AUSU Spectroscopy Lec IV

AL-ISTIQAMA UNIV.
SUMAILA
Faculty of Basic Health Sciences
2021/2022 Session
BCH2162: Instrumental Methods of Analysis

Lecture IV
UV / VISIBLE SPECTROSCOPIC TECHNIQUES

By
AJ Alhassan
11/20/2022 B AJ Alhassan 1
❖Spectroscopy
➢An instrumentation technic that deals with the
study of interaction of MATTER with light or
electromagnetic radiation.
✓Electromagnetic radiation consist of discrete

packages of energy known as photons.
✓A photon consists of an oscillating electric field

(E) & an oscillating magnetic field (M) which are
perpendicular to each other.
▪ Frequency (ν):
✓Is the number of times electrical field radiation
oscillates in one second. Its unit is Hertz (Hz).
1 Hz = 1 cycle per second
▪ Wavelength (λ):
✓Is the distance between two nearest crest or
troughs.
➢Speed of light (c) is the product of frequency and
wavelength that provide the relation between
wavelength & frequency (c = ν λ)
➢The electromagnetic energy E; E = h ν = h c / λ

▪Where;
✓h = Planck constant = 6.63 * 10-34 Js
✓v = frequency of radiation in hertz = c/λ
✓c = speed of light = 3 * 108ms-1
✓λ = wavelength of radiation in hertz = 1/ῡ
✓ῡ = wave number of radiation in waves cm-1
(Kaysers)
▪The units of wavelength includes centimeters (cm),

micrometers (µm), or nanometers (nm)
❖ The spectrum of electromagnetic radiation
➢ Visible spectrum of electromagnetic radiation
Violet 400 - 420 nm Yellow 570 - 585 nm

Indigo 420 - 440 nm Orange 585 - 620 nm
Blue 440 - 490 nm Red 620 - 780 nm
Green
11/20/2022
490 - 570 nm
B AJ Alhassan 7
❖ Principles of Spectroscopy
➢ It is based on the measurement of spectrum of a
sample containing atoms / molecules.
➢ The spectrum is a graph of intensity of absorbed

or emitted radiation by sample verses frequency
(ν) or wavelength (λ).
Fig: UV-visible spectrum of isoprene showing maximum absorption at 222 nm.
•Spectrometer is an instrument design to measure
the spectrum of a compound.
Scheme of a conventional single-beam spectrophotometer
❑ Type of Spectroscopy
▪ The electronic spectrum of an atom is due to changes
between major energy levels by the outer electrons.
▪ It occur in visible and ultraviolet regions, associated
with changes in rotational and vibrational energy
levels.
▪ Used routinely in for estimating concentration of a
component in a sample, based on the principle of
✓ visible and UV spectroscopy
✓ atomic/flame spectrophotometry.
✓ Though fluorescence spectra arises due to similar
transition it is apply on the principle of
spectrofluorimetry.
Fig. : Energy levels and transition of electrons
in Na atom
▪Absorption Spectroscopy:
An analytical technique concerns with the
measurement of absorption of electromagnetic
radiation.
•Examples
✓UV Spectroscopy (185 - 400 nm)
✓ Visible Spectroscopy (400 - 800 nm)
✓IR Spectroscopy (0.76 - 15 μm)
▪Emission Spectroscopy:
An analytical technique in which emission (of a
particle or radiation) is dispersed according to some
property of the emission & the amount of dispersion
is measured.
Example
✓Mass Spectroscopy
❖ Interaction of EMR with matter
➢ Electronic Energy Levels:
•At room temperature the molecules are in the
lowest energy levels E0.
•When the molecules absorb UV-visible light from

