Molecular Spectroscopy

UNIT 4 MOLECULAR SPECTROSCOPY

Structure

4.0 Introduction
4.1 Objectives

4.2 UV-VIS spectrometry

4.2.1 Origin of Spectrum

4.2.2 Characteristics of spectrum

4.2.3 Principle of UV-VIS spectrophotometry

4.2.4 Quantitative Methodology

4.2.5 Instrumentation

4.3 Fluorescence Spectrometry

4.3.1 Origin of spectrum: Jablonski Diagram

4.3.2 Excitation versus Emission Fluoroscence

4.3.3 Instrumentation

4.4 Vibration Spectroscopy

4.4.1 Origion of Raman Spectrum

4.4.2 Instrumentation

4.5 Applications of spectrometric methods in Environmental monitoring

4.6 Let Us Sum Up

4.7 Terminal Questions

4.0 INTRODUCTION
You would recall that spectroscopy is the study of interaction of electromagnetic
(EM) radiation with matter. The nature of the spectrum depends on the type of
electromagnetic radiation and the nature of matter e.g., atoms or molecules with
which it interacts. In this unit we are going to take up molecular spectroscopy
i.e., the study of interaction of EM radiation with molecular systems. We would
begin the unit with UV-VIS spectroscopy i.e., the study of interaction of UV-
VIS radiation with different species. Herein we would discuss the origin of the
spectrum in terms of the interaction of radiation with different types of absorbing
species. This will be followed by a discussion on the characteristics of the
spectrum and the instrumentation used for obtaining the spectrum. Then we
would take up the principle of UV-VIS spectrophotometry as an analytical tool.

The second molecular spectroscopic method would be fluorescence

spectrometry that is an emission spectroscopic method. Herein we would
discuss about the origin and characteristics of the fluorescence spectrum
and differentiate between absorption and fluorescence spectrum. We would 75
Spectroscopic Methods also explain the origin of phosphorescence spectrum and differentiate
between fluorescence and phosphorescence spectrum. Then we would
briefly explain chemiluminescence and then take up instrumentation used
in fluorescence spectrometry.

In the third part of the unit, we would take up vibration spectroscopy i.e.,
the study of interaction of EM radiation in the IR region with the molecules.
Herein, we would take up two types of interaction i.e., the absorption and
scattering of radiation. These give rise to IR and Raman spectra respectively.
We would explain the origin and characteristics of the IR and Raman spectra
and the instrumentation required for them. Having discussed these
molecular spectroscopic methods, we would outline their environmental
applications.

In the next unit we would take up atomic spectroscopic methods based on

absorption and emission of the radiation.

4.1 OBJECTIVES
After studying this unit you will be able to:

z explain the origin of UV spectrometry;

z explain the principle of UV spectrometry; and
z describe the instrumentation of fluoroscence spectroscopy.

4.2 UV-VIS SPECTROMETRY

UV-VIS spectrum results from the interaction of electromagnetic radiation
in the UV-VIS region with molecules, ions, or complexes. It forms the
basis of analysis of different substances such as, inorganic, organic and
biochemicals. These determinations find applications in research, industry,
clinical laboratories and in the chemical analysis of environmental samples.
It is therefore important to learn about the origin of the UV-VIS spectrum
and its characteristics. Let us try to understand the origin of the UV-VIS
spectrum.

4.2.1 Origin of UV-VIS Spectrum

The absorption of radiation in the UV-VIS region of the spectrum is dependent
on the electronic structure of the absorbing species like, atoms, molecules,
ions or complexes. You know that in an atom the electronic energies are
quantised and the electron in the atom can go to a higher energy level by
absorbing a suitable photon. Also, the excited atom can relax to a lower
energy level by emitting a photon. This in fact forms the basis of atomic
spectroscopic methods that we would take up in the next unit.

For now, it is important to note that the molecules are much more complex
and their electronic, vibrational and rotational energies are quantised. A
given electronic energy level has severalvibrational energy levels in it and
each of the vibrational energy level in turn has many rotational energy
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levels in it. A schematic energy level diagram for a molecule showing Molecular Spectroscopy
different quantised energy levels is given in Fig. 1.1.

The absorption of radiation

in the UV-VIS region of the
spectrum causes the
transitions amongst the
electronic energy levels.

Fig. 1.1: Schematic energy level diagram showing quantised electronic, vibrational,
and rotational energy levels.

When a photon of a given wavelength interacts with the molecule it may cause
a transition amongst the electronic energy levels if its energy matches with the
difference in the energies of these levels.In order to obtain a UV-VIS spectrum
the sample being studied is irradiated with the electromagnetic radiation varied
over a range of wavelength. A monochromatic radiation i.e., a radiation of a
single wavelength is employed at a time. This process is called scanning. The
amount of the radiation absorbed at each wavelength is measured and plotted
against the wavelength to obtain the spectrum. Thus, a typical UV spectrum is
a plot of wavelength or frequency versus the intensity of absorption.

In case of the samples in gaseous or vapour phase, the spectrum consists of a

number of closely spaced lines; Fig 1.2(a). However, in the solution phase,
the absorbing species are surrounded by solvent molecules and undergo constant
collisions with them. These collisions and the interactions among the absorbing
species and the solvent molecules cause the energies of the quantum states to
spread out. Therefore, the sample absorbs photons spread over a range of
wavelength. As a consequence the spectrum acquires the shape of a smooth
and continuous absorption peak. A typical UV-VIS spectrum in the solution
phase is depicted in Fig. 1.2 (b). Analyte: The chemical
species which is to be
determined in the sample
under investigation.

Fig. 1.2: Typical UV spectrum: a) in vapour phase and b) in solution phase

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Spectroscopic Methods 4.2.2 Characteristics of Spectrum
Have a look at Fig. 1.2 (b) again. The abscissa (x-axis) indicates the wavelengths
absorbed and therefore, is marked in wavelength though sometimes frequency
may also be used. The ordinate (y-axis) on the other hand represents the intensity
of absorption and is generally represented in terms of absorbance (explained
in Subsec. 1.2.3). The UV spectra of substances are characterised by three
parameters, namely, the position of the maximum of the absorption band called
Omax ; the intensity of the band and the width of the band.

Fig. 1.3: Characteristics of a UV-VIS absorption band

The Omax refers to the wavelength of the most absorbed radiation and is a
measure of the difference in the electronic energy levels involved in the
transition. The intensity on the other hand is indicative of the probability of
the transition i.e., whether the transition is allowed or not. It is also is a measure
of the concentration of the absorbing species. The relationship between the
intensity of absorption and the concentration is explained later.

The third characteristic of the spectral band is its width and is generally
expressed as full width at half maximum (FWHM) i.e., the width of the spectral
band at half its height (i.e., the intensity). Thewidth of the spectral band depends
on type of the instrument used and the intrinsic nature of the species being
analysed. The instrumental component can be minimised but not eliminated
whereas the natural line width due to the nature of the system cannot be
removed.

The first two characteristics of UV-VIS spectrum i.e., the position and the
intensity depend on the structure and concentration of the absorbing species
in solution. Therefore, these spectra are extensively used in the characterisation
and in the quantitative estimations of the analyte.

4.2.3 Principle of UV-VIS Spectrophotometry

In a typical UV-VIS absorption measurement, a monochromatic radiation is
made to fall on a sample taken in suitable container called cuvette. In such a
situation a part of the radiation is reflected, a part is absorbed, and a part is
transmitted. The intensity of original radiation, Po is equal to the sum of the
intensities of reflected (Pr), absorbed (Pa) and transmitted (Pt) radiation.

Po = Pr + Pa + Pt … (1.1)
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The effect of reflection can be compensated by passing equal intensities of Molecular Spectroscopy
beams through the solution and through the solvent contained in the same or
similar container and comparing the transmitted radiations. We can then write
the above equation as follows:

Po = Pa + Pt. … (1.2)

The intensity of the transmitted light is measured and is found to depend on

the thickness of the absorbing medium and the concentration, besides the
intensity of the incident radiation. This dependence forms the basis of
spectrometric determinations and is given in terms of two fundamental laws
i.e., Bouguer’s law or Lambert’s law, and the Beer’s law. The two laws are
combined to give Beer-Lambert’s law.Let us discuss these laws one by one.

