COLLEGE OF PHARMACY

ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

(An Autonomous College)
BELA (Ropar) Punjab

Name of Unit: UV Visible Spectroscopy and Fluorimetry

Course/Subject Name: Instrumental Methods of Analysis
Course/Subject Code: BP701T
Class: B. Pharm. Semester VII
Faculty: Dr. Monika Gupta
Email id: [email protected]
Mobile No. 8146891785

Learning Outcome of Module-I

LO Learning Outcome (LO) Course Outcome Code

LO1 To understand the interaction of matter with electromagnetic BP701.1, BP701.2
radiations and its applications in drug analysis.
LO2 To perform quantitative & qualitative analysis of drugs using BP701.1, BP701.5,
UV spectroscopy BP701.6
LO3 To perform quantitative & qualitative analysis of BP701.1, BP701.5,
drugs using Fluorimetery BP701.6
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

Table of Content

Topic

 Electromagnetic Radiation and Spectroscopy

 Ultraviolet–Visible Spectroscopy

 Types of Electronic Transitions

 UV-Spectrophotometer

 Applications

 Fluorimetry

 Factors affecting fluorescence and phosphorescence

 Instrumentation of Fluorometer

 Applications
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

Electromagnetic Radiation and Spectroscopy

The most challenging task of a chemist is to determine the chemical structure of an unknown
compound. There are many ways by which we can identify the unknown substance. A person
can use physical methods such as boiling point, melting point, spectroscopy as well as chemical
methods such as functional group testing and others to determine the structure of compounds.
Spectroscopy is one of the best methods to identify a substance, which may include UV, IR,
NMR, Raman and others. Here we will discuss about the various aspects of different
spectroscopic techniques and more specifically about UV-spectroscopy and its uses.
Electromagnetic spectrum covers a wide range of electromagnetic radiations, ranging from γ-
rays having wavelength which are the order of a fraction of an angstrom, to radio waves having
wavelength in meters or kilometers. The arrangement of all types of radiations in the order
of their increasing wavelength or decreasing frequencies is known as electromagnetic (EM)
spectrum. The electromagnetic spectrum includes radio and TV waves, microwaves, infrared,
visible light, ultraviolet, X-rays, γ-rays, and cosmic rays, as shown in the Figure 1.

Figure 1: Electromagnetic spectrum

Spectroscopy: Spectroscopy is the study of interaction of electromagnetic radiations with
matter. Electromagnetic radiations can interact with matter in various ways. Each
interaction gives us insights about certain properties of the matter and use of electromagnetic
radiations of different energies can give different information about the matter under study.
It is the motion of electrically charged particles that give rise to electromagnetic radiations.
There are various forms of electromagnetic radiation e.g. radio waves, X-rays, gamma rays,
infrared, visible, ultraviolet etc. All the types of radiations travel with the same velocity but
differ from each other in terms of frequency and wavelength. They do not require any medium
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

for their propagation and can travel through empty space as well as through air and other
substances. Each type of electromagnetic radiation has a dual nature- wave like nature and
particle like nature. The particle nature has been established by the fact that the energy of
particular radiation occurs in discrete packets or quanta known as photons. Each photon contains
a certain amount of energy. The different types of radiation are defined by the amount of energy
found in the photons. The energy associated with particular electromagnetic radiation is directly
proportional to its frequency. The photons with the highest energy correspond to the shortest
wavelengths.

1. Absorption of Electromagnetic Radiations by Organic Molecules

The energy required for these transitions is quantized. Excited molecules are unstable and
quickly come back to the ground state by releasing the energy they had received as
electromagnetic radiation. The wavelength and intensity of the electromagnetic radiation
absorbed or emitted can be recorded with the help of spectrometer to get a spectrum. The
energy required for the transition

Table 1: Energy changes depending on the type of Radiation

(E) from a state of lower energy (E1) to state of higher energy (E2) is exactly equivalent to
the energy of electromagnetic radiation that causes transition.
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

E1 – E2 = E = hν = hc/λ
Where E is energy of electromagnetic radiation being absorbed, h is the universal Planck’s
constant, 6.624 x 10-27 J sec and ν is the frequency of incident light in cycles per second (cps or
hertz, Hz), c is velocity of light 2.998 x 1010 cm s-1 and λ = wavelength (cm)
Higher is the frequency, higher would be the energy and on the other side, longer is the
wavelength, lower would be the energy. As we move from Gamma rays to ultraviolet region to
infrared region and then radio frequencies, we are gradually moving towards regions of lower
energies. However, almost all the parts of electromagnetic spectrum are used for understanding
the matter, in organic chemistry we are mainly concerned with energy absorption from only
ultraviolet and visible, infrared, microwave and radiofrequency regions.

