surface-active substrates on Al wafers for SEIRS analysis. This research can
be considered as a proof-of-principle, where SEIRS-active substrates can be potentially
used for trace drug analysis.

The presence of PAH/nanoparticle films on the substrate provided surface
enhancements as high as 33-fold in the absorbance of infrared peaks. It was verified that
the polymer does not contribute significantly to the surface infrared enhancement. Also,
spherical Au nanoparticles provided better SEIRS effect than the Au nanorods. The
surface modification with AgNPs resulted in more definitive peaks in the spectra, when
compared to AuNPs. There was also an increase in the surface infrared enhancement
with the increase in the number of bilayers; however, there was no further increase after
deposition of 3 bilayers. This may have been caused by decreased surface area due to the

aggregation of the nanoparticle systems.

5.2. Future Prospects
In this thesis, the work was performed on known concentrations of amphetamine

sulfate and methamphetamine hydrochloride samples. It has been used as a qualitative

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technique, which helps in determining the presence of pure drug samples. However,
analysis of street samples would be a little more complicated than the pure sample,
because of the presence of the cutting agents added to the sample. Thus, more detailed
analyses are required in addressing this challenge. In this case, it might be necessary to
analyze the effect of common cutting agents used in the preparation of amphetamine and
methamphetamine, such as sugars and caffeine, during the SEIRS analyses of these
compounds.

Since the work reported here is at a preliminary stage, there are a lot of
parameters that would have to be optimized before seeing the actual impact of the
technique on drug analysis. The size and shape of the nanoparticles may determine the
extent of infrared surface enhancement as reported earlier. When the analyses were
performed with Ag and Au nanoparticles, the sizes of these particles were different from
each other. It would be interesting to analyze similar sized particles of Ag and Au to
verify whether it is the composition or the size of the nanoparticles which has a major
impact on the enhancement effect. Thus, it would be necessary to investigate the effect
of both size and composition of nanoparticles such that much lower concentrations (in
micromolar levels) of the drug samples can be detected by this technique.

It would be also be interesting to see the application of this technique on
commonly used drug samples such as methylenedioxymethamphetamine (MDMA or
ecstasy) and methylendioxyamphetamine (MDA), other than amphetamine sulfate and
methamphetamine hydrochloride. Also, it would be necessary to expand to other classes

of illicit drugs so that this technique can be used as a universal analytical tool.

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5.3. Summary Statement

This thesis has established that the SEIRS technique may be used in the analyses
of amphetamine and methamphetamine, but it needs to be refined to allow analysis of
much lower analyte concentrations. This project is the first time where nanoparticles
embedded in polymer coatings have been used as SEIRS-active substrates for drug
analysis. A lot of future research must be conducted in order to employ this technique in
actual crime cases; however, this preliminary study has demonstrated the potential of this

technique in trace drug analysis.

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