The document discusses the principle and instrumentation of UV-Visible spectrophotometry, which uses light in the ultraviolet and visible ranges to analyze how much light is absorbed by a sample, providing information about molecular structure and concentration. It explains that UV-Visible spectrophotometry follows Beer's Law and describes the typical components of a spectrophotometer including the light source, monochromator, sample and reference cells, detector, and recording device. A variety of applications are listed such as quantitative and qualitative analysis of compounds, studying reaction kinetics, and determining molecular weights.

1. Agriculture University, Kota
College of Agriculture, Kota
(Rajasthan) -324001
Submitted to:-
Mrs. Yamini Tak
Assistant Professor
Department of Biochemistry
Submitted by:-
Anil Kumar Yadav
Ph.D. Scholar
Agronomy

2. • Principle-The Principle of UV-Visible Spectrophotometry is
based on the absorption of ultraviolet light or visible light by
chemical compounds, which produces distinct
spectra. Spectrophotometry is based on the interaction between
light and matter. When thematter absorbs the light, it undergoes
excitation and de-excitation, resulting in the production of a
spectrum.
• When matter absorbs ultraviolet radiation, the electrons present in
it undergo excitation. This causes them to jump from a ground state
(an energy state with a relatively small amount of energy associated
with it)
Principle and Applications of UV –Visible
Spectrophotometry

3. To an excited state (an energy state with a relatively large amount of
energy associated with it) or HOMO (highest occupied molecular
orbital) to a higher energy excited state LUMO (lowest unoccupied
molecular orbital).It is important to note that the difference in the
energies of the ground state and the excited state of the electron is
always equal to the amount of ultraviolet radiation or visible radiation
absorbed by it.
UV Visible spectrophotometry follows the Beer-Lambert Law. This law
states that absorbance is proportional to concentration and path length.
A ∝ CL, or A=ECL
IN which A= Absorption, E= Molar absorption coefficient ,C=
Concentration, L=Path length

4. Schematic Diagram of UV–Visible
Spectrophotometry

5. Instrumentation of UV–Visible
Spectrophotometry
UV-Visible Spectrophotometer following the six main components
1. Light Source
2. Monochrometer
3. Sample and Reference cell
4. Detector
5. Amplifier
6. Recording device

6. Light Source- supplies light in the UV range (200-400nm)- hydrogen
and deuterium lamp and visible range (400-800nm)-tungsten lamp.
Monochromator-Allows chosen wavelength and absorbs all the other
wavelengths and this happens for the complete chosen range of
wavelengths eg. prism, diffraction grating, etc.
Reference and Sample Cell – Specific light pass from the exit slit the
beam splitter splits the beam of light of each wavelength into halves of
equal intensities one goes to the test sample and the other to the blank
or reference sample, in the reference cell same intensity light pass no
absorption take place.
Detector- Difference of transmitted light ratio from reference and
sample cell are detected by the detector.

7.  Amplifier or Recording Device- Detector detect the reading in the
form of transmittance, the transmittance reading is converted into
absorbance, and through absorbance finds the concentration of the
sample.
• Transmittance of a sample increases the concentration of the sample
and decreases
• Absorbance of a sample increases the concentration of the sample also
increases
• Wavelengths which show sample solution maximum absorbance are
known as Lambda max (λmax)

8. • Quantitative analysis: Concentration determination.
• Qualitative analysis: Identification of compounds.
• Observing Structural Changes with UV-Vis Spectrophotometry-
Conformational Studies of Proteins.
• Analyzing reaction of kinetics.
• Detection of impurities in organic molecules.
• Structutre elucidation of organic compounds.
• Detection of functional groups
Application of UltraVoilet –Visible
Spectrophotometry

9.  Fixed Wavelength:
(A) Fixed wavelength application food and beverage
industries
(B) Fixed wavelength application in chemical industries
 Examination of polynuclear hydrocarbon.
 Molecular weight determination
 Determine the purity or concentration of biological
samples containing DNA or RNA.