Ultraviolet–Visible Spectroscopy
UV-Visible spectroscopy deals with the study of the electronic transitions of molecules as they
absorb light in the UV (190-400 nm) and visible regions (400-800 nm) of the electromagnetic
spectrum. The absorption of ultraviolet or visible radiation lead to transition among electronic
energy levels, hence it is also often called electronic spectroscopy.
As a rule, the energetically favored electron promotion will be from the highest occupied
molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). The resulting
species is said to be in an excited state. The wavelengths at which absorption occurs,
together with the degree of absorption at each wavelength is recorded by optical spectrometer.
A spectrum is obtained as a result. It commonly provides the knowledge about π-electron
systems, conjugated systems, aromatic compounds and conjugated non-bonding electron
systems etc.

Types of Electronic Transitions

The ground state of an organic molecule contains valence electrons in three principal types of
molecular orbitals, namely -sigma (σ) orbitals, pi (π) orbitals and filled but nonbonding orbitals
(n). Both σ and π orbitals are formed from the overlap of two atomic or hybrid orbitals. Each of
these molecular orbitals therefore has an antibonding σ* or π* orbital associated with it. An
orbital containing n electrons does not have an antibonding orbital because it is not formed from
two orbitals. Sigma bonding orbitals have lower energy than π bonding orbitals, which in turn
have lower energy than non-bonding orbitals. In the Electronic transitions, promotion of an
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

electron from one of the three Fig 2: Electronic Transitions in UV/VIS spectroscopy ground

states (σ, π, or n) to one of the two excited states (σ∗, or π∗) takes place. As a result, there are six
possible transitions- σ to σ∗, σ to π∗, π to π∗, π to σ∗, n to π∗, n to σ∗.

Fig 2: Electronic Transitions in UV/VIS spectroscopy

The most commonly observed transitions and their relative energies are summarized in
Figure 2. The exact energy differences between the orbitals depend on the nature of atoms
present and the nature of the bonding system. In each possible case, an electron is excited from a
low energy, ground state orbital into a higher energy, excited state anti-bonding orbital (Figure
2). Each transition requires a defined amount of energy. The larger the gap between the energy
levels, the greater the energy required to promote the electron to the higher energy level,
resulting in electromagnetic radiation of higher frequency, and therefore shorter wavelength,
being absorbed. The important modes of electronic transitions are described here.
1) σ to σ∗
A transition of electrons from a bonding sigma orbital to the antibonding sigma orbital is
designated as σ to σ∗ transition. These are high energy transitions as σ bonds are generally very
strong. Thus these transitions involve very short wavelength ultraviolet light (< 150 nm) and
usually fall outside the range of UV-visible spectrophotometers (200-800 nm). Alkanes can only
undergo σ to σ* transitions. Methane and ethane undergo σ → σ* transitions with an absorbance
maximum at 122 and 135 nm, respectively. Study of such transitions is usually done in an
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

evacuated spectrophotometer (< 200 nm) since oxygen present in air absorbs strongly at 200 nm
and below. Similarly nitrogen absorbs at ~150 nm and below.
2) π to π∗
The transition of an electron from a π bonding orbital to a π∗ antibonding orbital is designated as
π to π∗ transition. These types of transitions take place in compounds containing one or more
unsaturated groups like simple alkenes, carbonyl, aromatics, nitriles, nitro etc. These transitions
require lesser energy than n to σ∗ transitions. In non-conjugated alkenes, this type of transition
occurs in the range 170-190 nm e.g. ethane shows absorption maxima at 171 nm. Similarly, π to
π∗ transition in the range of 180-190 nm occurs in non-conjugated carbonyl compounds e.g.
acetone shows absorption maxima at 188 nm.

3) n to σ∗
The transition of an electron from a non-bonding orbital to the antibonding sigma orbital is
designated as n to σ∗ transition. Saturated compounds containing atoms with lone pairs (non-
bonding electrons) like saturated alcohols, amines, halides, ethers etc are capable of showing n
to σ∗ transitions.

Energy required for these transitions is usually less than σ to σ∗ transitions. Such compounds
absorb light having wavelength in the range 150-250 nm. For example the absorption maxima
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

for water, methyl alcohol, methyl chloride and methyl iodide are 167 nm, 174 nm, 169 nm and
258 nm, respectively.
4) n to π∗
The transition of an electron from a non-bonding orbital to a π∗ antibonding orbital is designated
as n to π∗ transition. This transition involves the least amount of energy in comparison to all
other transitions and therefore gives rise to an absorption band at longer wavelength.

Saturated carbonyl compounds show two types of transitions, low energy n to π∗ (270-300 nm )
and high energy π to π* (180-190 nm). The transition n to π∗ is of lowest energy but is of low
intensity as it is symmetry forbidden. Thus the most intense band for these compounds is always
due to π to π* transition.
1.
Selection Rule
Some electronic transitions, which are otherwise theoretically possible, are generally not
observed in the UV/VIS spectroscopy. Therefore there are some restrictions which govern the
observable transitions.
1. Transitions, which involve change in the spin quantum number of an electron during the
transitions do not occur, i.e. singlet-triplet transitions are not allowed.
2. Transitions between orbitals of different symmetry do not occur. For example,
transition
3. n to π∗ is forbidden because the symmetry of n and π∗ do not match.
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

