This document provides an overview of UV-Visible spectroscopy. It discusses the basic principles including electromagnetic radiation, spectroscopy, absorption of UV-Visible light, and Beer-Lambert's law. It describes the instrumentation of UV-Visible spectroscopy including light sources, wavelength selectors, sample compartments, detectors and basic components. It also discusses electronic transitions, shifts in absorption, and applications of UV-Visible spectroscopy in qualitative and quantitative analysis.

1. TOPIC :- UV VISIBLE
SPECTROSCOPY
Presented by :
Ashish J Hingnekar
(M.Pharm 1st year
QA)
Guided by :
Mr. Rushikesh
Relekar
Government College of Pharmacy,Amravati.

2. Contents ...
• Spectroscopy
• Principle of Spectroscopy
• Beer – Lamberts’s Law
• Electronic Transitions
• Absorption & Intensity Shifts
• Instrumention of UV Visible Spectroscopy
• Different types of Spectroscopy
• Applications
• References

3. SPECTROSCOPY
It is a branch of science that deals with the study of interaction
of matter with light.
OR
It is a branch of science that deals with the study of interaction
of electromagnetic radiation with matter.

5. • Electromagnetic radiation consists of discrete
packages of energy which are called as photons.
• A photon consists of oscillating electric field (E)
& an oscillating magnetic field (M) which are
perpendicular to each other.
Electromagnetic Radiation

6. Frequency (v):
It is defined as the number of times electrical field radiation oscillates in one second. The unit for
frequency is Hertz (Hz).1 Hz = 1 cycle per second
Wavelength (λ):
It is the distance between two nearest parts of the wave in the same phase i.e. distance between two
nearest crests or troughs.
The relationship between wavelength & frequency can be written as:
C = v λ
As photon is subjected to energy, So
E = h v = h c / λ
Electromagnetic radiation

8. PRINCIPLE OF SPECTROSCOPY
• The principle is based on the measurement
of spectrum of a sample containing atoms /
molecules.
• Spectrum is a graph of intensity of absorbed
or emitted radiation by sample verses
frequency (v) or wavelength (a).
• Spectrometer is an instrument design to
measure the spectrum of a compound.

9. 1. Absorption Spectroscopy:
An analytical technique which concerns with
the measurement of absorption of electromagnetic
radiation.
e.g. UV (185 - 400 nm) I Visible (400 to 800 nm)
Spectroscopy, IR Spectroscopy (0.76 to 15 um )
2. Emission Spectroscopy:
An analytical technique in which emission (of a
particle or radiation) is dispersed according to
some property of the emission & the amount of
dispersion is measured.
e.g. Mass Spectroscopy

10. Principle of UV- Visible Spectroscopy
• The UV radiation region extends from 10 nm
to 400 nm and the visible radiation region
extends from 400 nm to 800 nm.
Near UV Region: 200 nm to 400 nm
Far UV Region: below 200 nm
• Far UV spectroscopy is studied under vacuum
condition.
• The common solvent used for preparing
sample to be analyzed is either ethyl alcohol
or hexane.

12. Beers Lambert’s 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 as well as concentration of the
solution.
A = ε b c
A = Absorbance
ε = molar conductivity with units of L/mol.cm
b = path length of the sample (cuvette)
c = concentration of the compound in solution in mol /L

15. The possible electronic transitions are
σ – σ* transition
∏ – ∏* transition
n – σ* transition
n – ∏* transition
σ – ∏* transition
∏ – σ* transition

16. Terms used in UV- Visible Spectroscopy
Chromophore
The part of a molecule responsible for imparting color, are called
as chromopheres. OR
The functional groups containing multiple bonds capable of
absorbing radiations above 200 nm due to
n -∏ * & m - ∏* transitions.
e.g. NO2 , N=0, C=0, 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 .OR
The functional group with non-bonding electrons
that does not absorb radiation in near UV region but when attached to
a chromophore alters thewavelength & intensity of absorption.
E.g. Benzene λmax (255nm),Phenol (270nm ), Aniline (280nm ).

19.  Bathochromic shift (Red shift)
When absorption maxima (λmax) of a compound shifts to
longer wavelength.
 Hypsochromic shift (Blue shift)
When an absorption maxima (λmax) of a compound shifts to a
shorter wavelength.
 Hyperchromic shift
When absoprtion intensity (ε ) of a compound is increased
 Hypochromic shift
When absorption intensity (ε ) of a compound is decreased .