Lambert’s Law

Lambert (1760) and Bouguer independently studied the decrease in the intensity
of radiation when it passes through a substance and made the following
observations:

i) The amount of monochromatic light absorbed by a substance is proportional

to the intensity of the incident light i.e. the ratio of the intensity of the
transmitted and incident light is constant.

ii) The intensity of the transmitted light decreases exponentially when the
thickness of the substance, through which the light is passing, increases
linearly.

These observations, called Lambert’s law can be translated into a mathematical

expression as given below.

Let Po represent the radiant power of incident light and P represent the radiant
power of transmitted light after passing through a cuvette of thickness b. Now,
if consider a small slab of cuvette having thickness dx, then the change in
power (dPx), is proportional to the power of incident light (Px) multiplied by
thethickness (dx) of the slab through which the light is passed. That is,

dPxvPxdx or dPx = kPxdx … (1.3)

where k is the proportionality constant, and the negative sign indicates that
radiant power decreases with absorption. Eq. 1.3 can be rearranged to Eq. 1.4.
dPx
 k dx … (1.4)
Px
Integrating Eq. 1.4 within the limits of Po to P for intensity and 0 to b for the
thickness we can write,

p b
dPx
³p Px k ³ dx
0
… (1.5)
0

Solving, we get
P
ln  kb … (1.6)
Po
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Spectroscopic Methods Eq.1.6 is the mathematical expression for Bouguer-Lambert law or Lambert’s
law. Changing the logarithms in this equation to base 10 and rearranging we
get,
P k
log o b k 'b … (1.7)
P 2.303
Note that the ratio P/Po has been inverted to remove the negative sign.

Beer’s Law

In 1852, Beer and Bernard independently studied the dependence of intensity

of transmitted light on the concentration of the solution. It was found that the
relation between intensity of the transmitted light and concentration was exactly
the same as found by Lambert for the intensity of the transmitted light and the
thickness of the absorbing medium. Mathematically, the Beer’s observations
can be expressed as follows.

Consider a monochromatic radiation beam of powerPx traversing any thickness

of solution of concentration c. If c is changed by a small amount dc to c + dc,
the change in transmitted power dPx is proportional to the incident intensity Px
and dc. We can write,
dPx v Px dc
…(1.8)
dPx  k " Px dc
where k” is the proportionality constant and the negative sign indicates that
radiant power decreases with absorption. This equation can be rearranged to:
dPx
 k " dc …(1.9)
Px
On integration within the limits of Po to P for intensity and 0 to c for
concentration we get
P c
d Px
³ ³ dc
"
k
Po
Px o

P
or ln  k "c
Po … (1.10)
Po k"
or log 10 c
P 2.303

Eq. 1.10 is the mathematical expression for Beer’s law. The Lambert’s and
Beer’s laws are combined and are expressed as per Eq. 1.11.
P
log o abc … (1.11)
P
In this expression, ‘a’ is a constant (combining two constants k¢, k² and the
numerical factor) and is called absorptivity (earlier called extinction
coefficient) whose value depends on unit of concentration used and is a function
of wavelength of the monochromatic light used. The concentration is generally
expressed in terms of grams per dm3 and b in cm. Therefore, it has the units of
cm-1g-1 dm3.

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However, if the concentration is expressed as mol dm3 and b in cm then Molecular Spectroscopy
absorptivityis called molar absorptivity (earlier called molar extinction
coefficient) and is expressed as e. Its units are cm1 mol-1dm3. The modified
expression for the Beer-Lambert’s law then becomes,
P
log 0 Hbc … (1.12) Absorbance is defined as the
P logarithm of the ratio of the
The term log Po/P is called absorbance and is represented as ‘A’. intensities of the incident and
transmitted radiations.
P
A log 0 … (1.13)
P
The expressions for Beer-Lambert’s law then becomes,
P
log 0 A abc or Hbc ...(1.14)
P
The absorbance, A is related to another important term called transmittance
which is defined as the fraction of the incident radiation transmitted by the
absorbing medium. Mathematically,
P
Transmittance, T P … (1.15)
0

It is generally expressed as a percentage and is expressed as,

P
Percentage transmittance, %T P u 100% … (1.16)
0

It is related to absorbance as, A  log T …(1.17)

In typical measurements the radiant power transmitted by a solution is measured

and compared with that observed with solvent (also called a blank). The ratio
of transmitted powers through solution and that through blank or solvent is
called as transmittance. It is desirable that you get acquainted with the
expressions of the Beer-Lambert’s law and learn to use them. Let us take an
example illustrating the application of these expressions.

Example 1

The molar absorptivity of a substance is 1.0 × 104 cm1 mol1 dm3. Calculate the
transmittance through a cuvette of path length 2.0 cm containing 1.5 × 106mol
dm3 solution of the substance.

Solution

As per the Lambert Beer’s law, absorbance, A = e c b

Given:

e = 1.0 × 104 cm1 mol1 dm3, c = 1.5 × 106 mol dm3 and b = 2.0 cm

Substituting the values we get,

A = 1.0 × 104 × 1.5×105 × 2.0 = 0.3

From the Lambert-Beer’s law expression given above, you can note that
absorbanceis a direct measure of the concentration of the analyte if the thickness
of the absorbing medium is kept constant. This proportionality is exploited in
the determination of analyte concentration. However, the following factors
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Spectroscopic Methods affect the validity of the Beer’s law and need to be controlled to take up
quantitative determinations.

z Presence of Electrolytes

z Hydrogen Ion Concentration

z Complexation, Association or Dissociation

z Non-monochromatic Nature of the Radiation

z Concentration of the Analyte

z Temperature

UV-VIS spectrometry has limited applications in qualitative analysis of the

analytes; however, it is probably the most useful tool available for quantitative
determinations in diverse areas. It is due to its versatility, accuracy, and
sensitivity. It can be used for direct determination of many organic, inorganic,
and biochemical species accurately at low concentrations; viz., 104 to 105 M
or even lower. In addition to these, the convenience of conducting the
determination and its reasonable selectivity make it a method of choice for
quantitative determinations. Let us learn about the quantitative determination
methodology using UV-VIS spectrometry. However, before that answer the
following simple questions to assess your understanding.

SAQ 1

In a spectrophotometer set at the lmax of a sample the value of P0 (with

the solvent) was found to be 85.4 using cuvettes of 2.00 cm path length.
The value of P for a solution of the sample having a concentration of 1 ´
104M was measured in the same cuvette and was found to be 20.3.
Calculate the molar absorptivity of the sample.
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SAQ 2

A substance has a molar absorptivity of 14,000 cm-1 mol-1 dm3 at the

wavelength of its maximum absorption. Calculate the concentration of
the substance whose solution in a cuvette of path length 1 cm has an
absorbance of 0.85.
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4.2.4 Quantitative Determination Methodology

The methodology followed for the quantitative determinations using UV-VIS
spectrometry havethe following essential steps:
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A. Forming an absorbing species Molecular Spectroscopy

B. Selection of the measurement wavelength

C. Controlling factors that influence absorbance

D. Validation of Beer and Lambert’s law

Let us briefly discuss these, one by one.

A. Forming an Absorbing Species

Only a few analytes have a strong absorption in the UV or Visible region

and can be subjected to direct determinations. However, for most of the
analytes we mustfirst form an absorbing species by reacting them with a
suitable reagent. The reagent should be selective towards the analyte and
its reaction with the analyte should be quantitative i.e., 100%. For example,
in the determination of iron (II) in aqueous solutions, a tricyclic nitrogen
heterocyclic compound,1, 10-phenanthroline is used as the ligand that
reacts with it to form strongly coloured complex.

