Post on 24-Sep-2020
UNIVERZA V MARIBORU
FAKULTETA ZA NARAVOSLOVJE IN MATEMATIKO
Oddelek za fiziko
MAGISTRSKO DELO
Simon Hamler
Maribor, 2015
UNIVERZA V MARIBORU
FAKULTETA ZA NARAVOSLOVJE IN MATEMATIKO
Oddelek za fiziko
Magistrsko delo
VPLIV TEMPERATURE NA
POVRŠINSKO OJAČAN RAMANSKI SPEKTER
2,4,6-TRINITROTOLUENA
Master Thesis
INFLUENCE OF TEMPERATURE ON THE
SURFACE ENHANCED RAMAN SCATTERING
SPECTRA OF 2,4,6-TRINITROTOLUENE
Mentor: Kandidat:
doc. dr. Marko Jagodič Simon Hamler
Somentor:
dr. Hainer Wackerbarth
Maribor, 2015
ACKNOWLEDGEMENT
I would like to thank my mentor dr. Marko Jagodič and co-mentor dr. Hainer
Wackerbarth for help and guidance when writing this thesis. I would also like to thank
my family for always being there for me.
UNIVERZA V MARIBORU
FAKULTETA ZA NARAVOSLOVJE IN MATEMATIKO
IZJAVA
Podpisani Simon Hamler, rojen 4. 6. 1987, študent Fakultete za naravoslovje in
matematiko Univerze v Mariboru, študijskega programa Fizika, izjavljam, da je
magistrsko delo z naslovom Vpliv temperature na površinsko ojačan ramanski spekter
2,4,6-trinitrotoluena pri mentorju dr. Marku Jagodiču in somentorju dr. Hainerju
Wackerbarthu avtorsko delo. V magistrskem delu so uporabljeni viri in literatura
konkretno navedeni; teksti in druge oblike zapisov niso uporabljeni brez navedb
avtorjev.
Maribor, ______________ Podpis: ____________________
UNIVERZA V MARIBORU
FAKULTETA ZA NARAVOSLOVJE IN MATEMATIKO
Hamler S.: Vpliv temperature na površinsko ojačan ramanski spekter 2,4,6-
trinitrotoluena.
Magistrsko delo, Univerza v Mariboru, Fakulteta za naravoslovje in matematiko,
Oddelek za fiziko, 2015.
IZVLEČEK
Zaznavanje sledi eksplozivov, kot je trinitrotoluen (TNT), je pomembno področje pri
preprečevanju terorističnih napadov. Površinsko ojačana ramanska spektroskopija
(SERS) je postala močna detekcijska tehnika za identifikacijo majhnih količin analitov.
V magistrskem delu so predstavljeni podatki o TNT raztopini, naneseni na
nanostrukturirano zlato površino, ki je ogreta do 60 °C. Zaznane spremembe, ki jih
opazimo na podlagi mikroskopskih slik in SERS spektrov, razložimo s pomočjo
izhlapevanja, faznega prehoda in razgradnje TNT molekul. Vpliv temperaturne
odvisnosti na SERS učinek je bil raziskan na kemisorbiranem monosloju 4-
nitrothiophenol molekul. Da bi zmanjšali izhlapevanje TNT molekul, je bil med
plazmonsko površino in TNT vstavljen samosestavljiv monosloj mercaptoheksanola
(MCH).
KLJUČNE BESEDE: površinsko ojačana ramanska spektroskopija, eksplozivi,
temperaturna odvisnost, izhlapevanje, fazni prehod, molekulski razpad.
UNIVERZA V MARIBORU
FAKULTETA ZA NARAVOSLOVJE IN MATEMATIKO
Hamler S.: Influence of the Temperature on the Surface Enhanced Raman
Scattering Spectra of 2, 4, 6-Trinitrotoluene.
Master Thesis, University of Maribor, Faculty of Natural Sciences and
Mathematics, Department of Physics, 2015.
ABSTRACT
The detection of trace amounts of explosive like trinitrotoluene (TNT) is an important
issue in the prevention of terrorist attacks. Surface enhanced Raman scattering (SERS)
spectroscopy has become a powerful detection technique for identification of minute
amounts of analytes. This thesis presents data of TNT in solution, deposited on a
nanostructured gold surface, which is heated up to 60 °C. The observed changes in the
microscopy images and in the SERS spectra are explained by evaporation, phase
transition and decomposition of the TNT molecules. The impact of temperature
dependence of SERS effect is studied on a chemisorbed 4-Nitrothiophenol monolayer.
To minimize the evaporation of TNT molecules, a self-assembled monolayer of
mercaptohexanol (MCH) was inserted between plasmonic surface and TNT.
KEYWORDS: surface enhanced Raman spectroscopy, explosives, temperature
dependence, microscopy, evaporation, phase transition, decomposition.
CONTENTS
1 INTRODUCTION .................................................................................................. 1
2 RAMAN SPECTROSCOPY .................................................................................. 4
2.1 Energy units ..................................................................................................... 4
2.2 Degrees of freedom and molecular vibrations ................................................ 5
2.3 Basic theory ..................................................................................................... 7
2.3.1 Comparison of Raman and Fluorescence processes .............................. 12
2.4 Polarizability tensor ....................................................................................... 13
3 SURFACE ENHANCED RAMAN SCATTERING (SERS) .............................. 14
3.1 Electromagnetic Enhancement (EM) ............................................................ 15
3.1.1 Hot spots ................................................................................................ 21
3.2 Chemical mechanism .................................................................................... 21
4 EXPERIMENTAL SECTION .............................................................................. 23
4.1 Raman setup .................................................................................................. 23
4.2 Laser .............................................................................................................. 25
4.3 Spectrometer and CCD camera ..................................................................... 26
4.4 Plasmonic substrate ....................................................................................... 28
4.5 Microscope .................................................................................................... 28
4.6 Sample preparation ........................................................................................ 29
4.7 Data analysis ................................................................................................. 30
5 RESULTS AND DISCUSSION ........................................................................... 31
5.1 Microscopic observations .............................................................................. 31
5.2 SERS measurements of TNT ........................................................................ 32
5.2.1 Evaporation ............................................................................................ 35
5.2.2 Phase transition ...................................................................................... 35
5.2.3 Decomposition ....................................................................................... 36
5.2.4 Temperature dependence of the SERS effect ........................................ 37
5.3 TNT solution deposited on the substrate covered with mercaptohexanol
(MHC) monolayer .................................................................................................... 39
6 CONCLUSIONS .................................................................................................. 41
7 RAZŠIRJENI POVZETEK V SLOVENSKEM JEZIKU .................................... 43
REFERENCES ............................................................................................................ 46
1
1 INTRODUCTION
Owing to numerous attacks and attack attempts during the last years, the protection of
our society against terrorism has gained meaning. In this context, the detection of
explosives and their associated compounds is an important issue. This has led to
development of new detection technologies, especially in the field of homeland security,
to face the problems of hidden explosives at public places, such as airports, bus and
train stations. Many techniques have been investigated for this purpose. However, the
majority is not ideal for explosive detection, since they have disadvantages such as
invasiveness, detect only certain explosive, but fail to detect others, or require
complicated sample preparation. Vibrational spectroscopy has shown to be an excellent
technique for rapid, accurate quantitation and can be used for studying very wide range
of sample types and can be carried out from a simple identification test to an in-depth,
full spectrum, qualitative, and quantitative analysis [1].
Vibrational spectroscopy includes several different techniques. However, the most
important of them are infrared (IR) spectroscopy and Raman spectroscopy. Both of
these techniques can provide a complementary information about molecule vibrations
in many instances. Although both study the interaction of radiation with the molecule,
they differ in a manner in which photon is transferred to the molecule by changing its
vibrational state. IR spectroscopy measures transitions between molecular vibrational
energy levels, based on the direct absorption of light quanta. Absorption of photons
occurs, when the frequency of radiation by the polychromatic light matches that of a
vibration. Therefore, the molecule is prompted to a vibrational excited state. The loss
of this frequency of radiation from the beam after it passes through the sample is then
detected [1, 2]. On the other hand, Raman spectroscopy is based on a scattering
mechanism and requires monochromatic light for detection of molecular vibrations. A
portion of the incident photons will be scattered inelastically. Therefore, the energy of
scattered photons will differ from that of the incident photons. The energy difference
corresponds to the difference between the vibrational levels of the molecule.
The IR and Raman vibrational bands are characterized by their frequency (energy),
intensity (polar character or polarizability), and band shape (environment of bonds).
Since the vibrational energy levels are unique to each molecule, the IR and Raman
spectrum provide a ‘fingerprint’ of a particular molecule. The frequencies of these
molecular vibrations depend on the masses of the atoms, their geometric arrangement,
2
and the strength of their chemical bonds. Their spectrum provides information on a
molecular structure, dynamics, and environment [1]. In this thesis, the Raman
spectroscopy technique was used. There are two main advantages of Raman over IR
spectroscopy. The first one is that the samples can be confined or sealed in optical
transparent materials (glass, quartz), since they do not absorb the light. Because of this,
Raman spectroscopy is suited for the analysis of reactive or environmentally sensitive
compounds. The second advantage is that it is suitable for examining samples in
aqueous solution. Vibrations of water are weakly Raman active and hence does not
interfere the Raman signal, while in IR spectroscopy, the absorption of water is very
strong and thus often superimpose the signals of interest.
