Chapter 4.

ULTRAVOILET & VISIBLE SPECTROSCOPY

UV visible spectroscopy deals with the absorption of electromagnetic radiations in the visible
and UV region. It is also known as “Electronic Spectroscopy”. It involves the gain of energy by a
molecule and transfer of electrons from lower energy level to higher energy level.
RANGE:
UV-------------200-400 nm
Visible----------400-800 nm
PRINCIPLE OF UV-VIS SPECTROSCOPY:
Ultraviolet visible radiations being of higher energy than infrared radiation cause transition of
valence electrons from ground state to excited sate on their absorption by a sample.
THEORY:
A molecule absorb ultraviolet radiation of frequency (𝜗), the electron in that molecule
undergo transition from lower to higher energy level. The energy can be calculated by the equation,

E=h𝜗
A molecule process three types of energy
Et= electronic transitional energy (energy due to electronic transitions)
Er= rotational energy (energy due to rotation of a molecule about its center of gravity)
Ev = vibrational energy (energy due to vibration of atoms within a molecule with respect to each other)
Etotal= Etransitional+ Evibrotional+ Erotational
The energies decrease in the following order:
Et>Ev>Er
Vacuum UV Region:
 The UV region below 200nm cannot be studied by conventional UV spectrometer. Because O2
of air absorbs strongly in this region. However if, oxygen is expelled out of instrument with nitrogen,
the range of instrument can be extended down to about 150nm. The region below 200nm is therefore
usually known as “vacuum UV region”.
ELECTRONIC TRANSITIONS
The absorption of UV visible radiation by a sample results in transfer of electrons from one
energy state to another is known as electronic transitions.
Types of Transitions:
In U.V spectroscopy molecule undergo electronic transition involving σ, π and n electrons.
Four types of electronic transition are possible

• σ ⇾ σ* transition
• n ⇾ σ* transition
• n ⇾ π* transition
• π ⇾ π* transition
1. σ → σ* Transitions:

σ electron from orbital is excited to corresponding anti-bonding orbital σ*.The energy

required is large for this transition. E.g. Methane (CH4) has C-H bond only and can undergo σ → σ*
transition and spectrum range is below 150 nm.

2. π → π* Transition:

π electron in a bonding orbital is excited to corresponding anti-bonding orbital π*.Compounds

containing multiple bonds like alkenes, alkynes, carbonyl, nitriles, aromatic compounds, etc. undergo
π → π* transitions. E.g. Alkenes (CH2=CH2) and generally absorb in the region 160 to 250 nm.

3. n → σ* Transitions:

Saturated compounds containing atoms with lone pair of electrons like O, N, S and halogens
are capable of n → σ* transition. These transitions usually requires less energy than σ → σ* transitions.
The number of organic functional groups with n → σ* peaks in UV region is small (150 – 250 nm)

4. n → π* Transition:

An electron from non-bonding orbital is promoted to anti-bonding π* orbital. Compounds

containing double bond involving hetero atoms (C=O, C≡N, N=O) undergo such transitions.
n → π* transitions require minimum energy and spectral range is 275-295 nm.
Energy increase in order; σ< π < n

ABSORPTION & INTENSITY SHIFTS

Bathochromic Shift (Red Shift):
When absorption maxima (λmax) of a compound shift to longer wavelength, it is known as
bathochromic shift or red shift. The effect is due to presence of an auxochrome or by the change of
solvent.
[Link] auxochrome group like –OH, -OCH3 causes absorption of compound at longer wavelength.
Hypsochromic Shift (Blue Shift):
When absorption maxima (λmax) of a compound shift to shorter wavelength, it is known as
hypsochromic shift or blue shift. The effect is due to presence of a group causes removal of
conjugation or by the change of solvent.
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.
Hypochromic Effect:
When absorption intensity (ε) of a compound is decreased, it is known as hypochromic shift.

Chromophore:
The part of a molecule responsible for imparting color, are called as chromophore.
OR
The functional groups containing multiple bonds capable of absorbing radiations above 200
nm.
e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc.
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.
e.g. Benzene λmax = 255 nm
Phenol λmax = 270 nm
Aniline λmax = 280 nm

LAWS OF LIGHT ABSORPTION

Bouger or Lambert’s Law:
The law states that proportion of light absorbed by a transparent medium is independent of
intensity of the incident light and that each successive layer of medium absorb fraction of incident
light depending upon the thickness.
A= αb
b = thickness of layer
α = absorbance coefficient
Beer’s Law:
This law states that amount of light absorbed is proportional to the number of absorbing molecules
through which light passes.
A = αc
C = concentration
Combination of Two Laws:
Combining both laws, the absorbance is directly proportion to thickness both medium and
concentration of molecules.
A = αbc
This is known as Beer’s Lambert Law and is a fundamental relationship used in spectroscopy.

