Spectroscopic Methods

UV and Fluorescence Spectroscopy

Dr. Hitesh Pawar

DBT-ICT Centre for Energy Biosciences
Institute of Chemical Technology, Mumbai

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Spectroscopy
▪ Spectroscopy: A branch of science that deals with the study of interaction of matter with light.
OR
▪ A branch of science that deals with the study of interaction of electromagnetic radiation with matter

▪ Electromagnetic radiation….?
- Electromagnetic radiation consist of discrete
packages of energy which are called as photons.

- A photon consist of oscillating electric field (E) and

oscillating magnetic field (M) which are
perpendicular with each other.

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Characteristic of Electromagnetic Radiation
- Produced by oscillation of electric charge and magnetic field residing on the atom. The electric and
manganic field are perpendicular with each other and co-planer.

- These are characterized by their wavelengths or frequencies, or wavenumber.

- The energy carried by electromagnetic radiation is directly proportional to its frequency

- The emission or absorption of radiation is quantised, and each quantum of radiation is called photon.

- All type of radiation travel with the same velocity and no medium is required for their propagation, they
can travel through vacuum.

- When visible light is passed through a prism it is split up into seven colours which correspond to
definite wavelengths, the phenomenon is called as dispersion. The group of electromagnetic
radiation can be split into various components for analysis.
Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Principle of Spectroscopy
▪ Measurement of spectrum of a sample containing atom/molecule
▪ Spectrum is a graph of intensity of absorbed or emitted radiation by sample Vs. frequency (v) or
wavelength (λ)
▪ Spectrometer is an instrument design to measure the spectrum of a compound.

Types of spectroscopy
▪ Absorption spectroscopy
- An analytical technique which concerns with the measurement of absorption of electromagnetic
radiation.
- UV-Spectroscopy (185-400nm)/ Visible-Spectroscopy (400-800 nm), IR Spectroscopy (0.76-0.15 µm)
▪ Emission spectroscopy
- An analytical technique in which emission (of a particle or radiation) is dispersed according to some
property of the emission and the amount of dispersion is measured.
- e.g., Mass spectroscopy
Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Electromagnetic Radiation (EMR)

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 UV-Spectroscopy
▪ UV-Spectroscopy: It is measurement of attenuation of a beam of light after it passes
through a sample or after reflection from a sample surface. Absorption measurement can
be at a single wavelength or over an extended spectral range.

1. Detection of functional groups

2. Detection of impurities
3. Qualitative and quantitative analysis
4. Single compound without chromophore
5. Drug with chromophoric agent
6. To show relationship between different functional groups
7. Detection of conjugation in different compounds.

• UV region : 200-400 nm
• Far UV region: 100-200 nm
• Near UV region: 200-380nm
Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Principle of UV Spectroscopy
▪ UV and visible spectroscopy is a primarily used to measure the multiple bonds or aromatic
conjugations within the molecule.

▪ UV absorption spectra arise from transition of electron within the molecule from lower energy level to
higher energy level (electronic energy level).

▪ The UV region is extended from 100-400 nm.

▪ A molecule absorb UV radiation frequency, the electron in that molecule undergo transition from
lower to higher energy level.

▪ The energy can be calculated by the equation,

E1-Eo= hv
E total= E electronic+ E vibrational + E rotational

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Beer and Lambert Law
- German mathematician and chemist August Beer in 1852
- The Beer and Lambert Law is a relationship between the attenuation of light through a substance and
the properties of that substance.

It states that the absorptive capacity of a dissolved substance is directly proportional to its
concentration in a solution.

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 UV Absorption Spectrum
▪ When sample is exposed to light, energy that matches the energy difference between a possible
electronic transition within the molecule, a fraction of the light energy would be absorbed by the
molecule and the electrons would be promoted to the higher energy state orbital.

▪ A spectrometer records the degree of absorption by a sample at different wavelength and resulting
plot of absorbance (A) Vs. Wavelength (λ).

- λmax : Wavelength at which there is maximum absorption

- εmax : Intensity of maximum absorption

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Excitation of electron in a molecule due to light

• Molecule have a bond, the atom in a bond have their atomic orbital merged to form molecular orbital
which can be occupied by electrons of different energy levels.

• Ground state molecular orbital can be excited to antibonding molecular orbitals.

• Electron when imparted with energy in the form of light radiation get excited from the highest occupied
molecular orbital (HUMO) to lowest occupied molecular orbital (LUMO) and the resulting species known
as “excited state” or “antibonding state”.

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Types of Electronic Transition in UV

▪ Designation of various transitions

In UV spectroscopy molecule undergo electronic transition involving σ, π, and n electrons

Four types of electronic transition

are possible;

1. σ → σ* transition

2. n → σ* transition

3. n→ π* transition

4. π → π* transition

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 ▪ σ → σ* Transitions :
- A transition of electron from bonding σ orbital to antibonding σ*
orbital is designated as σ → σ* transition

- Sigma bond is very strong thus the energy required is large (very
short wavelength i.e., high energy UV ~150nm ) .

