Vibrational Spectroscopy 57 (2011) 163–176

Contents lists available at ScienceDirect

Vibrational Spectroscopy
journal homepage: www.elsevier.com/locate/vibspec

Review

Raman spectroscopy: Recent advancements, techniques and applications

Ruchita S. Das ∗ , Y.K. Agrawal
Gujarat Forensic Sciences University, Institute of Research and Development, Sector 18 A, Gandhinagar 382007, Gujarat, India

a r t i c l e i n f o a b s t r a c t

Article history: Vibrational spectroscopy has proven itself to be a valuable contributor in the study of various fields of
Received 14 March 2011 science, primarily due to the extraordinary versatility of sampling methods. Raman measurement gives
Received in revised form 12 July 2011 the vibrational spectrum of the analyte, which can be treated as its “fingerprint,” allows easy interpreta-
Accepted 9 August 2011
tion and identification. Over the last years, there has been tremendous technical improvement in Raman
Available online 17 August 2011
spectroscopy, as overcome by the problems like fluorescence, poor sensitivity or reproducibility. This
article reviews the recent advances in Raman spectroscopy and its new trend of applications ranging
Keywords:
from ancient archaeology to advanced nanotechnology. It includes the aspects of Raman spectroscopic
Vibrational spectroscopy
Raman spectrum
measurements to the analysis of various substances categorized into distinct application areas such as
Samples biotechnology, mineralogy, environmental monitoring, food and beverages, forensic science, medical and
Science clinical chemistry, diagnostics, pharmaceutical, material science, surface analysis, etc. Advances in the
Analysis instrumental design of Raman spectrometers coupled with newly developed sampling methodologies
Detection have also been described which enable trace level detection and satisfactory analysis.
© 2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
2. Basic principles: Mechanism and Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
3. Advanced Raman techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
3.1. Surface-enhanced Raman spectroscopy (SERS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
3.2. Confocal Raman microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
3.3. Coherent anti-Stokes Raman scattering (CARS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
3.4. Resonance Raman spectroscopy (RRS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3.5. Raman sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
4. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
4.1. Forensic science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
4.2. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
4.3. Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
4.4. Materials science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
4.4.1. Superconductors and Semiconductors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
4.4.2. Carbonaceous materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
4.4.3. Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
4.4.4. Environmental materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
4.4.5. Crystalline study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
4.4.6. Molecules and Molecular systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
4.4.7. Archaeological material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
4.5. Pharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
4.6. Nanotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

∗ Corresponding author. Tel.: +91 9328859853; fax: +91 7923256252.

E-mail address: das ruchita@yahoo.com (R.S. Das).

0924-2031/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.vibspec.2011.08.003
164 R.S. Das, Y.K. Agrawal / Vibrational Spectroscopy 57 (2011) 163–176
1. Introduction
In 1928, an Indian physicist Chandrashekhara Venkata Raman
discovered the phenomena of inelastic scattering of light, known
as the Raman effect. This explains the shift in wavelength of a small
fraction of radiation scattered by molecules, having different fre-
quency from that of the incident beam [1]. This shift in wavelength
depends upon the chemical structure of the molecules responsible
for scattering. Raman spectroscopy utilizes scattered light to gain
knowledge about molecular vibrations which can provide infor-
mation regarding the structure, symmetry, electronic environment
and bonding of the molecule, thus permits the quantitative and
qualitative analysis of the individual compounds [2]. This paper
is dedicated to the techniques and various applications related to
Raman spectroscopy in different fields of science, including a short
introduction of its principle and instrumentation, which will help
in better understanding of its analytical versatility.
2. Basic principles: Mechanism and Instrumentation
The irradiation of a molecule with a monochromatic light always
results in two types of light scattering, elastic and inelastic. In elastic
scattering, there occurs no change in photon frequency or without
any change in its wavelength and energy. Conversely, the other is
inelastic scattering which is accompanied by the shift in photon fre-
quency due to excitation or deactivation of molecular vibrations in
which either the photon may lose some amount of energy or gains
energy [3]. Thus, three types of phenomena can occur [4] (Fig. 1).
First, when light is incident on a molecule, it can interact with the
molecule but the net exchange of energy (E) is zero, so the frequency
of the scattered light is the same as that of the incident light (E = Eo ).
This process is known as Rayleigh scattering.
Second, the light can interact with the molecule and the net
exchange of energy is the energy of one molecular vibration. If the
interaction causes the light photon to gain vibrational energy from
the molecule then the frequency of the scattered light will be higher
than that of the incident light (E = Eo + Ev ), known as anti-Stokes
Raman scattering.
Third, if the interaction causes the molecule to gain energy from
the photon then the frequency of the scattered light will be lower
than that of the incident light (E = Eo − Ev ), this process is known as
Stokes Raman scattering.
A Raman spectrometer is composed of light source, monochro-
mator, sample holder and detector. The factors which affect the
analysis on Raman spectra may include high signal-to-noise ratio,
instrument stability and sufficient resolution. The development
of effective FT Raman spectrometers using NIR or red excitation
lasers solved the problem of avoiding fluorescence that affects
the Raman signals. On the other hand, the development of highly
Fig. 1. Mechanism of Raman scattering.
Fig. 2. Instrumentation.
sensitive detectors in conjunction with coupling of optical fibres
and microscopes enhanced the capacity of analysis [5]. Two major
technologies are used to collect the Raman spectra, Dispersive
Raman spectroscopy and Fourier transform Raman spectroscopy,
with difference in their laser sources and the way by which Raman
scattering is detected and analysed (Fig. 2). Both these techniques
have unique advantages and the method that best suit the sample
should be preferred [6–8].
Several type of lasers can be used as the excitation source, like
argon ion (488.0 and 514.5 nm), krypton ion (530.9 and 647.1 nm),
He:Ne (632.8 nm), Nd:YAG (1064 nm and 532 nm) and diode laser
(630 and 780 nm). Use of 1064 nm near-IR (NIR) excitation laser
causes lower fluorescent effect than visible wavelength lasers [9].
In the most basic, a molecule is Raman active when there is a
change in polarizability during the vibration. In addition, symmetry
of a molecule is also one of the basic requirements for obtain-
ing Raman spectra, as the symmetric stretches are more intense
in Raman spectra. Functional groups such as -C–X (X = F, Cl, Br
or I), –C–NO2 , –C–S–, –S–S–, –C C–, –C S–, –N N–, –S–H–, –CN,
etc., exhibit more polarizability changes, give strong Raman signals
[10]. Sometimes, Raman spectroscopy has been found coupled with
many hyphenated analytical techniques like high-performance liq-
uid chromatography [11], microchromatography [12], scanning
tunneling microscopy [13] atomic force microscopy [14], etc.; give
possibility for useful analysis at trace level studies. Thus, with the
revolutionary developments in Raman instrumentation, it is now
possible to acquire spectra more quickly on equipment which is
affordable and easier to use than in the past. The following will
summarise different techniques associated with the Raman spec-
troscopy and related applications.
3. Advanced Raman techniques
3.1. Surface-enhanced Raman spectroscopy (SERS)
SERS is one of the most sensitive tools for the detection of adsor-
bate molecules on roughened metal surfaces which produces a
large enhancement to the Raman scattering signal (Fig. 3). This
enhancement effect was studied independently by Jeanmaire et al.
[15] and Albrecht et al. [16] in 1977 and each proposed a differ-
ent mechanism for the observed enhancement. Accordingly, two
factors were assumed to be responsible for the enhancement of
signals:
 R.S. Das, Y.K. Agrawal / Vibrational Spectroscopy 57 (2011) 163–176 165

Fig. 3. Surface enhanced Raman scattering.

1. Electromagnetic enhancement (EM), associated with the rough-

ened metal surface [15].
2. Chemical enhancement (CHEM), due to the electronic cou-
pling of molecules adsorbed on the roughened metal surfaces
(or involves changes to the adsorbate electronic states due
to chemisorption of the analyte or chemical bond formation
between metal surface and molecules under observation) [16].

