OBJECTIVES OF THE STUDY

Vibrational spectroscopic techniques including FTIR spectroscopy, are potential

tools for non-invasive optical tissue diagnosis and protein conformations.
The applications of spectroscopic techniques in biological studies have
increased a great deal in recent years.
A considerably wide field of medical and biological studies has been covered
by spectroscopic methods in the past few years. It is strongly believed that in studies
related to spectroscopic techniques, both the reliable experimental procedure and
characterization of spectral peak positions and their assignment along with accurate
peak detection and definition are of great importance. Many scientists have used
different techniques. It appears that there is a remarkable parity in their spectral
interpretation of comparable ones in their collected spectra of different types of the
samples.
Due to these facts mentioned above, we have a plan to study this sophisticated
technique to apply to different fields of research.
We aim to provide a detailed database for the clinicians and researchers who are
working in the field of industry, biology, chemistry and geology.

BIOMEDICAL APPLICATION OF FOURIER TRANSFORM INFRARED

SPECTROSCOPY IN DIAGNOSIS OF DIFFERENT COMPLEX HUMAN
BODY DISORDERS
Introduction
The ability to diagnose the early onset of disease, rapidly, non-invasively and
unequivocally has multiple benefits. Some of the clinical findings currently in use are
not reliable. There are so many diseases covered under metabolic disturbances. It is
very important to measure metabolism directly.
One of the strategies employed by the emergent science of metabolism is
metabolic fingerprinting. It involves rapid, high-throughout global analysis to
discriminate between the different biological blood samples of complex body disorders.
It has been reported that whether clinical, biophysical or biochemical in nature,
the clinical tests have been inconsistent and contradictory. Many clinicians thought
these tests are unrealistic. Research continues for the optimum diagnostic test for the
1
complex body disorders. Fourier transform infrared spectroscopy was developed in
order to overcome the limitations encountered with dispersive instruments. The main
problem was very slow scanning process. A method for measuring all of the infrared
frequencies simultaneously was needed. The solution of this problem was developed
which employed a very simple optical device called n interferometer. The
interferometer produces a unique type of signal which has all the infrared frequencies
“encoded” into it. Signal is measured in one second only with no disturbances.
Fourier transform infrared spectroscopy has considerable potential as a rapid
high throughput screening method for the diagnosis of complex human body disorders.
Other options should be considered for the screening of the diseases. A more
holistic approach can be adopted. Method should be rapid, reagent free, non-
destructive, high throughput, relatively inexpensive and require a minimal amount of
background training. These features of the requirement could be provided by using
spectroscopy.
A routine spectroscopy should be portable and applicable to point of core
medicine. Infrared and Raman spectroscopy can solve the purpose of differential
diagnosis of human body disorders.

Fourier Transform Infrared Spectroscopy (FTIR)

In biological system, usually the rotational fine spectrum does not appear and
one is encountered with vibrational studies. Due to this fact we are giving our main
stress to Fourier Transform infrared Spectroscopic technique of spectroscopy.
FTIR spectroscopy is a well established and constantly developing analytical
method. It allows for rapid, high-throughput, non-destructive analysis of a wide range
of sample type [1, 2].
Chemical bonds absorb internal energy at specific frequencies (or wavelengths),
the basic structure of compounds can be determined by the spectral locations
determined by the spectral locations of their infrared absorptions.
If we plot a graph between compound’s infrared transmission and frequency, a
spectrum is obtained which is known as fingerprint. We can compare this spectrum
with reference spectrum and get a identification of the material.

