What is uv- visible spectroscopy?
It is molecular
spectroscopy in which we study interaction between molecules and UV – visible
radiation.
UV- Visible
spectroscopy is work on absorption phenomenon.
Basic:-
Ultra Violet
spectroscopy is concerned with the study of absorption of UV radiation which
range from 200nm to 400nm.
Visible spectroscopy
is concerned with the study of the absorption of visible radiation which range
from 400nm to 800nm.
In both
spectroscopy sample is absorbed radiation energy and valence electrons are goes
ground state to excited state.
There
are mainly three types of electron
- σ – electron :- These are
the one present in saturated compound. Such electron do not absorb near UV
but absorb vacuum UV radiation.
- π electron :- These electron
are present in unsaturated compound double or triple bond.
- N electron :- These are non bonded electron which are not involved in any bonding lone pair of electron like in S,O,N and Halogen.
PRINCIPLE
It is mainly work on
absorption phenomenon. We measure the absorption of light by molecules that are
in a gas or vapour state or dissolved molecules in spectrophotometry.
Investigation the
absorption of the difference substance wave length is 190 nm to 780 nm
Electronic
Transition And Excitation process :-
It was stated earlier
that σ,π, and n electron are present in a molecule and can be excited from
the ground state by the absorption of UV radiation.
There are mainly four
types of transition are seen
- σ—> σ∗
- Ï€ –> Ï€∗
- n —> Ï€∗
- n –> σ∗
Type of
electronic transition
1. n⇒Ï€∗ :- This electron transition required
lowest energy.
The peak of this
transition is called R band.
Wavelength is
approximately 200 nm.
This type of
peak can be seen in compound where n electron is present in a compound
containing double bond or triple bond eg. Aldehyde or ketones
2. Ï€⇒Ï€∗ :- This type of transition give B,E,
and k band.
Wavelength is
approximately more than 200 nm.
3. n⇒σ∗
In this
transition approximately 175 nm wavelength are seen.
When any hetero
atom is present in the saturated compound this transition is occur.
This transition
is also seen in water, chloroform, alcohol.
4.σ⇒σ∗
This type
transition required the highest energy.
The peak is seen
in 100 nm to 150 nm
Example of this
transition is hydrocarbon.
type of shift in UV Spectroscopy Chromophore, Auxochrome and
different
Chromophore
It is a
covalently unsaturated group which is responsible for absorption of UV or
visible radiation and may or may not impact in colour to the compound.
A compound which
contains chromophore it is called chromogen.
In unsaturated
linkage such as -c=c- ,
-N=N-,
the π electron are loosely bound. These loosely bound electron required
less energy for electronic transition and the absorption band occur in near UV
region.
Example
Acetylene possessed -C=C- in structure it λmax is 175-180 nm.
Auxo
chrome
It is saturated
and unsaturated group which consists of one or more pair of non-bonded
electron.
This group is
attached to Chromophore help in altering the wavelength by increasing the
intensity of absorption and increase λmax.
Example of
Auxochrome is -OH, -NH2,
-OR etc.
Type of
Shift
1.
Bathochromic shift ( Red shift ) :-
In this Types
the absorption maximum shift towards longer wavelength.
When two or more
chromophore present in a molecule is responsible for bathochromic shift.
Ex. Ethene which
contain unconjugated double bond show λmax at 170 nm.
2.
Hypsochromic shift ( Blue shift) :-
In this Type the
absorption maximum shift towards shorter wavelength. This shift is occur due to
removal of conjugation or due to change in the polarity of the solvent.
Ex. Aniline
show λmax at 280 nm because of on conjugation between lone pair of
electron between nitrogen and benzene.
3.
Hyperchromic shift :-
In this Type of
shift increase intensity with increase of absorption of radiation.
This is due to
the introduce Auxochrome.
Ex. Increase
intensity of the absorption of DNA due to denaturation at the λmax 260 nm.
4.
Hypochromic shift :–
In this Type of
shift decrease intensity of radiation because of decreased absorption.
It is due to
presence of certain group which may cause deformation of structure of compound.
UV-
Visible spectroscopy Principal and Factor affecting absorption
UV-VISIBLE
SPECTROSCOPY PRINCIPAL :-
- The principle is based on
the measurements of spectrum of a sample containing atom/molecules.
- Spectrum is a graph of
intensity of absorbed or emitted radiation by sample vs frequency or
wavelength.
- Spectrometer is an instrument
design to measure the spectrum of a compound.
- UV-Visible spectroscopy measure
the response of a sample to ultra Violet and visible range of EMR.
- Molecules have either σ,π
and n electron.
These electron
absorbed uv radiation and under goes transition from ground state to excited
state.
Factor
affecting absorption in uv-visible spectroscopy :-
1. Absorption compound
:-
It is increase
absorption of UV. Example is Chromophore and Auxochrome
2. Solvent effect :-
Solvent that absorbed
uv radiation are avoid.
Example is benzene ,
carbon tetrachloride, chloroform etc.
3. Temperature :-
Low temperature is
suitable for uv visible spectroscopy.
In,High temperature
absorption of UV radiation is increase.
4. Inorganic material
:-
Inorganic material are
increase the absorption.
Ex. Ag, Au etc.
ABSORPTION AND INTENSITY SHIFT :-
- Bathochromic shift :- when
absorption maximum of a compound shift to longer wavelength, it is known
as Bathochromic shift or red shift.
- Hypsochromic shift:- when
absorption maximum of a compound to a shorter wavelength it is called a
Hypsochromic shift or Blue shift.
- Hyperchromic shift :- when
absorption intensity of a compound is increased it is known as
hyperchromic shift.
