UNIT 1: SPECTROSCOPIC TECHNIQUES AND APPLICATIONS

Introduction: Spectroscopy is one of the most powerful tools available to study the structure of a
molecule. Also, it can be used for the analysis of the samples. It involves interaction of electromagnetic
radiation with matter. Electromagnetic radiation is a form of energy that is transmitted through space with
huge velocity in a straight line unless it is refracted or reflected. It has electric and magnetic components,
which oscillate in planes perpendicular to each other and perpendicular to the direction of propagation of the
radiation (Figure 1).

(a)

(b)
Figure 1 : Electromagnetic Wave

Electromagnetic radiation: The properties of electromagnetic radiation are as follows:

(1) Electromagnetic radiation is a form of energy. The energy which travels in the form of waves is
called Electromagnetic radiation.
(2) It has dual nature, exhibiting both wave and particle characteristics.
(3) It has both, electrical and magnetic components.
(4) The two components oscillate in planes perpendicular to each other and perpendicular to the
direction of propagation of the radiation.
(5) It can travel through empty space.
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 Wave properties of electromagnetic radiation:
(1) Wavelength: (λ) It is defined as the distance between two successive maxima of either electric or magnetic
components of the wave.
Unit: meters (m), centimeters (cm), nanometers (nm), angstrom (A○)
1 nm = 10-7 cm = 10-9 m = 10 A○

(2) Frequency: (v) It is the number of waves that pass through a fixed point in unit time.
Unit: cycles per second (cps) or Hertz (Hz) or Fresnel, kilocycles per second (k cps or kHz), megacycles per
second (M cps or MHz)
1 Hz = 1 cps
1 Frensel = 1012 Hz
1 MHz = 103 kHz = 106 Hz

(3) Wave number: ( ) It is the reciprocal of wavelength.

Unit: reciprocal of distance (generally cm-1)

(4) Velocity: (c) It is the product of wavelength and frequency of the wavelength in the given medium.
Wavelength x Frequency = Velocity
λxv = c
Velocity of light in a vacuum is denoted by c and is (3 x 108 m/s).

Particle properties of electromagnetic radiation: The electromagnetic radiation consists of a

stream of discrete packets (particles) of pure energy, called photons or quanta. They have definite energy and
travel in the direction of propagation of the radiation beam with the velocity equal to that of the light. The
energy of the photon (Ep) is proportional to the frequency of radiation given by the relationship,
hc
Ep = hv = ____
λ
Where Ep is the energy of photon in Joules.
v is frequency of photon.
h is the Planck’s constant (6.626 x 10-34 Joule-second or 6.624 x 10-27 erg-second).
c is the velocity of light in a vacuum.
λ is wavelength of radiation.
This relation shows that the frequency is directly proportional to energy and inversely proportional to
wavelength. The intensity of a beam of radiation is proportional to the number of photons per second which
are propagated in a beam. However, the intensity of a beam is not dependent on the energy of each photon.

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 The phenomena of refraction, reflection, reinforcement and destructive interference can be explained
by wave characteristics. The photoelectric effect can be explained by particle characteristics. Thus, the duality
of the electromagnetic radiation is useful for the quantitative description of the above mentioned phenomena.

Electromagnetic spectrum, its origin: When an electromagnetic radiation falls on matter, it interacts
with atoms or molecules constituting the matter. After interaction, there may occur variation in the intensity
of electromagnetic radiation with frequency or wavelength. This variation takes place either by absorption or
emission of radiation by the matter. The energy is absorbed or emitted by the matter in discrete amounts called
quanta. When the measurement of this absorption or emission radiation is carried out experimentally, it gives
a value for the change of energy involved, from which the set of possible energy levels of the matter can be
concluded. The technique in which the measurements of either absorbed or emitted radiation are done
experimentally, thereby producing a spectrum, involves the usage of spectroscopy.