2
 Unknown spectra can be analyzed to determine the base of the material of the
unknown by comparing their spectra to spectral spectra of known materials. We can
store these spectra in our computer based library.
The resulting spectra produce a profile of the sample, a unique molecular
fingerprint can be used to easily screen and scan samples for many different
components. This technique is used to detect functional groups and characterizing
materials.
Infrared spectroscopy has been a workhorse technique for material analysis in
the laboratory for several years. Infrared spectrum represents a fingerprint of a sample
with absorption peaks which correspond to the frequencies of vibrations between the
bonds of the atoms making up the material. Each different material is unique
combination of atoms, no two compounds produce the exact same infrared spectrum.
Infrared spectroscopy can result in a positive indication of every different kind of
material, size of the peaks in the spectrum is a direct indication of the amount of
material present. Infrared technique is an excellent tool for the quantative analysis these
days.
It is not possible to excite the vibrational levels of the molecule by the visible
light, even though it has high energy. We can however excite these levels in the
infrared region (4000 cm-1 to 400 cm-1). The atoms remain in unison against attractive
and repulsive forces existing in a molecule with the help of different types of bonds at
same distances. One needs the energy to affect or break bonds in stretching or for
altering the angle between them. Interaction with electromagnetic radiations leads to
transitions from lower energy state to higher energy state.
The major components of energy of a molecule are
(i) The vibration of the constituents.
(ii) The rotations of the molecules.
(iii) The motion of the electrons in the molecule.
Energy transition must satisfy the Bohr condition
F = − K (t − req. ) = − Kx (Hook’s law) …(1)
K stands for the force component.
r for the distance between the nuclei.
The energy of the vibration is

3
 1
E = kx 2 …(2)
2
The energy absorbed in transitions from state E1 to state E2 is
∆ = E2 − E1 = hν …(3)

If the system behaves like a harmonic oscillatory of mass µ R , its frequency in

Hertz will be

1 k
ν=
2π µR

ν 1 k
or =υ cm-1 …(4)
c 2π c µ R

M1M 2
µR = is reduced mass.
M1 + M 2
The vibrational energy is given by
 1  1  hc
Ev =  υ +  hυ = υ +  Joule …(5)
 2  2 λ
ν is the vibrational quantum number. The lowest energy will be at ν = 0 . The
constraints of selection rules allow only the heteronuclear diatomic molecules to give
vibrational spectra.
The force constant [3] is given by
k = 4π 2 c 2 µ Rν 2 Nm-1 …(6)
FTIR spectroscopy identifies chemical bonds in a molecule by producing an
infrared spectrum. FTIR generates an infrared spectral scan of samples that absorb
infrared light. Metals do not absorb infrared light. Polymers can be scanned with FTIR.
A material’s absorbance of infrared light at different frequencies produce a
unique identification based upon the frequencies at which the material absorb infrared
light and the intensity of those absorptions. The resulting spectrum is typically specific
to a general class of material.
Unknown spectra can be analysed to determine the base material of the
unknown by comparing their spectra with spectra of known materials.
The resulting spectra produce a profile of the samples. A unique molecular
fingerprint can be used to easily screen and scan samples for many different
compounds.
4
 FTIR spectroscopy is used to identify compounds or investigate sample
composition. It exploits the fact that molecules have specific frequencies at which they
rotate or vibrate corresponding to discrete energy levels (vibrational modes). These
resonant frequencies are determined by the shape of the molecular potential energy
surfaces, the masses of the atoms and by the associated vibronic coupling. If in a
molecule for a vibrational mode to be infrared active, thus it must be associated with
changes in the permanent dipole.
When the molecular Hamiltonian corresponding to the electronic ground state
can be approximated by a harmonic oscillator problem in the neighbourhood of the
equilibrium molecular geometry under the Born-oppenheimer and harmonic
approximations. The resonant frequencies may be determined by the normal modes
corresponding to the molecular electronic ground state potential energy surface. These
frequencies of resonance can be in a first approach related to the strength of the bond
and the mass of the atoms at either end of it. Vibrational frequencies can be associated
with a particular bond type.
Simple diatomic molecules have only one bond which may stretch. Complex
molecules have many bonds, and vibrations can be conjugated, leading to infrared
absorptions at characteristic frequencies that may be related to chemical groups.
In the organic compounds, the CH2 group is very common. This group contains
atoms and they can vibrate in different ways :
(a) Symmetrical and antisymetrical
(b) Stretching
(c) Scissoring
(d) Rocking
(e) Wagging
(f) Twisting
The technique of spectroscopy works exclusively on samples with covalent
bonds. Simple spectra can be obtained from the samples with few IR active bond and
high levels of purity. More complex molecular structures lead to more absorption bands
and more complex spectra. FTIR has been used for characterization of very complex
mixtures.
FTIR is useful for identifying chemicals that are either organic or inorganic. It
can be utilized to quantitative compounds of unknown mixture. It can be applied to the
5
analysis of solids, liquids and gases. It can be used to identify the types of chemical
bonds (functional groups). The wavelength of light absorbed is characteristic of the
chemical bond as can be seen in the spectrum. Infrared spectroscopy reveals
information about the vibrational states of a molecule. Intensity and spectral position of
IR absorption allow the identification of structural elements of molecule. Among them
are typical functional groups, hydrogen bonding, but also determination of
conformations or even investigation of chemical reactions.
Typical vibrations of functional groups make infrared spectroscopy also an
important analytical tool.
In the gas phase a rotational fine structure can often be observed from which the
moment of inertia (M.I.) of the molecules can be determined.
Infrared spectroscopy is widely used in both research and industry as a simple
and reliable technique for measurement, quality control and dynamic measurement.
This technique is used in forensic analysis in both criminal and civil cases.
By measuring at a specific frequency over time, changes in the character or
quantity of a particular bond can be measured. The functional groups within the sample
will absorb the infrared radiation and vibrate in one of a number of ways, either
stretching, bending, deformation or combination vibrations [4, 5]. These
absorptions/vibrations can be correlated directly to biochemical species. The resultant
infrared absorption spectrum can be described as an infrared “finger print” property of
any biochemical or chemical substance.
Researchers have concentrated on the mid IR port of the electromagnetic
spectrum (4000 cm-1 – 600 cm-1) for the diagnosis of complex body disorders.
In the biological terminology, the vibrations in the wave number region 2800
cm-1 – 3050 cm-1 can be ascribed to CH2 and CH3, stretching vibrations from fatty acids.
Vibrations in the region 1500 cm-1 – 1750 cm-1 are ascribed to C=O, OOH and C-N
from proteins and peptides [6]. This region is called amide I and II bands region. The
region 4000 cm-1 – 400 cm-1 is very interesting region called mid-infrared region of the
spectrum for the study of the organic compounds because the absorption bands are due
to the vibration of a particular functional grouping. The mid-infrared spectroscopy has
been used for the structural identification or quantitative determination of the finger
print of the organic compounds. Their functional groups display characteristic
vibrational absorption frequencies in the infrared region of electromagnetic spectrum.
6
The exact location of the corresponding bands depends on the influence of the rest of
molecules. Mid-infrared region is very useful and reliable in quantitative analysis
applications.
The intensities of the bands in the spectrum are proportional to the
concentration of their respective functional groups
Lambert-Beer’s law shows
I = I 0 eε bc