UV-Spectrophotometer
Spectrophotometer is a kind of spectrometer, which measures the transmittance or absorbance of
a sample as a function of wavelength, when light of certain intensity and frequency range is
passed through the sample. Unlike a spectrometer (which is any instrument that can measure the
properties of light over a range of wavelengths), a spectrophotometer measures only the intensity
of light as a function of its wavelength. The key components of a spectrophotometer are:
• Light source
• Monochromator
• Sample area
• Detector and Recorder

Light Source
The most suitable sources of light are
1. Deuterium lamp which emits the light in the UV-region (160-375 nm)
2. Tungsten lamp or tungsten-halogen lamp which emits
radiation in the Visible and near infrared regions (350-2500 nm)
3. Xenon arc lamp which emits radiation in the range 190-800 nm
4. Light emitting diodes (LED) which emits radiation in the visible range 400-800
nm.
The instruments automatically swap lamps when scanning between the UV and UV-VIS regions.

Monochromator
The main function of the monochromator is to disperse the beam of light obtained from the
primary source, into its component (Figure 3). The principle components of monochromator are
• An entrance slit
• A collimating lens
• A dispersing device
• A focusing lens
• An exit slit

The radiation emitted from the primary source (polychromatic radiation) enters the
monochromator through the entrance slit. The beam is collimated and then strikes the dispersing
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

element (Prism or grit) at a particular angle. Two types of dispersion devices viz. prisms and
holographic gratings (Figure 4) are commonly used in UV-visible spectrophotometers.

Figure 3: Functioning of Monochromator

Figure 4: Dispersion of white light through prism and grating

Light falling on the prism is reflected at different angles, depending on the wavelength. The
beam is split into its component colors. By moving the dispersing element or the exit slit,
radiation of only a particular wavelength can be obtained, which leaves from the exit slit and can
be used for the recording purpose (Figure 3). The beam selected by the slit is monochromatic
and further divided into two beams with the help of another prism, which then passes through
the sample and reference solutions.

Sample area
One of the two divided beams is passed through the sample solution and the other beam is
passed through the reference solution. Although, the samples for recording spectra are most
commonly liquids, but the absorbance of gases and even of solids can be measured. Both
sample and reference solutions are placed in a transparent cell, known as cuvette. The cuvettes
are rectangular in shape, and usually have an internal width of 1 cm (Figure 5).
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

Figure 5: A quartz cuvette

It is important that the material of cells must be transparent to the radiation throughout the region
under study. The cells are usually made of glass, plastic as well as silica or quartz. Of these, glass
cells cannot be used for the UV region as they absorb light in the UV region but can be used
satisfactorily in the visible region. Quartz is transparent in all (200-700 nm) ranges and is the best
choice and hence can be easily used in UV as well as visible region.
Detector and Recorder
A detector converts a light signal into an electrical signal. After the beams are passed through the
sample under study as well as the reference cell, the intensities of the respective transmitted
beams are then compared over the whole wavelength range of the instrument. Generally two
photocells are used as detector in UV spectrometer to record the spectra. One of the photocell
receives the beam from sample cell and second detector receives the beam from the reference.
The intensity of the radiation from the reference cell is stronger than the beam of sample cell.
This results in the generation of pulsating or alternating currents in the photocells.
Spectrophotometers consist of either a photomultiplier tube detector or a photodiode
detector.
The commonly used detector in UV-Vis spectroscopy is photomultiplier tube (Figure 6). It is
m consists of three components:

 a cathode which emits electrons when struck by photons of radiation

known as photo emissive cathode
 several dynodes which emit several electrons for each electron striking them
 an anode
In its functioning; when a photon of radiation strikes the cathode, emission of several electrons
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

take place. These emitted electrons are then accelerated towards the many dynodes. The first
dynode is 90V more positive than the cathode. The electrons strike the first dynode, causing the
emission of several electrons for each incident electron. These electrons are then accelerated
towards the second dynode, to produce more electrons which are accelerated towards dynode
three and so on. Finally all the electrons are collected at the anode. Several dynodes are
arranged between the anode and the cathode to produce an amplification effect. Each photon
usually produces 106-107 electrons, resulting in the amplified current that can be measured.

Figure 6: A photomultiplier tube

Photomultipliers are very sensitive to both UV and visible radiations and have fast response
times. It is significant to note that Intense light may damage photomultipliers, hence they are
limited to measuring low power radiation.
Photodiodes are increasingly being used as detectors in modern spectrophotometers.
Photodiode detectors have a wider dynamic range and are more robust than photomultiplier tube
detectors. In a photodiode, light falling on the semiconductor material allows electrons to flow
through it, thereby depleting the charge in a capacitor connected across the material. The
amount of charge that is required to recharge the capacitor at regular intervals is proportional to
the intensity of the light. The limits of detection are approximately 170–1100 nm for silicon-
based detectors.