B. Selection of the Measurement Wavelength

In the absence of interfering substances, the measurements are made at The wavelength chosen for
the wavelength of the maxima ( O max) of the largest peak in the spectrum quantitative work is called the
analytical wavelength.
of the analyte. At this wavelength the absorbance is most sensitive to the
concentration. However, in some cases where the reagent, metal and
products all absorb light, it may so happen that at O max there is not much
of difference in the absorbance value for pure reagent and the metal
complex. In such cases we need to identify such a wavelength at which
there is large difference in the absorbance values of pure reagent and the
complex.

C. Controlling Factors Influencing Absorbance Ordinarily the absorbance

should be measured at a
As stated before, several factors can affect the absorption spectrum of the wavelength where there is no
interference from other
analyte. It is, therefore, essential for a reliable quantitative determination absorbing species in the
that the conditions of the determination are so chosen that there is minimal solution.
effect of these factors.

D. Validation of Beer and Lambert’s law It is essential for a dependable

method that the A versus c
Let us rewrite the expression for the Beer and Lambert’s law as plot for the standard analyte
is a straight line.
log Po / P = A= abc orebc …(1.18)

According to this expression, if we know the values of a (ore) and b then we

can determine the concentration directly from the absorbance value. However,
generally the value of e is not known accurately for the species being determined
under the conditions of the determination. Therefore, in most of the methods,
a calibration curve is obtained by measuring the absorbance values for a series
of standard solutions of the analyte being determined at a fixed wavelength.
These solutions should be prepared under similar solution conditions and in
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Spectroscopic Methods the range of the concentration of the analyte. For the law to be valid, the plot
of absorbance, A versus the concentration, c for the standard solutions should
be a straight line passing through origin.

Fig. 1.4: Calibration curve: a) Standard solution method b) Standard addition

method

Such a curve is known as calibration curve and is handy in determination of

concentration of unknown solutions from a measurement of absorbance value
as shown in Fig. 1.4 (a). Sometimes, it may so happen that all the constituents
in the sample may not be known; so a standard solution with the same chemical
composition cannot be prepared. In such a case, a method called standard
addition method is used which eliminates any error arising from molar
absorptivities (e) being different in the standard solution from that in the sample
solution. In this method known amounts of the standard is added to identical
aliquots of the sample and the absorbance is measured. The first reading is the
absorbance of sample alone and the second reading is absorbance of sample
containing analyte plus, a known amount of analyte and so on. The readings
so obtained are then plotted to obtain the calibration curve. If the Beer’s law is
obeyed, i.e., a straight line is obtained; the unknown concentration of the
solution can then be obtained by the extrapolation of the calibration curve as
shown in Fig.1.4 (b).

Let us now take up the instrumentation required for measuring UV-VIS

spectrum. Why don’t you answer the following SAQ before proceeding further?

SAQ 3

What is the advantage of the standard addition technique as compared to a

calibration curve method?
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4.2.5 Instrumentation
Today a wide range of instruments are available for making molecular
absorption measurements in the UV-visible range. These vary from simple
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and inexpensive machines for routine work to highly sophisticated devices Molecular Spectroscopy
that can be used for specialised work and of course the routine jobs also.
However, the basic components of these instruments remain the same. The
five essential components of UV-VIS instruments are as follows

A. Stable radiation source (s)

B. Wavelength selector
C. Sample holder
D. Radiation detector or transducer, and
E. Signal processing and output device

The general layout of the essential components in a simple absorption

instrument is given in Fig. 1.5.

Fig. 1.5: General layout of the essential components in a simple absorption

instrument

Let us learn about these components one by one.

A. Radiation Sources

A radiation source must provide a stable high energy output over a broad range Glass prisms and lenses can
of wavelengths. There is no inexpensive source available that may provide be used in the visible region.
However, since glass absorbs
stable output over the entire UV-visible range (190 nm to 780 nm). For ultraviolet light, a quartz or
measurements in the UV region, electric discharge sources like hydrogen or a fused silica is a better choice
deuterium lamp are used. In these, the excitation of the gaseous molecules is for the material of the prism
because it can be used in both
brought about by the passage of electrons through the gas at low pressures. A the regions.
The gratings used for the
hydrogen lamp is commonly used in the spectrophotometers and gives light in
ultraviolet and visible region
the wavelength region of 160-375 nm. generally contain about 1200-
1400 grooves/mm.

Fig. 1.6 Radiation sources: a) deuterium lamp for UV range b) tungsten lamp for
visible range
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Spectroscopic Methods On the other hand, a tungsten filament lamp is used as the radiation source for
visible range. This consists of a thin, coiled tungsten wire that is sealed in an
evacuated glass bulb. This gives radiations in the range of 350-2200 nm.

B. Wavelength Selector: Monochromators

Monochromators are devices that can selectively provide radiation of a desired

wavelength out of the range of wavelengths emitted by the source. These are
of two types: the prism monochromators and grating monochromators. These
are described in the following paragraphs.

Prism Monochromators

You know that a prism disperses sunlight into seven different colours because
the radiations of different colours are refracted to different extent due to the
difference in the refractive index of glass for different wavelengths. Shorter
wavelengths are refracted more than longer wavelengths.

Fig. 1.7: Schematic diagram of the prism monochromator

In a prism monochromator, shown in Fig.1.7, a fine beam of the light from the
source is obtained by passing through an entrance slit. This is then collimated
on the prism with the help of a lens. The refracted beams are then focused on
an exit slit. The prism is then rotated in a predetermined way to provide the
desired wavelength from the exit slit.

Grating Monochromators

A grating is made by cutting or etching a series of closely spaced parallel

grooves on the smooth
reflective surface of a
solid material. The
intensity of radiation
reflected by a grating
varies with the
wavelength.In grating
monochromator,
Fig.1.8, a fine beam of
the light from the source
falls on a concave
mirror through an Fig. 1.8: Schematic diagram of a grating
monochromator
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entrance slit. This is then reflected on the grating which disperses it. The Molecular Spectroscopy
dispersed radiation is then directed to an exit slit.

The range of wavelengths isolated by the monochromator is determined by

the extent of dispersion by the grating and the width of the exit slit. Rotation
of the grating in a predetermined way can be used to obtain the desired
wavelength from the exit slit.

C. Sample holder

The UV-VIS absorption spectra are usually determined either in vapour phase
or in solution. Tomeasure the UV spectrum of the analyte it is taken in a cell
called a cuvette which is transparent to the wavelength of light passing through
it. A variety of quartz cuvettes are available for the spectral determination in
the vapour phase. These are of varying path lengths and are equipped with
inlet and outlets. For measurements on solutions in the visible region the
cuvettes made of glass can also be used. However, since glass absorbs the
ultraviolet radiations, these cannot be used in the UV region. Therefore, most
of the spectrophotometers employ quartz cuvettes, Fig 1.9 as these can be
used for both visible and UV region. Usually square cuvettes having internal
path length 1.0 cm are used, though cuvettes of much smaller path lengths say
of 0.1 mm or 0.05 mm are also available.

Fig. 1.9: Quartz cuvettes for measurements in solution and in vapour phase

D. Detectors

The detectors are used to convert a light signal to an electrical signal which The eye of a common person
can be suitably measured and transformed into an output. There are three types is quite sensitive to notice
differences in radiant power
of detectors which are used in modern spectrophotometers. These are described transmitted through two
in the following paragraphs. coloured solutions. We can
say that eye is a natural
1. Phototube photosensitive detector in the
visible range. Therefore, early
A phototube consists of a photoemissive cathode and an anode in an instruments used eye or
photographic plate as the
evacuated tube with a quartz window as shown in Fig.1.10 (a). These two
detector.
electrodes are subjected to high voltage (about 100 V) difference. When a
photon enters the tube and strikes the cathode, an electron is ejected and is
attracted to the anode resulting in a flow of current which can be amplified
and measured. The response of the photoemissive material is wavelength
dependent and different phototubes are available for different regions of
the spectrum.
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Spectroscopic Methods 2. Photomultiplier (PM) Tube

A photomultiplier tube (Fig. 1.10 (b)), consists of a series of electrodes,

called dynodes. The voltage of successive electrodes is maintained 50 to
90 volt more positive than the previous one.