Raman spectroscopy is already an established technique in analytical and forensic
science, collecting a unique chemical signature of molecules. Almost all explosives can
be identified by their Raman spectra. Therefore, neat explosives have been extensively
studied by Raman spectroscopy [3–5]. Further advantages are that the detection is rapid
and non-invasive. There are hardly any limitations to the sample, which can be present
in different physical states or as a composition of different compounds. Moreover, there
is hardly sample preparation.
However, Raman spectroscopy is not suitable for the detection of trace amounts, as it is
needed for the prevention of bomb attacks. Nevertheless, the inherently weak Raman
process can be greatly improved using surface enhanced Raman scattering (SERS).
SERS combines low detection limits with high information content about molecular
identity making it highly suitable for trace analysis [6, 7]. Based on SERS single
molecule, detection was achieved. The enhancement factors can be as high as ~1014-
1015, if SERS is combined with other effects like resonance Raman. However, the
substantial contribution to the enhancement comes from SERS [8-11]. The surface
enhanced Raman effect is a product of two mechanisms, the electromagnetic and
chemical enhancement. The electromagnetic mechanism is believed to be responsible
for the bulk of the enhancement (~104-108) and is based on the increase of the
electromagnetic field strength in the vicinity of nanostructured metal surface. The
enhancement is greatest when the surface plasmon frequency and the incident light are
in resonance. In comparison to electromagnetic effect, the chemical enhancement is
quite modest (~10-102) and arises from interaction between adsorbed molecule and the
metal surface. The charge transfer between adsorbate and metal increases the
3
polarizability of adsorbed molecules. Another advantage of SERS is the quenching of
fluorescence, which is a known obstacle in Raman spectroscopy of explosives [3].
The aim of this work was to study how increasing temperature affects the SERS spectra
of TNT, deposited on a nanostructured gold surface and also if at the elevated
temperature, SERS measurements are still possible. Chapter 2 and 3 include necessary
theoretical background of normal Raman and surface enhanced Raman scattering. The
experimental setup is introduced in Chapter 4, with detailed description of its major
components. In the following chapter, we start with the microscopic observations of
TNT deposited on nanostructured gold substrate and continue with the investigation of
the temperature dependence of the intensity of TNT SERS spectra. Similar study is also
performed for the 4-Nitrothiophenol adsorbed on a substrate and mercaptohexanol
(MCH), which is placed between the surface and the TNT molecules. In conclusions,
we summarize our results.
4
2 RAMAN SPECTROSCOPY
The phenomena of inelastic scattering of light was first predicted by Smekal in 1923
and first experimentally observed in 1928 by C.V. Raman and K.S. Krishnan in India
and, independently, by L. Mandelstam and G. Landsberg in the former Soviet Union.
Since then, this phenomenon is referred as Raman effect. In the original experiment,
sunlight was focused onto the sample by the telescope and the second lens, which was
placed by the sample, collected the scattered light. Using the system of optical filters,
they showed the existence of scattered light with a frequency different from that of the
incident light – the basic characteristic of Raman spectroscopy [2].
2.1 Energy units
Light is an electromagnetic radiation that can act like waves or like a stream of small
“packets” of energy called photons. This is also known as wave-particle duality. Figure
1 illustrates a wave of linearly polarized electromagnetic radiation propagating in the x-
direction. It consist of electric and magnetic field components, which always oscillate
in phase with each other and are perpendicular to one another to the direction of wave
propagation. Since the Raman effect does not involve the magnetic component, only
the former will be further discussed. The oscillation of electric field of strength (E) at a
given time (t) is expressed as [12]:
0 cos 2E E t , (1)
where 0E is the amplitude of the incident electric field and is the frequency of the
radiation. In EM radiation, the frequency and the wavelength , are inversely
proportional to each other [12]:
c
, (2)
where c is speed of light. In Raman spectroscopy, instead of the wavelength the
wavenumber is used and given by:
7-1 10
cmnmc
. (3)
5
Figure 1: Linearly polarized electromagnetic radiation [13].
A transfer of energy from electromagnetic radiation to the molecule occurs when
following condition is satisfied:
cE h h hc
, (4)
where E is the energy difference between two quantized states and h is Planck
constant ( 346.626 10 Js ). Thus, wavenumber is directly proportional to the energy
of the transition. For example, 1 eV corresponds to 1240 nm, 142.4 10 Hz, and
8065 cm-1 [12].
2.2 Degrees of freedom and molecular vibrations
Degrees of freedom describes the motion of the atoms in x, y, or z direction. An N-atom
molecule will therefore have a total of 3N degrees of freedom of motion. Three of these
degrees of freedom describe the translational motion of the molecule and three of them
describe the rotational motion of the nonlinear molecule about the three principal axis
of rotation, which go through the centre of gravity. A linear molecule only has two
rotational degrees of freedom, since rotation around its own axis is not considered a
degree of freedom of motion (no nuclei displacements are involved). After subtracting
translational and rotational degrees of freedom from total 3N degrees of freedom, the
net vibrational degrees of freedom (number of normal vibrations) is 3 6N for
nonlinear and 3 5N for linear molecule. This means that for diatomic molecule, we
will have only one vibration. In the case of H2O molecule, we have 3 3 6 3 normal
vibrations as shown in Figure 2. These modes of vibration are symmetrical stretch,
bending, and asymmetric stretch. The linear CO2 molecule has 4 modes of vibration.
6
However, the model discussed here has only three. The fourth mode is also bending
vibration but in a different plane as shown in Figure 2. Such pair of vibrations with the
same frequency, different only in their direction, are called doubly degenerate vibrations
[2, 12, 14].
Figure 2: Normal modes of vibration for CO2 (+ and – denote vibrations going upward
and downward, in direction perpendicular to the paper) and H2O. Based on [12].
For better understanding of molecular vibrations, which are responsible for
characteristic bands in Raman spectra, we consider a simple model of diatomic
molecule, as shown on Figure 3. Atoms, with masses 1m and 2m are connected with
chemical bond, which in this case can be regarded as massless spring, with force
constant, k. Displacement of atoms from their equilibrium position is 1x and 2x .
Displacement of each of the two masses varies periodically over the period of time as a
sine (or cosine) function. Atoms oscillate with different amplitudes, but with the same
frequency, thus both masses go through equilibrium position simultaneously. The
classical vibrational frequency for diatomic molecule is:
1 2
1 1 1
2k
m m
. (5)
It can be seen from the above equation that the frequency of diatomic oscillator is a
function of atomic masses of the two atoms, involved in the vibration and the force
constant k, which is a measure of bond strength between the two atoms. If the atoms are
connected with double or triple bond, the force constant will be bigger and consequently
the frequency will also be higher [1, 12].
7
Figure 3: Motion of a simple diatomic molecule [1].
2.3 Basic theory
The Raman effect is a light scattering phenomenon. When a monochromatic light of
energy 0h interacts with the molecule in a material, it can be scattered. The oscillating
electric field of light distorts (polarize) the electron cloud around nuclei and form a
“virtual’’ state, which is not necessarily a true quantum state of the molecule. Because
the state is not stable, the photon is quickly re-radiated [15].
In vibrational spectroscopy, the detected energy changes are those that require changing
the vibration of nuclei. The dominant scattering, also called the Rayleigh scattering, is
a process where only electron cloud distortion is involved. This scattering is referred as
elastic scattering, since the energy/frequency of the photon is the same as before
interacting with the molecule. On the other hand, Raman scattering is a weak process,
where only one in 106-108 of scatter photons are Raman scattered. This occurs when
incident light induce a change in the nuclear motion and energy will be transferred either
from molecule to scattered photon or from incident photon to molecule. This process is
referred as inelastic scattering, since the energy of scattered photons differs from that
of the incident photons. If a molecule at the ground vibrational state is excited by the
incident photon to the virtual state and relaxes to a higher vibrational excited state, the
molecule gains energy and the energy of scattered photon is smaller than that of incident
photon 0 mh . This process is called Stokes scattering. If the molecule is already
in an excited vibrational state due to the thermal energy, the scattered photon may gain
energy from the molecule 0 mh , leading to anti-Stokes scattering. Since most of
8
the molecules at room temperature are in the ground vibrational state, the majority of
Raman scattering is Stokes scattering [2, 15].
Figure 4: Diagram for Rayleigh and Raman scattering [16].
The classical description of Raman scattering can be explained by Eq. (6). As already
mentioned earlier, the oscillating electric field of light E interacts with a molecule and
distorts electron cloud, thereby inducing an electric dipole moment P in the molecule.