Figure 7: An array of photodiodes

ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

Some modern spectrophotometers contain an array of photodiode detectors instead of a single

detector (Figure 7). A diode array consists of a series of photodiode detectors positioned side by
side on a silicon chip. Each photodiode is connected to a transistor switch via a charged
capacitor When photons strike the diode, free electrical charge carriers are generated that
discharge the capacitors. The capacitors are recharged at regular intervals. The amount of charge
needed to recharge the capacitors is proportional to the number of photons detected by each
diode, which in turn is proportional to the light intensity. The absorption spectrum is obtained by
measuring the variation in light intensity over the entire wavelength range.

Types of UV/VIS Spectrophotometer

There are two types of spectrophotometers, namely, single beam spectrophotometer or double
beam spectrophotometer (Figure 8). Each has its advantages and disadvantages.

Figure 8: Types of Spectrophotometer

A single beam spectrophotometer has only one beam of light, which passes through the sample,
therefore it requires taking reading for the reference and sampling separately. On the other hand,
in a double-beam instrument, the light is split into two beams before it reaches the sample. The
two beams move simultaneously, one passing through a reference solution and the other through
the sample. The reference beam intensity is taken as 100% Transmission (or 0% Absorbance),
and the measurement displayed is the ratio of the two beam intensities.
Of the two types of spectrophotometer, double beam spectrophotometers are faster to operate
and in their performance. They provide more reproducible results because they perform an
automatic correction for the loss of light intensity as the beam passes through the sample and
reference solution.
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

Sample Preparation
The UV-Vis spectra are usually measured in very dilute solutions. Usually 1 mg of the
compound of molecular weight of 100-200 is dissolved in 100 ml of suitable solvent and only a
portion is used for recording the spectra. The solvent should not absorb radiation and must be
transparent over the desired range of wavelength. The solvents, which do not contain conjugated
system, are most suitable for recording the UV-spectra. The solvent should also be inert to the
sample. Some commonly used solvents are water, 95% ethanol, methanol and hexane.
Absorption Laws
When the radiation passes through a solution, some amount of light is absorbed. Suppose I0 is
the intensity of the incident radiation and I, the intensity of the transmitted radiation.
The amount of radiation absorbed can be
measured by Transmittance T = I/I0
% Transmittance = T×100
Absorbance A = Log10 I0/I=
Log10 1/T
If the entire light passes through a solution without any absorption, then
absorbance is zero. In this case, the percent transmittance is 100%.
If all the light is absorbed, then absorption is infinite and the percent transmittance is zero %.

Beer-Lambert Law: It gives a linear relationship between absorbance and concentration of an

absorbing species. This law states that the absorption is directly proportional to the
concentration of absorbing substance and the length of the path of radiation through the
sample. It is independent of the intensity of the incident light and each successive unit layer
absorbs an equal fraction of light incident on it.
A = ɛcb Log10 I0/I= ɛcb
Where A is the sample’s Absorbance value at specific wavelength or frequency
ε is the molar absorptivity or the molar extinction coefficient of the substance b is the path length
of the sample in cm
c is the concentration of the solute in mol L-1.
The value of ε absorptivity coefficient is constant for a particular material at a particular
wavelength.
ASBASJSM COLLEGE OF PHARMACY (AN AUTONOMOUS COLLEGE) BELA

Limitations of the Beer-Lambert Law

Under certain conditions Beer-Lambert law fails to maintain a linear relationship
between absorbance and concentration of the solution.
a) Deviations in absorptivity coefficients at high concentrations (>0.01M) due to
electrostatic interactions between molecules in close proximity.
b) Scattering of light due to particles present in the sample.
c) Chemical deviations due to the specific chemical species of the sample under
investigation.
d) Non-monochromatic radiation
e) Fluorescence or phosphorescence of the sample.

Absorption, intensity shift & UV spectrum

a) Bathochromic shift: It is also known as Red shift, in this case absorptionshift towards
longer wavelength (𝜆max).
b) Hypsochromic shift: It is also known as Blue shift, in this case absorptionshift towards
shorter wavelength (𝜆max).
c) Hyperchromic shift: Intensity of absorption maximum (𝜀max) increases.
d) Hypochromic shift: Intensity of absorption maximum (𝜀max) decrease.

Absorption & intensity shift

Chromophores & Auxochromes

a) Chromophores: Chromophore is a covalently unsaturated group absorbed electromagnetic
radiation in UV-visible region and impart color to the compound. For example, C=C,
 C≡C, benzene, NO2 etc.
b) Auxochromes: Auxochrome is a saturated group (containing lone pair of electron), when
auxochrome attach to the Chromophore absorption shift towards longer wavelength. For
example, -OH, -SH, -NH etc.

Factor affecting absorbance & intensity

a) Conjugation
Absorption shift towards longer wavelength, if double bonds (chromophore)present in the
molecule are in conjugation. For example,

In compound ‘A’, double bonds are in conjugation therefore ‘A’ possessing higher
wavelength (𝜆max) as compare to compound ‘B’ (non-conjugated derivative).
 We know that,
ℎ𝑐
E=
𝜆

As the conjugation increase transition energy (E) between the orbitals is decrease and therefore
wavelength (𝜆max) increase.
b) Effect of solvent:
In polar solvent, 𝜋 − 𝜋* transitions shift towards longer wavelength (Red shift), because
dipole interactions with polar solvent molecules lower the energy of excited state (𝜋*) than
that of the ground state (𝜋* orbital’s are stabilized by hydrogen bonding with polar solvent).

