Electromagnetic spectrum

The electromagnetic spectrum is the division of electromagnetic radiation based on the energy, frequency, or
wavelength of a photon.

Electromagnetic Spectrum
ENERGY ( kJ/mol) 1.2 x105 1.2 x107 12000 310 150 0.12 0.0012

Electronic excitation
e-

FREQUENCY (Hz) 1020 1018 1016 1014 1012 108

Cosmic

visible
γ rays X rays Ultra Infrared Radio
rays Microwave
violet waves

WAVELENGTH (m) 10-12 10-11 10-9 10-6 10-3 10-1

The strength of the radiation energy will interact with the molecules in different
ways:

▪ High energy sources produce breaking of bonds

Ex. X-Ray, γ Rays, …

▪ Medium energy sources excite electrons

Ex. UV / Visible Spectroscopy

▪ Low energy sources produce vibrations in chemical bonds

Ex. Infrared Energy

▪ Very low energy sources produce rotation of the chemical bonds

Ex. Microwaves and Radio waves
 UV-Vis Spectroscopy
(Principle, Instrumentation, and Applications)

Interaction of ultraviolet and visible light with matter

Working principle of UV-visible spectroscopy

When a chemical compound absorbs light, some excitation and de-excitation processes of electrons occur in atoms
which result in the production of the distinct spectrum.
Interaction of radiation and matter

In spectroscopic analysis, when radiations interact with a chemical species, they can cause transition at different
energy levels. The type of transition depends upon the energy of the radiation and the detection mode.

The transition of electrons always occurs from the ground state of low energy HOMO (highest occupied
molecular orbital) to a higher energy excited state LUMO (lowest unoccupied molecular orbital). The overlap of
atomic orbitals forms three types of molecular orbitals. This whole concept arises from molecular orbital theory
(MOT).

1. Bonding molecular orbitals of lower energy

2. Non-bonding molecular orbitals of intermediate energy

3. Anti bonding molecular orbitals are of higher energy

Energy order in electronic transitions
The electronic transitions that require the highest energy are σ-σ* electronic transitions, while the
transitions requiring the least energy are n-π* electronic transitions.

The energy order of different types of electronic transitions is as give below

σ-σ* > n-σ* > π-π* > n-π*
UV / Visible Spectroscopy

UV Radiation – Wavelength range 220 - 380nm Vibrational Energy Levels

Visible Radiation – Wavelength range 380 - 780nm Vibrational levels

Rotational levels
▪ Materials can absorb varying amounts S1
of UV and/or Visible radiation at
▪ particular wavelengths

▪ Coloured compounds absorb energy Effects of the energy levels depending on the

Absorption
in both UV and visible region of the nature of the energy received

Energy
electromagnetic spectrum.

Vibrational levels

Rotational levels
S0
Ground state
UV-Vis IR mW
 UV / Visible Spectroscopy - Theory
Lambert law
Absorbance ‘A’ of incident monochromatic light is directly proportional to the path length (cell length) ‘ℓ’. This
means that equal portions of absorbing material absorb equal fractions of incident light.

A∝ℓ
Beer law
Absorbance ‘A’ of incident light or electromagnetic radiation is directly proportional to the concentration ‘c’
of solution. This law gives the quantitative relationship between the intensity of radiation and the
concentrations of chemical species.
A∝c
Beer-lambert law
Collectively Beer and Lambert’s laws state that the absorbance ‘A’ of an incident monochromatic beam is directly
proportional to concentration ‘c’ of the solution and path length ‘ℓ’.

The rate of decrease in intensity of monochromatic light is proportional to the thickness of medium ‘ℓ’ and
concentration ‘c’ of absorbing substance in dilution.
A∝c.ℓ
A=ε.c.ℓ

where, ε is the molar absorptivity coefficient constant Or ε is the absorbance for a solution of concentration
1mole/dm-3 and a path length of 1cm.
Lambert showed the impact of Pathlength
on a UV-Vis spectrophotometric scan – the
longer the Pathlength, the higher will be
the absorption
Absorbance
It is the ratio of the intensity of incident electromagnetic radiation from the source to that of refracted
electromagnetic radiation detected by the detector.
A = Log Io / I
A=ε.c.ℓ
So,
Log Io / I = ε . c . ℓ
Transmittance
It is the ratio of power of electromagnetic radiation leaving the sample Pt to that of the incident radiation on the
sample from the source.
T= It / Io
% T = T × 100
Importance of the Beer-Lambert Law
% T = It / Io × 100
Absorbance  Concentration; should be linear
Conversion of absorbance to transmittance relationship

ABSORBANCE AT 300nm
o
A = – log T ; A = – log It / Io (Since T= It / Io)
o
A = log Io / It o
A = 2- log T % or % T = antilog (2 – A) o
o
CONCENTRATION (moles litre-1 )
Limitations of lambert beer law

▪ The light source used must be monochromatic.

