Ultraviolet–visible spectroscopy, Its working principle and Applications.

1. ULTRAVIOLET–VISIBLE SPECTROSCOPY
Presented by:
Mr. Sanjoy Chungkrang
Lecturer, Department of Pharmaceutical science
Northeast Frontier Technical University, Alo, Arunachal Pradesh

2. UV VISIBLESPECTROSCOPY

3. UVVISIBLESPECTROSCOPY
 DEFINATION :- UV- Visible spectroscopy is a
quantative analysis technique which is used to
measure the attenuation of a beam of light
(UV) after it passed through a sample or
reflectant from a sample surface.

4. Light source
Sample
Incident light
Transmitted
Light
(100 nm)
(60nm)
Absorption = Incident Light – Transmitted Light

5. PRINCIPLE
 The UV- Visible spectroscopy is work based on Absorption
spectroscopy.
 The Principle is explained by two laws –
 BEER’S LAW
 LAMBERT’S LAW

6. BEER’S LAW
 The decrease in the intensity of light is directly
proportional to the concentration.
OR
 The absorption of uv radiation is directly
proportional to the concentration

7. Light source
Sample
Incident light
Transmitted
Light
A α C (i)

8. LAMBERT’S LAW
 According to Lambert’s law the decrease in the
intensity of light is directly proportional to path
length travelled by the light.
OR
 The absorption is directly proportional to the
path length.

9. Light source
Sample
Incident light
Transmitted
Light
A α b (ii)
(Path length)

10. BEER’S LAMBERT’S LAW
 From Equation (i) and (ii) , we find that-
A α c (i)
A α b (ii)
Therefore,
A α C b
A= € C b
Here, A = Absorption of a given wave length.
€ = Molar absorbity Constant.
C = Concentration of the solution.
b = Path length, Distance light passed.

11. INSTRUMENTATION & IT’S WORKING
Monochromator

12. INSTRUMENTATION AND WORKING OF THE UV SPECTROMETERS CAN BE STUDIED
SIMULTANEOUSLY. MOST OF THE MODERN UV SPECTROMETERS CONSIST OF THE
FOLLOWING PARTS-
LIGHT SOURCE: Tungsten filament lamps and Hydrogen-Deuterium lamps are most
widely used and suitable light source as they cover the whole UV region. Tungsten
filament lamps are rich in red radiations; more specifically they emit the radiations of
375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm.
Deuterium lamp

13. MONOCHROMATOR- Monochromators generally composed of prisms and
slits. The most of the spectrophotometers are double beam spectrophotometers.
The radiation emitted from the primary source is dispersed with the help of
rotating prisms. The various wavelengths of the light source which are
separated by the prism are then selected by the slits such the rotation of the
prism results in a series of continuously increasing wavelength to pass through
the slits for recording purpose. The beam selected by the slit is monochromatic
and further divided into two beams with the help of another prism
PRISM

14. SAMPLE & REFERENCE CELL- One of the two divided beams is passed
through the sample solution and second beam is passé through the reference
solution. Both sample and reference solution are contained in the cells. These
cells are made of either silica or quartz. Glass can't be used for the cells as it
also absorbs light in the UV region.
I II III
Fig :- Cuvette / Sample

15. DETECTOR
 Generally two photocells serve the purpose of detector in UV
spectroscopy. One of the photocells receives the beam from
sample cell and second detector receives the beam from the
reference. The intensity of the radiation from the reference cell is
stronger than the beam of sample cell. This results in the
generation of pulsating or alternating currents in the photocells.

16. RECORDING DEVICES
Most of the time amplifier is coupled to a pen recorder which is
connected to the computer. Computer stores all the data
generated and produces the spectrum of the desired compound.

17. APPLICATIONOF UV- VISIBLE SEPCTROSCOPY-
 It is used in a quantative analysis.
 It is used to detect extended conjugation
Conjugated molecule are stable due to resonance
and have low energy, so it gives higher value of λ max
(absorption maximum) then Non- conjugated.
Example:- Penta-1,3-diene (conjugated)
CH2 = CH – CH = CH - CH3
Penta-1,4-diene (Non- conjugated)
CH2 = CH – CH – CH = CH2

18.  It is used to detect both Aromatic and Non-
Aromatic Compound.
 Aromatic compound are resonance stabilizer, so they
give higher value of λ max then Non- aromatic
compound.
Example :- Benzene
Cyclohexane
(High λ max)
(low λ max)

19.  It is useful to detect Geometrical Isomers:-
(a) Cis – Isomers
(b) Trans - Isomers
Cis isomers are less stable due to high energy and
repulsion than trans – isomers, so trans – isomers have higher
value of λ max than Cis – isomers.

20. Example :-
(High λ max) (Low λ max)

21.  And also the UV- Visible spectroscopy is used in
various activity such as –
Detection of Impurities ( Organic Solvent).
Drug Identification.
Bacterial Culturing.
Determination of Molecular weight by using
Beer’s Law.