Numericals based on energy of photon:

The energy of photon and Einstein: A photon can be defined as quantum of energy.
The energy of a photon (Ep) is given by, hv and
hc
hv = ____
λ
Where, h = Planck’s constant (6.626 x 10-34 J-sec or 6.624 x 10-27 erg-sec)
v = Frequency of radiation
λ = Wavelength of radiation
c = Velocity of light (3 x 108 m/s or 3 x 1010 cm/s)

The energy, E, of an Avogadro number (N) of photons is referred to as one einstein, given by,
Nhc
E = ______
λ
Where N = Avogadro number (6.023 x 1023)
Thus, Energy per einstein is also called as energy in one mole of photon.
Unit of energy is erg or J

Conversions:
1 cal = 4.184 J
l Kcal = 1000 cal and 1 KJ = 1000 J
1 mole = 6.023 x 1023
1 nm = 10-7 cm = 10-9 m = 10 A○

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 Solved Numericals:
(1) Calculate the energy associated with (a) one photon; (b) one einstein (or energy in one mole of
photon) of radiation of wavelength 8000 A°. (h = 6.62 x 10-27 erg-sec, c = 3 x 1010 cm/s, N = 6.023 x 1023).
Solution: 1 A° = 10-8 cm, ∴ 8000 A° = 8000 x 10-8 cm
hc 6.62 x 10-27 x 3 x 1010
(a) Energy of a photon = ____ = __________________ = 2.4825 x 10-12 erg
λ 8000 x 10-8

Nhc 6.023 x 1023 x 6.62 x 10-27 x 3 x 1010

(b) Energy per einstein, E = ____ = ______________________________ = 1.4952 x 1012 erg
λ 8000 x 10-8

(2) Calculate the energy associated with (i) one photon; (ii) one Einstein (or energy in one mole of
photon) of radiation of wavelength 7000 A°. Also, express the answer in Kcal/mole. (h = 6.626 x 10-34 J-sec,
c = 3 x 108 m/s, N = 6.023 x 1023).
Solution: 1 A° = 10-10 m, ∴ 7000 A° = 7000 x 10-10 m
hc 6.626 x 10-34 x 3 x 108
(i) Energy of a photon = ____ = __________________ = 2.8397 x 10-19 J
λ 7000 x 10-10

Nhc 6.023 x 1023 x 6.626 x 10-34 x 3 x 108

(ii) Energy per einstein, E = ____ = ______________________________ = 1.7104 x 105 J
λ 7000 x 10-10
(iii) As 1 cal = 4.184 J or 4.184 J = 1 cal
1.7104 x 105 x 1
1.7104 x 105 J = _______________ = 4.0727 x 104 cal
4.184
l Kcal = 1000 cal or 1000 cal = 1 Kcal
4.0727 x 104 x 1
4.0727 x 104 cal = ______________ = 4.0727 x 10 = 40.727 Kcal
1000
Thus, energy of wavelength = 40.727 Kcal/mole

(3) The energy for a quantum of light is 4.4 x 10-19 J. What is the wavelength and frequency of this
light? (Given: h = 6.626 x 10-34 J-s, c = 3 x 108 m/s).
Solution:
hc
(i) Energy of a quantum = ____
λ

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 hc 6.626 x 10-34 x 3 x 108
∴ λ = __________________ = ___________________ = 4.5177 x 10-7 m
Energy of a quantum 4.4 x 10-19
(ii) λ x v = c
c 3 x 108
∴ v = __ = __________ = 6.6405 x 1014 Hz
λ 4.5177 x 10-7
OR
E = hv
E 4.4 x 10-19
∴ v = __ = __________ = 6.6405 x 1014 Hz
h 6.626 x 10-34

Spectroscopy:
Definition: Spectroscopy is the branch of science which deals with the study of the interaction of
electromagnetic radiation with matter.
Principle: After interaction of electromagnetic radiation with matter, there may occur variation in the
intensity of electromagnetic radiation with frequency or wavelength. This variation takes place either by
absorption or emission of radiation by the matter. Accordingly, it is called as either absorption or emission
spectroscopy respectively. In absorption spectroscopy, the molecules undergo transition from ground to
excited state, whereas in emission spectroscopy the molecules undergo transition from excited to ground state.
The plot of absorption against wavelength is called absorption spectrum and the plot of emission against
wavelength is called emission spectrum.
Consider two molecular energy levels, a lower energy level (E1) and a higher energy level (E2). If a
photon of frequency v falls on a molecule in the ground state, then its energy hv is exactly equal to the
difference between the two molecular levels, i.e., ∆E = hv = E2 – E1
If the molecule undergoes a transition from lower energy level to the higher energy level with the
absorption of a photon of energy hv and the absorbed wavelength is measured, it is called absorption spectrum.
If the molecule undergoes a transition from higher energy level to the lower energy level with the emission of
a photon of energy hv and the emitted wavelength is measured, it is called emission spectrum. The
spectroscopic transitions between molecular energy levels is shown in Figure 2.