Whereas in polar solvent, 𝑛 − 𝜋* transitions shift towards lower wavelength (Blue shift),
because dipole interactions with polar solvent molecules lower the energy of ground state
(𝑛) than that of the excited state (𝑛 orbital’s are stabilized by hydrogen bonding with polar
solvent).

The Woodward-Fieser rules (Calculation of 𝝀max (nm) in conjugated dienes)

𝜆max (nm)
Base value:
214
Acyclic or heteroannular
dienesHomoannular dienes 253
Increments for:
Double bond extending conjugation 30
R alkyl substituent or ring residue 5
Exocyclic double bond 5
Polar groupings:
-OCOCH3 0
-OR 6
-Cl, -Br 5
-NR2 60

Explanation of terminology used in Woodward-Fieser rules

The Woodward-Fieser rules (Calculation of 𝝀 max (nm) in α,β-unsaturatedcarbonyl
compounds)

𝜆max (nm)
Base value:
215
Acyclic or 6-membered cyclic ketone
5-membered cyclic ketone 202

Aldehyde 210
39
Increments for: 30
Homocyclic diene component
𝛼 = 10, 𝛽 = 12, 𝛾 & ℎ𝑖𝑔ℎ𝑒𝑟 = 18
Double bond extending
conjugationR alkyl substituent or 5
ring residue Exocyclic double bond
Polar groupings:
𝛼 = 35, 𝛽 = 30, 𝛾 = 30
-OH
𝛼 = 35, 𝛽 = 30, 𝛾 = 17
-OR
𝛼 = 15, 𝛽 = 12, 𝛾 = 12
-Cl,
𝛼 = 25, 𝛽 = 30, 𝛾 = 25
-Br
𝛽 = 95
-NR2 𝛼 = 6, 𝛽 = 6, 𝛾 = 6
-OCOCH3
Application of UV spectroscopy

i) Extent of conjugation: As the double bond increase, absorption shift towards longer
wavelength.
ii) Distinction between conjugated and non conjugated compounds:

Both the above compound A & B are isomer of each other. Due to 𝑛 − 𝜋*
transition, compound A appear at longer wavelength than compound B.
iii) Study of geometric isomerism:

In isomer A, due to steric hindrance, absorption shift towards lower wavelength. We know
that, steric hindrance destabilize the compound means energy of the molecule increase
which decreases the wavelength.
iv) Used in the detection of impurities: Due to presence of impurities in the compound, give
additional peaks are observed in UV spectrum.
 Fluorimetry

Molecular luminescence spectroscopy is an analytical method which studies two types of

luminescence processes viz., fluorescence and phosphorescence.
.Luminescence refers to the emission of light by a substance (not resulting from heat which means
it is a form of cold body radiation). This means that only the luminescent analyte can be analyzed
by molecular luminescence spectroscopy.
Fluorimetry is an analytical method which measures the fluorescent light radiated by the unknown
sample and comparison is done with relation to fluorescent light radiated by the known sample
The terms flourimetry and fluorometry are used interchangeably in the chemical literature.
Similarly, phosphorimetry is an analytical method which measures the phosphorescence radiated
by the unknown sample and comparison is done with relation to phosphorescence radiated by the
known sample.

Principle
Collectively, fluorescence and phosphorescence are called photoluminescence methods. The
photoluminescence methods are the ones which result from absorption of photons. The absorption
of photon results in the excitation of molecule which results in luminescence (as when the excited
molecules or species revert back to the ground state, they give off light) and this type of
luminescence is therefore referred to as “photoluminescence’.
Before learning the principle of photoluminescence phenomena, let us first learn and understand
the terminology that we will use for excited states that lead to fluorescence and phosphorescence.
Singlet and Triplet States: When we talk about molecular systems, we refer to movement of
electrons on absorption of photons (A photon is a single quantum of electromagnetic energy). And
the electrons in molecular orbitals are paired, according to Pauli’s exclusion principle (no two
electrons in the same quantum state can have all four quantum numbers similar; they differ in their
spin – as you all know paired electrons in a molecular orbital have opposite spins). The electrons
are initially in their ground state (which is the most stable energy state where the electrons ar
paired). When an electron absorbs energy (i.e., when photons strike), it gets excited to a higher
energy state keeping its spin orientation intact. These excitations are not stable and the excited
electrons have a tendency to revert back to the stable ground state. And this reverse route from
excited state to ground state gives rise to the phenomena fluorescence and phosphorescence.
Let us now look into the electronic picture of this process:

Ground electronic state: It is the one where the electrons are paired with opposite spins.

