SPECTROSCOPY - INTRODUCTION

 Spectroscopy is the analysis of electromagnetic radiation scattered, absorbed or emitted by molecules.

 The interaction of light with matter results in physical changes (such as reflection, absorption, diffraction or
emission) or chemical changes (such as dissociation or isomerisation).
 During interaction, the energy is absorbed or emitted by the matter.
 Absorption and emission form the basis of spectral analytical techniques and the subject specialization is called
Spectroscopy.

Absorption and Emission Spectrum

Absorption spectrum results when an atom or molecule undergoes a transition from a lower energy level to a
higher one with absorption of a photon of energy hv. Provided hv is exactly equal to the energy difference,
(∆E = Ee - Eg) between the two levels.

Emission spectrum results when the atom or molecule falls from excited state to ground state with the emission of a
photon of energy hv.
Atomic spectrum is given by electronic transitions, whereas a molecular spectrum is given by vibrational, rotational
and electronic transitions.
Absorption law
Beer-Lambert’s law
When a monochromatic light of intensity I is passed through a solution of concentration, C molar and thickness, dx,
then intensity of transmitted light changes by dI. Then, probability of absorption of radiation is given by,
– (dI/I) = kCdx
On integrating the above expression,
Log (I0/I) = (k / 2.303) Cx
A = ξCx
This equation is known as Beer’s law.
where (Io/I ) = A (absorbance or optical density)
ξ = k / 2.303 (molar absorptivity coefficient)

Various types of Molecular spectra

Spectra Region Transition Range Criteria Source Sample Monochr Detector
(cm-1) form omator
Micro Rotation Between Microw Must have Klystron Gas / Optical Crystal
wave al rotational ave permanent valve vapour prism
energy (1 – dipole
levels 100) moment
Infrared Vibratio Between Infrared Dipole Nernst Gas / Optical Photoco
nal & vibrational (500 – moment glower liquid / prism nductivit
Vibratio energy 4000) must dilute made of y cell
nal levels change solution / NaCl, and
rotationa during solid+KBr KBr Photoele
l vibrations in the &grating ctric
form of detectors
disc
Raman Vibratio Between Infrared Inelastic Laser in Solid / Diffractin Charged
nal & vibrational (10 – scattering viscible iquid / gel g grating –coupled
Vibratio energy 4000) of photons range / slurries / device
nal levels powder / detector
rotationa films
l
UV- Electroni Between UV – Presence UV – UV – Gas / Prism / Photomult
Visible c electronic (25,000 of hydrogen dilute Grating iplier
energy – chromoph discharge solution
levels 70,000) ore in a lamp
Visible molecule Visible – Visible –
– Tungsten Gas / dilute
(12,500 filament solution
– lamp
25,000)

INFRARED SPECTROSCOPY (IR)

Principle
 Absorption of energy by a molecule in infrared region brings about changes in vibrational and
rotational energy levels of the molecule.
 IR spectroscopy is otherwise known as vibrational spectroscopy. A single vibrational energy change
is accompanied by a large number of rotational energy changes. Thus, vibrational spectra appear as
vibrational-rotational bands.
 Ordinary infrared region extends from 4000 - 667 cm-1.
 The region from 12500 - 4000 cm-1 is called near infrared.
 The region from 667 - 50 cm-1 is called far infrared.

Molecular Vibrations
Absorption of infrared radiations causes vibrations in a molecule. The fundamental vibrations in a
molecule are stretching and bending.

Molecular Vibrations
(a) Stretching vibration: Distance between two atoms increases or decreases but the bond axis remains
same.
Symmetric stretching: Movement of atoms w.r.to a particular atom in a molecule is in the same
direction.
Asymmetric stretching: One atom approaches the central atom while the other departs from it.

Symmetric Asymmetric
(b) Bending vibration: Bond angle changes but the bond length remains unaltered.
Scissoring: Two atoms approach each other.
Rocking: Movement of both atoms take place in same direction.
Wagging: Two atoms move up and below the plane with respect to central atom.
Twisting: One of the atoms move up the plane while the other moves down the plane with respect to the
central atom.