▪ This is not suitable for concentrated solutions i.e. It can only be applicable to dilute solutions.

▪ With an increase in dilution, the dissociation of weak acids occurs. The weak acids reach
equilibrium with their conjugate base. The acid (HA) and conjugate base (A–) cannot have the
same absorbance. Hence this law is not completely applicable to weak acidic solutions.
Instrumentation of UV-visible spectroscopy
The basic instrumentation of the UV-vis spectrometer comprises of
▪ Light source
▪ Diffraction grating
▪ Wavelength selector
▪ Sample container or cuvette
▪ Detector

Light Source
Light sources that lie in the ultraviolet and visible region are used as UV-visible spectrometer sources.
▪ Hydrogen & deuterium lamps range (160-380nm)
▪ Xenon arc lamps range (250-600nm)
▪ Tungsten halogen lamps (range 240-2500nm)

Wavelength selector: Instrument with narrow bandwidth would be better.

▪ Filters
▪ Monochromators
Monochromators
▪ A monochromator is an optical device that is used to select a narrow band of a wavelength of light. It may be a
quartz prism or grating.

▪ Used for spectral scanning i.e. varying wavelength of radiation over a range.

All monochromators are similar in mechanical construction. The essential components of a monochromator are:
• Slit
• Mirror
• Lense
• Grating/prism
Cuvette size
The most common cuvette size is 1 cm,
Sample container/cells or cuvettes although it can vary from 0.1-10 cm.
Quartz, Borosilicate, and Plastic

▪ Only quartz is transparent in both UV & visible regions (200-700nm range).

▪ Glass & plastic are suitable for the visible region only.
▪ Glass is not suitable for the UV region because it absorbs UV radiation i.e. it is not transparent in the UV
region.
▪ Plastic cells are not used for organic solvents.
Detectors
Detectors are devices that indicate the existence of some physical phenomenon.
▪ Human eye
▪ Transducers
▪ Photodetectors
▪ Photographic films
▪ Mercury level in thermometers (temperature detector)

Photodetectors
▪ Photo tubes
▪ Photomultiplier tubes
▪ Silicon diodes This process causes up to
▪ Photovoltaic cells 106 electrons collected for
each photon, striking the
first cathode.

Photomultiplier tubes are structured as repeated dynodes at particular angles. The emitted electrons strike on
different dynodes. Every dynode has a high voltage than the previous ones. This voltage difference accelerates the
electrons. These fast electrons knock out more electrons upon striking the next dynodes.
Spectral analysis in UV-VIS spectroscopy
▪ UV-VIS spectrum shows absorption at different wavelengths. The relative maxima are known as lambda max
(λmax).

▪ UV-Vis spectroscopic spectra are used to check the presence of different organic and conjugated chemical
species that have a wavelength in that range.

For example
Chlorophyll-a absorbs the regions of violet and orange wavelength regions. This is the fact that makes plants
unable to absorb green light making them appear green.

Chlorophyll-a

Amoxicillin is a famous
antibiotic

Quantitative analysis
: The absorbance at a certain wavelength of light is directly proportional to the
concentration by Beer-Lambert law.
Shifting of absorption band and change in intensity
Chromophores
The chromophore is an atom or group of atoms that are responsible for the absorption of UV-visible radiation.

Types of chromophores
There are two types of chromophores:

▪ Chromophores that can only contain π electrons.

They undergo π-π* transitions only e.g. ethylenic group (C＝C) and acetylenic group (C ≡ C) etc.

▪ Chromophores that contain π as well as n (nonbonding) electrons.

This type of chromophore contains lone pair(s) of electrons. So they are responsible for two types of transitions i.e.
n-π* and π-π* e.g. nitro group (-NO2 ), azo group (-N＝N-), nitro group (-NO3 ), carbonyl group ( >C＝O), nitrite
group (-ONO).