The magnitude of induced dipole moment P depends on the polarizability of the
molecule and the strength of the electric field E of the incident radiation. This can be
expressed as:
0 0cos 2P E E t . (6)
Polarizability is proportionality constant and can be described as the ease with which
molecular orbitals are deformed, by the presence of the external field. The more easily
the electron cloud of molecule is distorted, the bigger the polarizability and thus greater
the induced dipole moment of the given field. If the molecule vibrates with a frequency
m , the nuclear displacements q can be written as
0 cos 2 mq q t (7)
where 0q is the vibrational amplitude. Using a small amplitude approximation,
polarizability can be expressed as a linear function of displacement in the form of Taylor
series:
9
0
0q
(8)
Here 0 is the polarizability at the equilibrium position, and 0q
q
represents the
rate of change in polarizability with respect to the change in displacement from the
equilibrium position. If the derivative is equal to zero (no change in polarizability), the
vibration does not yield Raman scattering. Oscillations of polarizability cause the
induced dipole moment to oscillate at frequencies other than the incident frequency 0.
Combining Eq. (6) with Eq. (7) and Eq. (8), we obtain [12,15]:
0 0
0 0 0 0 0
0
0 0 0 0 0 0
0
0 0 0 0 0 0 0
0
cos 2
cos 2 cos 2
cos 2 cos 2 cos 2
1cos 2 cos 2 cos 2
2
q
m
q
m m
q
P E t
E t qE tq
E t q E t tq
E t q E t tq
(9)
The trigonometric identity 1
cos cos cos cos2
was used in the
final step of Eq. (9). According to classical theory, Eq. (9) demonstrates that the light
will be scattered at three different frequencies. The first term is the Rayleigh scattering
and represents an oscillating dipole which radiates light at the same frequency as the
incident light 0 . The second term corresponds to Raman scattering where oscillating
dipole radiates light at frequencies, which are different from the frequency of incident
beam that is 0 m (anti-Stokes) and 0 m (Stokes). The magnitude of these shifts
reflects the characteristic vibration of the molecule [12]. Some conclusions can be made
from Eq. (9):
1) As already stated before, if 0
0q
q
, the second term vanishes. The vibration
is not Raman active, since the molecular polarizability does not change during the
vibration.
2) If the vibration does not greatly change the polarizability, then the polarizability
derivative will be near zero and the signal from Raman scattering will be low.
10
Scattering intensity is proportional to the square of the induced dipole moment P,
which is proportional to the square of the polarizability derivative 2
q .
3) The equation also shows two possibilities to increase the Raman intensity. The one
is from the molecules with the larger polarizability and the other one is the stronger
electric field experienced by the molecules [17].
Thus, in Raman spectroscopy we measure the shift of the vibrational frequency m
from the incident beam frequency 0 . A Raman spectrum consist of scattered intensity
plotted vs. frequency shift between incident and scattered photons. The frequency shift,
also called Raman shift , is defined as [15]:
1 1
incident scattered
E
hc
(10)
where E is the energy difference between initial and final vibrational state of the
molecule. Raman shift is independent of wavelength of the incident beam incident , since
if we change incident , the wavelength of the scattered photons scattered changes in such
a way, that the remains the same.
As already mentioned in the beginning of this chapter, there are always more molecules
on the ground vibrational state than in the excited vibrational state at the room
temperature. This is why Stokes lines are much stronger than anti-Stokes lines. The
ratio of Stokes and anti-Stokes intensities depends on the population in ground and
excited vibrational states and can be obtained from the Boltzmann distribution [15]:
4
0
4
0
expmAS m
S Bm
I h
I k T
(11)
where Bk is the Boltzmann constant 231.38 10 J K . Measurement of this ratio can
also be used for temperature measurements. Since peaks from both lines are positioned
symmetrically with respect to the Rayleigh peak. Usual Raman spectrometers only
acquire Stokes spectra. A typical Raman spectrum, in this case CCl4, is illustrated in
Figure 5.
11
Figure 5: Raman spectrum of CCl4 [12].
The intensity of Raman scattering IR is given by [1]:
2
4
0RI I N tq
(12)
where is the frequency of the incident radiation, I0 is the intensity of the incident
radiation, N is the number of scattered molecules, is the polarizability of molecules,
q is the vibrational amplitude, and t is the acquisition time.
The above expression shows that increasing laser flux power or using shorter
wavelength excitation gives us a higher Raman intensity. However, since the molecules
usually have a bigger absorption cross section at lower wavelengths towards UV, the
fluorescence, which is a competing process and millions of times more efficient than
the Raman effect, is also higher and can thus overwhelm the Raman signal. Because of
such an inequality in signal strength, even the trace quantities of fluorescent materials
can mask the Raman signal of high-concentration analyte. This is why we usually use
excitation at longer wavelengths. Therefore, the wavelength selection is a balance
between minimizing the fluorescence and maximizing the signal strength [18].
12
2.3.1 Comparison of Raman and Fluorescence processes
Both Raman scattering and fluorescence produce photons with the frequencies different
from that of the incident photon, however, they are fundamentally different from each
other:
Raman scattering in a molecule is an instantaneous event in which an incoming
photon from the laser at 0 excites a molecular vibration m while emitting a
scattered photon at s o m . The incident photon does not need to be absorbed
and induce electronic transitions in the molecule, since Raman process can be
considered as an interaction with the ‘virtual state’, as depicted in Figure 4. Because
of this, Raman effects can take place at any frequency of the incident light, whereas
fluorescence is anchored at a specific excitation frequency.
Fluorescence, on the other hand is not an instantaneous, but a stepwise process. The
initial step involves the absorption of the incident light, where the system is
transferred from the ground single state S0 to a state in the vibrational substructure
of the first singlet state S1. The absorption process is shown in Figure 6a. Unlike in
Raman, the photon must have enough energy to reach S1 and start fluorescence
event. Once in the excited state, the molecule undergoes a series of vibrational
relaxations process, reaching the vibrational ground state of S1 (Figure 6b). After
certain amount of time (typically around few nanoseconds), the molecule relaxes
back to the vibrational levels of the ground state, thus emitting the photon (Figure
6(c)). In fluorescence, the emission process is completely independent of the initial
absorption, since both photons are not linked to each other in coherent and
instantaneous way like in Raman, where for each photon ‘taken’ from the laser,
there will be a scattered photon (one cannot exist without the other). However, in
fluorescence, there are situations where some potentially emitted photons from the
ground state of S1 go ‘missing’ (e.g. in non-radiative combination). Once the
molecule is excited to the S1 state, the best-case scenario is to ‘recover’ all the
photons that have been excited in the initial absorption process. However, small
fraction will usually be missing through a process that allows the molecule to relax
back to the ground state of S0 without emitting a photon. Therefore, the two
processes are effectively ‘disconnected’ in fluorescence (unlike in Raman) [19].
13
a) b) c)
Figure 6: Schematic presentation of fluorescence process as a sequence of events over
time. a) Fluorescence starts with the absorption of a photon. b) In the first electronically
excited state S1, the molecule undergoes vibrational relaxation and c) after few
nanoseconds, it relaxes back to the vibrational levels of the ground state S0, and thereby
emits a photon [19].
2.4 Polarizability tensor
To discuss Raman activity, we have to look more carefully at the polarizability . In
actual molecules, a nice linear relationship P E does not hold, since molecular
response to the applied electric field is not the same in every direction. Both P and E
are vectors, consisting of three components in x, y and z direction. Thus, Eq. (6) can be
written in the matrix form [12]:
x xx xy xz x
y yx yy yz y
z zx zy zz z
P E
P E
P E
. (13)
The first matrix on the right side is the polarizability tensor of second order. The tensor
is symmetric. The Raman scattering occurs when one of the components in
polarizability tensor changes during the vibration. For small molecules, it is easy to see
whether polarizability changes during the vibration. If we consider diatomic molecules
(e.g. H2) or linear molecules (e.g. CO2), the electrons are more polarizable (larger )
along the chemical bond than in direction perpendicular to it. Figure 7a shows changes
in polarizability from the vibrations of the CO2 molecule. Polarizability tensor is
graphically represented as the polarizability ellipsoid. This is a three-dimensional body,
whose distance from the electrical centre of the molecule is proportional to 1i, where
i is the polarizability in i-direction from the centre of gravity in all directions. When
14
xx yy zz polarizability ellipsoid will be a sphere and molecule is said to be
isotropic. For a completely anisotropic molecule, xx yy zz applies. The vibration
is Raman-active if the polarizability ellipsoid changes in its size, shape or orientation
and the intensity will depend on extent of this change. The 1 vibration is Raman-active
since polarizability changes in all directions. On the other hand vibrations 2 and 3
are Raman-inactive. Although in both cases the polarizability changes during the
vibration, the size and shape of the ellipsoid at +q and –q are identical by symmetry.
Note that the Raman activity is determined by the slope near the equilibrium position,
0q
q
(Figure 7b) [12].
a) b)
Figure 7: a) Changes in polarizability ellipsoid during three normal vibrations of CO2
molecule. b) The polarizability of CO2 as a function of displacement coordinate q for
1 and 3 vibrations (the function of the displacement is the same for 2 and 3 ) [12].