However, the electromagnetic effect is supposed to be more

dominant and sometimes it is also called as “first-layer effect,”
because it requires direct contact between analyte molecule and
metal surface [10,17]. Both these factors can be well understood
by the concept of surface plasmon which is located in the metals
like Ag (silver) or Au (gold). When this plasmon oscillate perpen-
dicular to the surface of the metal causes scattering, reflects the
roughness of the surface which can either be the physical rough-
ness or may be produced by some nano particles [18–20]. As the
signals obtained by normal Raman scattering is usually very weak,
therefore in order to get more detectable or increased signals, one
prefers SERS technique. This technique can yield information on
how molecules interact with surfaces, which allow detection of
very low concentrations of analytes. This specially prepared metal
surfaces like gold, silver and copper increase the intensity of the
Raman signal up to 104 to 106 fold which enables faster and higher
accuracy detection of biological and chemical samples [21].
The Raman signal enhancement is maximized when metal
grains are smaller than the incident laser wavelength with opti-
mized geometry [22]. Gold caps (nearly 50–400 nm in diameter)
were supposed suitable for exploiting the tip or surface enhanced
Raman scattering effects, since they assume the right size on
nanometer scale with almost ideal hemispherical shape as the sig-
nal enhancement relies on the geometry of the metal particles
[23]. Other concepts were also given for the enhancement of the
signals, suggest that the enhancement effect is mainly due to the
formation of nanojets, obtained when the laser focused on micro-
sphere of appropriate diameter. This may lead to highly localized
electromagnetic field causing enhancement [24]. SERS has been
applied successfully for the study of Ni (nickel) and Pt (platinum)
electrodes having different surface roughness, helps in obtaining
good quality surface Raman signals from transition metals. Thus
Raman spectroscopy will be further developed as a versatile mean
for characterizing interfacial processes on rough surfaces in elec-
trochemical and other surface environments for both fundamental
and practical applications [25].
Quantitative analysis of a fungicide thiram has been done by
using the substrate of silver (Ag+ ) nano-particles, prepared by radi-
olysis of Ag+ aqueous solution without addition of aggregating
or stabilizing substances, as it might limit the spurious bands in
Raman spectra. The detection thus obtained was at low concentra-
tion and without the interference of impurities of the medium [26].
Likewise, the Raman intensity of malachite green isothiocyanate
(MGITC) adsorbed on gold (Au3+ ) surface was increased nearly up
to 20 folds which explore the sensitivity of detection for adsorbed
species on single crystal surface [24].
SERS sensor allows the feasibility of detection of volatile organic
compounds (VOC), prepared by coating thiol with the substrate of
SERS mounted on thermoelectric cooler. The purpose of this coating
Fig. 4. Confocal Raman microscope.
was to protect the substrate from degradation, to provide an inter-
nal calibration standard and to attract contaminants of interest.
This can facilitate the detection of VOC such as chlorinated solvents,
methyl t-butyl ether (MTBE) and aromatics with their characteristic
SERS response [27].
Shell-isolated nanoparticle-enhanced Raman spectroscopy is
described for the amplification of Raman signals by gold nanopar-
ticles with an ultrathin silica or alumina shell. A monolayer of such
nanoparticles is spread over the surface, later probed in order to
obtain high quality Raman spectra. This can support the applica-
tion of SERS in material and life sciences, as well as in the field of
food safety, drugs, explosives and environment pollutants [28].
3.2. Confocal Raman microscopy
The first confocal scanning microscope was invented in 1955 by
Marvin Minsky, in order to avoid thin-slicing of brain tissues [29].
In confocal Raman microscopy, laser light from the probe-head is
made focused on the sample through microscope objective. The
backscattered Raman signal is refocused onto a pinhole aperture
that acts as a spatial filter. The filtered Raman signal then returns
to the spectrometer where it is dispersed on a CCD (charge cou-
pled device) camera to produce a spectrum [30] (Fig. 4). It must
be remembered that the fluorescence background should be suf-
ficiently low to affect weak Raman signals [31]. This technique
possesses a number of applications by providing three-dimensional
image of chemical composition with micrometer resolution and
clear image quality. The three-dimensional imaging through turbid
medium, high spatial resolution and rapid identification of microm-
eter sized biological specimens can be examined using a confocal
Raman microscope [32–34].
It is a non-invasive method to obtain detailed information about
the molecular composition of different tissues with high spatial
resolution, provides optical section of the tissues without physi-
cal dissection. This technique can also combine with other Raman
techniques to provide detailed information about molecular com-
positions of confocal images. This technique can also be combined
with other with other Raman techniques to provide detailed infor-
mation about the molecular composition in the confocal image.
166 R.S. Das, Y.K. Agrawal / Vibrational Spectroscopy 57 (2011) 163–176
The combination of confocal Raman microspectroscopy and con-
focal scanning laser microscopy (CSLM) has been presented as a
novel non-invasive method to obtain information about molecu-
lar composition in relation to skin architecture. In addition to this,
detail in vivo concentration profiles of water and natural moistur-
izing factors for stratum corneum has also been described which
are directly related to the skin architecture. Thus, the arrangement
of two different techniques can provide the basis for a wide range
of applications in fundamental skin research, as well as in phar-
macology, dermatology and cosmetics as well as for non-invasive
analysis of blood analytes [35].
Confocal Raman microscopy has been found to be very effective
in the analysis of the distribution of chemical moieties within poly-
meric coil coatings because it give strong Raman scattering which
provides characteristic profiling of pigment. Such kind of analysis
was performed with both dry and oil immersion objectives, and it
was observed that the resolution gets affected by both the intrin-
sic as well as extrinsic factors of the objective lens. It has also been
observed that the use of an oil immersion objective improves depth
resolution and minimised the refractive effect which favours multi-
ple analysis of samples. However, oil may contaminate the surface
coating. On the contrary, the dry method yields the lowest depth
resolution and allows non-destructive analysis [36].
Likewise, this technique can be used to measure the spatial and
temporal evolution of a drying coating film of paint. Comparison
between confocal Raman microscopy and nuclear magnetic reso-
nance (NMR) technique has done for the examination of two alkyd
paint coatings, one was organic solvent based and another was
water based. The NMR and confocal Raman microscopy have shown
good similarities in profiles of paint films which were obtained by
comparing the profiles of disappearance of the double bonds of
the unsaturated fatty acid side chains of alkyd molecules. Thus,
the technique can be used to analyse spatial variations of paint
properties within a thin film [37].
Moreover, this technique offers as an important alternative
to conventional spectroscopic techniques that provide elemen-
tal/atomic composition of hazardous components in cosmetic
products like eyeliner. Raman spectra of such cosmetic samples
have been measured between 150 and 3000 cm−1 at room tempera-
ture, showed the presence of lead (II) sulphide (PbS) which is a weak
Raman scatterer at room temperature and is therefore susceptible
to laser-induced degradation when intensely irradiated. The use of
confocal Raman microscopy exhibits superior rejection of fluores-
cence and thus the stray light is rejected, so only the desired Raman
signal is passed to detector which helps in proper analysis of the
sample [38].
3.3. Coherent anti-Stokes Raman scattering (CARS)
CARS was first reported by P.D. Maker and R.W. Terhune in
1965 and they called it ‘three wave mixing experiments’ [39]. But
the name coherent anti-Stokes Raman Scattering was assigned
by Begley et al. in 1974 [40]. This technique allows vibrational
imaging with high sensitivity, high spectral resolution and three-
dimensional sectioning capabilities. It is a nonlinear diagnostic
technique that relies on inducing Raman coherence in the tar-
get molecule using two lasers, probed by a third laser which
generates a coherent signal in the phase-matching direction at a
blue-shifted frequency. This phenomenon can be explained on the
basis of some equations. Incident radiation consists of two over-
lapping coherent monochromatic beams of frequencies (ωp1 ) and
(ωs ) with (ωp1 > ωs ). When (ωp1 − ωs = ωvib ) (molecular vibration
frequency), observed normal Raman scattering, but when probed
by a third laser (ωp2 ) generates a coherent laser-like signal in
the phase-matching direction, produces strong anti-Stokes signal
[ωas = (ωp1 + ωp2 ) − ωs ] with high spatial resolution [41] (Fig. 5).
Fig. 5. Coherent anti-Stokes Raman scattering.
This has been proven as a valuable tool for measuring temper-
ature and major species concentration in reacting flows [42]. An
overview about the advances made over the last few decades in
the development and applications of nanosecond, picosecond, and
femtosecond laser-based CARS spectroscopy in gas-phase reacting
flows has been demonstrated well [43]. CARS permits probing of
different molecular bonds, in various biological systems. Exam-
ples include, imaging of C–H stretching vibration present in the
lipid bilayer of the cell membranes [44,45], P–O vibration (at
1090 cm−1 ) in chromosomes and more recently, imaging of live
tissues [46].
Additionally, CARS microscopy has already been used for imag-
ing a number of delicate biological samples and processes. It
provides excellent sensitivity, high spatial resolution and inherent
chemical specificity, gives vibrational and spectral information at
low laser power also [47]. The detected signal in a CARS microscope
is a result of coherent superposition of the signal waves generated
in focus and can be used as an imaging tool for chemical map-
ping of biological cells and tissues with high sensitivity [48]. It is a
key technique in non-invasive cellular imaging as it permits imag-
ing of intrinsic biomolecules without chemical labelling or without
the complications of photobleaching [44,49]. It also has significant
potential for biomedical applications, since chemical imaging of live
skin tissues has been achieved [50].
The nanosecond laser based CARS technique is used in the
measurement of temperature and multiple species concentration
detection. On other hand, dual-pump CARS, triple-pump CARS,
dual-broadband CARS and dual-pump dual-broadband CARS were
applied mainly to detect multiple species in one experimental
setup. In picosecond CARS, the probe beam is temporally delayed
with respect to the pump and Stokes beams to suppress the non-
resonant background. Additionally, picosecond pulse laser works
above 800 nm, which avoids multiphoton damage of specimen and
allows deep penetration in thick samples. While temperature and
concentration measurements of diatomic and triatomic gas phase
molecules can be performed using a single femtosecond laser beam.
Thus, by tuning into characteristic vibrational resonance in sam-
ples, higher scanning speed and fast image acquisition time makes
CARS the technique of choice [51].
Furthermore, new developments in Electronic Resonance
Enhanced CARS (ERE-CARS) spectroscopy have paved the way for
detection of minor species such as NO [52,53], C2 H2 [54], etc. One
more approach is multiplex CARS (M-CARS) microscopy that has
been used to characterize neat liquids, polymer blends, domains
in lipid droplets and bacterial spores. In this technique a spectrally
broad femtosecond pulse is used for Stokes transition which can
provide sensitivity over 300–1500 cm−1 of spectral bandwidth at
each spatial pixel [55–57].
 R.S. Das, Y.K. Agrawal / Vibrational Spectroscopy 57 (2011) 163–176 167