(a) (b)
Fig. 1.10: Detectors of UV-VIS radiation; a) Phototube and b) Photomultiplier tube

When a radiation falls on the cathode an electron is emitted from it. This is
accelerated towards the next photoemissive electrode by the potential difference
between the two. Here, it releases many more secondary electrons. These, in
turn are accelerated to the next electrode where each secondary electron releases
more electrons. The process continuous upto about 10 stages of amplification.
The final output of the photomultiplier tube gives a much larger signal than
the photocell.

3. Diode Array Detector

These detectors employ a large number of silicon diodes arranged side by

side on a single chip. When a UV-VIS radiation falls on the diode, its
conductivity increases significantly. This increase in conductivity is
proportional to the intensity of the radiation and can be readily measured.
Since a large number of diodes can be arranged together, the intensity at a
number of wavelengths can be measured simultaneously. Though the
photodiode array is not as sensitive as the photomultiplier tube, the
possibility of being able to measure many wavelengths makes it a detector
of choice in the modern fast instruments.

E. Signal Processing and Output Devices

The electrical signal from the detector is suitably amplified or processed before
it is sent to the recorder to give an output. The subtraction of the solvent
spectrum from that of the solution is done electronically. The output plot
between the wavelength and the intensity of absorption is the resultant of the
subtraction process and is characteristic of the absorbing species.

Having learnt about different components of UV-VIS instruments and their

importance; you are now equipped to learn about the types of instruments
used.
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 Molecular Spectroscopy
4.3 FLUORESCENCE SPECTROSCOPY
You would recall from above that when a radiation in the UV-visible region
The excitation
interacts with a molecule, it causes transition among the quantised electronic Thetoterm
a triplet state
‘fluorescence’ was
requires a reversal of the
energy levels. Have you ever thought what happens to the excited molecules coined by
electron spin and such a
G. G. Stokes in 1852
on the name of the mineral
so obtained? These relax back to the ground state by using different processes.
transition fluorspar
is forbidden(CaF 2by
) that emits
quantum theory. Accordingly,
In some of the processes the absorbed energy is given off as heat to the visible light on illumination
the probability of such a
surroundings and in some it is emitted as radiation. When such a relaxation is with the UV light.
transition is very low.
accompanied by the emission of a radiation it is called luminescence. There
are two types of luminescence phenomena namely, fluorescence and
phosphorescence that arise from the excited state generated by absorption of
electromagnetic radiation. As the excitation of the molecule is due to the
absorption of a photon (light), these types of luminescence are called
photoluminescence.The processes involved in the excitation by radiation and
the radiative emissions can be understood with the help ofJablonski diagram.
Let us learn about Jablonski diagram and see how it explains the phenomena
of fluorescence and phosphorescence.

4.3.1 Origin of Spectrum: Jablonski Diagram

The Jablonski diagram gives a representation of ground and different excited
electronic states of a molecule and the processes associated with absorption
and emission (radiative and nonradiative) of energy. A typical Jablonski diagram
is given in Fig. 1.14.The set of lines at the bottom of the figure represents the
ground state and the ones in the upper portion indicate the excited electronic
states. To begin with, the molecule is in the electronic ground state. In this
state, the molecular orbitals are occupied by two electrons. You would recall
from your earlier knowledge that according to Pauli’s principle, the spins of
the two electrons in the same orbital must be antiparallel. This implies that the
total spin, S, of the molecule in the ground state is zero [½ + (- ½)]. This
energy state is called “singlet state” and is labeled as S0.

Fig. 1.14: The Jablonski diagram showing the phenomena of fluorescence and
phosphorescence
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Spectroscopic Methods The electron spins in the excited state achieved by absorption of radiation
may either be parallel or antiparallel. Accordingly, this may be a triplet (parallel)
or a singlet (antiparallel) state. These are designated as T1or S1, respectively as
the case may be. The spin states are schematically depicted in Fig.1.15.

Fig. 1.15: Possible electronic spin states of the molecules

The energies of the two excited spin states are also different due to the difference
in the interactions between electrons: the energy of the triplet state usually
being lower than that of the singlet state. In the Jablonski diagram two excited
singlet states (S1 and S2) and a triplet state (T1) are shown.

The absorption of a photon of suitable energy causes the molecule to get excited
from the ground state to one of the excited states. This process is called as
excitation or activation and is governed by Franck-Condon principle.
According to this principle, the electronic transition takes place so fast (~1015s)
that the molecule does not get an opportunity to execute a vibration, i.e., when
the electrons are excited the internuclear distance does not change. The basis
for the principle is that the nuclei are very massive as compared to the electrons
and therefore move very slowly. The implication of Frank -Condon principle
is that the transition from the ground state to excited state can be represented
by vertical arrows in the Jablonski diagram.

The electronic transition takes place from the state of lowest vibrational energy
of an electronic ground state to any of the vibrational levels of the excited
electronic states as shown by the vertical upward arrows in the diagram. The
excited state achieved would depend on the energy of the photon absorbed. In
the diagram the two excited states as S1 and S2 have been shown. These are
obtained by the absorption of the radiation in the region of wavelength O1 and
O2 respectively.

Once excited, the molecule can undergo a number of relaxation processes

during the time it spends in the excited state. The deactivation processes can
be broadly categorised into two groups given below.

z Nonradiative deactivation

z Radiative deactivation

Let us learn about these.

90
z Nonradiative Deactivation Molecular Spectroscopy

As the name suggests, these deactivation mechanisms are not accompanied

by any radiative emission. There are three different nonradiative means of
deactivation. These are described in the following paragraphs.

Vibrational Relaxation

In the higher vibrational levels of an excited state, the molecule rapidly loses

(in < 10-12 s) its excess vibrational energy by collision with other molecules
and falls to the lowest vibrational level of the excited state. This nonradiative
mode of relaxation is called vibrational relaxation,and the energy lost in this
process is dissipated as heat to the surroundings.

Internal Conversion
If the intensity of exciting
Once the molecules that are excited to an electronic state (say S2)higherlightthanis kept constant as its
the S1 state reach the vibrational ground state in the electronic level, these can
wavelength is changed, the
plot
pass to a higher vibrational level of a lower excited state (S1) which has the of emission against
exciting wavelength is
same energy. This process is referred to as internal conversion. The moleculeknown as the corrected
can continue to lose energy in this state in a nonradiative way (vibrational excitation spectrum.
relaxation) until it reaches the lowest vibrational level in this excited state.

Intersystem Crossing

In rare occasions, the molecule in the vibrational states of a singlet excited

state may cross over to a vibrational level of a triplet state if the two have same
energy. This process is called intersystem crossing.

z Radiative Deactivation

When the molecule in the excited state (S1) relaxes down to the lowest
vibrational level it may emit a photon and come down to the electronic ground
state (S0). This process is called fluorescence and takes about 109 s. Another
radiative relaxation process can arise from the excited molecule that had crossed
over to the triplet excited state by intersystem crossing and has relaxed to the
vibrational ground state in the triplet excited state. In such a case the molecule
emits a photon and comes down to a vibrational mode of the electronic ground
state, S0. This phenomenon is called phosphorescence. As the transition from
a triplet state to a singlet state is theoretically forbidden, it does not take place In fluorescence, the spin
readily. Thus, while the fluorescence emission can take place within 109"106 multiplicities of the ground
and emissive excited states
seconds, the transition from an excited triplet state to the ground state in case are the same whereas for
of phosphorescence requires at least 104 seconds and may take as long as 102 phosphorescence, the excited
seconds. and ground states have
different spin multiplicities.
Since the fluorescence emission takes place only after the excited molecule
has relaxed to the vibrational ground state of the S1 state i.e., after having lost
part of its energy, the energy of the emitted radiation is lower than that of the
excitation radiation. This means that the wavelength of fluorescence emission
would be greater than the excitation wavelength. Further, since the energy of
the triplet excited state of the molecule is lower than that of the associated
91
Spectroscopic Methods singlet state; the transitions to the ground state are associated with the emission
of light of still lower energy. As a consequence the phosphorescence occurs at
longer wavelengths than fluorescence. Fig.1.16 shows the excitation,
fluorescence and phosphorescence spectra of phenanthrene