- e.g., Methane (C-H bonds, undergo σ → σ* transitions) shows an

absorbance maximum at 125 nm.

- Absorption maxima due to σ → σ* transitions are not seen in typical UV-Visible spectra (200-700 nm).

- The study of such transition is done in vacuum UV region because below 200nm oxygen present in air
also absorbs UV.

- There is retention of electron spins. This is less informative transition

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 ▪ n → σ* Transitions - Saturated compounds containing hetero atoms with lone pairs are
capable of n → σ* transitions.

- These transitions usually need less energy than σ→ σ* transitions.

- Can be initiated by light whose wavelength is in the range 150 - 250

nm.

- The number of organic functional groups with n → σ* peaks in the

UV region is small.
e.g., H2O (167nm) CH3OH (174nm) CH3Cl(169), CH3I (258nm).

- In R-X energy required for transition decreases with increase in size (decrease in electronegativity) of
halogen atom. (R-F > R-Cl > R-Br > R-I).

- Saturated halides, alcohols, ethers, aldehydes, ketones etc., shows n → σ* transition.

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 ▪ π → π* Transitions

- Most absorption spectroscopy of organic compounds is based on transitions of n or π electrons to

the π* excited state.
- Compounds with unsaturated centres e.g. C=C, aromatic carbonyl compounds etc.
- This transition required less energy than n → σ*
- In simple unconjugated alkene CH2= CH2 (170-190nm),saturated ketones (150nm)

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 ▪ n→ π* Transitions

- In this transition an electron of unshared electron pair on hetero atom is excited to π* anti-bonding
orbital.

- This transition required least energy among all the transitions thus it gives an absorption band at longer
wavelength.

- Saturated aliphatic ketone n→ π* transition 280nm is the lowest energy transition. This n→ π*
transition is “forbidden” by symmetry consideration, thus the intensity of the band due to this is low
although at longer wavelength (low energy).
Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 e-
e-
e-
e-

e- e- e-

e-
n → π*
σ → σ* n→σ* π → π*
Easy to do (medium ε)
Hard to do (small ε) Hard to do (small ε) Easy to do (large ε)
medium energy photon
and high energy photon and low energy photon pretty low energy photon

σ → σ* > n → σ * > π → π* > n → π*

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Chromophore
▪ Chromophore
- Any functional group which exhibit absorption of electromagnetic radiation in a visible or ultra-
visible region.

- A chromophore is a covalently unsaturated group responsible for electronic absorption e.g., C=C, C=O
and NO2

- e.g., simple compound ethylene, benzene and acetone etc.

- It may or may not impart any colour to the compound

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Auxochrome
▪ Auxochrome: A saturated group, which when attached to a chromophore changes both
intensity as well as the wavelength of absorption. e.g., -OH, -NH2, -OR, -NHR, and –NR2

- When the auxochrome –NH2 group is attached to benzene ring, absorption shifted from,
λmax 225 (ɛmax 203) to → λmax 280 (εmax 1430)

- All auxochromes have one or more non-bonding pairs of electrons.

- If an auxochromes is attached to a chromophore, it extends the conjugation by sharing of non-

bonding pair of electrons.

Crystal Violet,
Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Shift of absorption maxima (λmax) in UV

▪ Red Shift

- A shift of absorption to a longer wavelength called as “Red shift”

OR “ Bathochromic Shift”.
- e.g., presence of –NH2 in aniline λmax 225 (ɛmax 203) to → λmax 280
(εmax 1430)

- Peaks resulting from n→ π* transitions are shifted to longer

wavelengths (red shift) with increasing solvent polarity.

- This is caused by attractive polarisation forces between the solvent

and the absorber, which lower the energy levels of both the excited
and unexcited states.

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 ▪ Blue Shift

- A shift of absorption to a shorter wavelength called as “Blue shift” OR “ Hypsochromic Shift”

- Peaks resulting from n→ π* transitions are shifted to shorter wavelengths (blue shift) with
increasing solvent polarity. This arises from increased solvation of the lone pair, which lowers
the energy of the n orbital.

Red shift

Blue shift

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Shift of absorption intensity (εmax) in UV
▪ Hyperchromic Shift
- The increase in absorption intensity is called as “ Hyperchromoic Shift”
- Introduction of auxochrome usually increases intensity of absorption.
- The B-band for pyridine 257nm (εmax 2750) shifted to 262nm (εmax 3560) for 2-methyl pyridine

Pyridine 2-methyl pyridine

257nm(εmax 2750) 262nm (εmax 3560)

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 ▪ Hypochromic Shift

- The decrease in absorption intensity is called as “ Hypochromoic Shift”

- The biphenyl 250nm (εmax 19000) shifted to 237nm (εmax 10250) for 2-methyl biphenyl
- Introduction of methyl group distorts geometry of molecule thus cause decrease in absorption
intensity

Biphenyl 250nm 2-methyl biphenyl 237nm

(εmax 19000) (εmax 10250)

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 General Applications of UV Spectroscopy
1. Detection of Functional group
- The UV absorption spectroscopy is used for detection of chromophore in the compound.