3 SURFACE ENHANCED RAMAN SCATTERING (SERS)
Surface enhanced Raman scattering (SERS) was discovered by Fleischmann and co-
workers in 1974 when they obtained an unusually strong Raman signal from pyridine
adsorbed on electrochemical roughened silver electrode. The reason to roughen the
electrode was to increase the surface area and thus the number of adsorbed molecules.
In 1977, Jeanmarie et al. and Creighton et al. confirmed the results and pointed out that
the Raman signal of molecules adsorbed on metal was enhanced by a factor of ~106,
15
compared to a signal from molecules in absence of metal. They reported that such
enhancement cannot be explained just by an increase in surface alone, but is also related
to an intrinsic surface enhancement effect. 40 years since discovery, improvements in
instrumental capabilities, better understanding of the enhancement effect and advances
in nanotechnology, made SERS spectroscopy a powerful analytical tool, used in various
fields, including physics, chemistry, and biology. It exploits the interaction of light,
molecules and metal nanostructured surface to enhance the Raman signal, in some cases
even to 14 orders of magnitude, thus allowing detection of single molecules.
The total enhancement is a product of two mechanisms, electromagnetic and chemical
or electronic enhancement. The dominant effect is electromagnetic enhancement (~104-
108, depending on the nanostructured surface) and is associated with magnification of
both incident and Raman-scattered fields, while chemical enhancement (≤102) arises
from electronic interaction between metal and adsorbed molecules. Both mechanisms
will be discussed in detail in the next chapter [20].
3.1 Electromagnetic Enhancement (EM)
The electromagnetic enhancement effect occurs at the metal-air interface. When
electromagnetic wave interacts with the metal surface, it causes collective oscillations
of the conduction electrons in metal - surface plasmons (SP). A plasmon is a quantum
of plasma oscillation. The plasmon can be consider a quasiparticle since it arises from
the quantization of plasma oscillations, just as phonons are quantizations of mechanical
vibrations. Thus, plasmons are collective oscillations of the free electron gas density,
for example, at optical frequencies. Surface plasmons are those plasmons that are
confined to surfaces and that interact strongly with light resulting in a polariton. If the
incident light is in resonance with plasmon frequency, the electromagnetic field at the
surface is enhanced. The electric field of SP can be expressed as
0
x zi k x k z tE E e
(14)
with for 0z , for 0z and where x is direction of propagation parallel to the
surface, z direction perpendicular to the surface, xk and zk are wave vectors
components along the x- and z-axis and is the frequency of the longitudinal
oscillation. The electromagnetic field disappears at z and is the strongest when
0z , which is typical for surface waves.
16
The solution of the Maxwell equations for the electric field from Eq. (15) at the metal-
dielectric interference with dielectric constants M and D yields the dispersion relation
SP of SP [21]:
M DSPSP
M D
c k
, (15)
where SPk is the wavevector of SP. The dielectric constant of metals is expressed as a
complex value ' ''
M M Mi . The real part '
M is associated with the polarizability of
incident light and imaginary part ''
Mi with the absorption. The complex function is
usually frequency dependent. In order to achieve the enhancing effect of the plasmons,
the electric field of the incident photon must oscillate parallel to the plane of the
incidence, so that its components lies in the direction of SP propagation. For a resonant
coupling of SP and photons, the energy E and the momentum p k has to be
conserved. The wavevector component of incident light ,Ph xk depends on the incident
angle and can be described with the help of the dispersion relation of the incident
photon Ph Ph
D
c k
, where Phk is a wavevector of an incident photon with the
following equation [21]
, sinPhPh x Dk
c
(16).
For resonance, the conservation of energy is given when Ph SP and the
following equation applies
, sin M DPh x D SP
M D
k kc c
(17).
The Eq. (17) relationship is shown graphically in Figure 8 [21].
17
Figure 8: a) Wavevectors components of incident photon, Phk and surface plasmons,
SPk , along a smooth metal surface. b) Dispersion relation of incident photon and surface
plasmons [based on 21].
Figure 8b shows that SP dispersion relation curve never intersects with the dispersion
relation line of a light in air with a dielectric constant, 1D . Consequently, on a
smooth metal surface, SP cannot be excited directly by just free-space light. The SP in-
plane wavevector is greater than that of incident light and since the momentum must be
conserved, the SP cannot radiate to the surrounding media. To ‘turn’ a light line to the
point, where it intersects with dispersion relation curve of SP to get a resonant effect,
and thus enhancement, the dielectric constant of the surrounding medium has to be
bigger than 1. This is usually achieved using a coupling medium such as prism.
Another possibility for resonant excitation of SP is roughening (usually
nanostructuring) the metallic surface to get a grating. In this way, ,Ph xk is matched with
SPk , by increasing the parallel wavevector component of the incident light with the
wavevector of the grating ,ph xk . Dispersion relation in this case is fulfilled by the sum
, ,
2sin sinPh x ph x SPk k n k
c c a
(18)
where n is the integer and a is the grating constant. , 0ph xk gives no solution to
dispersion relation. This is shown schematically on a Figure 9 [21].
18
Figure 9: a) Wavevectors components of incident photon Phk and surface plasmons SPk
along a nanostructured metal surface. b) Dispersion relation of incident photon and
surface plasmons [based on 21].
Due to the direction of SP, modes involving changes in molecular polarizability with a
component along the surface are the most enhanced. The most often used metals in
SERS are Ag, Au and Cu, since those materials have a negative real and small positive
imaginary dielectric constant (proportional to the damping of surface plasmons). Also
these materials fulfill the resonance condition in the visible or NIR frequency range. SP
can either be propagating in the x- and y-direction (~10-100 µm) along the metal-
dielectric interface and decay evanescently in the z-direction (~200 nm), or can be
localized on spherical particle for example. In the latter case, we talk about localized
surface plasmons (LSP). Since both, SP and LSP, are sensitive to the surrounding
dielectric environment, both are also used for SERS sensing experiments [8, 20, 22].
Figure 10: Illustration of the a) SP and the b) LSP [8].
A simplified schematic diagram for understanding the concept of electromagnetic SERS
enhancement is shown in Figure 11. The metallic ‘nanostructure’ is a small sphere with
the dielectric constant in a surrounding medium with a dielectric constant 0 . Since
19
the radius of the sphere is much smaller than the wavelength of light 0.05r , the
electric field is uniform across the particle and the Rayleigh approximation can be used
[23].
Figure 11: Simple schematic diagram for understanding the concept of EM SERS
enhancement.
A molecule in the vicinity of the sphere (distance d) is exposed to a field EM, which is
the superposition of the incident field E0 and the field of a dipole Esp induced in the
metal sphere, therefore 0M spE E E . The magnitude of the dipolar field Esp is given
as [23]:
3
0
0
02sp
rE E
r d
(19).
The field enhancement factor A is the ratio of the field at the position of the
molecule and the incident field
3
0
0 02
ME rA
E r d
(20).
A is particularly strong when the real part of the denominator is zero (i.e., real part
of is equal to 02 ). Additionally, the imaginary part of the dielectric constant
needs to be small for a strong electromagnetic enhancement. This condition describes
the resonant excitation of surface plasmons of the metal [23].
In an analogous way to the incident light, the scattered Stokes/anti-Stokes is enhanced.
Taking into account enhancement of the incident and the Stokes light, the
electromagnetic enhancement factor SG for Raman signals can be written as:
20
2 2 122 2 0 0
0 02 2
L S
em S L S
L S
rG A A
r d
(21).
The above equation shows that the enhancement scales as the fourth power of the local
field of the metallic nanostructure and that is particularly strong when both incident
light is in resonance with the surface plasmons and the inelastically scattered light is
close to this resonance [23].
The Eq. (21) also indicates that electromagnetic SERS enhancement is a long range
effect, which means that the adsorbate is not required to be in direct contact with the
surface of the metal. Enhanced EM fields generated by the surface plasmon resonance
(SPR) enables the detection of molecules even few nanometers from the surface of the
substrate. Long range effect of EM mechanism differs from the chemical enhancement
mechanism, where molecules have to be in direct contact with the surface. The detection
of the molecules nearby to the surface, which are not necessarily bound to it, can be
very useful in SERS applications, since many analytes have low or no affinity to Ag or
Au. In such cases, the surface can be modified with adlayers to improve specificity of
the analytes. The field enhancement around metal sphere decays with the growing
distance, described by the decay of the field of a dipole over the distance 3
1 d , as
shown in Eq. (20), to the fourth power, resulting to the 12
1 d (see Eq. (21)) [8].
The power of SERS signal is proportional to the following parameters:
2 2 R
SERS L L S adsP N I A A (22).
The power of the Raman signal SERSP depends on the number of molecules N, laser
intensity IL, enhancement factors of excitation LA , the scattered field SA , and on
the Raman cross section of the adsorbed molecule R
ads [20].
The increase in Raman intensity when the molecule is adsorbed on a SERS active
substrate is described by the enhancement factor (EF). The average EF for a SERS
system, which is evaluated at the single excitation frequency and the same acquisition,
is given as
SERS surf
NRS vol
I NEF
I N
(23)
21
where SERSI is a surface-enhanced Raman intensity, surfN is the number of molecules
adsorbed to the metallic surface that contribute to the SERS signal, NRSI is the intensity
of normal Raman scattering and the volN is the number of molecules in the excitation
volume [24].