Fig. 1.16:The excitation (E), fluorescence (F) and phosphorescence (P) spectra of
phenanthrene

4.3.2 Excitation versus Emission Fluorescence

You have learnt that the fluorescence arises when the electronically excited
molecule relaxes back from S1 state to S0 state accompanied by an emission of
radiation. You would have noticed from Fig.1.15 that when this radiative
relaxation takes place, the molecule can come to any of the vibration levels of
S0 state. This implies that the emitted radiation is constituted of different
wavelengths. A plot of the emitted radiation as a function of wavelength for
any given excitation wavelength is known as the emission fluorescence
spectrum. In Fig. 1.15, the excitation radiation is shown to have caused the
transition from S0 to S 2 state. What do you think would happen to the
fluorescence emission if we change the wavelength of the excitation radiation?
Yes, you thought it right; the emission spectrum would still remain the same

Fig. 1.17:The excitation and emission fluorescence spectra of 9-methylanthracene

92
because the fluorescence emission generally occurs only from the first excited Molecular Spectroscopy
singlet state irrespective of the excited singlet state produced initially. You
would observe in Fig. 1.15 that the fluorescence emission remains the same
for both the excitations viz., S0oS1 and S0oS2. However, the intensity of the
emission would be expected to change because the excitation radiation is
absorbed to different extents (recall the UV-VIS spectra). Now if we change
the wavelength of the exciting light and plot the emission from the sample at
a given emission wavelength against the wavelength of exciting radiation, the
result is known as the excitation spectrum. The emission fluorescence and
excitation spectra of 9-methylanthracene is given in Fig. 1.17.

Since a given analyte can fluoresce only after it has absorbed radiation, an
excitation spectrum consists of the wavelengths of light that the analyte is
able to absorb. In other words generally the excitation spectrum of a molecule
is the same as its UV-VIS absorption spectrum. Generally, the maximum in
the fluorescence spectrum of a compound occurs at longer wavelength than
the maximum in the absorption spectrum. The wavelength difference between
the absorption and fluorescence maxima is called the Stokes shift.

Having learnt about the origin of fluorescence and phosphorescence spectra

and the relation between absorption and fluorescence emission let us learn
about the instrument used for fluorescence measurement. However, before
that you can test your understanding by answering the following simple
questions.

SAQ 4

Which of the statements that follow the following incomplete statements

can be used to correctly complete it?

a) Fluorescence occurs when ………………..

i) a molecule returns to the electronic ground state from an excited

triplet state by losing its excess energy as a photon.

ii) a molecule returns to the electronic ground state from an excited

singlet state by losing its excess energy as a photon.

iii) a molecule lowers its vibrational energy by losing its excess energy
as a photon.

b) Intersystem crossing refers to…………………..

i) the reversal of the spin of an excited electron, changing the state of

the molecule (from singlet state to triplet state or vice versa).

ii) the loss of excess energy by the molecule by emitting a photon.

iii) the conversion of the excess electronic energy by the molecule to

vibrational energy.

93
Spectroscopic Methods
SAQ 5

Why does the fluorescence occur at longer wavelengths than the

absorption?
.................................................................................................................
.................................................................................................................
.................................................................................................................
.................................................................................................................

4.3.3 Instrumentation
All fluorescence instruments use essentially the same components as are used
in absorption spectrophotometers. However, the geometric arrangement of the
components is somewhat different. This is due to the reason that any transmitted
radiation is not measured along with the fluorescence. The absorption and
transmission of radiant energy occur only along the direction of the incident
light whereas the fluorescence radiation emanates in all directions. The
detection of transmitted radiation is avoided by placing the detector at a right
angle to the transmitted beam, as shown in Fig. 1.18.

Fig. 1.18: A schematic layout of a fluorimeter

Let us learn about the essential components of a fluorimeter

A. Sources

The source in fluorescence spectrometer must be more intense than that required
for UV-VIS absorption spectroscopy because the fluorescence intensity, IF is
directly proportional to the source power. An increase in P0 will produce a
94
larger signal for a given concentration and thereby improve sensitivity. The Molecular Spectroscopy
tungsten filament and deuterium lamps used in absorption spectrophotometers
are generally not suitable for fluorescence instruments as they lack the desired
intensity.

The modernspectrofluorometersare often equipped with a 75-450 W high-

pressure xenon arc lamp. These produce an intense continuum between about
250 and 600 nm. As the xenon arc lamp produces lot of heat, the lamp assembly
needs to be cooled therefore, these instruments cannot be used for routine
work.For certain applications, it is preferable to use a laser excitation source.
A tuneable dye laser, using a pulsed nitrogen laser as the primary source can
produce monochromatic radiation between 360 and 650 nm. A few fluorescence
spectrometers using laser sources are commercially available; most such
instruments are intended for highly specific applications.

B. Wavelength Selectors

The low-cost instruments designed for routine determinations are simple filter
fluorimeters. Such instruments are used when it is sufficient to measure fluorescence
intensity at a single excitation and emission wavelength. These employ fixed filters
to isolate both the excitation and emission wavelengths. In order to isolate one
particular wavelength from a source what we need is just a pair of cut-off filters.
Absorption filters are comprised of a suitably absorbing substance or substances
dispersed in gelatin, glass or plastic. The filter fluorimeters are used primarily in
environmental field screening, hospital or clinical settings and other applications
in which low cost and small size are crucial.

Most modern fluorimeters used in analytical laboratories generally use

diffraction grating monochromators about which you have learnt above. Such
a fluorescence spectrometer is capable of recording both excitation and emission
spectra and therefore makes full use of the analytical potential of the technique.

C. Sample holder

The majority of fluorescence measurements of the analyte are carried out in

solution. For this the sample is taken in a cuvette or in a flow cell. The cuvettes
generally are circular, or square shaped. These are constructed of a material
that will transmit both the incident as well as the emitted light. Glass and
quartz both qualify this criterion. However, the quartz cuvettes are generally
used when the radiation belongs to the UV region. The square cuvettes are
most common and are found to be quite precise as the parameters of a square
cuvette are easier to maintain during manufacture. As fluorimetry is a very
sensitive technique, the following precautions should be followed without
exception while handling the cuvettes.

z The optical surfaces of the cell should not be touched with hand as it
invariably leaves an invisible film that may change the light transmission
and reflection characteristics of the cell, especially in the ultraviolet region.

z The cuvettes should be handled only at the top portions of the side plates
that do not face the optical axis.
95
Spectroscopic Methods z The samples should be transferred to the cuvettes with the help of a dropper
or a pipette.

z The cuvettes should be rinsed with the analyte solution before filling and
the overfilling should be avoided.

D. Detectors

The fluorescence signal for an analyte present at low concentration is weak. It

is partly due to low photoluminescence efficiencies and partly because only a
small fraction of the fluorescence radiation reaches the detector. Thus, the
basic requirement for the detector is that it should be able to detect weak optical
signals. Therefore, photomultiplier tubes with their high sensitivity and low
noise are generally used. You have learnt about PM tubes above.

E. Output Devices

The output from the detector is suitably amplified and displayed on a read out
device like a meter or digital display. The sensitivity of the amplifier can be
changed so as to be able to analyse samples of varying concentrations. In
some instruments the display can be adjusted to directly give the output in
terms of the concentration. Nowadays, the instruments have microprocessor
controlled electronics that provides outputs compatible with the printers and
computers whereby minimising the possibility of operator error in transferring
data.

Let us now more on to yet another important molecular spectroscopy viz.

vibration spectroscopy. Herein we would talk about IR spectroscopy and Raman
spectroscopy. Howeverbefore proceeding further, answer the following simple
questions to assess your understanding.