Summary of absorptions of bonds in organic molecules

Finger Print Region: The region to the right-hand side of the diagram (from about 1500 to 500
cm- 1) usually contains a very complicated series of absorptions. These are mainly due to all
manner of bending vibrations within the molecule. This is called the fingerprint region. It is
much more difficult to pick out individual bonds in this region than it is in the "cleaner" region at
higher wavenumbers. The importance of the fingerprint region is that each different compound
produces a different pattern of troughs in this part of the spectrum.
Using the fingerprint region: Compare the infra-red spectra of propan-1-ol and propan-2-ol.
 Both compounds contain exactly the same bonds. Both compounds have very similar troughs in
the area around 3000 cm-1 - but compare them in the fingerprint region between 1500 and 500
cm-1.

Number of Fundamental Vibrations

Polyatomic molecules may exhibit more than one fundamental vibrational absorption bands. Number
of these fundamental bands is related to the degrees of freedom in a molecule. The total number of degrees
of freedom of a molecule will be equal to 3n where n is the number of atoms in a molecule.
3n degrees of freedom = Translational + Rotational + Vibrational
In the case of linear molecule there are only two degrees of rotation.
Total degrees of freedom = 3n
Translational degrees of freedom = 3
Rotational degrees of freedom =2
Vibrational degrees of freedom = 3n-3-2 = 3n - 5

In the case of non-linear molecule, there are three degrees of rotation as the rotation about all the three
exes will result in a change in position of the atoms. So, for non-linear molecule,
Total degrees of freedom = 3n
Translational degrees of freedom = 3
Rotational degrees of freedom =3
Vibrational degrees of freedom = 3n-3-3 = 3n-6

Selection Rules
Infra-red light absorbed only when a change in dipole character of molecule takes place. If a molecule
has a centre of symmetry, then vibrations are centro symmetric and inactive in the infra-red but are active
in the Raman. The vibrations which are not centro symmetric are active in infra-red but inactive in
Raman.

Block diagram of IR spectrometer

Instrumentation
Radiation source
Eg: Nernst glower, consists of a rod of sintered mixture of oxides of Zr, Y and Er. This rod is electrically
heated to 1500° C to produce IR radiation.
Monochromator
It allows the light of a particular wavelength to pass through it.
Eg: Optical prism made of NaCl, KBr and grating.
Sample cells
These must be transparent to IR radiation.
Detectors & Recorder
These convert thermal radiant energy into electrical energy.
Eg: Photoconductivity cell and Photoelectric detectors.

Working
Light from the source is split into two beams. One of the beams which passes through sample is
called sample beam and the other beam is called reference beam.
When the beam passes through the sample, it becomes less intense due to the absorption of certain
frequencies. Now, there will be a difference in intensity of the two beams. When the two beams
recombine, they produce an oscillating signal, which is converted and measured as electrical signal with
the help of detector. The signal from detector is passed to the recording unit and recorded.

Applications
 To identity compounds
 In detecting impurities in a sample
 To determine hydrogen bonding in a molecule
 It provides valuable information of molecular symmetry, dipole
moments, bond length, etc.
 To detect functional groups
 To distinguish positional isomers of a compound
Applications of IR-Spectroscopy

Infrared spectroscopy is widely used in industry as well as in research. It is a simple and reliable
technique for measurement, quality control and dynamic measurement. It is also employed in
forensic analysis in civil and criminal analysis.
Some of the major applications of IR spectroscopy are as follows:

1. Identification of functional group and structure elucidation

Entire IR region is divided into group frequency region and fingerprint region. Range of group
frequency is 4000-1500 cm-1 while that of finger print region is 1500-400 cm-1.
In group frequency region, the peaks corresponding to different functional groups can be
observed. According to corresponding peaks, functional group can be determined.
Each atom of the molecule is connected by bond and each bond requires different IR region so
characteristic peaks are observed. This region of IR spectrum is called as finger print region of
the molecule. It can be determined by characteristic peaks.

Identification of substances
IR spectroscopy is used to establish whether a given sample of an organic substance is identical
with another or not. This is because large number of absorption bands is observed in the IR
spectra of organic molecules and the probability that any two compounds will produce identical
spectra is almost zero. So if two compounds have identical IR spectra then both of them must be
samples of the same substances.
IR spectra of two enatiomeric compound are identical. So IR spectroscopy fails to distinguish
 between enantiomers.
For example, an IR spectrum of benzaldehyde is observed as
follows. C-H stretching of aromatic ring- 3080 cm-1
C-H stretching of aldehyde- 2860 cm-1 and 2775
cm-1 C=O stretching of an aromatic aldehyde- 1700 cm-1
C=C stretching of an aromatic ring- 1595 cm-1
C-H bending- 745 cm-1and 685 cm-1
No other compound then benzaldehyde produces same IR spectra as shown above.