I
= A = ε bc
I0

where, I 0 = Incident radiation; I = Transmitted radiation

A is the absorption of the band
b is the path length
ε is the molar proportionality constant called molar absorptivity. It is the
characteristic of each functional group.
c is the concentration of the functional group.
Lipid study is a subject that may strongly profit from FTIR, because they are
composed of functional groups showing characteristic absorption bands in this region
of electromagnetic spectrum [7].
FTIR spectroscopy requires only small amounts of proteins (1 mM) in a variety
of environments. High quality spectra can be recorded relatively easily without the
problem of background fluorescence, light scattering and problems related to size of the
proteins. By the application of mathematical approach, water absorption may be
subtracted.
The peptide group, the structural repeat unit of proteins gives characteristic
bands of nine types. These are amide A, amide B, amide I, amide II, amide III,
amide IV, amide V, amide VI, amide A band (about 3500 cm -1) and amide B (about
3100 cm-1) originate from a Fermi resonance between the first overtone of amide II and
the N-H stretching vibration. Amide I and amide II bands are two major bands of the
protein infrared spectrum.
The amide I band (between 1600 cm-1 and 1700 cm-1) is associated with the
C=O stretching vibration (70-85%). Due to this fascinating property this is directly
related to the backbone conformation.

7
 Amide II band results from the N-H bending vibration (40-60%) and from the
C-N stretching vibration (18-40%). This band is conformationally sensitive.
Amide III and amide IV are very complex bands resulting from a mixture of
several coordinate displacements.
The out of plane motions are found in amide V and VI bands.
Amide A is with more than 95% due to the N-H stretching vibration. This mode
of vibration does not depend on the backbone conformation but is very sensitive to the
strength of a hydrogen bond. It has wave numbers between 3225 cm-1 and 3280 cm-1 for
hydrogen bond lengths between 2.69 to 2.85 Å [10].
Amide I is the most intense absorption band in proteins. It is governed by the
stretching vibrations of the C=O (70-85%) and C-N groups (10-20%). Its frequency is
found in the range between 1600 cm-1 – 1700 cm-1. The exact band position is
determined by the backbone conformation and the hydrogen bonding pattern.
Amide II is found in the region between 1510 cm -1 to 1580 cm-1. This region is
more complex than amide I. Amide II derives mainly from in-place N-H bending (40-
60% of potential energy). The rest of the potential energy arises from the C-N (18-
40%) and the C-C (about 10%) stretching vibrations. Amide III, V are very complex
bands dependent on the details of the force field, nature of the side chains and hydrogen
bonding. We may use these bonds for the limited use in the structural information.
The rapidity and reproducibility of FTIR can not be overstressed and due to its
holistic nature, this technique has been recognized as a valuable tool for metabolic
fingerprinting. Some of the researchers [11, 12] have reported in the literature
carbohydrates, amino acids, fatty acids, lipids, proteins and polysaccharides can be
analysed simultaneously.
FTIR spectroscopy is a vibrational spectroscopic technique that can be used to
optically probe the molecular changes associated with diseased samples [13, 14, 15].
FTIR mainly deals with non-aqueous samples. The physical effect of infrared is
created by absorption and mainly influences the dipole and ionic bonds such as O-H,
N-H and C=O. FTIR spectroscopy is due to changes in dipole moment during
molecular vibration.

Brief Summary of Experimental Research

A wide range of biological studies have been covered by FTIR analysis.

8
 These studies include cervix [16-24], lungs [20-27], breast [28, 29, 32], skin
[33-37], gastro-intestinal tissue [38-41], brain [42-44], oral tissue [45], lymphoid tissue
[46], lymphocytes (childhood leukemia) [47], non Hodgkin’s lymphoma [48], prostate
[49, 50], colon [51-54], fibroblasts [55], bacteria [56, 57], tumor cells [58], DNA [59],
anti-cancer drug [60], tissue processing [61], cancer detection [62], tissue preservation