- Hypochromic shift :- when
absorption intensity of compound is decreased it is called hypochromic shift.
COLLIMATING SYSTEM
MONOCHROMATOR
FILTER
SAMPLE CELL
DETECTOR
- RADIATION SOURCE :- it is
required ideal condition that are given below :-
- It should be stable and
not allow fluctuations.
- It should be continuing
emitting light.
- It should be not show fatigue
on continuing use.
- It should provide incident
light of sufficient intensity for the transmitted energy to be detected at
the end of optical path.
There
are following type of radiation source are used in UV-VISIBLE SPECTROSCOPY
1. Tungsten Halogen
lamp:-
It is construction is
similar to house hold lamp. The bulb contain a filament and it is filled
with inert gas like He, Ar etc.
Filament heated at
3000k temperature after 3000k temp. It is started to generate radiation.
It is used when
polychromatic light are required.
2.
Hydrogen Discharge lamp :-
In Hydrogen Discharge
lamp are pair of electrodes that are enclosed in glass tube.
This glass tube is
filled with H2 gas.
When current is passed
through these electrod at high voltage, discharge of electron
This electron are
responsible for the excited hydrogen molecules which in turn cause emissions of
U.V.
3.
Xenon Discharge Lamp :-
It possessed two
tungsten electrod separated by some distance.
These are enclosed in
a glass tube and xenon gas is filled under pressure.
An intense arc
is formed between electrod by applying high voltage.
This is a good
source of continuous plus additional intense radiation.
It intensity is
higher than the Hydrogen discharge lamp.
4.
Mercury arc lamp :-
In Mercury arc
lamp , Mercury vapour is stored under high pressure and excitation of Mercury
atom is done by electric discharge.
Collimating
system :-
The radiation
emitted by the radiation source is collimated by lense, mirror and slits.
Lenses:-
Materials used
for the lenses must be transparent to the radiation being used.
Ordinary
silicate glass transmit between 350 to 300 nm and is suitable for visible and
near IR region.
Quartz or fused
silica is used as a material for lenses to work below 300 nm.
Mirrors:-
These are used
to reflect focus or collimate light beam in spectrophotometer.
Slits
:-
Slits is an
important device in resolving polychromatic radiation into monochromatic
radiation. To achieve this entrance slit and exit slit are used.
Monochromator
:-
It is
device used to isolate the radiation of the desired wavelength from wavelength
of the continuous spectra.
MONOCHROMATOR
has following unit
1.
Entrance slit ( to get narrow source)
2.
Dispersing slit (disperses
the heterochromatic radiation into it components )
3. Exit
slit ( Allow
the nominal wavelength together with a bond of wavelength on either side of it)
1. Prisms :- The
prism disperse the light radiation into individual colour or wavelength.
The band pass is
lower than that of filter and hence it has better resolution.
2. Grating :-
Grating are the most efficient one in converting a polychromatic to
monochromatic light.
It is used to
achieve 0.1 nm resolution.
Grating
are of two types
1.
Diffraction grating :-
Grating are
nothing but ruling made on some material like glass , quartz, or alkyl halide
depending upon the instrument whether it is visible / IR/UV spectrophotometer
2.
Transmission grating :-
Transmission
grating is similar to diffraction grating, but refraction take place instead of
reflection.
Sample
cell :-
Sample cell are
used to hold a sample solution. The material of sample cell should not absorb
at the wavelength being observed. Sample cell are cylindrical or rectangular in
shape.
The pathlength
the cells are 10 mm or 1 cm.
Detectors
:-
Detector are the
device which convert light energy into electrical signal that are display on
the read out device.
There are mainly
three types of detector are used in UV-VISIBLE SPECTROSCOPY
1. Photo
multiplier tube :-
is quite sensitive cathode and about 10 anode which are maintained at a potential
of 75- 100 volta.
Working :- It is
work on principal of multiplication of the photo electrons by secondary
emissions of electron.
When a beam of
light fall on the photo cathode, photo electrons are generated which are
accelerate towards the anode. At each stage of their accelerate from one anode
to another anode the emissions of electron is increase by a factor 4 to 5 due
to secondary emissions of electron.
2. Photo
voltaic cell :-
It is consists
of a photo cathode which is a thin metallic layer coated with gold or silver.
It is also contain a metal base which act as anode. Between these two electrod
is a semi conductor layer of selenium.
Working :- while
light ray falls on the selenium layer electron are generated which are taken by
the photo cathode.
Because of the
poor electrical conductivity of the selenium the electron he accumulated on the
cathode leading to the development of the potential difference across the two
electrod which result to generate electric current.
3. Photo
tube :-
It consists of a
hollow glass tube with a photo cathode and a collector anode. The surface of
the photo cathode is coated with a layer of element like cesium, potassium,
silver oxide.
Working
:-
When light fall
on the photo cathode , electron are produced which flow towards the collect or
anode and generate current. The amount of current generate is directly
proportional to the intensity of the light radiation.
Their
are mainly two types of UV-VISIBLE INSTRUMENTS
1.
Single beam
2.
Double beam
Single
beam UV-VISIBLE :-
In a single beam
UV-VISIBLE spectro-photometer light from the radiation source is passed through
out the monochromator enters the sample cell containing the sample cell
solution.
A part of the
incident light is absorbed by the sample and remaining get transmitted .
The transmitted
light strikes the detector and produced electrical signal.
The signal
produced by the detector is directly proportional to the intensity of the light
beam striking it surface.
The absorbance
reading both the standard and unknown solution are recorded.
After adjust the
instrument to 100% transmission with a blank solution each time when even the
wavelength is charged.
If diagrams are present then it would be more helpful .
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