3. Studying the progress of the reaction

Progress of chemical reaction can be determined by examining the small portion of the reaction
mixure withdrawn from time to time. The rate of disappearance of a characteristic absorption
band of the reactant group and/or the rate of appearance of the characteristic absorption band of
the product group due to formation of product is observed.

4. Detection of impurities
IR spectrum of the test sample to be determined is compared with the standard compound. If any
additional peaks are observed in the IR spectrum, then it is due to impurities present in the
compound.

5. Quantitative analysis
The quantity of the substance can be determined either in pure form or as a mixure of two or
more compounds. In this, characteristic peak corresponding to the drug substance is chosen and
log I0/It of peaks for standard and test sample is compared. This is called base line technique to
determine the quantity of the substance.

ULTRAVIOLET AND VISIBLE SPECTROSCOPY (UV-VISIBLE)

Principle
Molecules containing π-electrons or non-bonding electrons (n-electrons) can absorb energy in the
form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular orbitals. The
more easily excited the electrons, longer the wavelength of light it can absorb.

Types of Electronic Transitions

When a molecule absorbs energy in UV/visible region, its electrons are promoted from bonding
molecular orbital to anti-bonding molecular orbital.
The electronic transitions involved in UV and visible region are,

(i) σ – σ* transitions: These

transitions occur by the promotion of
sigma electrons from bonding to
antibonding (σ*) orbitals. It requires
high energy and thus absorbs radiation
in far region (126 nm-136 nm).
(ii)
(iii) n – σ* transitions: These transitions are seen in saturated compounds, containing heteroatom in
addition to σ – σ* transitions. The energy required is less than that for σ – σ* transitions.
(iv) π - π* transitions: The electron transfer occur from bonding π orbital to antibonding π* orbital. It is
only associated with compound containing double or triple bond and aromatic compounds.
The energy required for various transitions obey the following order:
σ – σ* > n – σ* > π - π* > n - π*

Solvent Effect:
The solvent in which the absorbing species is dissolved also has an effect on the spectrum of the species.
Peaks resulting from n - Π* transitions are shifted to shorter wavelengths (blue shift) with increasing
solvent polarity. This arises from increased solvation of the lone pair, which lowers the energy of n orbital.
Often the reverse (i.e. red shift) is seen for Π - Π* transitions. This is caused by attractive polarization
forces between the solvent and the absorber, which lower the energy levels of both the excited and
unexcited states. This effect is greater for the excited state, and so the energy difference between the
excited and unexcited states is slightly reduced - resulting in a small red shift. This effect also influences n
- Π* transitions but is overshadowed by the blue shift resulting from solvation of lone pairs.

Block Diagram of UV- visible spectrometer

Instrumentation
Radiation source
Hydrogen or deuterium lamp.
Monochromator
Selects appropriate wavelength of light (Prism / Grating).
Cells (sample and reference)
It is made out of quartz or fused silica.
Detectors
Photomultiplier is commonly used detector which converts light radiations into electrical signals.
Recorder

Working
 Radiation from a hydrogen or deuterium lamp is allowed to pass through monochromator unit, which
allows a narrow range of wavelength to pass through it.
 The beam of light coming out of monochromator is split into two halves. One-half of the beam
(sample beam) is directed through a transparent cell containing a solution of sample under analysis.
 The other half (reference beam) is directed through an identical cell containing the solvent.
 The instrument is so designed that it compares intensities of sample beam and reference beam.
 If the sample absorbs light at a particular wavelength, then the intensity of sample beam I will be less
than the intensity of reference beam Io.
 The instrument gives output graph - a plot of wavelength vs absorbance A, and is known as the
absorption spectrum.
Applications
• In determining the structure of several vitamins
 • In detecting steric hindrance
• To study rates of reactions
• In determination of dissociation constants of acids and bases.