The molecular electronic state in which the electrons are paired is referred to as Singlet State
(𝑀𝑢𝑙𝑡𝑖𝑝𝑙𝑖𝑐𝑖𝑡𝑦 = 2𝑆 + 1 = 1; 𝑑𝑖𝑎𝑚𝑎𝑔𝑛𝑒𝑡𝑖𝑐). So, the ground state is always spin paired and is a
singlet state.
Excited electronic states: (a) If the spins remain paired in the excited state, the molecule is in an
excited singlet state (𝑀𝑢𝑙𝑡𝑖𝑝𝑙𝑖𝑐𝑖𝑡𝑦 = 2𝑆 + 1 = 1; 𝑑𝑖𝑎𝑚𝑎𝑔𝑛𝑒𝑡𝑖𝑐). (b) If the spins become
unpaired, the molecule is in an excited triplet state (𝑀𝑢𝑙𝑡𝑖𝑝𝑙𝑖𝑐𝑖𝑡𝑦 = 2𝑆 + 1 = 3;
𝑝𝑎𝑟𝑎𝑚𝑎𝑔𝑛𝑒𝑡𝑖𝑐).

After learning the singlet and triplet terminology, let us now look into the energy level diagram for
photoluminescence molecules and understand the processes that take place on absorption of
photons. This diagram is referred to as Jablonski Diagram.
Figure 1: Jablonski diagram representing absorption, fluorescence and phosphorescence

Jablonski Diagram is named after physicist Aleksander Jablonski. It is a diagram used in molecular
spectroscopy for illustrating the electronic states in a molecule and the transitions that occur
between them. The electronic states are arranged vertically by energy and grouped horizontally by
spin multiplicity.

When an electron is transferred from a singlet energy level into a triplet energy level, by
a processcalled inter-system crossing, it is accompanied by a flip spin.

Also, the probability of a singlet to triplet transition is much lower han a singlet to singlet
transition.Therefore, the intensity of the emission from a triplet state to singlet state is
much lower than the emission intensities from a singlet state to a singlet state.

Absorption of UV-Vis radiation is necessary for exciting the molecules from the ground state
to one of the excited states. At room temperature, essentially all electrons in a molecule
occupy the ground state energy level (S0). This absorption of light populates number of
vibrational energy levels of excited electronic states (S1, S2, ……..). After an electron has
been excited to higher energy levels, the energy is eventually dissipated to the environment
and the electron reverts to the ground state. This energy dissipation can take place in a number
of ways and is responsible for fluorescence and phosphorescence. The dissipation of absorbed
energy can occur by a number of electronic process which can be categorized as radiation-less
transitions and radiative transitions.
 Radiation-less transitions (Heat) Radiative transitions (Photoluminescence)
a. Vibrational relaxation a. Fluorescence
b. Internal Conversion b. Phosphorescence
c. Inter-system crossing c. Delayed fluorscence
d. Energy transfer during molecular
collisions (External Conversion)

Both fluorescence and phosphorescence processes are basically nothing but relaxation of an
excited state via light emission. Let us learn now more about these processes.
The processes that take place on absorption of photons are explained below in the table which also
explains the Jablonski diagram.

Term Process Effect

Absorption Analyte molecule absorbs photons (very fast ~ 10-14 – 10-15 Excitation
s). An electronic transition in molecules after absorbing
photons from an excitation beam of light in the UV-Visible
range. The transition is typically from the lower energy level
that is the lowest vibrational level of the ground singlet state
(S0). The electronic transition is from the low to high energy
level, i.e. excited singlet state (S1, S2, …..).
Slightly different wavelengths excite to different vibrational
energy levels.
Vibrational Collisions of excited state analyte molecules with other Deactivate,
Relaxation molecules loss of excess vibrational energy and relaxation Radiationless
to lower vibrational levels (within the excited
electronic state).
Internal Molecule passes to a lower energy state – vibrational energy Deactivate,
conversion levels of the two electronic states overlap and molecules Radiationless
passes from one electronic state to the other.
Fluorescence Emission of a photon via a singlet to singlet transition (short Deactivate,
– lived excited state ~10-7 – 10-9 s). Emission of

Intersystem Spin of electron is reversed leading to change from singlet Deactivate,

Crossing to triplet state. Occurs more readily if vibrational levels of Radiationless
the two states overlap. Common in molecules with heavy
atoms (e.g., I or Br).
In inter-system crossing, the molecules transit from the
forbidden transition state to an overlapping triplet state. In
the triplet state, the molecules may go through vibrational
relaxation from the excited state to the lowest vibrational
level of T1 or may return to S0 by intersystem crossing

External Collisions of excited state analyte molecules with other Deactivate,

Conversion molecules molecule relaxes to the ground state without Radiationless
emission of a photon.

