Muhammad Abdullah

Department of BioMedical Engineering

University of Engineering and Technology New Campus
Kala Shah Kaku
CONTENTS:

• History and introduction to spectroscopy

• Basic principles
• The law of absorption
• UV visible spectroscopy
• Instrumentation
• Application
• Conclusion
• Reference
HISTORY OF
SPECTROSCOPY:
• Spectroscopy began with Isaac Newton's
optics experiments (1666–1672). Newton applied
the word "spectrum" to describe the rainbow of
colors .
• During the early 1800s, Joseph von Fraunhofer
made experimental advances with
dispersive spectrometers that enabled
spectroscopy
to become a more precise and quantitative
scientific technique.
• Since then, spectroscopy has played and
continues to play a significant role
in chemistry, physics and astronomy.
SPECTROSCOPY:
• Spectroscopy is the branch of science dealing the study
of interaction of electromagnetic radiation with
matter.
• Spectroscopy is the most powerful tool available
for the study of atomic & molecular
structure and is used in the analysis of a
wide range of samples .
it s two main two type
• Atomic Spectroscopy; This Spectroscopy is
concerned with the interaction of
electromagnetic radiation with atoms are
commonly in the lowest energy state
called as grown state .
• Molecular Spectroscopy ; This Spectroscopy deals
with the interaction of electromagnetic radiation with
molecule.
BASIC PRINCIPLES:
• Light is supposed to duel
characteristic, corpuscular and
waveform
• Thus a beam of light may be understood as
electromagnetic waveform photons of energy
propagated at 3*108 m/s i.e., speed of light
• The term electromagnetic in a precise description
of the radiation in that the radiation is made up
of electrical & a magnetic wave which are in
phase & perpendicular to each other & to the
direction of propagation.
• A beam of light form a bulb consists of
many randomly oriented plane polarised
component being propagated in same
direction.
• The distance along the direction of
propagation for one complete cycle is known
as wavelength.
THE LAWS OF
ABSORPTION:
The absorption of light by any
absorbing material is governed by
two laws .
 Bouger-Lambert law
 Beer’s law
Bouger-Lambert law
This law is suggested by Picre Bouguer
in 1729, its often attributed to
Johann
Heinrich Lambert .
THE LAWS OF
ABSORPTION:
• This law is states that “ The amount of
the light absorbed is proportional to the
thickness of the absorbing material & is
independent of the intensity of the
incident light "
THE LAWS OF
ABSORPTION:
12.5% 6.25% 3.125%
100% 50% 25%

b
I – Intensity of transmitted light
- initial intensity of incident
light b – thickness (path –length)
k – linear absorption co-efficient

The power term can be removed by converting to the

log form.

ln(I/ )=-

/
kb ln(

I )=kb

Changing
to common
logarithms
we get,
Second law – Beer’s law
It states that, the amount of light absorbed by a material is proportional to
the number of
Absorbing
molecules(concentration) Again it
can be represented –
2.303 log( /I) = k’c
K’=absortivity constant
c= concentration

K and k’ merge
together = a

Log = /I = a b c

a = k & k’
b = thickness
C = concentration
This combined law states that the amount of light absorbed is
proportional to the Concentration of the absorbing substance & to the
thickness of the absorbing material
(path – length)
The quantity /I it is absorbance (O.D – optical density)
The two terms are mathematically commutable i.e., one can be calculated from
the
other
A=log - log I
= 100%
Log 100 =
2
=2-log
I Or
O.D is dirctly proportional to the concentration if path is
constant So if we know the
Unknown(sample) O.Dvalue of unknown
of the O.D concentration can beof std
x concentration
= of the
calculated Concentration
O.D of the std
 Terms describing UV absorptions
1. Chromophores: functional groups that
give electronic transitions.
2. Auxochromes: substituents with unshared
pair e's
like OH, NH, SH ..., when attached to π
chromophore they generally move the absorption
max. to longer λ.
3. Bathochromic shift: shift to longer λ, also called
red shift.
4. Hysochromic shift: shift to shorter λ, also called
blue shift.
5. Hyperchromism: increase in ε of a band.
6. Hypochromism: decrease in ε of a band.
UV-VISIBLE
Spectroscopy :
Uv-vis spectroscopy is also known as
electronic spectroscopy. In which the amount
of light absorbed at each wavelength of Uv
and visible regions of electromagnetic
spectrum is measured. This absorption of
electromagnetic radiations by the molecules
leads to molecular excitation.
ELECTRONIC
SPECTROSCOPY:
• Ultraviolet (UV) and visible
(VIS) spectroscopy
• This is the earliest method of
molecular spectroscopy.
• A phenomenon of interaction of
molecules with ultraviolet and visible
lights.
• Absorption of photon results in
electronic transition of a molecule, and
electrons are promoted from ground
state to higher electronic states.
• The first discovery of electromagnetic waves other than
light came in 1800, when William Herschel
discovered infrared light. He was studying the
temperature of different colors by moving a
thermometer through light split by a prism.
• The types of electromagnetic radiation are
broadly classified into the following classes
• Gamma radiation
• X-ray radiation
• Ultraviolet radiation
• Visible radiation
• Infrared radiation
• Terahertz radiation
• Microwave radiation
• Radio waves
• Ultraviolet: 190~400nm
• Violet: 400 - 420
nm
• Indigo: 420 - 440
• nm
Green: 490 - 570 nm
•• Blue: 440 -- 585
Yellow: 570 490 nm
nm
• Orange: 585 - 620 nm
• Red: 620 - 780
nm
ULTRAVIOLET
RAYS
Shorter wavelength and higher frequency than
visible light Carry than visible light
ELECTRONIC
TRANSITIONS:
There are three types of electronic
transition which can be considered;
• Transitions involving p, s, and n
electrons
• Transitions involving charge-
transfer electrons
• Transitions involving d and f
electrons
 ABSORBING SPECIES
CONTAINING P, S,
AND N ELECTRONS:
• Absorption of ultraviolet and visible
radiation in organic molecules is
restricted to certain functional
groups (chromophores) that contain
valence electrons of low excitation
energy.
    Transitions
• An electron in a bonding s orbital is excited to
the corresponding antibonding orbital. The
energy required is large. For example, methane
(which has only C-H bonds, and can only
undergo    transitions) shows an
absorbance maximum at 125 nm. Absorption
maxima due to    transitions are not
seen in typical UV-VIS spectra (200 - 700
nm)
 n   Transitions
• Saturated compounds containing atoms with
lone pairs (non-bonding electrons) are
capable of n   transitions. These
transitions usually need less energy than  
  transitions. They can be initiated by light
whose wavelength is in the range 150 - 250
nm. The number of organic functional groups
with
n   peaks in the UV region is small.
 n   and    Transitions
• Most absorption spectroscopy of organic
compounds is based on transitions of n or
 electrons to the  excited state.
• These transitions fall in an experimentally
convenient region of the spectrum (200 -
700 nm). These transitions need an
unsaturated group in the molecule to
provide the  electrons.
 INSTRUMENTA