Application of UV-Spectroscopy
• Detection of Impurities: UV absorption spectroscopy is one of the best methods for
determination of impurities in organic molecules. Additional peaks can be observed due to
impurities in the sample and it can be compared with that of standard raw material. By also
measuring the absorbance at specific wavelength, the impurities can be detected. Benzene
appears as a common impurity in cyclohexane. Its presence can be easily detected by its
absorption at 255 nm.
• Structure elucidation of organic compounds: UV spectroscopy is useful in the structure
elucidation of organic molecules, the presence or absence of unsaturation the presence of
heteroatoms.
From the location of peaks and combination of peaks, it can be concluded that whether the
compound is saturated or unsaturated, hetero atoms are present or not etc.
• Quantitative analysis: UV absorption spectroscopy can be used for the quantitative
determination of compounds that absorb UV radiation. This determination is based on Beer’s law
Qualitative analysis: UV absorption spectroscopy can characterize those types of compounds
which absorbs UV radiation. Identification is done by comparing the absorption spectrum with the
spectra of known compounds. UV absorption spectroscopy is generally used for characterizing
aromatic compounds and aromatic olefins.
• Dissociation constants ofacids and bases.
• pH=pKa+log -
[A ]/ [HA]
• From the above equation the pKa value can be calculated if the ratio of [A-] /
[HA] is known at a particular PH. and the ratio of [A-] / [HA] can be determined
spectrophotometrically from the graph plotted between absorbance and wavelength at different PH
values.
• Chemical kinetics: Kinetics of reaction can also be studied using UV spectroscopy. The UV
radiation is passed through the reaction cell and the absorbance changes can be observed.

• Quantitative analysis of pharmaceutical substances: Many drugs are either in the form of
raw material or in the form of formulation. They can be assayed by making a suitable solution of
the drug in a solvent and measuring the absorbance at specific wavelength. Diazepam tablet can be
analyzed by 0.5% H2SO4 in methanol at the wavelength 284 nm.
• Molecular weight determination: Molecular weights of compounds can be measured
spectrophotometrically by preparing the suitable derivatives of these compounds. For
example, if we want to determine the molecular weight of amine then it is converted in to amine
picrate. Then known concentration of amine picrate is dissolved in a litre of solution and its
optical density is measured at λmax380n.Afterhis conetrai ofhemax 380 nm. After this the concentration of the solution in
gm moles per litre can be calculated by using the following formula

RAMAN SPECTROSCOPY
Raman spectroscopy (Named after Indian physicist Sir C. V. Raman) is spectroscopic technique used to
observe vibrational, rotational, and other low-frequency modes in a system.[1] Raman spectroscopy is
commonly used in chemistry to provide a structural fingerprint by which molecules can be identified.
Raman spectra relate to vibrational and /or roatational transitions in molecules but in different manner.In
this case we measure the scattering and not the absorption of radiation.An intense beam of monochromatic
radiation in the visible region is allowed to fall on a sample and the intensity of scattered light is observed
at right angles to the incident beam.
Raman spectroscopy reveals the chemical and structural composition of samples. Generally, all materials
produce Raman spectra, with the exception of pure metals.

Raman scattering

Raman scattering occurs when light interacts with molecular vibrations. This is similar to the more widely
known infrared absorption spectroscopy, but different rules apply. A change in molecular polarisability is
required during the vibration for the Raman effect to occur.
You will see some vibrations in the Raman spectrum that are not visible in the infrared spectrum, and vice-
versa, because of the different selection rules. For example, Raman spectroscopy is superb for studying the
carbon atoms that make up the structure of diamond, unlike infrared absorption spectroscopy.

Scattered light
The first step in producing a Raman spectrum is to illuminate your sample with a monochromatic light
source, such as a laser.
Most of the light that scatters off is unchanged in energy ('Rayleigh scattered'). A minute fraction—
perhaps 1 part in 10 million—has lost or gained energy ('Raman scattered'). This Raman shift occurs
because photons (particles of light) exchange part of their energy with molecular vibrations in the material.
Where energy is lost the Raman scattering is designated as 'Stokes'; where energy is gained the Raman
scattering is designated as 'anti-Stokes'. We rarely use anti-Stokes Raman light as it is less
intense than the Stokes, however it does represent equivalent vibrational information of the molecule.
Vibrating atoms
The change in energy depends on the frequency of vibration of the molecule. If it is very fast (high
frequency)—light atoms held together with strong bonds—the energy change is significant. If it is very
 slow (low frequency)—heavy atoms held together with weak bonds—the energy change is small.