The SERS enhancement factor also strongly depends on the orientation of the adsorbed
molecules with the respect to the metal surface. Enhancements are stronger if the
vibrations of the adsorbate are parallel to metal surface.
3.1.1 Hot spots
Hot spots are highly localized regions of intense local field enhancement, which are
caused by local surface plasmon resonances (LSPR). Hot spots occur when intense
electromagnetic field of two nanostructures superimpose. Nanostructures can be
nanoparticles or structures with a gap in-between. This phenomenon is strongly
dependent on the excitation wavelength, particle size, shape, and separation as well as
arrangement with respect to the polarization direction of the incident light. Many
articles in literature describe hot spots created between two nearby nanoparticles (gap
junctions) that line up their induced field with the external field. In other words, the
incident field induces two in-phase dipoles along the direction of the incoming field. If
d a , where d is the distance between particles and a is a particle diameter, the near-
field interactions will dominate and fall according to 3d . Therefore, in order to create
extremely high field confinements (hot spots), it is important to produce very small (few
nanometers) gap junctions, i.e. d a . When optical excitation is localized in such
small area, extremely large electromagnetic SERS enhancement up to 1210 can be
generated, thus allowing observations of single molecules [23, 25].
3.2 Chemical mechanism
Chemical or electronic SERS effect is a common name for different mechanisms, which
require direct contact between molecule and metal surface. Early researchers found out
that the SERS intensities between molecules of CO and N2 differ by a factor of 200
under the same experimental conditions. This result was very hard to explain just with
electromagnetic mechanism, since the polarizabilities of the molecules are nearly
22
identical and even the most radical variation in orientation upon adsorption could not
produce such large differences [26].
Chemical enhancement mechanism can be explained by the electronic coupling
between metal and a molecule, adsorbed on the metal surface. This adsorbate-surface
formation produces an increase of Raman cross section of the adsorbed molecule,
compared to the cross section of molecules in ‘normal’ Raman experiment. Other
possible explanation involve resonance Raman effect, which can occur due to shifted
and broadened electronic levels in the adsorbed molecule (compared to the free one) or
due to the new electronic transition in metal-molecule system. The later occur through
photon driven charge transfer process (PDCT) between metal and adsorbates, which is
shown in Figure 12, where HOMO and LUMO denote for the highest occupied
molecular orbital and the lowest unoccupied molecular orbital of the adsorbate,
respectively. The energies of HOMO and LUMO are approximately symmetric relative
to the Fermi level of the metal. The whole process can be described by the following
four steps [23, 26]:
Step 1: An electron-hole pair of the metal is created by the incident photon with energy
0h and the electron is excited to the hot-electron state.
Step 2: So-called ‘hot’ electron tunnels into the LUMO of the adsorbed molecule,
generating a charge transfer excited state.
Step 3: ‘Hot’ electron tunnels from LUMO (with changed normal coordinates of some
internal molecular vibrations) back to the metal.
Step 4: The electron recombines with the hole created in the step 1, which leads to a
vibrationally excited neutral molecule and to a emission of a Raman shifted photon,
with the energy h .
23
Figure 12: Schematic diagram of the photon-driven charge transfer model for a
molecule adsorbed on a metal [27].
Chemical enhancement mechanism is a short-range effect (0.1–0.5 nm), limited only to
first layer of adsorbed molecules. It depends on the geometry of bonding, the adsorption
site and the energy levels of the adsorbate molecule. The contribution of chemical
enhancement to the SERS intensity is estimated to be approximately 10-102. However,
is generally agreed that the electromagnetic enhancement is significantly larger in
magnitude. Although the chemical enhancement is not the general mechanism and is
restricted only to specific adsorbate-metal systems, it can still provide us useful
information on chemisorption and hence interactions between adsorbate and metal [20].
SERS spectrum can show some deviations in relative intensities compared to a normal
Raman spectrum of the same molecule. Interactions between molecule and metal may
cause, that the Raman lines are slightly shifted in frequency and changed in line width
compared with a ‘free’ molecule. Despite small changes, which can occur in SERS
spectrum, it still provides us a very clear ‘fingerprint’ of the molecule [23].
4 EXPERIMENTAL SECTION
4.1 Raman setup
The Raman setup used in our experiments consists of four major components:
excitation source (laser), light focusing and collecting system (in our case a fiber optic
probe head), spectrometer and CCD detector. A schematic of the apparatus is shown in
Figure 13.
24
Figure 13: Schematic presentation of major components in our Raman setup [28].
The SERS spectra were collected with a standard system (Kaiser Optical System Inc.,
Ann Arbor, MI, USA); the 785 nm (linewidth 0.06 nm) GaAlAs diode laser (Invictus,
Kaiser Optical Systems, Inc.) beam was focused onto the sample. The excitation and
scattered light were guided onto/from the sample by a multimode optical fiber equipped
with the probe head. The incident power of the laser emission was about 100 mW at a
probe head for 5 s recording with 1 accumulation on the detector. The scattered light
was coupled into the optical fiber by a confocal aperture and guided to the spectrograph
(PhAT SystemTM, Kaiser Optical Systems Inc.), which uses Volume Phase Holographic
(VPH) transmission gratings to perform filtering and dispersion functions. Prior to
entering the spectrograph, the scattered light goes through a holographic notch filter,
which cuts off photons at the laser frequency (i.e. Rayleigh scattering). The diffracted
light was recorded with a CCD camera (iDus, Andor Tecnology plc.) with a spectral
resolution of 5 cm-1. The most important prerequisite when comparing Raman shifts is
the reproducibility (or repeatability) of the experiment. Kaiser Optical Systems provide
a Raman shift tolerance between ± 0.5 and ± 1.0 cm-1, the individual system
performance will not vary to this extent. Upon calibration, a system should yield Raman
shift values reproducible to ± 0.1 cm-1 [7].
25
Figure 14: Front view of the Raman spectrometer.
4.2 Laser
Lasers are ideal excitation sources for Raman spectroscopy, since they provide a
monochromatic light with narrow bandwidth and high intensities necessary for
generation of a sufficient amount of Raman scattered photons. In addition, laser beams
have a small spot diameters that can be further reduced using optical lenses (smallest
possible diameter is approximately equal to the laser wavelength) for higher photon flux
at the measurement zone. Since the scattering intensity scales with the fourth power of
laser frequency (Eq. (12)), the most logical thing for improving Raman sensitivity
would be to use highest possible frequency. However, the problem that arises with the
use of high frequencies (or short wavelengths) is the emergence of fluorescence, which
can cover the Raman signal. The presence of the fluorescence can be reduced by using
longer wavelengths. Nowadays, the most common light sources for Raman
spectroscopy are diode lasers operating at near-infrared (NIR) wavelength.
Fluorescence at such wavelengths is not completely absent, but it is significantly
suppressed. The Raman intensity is however weaker, since the energy of radiation is
lower and the fourth power law applies.
We used a continuous wave (cw), Invictus 785 nm NIR diode laser, with a maximum
output power of 450mW. It is rated as class IIIb laser, meaning, that eye damage can
occur upon direct exposure to the laser beam. This class applies for laser with no more
than 500 mW of radiant power. It uses external cavity design to provide a narrow
26
linewidth and excellent wavelength stability. The Invictus laser also has integrated
holographic bandpass filter, which rejects any spontaneous emission from the diode that
is not at the lasing wavelength [29].
The probe head uses non-contact optics, which are optimized for incident NIR radiation.
The working distance is 1 cm, while the aperture ratio is f/2.0 [30].
4.3 Spectrometer and CCD camera
The spectral separation of Raman scattered light was performed using PhAT SystemTM
spectrograph from Kaiser Optical Systems Inc. Instead of a classical surface relief
reflection grating (usually in Czerny-Turner design) (Figure 15a) as dispersive element,
the PhAT SystemTM spectrograph uses Volume Phase Holographic (VPH) transmission
grating to perform filtering and dispersion functions (Figure 15b). VPH grating is made
from a layer of transparent material, usually dichromated gelatin, which is sandwiched
between two layers of clear glass or fused silica. When the light passes through the
optical thin film that has a periodic differential hardness or refractive index, its phase is
modulated. Hence the term ‘Volume Phase’. This is the biggest difference in
comparison to conventional reflection gratings, where the phase of the incident light is
modulated by the depth of a surface relief patterns. As in the conventional reflection
gratings, the spectral dispersion or angular separation of wavelength components in
diffracted light is determined by the spatial frequency of the periodic structure [31].
a) b)
Figure 15: a) Classical surface relief reflection grating. b) VPH transmission grating
[31].
Spectrally separated light is collected with the iDus DU420-BRDD charge-coupled
device (CCD) camera, which consists of rectangular two-dimensional arrays of 1024 ×
27
256 photosensitive elements (pixels). The pixel site is 26 × 26 µm, while the image area
is 26.6 × 6.6 mm. For readout, the detector uses so-called ‘Full Vertical Binning (FVB)’
method. Collected signal is converted into a 16-bit grayscale image. The operation
temperature of the camera was at 66 °C.