With the advent of time there has been remarkable develop-

ment in the CARS technique. Recently with the use of pulse train,
the time required for obtaining broadband CARS signals is reduced
to one third as compared to previous studies without using pulse
trains. The pulse train was created by shaping optical pulses with
a pulse shaper and their waveforms were measured by a cross-
correlation frequency-resolved optical gating method. Pulse train
was generated from a photonic crystal fibre (PCF) with different
centre wavelengths and used as Stokes optical pulses in CARS which
can be used to measure broadband CARS signals. Such kind of study
has been done to create pulse train by a pulse shaper to gener-
ate multiple soliton pulses with different centre wavelengths and
different delay times from a PCF. This helped in obtaining five soli-
ton pulses necessary for broadband CARS spectroscopy; generated
using only two phase patterns and the time required for the mea- Fig. 6. Fibre Raman sensor.
surement was reduced [58].
These pulses can also be used as Stokes pulses in broadband
CARS (B-CARS) measurements. The B-CARS imaging technique is Resonance Raman scattering has been extensively exploited
used to measure spectral signatures of individual cells at least five- in the analysis of various chromophoric biological samples like
fold faster than spontaneous Raman microspectroscopy and can enzymes, various parts of biomolecules and protective pigments
be used to generate maps of biochemical species in cell. It offers of photosynthetic organisms. Similarly, it can also be used for
the same inherent chemical contrast as spontaneous Raman sig- obtaining high quality pre-resonance Raman spectra of bacte-
nal but with increased acquisition rate. B-CARS imaging technique riochlorophyll chromophores in photosynthetic proteins from
has been studied to obtain complete resonant vibrational spectra of purple bacteria without sample degradation [64]. Discussion and
a single cell with 50-ms individual pixel dwell times. This acquisi- comparison of RRS and normal Raman spectroscopy has been
tion time is at least fivefold faster than spontaneous confocal Raman reviewed well. This has explored the utility of RRS as a reliable
microspectroscopy of cells at similar spatial resolution. The spectra and versatile analytical technique, as in the analyses of carotenoids
thus obtained were in the range of 600–3200 cm−1 . This covers both in biological matrices, analyses of pigments and dyes in forensic
fingerprint and CH-stretch regions that provide both morphologi- investigations, in bioanalytical and life sciences, study of met-
cal as well as rich biochemical information. This improved spectral alloproteins, etc. Furthermore, this technique is made to couple
range and signal intensity opens the door for more widespread use with other separation (chromatographic and electrophoretic) and
of vibrational spectroscopic imaging in biology and clinical diag- detection (UV) techniques [65]. Sometimes the extreme enhance-
nostics [59]. ment factors can be realized by combining RRS and SERS to SERRS
Furthermore, the non-resonant background can easily over- as SE(R)RS (Surface Enhanced Resonance Raman Spectroscopy),
whelm less intense signals from other molecular vibrations which where the laser excitation wavelength not only coincide with the
contain important information about the vibrational energy lev- plasmon band, but also with the absorption wavelength of the ana-
els of molecule or sample specimen. This non-resonant scattered lyte [66].
light can interfere coherently with the resonant signal which may
distort the band shapes. Many approaches have been established
3.5. Raman sensing
for reducing the non-resonant background, like epi-detection, tem-
poral delay of probe with respect to the pump and Stokes fields
Advances in low-cost fibre optics and miniaturized detectors led
[60], and polarization control of the CARS signal [61]. One more
to rapid increase in the use of Raman spectroscopy with greatest
technique has been given for interferometric suppression of the
advantage of remote sensing. This technique is basically associ-
non-resonant background based on interferometric mixing of the
ated with optical fibres to transport Raman signals by collecting
CARS signal with a local oscillator, generated within the sample. In
the scattered photons, simultaneously filtering out Rayleigh scat-
this technique liquid benzonitrile was selected as a model system
tering (Fig. 6). Fibre system includes single-fibre and multiple fibres
for removing the non-resonant signals, which gives prominent peak
in which the laser excitation is transmitted along one fibre and the
throughout the vibrational spectrum, provides good sensitivity and
scattered radiations are transmitted to the detector along different
chemical species identification [62].
fibres [4]. Thus, gives the feasibility of online monitoring of process
streams and hazardous reactions [67].
Background discrimination, sample volume, and probe sensi-
3.4. Resonance Raman spectroscopy (RRS)
tivity has been investigated as a function of laser source and fibre
design [68,69]. Other studies have focused on coupling efficiency,
Resonance Raman spectra obtained when the energy of photon
damage threshold, and sensitivity for UV Raman fibre probes in
of an exciting laser beam matches approximately with the energy
the presence of adsorbing materials [70]. Fibre-optic Raman can be
require for electronic transition (Fig. 1). The vibrations which cause
used to determine the amount of organic vapour that partitions into
enhancement in Raman bands fall into two or three general classes.
solid-phase extraction medium [71]. Remote micro imaging has
The most common class is Franck-Condon enhancement, in which
also been accomplished using a coherent optical microfiber array
a component of normal coordinate of the vibration is in the direc-
as a probe [72].
tion of molecule which expands during an electronic excitation. The
more the molecule expands along the axis (when it absorbs light),
the larger enhancement factor occurs. Vibrations which couple 4. Applications
two electronic excited states produce enhanced resonance sig-
nal. This mechanism is called vibronic enhancement. In both cases Raman spectroscopy is becoming increasingly important in a
enhancement factors roughly follow the intensities of the absorp- broad range of scientific disciplines. Brief discussion of these appli-
tion spectrum [63]. cations in various field of science have been discussed below.
168 R.S. Das, Y.K. Agrawal / Vibrational Spectroscopy 57 (2011) 163–176
4.1. Forensic science
The recent technological advancements in Raman spectrome-
ter have provided a reason for exploring its use in forensic science.
Analysis of fibres, explosives [73], drugs [74], paints [75,76], inor-
ganic fillers, lipsticks [77] and other materials have been done
successively by using Raman techniques.
Confocal Raman Microscopy is used for the examination of var-
ious kinds of biological fluids like dry traces of semen, vaginal
fluid, sweat, saliva and blood. These fluids can be differentiated
from one another by visual comparison of their Raman spectra,
as each body fluid has its own composition that can give spe-
cific Raman signal. An even dry trace of human and canine semen
exhibits different Raman signatures. This can help in better inter-
pretation of crime scene exhibit especially body fluids, as these
analyses are non-destructive in nature and offers the possibility
of further testing. This study was done on only one sample of each
body fluid and did not taken into account any variations that may
occur between different donors of the same fluid [78]. However, in
another study heterogeneity was obtained within a sample as well
as among multiple donors for human semen samples. NIR is used to
measure spectra of purely dried human semen samples from mul-
tiple donors. Statistical analysis of spectra obtained from samples
showed that the dry semen is heterogeneous and its Raman spectra
can be presented as a linear combination of a fluorescent back-
ground with three different spectral components varies with donor.
Thus, no single spectrum could effectively represent an experimen-
tal Raman spectrum of dry semen in a quantitative way. However,
combination of three spectral components can be considered to
be a spectroscopic signature for semen during forensic analysis
[79].
Examination of ink is very important aspect for the investiga-
tion of questioned documents. Sometimes it is necessary to apply
chemical methods that normally cause partial destruction of the
examined material, but Raman spectroscopy provides direct iden-
tification of inks on documents for determination of sequence of
the handwritten lines. The investigations are mostly often aimed