EMR, one of the outermost bond / lone pair
electron is promoted to higher energy state such as
E1, E2, …En, etc is called as electronic transition
and the difference is as:
∆E = h ν = En - E0 where (n = 1, 2, 3, … etc)
∆E = 35 to 71 kcal/mole
➢Vibrational Energy Levels:
▪Are less energy level than electronic energy levels.
▪The spacing between the energy levels are

relatively small (0.01 to 10 kcal/mole).
•e.g. when IR radiation is absorbed, molecules are

excited from one vibrational level to another or it
vibrates with higher amplitude.
➢Rotational Energy Levels:
•These energy levels are quantized & discrete.
•The spacing between the energy levels are even

smaller than vibrational energy levels.
∆Erotational < ∆Evibrational < ∆Eelectronic
❖ Beers Lambert’s Law (Laws of light absorption)
▪ For a uniform absorbing medium the proportion
of the radiation passing through it is called
transmittance, T T = I/Io
✓ Where; I = intensity of transmitted radiation
Io = intensity of incidence radiation
▪ Extent of absorption is known as extinction (E) or
absorbance (A) is equal to the logarithm of the
reciprocal of transmittance
A =E = log 1/T = log Io/I
✓ The transmittance is in the range 0 - 100%,
✓ The Extinction/absorbance varies from 0 - ∞.
➢ Beers – Lambert law states that the extinction is
proportional to the concentration of absorbing
substance and the to the thickness of the path
length
✓ E = ϵλcd
✓ Where ϵλ= molar extinction coefficient at
wavelength λ (dm3mol-1 cm-1)
✓ c = concentration of the absorbing solution
✓ d = light path in the absorbing material (cm)
▪ The ϵλ quoted for a compound is that given by a

1cm thick sample of 1% solution of the
compounds (E1%1cm)
❖ Terms used in UV / Visible Spectroscopy
➢Chromophore
▪ Is the part of a molecule responsible for imparting
color
OR
▪The functional groups containing multiple bonds
capable of absorbing radiations above 200 nm due
to n → π* & π → π* transitions.
e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc
To interpretate UV – visible spectrum following
points should be noted:
1. Non-conjugated alkenes show an intense

absorption below 200 nm & are therefore
inaccessible to UV spectrophotometer.
2. Non-conjugated carbonyl group compound give a

weak absorption band in the 200 - 300 nm region.
Chromophore
e.g. Acetone which has λmax = 279 nm
O O
C
H3C CH3
cyclohexane has λmax = 291 nm.
When double bonds are conjugated in a compound λmax is shifted to longer

wavelength.
e.g.
CH2 CH3
H2C H3C
1,5 - hexadiene has λmax = 178 nm 2,4 - hexadiene has λ = 227 nm
max
➢Auxochrome
The functional groups attached to a chromophore
which modifies the ability of the chromophore to
absorb light , altering the wavelength or intensity of
absorption.
OR
The functional group with non-bonding electrons
that does not absorb radiation in near UV region but
when attached to a chromophore alters the
wavelength & intensity of absorption.
Auxochrome
e.g. Benzene λmax = 255 nm
OH
Phenol λmax = 270 nm
Aniline λmax = 280 nm
NH2
➢ Absorption & Intensity Shifts
APPLICATIONS
❖ Applications of UV / Visible Spectroscopy
➢ Qualitative & Quantitative Analysis:
•It is used for characterizing aromatic compounds
and conjugated olefins.
•It can be used to find out molar concentration of
the solute under study.
➢Detection of impurities:
•It is one of the important method to detect
impurities in organic solvents.
➢Detection of isomers are possible.
➢Determination of molecular weight

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AL-ISTIQAMA UNIV.

BCH2162: Instrumental Methods of Analysis

UV / VISIBLE SPECTROSCOPIC TECHNIQUES

✓Electromagnetic radiation consist of discrete

✓A photon consists of an oscillating electric field

➢The electromagnetic energy E; E = h ν = h c / λ

▪The units of wavelength includes centimeters (cm),

Violet 400 - 420 nm Yellow 570 - 585 nm

➢ The spectrum is a graph of intensity of absorbed

Scheme of a conventional single-beam spectrophotometer

•When the molecules absorb UV-visible light from

▪The spacing between the energy levels are

•e.g. when IR radiation is absorbed, molecules are

•The spacing between the energy levels are even

∆Erotational < ∆Evibrational < ∆Eelectronic

▪ The ϵλ quoted for a compound is that given by a

e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc

1. Non-conjugated alkenes show an intense

2. Non-conjugated carbonyl group compound give a

cyclohexane has λmax = 291 nm.

When double bonds are conjugated in a compound λmax is shifted to longer

Aniline λmax = 280 nm

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