(a) (b)
Absorption spectrum Emission spectrum
Figure 2 : Spectroscopic transitions between Molecular energy levels

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 Classification of spectroscopy: Spectroscopy is classified as atomic and molecular spectroscopy
depending on the types of energy changes those take place within the molecule. The types of energy present
in molecules are as follows:
(i) Translational energy (Et): It is considered with the overall movement of the molecules along the
three axes.
(ii) Electronic energy (Ee): It involves changes in the distribution of electrons to higher levels on
absorption of energy.
(iii) Vibrational energy (Ev): It is associated with vibrations within the molecule such as stretching
and bending of bonds.
(iv) Rotational energy (Er): It involves the spinning of molecules about the axes passing through the
centre of gravity.
The translational energy levels of a molecule are so close that they are considered as continuous.
Hence, they are not quantised. The other three types of energy are quantised. Hence, the internal energy (E)
of the molecule is due to the sum of electronic (Ee), vibrational (Ev) and rotational (Er) motions within the
molecule.
∴ E = Ee + Ev + Er
When a molecule absorbs electromagnetic radiation, the change in the internal energy of the molecule
may be electronic, vibrational or rotational energy. The types of energy levels and energy changes are shown
in Figure 3(a) and 3(b) respectively.

Figure 3(a) : Types of energy levels in a molecule

(a) Electronic (b) Vibrational (c) Rotational

Figure 3(b) : Types of energy changes in a molecule

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 Atomic spectroscopy: This deals with the interaction of electromagnetic radiations with atoms which
are most commonly in their lower energy state called the ground state. The electronic absorption of
electromagnetic radiation can occur only if the photon has an energy which is equal to the energy difference
between two quantized energy levels, i.e.,
∆E = hv
where ∆E is the energy difference between two quantum levels, h is the Planck’s constant and v is frequency
of photon.
Monoatomic substances normally exist in the gaseous state and are able to absorb electromagnetic
radiation, resulting in transition of electrons from one electronic level to another.
Atomic spectroscopy provides information about the qualitative and quantitative analysis of an
element. It includes atomic absorption as well as emission spectroscopy.

Molecular spectroscopy: This deals with the interaction of electromagnetic radiations with
molecules. This results in transitions between rotational and vibrational energy levels in addition to electronic
transitions.
Molecular spectroscopy provides information about the structure of the molecule. It includes various
types, depending on their principles such as ultraviolet, infra-red spectroscopy.

Differences between atomic and molecular spectra is as follows:

Sr. No. Atomic spectra Molecular spectra
1. It is obtained from the interaction of atoms and It is obtained from the interaction of molecules
electromagnetic radiation. and electromagnetic radiation.
2. It is due to electronic transition in an element. It is due to electronic, vibrational and rotational
transitions in an element.
3. It is a line spectra. It is a complicated spectra.
4. It determines the type of atoms present in a It determines the structure of molecules present
given sample. in a given sample.

Types of Spectroscopy: (Include in classification also)

(i) Absorption spectroscopy: When light is initially passed through an absorbing substance and then
absorbed through a spectroscope, it will be observed that certain lines are missing in the spectrum which leaves
dark lines or bands at their places. As the light of wavelength corresponding to black bands is absorbed by the
substance through which the light is passed, the spectrum so obtained is known as absorption spectrum. (Refer
Figure 2(a) and write about absorption spectrum.)
(ii) Emission spectroscopy: When a substance is heated to a high temperature by thermal or electric
method, light is emitted. When the light coming after passing through a prism or a grating is examined with a

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spectroscope, the spectrum so obtained is known as emission spectrum. (Refer Figure 2(b) and write about
emission spectrum.)
(iii) Scattering spectroscopy: It is based on scattering phenomenon. It differs from absorption and
emissions spectroscopic techniques as scattering is much faster than absorption and emission process. It can
be of two types, elastic and inelastic. When wavelength of scattered light is same as that of incident light, it is
elastic scattering and when wavelength of scattered light is different as that of incident light, it is inelastic
scattering.

