The document discusses UV-visible spectroscopy, a technique that uses electromagnetic radiation to analyze the atomic and molecular structure of matter by measuring light absorption at specific wavelengths. It explains the components of a UV-visible spectrophotometer, the principles of Beer’s and Lambert’s laws, and highlights the applications, advantages, and limitations of this method across various scientific fields. UV-visible spectroscopy is notable for its ability to analyze small sample sizes and its widespread use in industries such as pharmaceuticals, forensics, and biochemistry.

2. Introduction
Electromagnetic Radiations
Electromagnetic Spectrum
UV-Visible Spectroscopy
Principle
UV-Visible Spectrophotometer
Radiation source
Monochromator
Sample cell
Detector
Recorder
Sample preparation
Applications
Advantages
Limitations

3.  Spectroscopy is the interaction of electromagnetic radiation with matter.
 After interaction, may variation in intensity of electromagnetic radiation with
frequency.
 The device which is used to measure variation of EMR is called as
spectrophotometer or sometime called as spectrometer.
 Spectroscopy is an scientific tool which is used for the study of atomic as well
as molecular structure of matter.

4. Explain
EMR The radiation constitutes electric and magnetic field is known as
electromagnetic radiation (EMR).
 EMR may be produced by changing electric and magnetic field
simultaneously which act at right angle to each other and also perpendicular
to the direction of propagation of electromagnetic waves.
Properties of Electromagnetic Radiations
 Electromagnetic waves are transverse in nature.
 These waves can propagate in vacuum also.
 They may produced optical effects.
 They can be polarized.

5.  The electromagnetic spectrum is bunch of energies which are arranged in
order of increasing wavelength and decreasing frequency.
 With increasing wavelength, gamma rays situated in first position with low
wavelength and highest frequency and it has more energy as compare to other
electromagnetic waves.
 Cosmic rays having shorter wavelength and highly energetic.
 Radio waves are longer wavelength, hence less energetic.

6. Diagram of electromagnetic
radiation

7. UV visible
spectroscopy.
UV-visible spectroscopy uses electromagnetic radiations of UV and Visible
region.
UV rays ranging from 180 to 380 nm and visible region ranging from 380 to
720 nm of electromagnetic spectrum.
UV-Visible spectroscopy refers to absorption spectroscopy.
Molecules containing bonding and non-bonding electrons can be absorbed in
the form of UV or visible light and gets excited into higher molecular orbital.
It is also knows as electron spectroscopy.

8. Princip
al Many molecules absorb UV visible light.
 The problems of solution increases as attenuation of the beam increases.
 Absorbance directly proportional to the path length (l) and concentration (c) of
absorbing species.
 The intensity of absorption is governed by two fundamental laws of absorption
known as Beer and Lambert law.
 Beer's and Lambert‘s law: when a beam of light passes through a transparent sample
cell containing a solution of absorbing substance the intensity of light reduces.

9.  This is due to :-
 reflection of the inner and outer surface of cell.
 scattering by particle of solution.
 absorption of light by molecule in the solution.
(Note: Since it is not possible to directly measure the amount of radiation absorbed by
partially transparent substance it may be measured by calculating the difference in intensity
between original and transmitted radiation falling on the sample).
Iabsorbed = Io – It
(Where asIo is the original intensity falling on the cell and It total intensity)

10.  Beers law :- When rays of monochromatic light passes through and absorbing
medium, its intensity decreases export exponentially as the concentration of
absorbing material increases.
 Lambert’s law :- When the ray of monochromatic light passes through an
absorbing medium its intensity decreases exponentially as a length of
absorbing material increases.

11. UV visible spectrophotometer is a devices which is used to measure
absorbance of samples.
Spectrophotometer available in market with two type; one is single beam in
which only one sample can be investigate and another is double beam
spectrophotometer in which two samples can be investigate (questioned &
reference sample).
It consist of following parts;
1) Radiation source.
2) Monochromator .
3) Sample cell.
4) Detector.
5) Data output and recording device.

13.  The radiation source is Deuterium lamp used for UV region having
wavelength range of approximately 190-450nm.
 For the visible region Tungsten filament bulb is used having wavelength
range from about 332 – 850 nm.
 Xenon arc lamp also is used in entire UV visible region.
 The condensing mirror is rotated manually to focus the light emitted from
other source into the entrance slit of the monochromator

14.  It is a device for isolating monochromatic or narrow band of radiant energy
from the source.
 It will allow to pass radiant energy of particular wavelength.
 Quartz prism was used in earlier photometer but now they are
completely replaced by diffraction grating.
 It mainly consists two parts;
1) Slit
2) Dispersion

15.  Pyrex glass in visible region is satisfactory material for sample cell but
below 350 nm (UV region) wavelength quartz i.e. glass of pure silicon
dioxide must be used.
Fig. Various types of Sample Cells

16. Detecto
rThe detector measures the quantity of radiation that passes through the
sample converting it into the electric signal.
The most common detector used are vacuum photo cells and this is based on
photoelectriceffect.
Receiving signal is obtained when a photon of sufficient energy strikes the
metals of face cause the election of an electron.
This is determined by quantitative measurement of amount of light passing
through instrument by meansof detector.

17. Another type of detector is photomultiplier tube (PMT) which is frequently
use for the detection of absorbance in spectroscopy.
Fig. Photomultiplier tube

18. Sample signal output can be analog absorbance or transmittance meter where
the data can be read, recorded and processed.
 Some spectrophotometer having attached computer for monitoring the
instrument and record the absorbance.

19. UV-Visible spectroscopy allows transparent solid, liquid and gaseous
sample.
Very small amount such as microgram or microliter sample is sufficient for
detection.
There are some point to be consider while preparing the sample;
 High purity of transparent solvent should be used.
 Aqueous system often requires for the use of buffer system.
 Sample clean up may be required before investigation.
 Turbid sample should be filtered to reduced light scattering.

20.  UV-Visible spectroscopy used to detect conjugations.
 It also used to detects isomers.
 Detection of functional group can be possible in this technique.
 It is also use for identification and quantification of organic as well as inorganic
compound.
 It is also use for research and development of science and technology.
 It also play key role in ink and fibers in Forensic science.
 UV-Visible spectroscopy is widely used as a significant tool for both qualitative and
quantitative drug analysis .

21. UV-Visible spectroscopy is an quick and inexpensive technique available for
identification of certain class of material.
It can detect minute amount of sample such as microliter or microgram.
Quantity of sample can be done precisely and reproducibly.

22. Extensive sample preparation is required.
 The sample should be purify before analysis.
Broad band obtained in this technique. Sometime definite identification is
not possible for this band broadening.

23. UV-Visible spectroscopy is one of the most important tool used for the
purpose of identification and quantification. It has wide applications in
various field such as Chemistry, Forensic Science, Biochemistry,
Pharmaceutical industry, Cosmetics Industry, Petroleum Industry, Food
Industry and many more. It has wide used because most of the molecules
present in the universe shows absorption in UV-Visible region of
electromagnetic spectrum. It acts as a screening technique in most of the cases
to identify molecule before performing other highly sophisticated technique
of that sample molecule. Other advantages with this technique is that it is
simple to perform and inexpensive. But sometime sample preparation is
difficult and it will depends upon the nature of sample.