Auxochromes
An auxochrome is an atom or group of atoms that do not give rise to an absorption band of its own but change
the absorption characteristics of a chromophore. Auxochromes may change both intensity and wavelength of
chromophore when added to it. It is also called a color-enhancing group. The replacement of hydrogen on a basic
chromophore changes its absorption characteristics. i.e. Methyl (-CH3,), chloride (Cl–), hydroxyl (OH–), amino (-
NH2), alkoxy (CH3O–), etc.
There exist four types of shifts corresponding to auxochromes
Bathochromic shift or redshift
▪ The bathochromic shift is the change of position of the absorption
band towards the longer wavelength.
▪ This change occurs in the presence of auxochrome or change in the solvent.

Hypsochromic shift or blue shift

▪ The hypsochromic shift is the change in the position of the spectral
band towards a shorter wavelength.
▪ It occurs due to the removal of conjugation or change in polarity of the
solvent.

Hyperchromic shift
▪ Hyperchromic shift is the increase in the intensity of the absorption
band i.e. εmax.
▪ A hyperchromic shift occurs due to the presence of an auxochrome.

▪ Hypochromic shift
▪ A hypochromic shift is a decrease in the intensity of the absorption
i.e. εmax band.
Factors affecting change in the absorption and wavelength
Effect of conjugation
According to MOT, as the number of pi electrons increases, delocalization increases. Due to this increase in
delocalization, the molecules of the sample get stabilized and hence reach a state of lower energy. This lowering of
energy causes a change in wavelength towards a higher wavelength known as redshift.
Effect of additive characteristics
When a molecule contains two or more chromophores separated by more than one single bond the total
absorption is equal to the sum of absorption characteristics of each chromophore.

For example,
Ethylene and 1,5 hexadiene absorb the same wavelength. But the amount of radiation absorbed for similar
concentrations is almost doubled for 1,5 hexadiene than ethylene.

Effect of the aromatic ring

The aromatic ring especially when two or more rings in conjugation (polycyclic compounds) absorbs a higher
wavelength in the visible region, it alters the spectrum of absorption.

For example
Naphthalene (C6H6 ) absorbs at 268nm
Anthracene absorbs at 311nm
Tetracene absorbs at 476nm
Effect of substitution of auxochrome

Benzene is a less effective chromophore, Auxochrome Compound λ(max) ε(max)

the substitution of a polar group to it --- Benzene 256 250
causes an increase in λmax in the visible
-NH2 Aniline 280 200
region and hence εmax value increase.
-Cl Chlorobenzene 265 360

Effect of the polarity of the solvent

Polarity causes a pronounced effect on the position and intensity of absorption bands. This increase is due to the n-
π* and π-π* transitions.

In the presence of polar hydrolytic solvent (i.e. water ) hydrogen bonds form with the lone pair of electrons of
auxochrome. As a result, the auxochrome’s energy lowers to an equal amount of the bond formation energy, and
hence the energy gap between HOMO and LUMO increases, so, a hypsochromic shift is observed for n-π* transition.
For example
Absorption
Solvent
wavelength

n-π* Transitions Hexane 279nm

Methanol 270 nm
Water 264 nm
While for π-π* transition, the π* orbital is more polar than π orbital therefore it is stabilized to a greater extent
in the presence of a polar solvent. This will cause a bathochromic shift because the energy gap between π-
π* is reduced due to the stability of the π* orbital.

Absorption
Solvent
wavelength
π-π*
Transitions Hexane 230 nm
Water 243 nm
Types of UV-Vis spectrometers
1.Single beam UV-Vis spectrometer
2.Double beam UV-Vis spectrometer

Single beam
UV-Visible spectrophotometer
Light source, Lens, Gratings, Wavelength selector,
Sample container/cuvette, Detector, Digital
meter/Recorder

Double beam
UV-Visible
spectrophotometer
Applications of UV-Vis Spectroscopy

▪ Used to identify organic and inorganic species present in a solution.

▪ Concentration of the unknown solution.
▪ For the determination of structure along with other data such as bands and intensities of functional
groups.
▪ To study of chemical kinetics i.e. disappearance of one functional group and appearance of another
functional group.
▪ To study isomers, i.e. in geometric isomerism, the trans-species absorb a high wavelength with a large
molar absorptivity ‘ε’ value than the cis-species.
▪ Detection or presence of conjugation.
▪ Drug analysis.
▪ Petrochemical industries.
▪ Water quality
▪ Forensic labs.
▪ Investigation of chemical and biological plants.
▪ For qualitative analysis maximum absorption is used and for quantitative analysis, Lambert beer’s
law is used.
How to calculate Lambda
maxima (λmax)?