at authenticating the document or at determining its age or origin.
This could be possible due to some characteristics of the ink such
as luminescence in visible or in infrared light. A good discrimina-
tion between inks has been obtained using Raman spectroscopy,
possibly used to differentiate between inks of the same colour [80].
Fibres are also one of the commonly encountered evidence dur-
ing forensic investigation especially black/grey and blue cotton
type fibres. The fibres are generally coloured by reactive dyes, as
dyes can able to form non-polar bonds with fibres, which can pro-
duce difficulty in extraction during analysis. Raman spectroscopy
measures the vibrational states of non-polar bonds through the use
of high intensity lasers and can be used as analytical tool for fibre
examination [81,82].
Different aspects of fibre analysis were studied via Raman Spec-
troscopy, observed variations due to the method of mounting
the dye particles, spectral degradation, fluorescence, resonance
enhancement and laser wavelengths. Results have shown good
spectra of dyed cotton fibres without interference from cotton sub-
strate, thereby provides molecular information about the dye used
in the fibre [83]. Similarly, Raman measurements can be performed
directly on mounted fibres without encountering much interfer-
ence from the mounting medium. Forensic cases were described
where the comparison of Raman spectra of reference fibres and
suspect fibres has been discussed in detail. The results suggested
Raman spectroscopy as a powerful tool for Forensic fibre exam-
ination (Fig. 7) [84]. Some studies have also explored the use
of resonance, non-resonance, and surface-enhanced Raman spec-
troscopy for forensic fibre examination, including both dyed and
Fig. 7. Baseline corrected Raman spectra of (a) the brown cotton fibres found on the
suspect, and (b) those found on the victim.
non-dyed textile fibres, as it can provide detailed vibrational pro-
files which gives characteristic “signature” to dyes [85].
Raman spectroscopy has been proved as a good complemen-
tary method for detection of drugs of abuse in fingerprints. This
kind of study was done on five drugs of abuse that is codeine phos-
phate, cocaine hydrochloride, amphetamine sulphate, barbital and
nitrazepam. Detection of these drugs was successfully achieved
and clearly distinguished using their Raman spectra obtained from
the substances in cyanoacrylate-fumed fingerprints (polymer is
deposited on the fingerprint material for enhancing the visibility).
Interestingly, it was observed that the spectra obtained from the
fingerprints were of a similar quality to the spectra obtained from
the substances under normal sampling conditions [86]. Likewise,
quantitative determination of caffeine in different energy drinks
has been achieved by FT-Raman spectroscopy, provides fast and
alternative mean to chromatographic method with higher sam-
pling frequency. In order to quantify caffeine, spectra were obtained
directly between 3500 and 70 cm−1 with Raman bands between
573 and 542 cm−1 (corrected using a baseline between 580 and
540 cm−1 ), obtained limit of detection as 18 mg/l [87].
Raman spectroscopy can also offer a good flexibility in
the analysis of hazardous environmental samples. Detection of
cyclotrimethylenetrinitramine (RDX) is achieved by SERS, extend-
ing its broad application areas in the examination of other
explosives also. Analysis of RDX was done with gold (Au) nanoparti-
cles (90–100 nm in diameter) as SERS substrates, with the detection
level of 0.15 mg/l from contaminated groundwater sample. Thus,
it can be potentially used as a valuable tool for rapid screening
and characterization of energetics in the environment, such as tri
nitro toluene (TNT), perchlorate, pertechnetate, and uranium in
groundwater at low concentrations [88]. SERS has also been used
to detect and distinguish explosives in the solution using azo dyes.
These dyes contain electron-donating moieties give efficient diazo
coupling and have a strong silver complexing group to attach the
product molecule to SERS substrate, allows detection up to nM
(nano molar) concentration [89]. Thus, it can explore the studies
related to environmental monitoring, security and biological mate-
rials like nuclear waste testing [90]. Raman has also been proved
as a powerful tool in the analysis of adulterated fuel samples like
diesel or biodiesel blends with vegetable oil, used in remote qual-
ity control. Calibration models based on multivariate analysis such
as principal component regression and partial least square regres-
sion in combination to vibrational spectroscopy has been applied
for examining the adulteration with complete accuracy; especially
with the help of appropriate choice of spectral regions [91].
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4.2. Biology
The greatest advantage of using Raman spectroscopy in bio-
analysis is the wealth of information contained in each spectrum.
Even Raman micro-spectroscopy can provide useful biochemical
information regarding live cells, without the need of fixatives,
markers or stains [92]. This can be related to the interactions with
toxic agents or drugs, disease, cell death and differentiation. Raman
spectrum of a cell can produce “fingerprint” of its biochemical com-
position so, if any toxic agent causes biochemical changes it appears
in the Raman spectra [93]. It has been proved that the Raman spec-
tra of biological macromolecules arise from the molecular vibration
of either the backbone chains or the side chains. The wavenumbers
of the Raman bands lie in a region between 200 and 3000 cm−1 .
Even conformation or secondary structure can also be determined
by analysing Amide I and Amide III vibrations for polypeptides and
protein backbone chain. Similarly, in polynueleotides and nucleic
acids the wavenumbers of phosphate diester stretch of phosphate
furanose chain varies between 814 cm−1 for A conformation and
790 cm−1 for B conformation [94].
Hemoglobin-oxygen saturation in living tissue is determined
by measuring the ratio of intensities of resonance Raman bands
aroused from oxygenated and de-oxygenated hemoglobin [95].
Similarly, CARS also provides a non-invasive determination of the
blood oxygenation (oxygenation state of hemoglobin) in individ-
ual vessel inside bulk tissues [96]. It can also be used for detection
of bacterial spores by obtaining the molecular specific signals, as
a marker molecule (dipicolinic acid) for bacterial spores [97]. In
addition, CARS has provided a non-destructive analysis of embry-
onic stem cells within a growing culture. It has also given specific
backbone vibrations for DNA (788 cm−1 ), RNA (811 cm−1 ) and pro-
teins [98]. Even, Raman spectra can provide information about the
structure, function and kinetics of protein by identification of its
vibrational bands. The high sensitivity of deep UV resonance Raman
spectroscopy makes it a valuable tool for studying biological sys-
tems under different physiological conditions [99–102]. It is also
possible to determine the metabolic activity of cell mitochondria.
The Raman band at 1602 cm−1 reflects respiratory activity of mito-
chondria in living cells and it was observed that the intensity of the
band decreases with time after addition of sodium azide, act as a
respiratory inhibitor [103].
Study of these biomolecules with Raman spectroscopy helps
in evaluation of the quality of natural food products, as it pro-
vides structural information about the change in proteins, water
and lipids of muscle food which occurs during deterioration. As
compared to the traditional techniques like protein solubility, vis-
cosity, water holding capacity, instrumental texture methods and
peroxide value calculation which were used for the determination
of quality of muscle foods were destructive in nature. In con-
trast, Raman spectroscopy provides valuable and non-destructive
analysis of samples [104]. Due to the extraordinary versatility of
sampling methods vibrational spectroscopy has proven itself to be
a valuable contributor in the study of biophysical interfaces, which
can help in obtaining the spectra of oriented monolayer films.
Advances in the Raman techniques coupled with newly developed
sampling methodologies have enabled to probe ever-smaller and
ever-thinner samples. This will allow the examination and identifi-
cation of unenhanced bio-membrane monolayer spectra, by giving
molecular structure of membrane [105].
Advances in instrumentation have increased the performance
of Raman instruments in bioanalysis. The specificity of the tech-
nique makes it an attractive tool for biomedical analysis which can
provides information applicable to almost any biomolecule, that
too without labelling [106]. It can also become a useful tool in the
early detection of cells exposed to human papillomavirus (HPV)