Classification of spectroscopy can be represented as follows:

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 Table of relation between electromagnetic spectrum, types of spectroscopy and energy changes:
Electromagnetic spectrum is an ordered arrangement of electromagnetic radiation over the entire range
of wavelength (Figure 4). It ranges from the gamma region (left) to the radio region (right), in order of
increasing wavelength and decreasing frequency. There is an inverse relationship between wavelength and
frequency. (As one goes up, the other goes down.)

Figure 4 : Electromagnetic spectrum

The relation between types of spectroscopy, wavelength range photon energy and molecular effects is
represented as follows:

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 The relation between types of spectroscopy, frequency, wavelength range and photon energy is given
in Table-1.
Table-1 : Electromagnetic spectrum
Sr. Type of Frequency (Hz) Wavelength Photon energy (J/mol)
No. spectroscopy range
1. Mossbauer (γ- 3 x 1018 – 3 x 1020 Less than 0.01 nm 109 – 1011
ray)
2. X-ray 3 x 1016 – 3 x 1018 0.01 nm – 10 nm 107 – 109
3. Ultraviolet or 3 x 1014 – 3 x 1016 10 nm – 400 nm 105 – 107
Electronic
4. Visible 400 nm – 750 nm
5. Raman 3 x 10 – 3 x 10
12 14
400 nm – 750 nm 103 – 105
6. Infrared 750 nm – 1 mm
7. Rotational 3 x 1010 – 3 x 1012 1 mm – 1 m 101 – 103
(Microwave)
8. Electron Spin
Resonance
(ESR)
9. Nuclear 3 x 106 – 3 x 1010 1 m – 105 km 10-3 – 101
Magnetic
Resonance
(NMR)
10. Nuclear
Quadrapole
Resonance
(NQR)

The information regarding the type of spectroscopy and energy changes associated with particular
spectroscopy is given in Table-2.
Table-2 : Type and Energy changes of spectroscopy
Sr. No. Type of spectroscopy Energy changes
1. Mossbauer (γ-ray) The transitions occur between the nuclear spin energy levels
of a molecule due to absorption of gamma (γ) photons.
2. X-ray Electronic transitions occur.
3. Ultraviolet or Electronic Mainly electronic transitions occur, invariably accompanied
4. Visible by vibrational and rotational transitions.
5. Raman Vibrational-rotational energy level changes accompanied by
change in the polarizability of the molecule.
6. Infrared Vibrational-rotational energy level changes, accompanied by
change in the dipole moment.
7. Rotational (Microwave) Rotational energy level changes, accompanied by change in
the dipole moment.
8. Electron Spin Resonance (ESR) The transitions occur between the electron spin energy levels
of a molecule in an applied magnetic field.
9. Nuclear Magnetic Resonance The transitions occur between the nuclear spin energy levels
(NMR) of a molecule in an applied magnetic field.
10. Nuclear Quadrupole Resonance The transitions occur between the nuclear spin energy of a
(NQR) molecule arising from the interaction of the unsymmetrical
charge distribution in nuclei with the electric field gradient
which arises from the bonding and non-bonding electrons in
the molecule.

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 Flame Photometry: Flame photometry is based on the principle of emission spectroscopy. Flame
photometry is also called as flame atomic emission spectroscopy. It is based on the measurement of intensity
of the light emitted when a metal is introduced into a flame. The wavelength of light emitted is useful for the
identification of the element and the intensity of the light emitted informs about the concentration of the
element.

Principle: The principle of the technique involves introduction (or aspiration) of the liquid sample to
be analysed, containing a metallic salt solution into a flame. The sample solution gets converted into an aerosol
by atomizer principle. Due to hot flame, the solvent evaporates, sublimates and atomizes the metal as shown
below.
Na+ + e- Na
The heat of the flame excites a valence electron/s from lower energy state (E0) to a higher energy state
(E1). The excited electron returns to the ground state by emitting the light energy, hv, at characteristic
wavelength for each metal, which is selected by the optical filters. The intensity of emitted light is directly
proportional to the concentration of sample. The radiation emitted during the process is given by,
E1 – E0 = hv ………(1)
where h is the Plank’s constant and v is the frequency of the emitted light, defined as,

c
v = ____
λ

Substituting for v in equation (1),

hc
E1 – E0 = _____
λ

hc
∴ λ = _______ ………..(2)
E1 – E0

From equation (2), wavelength of the emitted light can be calculated, which is the characteristic of a
particular element.
The quantity of atoms excited depends upon the temperature of the flame. Hence, more number of
atoms get excited if the temperature of the flame is increased. Therefore, the temperature of the flame needs
to be controlled and should be uniform while performing the experiment.

