Uv Visible Spectros

UV-VISIBLE
SPECTROSCOPY
Group Members:
Sidra Rasheed
Asma Muhammad
Raheel Qamar Butt
Kamran Haider
Muzammil Zaidi
Contents
Introduction and principle
Quantitative measurment and
electronic Transitions
Instrumentation and the experiment
Applications
Limitations
What is Spectroscopy??
Spectroscopyis the use of the
absorption, emission, or scattering of
electromagnetic radiation by matter
to qualitatively or quantitatively
study the matter or to study physical
processes.
The matter can be atoms, molecules,
atomic or molecular ions, or solids.
Types of spectroscopy
Emission Spectroscopy:
A method ofchemical analysisthat
uses the intensity of light emitted from
a flame or spark at a particular
wavelength to determine the quantity
of element in a sample.
Absorption spectroscopy
Itis the analytical technique based
on measuring the amount of light
absorbed by a sample at a given
wavelength.
1. Atomic absorption Spectroscopy
2. Molecular Absorption Spectroscopy
UV-Visible Spectroscopy
It is type of Molecular Absorption
Spectroscopy in which UV and Visible
Radiations are used for quantitative
and qualitative analysis of
compounds especially conjugated Pi
systems.
The wavelength range is from 200nm
to almost 800nm.
Electromagnetic Radiation
Violet
Indigo
Blue
400 - 420
nm
420 - 440
nm
440 - 490
nm
Yellow
Orange
Red
570 - 585
nm
585 - 620
nm
620 - 780
nm
Interaction with
Matter
Interaction with matter

When Electromagnetic Radiations
Interact with molecules, it imparts
energy to the electrons, which get
excited.
Electrons will only be excited if the
wavelength of incoming photon is
comparable to the energy gap
between two energy levels.
Interaction of EMR with

matter
1. Electronic Energy Levels:
. At room temperature the molecules
are in the lowest energy levels E0.
The absorption of
energy excites the
electron from the
ground state to an
electronic excited
state
E = 35 to 71 kcal/mole

matter
2. Vibrational Energy Levels:
. A given electronic energy level has a
number of vibrational energy levels in
it.
The
spacing
between energy
levels
are
relatively small
i.e. 0.01 to 10
kcal/mole.

matter
3. Rotational Energy Levels:
. Each of the vibrational energy level
has a number of rotational energy
levels in it.
. The spacing between energy levels
are even smaller than vibrational
energy levels.
Erotational < Evibrational < Eelectronic
Physics Behind the color of a

material
When white falls on a
material, some portion of
light is absorbed.
The other portion is reflected
back and this is what we see
as the color of that
substance.
The excited state of an
electron is unstable.
It has a lifetime of about
10^-8s
When electron gets deexcited it emits
radiation(reflection) of
specific frequency(color) in
the visible Range.
Quantitative Analysis
Lamberts Law
When a monochromatic radiation is passed through a
solution, the decrease in the intensity of radiation with
thickness of the solution is directly proportional to the
intensity of the incident light.
Let I be the intensity of incident radiation.
x be the thickness of the solution.
dI
I
dx
Beers Law
Includes
concentration
A=.C.L
Where; A =
absorbance
= absorptivity or
extinction co-efficient
L= the path length
C = Concentration of
the absorbing specie
Electronic Transitions
The possible electronic transitions

can be graphically shown as:
Generally the possible

Electronic Transitions are:
Possible Transitions in UV-Vis

Allowed transitions and forbidden
transitions
Thus, n * & * electronic
transitions show absorption in region
above 200 nm which is accessible to
UV-visible spectrophotometer.
Instrumentation
UV-Visible Spectrometer
Spectrometer consists of following
parts:
1. Radiation source
2. Monochromator
3. Sample container
4. detector
Radiation Source
Hydrogen or deuterium Lamps
are used as UV radiation.
The mechanism for this involves
formation of an excited molecular
species, which breaks up to give
two atomic species and an
ultraviolet photon.
wavelength range 160 - 375 nm
The tungsten filament lamp is
commonly employed as a source
of visible light.
wavelength range of 350 - 2500
nm
Monochromator
All monochromators contain the
following component parts;
1. An entrance slit
2. A collimating lens
3. A dispersing device (usually a
prism or a grating)
4. A focusing lens
5. An exit slit
Monochromator
Cuvettes/Sample container
The containers for the sample and
reference solution must be transparent
to the radiation which will pass through
them.
Quartz or fused silica cuvettes are
required for spectroscopy in the UV
region.
These cells are also transparent in the
visible region.
Silicate glasses can be used for the
manufacture of cuvettes for use
between 350 and 2000 nm.
Detector
The photomultiplier tubeis a
commonly used detector in UV-Vis
spectroscopy.
It consists of aphoto-emissive
cathode,
severaldynodes(which
emit several electrons for each
electron
striking
them)
and
ananode.
Photomultipliers are very sensitive
to UV and visible radiation. They
have fast response times. Intense
light damages photomultipliers;
they are limited to measuring low
power radiation.
Sample preparation
Sample must always be clear
solutions
Sample cell should not be touched by
hands
Absorbance due to solvent should be
less than 1A.
The dilution Factor must be
estimated
Be sure there are not air bubbles
inside the cell.
The
Experiment
Reference cell
A measurement of the absorbance
of the reference cell ( not containing
the absorbing specie) is made.
The exact value of the absorption of
the sample is obtained by
subtracting the absorbance of the
reference cell from that of the whole
sample.
Chlorophyll
Applications of U.V. Spectroscopy:

1. Detection of Impurities
.
UV absorption spectroscopy is one of the best methods for

determination of impurities in organic molecules. Additional
peaks can be observed due to impurities in the sample and it
can be compared with that of standard raw material. By also
measuring the absorbance at specific wavelength, the
impurities can be detected.
2. Structure clarification 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.
3.Chemicalkinetics
Kinetics of reaction can also be studied using
UV spectroscopy. The UV radiation is passed
through the reaction cell and the absorbance
changes can be observed.
4.Detection of Functional Groups

This technique is used to detect the
presence or absence of functional

group in the compound
Absence
of
band
at
particular
wavelength regarded as an evidence

for absence of particular group
5. Quantitative analysis of pharmaceutical

substances
Many drugs are either in the form of raw material or

in the form of formulation. They can be assayed by
making a suitable solution of the drug in a solvent
and measuring the absorbance at specific wavelength.
Diazepam tablet can be analyzed by 0.5% H2SO4 in
methanol at the wavelength 284 nm.
LIMITATIONS ON UV-VIS
SPECTROSCOPY
There are three types of deviations from
usual behaviour
Real Deviations These are fundamental
deviations due to the limitations of the law
itself.
Chemical Deviations These are deviations
observed due to specific chemical species of the
sample which is being analyzed.
Instrument Deviations These are deviations
which occur due to how the absorbance
measurements are made.
Real Limitation and Deviation of Beer-Lambert

Law
Beer law and Lambert law is capable of describing absorption behavior of solutions
containing relatively low amounts of solutes dissolved in it (<10mM). When the
concentration of the analyte in the solution is high (>10mM), the analyte begins to
behave differently due to interactions with the solvent and other solute molecules
and at times even due to hydrogen bonding interactions.
At high concentrations, solute molecules can cause different charge distribution on
their neighboring species in the solution. Since UV-visible absorption is an
electronic phenomenon, high concentrations would possibly result in a shift in the
absorption wavelength of the analyte.
At times, even electrolyte concentrations (such as those present in buffers) play
an important role in altering the charge distributions and affecting UV-visible
absorbance. Some large ions or molecules show deviations even at very low
concentrations. For e.g. methylene blue absorptivity at 436 nm fails to observe
Beer Lambert law even at concentrations as low as 10M.
High analyte concentrations can also possibly alter the refractive index () of the
solution which in turn could affect the absorbance obtained. If the addition of solute
causes a significant change in the refractive index of the solution a correction to
the
Beer
Lambert
formula
can
be
placed
as:
A = bc (2+ 2)2
Chemical Deviations and Limitations to BeerLambert Law

Chemical deviations occur due to chemical phenomenon
involving the analyte molecules due to association,
dissociation and interaction with the solvent to produce a
product with different absorption characteristics. For example,
phenol red undergoes a resonance transformation when
moving from the acidic form (yellow) to the basic form (red).
Due to this resonance, the electron distribution of the bonds
of molecule changes with the pH of the solvent in which it is
dissolved. Since UV-visible spectroscopy is an electron-related
phenomenon, the absorption spectrum of the sample changes
with the change in pH of the solvent.
Acid and Base forms of phenol red along with their UV spectra
at different pH demonstrates chemical deviations of BeerLambert law in UV-Visible spectroscopy
Instrumental Deviations and Limitations to

Beer-Lambert Law
Beer-Lambert law is strictly followed when a monochromatic
source of radiation exists. In practice, however, it is
common to use a polychromatic source of radiation with
continuous distribution of wavelengths along with a filter or
a grating unit (monochromators) to create a monochromatic
beam from this source.
For example consider a molecule having molar
absorptivities and at wavelengths and .
When the molar absorptivities are the same at both
wavelengths (i.e. = ), the relationship between
absorbance and concentration follows Beer-Lambert law to
obtain a straight line. However, as the difference between
and increases, the deviations from linearity also
increases.
TESEKKUR
EDIRIM
(Thank you
very much)

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UV-VISIBLE

Interaction with matter

Interaction of EMR with

Interaction of EMR with

Interaction of EMR with

Physics Behind the color of a

The possible electronic transitions

Generally the possible

Possible Transitions in UV-Vis

Applications of U.V. Spectroscopy:

UV absorption spectroscopy is one of the best methods for

2. Structure clarification of organic compounds.

UV spectroscopy is useful in the structure elucidation of

organic molecules, the presence or absence of

From the location of peaks and combination of peaks, it can

be concluded that whether the compound is saturated or

4.Detection of Functional Groups

presence or absence of functional

wavelength regarded as an evidence

5. Quantitative analysis of pharmaceutical

Many drugs are either in the form of raw material or

Real Limitation and Deviation of Beer-Lambert

Chemical Deviations and Limitations to BeerLambert Law

Instrumental Deviations and Limitations to

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