The silica based CCD sensors of the DU420-BRDD CCD camera have the highest
quantum efficiency in the NIR region (Figure 16). Since we used lasers with an
excitation wavelength of 785 nm, our spectral range of interest ranges from 785-915
nm, which corresponds to approximately 1800 cm-1 (Eq. (10)). In this region, quantum
efficiencies from 50 to 90% can be achieved. Figure 16 also shows a decrease in
quantum efficiency for wavelengths above 750 nm. The reason for the decrease lies in
the wavelength dependant absorption of photons in silica, which is why excitation
wavelengths longer than 785 nm can be unfavourable for detection with a CCD camera,
since weak Raman signals cannot be detected [21].
The so called “deep depletion“ technologies enables high quantum efficiencies in the
NIR. Devices manufactured with this technology have thicker photosensitive silicon
layers, which offer longer absorption path to photons with longer wavelength and thus
increase the probability for creation of excited electron-hole pairs [21].
Figure 16: Quantum efficiency of iDUS DU420-BRDD CCD camera [adapted from
21].
28
4.4 Plasmonic substrate
All SERS spectra were recorded on a commercially available nanostructured gold
substrate (Klarite®, Renishaw Diagnostics). The size of the substrate is 6 mm 10 mm,
with an active area of 4 mm 4 mm. The active area of the substrate consists of gold-
coated periodic square lattice of inverted pyramid pits (~1.4 μm wide and ~1 μm deep),
shown on Figure 17. The pyramid pits were produced using conventional optical
lithography on a (100) oriented silicon wafer followed by an anisotropic chemical
etching [6]. The substrate was opened from a vacuum-sealed package just prior to
experiment, to prevent any possible surface contamination.
Figure 17: SERS substrate with visible active area (left) and scanning electron
microscope images of the nanostructured gold surface (middle), and inverted pyramid
pits (right) [6].
The substrate was heated with Peltier element, with the size of 25 × 25 mm and
maximum working temperature of 138 °C.
4.5 Microscope
TNT solution on the SERS substrate was examined by Carl Zeiss El-Einsatz Axioskop
microscope We used a 100× microscope objective, with the numerical aperture of 0.90
and dark field illumination technique. Images were taken by the Lumenera’s
INFINITY2-2 digital CCD camera.
29
Figure 18: Carl Zeiss El-Einsatz Axioskop microscope.
4.6 Sample preparation
In SERS measurements, it is essential that the molecules are delivered to close
proximity of a metal surface. The molecules were analysed as thin films by dropping a
small volume of solution on the substrate. We used TNT solved in methanol/acetonitrile
(1 mg/ml), which was purchased from AccuStandard, Inc. (New Haven, USA). TNT is
a nitroaromatic compound (Figure 19a), which is mostly used for military and industrial
explosives applications. The melting point of TNT is at 80 °C and is thus far below the
temperature at which it will spontaneously detonate. It is also relatively insensitive to
shock and friction and the explosive cannot be initiated without a detonator.
Self-assembled monolayers of 4-Nitrothiophenol (technical grade from Sigma-Aldrich)
were generated by soaking the substrates in a 13 mM nitrothiophenol / ethanol (p.a.
from Sigma-Aldrich) solution for 24 h (Figure 19b). Before SERS measurements, the
substrate was rinsed with ethanol and left to dry in air for 10 min.
In the same way, by soaking the substrate in a 1 mM mercaptohexanol (MCH) / ethanol
solution for 24 h, was also made a self-assembled monolayer of MCH (technical grade
from Sigma-Aldrich) (Figure 19c).
30
a) b) c)
Figure 19: a) Chemical structure of TNT, b) 4-Nitrothophenol and c) MCH adsorbed
on a gold surface [6].
4.7 Data analysis
When recording a Raman or SERS spectra, in addition to the signal from the sample we
also detect some background noise, which can alter the profile of Raman bands.
Background noise can emerge because of different reasons; from not-fully suppressed
Rayleigh light from the laser, fluorescence or stray light. Because of this, background
subtraction or so-called baseline correction (Figure 20) is performed for each TNT
SERS spectrum. To perform a baseline correction it is necessary to create a baseline
based on a recorded spectrum, which is then subtracted from the spectrum. The baseline
was calculated with the help of local and global minima. Here, spectrum is divided into
intervals and in each of these intervals local, and global minima are searched. Per
interval, we have one support point or a node. These nodes are connected together with
the cubic spline, resulting in a baseline, which can be seen as a red curve in the Figure
20. The baseline is then subtracted from the recorded spectrum and we get the correct
TNT spectrum without a background noise (blue line in Figure 20).
Baseline subtraction on all spectra was performed with Origin 9.0.
31
Figure 20: Principle of the baseline correction. From a recorder TNT spectrum (black)
we subtract baseline (red), calculated on the basis of a local on global minima. Result
is a TNT spectrum without a background noise (blue).
5 RESULTS AND DISCUSSION
5.1 Microscopic observations
TNT diluted in methanol/acetonitrile was dropped on a nanostructured gold surface.
Even at small volumes, the solvents spread across the surface and evaporate, leaving
molecules adsorbed on the surface. Before and after the SERS measurements, in which
the substrate was heated to 60 °C, we examined the nanostructured surface with the
microscope. One corner of the substrate was chosen to make sure we would observe the
same area during the experiments. Figure 21a shows the nanostructured surface with
inversed pyramid pits after depositing a drop of solution on it. The border to the
unstructured area is also clearly seen. At first, it appears that observed spheres on the
surface could be liquid bubbles of the solution, but after being stable for 30 minutes, we
can conclude that all volatile solvents already evaporated. We assume that the flattened
spheres are pure TNT crystals. This agrees quite well with the SERS measurements
recorded immediately after microscope observations, which did not show any signs of
32
methanol or acetonitrile in the spectra. The distribution of TNT crystals on this substrate
is heterogenic and the crystal sizes are in the range from ~2 µm to ~12 µm.
Figure 21b shows the microscope image after heating it up to 60 °C and then cooling it
back to 20 °C. Almost all of the bright spheres on a substrate have disappeared and dark
patches are seen on the gold surface, where the large bright spheres were located before.
The smaller bright spheres disappeared completely, indicating evaporation of the TNT
microcrystals.
Figure 21: Microscope images of a solution on the nanostructured gold substrate a)
before and b) after heating to 60 °C.
5.2 SERS measurements of TNT
After the microscope examination, we immediately started the SERS measurements.
The temperature dependence of the intensity of the TNT Raman bands was studied. We
started at 20 °C and continued to 60 °C in 5 °C intervals. The acquisition time was 5 s.
A typical SERS spectrum of TNT is shown on Figure 22. The characteristic bands of
TNT are in the range between 200-1800 cm-1. Moreover, we observed peaks in the
region around 3000 cm-1, which can be assigned to different C-H vibrations.
At each temperature, the SERS spectrum was obtained for three random spots on the
substrate surface (Figure 23). Different intensities of the TNT bands for different spots
can be explained by the heterogenic distribution of TNT crystals in the excitation focus
on the nanostructured surface. Each spectrum for a given temperature is the average of
three spectra at different locations on the surface.
33
Figure 22: SERS TNT spectrum at 20 °C.
Figure 23: Three SERS spectra of TNT at 20 °C, obtained at three random spots on the
substrate surface.
34
TNT can be easily identified by the vibrational modes at the following frequencies: 323
cm-1 (2,4,6 C-N in plane torsion, ring in-plane bend), 792 cm-1 (ring in-plane bend, C-
CH3 stretch), 824 cm-1 (nitro group scissoring mode), 1207 cm-1 (ring breathing), 1356
cm-1 (4-NO2 symmetric stretching, C-N stretch), 1542 cm-1 (NO2 asymmetric
stretching) and 1616 cm-1 (phenyl modes) [32].
TNT SERS spectra from 20 °C to 60 °C are shown in Figure 24. Due to the temperature
change of the SERS substrate, a red-shift of the position of the frequencies up to 12 cm-
1 arises between SERS spectra of 20 °C and 60 °C. In Figure 25, the behaviour of the
most dominant band of the TNT spectrum at 1356 -1cm (NO2 symmetric stretching
vibration) is shown. The intensity decreases by a factor of 5. In the following,
contributions of evaporation, phase transition, decomposition, and temperature
dependence of the SERS effect are discussed on the base of the microscopy and SERS
results.
Figure 24: SERS spectra of TNT from 20 °C to 60 °C in 5 °C interval.
35
Figure 25: Intensity of the dominant TNT band (1356 cm-1) at different temperatures.
5.2.1 Evaporation
Evaporation of TNT molecules from the surface is a major contribution for the decrease
of the signals, as indicated by the microscopic images, in which some of the shiny
spheroids are disappeared after the heating. However, evaporation could not explain
dark patches and the changes in the spectra.
