UV - Visible

Prof. N. A. Ochekpe

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Outline

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Introduction - Spectroscopy

• It is the branch of science that deals with the study of

interaction of molecules (matter) with light.

OR

• It is the branch of science that deals with the study of

interaction of electromagnetic radiation with matter

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SPECTROSCOPY
• Interaction of Radiation with a sample

• The study of molecular or atomic structure of a

substance by observation of its interaction with
electromagnetic radiation

• QUANTITATIVELY - For determining the amount of

material in a sample

• QUALITATIVELY – For identifying the chemical structure

of a sample

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THE ELECTROMAGNETIC SPECTRUM

• Most of us are aware of many different ways of

transmitting energy and these phenomena come together in
one physical entity called the ELECTROMAGNETIC
SPECTRUM
• The difference between these sources of radiation is the
amount of energy they radiate.
• The radiation from these and other sources covers a range
of energies
 

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Introduction – Electromagnetic Radiation
• Electromagnetic radiation
consist of discrete packages
of energy which are called
as photons.
OR
• A photon consists of an
oscillating electric field (E)
& an oscillating magnetic
field (M) which are
perpendicular to each other.
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Electromagnetic Radiation

• Frequency (ν):
• It is defined as the number of times electrical field radiation oscillates
in one second.
• The unit for frequency is Hertz (Hz). 1 Hz = 1 cycle per second.

• Wavelength (λ):
• It is the distance between two nearest parts of the wave in the same
phase i.e. distance between two nearest crest or troughs.

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The Electromagnetic Spectrum

Radio waves

Microwave

Infra-red

Visible

Ultraviolet

X-rays

Gamma rays
Long Wavelength Shor
Energy t
Low
Frequency High
Low
High

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RADIATION IS TRANSMITTED IN A WAVEFORM

• LOW ENERGY RADIATION has a LONG WAVELENGTH

• HIGH ENERGY RADIATION has a SHORT WAVELENGTH

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Radiation Energy

• The strength of the radiation energy will interect with the molecules in
different ways:
• High energy sources produce breaking of bonds
• X-Ray, γ Rays, …

• Medium energy sources excite electrons

• UV / VISIBLE Spectroscopy

• Low energy sources produce vibrations in chemical bonds

• Infrared Energy

• Very low energy sources produce rotation of the chemical bonds

• Microwaves and Radio waves

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EFFECT OF ENERGY ON A MOLECULE

ELECTROMAGNETIC SPECTRUM
ENERGY
1.2 x105 1.2 x107 12000 310 150 0.12 0.0012
( kJ/mol)

Electronic excitation
e-

FREQUENCY
(Hz) 1020 1018 1016 1014 1012 108

visible
Cosmic γ Ultra Radio
Infrared Microwave
rays rays rays violet waves
WAVELENGTH
(m) 10-12 10-11 10-9 10-6 10-3 10-1

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VISIBLE SPECTROSCOPY
WHAT IS COLOUR?

Colour is a sensation which occurs when light enters the eye and focuses on the retina
at the back of the eye. The light actually initiates a photochemical reaction in the
nerve cells attached to the retina. These transmit impulses to the brain and stimulate
our sense of colour
 
CONES - Give colour and RETINA

three types which pick up

red, blue and green  BRAIN

RODS - Give grey/black

and also used for night = CONES

vision.
= RODS

All the colours we actually sense are made up of these three

colours together with white and grey and black.

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VISIBLE SPECTROSCOPY
COMPOSITION OF WHITE LIGHT
• Sunlight is white light and covers a wavelength range of 380-750nm. A simple physics experiment
shows that white light is actually a composition of a range of colours i.e., light of different
energies and hence wavelengths.

When white light falls on an

Red
object the colour detected by O range
WHITE
the eye will depend upon the LIGHT
Yellow
G reen
ABSORPTION/REFLECTION B lue
properties of the material Indigo
Violet
in the object; 

• If the material completely REFLECTS all light it appears WHITE

• If the material absorbs a constant fraction of the light across the spectrum it appears GREY.
• If the material completely ABSORBS all the light it appears BLACK

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VISIBLE SPECTROSCOPY
When a sample only absorbs light of a single wavelength the eye sees
COMPLEMENTARY colours.

Wavelength Range Absorbed Colour Absorbed Colour Seen By Eye

380 - 430 Violet Yellow - Green
430 - 480 Blue Yellow
 
480 - 490 Green - Blue Orange

 
490 - 500 Blue - Green Red
500 - 560 Green Purple
560 - 580 Yellow - Green Violet
580 - 590 Yellow Blue
590 - 610 Orange Green - Blue
610 - 750 Red Blue - Green

LOW HIGH

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 Vibrational Energy Levels
Vibrational levels

rotational levels
S1

Effects of the energy levels depending

on the nature of the energy received
absorption
Energy

Vibrational levels

rotational levels
S0
Ground state
UV-Vis IR mW

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UV / VISIBLE SPECTROSCOPY
UV Radiation – Wavelength range 220 - 380nm

VISIBLE Radiation – Wavelength range 380 - 780nm

Substances can absorb varying amounts of UV and/or Visible radiation at

particular wavelengths – Coloured compounds absorb energy in both UV
and visible region of the electromagnetic spectrum.