[107]. Likewise, Raman spectra of other viruses have also been stud-
169
ied in an effort to elucidate the mechanism of replication in the
host organism where the side chains and secondary structure of
proteins were clearly observed [108]. Analysis of individual bac-
teria, complex mixture of spores and vegetative cells can be done
by Raman chemical imaging (RCI). One of the major advantages of
this technique is that it resides primarily during analysis of samples
in complex backgrounds without the need of physical isolation or
purification of the sample [109].
A study of interactions between lysozyme and whey protein
indicates the presence of hydrophobic interactions. This can be
evidenced by intensification of spectral bands assigned to CH and
CH2 bending vibrations which arose with the changes in disul-
fide vibrational frequency and through lowering in R-helix and
alpha-sheet contents [110]. The structure of lipid-cholesterol vesi-
cles (as a function of pressure) have been investigated using Raman
wavenumbers, band shapes and splittings in the lipid bilayer [111].
Likewise, Filipin (which binds specifically to cholesterol) is used as
an extrinsic Raman probe molecule for determination of cholesterol
distributions within rat eye lenses [112]. Intake of glucose into the
cell could be determined by attaching quinoline red dye with cell
membranes. Quinoline was used as labelling agent to bind with the
analyte of interest. Thus, results in increasing Raman cross-section
of weak scatterers because peak position of dye spectrum changes
when it binds to the cell. Moreover, when the cell membrane gets
energized and glucose is transported into cell there occurs with
change in the intensity of the spectra. Hence, use of Raman labels
can be applied for studies at molecular level [100].
It was believed that the physical properties of nails changes
because of change in keratin structure. Thus, examination of change
in molecular structure of intact moisten nails has been stud-
ied through NIR-FT-Raman spectroscopy. The results obtained
revealed that nails have water holding capacity, which causes
increase in intensity ratio of the X(OH)/X(CH2 ) bands. This imply
about the water–protein interaction which leads to change in pro-
tein geometry [113]. The Interaction of calf-thymus DNA with
aspirin was investigated by Fourier transform Infrared (FTIR) and
laser Raman difference Spectroscopy, provided information about
the drug binding sites, sequence preference and changes in sec-
ondary structure of DNA. In addition to this, structural variations
of aspirin-DNA complexes in aqueous solution have also been
determined. Spectroscopic evidence showed that low aspirin con-
centration was helpful for drug-DNA interaction which is mainly
through the backbone PO2 groups and the AT base pairs, obtained
phosphate vibration at 1227 cm−1 and A-T bands at 1663 and
1609 cm−1 , with no major helix destabilisation [114].
Macroscopic resolution allows examination of the chemistry of
individual cells by mapping their images. These images can con-
tain full spectral information at each pixel so that the distribution of
components within the cell can be visualized based on their Raman
signature. This is extremely valuable to researchers as biochem-
ical changes can be observed during a cell’s life cycle or when a
cell is damaged or cancerous. Using confocal Raman microscopy,
the changes in a variety of cells, including bacteria and eukaryotes
can be monitored over time and comparison between healthy and
diseased tissue states can be done easily [115].
4.3. Diagnostics
Raman spectroscopy has widely showed its utility as a diagnos-
tic technique, offering various advantages for analysis of variety
of biomedical materials. NIR provides a rapid and non-destructive
analysis to assess the thyroid stimulating Hormone (TSH) in blood,
given good reliability to the experiment [116]. FT Raman spec-
troscopy is used for examining and characterising the outermost
layer of the human skin, stratum corneum provided valuable data
related to the nature of healthy and diseased skin, transdermal
170 R.S. Das, Y.K. Agrawal / Vibrational Spectroscopy 57 (2011) 163–176
Fig. 8. Raman spectra of (a) non-pathologic, (b) atheromatous and (c) calcified artery
tissues.
drugs, pollutant permeation, mechanism action of penetration
enhancers, etc. [117]. Histochemical analysis of biological tissues
have been reviewed, explores the advantages of Raman spec-
troscopy in endoscopic imaging and quantisation of biochemical
constituents during clinical studies. This involves disease diagnosis
which involves analysis of tissue proteins, lens, cornea, blood con-
stituents, biological stones, hard tissues, arterial disease etc. [31].
Cancer studies in gynaecological tissues, soft tissues, breast, colon,
bladder and brain has also been summarized [118].
A method had been reported on the use of near-infrared (NIR)
Fourier transform (FT) Raman spectroscopy to analyse normal
human epidermal keratinocytes before and after malignant trans-
formation. Raman spectral differences between isolated DNA of
immortalized and malignant transformed cell indicates some spe-
cific alteration during malignant transformation. Thus this make
possible to observe progressive structural changes in either DNA or
protein components of cancerous tissues. These changes in Raman
spectrum can build area of interest in the evaluation of newer anti-
cancer agents [119].
Raman spectra of various tissues were used to differentiate
between normal and diseased ones, as well as different chemical
states of organs and cells. Even white and grey matter of the brain
can be easily differentiated with micrometer resolution in spite
of being complex and heterogeneous. This can furnish an open-
ing in the diagnosis of brain related disorders like Parkinson’s and
Alzheimer’s disease by examining the differences between healthy
and diseased tissues [120]. Studies have also revealed the facts, that
the Raman band at 1628 cm−1 indicates the presence of b-amylose
proteins obtained from post-mortem paraffinated brain tissues,
thought to be responsible for the onset of Alzheimer’s disease [121].
In vivo diagnosis of atherosclerosis in human arteries has
been done by using Near-infrared Raman spectroscopy (NIRS). It
develops with the deposition of calcified mineral layers which
decreases the elasticity of the artery. Each one of these biochemical
depositions give a well distinct Raman spectrum which provides
the information to differentiate between calcified artery tissue,
atherosclerotic and non-pathological calcified mineral layer (Fig. 8).
Thus, it is possible to obtain satisfactory tissue classification for
in vivo clinical applications [122]. A similar kind of study has been
Fig. 9. Raman spectra of stomach mucosal tissues: (a) mean spectra of (—) normal
and (- - -) malignant tissues.
performed in vivo using optical fibre technology. The Raman spec-
tra were collected from normal and atherosclerotic coronary artery
samples in different stages of disease progression from explanted
transplant recipient hearts. This provides information about the
morphologic composition of intact human coronary artery without
the need of excision and microscopic examination [123]. Moreover,
it can also provide a new and important means for analyzing the
chemical composition (lipid and calcium salt content) of the arte-
rial wall, which may help in selecting appropriate treatment [124].
Similarly, Raman technique can be applied to quantitative analy-
sis of cholesterol and cholesteryl esters in human atherosclerotic
lesions [125].
Raman techniques can be used as an alternative tool to can-
cer detection, as it has been used for determination of the nature
of tumour (as benign and malignant tumour has different spec-
tra) and examination of paraffin-embedded skin biopsies. Raman
spectra provide wealthy information about the chemical composi-
tion of biological samples by admitting Raman signature to every
constituent. This can also help in building proper strategies dur-
ing treatment of cancer by targeting the specific site. [126–131].
Moreover, the feasibility of discriminating normal and malignant
stomach mucosal tissues has been studied. The mean Raman spec-
tra of normal and malignant tissues exhibit significant differences
in amide I, -CH2 , and amide III regions. The major spectral variation
of normal tissue with respect to malignant tissue was observed
due to weak amide I, slightly red shifted -CH2 , an intense band at
1303 cm−1 and hump at 1276 cm−1 . It has also been observed that
the normal tissue has high lipid content, while malignant tissue is
rich in protein, which can help in differentiation (Fig. 9) [132].
The Raman spectroscopy method was also validated for the dis-
crimination of normal and malignant tissues in cervical cancers.