Phosphoresc Emission of a photon via a triplet to single transition (long– Deactivate,

ence lived excited state ~ 10-4 – 101s) Emission
of

So now with this understanding of Jablonski diagram, we can talk about fluorescence and
phosphorescence.
Fluorescence: This photoluminescence phenomenon is observed when an electron relaxes from
its first singlet electronic excited state to its singlet electronic ground state. The return to ground
 state produces a photon emission of longer wavelength. Fluorescence is observed in simple as
wellas in complex gaseous, liquid, and solid chemical systems. It is of two types:
Resonance fluorescence is the one where the absorbed radiation is remitted without a change in
frequency = excitation
Molecular fluorescence is the one where the bands are found centered at wavelengths that arelonger than
the resonating wavelengths. This shift toward longer wavelengths, or lower energies,is termed the Stoke’s
shif

Figure 2: Fluorescence excitation and emission spectra for a solution of quinine

Phosphorescence: This photoluminescence phenomenon is observed when an electron relaxes from its
first triplet electronic excited state to its singlet electronic ground state. It occurs via conversion of an
excited singlet state to an excited triplet state through the process of intersystem crossing (facilitated by
presence of non-bonding electrons as well as heavy atoms). The return to ground state produces a
photon emission of longer wavelength in comparison with fluorescence. Phosphorescence is not

common in solutions at room temperature but it can be easily observed at liquid nitrogen temperature or
in solid state. This kind of emission is ordinarily observed only at low temperatures, in highly viscous
media or by molecules that are adsorbed on solid.

Delayed fluorescence: An electron in the excited triplet state can cross back to the singlet excited
state and can result in a photon as fluorescence but at a much longer time than prompt fluorescence.
This process is referred to as delayed fluorescence and has similar characteristics as prompt
fluorescence except for longer lifetime. Actually, the corresponding lifetime is intermediate
between fluorescence and phosphorescence.

The difference between fluorescence and phosphorescence is that phosphorescence

occurs at longer wavelength because of the triplet stage as it represents a lower
energy level in comparisonto the overlapping excited singlet stage.

Now, before taking up the instrumentation for fluorimeter and phosphorimeter, let us learn about
luminescence spectra.

Luminescence spectra

The data obtained from a luminescence spectrophotometer is represented in the form of excitation or
emission scan. The excitation scan is a spectral profile of excitation wavelengths which are produced
at a fixed emission wavelength whereas the emission scan is a spectral profile of emission
wavelengths which are produced by a fixed excitation (i.e. absorbance) wavelength.

The transition from the lowest vibrational level in the electronic ground state to the lowest vibrational
level in the first excited electronic state, called the 0 - 0 transition, is common to both the excitation
and emission process. Thus, the emission spectrum would be expected to overlap the excitation
spectrum at the wavelength corresponding to the 0 - 0 transition and the rest of the emission spectrum
to be of lower energy and hence a longer wavelength. In practice the 0 - 0 transition for the
excitation and emission spectra of a molecule rarely coincide exactly. This difference is
representative of the small loss of energy through the interaction of the absorbing molecules with the
surrounding solvent molecules
 Figure 3: 0 - 0 transition overlap in the excitation and emission process

Let us now briefly look into the factors that affect fluorescence and phosphorescence phenomena.
Both molecular structure and chemical environment influence whether a substance will or will not
luminesce; these factors also determine the intensity of emission when luminescence does occur.

Factors affecting fluorescence and phosphorescence:

o Solvent- Modification in polarity of the solvent will have substantial effect on the
fluorescent behavior of the sample. Apart from solvent polarity, the viscosity of the solvent
also effects the fluorescence and phosphorescence. Increase in viscosity will increase the
fluorescence because chances of deactivation is less.
o Temperature- As the temperature changes the viscosity of the medium also changes
which causes change in collisions of sample molecules present in solvent, which results in
escalation in chances of deactivation due to vibrational relaxation and internal conversion.
Therefore, increase in temperature will result in decrease in fluorescence and vice-versa.
o Dissolved oxygen- It is a paramagnetic substance which reduces fluorescence and will
cause intrusion in fluorimetry analysis. Dissolved oxygen stimulates intersystem crossing.
Dissolvedoxygen also influences phosphorescence.
o pH- Any change in pH effects the intensity of fluorescence and spectral features of
fluorescence. Presence of basic or acidic functional groups in molecules effects the pH of
the medium which changes the degree of ionization of functional groups. This will result in
changes in molecules in terms of conjugation and aromaticity that affects fluorescence.
 Apart from these factors adsorption, intensity, oxidation also affects fluorescence and
phosphorescence.

Spectro-fluorometer
Fluorescence is measured by Spectro-fluorometer and Fluorometer. Following are the
basiccomponents of Spectro- fluorometer and Fluorometer-
 Excitation source
 Excitation Monochromator
 Cuvette
 Emission Monochromator
 Detector

Figure 3- Flow diagram of fluorometer

 Excitation source: 300-700 nm is the range of interest for most of the fluorescent compounds.
The excitation sources used in Spectro- fluorometer and fluorometer include xenon lamps,
lasers, mercury arc lamps and quartz halogen lamps. Xenon lamps are one of the known
excitation source and it provides continuum high intensity radiant energy in 250-800 nm
spectral region. Because of high energy output of xenon lamp, stability of lamp flashes and
higher Ultra-violet and visible spectral productivity, it is widely used in fluorometer. Lasers
are also used as an excitation source because of its high intensity and is well focused.
  Excitation and Emission Monochromator: In fluorometers and spectro-fluorometers, the
monochromator consist of filters (interference and colored glass filter), prisms and gratings.
Colored glass filters selectively absorbs particular wavelength, this type of filter is generally
used for both excitation and emission selection of wavelength. But the only limitation is that
colored glass filters are vulnerable for transmitting stray light and undesirable fluorescence.
Grating monochromators are used for isolation of selective regions of UV-Visible spectrum.