[64], cytotoxicity and heating [64], plant tissue [65], gall stones [66], glucose
measurement [67] and bones [68].
Wood et al. [16] has reported on FTIR spectroscopy as a biodiagnostic tool for
cervical cancer. The spectra of the normal epithelial cells provide intense glycogen
bands at 1022 cm-1 and 1150 cm-1. A pronounced symmetric phosphate stretch was also
found at 1078 cm-1. Dysplastic or malignant transformations were pronounced
symmetric and asymmetric phosphate modes and a reduction in the glycogen bond
intensity. The potential of automated FTIR cervical screening technology in the clinical
environment has been demonstrated from the study of Wood et al. [16].
Chao et al. [42] applied infrared spectra of human central nervous system tissue
for diagnosis of Alzheimer’s disease (AD).
Jalkanen et al. [59] used vibration spectroscopy to study protein and DNA
structure, hydration and binding of biomolecules, as experimental approach.
Sahu and Mordechai [62] studied FTIR spectroscopy in a cancer detection. The
area of focus were the distinction of premalignant and malignant cells and tissues from
their normal state using specific parameters obtained from the spectra.
Lucassen et al. [35] and Barry et al. [37] used FTIR spectoscopy to measure
hydration of the stratum corneum. The determination of the hydration state of the skin
can provide the basic knowledge about the penetration and loss of water in the skin
stratum corneum.
Yoshida et al. [44] measured the FTIR spectra of brain microsomal membranes
prepared from rats fed under two dietary oil conditions with and without brightness-
discrimination learning tasks : one group fed α -linolenate deficient oil and the other
group fed the perilla oil.
Mossoba et al. [56] made an investigation on printing microarrays of bacteria
for identification by infrared microscopy.
Jalkanen et al. [59] used vibrational spectroscopy to study protein and DNA
structure, hydration, and binding of biomolecules, as a combined theoretical and
9
experimental approach. Amino acids, peptides and a variety of small molecules were
studied systematically.
It is believed that accurate peak definitions can have significant influence on
reliability of the results provided through different spectroscopic techniques. The
review of the literature indicate that most of the scientists have mainly used the
previously published research articles in order to define the data acquired from the
spectra collected. In biological studies, for instance, where a wide range of chemical
bonds and functional groups can be attributed to every single peak, finding appropriate
meanings, which can demonstrate the clinical importance of the technique and achieved
results, can be one of the most important steps in finalizing a spectroscopic research
work.
The technique of FTIR spectroscopy has become a technique of choice for
scientists interested in finding the chemical structural properties of natural and
synthetic tissues.
David and Royston have studied metabolic fingerprinting in disease diagnosis :
biomedical applications of infrared and Raman spectroscopy [13].
Eysel et al. [69] demonstrated the ability to determine a number of differences
in synovial fluids resulting from disease processes and moreover, associated specific
subregions of the infrared spectra with significant discriminatory power. CH x stretching
vibrations were dominated in 3500-2800 cm-1 range.
FTIR spectra of synovial fluids could be used as a diagnostic aid for arthiritic
disorders [69, 70, 71].
Infrared spectroscopy is generally used to generate holistic measurements of
biological samples, followed by some multivariate analysis. Thus, spectroscopy can be
used to detect specific chemicals.
Hyalurenic acid can be measured by the method of spectroscopy. This acid
leaks from the joint’s synovial fluid in osteoarthritis [69].
FTIR has been used for the examination of bacteria to the subspecies level [72],
differentiation of clinically relevant species [73].
Under the strategies of analysis of multivariate, FTIR has considerable potential
as a metabolic fingerprinting tool for the detection and diagnosis of disease or
dysfunction. Diem et al. [74] have reported that the significant number of studies have