Substances can be liquids or solids and measurements are made with

instruments called SPECTROPHOTOMETERS or SPECTROMETERS.

Modern instruments can be coupled to microscopes which allow solid

samples and very small samples of solids and liquids to be analysed both
qualitatively and quantitatively.

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Principles of Spectroscopy

• The principle is based on the measurement of spectrum of a

sample containing atoms / molecules.

• Spectrum is a graph of intensity of absorbed or emitted

radiation by sample verses frequency (ν) or wavelength (λ).

• Spectrometer is an instrument design to measure the

spectrum of a compound

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Principles of Spectroscopy
1. Absorption Spectroscopy: 2. Emission Spectroscopy:

• An analytical technique which • An analytical technique in which

concerns with the measurement emission (of a particle or
of absorption of electromagnetic radiation) is dispersed according
radiation. to some property of the emission
& the amount of dispersion is
measured.
• e.g. UV (185 - 400 nm) / Visible
(400 - 800 nm) Spectroscopy, IR
Spectroscopy (0.76 - 15 μm) • e.g. Mass Spectroscopy

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Interaction of EMR with Matter
1. 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
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Interaction of EMR with Matter
2. Vibrational Energy Levels:

• These are less energy level than

electronic energy levels.

• The spacing between energy levels

are relatively small i.e. 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.
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Interaction of EMR with Matter
3. Rotational Energy Levels:
• These energy levels are quantized &
discrete.

• The spacing between energy levels

are even smaller than vibrational
energy levels.

∆Erotational < ∆Evibrational <

∆Eelectronic

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Experimental Set Up
• Various designs of spectrophotometers :
• Fixed and variable wavelength; Single, split and double beam
• Cuvettes:
- Glass or plastic - visible
- Quartz – visible, UV
- Acrylic – visible, UV

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Monochromator and Instrumentation

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UV / VISIBLE SPECTROSCOPY - THEORY
INCIDENT LIGHT TRANSMITTED LIGHT
254nm 254nm
SAMPLE
Intensity (I o ) Intensity (I t )

• If a particular wavelength of UV or Visible radiation can be isolated from the source and
passed through a sample which can ABSORB some of the radiation then the TRANSMITTED
light intensity (It) will less than the INCIDENT light intensity (Io).

• The amount of light transmitted with respect to the incident light is called TRANSMITTANCE
(T) i.e.,

T=
It

Io
• Sample can absorb all or none of the incident light and therefore
• transmittance often quoted as a percentage eg.,

It
% T= X 100
Io

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UV / VISIBLE SPECTROSCOPY - THEORY

ABSORBANCE A = - log10 T
It 2
A = - log10
B
Io
A
A = log10 Io 0
It 220 Wavelength(nm) 380

For of %T = 0 and 100 the corresponding absorbance

values will be 0 and 2 respectively

By plotting Absorbance vs wavelength an ABSORBANCE SPECTRUM is

generated. The absorbance spectra for the same compounds A and B are
shown.

With the advantage that absorbance measurements are usually linear with
Concentration, absorbance spectra are now used

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 THE LAWS OF SPECTROPHOTOMETRY
There are two very important basic laws and a third one which is a
combination of the two.

LAMBERTS LAW – ABSORBANCE (A) proportional to the PATHLENGTH (l)

of the absorbing medium.

BEERS LAW - ABSORBANCE (A) proportional to the CONCENTRATION (c)

of the sample.

BEER- LAMBERT LAW - ABSORBANCE (A) proportional to c x l

A  cl
A = Ecl (A is a ratio and therefore has no units)

The constant E is called the MOLAR EXTINCTION COEFFICIENT

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 UV / VISIBLE SPECTROSCOPY - THEORY
UNITS OF THE MOLAR EXTINCTION COEFFICIENT
• CONCENTRATION (c) - Moles litre-1
• PATHLENGTH (l) - cm
A = Ecl Hence E= A
cl
E = 1 ˛

mole litre-1 x cm
E = mole-1 litre x cm -1
But 1 litre = 1000cm3
E = 1000 mole -1 cm3 x cm -1
Hence Units of E = 1000 cm2 mole -1

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UV/VISIBLE SPECTROSCOPY - THEORY

IMPORTANCE OF THE BEER LAMBERT LAW

A = Ecl but if E and l are constant

ABSORBANCE  CONCENTRATION and should be linear relationship

Prepare standards of the analyte to be quantified at known concentrations

and measure absorbance at a specified wavelength.

Prepare calibration curve.