SAQ 7

Complete the following sentences with appropriate words.

a) The fluorescence power or intensity is directly proportional to the

…………. .

b) Photomultiplier tubes with low noise and high sensitivity are preferred
over simple photo tubes so as to detect …………... signals.

4.4 VIBRATION SPECTROSCOPY

You would recall from Unit 2 of the MEV 13 course that the absorption of IR
radiation causes transitions amongst the quantised vibrational energy levels
of the molecules. This gives rise to vibrational or IR spectrum that can be
used for structure elucidation and identification of a large variety of organic,
inorganic and biological samples. This finds applications in diverse areas like
drugs and pharmaceuticals, clinical and biomedical determinations, and
environmental analysis etc.

The Raman spectroscopy also involves vibrational energy levels but it differs
96
from IR spectroscopy on many counts. These include the nature of radiation Molecular Spectroscopy
used, the mechanism of interaction between the radiation and matter, the
necessary condition for the interaction, required instrumentation and the
resulting spectra etc. Further, even the information available from it is different;
in fact it is complimentary to the one available from IR spectroscopy. The UV-
VIS spectroscopy discussed above involved absorption of radiation whereas
the fluorescence spectroscopy is based on emission of radiation. The Raman
spectroscopy on the other hand involves scattering of radiation.

An electromagnetic radiation when passed through a transparent medium

C.V. Raman the discoverer of
interacts with the particles (e.g., molecules, atoms, or ions) constituting it. If Raman Effect in 1928. He was
the dimensions of the particles are equal to or smaller than that of its wavelength awarded the 1930 Nobel prize
then it undergoes scattering. It has been observed that most of the scattered in Physics.
Like IR spectrum, theThe significance of
Raman
spectra are also given in be gauged
his discovery can
radiation has the same wavelength as that of the incident radiation. Such a from the fact that it holds the
wavenumber units. This is
scattering is referred to as Rayleigh scattering. However, a very small fraction
convenient record for the the
because shortest time
(to the tune of about 1 in 10 ) of the scattered radiation is found to have
7
a of other scatteredto awarding
position from a discovery
of the Noble
radiations (Raman prize.
scattering)
wavelength different from that of the incident radiation. This is called Raman
can be conveniently expressed
scattering and the existence of Raman scattering is called Raman effect. Let of Raman shifts.
in terms
us learn about the origin and significance of Raman spectrum.

4.4.1 Origin of Raman Spectrum

The origin of Raman effect and hence the Raman spectrum can be explained
in terms of following two theories:

z Quantum or particle theory

z Classical or wave theory

Let us discuss these one by one.

Elastic collisions: The
Quantum or Particle Theory collisions that do not involve
any exchange of energy.
According to the quantum theory of radiation, an electromagnetic radiation is
Inelastic collisions: The
considered to be consisting of a stream of particles called photons. The photons collisions that involve
constituting a radiation of frequency Q would have energy equal to hQ where exchange of energy.
h is the Planck’s constant. The interaction of the radiation with the interacting
species can be visualised in terms of collisions between them and the photons.
If the collisions are elastic (i.e., they do not involve any exchange of energy)
the photons would be scattered (deflected) with their incident frequency
remaining unchanged. This explains the observance of Rayleigh scattering.
Since most of the collisions are elastic in nature, most of the scattered photons
would have same frequency as the incident frequency and the Rayleigh line is
observed to be quite intense.
Though the inelastic
A small fraction of the collisions between the photons and particles of matter collisions can bring about
is found to be inelastic in nature i.e., these involve exchange of energy. When transitions amongst vibra-
tional (and/or rotational)
the photons constituting the radiation undergo inelastic collisions with the
energy levels we would
absorbing species, they either gain or lose energy. These energy exchanges consider only vibrational
bring about transitions in the quantised energy levels of the molecules. In such transitions.
an event of inelastic collisions, the molecules are either vibrationally (and/or
97
Spectroscopic Methods rotationally) excited or they may undergo vibrational (and/or rotational)
relaxation. In both the cases the photons get scattered with a frequency different
from their initial frequency. In former case i.e., when the molecules undergo
excitation the scattered radiation is of lower frequency and the spectral lines
are called Stokes Raman lines. In the latter case, where the molecules undergo
relaxation, the scattered photons are of a higher frequency and the spectral
lines are called anti-Stokes Raman lines. Fig 1.19 gives the spectra of the
scattered radiation obtained when a sample of CCl4 was interacted with a laser
beam having a wavelength of 488.0 nm.

Fig. 1.19: Raman spectra for CCl4 using 488 nm laser source

You may note that the intense peak in the centre of the spectrum has the same
wavelength (or wavenumber) as that of the incident radiation. This is the
Rayleigh peak and the other signals on either side of the Rayleigh peak are the
Raman lines. The lines to the left of Rayleigh peak and having lower value of
wavenumber are the Stokes lines while the ones to the right and having higher
value of wavenumber are the anti-Stokes lines. Stokes lines are at lower energy
while the anti-Stokes lines are at energy greater than the Rayleigh peak. You
may note two more features in the spectrum firstly, the Stokes and anti-Stokes
lines are equidistant from the Rayleigh line and secondly, the Stokes lines are
much more intense as compared to the anti-Stokes lines.

The positions of Raman lines are expressed in terms of a parameter called

Raman shift, 'Q which is defined as per the following equation.

'Q ( Q s  Q 0 ) cm 1 …(1.19)

Where, Q s and Q 0 are the wavenumbers of the source (or incident) radiation
and the observed scattered lines respectively. It is obvious that the Raman
shifts of the Stokes lines would be positive while for anti-Stokes lines, these
would be negative.

Classical or Wave Theory

When a molecule is placed in a static electric field, the positive and negatively
98
charged particles constituting the molecule get attracted to the opposite poles Molecular Spectroscopy
of the applied field. This leads to a charge separation and consequently to the
development of an induced dipole moment. In simple words, we say that the
molecule has become polarised. This tendency of the molecule to get polarised
is called polarisability i.e., the ability to get polarised. The magnitude of the
induced dipole moment is proportional to the strength of the applied field and
is given by the following equation.

P DE … (1.20)

The proportionality constant D is called polarisability- a measure of the ease The polarisability (D) of the
with which the molecule (or a bond in it) can get polarised. The polarisability molecule depends on the
bond length; shorter bonds
can be anisotropic, i.e., the electrons of a bond may be polarised to different being difficult to polarise
extents when the field is applied in different directions. For example, the than longer bonds.
hydrogen molecule gets polarised to different extents when the field is applied
in the direction of the bond or in the direction perpendicular to it, as shown in
the margin. What would happen if we place a molecule in an oscillating electric
field like that of an electromagnetic radiation? Let us see.

The electric field associated with a radiation having a frequency Q ex varies as

per the following equation.

E E0 cos( 2SQ ext ) …(1.21)

where E is the electrical field strength at any time t and E0 is the amplitude of
the wave. When such a radiation is made to interact with a molecule it would
induce a dipole moment in it. Further, as the electrical field of the radiation is
oscillating the resulting induced dipole moment would also oscillate at the The necessary condition to
same frequency as that of the radiation. That is, observe vibration Raman
spectrum is that the
P DE DE0 cos( 2SQ ext ) … (1.22) polarisability of the bond
must change in the course of
This oscillating dipole would generate a radiation of the frequency, Q ex i.e vibration
Rayleigh scattering.

As you are aware, the molecules are never stationary and always undergo
vibration (and may be rotation) motions. These motions may cause a change
in the polarisability of the molecule. When such a system vibrating with a
frequency of Q vib interacts with the oscillatory electric field of the
electromagnetic radiation then the scattered radiation may have three different
components. One of these has frequency equal to the incident frequency ()
while the frequencies of the other two are equal to and respectively. The first
component with a frequency of is the reason for the Rayleigh scattering. The
other two components are the sources of the anti-Stokes and Stokes lines
respectively.