Raman spectrometers
It consist of: single or multiple lasers, from UV (244 nm) to IR (1064 nm) – switch with a single click
•
high quality objective lenses, from high confocal 100× to long working distance andimmersion options
•
custom designed motorised spectrometer lenses - automatically align for each configurationlaser-line-specific
Rayleigh filters with a dual filter arrangement to optimise sensitivity highest quality master diffraction
gratings provide exceptional dispersion and longevity thermoelectrically cooled (- 70 ºC) CCD detector –
stable and sensitive high specification multi-core PC for data collection and analysis

•
Energy diagram showing absorption of light and the processes involved in the emission of
light as fluorescence and phosphorescence

Vibration frequencies
The frequencies of vibration depend on the masses of the atoms involved and the strength of the
bonds between them. Heavy atoms and weak bonds have low Raman shifts. Light atoms and
strong bonds have high Raman shifts. When a sample is illuminated by a laser, both Raman
scattering and photoluminescence (PL) can occur. The latter can be many times stronger than the
former and can prevent successful Raman analysis.
PL comprises both fluorescence and phosphorescence processes and originates from an
absorption/emission process between different electronic energy levels in the material. The amount and
type of PL depends on which material you are studying and which laser wavelength you are using.
Unwanted fluorescence interference can normally be avoided by choosing an appropriate laser wavelength
•
Raman spectroscopy uses intense light from a laser to probe the chemical bonds in a
substance, generating a spectrum that acts as a fingerprint which can be used to characterize or
identify the substance. This can be used to authenticate materials or assess their quality, and
even for medical diagnostics. But how does a Raman spectrometer work?
How Raman Spectrometers Work
A Raman spectrometer system requires three primary components:
 A high-intensity laser
 A sample interface
 A spectrometer

Applications of Raman Spectroscopy

1 Carbon Materials
• Purity of Carbon nanotubes(CNTs)
• Sp2 and sp3 structure in carbon materials and Diamond like carbon coating properties
• Defects and disorder analysis in carbon materials
2 Pharmaceuticals and Cosmetics
• Compound distribution in tablets.
• Polymorphic forms
• Contaminant identification and Powder content and purity
3 Life Sciences
• Bio-compatibility and DNA/RNA analysis
• Drug/cell interactions and Disease Diagnosis
• Bone Structure
4 Geology and Minerology
• Gemstone and Mineral identification
• Mineral behaviour under extreme conditions.
• Phase Transitions and Fluid inclusion
5 Semiconductors
• Alloy composition
• Defect analysis

• Contamination identification and Doping effects

Fluorescence spectroscopy
Fluorescence spectroscopy is a rapid, sensitive method for characterizing molecular
environments and events samples. Fluorimetry is chosen for its extraordinary sensitivity, high
specificity, simplicity and low cost as compared to other analytical techniques. It is widely accepted
and powerful technique that is used for a variety of environmental, industrial, medical diagnostics,
 DNA sequencing, forensics, genetic analysis and biotechnology applications. It is a valuable
analytical tool for both quantitative and qualitative analysis
Fluorescence and phosphorescence are photon emission processes that occur during molecular
relaxation from electronic excited states. These photonic processes involve transitions between electronic and
vibrational states of polyatomic fluorescent molecules (fluorophores).
Fluorophores play the central role in fluorescence spectroscopy. Fluorophores are the components in
molecules that cause them to fluorescence. Majorly fluorophores are the molecule which contains aromatic
rings such as Tyrosine, Tryptophan, and Fluorescein etc.
Fluorescence spectroscopy is a sensitive optical emission technique in which sample molecules are excited with
a photon source. Those molecules that relax by radiant emission can be subsequently detected by measuring the
intensity of that emission.