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 The steps are shown as follows:

Liquid sample
↓
Formation of droplets
↓
Fine residue
↓
Formation of neutral atoms
↓
Excitation of atoms by thermal energy
↓
Emission of radiation of specific wavelength
↓
Intensity of emitted radiation measured

Instrumentation: A flame photometer comprises a source of heat (flame), i.e, burner, a nebulizer, a
monochromator (an optical system, i.e., lens), slit system, filter, a detector and a recorder. The instrumentation
of the technique is shown in Figure 5.

Figure 5 : Flame photometer

(i) Burner: It is the source of flame. It can be maintained at a constant temperature. The temperature
of the flame is one of the critical factors in flame photometry.
Example: Total consumption burner and Laminar flow burner.
Total consumption burner - Due to the high pressure of fuel and oxidant, the sample solution is aspirated
through capillary and burnt at the tip of burner. Hydrogen and oxygen are generally employed as fuel and

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oxidant. The advantage over other is the entire consumption of sample. Its disadvantage is the production of
non-uniform flame and turbulent.
Premix burner - In this burner, the sample, fuel and oxidant are thoroughly mixed before aspiration and
reaching to flame. The main advantage is the uniformity of flame produced. The main disadvantage is the
heavy loss of mix up to 95%.
(ii) Nebuliser: It is used to send homogeneous solution at a balanced rate.
(iii) Optical system: It consists of convex mirror and convex lens. The convex mirror transmits the
light emitted from the atoms and helps to focus emissions to the lens. The lens helps to focus light on the slit.
(iv) Filter: The reflections from the mirror pass through the slit and reach the filters. Filters will isolate
the wavelength to be measured from that of irrelevant emissions. A simple flame photometer contains a filter
wheel and when a particular element has to be analysed the specific filter is selected.
(v) Photodetector: The intensity of radiation emitted by the flame is measured by photodetector. The
radiation emitted by the elements is mostly in the visible region. Hence, conventional detectors like photo
voltaic cell or photo tubes can be used. In a flame spectrophotometer, photomultiplier tube is used as detector.
These electrical signals are directly proportional to the intensity of light.

Working: The sample to be analysed is taken in a sample container. The burner is the source of flame
which is produced by burning of fuel, such as acetylene or natural gas or propane-butane gas, in the presence
of air or oxygen. The sample is sucked up by air and is passed into the nebulizer. Nebulizer or aspirator is a
device that introduces the sample solution in the flame at the balanced rate. The gases help in aspiration and
making the mist of the sample, called aerosol.
The temperature of about 2000-3000°C is obtained due to which excitation of valence electron/s takes
place. The molecules and ions of the sample get decomposed and reduced to give atoms. The characteristic
wavelength of the light is emitted. The emitted wavelength is selected by the monochromator. In some cases,
optical filters are also used to select the emission wavelength. The emitted light is allowed to fall on the
detector. The detector is photomultiplier detector, which produces an electric signal proportional to the
intensity of emitted light.

Applications: Flame photometry is suitable for analysis of elements both qualitatively as well as
quantitatively. Hence, it finds applications in various fields such as medicine, agriculture.
(1) Qualitative analysis: As each element emits its own characteristic wavelength, the method is useful
for the qualitative determination of group I and II elements such as Na, K, Rb, Cs. Thus, the alkali and alkaline
earth metals can be detected by flame photometry.
(2) Quantitative analysis: As the intensity of emitted light is proportional to the concentration of the
element, the method can be significantly useful for determination of concentration of unknown sample. The
method is discussed below.