5.2.2 Phase transition
The change of the shiny TNT crystals to dark patches (Fig. 21) can be explained by a
phase transition. The intensity of the band at 1356 cm-1 decreases between 20 and 60
°C (Figure 25). Above 35 °C the decrease in intensity is steeper than at lower
temperatures, whereas above 55 °C the intensity appears to be constant. The melting
point of bulk TNT is at 80 °C. However, the melting point is size-dependent. Therefore,
the drop of the melting point can be explained by the small size of the TNT crystals on
the surface [33]. Moreover, volatile solvents can also have some effect on crystallization
of TNT, since the crystals could be formed differently, which may affect their quality,
resulting also in a melting point depression. Considering the heterogenic size
distribution of TNT crystals and their size dependent melting points, the temperature
dependent behaviour of the SERS intensities resembles conceivably a sigmoidal
36
melting curve, supporting the phase transition. The change of the shiny crystals and the
sigmoidal curve shape of the temperature dependence indicate melting of the TNT
crystals upon the heating. What exactly are these dark patches and in which phase are
they, is unknown.
5.2.3 Decomposition
Decomposition of the molecules is an issue in SERS spectroscopy, in particular as the
laser beam is focused on a small area at the surface. However, we have not observed
decomposition of TNT on such a substrate at room temperature under these conditions
(laser power, acquisition time) before. After heating the substrate to 60 °C, we cooled
it back to 20 °C and recorded a spectrum. The comparison of SERS spectra at 20 °C
before and after heating to 60 °C is shown in Figure 26. The heating and recording of
SERS spectra result in a non-reversible chemical process.
Figure 26: Comparison of TNT spectra at 20 °C at the beginning of SERS measurements
(black) with spectra at 20 °C, after cooling it down from 60 °C (red).
Characteristic TNT bands at 323 cm-1, 792 cm-1, 824 cm-1, 1207 cm-1 and 1542 cm-1
have totally vanished. The intensity of the dominant band at 1356 cm-1 has dropped to
approximately 24% of the original value and has also shifted by 12 cm-1 to a lower
37
frequency. Moreover, the band at 1617 cm-1 is shifted to 1607 cm-1. In contrast, the band
at 1128 cm-1, which had a low intensity at the beginning of the measurements, increases
for about four times. This clearly indicates that we can observe newly generated
unknown chemical species. A simple elimination of a nitro group which would
decompose TNT to DNT could not be explanation, as DNT can be identified by two
characteristic bands at 834 cm-1 and 1327 cm-1 [34]. Consequently, it looks like that
melting of the crystals is followed by the decomposition of the TNT molecules by
heating the plasmonic surface.
5.2.4 Temperature dependence of the SERS effect
Temperature dependence of the SERS effect could be further explanation for the
decreasing intensities in the SERS spectra. Pang et al. have published a study about the
temperature dependence of the SERS effect [35]. These authors found a decrease of the
SERS effect with rising temperatures. From 15 K to 300 K the enhancement drops by a
factor of approximately 3. To verify the impact of the temperature dependence of the
SERS effect, we adsorbed 4-Nitrothiophenol on the surface. These molecules form a
covalent gold-sulfur bond with the nanostructured substrate leading to a defined and
stable monolayer. Thus, evaporation and phase transition effects should not be relevant.
Spectra were recorded from 20 °C to 80 °C in 5 °C intervals; spectra were taken at three
different positions on the surface. Figure 27 shows SERS spectra of 4-Nitrothiophenol
between 20 °C and 80 °C.
The characteristic bands of 4-Nitrothiophenol are at 1081 cm-1 (C─S stretching
vibration), 1343 cm-1 (N─O symmetric stretching mode) and 1570 cm-1 (C═C
stretching mode of benzyl ring) [36,37]. The position of the most dominant peak shifts
slightly from 1344 cm-1 to 1342 cm-1 (Figure 27 inset).
The temperature dependence of the intensity of this band is shown in Figure 28. The
behaviour can be described by a linear decrease (Figure 28 inset). The overall decrease
in intensity between 20 °C and 80 °C is approximately 11 %. Besides this small drop in
intensity, we cannot find any significant changes in the spectra. The small decrease
could nevertheless be a consequence of desorption of the molecules caused by the
heating. On the other side, the drop can also be caused by the temperature dependence
of the SERS effect. However, the effect is small compared to the drop of the TNT
intensities by heating. It may contribute to rather small extent to the decrease. Moreover,
38
we can conclude that the used SERS substrate is suited for applications up to 80 °C.
That means that detection by SERS at elevated temperature is possible.
Figure 27: SERS spectra of 4-Nitrothiophenol recorded at 20 °C (black), 50 °C (red)
and 80 °C (blue).
Figure 28: Intensity of the dominant 4-Nitrothiophenol band at 1343 cm-1.
39
5.3 TNT solution deposited on the substrate covered with
mercaptohexanol (MHC) monolayer
MCH was adsorbed on a gold surface, where the molecules formed a defined and stable
self-assembled monolayer, similar as with 4-Nitrothiophenol. Then we dropped the
TNT solution on top of the formed monolayer. The main idea of inserting a layer of
MCH between the gold surface and the TNT was to diminish the evaporation of the
TNT molecules by the formation of hydrogen bonds between hydroxyl group of MCH
and the nitro group of the TNT.
Before the SERS experiment, we have taken microscope images with just MCH
monolayer adsorbed on the surface. As seen on Figure 29a, the MCH monolayer is not
visible and we can see just a nanostructured gold surface with some impurities on it
(bright yellow dots). After measuring the spectra of the MCH at 20 °C, we dropped the
TNT solution on top of the monolayer (Figure 29b). In comparison to Figure 21a, there
are not visible any TNT crystals in the shape of the flattened spheres. Noticeable are
just dark patches on the surface. Since on the planar gold surface area, TNT crystals are
clearly seen, we can assume that the added monolayer of MCH had an effect on the
adsorption of the TNT molecules on the surface. The formation of dark patches was
also observed after heating the uncoated substrate. Obviously, the heating and the MCH
layer cause the formation of a non-crystal phase of TNT molecules on the surface. The
substrate was then heated to 60 °C. Figure 29c shows a nanostructured surface after
cooling it back to 20 °C. Dark patches that were visible before heating are now
disappeared, which indicates that the TNT molecules evaporate from the surface.
a) b) c)
Figure 29: Microscope images of a) MCH monolayer and b) MCH monolayer with
added TNT on it, before heating to 60 °C. c) MCH monolayer with added TNT after
cooling it back to 20 °C.
40
Figure 30 shows comparison of a spectrum of TNT dropped directly on the surface of
the substrate (red) and spectrum of TNT, dropped on the MCH monolayer (black). Both
are measured at 20 °C with acquisition time of 5 s. Intensity of the most dominant band
of TNT, which is dropped on the MCH, is lower by approximately factor of 6. Decrease
in intensity because of MCH layer was expected, as the SERS effect depends strongly
on the distance between the molecules and the surface. Moreover, the MCH layer effects
the position of the TNT main bands. These are slightly (0-2 cm-1) shifted to the lower
frequency, which indicate interaction between the TNT-Au surface and TNT-MCH
layer.
Figure 30: Comparison of TNT spectra, where TNT was dropped directly onto the
surface (red) and onto the MCH monolayer (black).
Figure 31 contains the spectra of MCH (red) and TNT deposited on MCH layer before
(black) and after (blue) heating to 60 °C. Acquisition time was 30 s. Interestingly, the
signal from MCH layer is completely hidden under both of TNT spectra, since its bands
are hardly visible in TNT spectra, before and after heating. The most dominant peak is
around 1100 cm-1 and is attributed to the C-C stretching vibration. MCH forms a stable
and defined monolayer, in which the molecules are adsorbed in almost perpendicular
orientation to the surface. This might explain the low intensity of the band, as vibrations
oriented perpendicular are less enhanced by the SERS effect in contrast to parallel
41
oriented vibrations. Developing this argument further, the TNT should lay flat on the
MCH layer. A flat orientation is also assumed for the uncoated nanostructured surface,
which is in line with a similar intensity distribution of the Raman bands.
In conclusion, MCH does not prevent evaporation of the TNT molecules. The intensity
of the TNT band decreased with increasing distance from the plasmonic surface caused
by the spacer MCH. However, the MCH layer changed the adsorption of the TNT
molecules.
Figure 31: SERS spectra of MCH monolayer (red) and TNT deposited on MCH layer
before (black) and after (blue) heating to 60 °C. All spectra were recorded with
acquisition time of 30 s at 785 nm excitation.
6 CONCLUSIONS
Detection of trace amount of explosive materials such as TNT has recently gained the
highest importance in homeland security, environmental safety, and protection. The
surface of enhanced Raman scattering has become powerful technique for trace analysis
with its high sensitivity. It exploits the interaction of light, molecules and metallic nano-
scale roughened substrate, which greatly enhance the Raman signal.