The three components in the scattered radiation are observed only when the
vibration causes a change in the polarisability. If the polarisability does not
change in the course of vibration only Rayleigh scattering is observed.
Therefore, we can conclude that in order to observe Stokes and anti-Stokes
lines the polarisability of the bond must change in the course of vibration.
99
Spectroscopic Methods Let us raise a question. Since both the IR and Raman spectra involve transitions
amongst the same vibrational levels, would the two spectra be the same? As a
first response, the answer to the question about the similarity of IR and Raman
spectra raised above, could be in a firm affirmative or one may respond, ‘may
be’. However in actual practice the two spectra are generally not identical and
in case of a number of molecules, the spectra in fact do not have anything in
common. The Raman and IR spectra for a simple molecule CCl4 is given in
Fig. 1.20. You may note that only some of the signals are common in the two
spectra and also the common signals are of different relative intensities.

Fig. 1.20: IR and Raman spectra of CCl4; note the similarities and the differences

You would recall that only those modes of vibration are IR active which have
an associated change in dipole moment. Similarly only those vibrational modes
are Raman active which are associated with a change in polarisability.

The Raman activity of different vibrational modes can be predicted on the

basis of whether or not the polarisability changes during the vibration. However,
it is not very simple and in many cases requires a detailed analysis. We would
not get into these details. It would be suffice to mention here that in addition to
the CCl4 example where only some modes have IR as well as Raman activities,
we may have situations where all the vibration modes are active in IR as well
as in Raman and in some cases there is no mode which is active in both.

The IR and Raman activities of different modes of vibration of a molecule

depend on its symmetry properties. An analysis of the IR and Raman spectra
of a large number of molecules has led to an extremely important general rule
known as the rule of mutual exclusion. It states that, “for a molecule having
a centre of symmetry the Raman active vibrations are IR inactive and vice
versa”. However, if the molecule does not have a centre of symmetry then
some vibration may be Raman as well as IR active. The application of this
rule can provide very useful structural information. For example, the fact that
the Raman and IR spectra for N2O have signals at same wavenumber suggests
that the molecule does not have a centre of symmetry. This information can be
used to conclude that the structure of the molecule is N–N–O and not N–O–N.

100
4.4.2 Instrumentation for Raman Spectroscopy Molecular Spectroscopy

Like the IR instruments, the Raman spectrometer, has the following basic
components.

z An intense laser source

z Sample handling unit
z Monochromator or interferometer
z Detector
z Signal processing and output device

The schematic representations of the general set up of the monochromator

based dispersion Raman spectrometer is given in Fig. 1.21.
Lasers used for Raman
spectroscopy must exhibit
good wavelength stability and
low background emission.

As Raman experiments
always involve the
measurement of small energy
shifts of the order of 100 to
3000 cm1 from the excitation
energy, a monochromatic
source is highly desirable and
lasers meet this requirement
the most.

Fig. 1.21: Schematic representation of the experimental set up of a dispersion Raman Unlike most chemical
spectrometer analysis techniques, Raman
spectrometry does not require
You may find the arrangements to be similar to that of the corresponding IR any special preparation of the
sample. Since Raman
instruments with the only difference being that the scattered radiation is spectrometry involves only
collected at right angles to the radiation and it is passed through a filter before illuminating a sample with a
sending to a transducer for detection. The Raman spectrometers differ from laser and collecting the
scattered photons, no contact
the IR instruments in terms of the sources, the sample handling devices and with the sample is needed at
the transducers used for detecting the scattered radiation. The first ever Raman all. This makes Raman
instrument was constructed in 1928 which used the Sun light focused by a spectroscopy a non-
destructive technique.
telescope to achieve a high enough intensity in the scattered signals. The
spectrometers that followed used mercury arc lamps as a light source. With
the advent of lasers with highly desirable properties of high intensity, single
wavelength and coherence, the modern spectrometers almost exclusively use
laser sources.

Raman spectrometer has a distinct advantage over IR spectrometry because it

permits the use of glass for holding aqueous solutions. The possibility of using
aqueous samples is particularly important as it allows ascertaining water
pollutants besides biological and inorganic samples. More so, since the laser
source provides a focused coherent radiation, the sample container can be
101
Spectroscopic Methods very small which in turn requires exceedingly smaller sample volume. For a
typical measurement of the spectrum a solid sample is ground to a fine powder
and packed into a small cavity to be kept in the path of incident radiation. The
liquid samples on the other hand are taken in a fine capillary which is about 5
cm long and has an outer diameter of about 1 mm.

For analysing gaseous samples, the cuvette consists of a cylindrical glass tube
with mirrors on both the ends; one of the mirrors has a small window to let the
incident radiation to pass through. When the cuvette containing the sample is
placed in the radiation path the incident laser beam enters the sample through
the window and undergoes multiple reflections in the sample. The scattered
light at the right angles to the sample is then suitably collected and measured.

The original Raman instrument that lead to the discovery of the Raman effect
used eyes as the detector. In initial versions of the spectrometers, photographic
plates were used to detect the scattered radiation. This was followed by more
sensitive photomultiplier tubes (PMTs) that allowed electronic data collection
and manipulation. However, today most of the Raman spectrometers are FT
instruments which use cooled germanium photoconductors as transducers.

We hope you would have understood the basic features of a Raman

spectrometry. Let us now take up the environmental applications of the
spectroscopic methods discussed in this unit.However, before that why don’t
you check your understanding of Raman spectrometry by answering the
following simple questions?

SAQ 7

What is Raman shift? The Raman shift positions of Stokes and anti-Stokes
lines are equal but opposite. Comment.
...................................................................................................................
...................................................................................................................
...................................................................................................................
...................................................................................................................
...................................................................................................................

SAQ 8

State whether the following statements are true or false.

I. The intensities of the Raman lines depend on the frequency of the

exciting radiation.

II. The Raman spectra are generally taken for aqueous solutions only.

SAQ 9

Fill up the blanks in the following.

I. When the collisions between the photons and molecules are ……….,
the photons would be…………..with the same frequency.

102
 II. If in the event of an inelastic collision with photons the molecules get Molecular Spectroscopy

vibrationally excited, the frequency of the scattered photon will be

………….than the incident photon.

III. In order to observe Raman scattering the …………… of the bond

must change in the course of vibration.

4.5 APPLICATIONS OF SPECTROSCOPIC

METHODSIN ENVIRONMENTAL
MONITORING
UV-VIS spectrometry is highly adaptable, and versatile technique that provides
an affordable and reliable method of analysis. It finds applications in most of
the areas of environmental concern viz., air, water and soil. A number of
important UV/Vis methodologies have been developed for the determinationof
organic, inorganic and complex ionic structure in various environmental
matrices. Some of the general and specific applications of UV-VIS spectrometry
are listed below.

z Determination of heavy metals by coloured complex formation with

selected ligands.

z Identification of petroleum products in marine environment; useful in cases

of oil spills. Detection of bacteria in water samples by monitoring the
change in transmittance as a function of time.

z Determination of pesticide residues in soil samples by suitable

complexation followed by absorbance measurement.

z Determination of fluoride content in water samples using Alizarin red

method. In this method, the water sample is treated with the reagent and
the complex is extracted with pentanol and the absorbance is measured at
430 nm.

z Determination of nitrite in water and soil samples by reacting with p-

nitroaniline in acid medium to form diazonium ion followed by treatment
with ethyl cyanoacetate in basic medium to form an azo dye. The dye is
then monitored by measuring absorbance at 465 nm.

z Determination of sodium dodecyl sulfate (SDS)-a component of non-

biodegradable surfactants in wastewater by forming a yellow-colored
complex with acridine orange. The complex is then extracted using toluene
and the concentration is obtained by measuring absorbance at 467 nm.

z Determination of ozone in ambient air by passing it in a solution of indigo

disulfonate (IDS) in a special double sintered disc absorber and following
the reduction in the concentration of IDS by measuring absorbance at 610
nm.

103
Spectroscopic Methods
4.6 LET US SUM UP
In this unit we have discussed about molecular spectroscopy i.e., the study of
interaction of EM radiation with molecular systems. We began the unit with a
discussion on UV-VIS spectroscopy wherein we discussed the origin of the
UV-VIS spectrum. Then we discussed the characteristics of the spectrum i.e.,
the position of the signal, its intensity and width. This was followed by the
description of the instrumentation used and the explanation of the principle of
UV-VIS spectrophotometry as an analytical tool.