It was observed that normal cervix tissues were characterized by
strong, broad amide I, broader amide III and strong peaks at 853
and 938 cm−1 , which can be attributed to structural proteins such
as collagen. While the malignant tissue spectra with respect to
normal tissue was relatively weaker and sharper amide I, minor
red shift in -CH2 and sharper amide III, indicated the presence of
Deoxyribonucleic acid (DNA), lipids and non-collagenous proteins
[133]. It has also shown good results during the evaluation of renal
tumours, provided complete and accurate information of differ-
entiation between normal and tumoural renal tissue, low-grade
and high-grade renal tumours, and histologic subtype of renal cell
carcinoma [134].
Even kidney stones which form due to the reduction of cys-
tine to cysteine, can be well diagnosed with the help of Raman
instruments [135]. Analysis of calcium oxalate-type kidney stones
is also possible using Raman techniques [136]. Thus, Raman spec-
troscopy can provide important information about the molecular
and supramolecular structure of living tissues including hard tis-
 R.S. Das, Y.K. Agrawal / Vibrational Spectroscopy 57 (2011) 163–176
sues like bone [137–139]. SERS has been proved useful in detecting
the specific Anthrax biomarker, protective antigen (PA), after using
a short 16-amino acid peptide chain in place of an antibody as it
has been observed that the peptides are more stable than antibod-
ies under various biological conditions and are easily synthesized
for a specific target. Thus, the peptides were made conjugated
on gold nano-particles labelled with a Raman tag (dithiobis-N-
succinimidyl propionate, DTSP) to specifically recognize target
biomarkers against biological and environmental pathogens [141].
4.4. Materials science
Among different vibrational techniques, Raman spectroscopy
appears as privileged method for the analysis in material sci-
ence. This field provides a varied and challenging array of samples
ranging from Super-conductors to Archaeological materials. Being
as indispensable characterization procedure, Raman technique
is readily applicable to any material system. Thus, this section
explores the tremendous possibilities of Raman signature in the
area of material science [140].
4.4.1. Superconductors and Semiconductors
Semiconductors are materials that have intermediate electrical
conductivity between that of metals (conductors) and insulators
(non-conductors). Raman measurements have been reported effec-
tive for structural characterization and investigation of properties
of semiconductor [141,142]. The effect of reduced dimensionality
on the shape, size and position of the first order phonon bands of
semiconductors can also be described through their Raman spec-
trum. The same has been used for the study of non-destructive
measurement stress, crystal lattice disorder, phase-separation of
supersaturated solid solutions and homogeneity of materials [143].
Likewise, superconducting materials have also been widely studied
through Raman spectroscopy [144,145]. Study of semiconduc-
tors such as nanowires and nanocones revealed that the Raman
enhancements are diameter, wavelength and polarization depen-
dent, which can play an important role for engineered photonic and
sensing applications [146].
4.4.2. Carbonaceous materials
Natural carbon exhibits its distinct properties in two forms; the
first is diamond which is an insulator having the greatest mechan-
ical strength and the second is graphite, a brittle material that
conducts electricity at room temperature. While recently synthe-
sized carbon form that is carbon nanotube (CNT), contains both
these properties (strength and conductivity). Raman scattering is
one of the prime techniques for the characterization of carbon
nanotube, which includes study related to their vibrational, elec-
tronic and optical properties. Even metallic and semiconducting
tubes can be distinguished from their high-energy Raman spec-
tra that can also help in their characterization [147,148]. A sharp
Raman band for the cubical structure of diamond was also observed
at 1332 cm−1 [149]. Thus, it is also possible to study the defec-
tive structures of diamonds and other similar materials. Likewise,
spectra of carbon nanotubes and fullerene type carbonaceous mate-
rials can provide various information regarding their structure and
properties [150].
Raman spectra of nine different carbonaceous materials of astro-
nomical relevance have also been determined. In which eight out
of nine samples showed two main bands falling around 1600 and
1350 cm−1 . This implies that all the materials are composed of ran-
domly oriented structural units varying from 50 to 80 Å in size.
Some bands were also observed between 800 and 1700 cm−1 which
is of C C or CHn (n = 1, 2, 3) functional groups, provides informa-
tion about astronomically relevant materials [151]. Laser-Raman
171
spectra of carbonized coals, carbons, and graphite have also been
observed near 1590 and 1360 cm−1 [152].
4.4.3. Polymers
Characterization of the structure, environment and dynamics
of polymeric materials has also been obtained through Raman
spectroscopy [153]. Study of the chemical composition and struc-
ture of polymer materials such as Kevlar, involves illuminating
the surface with an incident laser light. This helped in studying
its characteristics scattering including the effect of incident wave-
length, polarization and laser power. This allows the development
of non-destructive evaluation technologies based on the interac-
tion of laser light with Kevlar for detection of in situ deterioration
[154]. Raman characterization of plastic explosive materials (tri-
nitro toluene and di-nitro toluene) have been reported by SERS
using 3D alumina membrane with cylindrical nanopores which
allows molecular level detection of trace amount of explosives and
other relevant chemical compounds [155].
Raman spectra and cross sections of spider silk fibres produced
by two different species were measured as one of the characteris-
tic feature to differentiate between species. Comparison of polymer
fibres formed by electro-spinning (technique which produces con-
tinuous fibres from a polymer solution through the action of an
electrical field) from mixture of polymer solutions produced homo-
geneous Raman spectra [156]. The spectra can be obtained even in
cases of immiscible polymers through confocal Raman microscopy
for analyzing the structure of electrospun fibres [31].
4.4.4. Environmental materials
Hazardous materials in trace amount can find their way in the
water system causes very severe effects. Resonance Raman spec-
troscopy can be applied for on-line monitoring of NO2 − /NO3 − in
wastewater (using such excitation radiation practically eliminates
fluorescence from other species present in the wastewater) with
the detection limits below 200 ppb for both the analytes [157]. SERS
can be applied to detect contaminants at femtomolar concentra-
tions over practical time scales in highly controlled environment.
Qualitative identification of TNT (2,4,6-trinitrotoluene), fullerenes
and numerous other compounds often rely on detection of a specific
SERS spectrum to infer the presence of contaminants in environ-
mental samples [158]. Even uranium can be detected in aqueous
media by SERS technique. A method has been developed for the
rapid screening of uranium in environmental samples, using a new
SERS substrate, based on (aminomethyl)phosphonic acid (APA)-
modified gold nanoparticles. The intensity of uranyl band was
observed at 830 cm−1 which is proportional to the concentration of
uranium in solution, achieved detection limit close to 8 × 10−7 M
with good reproducibility. This allows the detection of uranium
in a highly contaminated groundwater with dissolved salts [159].
Similarly, the same technique has been reported for the detection
and monitoring of perchlorate (ClO4 − ) in groundwater and surface
water with detection limit of 10–100 ␮g/L [160].
Laser spectroscopy with tunable IR laser source is used as
sensitive techniques for the detection of various molecules in
atmosphere. This was made possible by photoacoustic Raman spec-
troscopy (PARS), where the thermal relaxation of molecules from
the upper vibrational state was detected as an acoustic wave.
Accompanied with two visible lasers with frequencies (ωp ) and
(ωs ) were used instead of an IR laser, and the frequency difference
(ωp − ωs ) was tuned equal to the Raman shift frequency (ωR ) [161].
Likewise, nonlinear Raman spectroscopy can be applied for
monitoring various kinds of factories, chemical plants, gas
pipelines, any leakages of inflammable gases such as H2 , CH4 , and
H2 S. This gives the possibility to excite the vibrational level of
gas molecules in the IR region without the use of tunable IR laser
[162]. Optical detection methods in combination with Raman spec-