1.Principle of fluorescence spectroscopy

Absorption of UV or visible radiation causes transition of electrons from singlet ground state to the singlet
excited state. As this state is not stable, it emits energy in the form of UV or visible radiation and returns to
singlet ground state. Fluorescence emission occurs as the fluorophore decay from the singlet electronic
excited states to an allowable vibrational level in the electronic ground state. The fluorescence excitation and
emission spectra reflect the vibrational level structures in the ground and the excited electronic states
respectively.
Nature of substituent groups
Electron donating groups like amino, hydroxyl groups enhance fluorescence activity. Electron
withdrawing groups like Nitro, carboxylic group reduce fluorescence. Groups like SO3H or on
NH4+ have no effect on fluorescence intensity.
Effect of temperature
Increase in temperature leads to increase in collisions of molecules and decrease in fluorescence
intensity while decrease in temperature leads to decrease in collisions of molecules and increased
fluorescence intensity.
Viscosity
Figure 1: Jablonski diagram
2. Instrumentation of spectrofluorometer

Figure 2: Schematic diagram of a spectrofluorometer

Spectroflourometer mainly consists of
A. Source of light
 Mercury vapour lamp
 Xenon arc lamp
 Tungsten film
B. Filters and monochromators
 Primary filters and secondary filters
 Excitation monochromators and Emission monochromators
C. Sample cells, Detectors
3. Factors affecting fluorescence
• Conjugation
Molecule must have unsaturation i.e. it must have πelectrons so that UV/vis radiation can be
absorbed. If there is no absorption of radiation, there will not be fluorescence.
• Rigidity of structures
Rigid structures will produce more fluorescence, while flexible structure will produce
less fluorescence.
Increase in viscosity leads to decreased collisions of molecules which will
enhance fluorescence intensity while decrease in viscosity causes increased collisions of
molecules which cause decreased fluorescence intensity.
• Oxygen
Oxygen decreases the Fluorescence intensity in two ways: Oxidises fluorescence
substances to non fluorescence substances. It quenches fluorescence because of paramagnetic
properties.
• Effect of pH
a. Aniline: Neutral or alkaline medium shows visible fluorescence while acidic conditions give
fluorescence in UV region only.
 b.Phenols : Acidic conditions do not give fluorescence while alkaline conditions gives good
fluorescence.[2,4]

4. Effect of concentration on fluorescence intensity

Fluorescence intensity = Q x Ia
Where,
Q = Fluorescence efficiency
Ia = Intensity of absorbed light
Fluorescence intensity is directly proportional to the substances.
5. Advantages
o It’s one of the newer methods and its potentialities are
 still largely unexplored.
o It also affects precision. Up to 1% can be achieved easily in Flourimetric.
o The method is very sensitive and also possesses specificity because there is a choice of wavelength not
only for the radiation emitted, but also for the light which excites it.
6. Limitations
o Careful buffering is necessary as fluorescence intensity may be strongly dependent
o Ultraviolet light used for excitation may cause photochemical changes or destruction of the fluorescent
molecule .
o The presence of dissolved oxygen may cause increased photochemical destruction.
o Traces of iodide and nitrogen oxides are efficient quenchers and therefore interfere.
o The method is not suited for determination of major constituents of a sample, because the accuracy is
very less for large amounts.
o The extent of applicability of this technique is limited, because of the fact that all elements and
compounds are unable to exhibit fluorescence.[13,15]

7. Fluorescent indicators
The intensity and colour of many fluorescent substances depends on pH. Some substances are
so sensitive to pH that they can be used as pH indicators. These are termed as fluorescent or
luminescent indicators. Those substances which fluorescence in ultra violet light and change in
colour or have their fluorescence quenched with change in pH can be used as fluorescent
indicators in acid base indicators .The merit of such indicators is that they can be employed in
the titration of coloured solution in which the colour change of usual indicators would be masked
Table 3: Some important fluorescent indicators [2,7]

Indicators pH Colour change

Fluorescence 4.0 -6.0 Colourless to
green
Quinine 3.0 -5.0 Blue to violet
sulphate
2 naptha 4.4 -8.3 Blue to colourless
quinine
Fluorescence processes find application in the following:
 Fluorescent lamps
 Biological detectors
 Spectroscopy/chemical sensors
 Fluorescent labeling
 Mineralogy
 Forensic applications