Then we took up fluorescence spectrometry-an emission spectroscopic method.

In this context we discussed about the origin and characteristics of the
fluorescence spectrum. We also explained the origin of phosphorescence
spectrum and differentiated it from the fluorescence spectrum. We briefly
explained the phenomenon of chemiluminescence and discussed about the
instrumentation used in fluorescence spectrometry.

Having discussed about the UV-Vis and fluorescence spectroscopic methods

involving electronic energy levels, we took up vibration spectroscopy i.e., the
study of interaction of EM radiation in the IR region with the molecules. In
this context we explained the origin and characteristics of the IR and Raman
spectra and the instrumentation required for them. Towards the end of the unit
we have outlined the environmental applications of the discussed molecular
spectroscopic methods.

4.7 TERMINAL QUESTIONS

1. Outline the factors that may cause deviations from Beer and Lambert’s
law.

2. The absorbance of a solution of ferrous 1, 10-phenanthroline complex

containing 1.00 microgram of iron (II) in 10 cm3 of solution was measured
at 515 nm in 1 cm cuvette. It was found to be 0.0200. Calculate the molar
absorptivity of the complex.

3. What are monochromators? Describe the functioning of a grating

monochromator.

4. Why do we need to take UV spectra in quartz cuvettes?

5. Enlist the essential steps of quantitative determination methodology.

6. Differentiate between internal conversion and inter system crossing.

7. What do you understand by the spin multiplicity of an electronic energy

level? What are the multiplicities of the singlet and triplet state?

8. What is the difference between chemiluminescence and photo-

luminescence?

9. State Franck Condon principle. What is its significance?

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10. Describe briefly different mechanisms of nonradiative relaxation of an Molecular Spectroscopy
excited electronic state.

11. The Raman spectrum of an amino acid cystine was obtained by using a
488 nm laser. The observed Raman shift of the most intense Stokes line is
105 cm1. Compute the positions of the most intense Stokes and the
corresponding anti-Stokes lines.

12. State mutual exclusion principle for deciding IR and Raman activities.

ANSWERS
Self Assessment Questions

1. P0 = 85.4 b = 2.00cm

P = 20.3 c = 1 × 10-4M e =?

A = log Pî/P = A = log 85.4/20.3 = 0.624

Since A= e b c; º = A/b.c
0.624
Substantially the value H
2.0 cm u1 u104 M
1 1 3
=> 3120 cm mol dm
1 1 3
2. º =14000 cm mol dm

c= ?b = 1cm A= 0.85

A = ecbc = A/ e.b

c = 0.85/14000 × b

= 6.07 ×10-5M

3. The standard addition method allows the determination of the concentration

of the species in such solution also in which the other constituents are not
fully known.

4. A diode array detector is better than a photomultiplier tube as it allows

simultaneous determination of the intensity of several electromagnetic
radiations of different wavelengths.

5. a) ii)

b) i)

6. The fluorescence emission takes place only after the excited molecule has
relaxed to the vibrational ground state of the S1 state i.e., after having lost
part of its energy. As the energy of the emitted radiation is lower than that
of the excitation radiation it is observed at a longer wavelength than that
of the excitation radiation

7. a) source power

b) weak optical
105
Spectroscopic Methods 8. Raman shift refers to shift in the observed scattered frequency from the
frequency of the excitation radiation. Raman shift, 'Q is defined as
Δν ( νs  ν0 ) cm 1
Both the Stokes lines and anti-Stokes lines arise from inelastic collisions
with the incident radiation. A Stokes line arises when the radiation excites
a given vibration mode whereas the corresponding anti-Stokes line arises
due to relaxation of the same vibration. The Raman shifts of the two are
equal because they involve same vibration and are opposite because one
involves excitation while the other concerns relaxation.

9. a) True

b) False

10. a) elastic, scattered

b) lesser

c) polarisability

Terminal Questions

1. Some of the factors responsible for the deviation from Beer and Lambert’s
law are as follows:

z Presence of Electrolytes

z Complexation, Association or Dissociation

z Non monochromatic nature of the radiation

z Concentration of the Analyte

z Temperature

2. The atomic weight of iron is 56, therefore the molar concentration of the
solution containing 1.00 microgram of iron (II) present in 10 mL of solution
will be
1u 10 6 g 1000mL
1.786 u10 6 mol per dm .
3
u
56 10mL

0.02
H A / b.c Ÿ
1.786 mol dm 3 u 1 cm
= 1.12 × 104

3. A monochromator is a device that can selectively provide radiation of a

desired wavelength out of the range of wavelengths emitted by the source.
In grating monochromator, a fine beam of the light from the radiation
source is made to fall on a concave mirror through an entrance slit. After
reflection, it falls on the grating which causes it to disperse. The dispersed
radiation is then emerges out of an exit slit. The range of wavelengths
isolated by the monochromator is determined by the extent of dispersion
by the grating and the width of the exit slit. Rotation of the grating in a
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 predetermined way used to obtain the desired wavelength from the exit Molecular Spectroscopy
slit.

4. In order to take the UV absorption spectra the analyte is taken in a quartz

cuvette because it is transparent to the electromagnetic radiations of the
UV range. As glass absorbs the ultraviolet radiations, these cannot be used
for the purpose.

5. The essential steps of the quantitative determinations methodology are,

z Forming an absorbing species

z Selection of the measurement wavelength

z Controlling factors influencing absorbance

z Validation of Beer and Lambert’s law

6. The internal conversion refers to the transfer of the molecule from an

excited singlet state to another whereas in case of intersystem crossing the
molecule in the vibrational states of a singlet excited state crosses over to
a vibrational level of a triplet state (rarely from a triplet to singlet). However,
in both the cases the energies of the initial and final level must be the
same.

7. Spin multiplicity of an electronic state refers to the number of possible

quantum states of the system. It is computed as (2S+1) where S is the total
spin quantum number for the electronic state.

The spin multiplicities of the singlet and triplet states are 1 and 3,
respectively.

8. The difference between chemiluminescence and photoluminescence lies

in the way the excited states are achieved. In chemiluminescence, the energy
required for the excitation comes from the chemical reaction whereas in
photoluminescence the energy is provided by a photon (light). The principle
explains the phenomenon of activation process during the fluorescence
and phosphorescence of a molecule.

9. According to Franck Condon principle the electronic transition takes place

so fast that the molecule does not get an opportunity to execute a vibration.
Therefore, in the Jablonski diagram, the transition from the ground state
to excited state can be represented by vertical arrows.

10. The nonradiative relaxation refers to the deactivation of the molecule in

the excited state without emission of radiation. This may occur by any of
the three mechanisms viz., vibrational relaxation, internal conversion or
inter-system crossing.

z In vibrational relaxation, the molecules rapidly lose their excess

vibrational energy by collision with other molecules and the energy is
dissipated as heat to the surroundings.

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Spectroscopic Methods z In internal conversion the molecule is transferred from an excited
singlet state to another singlet state of the same energy.

z In intersystem crossing the molecule is transferred from an excited

singlet state to a vibrational level of a triplet state (or vice-versa).

11. We know that for a photon,

hc 1 1
E hQ hcQ or ν (cm )
O λ(cm)
Given wavelength = 488 nm =488 × 10-9 m
= 488 × 109 × 102 cm = 488 × 107cm

Substituting it in the above formula we get, wave number of the 488 nm

laser as.
ν (cm 1 ) = 1 × 107 / 488 = 20491 cm-1

Since the Raman shift of the most intense Stokes line is at 105 cm-1 therefore
its wavenumber would be

20491-105 = 20386 cm-1

The corresponding anti-Stokes line would be observed at

20491 + 105 = 20596 cm-1.

12. The mutual exclusion principle states that, “for a molecule having a center
of symmetry the Raman active vibrations are IR inactive and vice versa”.

108