10
been done so far on tissues, cell and biofluids in an emergent area of research termed as
infrared pathology.
Goodacre et al. [75] have referred the use of this sophisticated technique as
metabolic finger printing.
Several researchers have used FTIR to detect scrapie and BSE in serum [76, 77-
80].
Kneipp et al. [77, 78] have used FTIR technique to detect disease associated
molecular changes in Central Nervous System (CNS) tissue acquired from case and
control hamsters. Complex histological and molecular differences occur in TSF-
affected nervous tissue, such as changes in protein expression, alterations in gene
expression, and composition of membrane systems [78]. We can detect the changes in
multiple biomolecules by using FTIR technique.
Petibois et al. [81-86] have used this technique and studied the metabolic
profiling of athletes. They have used serum, blood and plasma for FTIR spectroscopy.
The toxicity of drug can be measured with the help of FTIR.
Narasimhan et al. have studied the diagnosis of renal stones with underlying
metabolic abnormalities using FTIR spectroscopy in children [87]. They found that this
tool of spectroscopy gave a clue to the nature of stones.
Le et al. [88] have studied this spectroscopic technique for the diagnosis of
gastric inflammation and malignancy in endoscopic biopsies based on Fourier
Transform Infrared Spectroscopy. They found that in the endoscopic biopsies, the
majority of spectral peaks were in the 1000 cm-1 to 1800 cm-1 region. These spectral
features were related to the changes in structure and composition of biological
molecules such as proteins, nucleic acids and fats.
Graham et al. [89] have studied the use of infrared spectrophotometry for
measuring body water spaces. They have determined the enrichments of the body fluids
from FTIR spectra in the range 1800 cm-1 – 2800 cm-1. This improved infrared method
for measuring deuterium enrichment in plasma and saliva requires no sample
preparation, is rapid, and has potential value to the clinician.
Melmiciuc et al. [90] have studied FTIR spectroscopy for the analysis of
vegetable tanned ancient leather. They found that the IR spectra of the leather extract
contains in addition to a series of bands that are common to those found for the oak
extract.
11
 Hameed et al. [91] have studied the applications of FTIR spectroscopy in the
determination of antioxidant efficacy in sunflower oil and reported that an atmospheric
oxygen can react spontaneously with lipids and other organic compounds causing
structural degradation, which is ultimately responsible for the loss of quality in several
chemical or natural products of industrial importance. We can prevent or retard this
phenomenon by the addition of synthetic or natural antioxidants. FTIR spectroscopic
technique has been used for the evaluation of antioxidant activity in sunflower oil.
They have also found that the intensity of the hydroperoxide absorption band in the
infrared spectra was increased proportionally with the increase of hydroperoxide
concentration. The activity of an antioxidant can be assessed by calculating its ability to
inhibit hydroperoxide formation.
Bruchard et al. [92] have studied formation of insulin amyloid fibrils followed
by FTIR simultaneously with CD and electron microscopy. They have observed
changes in the shape and the frequency of the amide I band as a function of time. The
amide I band was very sharp and located at 1,651 cm-1. The shape and position of the
band are consistent with the presence of largely helical or disordered structure studied
by Klimon and Bandeko [10], Arrondo et al. [93], Vecchio et al. [94]. FTIR results
were in good agreement and indicative of the partial unfolding of the protein. They
have also suggested that the short structure in the insulin fibrils could be predominantly
parallel rather than antiparallel on the molecular level. FTIR findings show that insulin
prior to heat treatment has substantially native like α -helical characteristics.
Movasoghi et al. [95] have reviewed the literature and supplied the relevant
information regarding the peak position intensities and frequencies of the bands which
are useful in the field of research and industries globally.
Jackson and Mantsch [96] have studied the use and misuse of FTIR
spectroscopy in the determination of protein structure. This technique can be used as a
tool for the structural characterization of proteins.
Many researchers such as Elliot and Ambrose [97], Ambrose and Elliot [98] and
Elliot [99] demonstrated that the IR spectroscopy can supply the information regarding
secondary structure of proteins. They showed that an empirical correlation holds
between the frequency of amide I and amide II absorption of a protein.
Most of the research scientists have found that the amide I absorption is more
useful for the determination of secondary structure of the protein.
12
 Devbeshko et al. [58] have studied FTIR reflectance study on surface enhanced
IR absorption of nucleic acids from tumor cells. Studies revealed some possible
peculiarities of their structural organization, such as the appearance of unusual sugar
and base conformations, modification of the phosphate backbone, and redistribution of
the H-band net.
Rumana et al. [100] have studied Fourier transform infrared spectroscopy to
distinguish wood of trees from different growth habitats for wood certification.
Crow and Mantesch [101] have studied vibrational biospectroscopy from plants
to animals to human. They have given a historical aspects of FTIR in different living
plants and humans and animals.