ABSORBANCE AT 300nm
x
From measuring absorbance of sample
x
Concentration of analyte in sample x
can be obtained from the calibration curve x

E can be obtained from the slope of the x

CONCENTRATION (moles litre-1 )
calibration curve for a given wavelength ()
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UV/VISIBLE SPECTROSCOPY - THEORY
RULES FOR QUANTITATIVE ANALYSES
x

ABSORBANCE AT 300nm
At high concentrations the calibration curve may
deviate from linearity – Always ensure your x
concentration of the sample falls within the linear
range – if necessary dilute sample x

x
Absorbance not to exceed 1 to reduce error*
x
CHOOSE CORRECT WAVELENGTH CONCENTRATION (moles litre-1 )

An analyte may give more than one absorbance A

maxima (max) value. C max
0.6
B

Many compounds absorb at 220-230nm hence do not

use A

Need to choose wavelength more specific

to compound (SELECTIVITY) and if more 0
than one select one with highest absorbance 220 Wavelength (nm) 380
as this gives less error – hence use C
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 Let’s Review!
A couple of things to take into account…

The intensity of incident light from the light source is always 110.0 photons/sec

You have to calculate Transmittance T= It / Io and Absorbance A=–Log T by

yourself and supply the website with the values you obtain

Now you can play with the virtual spectrophotometer changing the
path length, concentration, calculate the Molar Absorptivity (or
Molar Extinction Coefficient)
And run a calibration curve….

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Example of calculations for photometry
Given the following set of data for a compound C:
Can you give the least square equation better fitting the curve?
(Conc=X, Abs=Y)

0 .7
Conc (M) Abs 0 .6 y = 1.0 137x + 0 .1378
R2 = 0 .997
0.1 0.2322 0 .5
0 .4

Abs
0.2 0.3456
0 .3
0.3 0.4532 0 .2
0.4 0.5331 0 .1
0.5 0.6453 0
0 0 .2 conc 0 .4 0 .6

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Is the fitting of the curve to the equation
acceptable? How can you tell?

What is the concentration of C when we obtain an

Absorbance of 0.3321?

The concentration is: Abs= 1.0137 * Conc + 0.1378

Abs= 0.3321 – Abs blank= 0.3321- 0.13800 = 0.1941

Conc= Abs – 0.1378 = 0.1941 – 0.1378 = 0.055 M

1.0137 1.0137

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The possible electronic transitions can graphically shown as:

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• σ electron from orbital is excited • π electron in a bonding orbital is
to corresponding anti-bonding excited to corresponding anti-
orbital σ*. bonding orbital π*.

• The energy required is large for • Compounds containing multiple

this transition. bonds like alkenes, alkynes,
carbonyl, nitriles, aromatic
compounds, etc undergo π → π*
• e.g. Methane (CH4 ) has C-H
transitions.
bond only and can undergo σ → σ*
transition and shows absorbance
maxima at 125 nm. • e.g. Alkenes generally absorb in
the region 170 to 205 nm.
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• Saturated compounds containing • An electron from non-bonding
atoms with lone pair of electrons orbital is promoted to anti-
like O, N, S and halogens are bonding π* orbital.
capable of n → σ* transition.
• Compounds containing double
• These transitions usually bond involving hetero atoms
requires less energy than σ → σ* (C=O, C≡N, N=O) undergo such
transitions. transitions.

• The number of organic functional • n → π* transitions require

groups with n → σ* peaks in UV minimum energy and show
region is small (150 – 250 nm). absorption at longer wavelength
around 300 nm.
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•These electronic transitions are forbidden transitions & are
only theoretically possible.

•Thus, n → π* & π → π* electronic transitions show absorption in

region above 200 nm which is accessible to UV-visible
spectrophotometer.

•The UV spectrum is of only a few broad of absorption.

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Terms used in UV -Visible
Chromophore:
• The part of a molecule responsible for imparting color, are called as
chromospheres.

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

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Chromophore…
• To interprete 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.
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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.

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Absorption Intensity and Shift

• Bathochromic shift (Red Shift)

• Hypsochromic shift (Blue Shift)
• Hyperchromic effect
• Hypochromic effect

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Bathochromic Shift – Red Shift
• When absorption maxima (λmax) • In alkaline medium, p-nitrophenol
of a compound shifts to longer shows red shift. Because
wavelength, it is known as negatively charged oxygen
bathochromic shift or red shift. delocalizes more effectively than
the unshared pair of electron.
• The effect is due to presence
of an auxochrome or by the
change of solvent.

• e.g. An auxochrome group like –

OH, -OCH3 causes absorption of
compound at longer wavelength.
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Hypsochromic shift (Blue Shift)
• Aniline shows blue shift in
• Absorption maxima (λmax) of a acidic medium, it loses
compound shifts to shorter conjugation.
wavelength, it is known as
hypsochromic shift or blue
shift.

• The effect is due to presence

of an group causes removal of
conjugation or by the change of
solvent.

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Hyperchromic Effect
• When absorption intensity (ε) of a compound is increased, it is
known as hyperchromic shift.
• If auxochrome introduces to the compound, the intensity of
absorption increases.

Pyridine 2-methyl pyridine

λmax = 257 nm λmax = 260 nm

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Hypochromic Shift
• When absorption intensity (ε) of a compound is decreased, it is
known as hypochromic shift.

• Naphthalene 2-methyl naphthalene

• ε = 19000 ε = 10250

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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 using Beer’s law.

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 GOODLUCK!

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