172 R.S. Das, Y.K. Agrawal / Vibrational Spectroscopy 57 (2011) 163–176
troscopy, fluorescence spectroscopy and digital imaging have been
experimented to detect the trace levels of harmful biological agents
like Bacillus anthracis, Yersinia pestis, and Francisella tularensis by
their Raman signatures [163].
4.4.5. Crystalline study
Micro-Raman spectroscopy has been used to study the spatial
variation of internal stress in synthetic diamond, including single
crystal and polycrystalline specimens. It also provides direct infor-
mation on the state of stress and was successfully employed to
examine the stress distribution around cracks produced by inden-
tation in the diamond [164]. Coherent Raman measurements of
nanoshock in solids have been reported for the determination of
shock-induced deformation in materials [165].
Specimens of sintered TiO2 ceramics were investigated on the
basis of IR Raman signals for detecting the impure grains of Al2 O3
(including composition, size and shape variations). Micro-Raman
configuration can offer the ability to monitor changes in chem-
ical composition and morphology at microscale which helps in
investigation and characterization of the materials. Moreover, this
technique has the ability to recognise oxidizing and reducing
production conditions, potentially save costs and optimise raw
material consumption [166]. Temperature-dependent characteris-
tic spectra of a BSO crystal were studied by Raman spectroscopy.
It has been observed that the vibration mode of the longest bond
Bi-O (1) in crystal shifts from 542 to 512 cm−1 when tempera-
ture was increased (from room temperature to 1123 K). In addition,
the 58 cm−1 mode of Bi atoms (in crystal lattice) decreases rapidly
when the temperature is higher than 873 K, indicates the breaking
of crystal at high temperature [167]. Study of Raman spectra of crys-
talline domoic acid (DA) confirms its existence in zwitterionic form.
DA is a natural occurring neurotoxin present in the marine ecosys-
tem. The variations in the spectra were also observed attributed by
hydration, the degree of protonation and crystallinity [168].
4.4.6. Molecules and Molecular systems
By plotting the excitation profile of Raman bands can help in the
determination of coupling of electronic and vibrational motions
of the molecule. In addition, structural changes results from a
change in the electronic state of the scattering species and multiple
metal–metal (MM) bonding can also be studied. Unique molec-
ular information regarding oxygen–nitrogen, dinitrogen bridged
complexes, and linear chain halogen-bridged complexes have also
been examined successfully [169]. Apart from these applications it
was also used to examine the solubility mechanism of fluorine in
depolymerized silicate liquids, quenched glasses in CaO–CaFr–SiO
system as an evidence for concomitant polymerization of the liq-
uid [170]. Moreover, Raman scattering is a very sensitive technique
to probe local atomic environments. Indeed, the properties of the
vibrational modes are basically determined by the mass, bond type
and symmetry of constituting atoms in the elemental unit [171].
Laser Raman spectroscopy holds great promise for trace level detec-
tion of surface planetary minerals especially oxy-anionic mineral
such as silicates, carbonates, sulphates and phosphates [172].
A wide range of coloured main group metals, transition metal
co-ordination and transition metal organometallic complexes has
been studied by FT-Raman spectroscopy which gives good quality
spectra in less time. This suggests its utility as a routine spectro-
scopic tool for inorganic as well as organic research and teaching
laboratories [173]. The structure of supercritical H2 O has also been
studied through Raman spectroscopy [174].
4.4.7. Archaeological material
Analysis of archaeological objects by Raman spectroscopy is a
rapidly developing technique, provides non-destructive examina-
tion and investigation of such invaluable objects. Non-destructive
analysis of ancient skin tissue samples has been extensively stud-
ied by Raman spectroscopy. Partially desiccated or wet samples of
skin were analysed when subjected to varying degrees of degrada-
tion (depending on the burial environment). Comparisons of Raman
spectra between healthy and diseased contemporary skin speci-
mens make possible to detect whether the skin preservation used
were natural or assisted by chemicals (used in mummification pro-
cesses). This can either represent a natural process of drying, which
is often seen for mummies found in hot or cold deserts, or an
artificial process where the body has been treated with different
substances to promote preservation. In all the spectra of mummi-
fied skin observed changes due to degradation in protein structure.
A progressive loss of protein amide I (1640–1680 cm−1 ) and amide
III (1220–1290 cm−1 ) band intensities indicates loss of protein and
changes in the secondary protein structure. This implies that most
of the changes in molecular structure of the skin took place in a
relatively short time interval during the natural mummification
process. Such information is of great importance in archaeological
conservation and can shed light on historical practices also [175].
Similar kind of study has been done for characterization of
about 5200 year old skin sample. Contemporary skin was used for
comparison in freeze-dried condition (for giving the similar envi-
ronment) and its molecular structure was compared with that of
Iceman skin. The results showed that the proteinaceous moiety
of the ancient skin have degraded considerably, although olefinic
bonds might oxidised with less or no alteration in the lipoidal com-
ponent. It has been observed that there were significant alterations
in the nature of lipid component of ancient skin. The spectral region
3200 cm−1 to 2700 cm−1 provides C–H stretching modes of the lipid
alkyl chains, observed with reduced intensity and reduced width in
case of old skin sample indicates that the tissue has lost some of its
components. In addition to this the loss of olefinic C–H stretching
(around 3060 cm−1 ) indicates that the unsaturated lipid compo-
nent of older skin has degraded [176].
Pigment identification in artefacts has been successfully studied
by obtaining their Raman spectra in order to identify and differen-
tiate ochres (most important earth pigments) found extensively
in Byzantine hagiography. Compared to other techniques such as
X-ray fluorescence, particle-induced X-ray, gamma-ray emission
and scanning electron microscopy, Raman spectroscopy has proved
to be more significant particularly with respect to analysis time,
sensitivity, specificity, amount of sample, spatial resolution, and
immunity to interference [177]. In addition, pigment structure
and degradation of manuscript has been identified using Raman
techniques [178,179]. A significant effect was observed in the con-
struction of novel micro-Raman-dedicated spectrometers united
with holographic notch filters and charge coupled device detectors.
This made it a powerful technique for studying variety of materials
ranging from epitaxial semiconductor thin films to optoelectronics
devices and also from biomaterials of interest to medical science
[171].
4.5. Pharmaceuticals
Mid-IR and Raman spectroscopy are versatile tools in pharma-
ceutics and bio-pharmaceutics, with a wide field of applications
ranging from characterization of drug formulations to elucidation
of kinetic processes in drug delivery (Fig. 10). It could also provide
fast detection and identification of counterfeit medicines. Since
many blister package materials provide suitable spectral windows
for both exciting and scattering radiations [180].
New developments in applications of Raman spectroscopy for
studying drug delivery systems, in particular topical drug deliv-
ery have been reviewed [181]. Well-established standard methods
coupled with Raman spectroscopy enables to study drug release in
semisolid formulations, drug penetration, and influence of penetra-
 R.S. Das, Y.K. Agrawal / Vibrational Spectroscopy 57 (2011) 163–176
Fig. 10. An FT-Raman spectrum for characterization of antidepressant drugs belongs
to the same class (selective serotonin reuptake inhibitors (SSRIs)): (A) Citalopram,
(B) Sertraline.
tion modifiers. This approach is also applicable for in vivo studies
and in characterizing the structure of colloidal drug carrier systems.
The interaction of therapeutic drugs and their target biomolecules
is of great interest in pharmaceutical research. Shift in Raman bands
and changes in band intensity are used to study the kinetics and
mechanism of these drug-target reactions [182–185].
Quality testing for tablet composition and uniformity are exces-
sively critical issues at manufacturing stage in pharmaceutical