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 The solutions of known concentrations of the sample to be analysed are prepared. These solutions are
aspirated into the flame one after the other form lower to higher concentration. The intensity of emission
radiation is recorded and a calibration curve is plotted as intensity against the concentration of the analyte
(Figure 6). A sample solution of unknown concentration is then aspirated into the flame and the intensity
reading is recorded. The intensity reading is plotted in the calibration graph and the concentration of the
unknown sample corresponding to the intensity reading is noted. Thus, the concentration of the unknown
sample can be determined.

Figure 6 : Graph of finding concentration of the analyte in unknown sample

This method has an advantage that the interference of material that present in the sample but does not
respond to flame photometry, is automatically eliminated.
(3) Application in clinical laboratory: Flame photometry is used in clinical laboratory to determine the
concentrations of sodium and potassium in biological fluids like serum, urine and also in body tissues.
(4) Application in agriculture: In agriculture, the fertilizer requirement of the soil is analysed by flame
photometric analysis of the soil.
(5) Application in water technology: Concentration of calcium is determined in hard water using flame
photometry.
(6) Application in metallurgy: In metallurgy, sodium is determined in aluminium and aluminium
alloys.

Interferences: In determining the amount of a particular element present, other elements can also
affect the result. Such interferences are as follows:
(1) Spectral interferences: It occurs when the emission lines of two elements cannot be resolved or
arises from the background of flame itself. They are either too close, or overlap, or occur due to high
concentration of salts in the sample.
(2) Ionic interferences: High temperature flame may cause ionisation of some of the metal atoms.
Example: Sodium. The Na+ ion possesses an emission spectrum of its own with frequencies, which are
different from those of atomic spectrum of the Na atom.

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 (3) Chemical interferences: The chemical interferences arise out of the reaction between different
interferents and the analyte. It includes,
(i) Cation-anion interference – The presence of certain anions, such as oxalate, phosphate, sulphate, in
a solution may affect the intensity of radiation emitted by an element.
Example: Calcium and phosphate ions form a stable substance, as Ca3(PO4)2 which does not decompose easily,
resulting in the production of lesser atoms.
(ii) Cation-cation interference – These interferences are neither spectral nor ionic in nature.
Example: Aluminium interferes with calcium and magnesium.

Disadvantages: They are as follows:

(1) In flame photometry, the energy produced due to flame is sufficient for the excitation of alkali and
alkaline earth metals. However, the energy produced due to flame is insufficient for the excitation of transition
metals and most of the metals in the periodic table. Hence, this method is not suitable for the analysis of these
elements, which require more energy for excitation.
(2) It cannot directly detect and determine the presence of inert gases.
(3) Though this technique measures the total metal content present in the sample, it does not provide
the information about the molecular structure of the metal present in the sample.
(4) Only liquid samples may be used. Also sample preparation becomes lengthy in some cases.
(5) Flame photometry cannot be used for the direct determination of each and every metal atom. A
number of metal atoms cannot be analysed by this method. The elements such as carbon, hydrogen and halides
cannot be detected due to their non-radiating nature.

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 Jablonski diagram: Molecules absorb light radiations and get excited. The molecules in the excited
state are highly unstable. When the molecules return to the ground state, the extra energy is given out by
different processes. The dissipation of the energy can take place by either radiative or non-radiative mode.
Jablonski diagram represents how the extra energy is dissipated while the molecules return to the ground state
(Figure 7).
(The Foundation of a typical Jablonski Diagram is given for understanding)

(The Foundation of a typical Jablonski Diagram)

Figure 7 : Jablonski diagram

Non-radiative transitions:
(1) Vibrational relaxation: During the excitation, the molecules may go to any vibrational levels in the
excited state S1. The excited molecules in the vibrational levels higher than the lower vibrational levels in S1

16
rapidly give up energy in small amounts and come down to the lowest vibrational level (v = 0) of S1. It is
called as vibrational relaxation or energy cascade. The energy is lost by collision with other molecules.
(2) Internal conversion (IC): The molecules from S1 (or S2) can return to ground state of vibrational
level (v = 0) of S0 (or S1) by giving energy to other vibrations or to solvent. This is called an internal
conversion. The excess energy is lost through molecular collisions and heat is produced. It is a slow process.
It may also result in a photochemical reaction.
(3) Intersystem crossing: In other case, S1 to T1 transition takes place. T1 has a lower energy level and
much longer life-time than S1. The process of intersystem crossing involves the spin inversion.