INSTRUMENTATION OF ULTRA-VIOLET VISIBLE

SPECTROSCOPY:
Spectrophotometers can be divided in to

• Single beam spectrophotometer

• Double beam spectrophotometer

Single beam spectrophotometer:

Practically, all U.V spectrophotometers possess five essential components

• Radiation source
 • Monochromator
• Cell
• Detector
• Recorder
1. Source:

In U.V spectrophotometer, the source of radiant energy used is the hydrogen discharge lamp .It is
composed of a tube with two electrodes and hydrogen gas at low pressure. Bombardment of H2 and
return of excited molecules to ground state results in emission of continuous spectrum. For shorter
wavelength, the temperature of a light source should be higher. For visible spectrophotometer, source
is usually a tungsten filament lamp

2. Monochromator:

The devices employed to isolate desired wavelength are called as monochromators.

Types of Monochromators:
Depending upon the type of dispersion element, there are three types of Monochromators.

• Prism type
• Grating type
• Filters

a) Prism type:

A prism is used to get monochromatic light .For visible region glass prism is used and for u.v region
quartz prism is used.

b) Grating type:

Grating type can also be used in which a large number of equi-spaced lines ruled on a glass coated
with thin film of aluminum.

c) Filters:

Filters may be used to filter out the light of undesirable wavelength. Filters may be of following types

• Glass filter:

These are the pieces of colored glasses which transmit limited wavelength
 • Gelatin filters:

It is consist of thin gelatin sheets between a pair of glass plates.

3. Cells:

A solution of the compound is contained in the cell, which range from a simple test tube to expensive
cuvettes. In most cases cells are rectangular with a path length of [Link] visible region glass cells are
used and for UV region quartz cells are used.

4. Detectors:

Several types of detectors have been used in spectrophotometers. But phototubes and
photomultiplier tubes are used mostly. Photomultiplier tubes are used more often than phototubes
because it can detect lower intensity radiations.

5. Recorders:

The output from detector is amplified and is observed on a recorder.

Working:
For recording the UV spectrum, the given sample is dissolved in a suitable solvent which itself does
not absorb light. The commonly used solvents are 95 percent ethanol, methanol, hexane etc. A quartz
cell of path length 1cm is used as a container for the sample solution. The solution is exposed to UV-
visible light by the prism and detected through the detector and amplified at recorder.
Double beam spectrophotometer:
In research purpose we use double beam spectrophotometer. In this case monochromatic radiations
are split into two beams of equal intensity. One beam is passed through the reference cell while other
is passed through the sample cell. After passing through the detector beam of both cells are matched.
SPECTROPHOTOMETRIC DETERMINATION:
Spectrophotometric determination is used for the analysis of drugs. Suppose we want to determine
the amount of substance in its solution by spectrometric determination; then the procedure will be as
follow:
1- Prepare a series of solution of substance of known concentrations and place the first one
concentration in a quartz cell and place it on sample holder in spectrophotometer.
2. Now, go to wavelength button and select a wavelength value range on which you want to take
readings. Press the scan button and get the scan of your sample and determine the lambda max from
it.
3. If u want to determine the unknown concentration then put the solution of different
concentrations one by one in a cell and select lambda max value for each concentration as concluded
from step 2 and measure the absorbance of each of the solution
4. Now plot a graph between different values of absorbance and corresponding concentration. This
graph is known as standard graph
5. Measure the absorbance of given solution and find out the concentration of unknown sample by
using the graph.
APPLICATIONS OF UV-VISIBLE SPECTRUM:

U.v-visible spectroscopy is used for both qualitative and quantitative analyses which are as follows:

1) DETERMINATION OF STRUCTURE OF ORGANIC COMPOUNDS:

UV visible spectroscopy is used for determination of conjugation in many organic compounds.

For example:
Ethane, buta1, 3 diene etc.
2) DETERMINATION OF UNKNOWN CONCENTRATION OF A SUBSTANCE:
With the help of this technique we can determine the unknown concentration of active ingredients
in any sample.
3) DETERMINATION OF RATE OF REACTION:
Rate of reactions for various chemical reactions can also be determined with the help of u.v-visible
spectroscopy as the absorbance decreases when the reactants are absorbing species and increases
when the products are absorbing species.
4) DETERMINATION OF PURITY:
Purity of compound is determined by its absorbance characteristics by using the formula:
Percentage purity= concentration found/total concentration taken × 100
5) DETERMINATION OF IONIZING CONSTANT:
Ionization constant of an acids or bases can be determined by measuring the absorbance at with the
help of vu-visible spectroscopy.
6) DETECTION OF FUNCTIONAL GROUP:
Various functional groups can be determined in a compound with the help of u.v-visible spectroscopy.
7) ANALYSIS OF BODY FLUID:
Different body fluids can be analyzed by u.v-visible spectroscopy .for example blood glucose content
can be determined in diabetic patients.
8) ANALYSIS OF MIXTURE OF COMPOUNDS:
Mixture of two or more compounds or body fluids can be analyzed by using u.v-visible spectroscopy
by using absorbance values having similar or dissimilar spectra.