TION
Light source:
UV - Hydrogen lamp ( hydrogen stored under
pressure) , Deuterium lamp and Xenon lamp-
it is not regularly used becos of unstability and
also the radiation of UV causes the generation
of ozone by ionization of the oxygen
molecule.
VIS – Tungston filament lamp , Tungston
halogen lamp and carbon arc lamp.
 Waveselectors are mainly either filters or
monochrmators.
 Filters : Gelatin filters are made using a
layer of gelatin coloured with organic dyes
that are sealed between glassplates. This
filters resolve polychromatic light into a
relatively wide band width of about 40 nm
and these are commonly used in
colorimeters since they have low
transmittance i.e. 5 – 20 %.
 Monochromators :
Consists of an entrance slit which admits the polychromatic light
from the source.
 A collimating device – lens or mirror which helps in reflecting
the polychromatic light to the dispersion device.
 A wavelength resolving device - prism or grating.
 A focussing lens or mirror
 Exit slit
 Sample holder/ containers :
 Cuvettes – Quarts or fused silica , ordinary glass is known to absorb
uv
rad.
 for IR – samples are ground with potassium bromide and pressed
into a pellet, if aqueous solution silver chloride is coated inside the
cell. While preparing samples selection of solvents is imp. , becos
they do
absorb light.
 INSTRUMENTATION:Single
and Double Beam
Spectrometer
• Single-Beam: There is only one light beam
or optical path from the source through to
the detector.
• Double-Beam: The light from the source,
after passing through the monochromator, is
split into two separate beams-one for the
sample and the other for the reference.
 VISIBLE
LIGHT
• Shorter wavelength and higher frequency than infrared
rays.
• Electromagnetic waves we can see
•Longest wavelength= red light
Shortest wavelength= violet (purple)
light
Single beam spectrophotometer
A single beam of radiation pass
through a single cell, the reference cell is
used to set the absorbance scale at zero for
the wavelength to be studied. It is then
replaced by sample cell to determine the
absorbance of the sample at that
wavelength . This was the earliest design
and is still use in both teaching and
industrial labs.
 Double beam spectrophotometer

• The instrument used in ultraviolet-visible spectroscopy is called a

UV/Vis spectrophotometer. It measures the intensity of light passing
through a sample (I), and compares it to the intensity of light before
it passes through the sample (I). The ratio is called the
transmittance, and is usually expressed as a percentage (%T). The
absorbance, (A). is based on the transmittance.
• A= -log(%T/100%)
• Detection devices :
• UV-VIS detectors –
• 1. Photocells made of cadmium sulphide ,
silicon and selenium. Steel base coated
with silver film then finally thin coating of
selenium. Electrons pass through
selenium to silver and silver acts as the
collecting electrode and steel plate as
another electrode, the current flowing
between two electrodes is then measured
by a micro-ammeter.
• 2. Phototubes : glass envelop with a quartz
window, centrally situated metal wire acts as
anode and a semi-circle cathode.
• The energy of the photon is transferred to
the loosely bound electrons of the cathode
surface. The electrons excited move towards
anode causing to flow in the circuit.
Phototube currents are quite small and
require amplification, then it is recorded.
• 3. photomultiplier : these are designed to
amplify the initial photoelectric effect and are
suitable for the use at very low light intensities.
• This consists of an evacuated glass tube into
which are sealed the cathode and anode and
an additional intervening electrodes known as
dynodes. As the radiation strikes the cathode
electrons are liberated and the applied
potential difference accelerates the electrons
towards the first dynode. Each successive
dynode is at higher electrical potential acts as
amplifier.
• 4. photodiodes : are semiconductors
that
charge their charged voltage upon being
striked by the radiation, the voltage is
converted to current and it is measured.
 Instrumentation
UV-VIS and INFRARED
Spectrophotometer