Radiative transitions:
(1) Fluorescence: The molecules in the lower vibrational state (v = 0) of S1 return to any one of the
vibrational levels in S0. It can take place by giving the excess energy in the form of radiation. The emission
takes place without spin inversion and the process is called as fluorescence.
(2) Phosphorescence: The molecules in the lower vibrational state (v = 0) of T1 return to any one of
the vibrational levels in S0 by giving the excess energy in the form of radiation. The process is called as
phosphorescence.

Introduction to fluorescence and phosphorescence: Fluorescence and Phosphorescence are

photophysical processes. Some molecules absorb radiations in the visible and ultraviolet regions of the
electromagnetic spectrum and get excited. These radiations if not utilized, are re-emitted as fluorescence and
phosphorescence.
During excitation, the electrons move from ground to excited state. The electrons may have singlet or
triplet spin states. A singlet state is a system in which all electrons are paired (have anti-parallel spins) and
triplet state is one in which electrons are not paired (have parallel spins).

Fluorescence: Certain molecules when exposed to light radiation of short wavelength, emit light of
longer wavelength and the emission stops as soon as the incident radiation is cut off. The process is called
fluorescence and the substance that exhibits fluorescence is called fluorescent substance. The emission of light
stops after the removal of source of excitation.
The path involved in fluorescence process is,
Ground singlet state (S0) Excited singlet state (S1) Vibrational relaxation
Ground singlet state (S0)
It involves transition from singlet ground state (S0) to singlet excited state (S1) and do not involve
change of the spin state. It is observed in solution at room temperature.
Examples of fluorescence are as follows:
(i) A solution of quinine sulphate exhibits blue colour fluorescence.
(ii) A solution of chlorophyll in ether shows blood red fluorescence.

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 Phosphorescence: Certain molecules when exposed to light radiation of short wavelength, emit light
of longer wavelength and the emission continues even after the incident radiation is cut off. The process is
called phosphorescence and the substance that exhibits phosphorescence is called phosphorescent substance.
The path involved in fluorescence process is,
Ground singlet state (S0) Excited singlet state (S1) Intersystem crossing
Excited triplet state (T1) Ground singlet state (S0)
It involves transition from singlet ground state (S0) to singlet excited state (S1) to triplet excited state
(T1) and involves change of the spin state. It cannot be observed in solution at room temperature.
Examples of phosphorescence are as follows:
(i) Sulphates of calcium, barium exhibit phosphorescence.
(ii) Fluorescein in boric acid shows phosphorescence in the blue region at 5700A° wavelength.

Comparison of fluorescence and phosphorescence is as follows:

Sr. Fluorescence Phosphorescence
No.
1. The process involves absorption of light The process involves absorption of light
followed by immediate emission of light. followed by delayed emission of light.
2. The emission of light stops after the The emission of light continues after the
removal of source of excitation. removal of source of excitation.
3. The excited atom has comparatively short The excited atom has comparatively long
life time before its transition to ground life time before its transition to ground state.
state.
4. The time interval between absorption andThe time interval between absorption and
emission of energy is very short. emission of energy is comparatively long.
5. It involves transition from singlet ground
It involves transition from singlet ground
state (S0) to singlet excited state (S1) and
state (S0) to singlet excited state (S1) to
does not involve change of the spin state.
triplet excited state (T1) and involves
change of the spin state.
6. It is observed in solution at room It cannot be observed in solution at room
temperature. temperature.
7. Energy released is higher compared to Energy released is lower compared to
phosphorescence. fluorescence.

Applications of fluorescence in medicine: The applications of fluorescence in medicine are as

follows:
(1) It is used for the direct determination of sulphur in protein. The sulphur content of each of the many
different forms in which protein exists in human blood varies considerably. X-ray fluorescence indicates
protein distribution and provides a diagnostic link for the medical practitioner.
(2) It is used for the determination of chloride in blood serum.
(3) It is used for the determination of strontium in blood serum and bone tissue.
(4) It is useful for the elemental analysis of tissue, bones and body fluids.
(5) It is useful for the diagnosis of human diseases such as cancer, skin and eye diseases and
atherosclerotic cardiovascular disease.
(6) It is a potential diagnostic tool for microorganisms.
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