CHIEF PURPOSE AND GOAL OF THE PURPOSE

The chief goal of our project is to interpret the experimentally measured
vibrational spectra for the molecules to the greatest extent possible and to understand
the structure, function and electronic properties of these molecules in their various
environments. It is also believed that the application of different spectroscopic
techniques to biophysical and environmental assays is expanding day by day, and
therefore a true understanding of the phenomenon from rigorous theoretical and
experimental verification is also required.
In biological studies, where a wide range of chemical bands and functional
groups can be attributed to every single peak, finding appropriate meanings, which can
demonstrate the clinical importance of the technique and achieved the results, can be
one of the greatest importance of the steps in finalizing a spectroscopic research work.
As a result, and with the goal of putting these shortage aside, we would like to carry our
work on this sophisticated FTIR technique.
However, it would be very useful to have further and continuous review of this
field to improve the work of Fourier transform infrared spectroscopy on regular basis
and to keep it updated to prepare an experimental database that can be used for
different researchers in the areas related to biomaterial, science, chemistry, geology,
plants and tissue engineering.
We wish to carry our work on Fourier Transform Infrared Spectroscopy because
various chain conformations of polypeptides and proteins yield different types of
absorption spectra. The significant difference in the absorption spectra may give a lot

13
of valuable information. The correlations can be applied for explaining the chain
conformations of a number of polypeptides or proteins by FTIR.

14
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