22. Componentsof UV Spectrophotometer
Recorder
Detector
Sample Compartment
Monochromator
Filter
Source

24. Basic Optical Instrument Components
1) Source : A stable source of radiant energy at the desired
wavelength or range).
2) Wavelength Selector : A device that isolates a restricted
region of the EM spectrum used for measurement(monochromators,
prisms & filters).
3) Sample Container : A transparent container used to hold the
sample (cells, cuvettes, etc).
4) Detector/Photoelectric Transducer : Converts the
radiant energy into a useable signal (usually electrical).
5) Signal Processor & Readout : Amplifies or attenuates the
transduced signal and sends it to a readout device as a
meter, digital readout, chart recorder, computer, etc.

25. LIGHT SOURCES
Various UV radiation sources are as follows
a. Deuterium lamp
b. Hydrogen lamp
c. Tungsten lamp
d. Xenon discharge lamp
e. Mercury arc lamp
Various Visible radiation sources are as follows
a. Tungsten lamp
b. Mercury vapour lamp
c. Carbonone lamp

26. Wavelength Selectors
Wavelength selectors limited, narrow, continuous group of
wavelengths called a band.
Two types of wavelength selectors:
A) Filters
B) Monochromators
A)Filters -
Two types of filters:
a) Interference Filters
b) Absorption Filters

27. B) Monochromators
 Wavelength selector that can continuously scan a
broad range of wavelengths.
 Used in most scanning spectrometers including UV, visible, and IR
instruments.
• Refractive
• Reflective
Prism
type
• Diffraction
• Transmission
Grating
type

28. Sample Compartment
 Spectroscopy requires all materials in the beam path other than
the analyte should be as transparent to the radiation as possible.
 The geometries of all components in the system should be such as
to maximize the signal and minimize the scattered light.
 The material from which a sample curette is fabricated controls
the optical window that can be used.
 Some typical materials are:
Optical Glass - 335 - 2500 nm
Special Optical Glass — 320 - 2500 nm
Quartz (Infrared) — 220 - 3800 nm
Quartz (Far-UV) — 170 - 2700 nm

29. Detector
 After the light has passed through the sample, we want to be able
to detect and measure the resulting tight.
 These types of detectors come in the form of transducers that are
able to take energy from light and convert it into an electrical
signal that can be recorded, and if necessary, amplified.
Requirements of an ideal detector:-
a. It should give quantitative response.
b. It should have high sensitivity and low noise level.
c. It should have a short response time.
d. It should provide signal or response quantitative to wide
spectrum of radiation received.
 Three common types of detectors are used
a) Barrier layer cells
b) Photo emissive cell detector
c) Photomultiplier

30. 1. Barrier layer cell/Photovoltaic cell:
The detector has a thin film metallic layer coated with silver or gold
and acts as an electrode.
 It also has a metal base plate which acts as another electrode.
 These two layers are separated by a semiconductor layer selenium.

31. 2. Photoemissive cell / Phototube
 Consists of a evacuated glass tube with a photocathode and a
collector anode.
 The surface of photocathode is coated with a layer of
elements like cesium, silver oxide or mixture of them.
 When radiant energy fails on photosensitive cathode,
electrons are emitted which are attracted to anode causing
current to flow.
 More sensitive compared to barrier layer cell and therefore
widely used.

32. 3.Photomultiplier tube
 The principle employed in this detector is that, multiplication
of photoelectrons by secondary emission of electrons.
 In a vacuum tube, a primary photo-cathode is fixed which
receives radiation from the sample.
 Some eight to ten dynodes are fixed each with increasing
potential of 75-I0OV higher than preceding one.
Near the last dynode is fixed an anode or electron collector
electrode.
Photo-multiplier is extremely sensitive to light and is best
suited where weaker or low radiation is received.

33. Single Beam UV Spectrophotometer

34. Double Beam UV Spectrophotometer

35. Different UV - visible spectrophotometer
methods
For multicomponent analysis
(a) Simultaneous equation method
(b) Absorbance ratio method
(c) Geometric correction method
(d) Orthogonal polynomial method
(e) Derivative spectrophotometry
(f) Difference spectrophotornetry

36. Applications
Qualitative & Quantitative Analysis:
 It is used for characterizing aromatic compounds and
conjugated olefins.
 It can be used to find out molar concentration of the solute
under study.
Detection of impurities:
 It is one of the important method to detect impurities in organic
solvents.
Detection of isomers are possible.
Determination of molecular weight using Beer's law.

37. Reference Books
1) Instrumental Method of Analysis : G.R. Chatwal, S K.
Anand.
2) Introduction to Spectroscopy : Donald A. Pavia
3) Elementary Organic Spectroscopy : Y.R. Sharma
4) Physical Chemistry : Puri, Sharma & Pathaniya