industries because composition and uniformity are the main con-
cerns. A non-destructive and cost-effective analysis by Raman
technique played an important role for increasing the quality
of products. Thus, Raman spectroscopy reduces time to anal-
yse active ingredient composition of tablets while increasing the
number of tablets tested with increased confidence level [186].
The crystal forms present in the drug products can be identi-
fied by NIR FT-Raman spectroscopy. Determination of polymorphic
forms in a number of commercial drug products have been
obtained containing polymorphic drug compounds like sorbitol,
mannitol, famotidine, acemetacin, carbamazepine, meprobamate
and phenylbutazone. The identification of crystal forms in drug
products can be carried out on basis of comparison of peak posi-
tions, intensities and shapes of reference spectra with the spectra
obtained from the drug product by their varying peak positions.
Thus, supports the feasibility of analytical assays to identify crys-
tal forms in drug product and can be easily exploited routinely for
monitoring phase changes [187]. The quantitative measurements
for solid state of pharmaceutical compounds have also been stud-
ied by Raman spectroscopy. Most of the organic molecules exhibits
clear and well-resolved bands offer possibility of both qualita-
tive as well as quantitative analysis. This feature is essential for
pharmaceutical applications where final dosage forms are typically
complex blends of numerous additives and excipients. Thus, Raman
spectroscopy supports as a reliable tool in various industrial appli-
cations for monitoring and controlling solid elaboration processes
[188].
Quantitative analysis of active pharmaceutical ingredient (API)
in pharmaceutical products is the most essential part of pharma-
ceutical analysis. A simple linear regression method is developed
and statistically validated using FT-Raman spectroscopy for the
direct and non-destructive quantitative analysis that too without
sample preparation of the active pharmaceutical ingredient (API),
173
Fig. 11. Example of API peak detection and tablet peak detection which has been
correctly identified and symbolised, reflects the genuine tablet, not the counter-
feited.
medroxyprogesterone acetate (MPA) in an aqueous pharmaceu-
tical suspension. The results thus obtained were compared with
simple linear regression model of HPLC. The results suggested the
reliability of the FT-Raman over the HPLC method [189]. Similarly
quantitative analysis of amiodarone hydrochloride based on solu-
tions with known concentrations has also been done, obtained limit
of detection (LOD) = 2.11 mg/ml. However, this limit is much higher
than the corresponding limit obtained from HPLC methodology, but
the proposed method was also validated on Raman spectroscopy
by evaluating the linearity of the calibration line as well as its
accuracy and precision [190]. Raman spectroscopy has also shown
its utility in Process analytical technologies (PAT) commonly used
for manufacturing processes, for monitoring the synthesis of API,
identification of raw materials and quantitative determination in
finished dosage forms [191]. Likewise, the analysis of pharmaceu-
tical solid dosage forms can also be carried out easily. The study
has been made for identification of tablets with the applications in
release of final products in quality control and detection of coun-
terfeits. Extending, it also gives application in the development of
calibration strategies for automatic identification of tablets from
their API peak stored in database spectra collection. This can per-
mit the identification of tablets and other pharmaceutical products
in future to discriminate counterfeits from genuine stuff (Fig. 11)
[192].
Determination of diphenhydramine hydrochloride (DPH), the
active ingredient of Benadryl has been studied through FT-Raman
spectroscopy. The reliability of this method was verified by testing
it against the conventional HPLC, obtained with satisfactory results
even in the presence of numerous excipients. The main difference
between these two techniques was with their LOD = 0.81 ␮g/ml
for HPLC, while LOD = 0.14 mg/ml for FT-Raman spectroscopy, but
involves tedious sample preparation in former process [193]. Stud-
ies related to structure and interactions of the drug molecules can
be well resolved with Raman spectroscopy by examining the shift
in characteristic vibrational frequencies. This involves drug reactiv-
ity, such as hydrogen bonding, proton transfer, charge transfer and
ion-molecule attraction, allows better understanding of the binding
properties of drug in biological medium [194].
Other than drug analysis, there are also various substances
which have profound use in pharmaceutical industries like sur-
factants, detergents, cosmetic products, etc. Their analysis is of
particular interest with Raman spectroscopy as they contain water,
which is a weak Raman scatterer and does not contribute signif-
icantly to solvent effects or band broadening phenomena. Thus,
solutes can be measured in aqueous solutions facilitating more
174 R.S. Das, Y.K. Agrawal / Vibrational Spectroscopy 57 (2011) 163–176
Fig. 12. (A) Typical Raman spectrum of C60 molecule measured with 514.5 nm laser excitation; (B) typical Raman spectrum of single-walled carbon nanotubes.
effective spectral subtractions or manipulations and quantitative
analysis without sample preparation [195].
4.6. Nanotechnology
Raman spectroscopy undoubtedly emerged as a relevant tool
for probing and characterization of nanomaterials like nanosensor,
nanotube and nanowire, as these are intensively studied in vari-
ous areas of science due to their unique mechanical, electrical and
chemical properties. For better understanding of nanomaterials like
nanoceramic, nanocomposite, glassy material and semiconductor,
techniques like micro Raman spectroscopy have been successfully
applied for their better characterization [141,196,197].
Based on theoretical considerations and comparisons with
absorption and luminescence data RRS cross-sections were derived
and used to estimate the relative abundance of different species
in the sample. It was observed that the total level of semiconduc-
tive single wall carbon nano tubes (SWCNTs) was about 11 times
higher than that of the metallic species [147]. The same approaches
was used to test the selectivity of DNA wrapping as a purification
method which has given a mixture enriched with semiconductive
SWCNTs [198].
Raman spectroscopy is one of the leading investigation
techniques for the characterization of covalently functionalized
metallic and semiconducting carbon nano tubes (CNTs). However,
CNTs need to be functionalized for handling, sorting (according
to electronic or structural characteristics) and property adjust-
ments [199,200]. Semi-empirical quantum simulations of water
or methanol absorption on CNTs and fullerenes have been stud-
ied profoundly with experimental Raman spectra, exploring the
applications of CNTs and fullerenes in selectively absorbing nano-
filters and substrates. The Raman spectrum of both the species can
provide valuable information regarding the structure and proper-
ties of carbon species (Fig. 12) [201]. In addition, one of the latest
applications of Nanotechnology is the development of improved
chemical and biological sensors. Recent developments in the design
and application of two types of optical nanosensors have been
reviewed. It was based on localized surface plasmon resonance
(LSPR) spectroscopy which measures change in local refractive
index and surface-enhanced Raman scattering (SERS) by measuring
the vibrational bands of the target molecule [202].
5. Conclusion
Vibrational spectroscopy is an excellent method for identify-
ing substances by providing unique fingerprint spectra. Raman
spectroscopy is one of the fastest and non-destructive analyti-
cal technique that give the vibrational spectrum and physical or
chemical information of virtually any matrix in any state of matter.
Thus, in this article we have discussed with the various possibilities
and potentials of Raman measurements, with special emphasis on
recent technical developments. In recent years, this technique has
been explored with much technical advancement like wide range
of laser wavelengths, sampling conditions, instrumentation and
data-processing methods, applicable in various fields of science.
Additionally, Raman signals can be collected with a small probe
head linked with the (portable) apparatus by optical fibre gives the
flexibility for online process monitoring. The growing interest in
Raman spectroscopy is probably due to its major advantages as it
brings direct results of the analysis without sample preparation and
easy interpretation, making it as a time saving and cost-effective
technique.
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