1. Chemistry of bioluminescence
Determination of boron in steel
Determination of aluminium in alloys
Determination of chromium and manganese in steel
2. Applications
Applications in inorganic chemistry
Determination of ruthenium
Determination of uranium salts
Estimation of rare earth terbium
Estimation of bismuth
Determination of beryllium in silicates
Estimation of 3,4 benzpyrene
Determination of zinc
Determination of cadmium
Application in organic chemistry
Assay of thiamine:
Estimation of quinine sulphate by fluorimetry
Special fluorimetric applications
Investigation of chemical structures and processes
Chemical analysis
Laser induced fluorescence spectroscopy of human tissues for cancer diagnosis
Cancer is one of the most dreaded disease. Laser spectroscopic techniques have the potential for in-
situ, near real time diagnosis and the use of non-ionizing radiation ensures that the diagnosis can be made
repeatedly without any adverse side effects. Laser Induced Fluorescence (LIF) has been used for
diagnosing cancer in two ways.
Study of marine petroleum pollutants
Accurate determination of glucose
Glucose is considered as a major component of animal and plant carbohydrates in
biological systems. Furthermore, blood glucose levels are also an indicator of human health
conditions. The abnormal amount of glucose provides significant information of many diseases
such as diabetes or hypoglycemia. Fluorophotometry was used widely owing to its operational
simplicity and high sensitivity .Recently bio-molecule-stabilized Au nanoclusters were
demonstrated as a novel fluorescence probe for sensitive and selective detection of glucose.
MICROWAVE SPECTROSCOPY
In a microwave spectrometer a microwave source, usually a klystron valve, produces a beam that is
passed through a gaseous sample. The beam then impinges on the detector, usually a crystal detector, and
the signal (wavelength against intensity) is displayed, either as a printed plot or on an oscilloscope.
PRINCIPLE
A microwave spectrum is obtained by sweeping the microwave source over a range of frequencies and
 observing the small variations in power at the detector. An absorption line appears as a sharp dip on the
recording system. To increase the sensitivity of a microwave spectrometer, Stark modulation may be
added. It is based on the principle that microwave radiation (see microwaves) causes changes in the
rotational energy levels of molecules and absorption consequently occurs at characteristic
frequencies.

Selection rules of Microwavespectroscopy

In order for a molecule to give rise to rotational spectrum, it becomes essential that the molecule must
have a dipole moment but all transitions are not permitted. selection rule which is given as ΔJ=±1

From the above rule, It is evident that that only those transitions are permitted in which there is an
increase or decrease by unity in the rotational quantum number. It means that J=0 J=2 J=4…transitions
are not possible
. In other word,these transitions are spectroscopically forbidden.  When a rotational spectrum is
recorded in the presence of a strong electric field E, the lines will in general be split and shifted. The
effect was first of all observed by Stark in atomic spectra and is known as Stark effect.

Degrees of Freedom
Polyatomic molecules may exhibit more than one fundamental vibrational absorption bands. Number
of these fundamental bands is related to the degrees of freedom in a molecule. The total number of degrees
of freedom of a molecule will be equal to 3n where n is the number of atoms in a molecule.
3n degrees of freedom = Translational + Rotational + Vibrational
In the case of linear molecule there are only two degrees of rotation.
Total degrees of freedom = 3n
Translational degrees of freedom = 3
Rotational degrees of freedom =2
Vibrational degrees of freedom = 3n-3-2 = 3n - 5

In the case of non-linear molecule, there are three degrees of rotation as the rotation about all the three
exes will result in a change in position of the atoms. So, for non-linear molecule,
Total degrees of freedom = 3n
Translational degrees of freedom = 3
Rotational degrees of freedom =3
Vibrational degrees of freedom = 3n-3-3 = 3n-6

Each of these degrees of freedom is able to store energy. However, In the case of rotational and vibrational
degrees of freedom, energy can only be stored in discrete amounts. This is due to the quantized break down
of energy levels in a molecule described by quantum mechanics. In the case of rotations the energy stored is
dependent on the rotational inertia of the gas along with the corresponding quantum number describing the
energy level.
The essential elements of a microwave spectrometer are a microwave source, absorption cell, detection
system, and a system for measuring the source frequency.
 APPLICATIONS
Microwave spectroscopy is suitable for studying chemically and physically very interesting
molecular systems, including weakly bound complexes, radicals, ions, and other transient species.
Information on molecular structure, internal motions and intermolecular interactions are easily obtained.

Limitations
The chief disadvantage of microwave spectroscopy for gas-phase analytical applications is that its sensitivity
is not as high as for some other methods (such as laser fluorescence or mass spectrometry).