The spectral resolution of the light passing from the slit is a function of width of the slit and
grating resolution. To obtain higher excitation intensity the spectro-fluorometers generally
uses large slit width. Grating monochromators are of great use because it provides selective
emission and excitation wavelengths. The grating rotations are digitally controlled. For
elimination of extra order of emission, laser is used as both monochromator and excitation
light source.

 Cuvette: Cuvettes are used to hold the sample to be examined. The shape of cuvettes used in
fluorescence instruments are generally of rectangular and square shape and the material used
for constructing these cuvettes allows the excitation and emission light to pass through it. Glass
and plastic materials are used for visible light and for Ultra Violet light, quartz is used. Glass
and plastic is not used for UV light because instead of passing the UV light it absorbs the UV
light. The position of the cuvette and the excitation beam is important for establishing the
optical geometry for the fluorometers. Right angle detector approach is used by the majority
of modern fluorometers and spectro- fluorometers because of its property of minimizing
background signal used for limiting analytical sensitivity. To accommodate various types of
geometries, the sample cell is placed at different angles according to the excitation source and
detector.

 Photodetector: Detectors used in spectro- fluorometers and fluorometers are same as detectors
used in spectrophotometers, for instance- photomultiplier tube (PMT) and charged couple
detectors (CCD). Visual observation is also used.

 Photomultiplier Tube (PMT): Photomultiplier tube is commonly used in spectrofluorometers

and fluorometers for quantitative analysis. The most important characteristics of PMT for
 fluorescence analysis are
i. Wide range of spectral responses
ii. Highly sensitive
iii. Quick photon response time

At high light intensities, analog techniques are used for measuring PMT current, then this
analog signal is converted into digital signal for computer use and displaying the result. At low
light level, at cathode of PMT spikes are counted. The number of pulses generated per unit
time is directl

roportional to intensity of fluorescent light emitted by the sample which are striking the PMT.
This techniques is called as photon counting.

 Charged couple detectors (CCD): These are multichannel devices which have vast range and a
signal to noise ratio, which is higher in comparison with photomultiplier tube. These are solid
state devices which are composed of photon detecting shift registers and these registers reads
horizontally and vertically. They are used for fluorescence applications because they can
detect very low intensity of light and can measure low concentration of fluorescent molecules.
A 30W mercury lamp is used when fluorometer is used with charged couple detectors.

Long answer type Questions (10 Marks)

1. Find out the 𝜆max of following molecules

 2. Give principle and detailing of various components of double beam UV-
visible Spectrophotometer.
3. Explain various electroic transitions taking place in UV Spectroscopy.
4. Discuss the instrumentation of UV Spectrophotometer
5. Explain Beer’s law Describe factors responsible for deviation from Beer’s
law.
6. Describe various detectors used in UV spectroscopy.
7. Write a note on the various factors affecting Flouresence
8. What are the structural requirements of flourophore? What are the factors
affecting Flouresence?

Short answer type questions (5Marks)

1. Write the short note on electronic transitions.

2. Write notes on application of UV-visible spectroscopy.
3. Explain the term Hypsochromic shift and Bathochromic shift.
What structural features may lead to these shifts in organic molecules?
4. Explain the effect of polar solvent on 𝜋 − 𝜋* and 𝑛 − 𝜋* transition.
5. How 1,3-pentadiene and 1,4-pentadiene can be distinguished by UV
spectroscopy?
6. Why aniline behaves like that of benzene in acidic medium?
7. Two isomeric dienes ‘A’ and ‘B’ having the molecular formula C5H8 absorb
at 𝜆max 225 nm and 𝜆max 180 nm respectively. Write the structure of the
two isomers.
8. Discuss the applications of Flourimetry
9. Describe the factors affecting fluoresence intensity of compound in
flourmetric analysis.

Very Short answer type questions (2 Marks)

1) What is electronic spectroscopy?

2) What is Chromophore? How does it differ from auxochrome?
3) Discuss how conjugation affects the intensity of absorption band.
4) Enlist elements of process validation.
5) Write the limitations of process validation.
6) Name the parameters for calibration of GC.
7) Name the parameters for calibration of HPLC.
8) Name the parameters for calibration of UV Visible spectrophotometer.
9) What is quenching?
10) Explain triplet excited state of molecule giving diagram
11) Define electronic transition.
12) Give the principle involved in Flourimetry?
13) Difference between flouresence and phosphorescence?