- The absence of UV absorption band at particular wavelength providence for presence or absence
of chromophore.

- If the spectrum is transparent above 200nm the it shows the absence of;
a) Conjugation
b) Carbonyl group
c) Benzene or aromatic compound
d) Br of I atom

2. Detection of Functional group

- The extension of conjugation can be determined
- Addition of double bond increase the UV absorbance

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 3. Structure elucidation of organic compounds.

- UV spectroscopy is useful in the structure elucidation of organic molecules, the presence or absence of
unsaturation, the presence of hetero atoms.

- From the location of peaks and combination of peaks, it can be concluded that whether the compound is
saturated or unsaturated, hetero atoms are present or not etc.

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Fluorescence Spectroscopy

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 ▪ Emission Spectroscopy:
- Emission spectroscopy is a spectroscopic technique which examines the wavelength of photons
emitted by an atom or molecule during their transition from an excited state to lower energy state.

- An emission spectrum is produced by emission of radiant energy by

excited atom.

- The excitation of an atom can be brought thermally or electrically.

- When electric charge is passed through vapour of the substance, energy is absorbed the electrons in the
ground state are promoted meta-stable state.

- When electron from meta stable state jump to lower energy state, then some energy of definite
frequency is released as a radiation.

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 ▪ Luminescence:
- Luminescence is emission of light by a substance not resulting from heat is thus a form of cold
body radiation.
- It can be caused by chemical reactions, electrical energy, subatomic motions, or stress on a
crystal.
There are two pre-requisites for luminescence:
- The luminescent material must have a semiconductor structure with a nonzero band gap. [Metals
do not provide luminescence if they have no band gap].
- The energy must be imparted to this material before luminescence can take place.

▪ Types of luminescence:
a) By Mechanism: i) Fluorescence ii) Phosphorescence
b) By Excitation Source: i) Chemiluminescence ii) Cathodoluminescence iii) Electroluminescence
iv) Photoluminescence

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Florescence Spectroscopy

▪ Fluorescence spectroscopy: It is a type of electromagnetic spectroscopy which analyzes florescence

from a sample. It is also called as fluorometry or spectrofluorimetry.

- It involves use of beam of light usually UV light, that excites the electrons in a molecule of certain
compound and causes them to emit light of lower energy, typically, but not necessarily, visible light.
This shift to longer wavelength is called the Stokes shift.

- The device that measures florescence are called fluorometers or fluorimeters.

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Theory of Molecular Fluorescence
▪ Theory of Molecular fluorescence: Molecular fluorescence is measured by exciting the sample at the
absorption wavelength, also called the excitation wavelength and measuring the emission at a longer
wavelength called the emission or fluorescence wavelength .
- The first observation of florescence from a quinine solution in sunlight was reported by Sir John
Frederick William Herschel in 1845.
- Quinine is having the phenomenon of emitting light of a different wavelength.

- The quinine in tonic water is excited by

the ultraviolet light from the sun. Upon
return to the ground state the quinine
emits blue light with a wavelength
450nm.

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 ▪ Relaxation Process: Once the molecule is excited to S1 or S2 several processes can occur that cause
the molecule to lose its excess energy.
Jablonski diagram
- Various mechanism of relaxation are.
a. Fluorescence
b. Phosphorescence
c. Vibrational relaxation
d. Internal conversion
e. External conversion
f. Inter system crossing

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Average timescales for radiative and non-radiative processes

Transition Time Scale Radiative Process?

Internal Conversion 10-14 - 10-11 s no

Vibrational Relaxation 10-14 - 10-11 s no

Absorption 10-15 s yes

Phosphorescence 10-4 - 10-1 s yes

Intersystem Crossing 10-8 - 10-3 s no

Fluorescence 10-9 - 10-7 s yes

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Instrumentation of Fluorometer

▪ Light Source:
- Xenon lamps
- Mercury vapour lamps

▪ Filters and/or monochromators

- Grating monochromators

▪ Detector
- Photomultiplier tube

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Factors interfering with florescence intensity

- Following factors affecting the intensity of florescence.

a. Concentration

b. Transition type in fluorescence

c. Structure

d. Temperature and solvent

e. Impurities present in the solution

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Few Molecules with Florescence

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai
 Applications

- Determination of florescent drug in low-dose formulations in the presence of non- florescent

excipients.

- In carrying out the analysis limit test where the impurity is florescent.

- Useful for the study of drug binding to the components in complex formulation.

- Bioanalysis for measuring small amounts of drugs and for the study of drug-protein binding.

Applied Chemistry Spectroscopic Methods (UV and Florescence) Institute of Chemical Technology Mumbai