42
In this master thesis, we have studied the influence of heating the plasmonic substrate
on the SERS spectra of the adsorbed TNT molecules. TNT in solution was deposited
on a nanostructured gold surface. After evaporation of the solvent, small TNT crystals
were formed in the shape of the flattened spheres. After heating the substrate to 60 °C
a part of TNT crystals disappeared, which agrees with the intensity decrease of the most
dominant TNT band, indicating evaporation of TNT.
However, evaporation cannot explain all of our observations. The change of the shiny
spots to dark patches in the microscopic images and the sigmoidal decrease of SERS
intensities of the dominant TNT band indicate melting of the TNT crystals by heating.
Finally, the changes in the SERS spectrum at 60 °C to the spectrum at 20 °C cannot be
explained solely by evaporation and melting. Disappearance of characteristic TNT band
and appearance of new band also points to the decomposition of TNT to other chemical
species. Moreover, we have studied the temperature dependence of the SERS effect.
Therefore, 4-Nitrothiophenol was chemisorbed to the nanostructured substrate. The 4-
Nitrothiophenol SERS spectra showed only a small decrease of the intensities,
indicating that if ever, the decrease of the enhancement up to 80 °C is small. In order to
reduce the evaporation of the TNT from the substrate, we immobilized a self-assembled
monolayer of mercaptohexanol (MCH) on the plasmonic surface. The MCH should
bind the TNT molecules stronger to the substrate. We indeed find a change in the
adsorption. The TNT does not form crystals on the MCH layer and the SERS signals
of TNT molecules are significantly reduced. However, upon heating the substrate the
SERS signal decrease again significantly, indicating that MCH cannot prevent the
evaporation of the TNT molecules.
The study shows that SERS at slightly elevated temperatures is still possible. The
enhancement of the plasmonic substrate is hardly affected up to 80 °C. However,
heating of small amounts of substance can lead to melting and decomposition. Even
when the bulk material is stable under such conditions. Explosive detection by SERS is
a field, in which these results should pave the way for new devices.
43
7 RAZŠIRJENI POVZETEK V SLOVENSKEM JEZIKU
Z naraščajočim številom terorističnih napadov v zadnjih letih je detekcija eksplozivov
in njim sorodnih spojin postala zelo pomembno področje. Ramanska spektroskopija je
že uveljavljena metoda pri analitičnih in forenzičnih raziskavah, saj je večino
eksplozivov mogoče identificirati na podlagi njihovih ramanskih spektrov, ki
predstavljajo kemijski ''podpis'' molekul.
Ramansko sipanje je neelastično sipanje svetlobe oziroma fotonov. Ko monokromatska
svetloba z energijo 0h interagira z molekulami v snovi, se lahko siplje. Pri tem
molekula iz osnovnega energijskega stanja preide v virtualno (navidezno) stanje, ki ni
pravo kvanto stanje molekule, zato se le-ta hitro vrne v (največkrat) osnovno stanje, pri
čemer emitira (sipa) foton. Običajno je frekvenca sipanega fotona enaka frekvenci
vpadnega, zato govorimo o elastičnem ali Rayleighovem sipanju, kjer se vibracijska
energija molekule ne spremeni. Pri majhnem deležu sipanih fotonov (en na 10
milijonov) pa je energija različna od vpadne. V tem primeru govorimo o neelastičnem
sipanju. Če molekula iz virtualnega stanja ne pade nazaj na osnovno stanje, ampak
zasede višje vibracijsko stanje, govorimo o Stokesovem sipanju. Vibracijska energija
molekule se poveča za razliko energije vpadnega in sipanega fotona 0 mh . V
primeru, da je pred sipanjem molekula že v vzbujenem vibracijskem stanju, sipani foton
pridobi energijo molekule 0 mh , kar vodi do anti-Stokesovega sipanja. Gledano
iz vidika klasične mehanike, nihajoče električno polje svetlobe deformira elektronski
oblak okoli jedra molekule, s čimer se inducira električni dipolni moment v molekuli,
ki je enak produktu polarizibilnosti molekule in jakosti električnega polja.
Polarizabilnost je odvisna od medatomskih razdalj, ki pa se zaradi nihanja molekul
nenehno spreminjajo. Polarizabilnost tako periodično niha s frekvenco nihanja
molekule. Zaradi pospešenega gibanja elektronov nihajoči električni dipolni moment
seva. Tako dobimo svetlobo, katere energija oziroma frekvenca se razlikuje od
energije/frekvence vpadne svetlobe.
Zaradi šibkosti signala pa ramanska spekstroskopija ni primerna za detektiranje zelo
majhnih količin vzorcev. Ramanski proces lahko znatno ojačamo z uporabo metode
površinsko ojačenega ramanskega sipanja (ang. surface enhanced Raman scattering –
SERS) tako, da molekule nanesemo na hrapavo (običajno nanostrukturirano) kovinsko
površino. SERS je posledica dveh mehanizmov, elektromagnetnega in kemičnega.
44
Slednji izhaja iz elektronskih interakcij med kovino in adsorbirano molekulo (≤102).
Večinski delež pri ojačitvi prispeva elektromagnetni mehanizem (~104-108), ki je
povezan z ekscitacijo lokalnih površinskih plazmonov in s tem ojačitvijo tako vpadnega
kot ramansko sipajočega elektromagnetnega polja.
Uporabljen ramanski merilni sistem je bil sestavljen iz štirih glavnih komponent: laserja
(785 nm), optičnega vlakna za fokusiranje in zbiranje sipane svetlobe, spektrometra in
CCD detektorja. V magistrskem delu smo raziskovali vpliv temperature plasmonskega
substrata na SERS spekter adsorbiranih TNT molekul. TNT, razredčen v
metanolu/acetonitrilu smo s pipeto nanesli na nanostrukturirano pozlačeno površino. Po
izhlapetju topila so se na površini substrata formirali majhni kristali TNT-ja, v obliki
sploščenih kroglic. Pred in po koncu SERS meritev smo na enakem mestu na
nanostrukturirani površini izvedli mikroskopsko analizo. Po opravljenih SERS
meritvah, kjer smo segrevali substrat od 20 °C do 60 °C s 5 °C intervali, večina TNT
kristalov izgine. To se ujema z intenziteto najbolj dominantne ramanske črte TNT, ki
se zmanjša za faktor 5, kar nakazuje na izhlapevanje. Vendar pa samo izhlapevanje ne
razloži vseh naših opazovanj. Pojav temnih lis na mestih, kjer so prej bili svetleči kristali
na mikroskopskih slikah, ter sigmoidni padec SERS intenzitete dominante črte TNT
nam nakazuje tudi taljenje TNT kristalov. Čeprav je temperatura tališča TNT pri 80 °C,
pa je ta odvisna od količine oziroma velikosti snovi. Znižanje temperature tališča lahko
razložimo z majhnostjo kristalov TNT na površini. Spremembe v SERS spektru pri 60
°C v primerjavi s spektrom pri 20 °C, pa ne moremo razložiti zgolj s izhlapevanjem in
taljenjem. Razgradnja molekul pri SERS spektroskopiji je lahko težava, zlasti ker je
laserski žarek usmerjen na majhno območje na površini. Izginotje karakteristične TNT
črte in pojav nove črte jasno kaže na razgradnjo TNT v drugo kemično spojino. Prav
tako smo preučevali temperaturno odvisnost na SERS učinek, saj bi ta prav tako lahko
vplivala na znižanje intenzitete pri SERS spektrih. Tako smo na substrat adsorbirali 4-
nitrothiophenol. Te molekule tvorijo kovalentne vezi med zlato nanostrukturirano
površino in žveplovim atomom, kar vodi do stabilnega monosloja. V tem primeru
izhlapevanje in fazni prehod ne bi smela imeti vpliva. Substrat smo ogreli do 80 °C.
SERS spektri 4-Nitrothiophenola so nam pokazali le majhno izgubo intenzitete, ki je
približno 11 %. Da bi zmanjšali izhlapevanje TNT-ja, smo na substrat absorbirali
mercaptohexanol (MCH), ki tvori stabilen samosestavljiv monosloj, podobno kot 4-
Nitrothiophenol. Na monosloj smo nato kapnili TNT raztopino. S tem so se tvorile
vodikove vezi med hidroksilno skupino MCH in nitro skupino TNT. Na ta način naj bi
45
MCH vezal TNT molekule močneje k površini. Mikroskopska opazovanja pred SERS
meritvami so razkrila spremembo v adsorpciji (TNT molekule namreč niso tvorile več
kristalov na MCH sloju). Tudi SERS signal TNTja je bil znatno manjši, saj je SERS
učinek močno odvisen od razdalje med molekulami in površino. Vendar pa se je ob
segrevanju substrata SERS signal ponovno bistveno zmanjša, kar kaže na to, da MCH
ne more preprečiti izhlapevanje TNT molekul.
Pokazali smo, da so SERS meritve pri višjih temperaturah še vedno možne, saj je
ojačitveni učinek plasmonskega substrata skorajda nespremenjen vse do 80 °C. Vendar
pa lahko segrevanje majhnih količin snovi vodi do taljenja in razgradnje le-teh tudi v
primeru, če je večja količina te snovi stabilna pri enakih razmerah.
46
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