Ultraviolet-visible (UV-Vis) spectrophotometry is a technique used to measure light absorbance across the ultraviolet and visible ranges of the electromagnetic spectrum. When incident light strikes matter it can either be absorbed, reflected, or transmitted. The absorbance of radiation in the UV-Vis range causes atomic excitation, which refers to the transition of molecules from a low-energy ground state to an excited state.
3. Introduction
A spectrophotometer is a photometer (a device
for measuring light intensity) that can measure
intensity as a function of the color (or more
specifically the wavelength).
UV-visible spectroscopy or UV-visible
spectrophotometer involves the spectroscopy of
photons in the UV – visible region.
This means it use light in the visible and
adjacent (near ultraviolet and near infrared)
range.
5. Absorption laws
When a beam of monochromatic light passed
through a solution it may transmitted as a such or
some of may be absorb.
Proportional the transmitted light can be represented
by to the intensity of the incident radiation.
T=I/Io
Absorbance (A) of light through a solution in
inversely proportional to log 10 of %T
A=Log (1/T)
=Log I/Io
Where;
I= INTENSITY OF Transmitted light
Io= Intensity of incident light
6. The quantitative determination of compounds by
spectrometric technique is
based on two law.
1 lamberts law
2 beers law
1 lamberts law- its state that light absorbed by solution
is directly proportional to length of the light through
the solution.
hence
A=log(Io/I)= Є.C
where A= absorbance
Є=molar absorptivity coefficient
l= path length of the sample (usually 1cm)
7. 2- Beers law-its state that the amount of light absorbed is directly
proportional to concentration of absorbing solute in the solution.
Thus
A=log(Io/I)= Є.C
where C=concentration of solution moles liter-1
combining equation
A=log(Io/I)= Є.C.l
it a standard cuvette with light path of 1.cm is used.
A=log(Io/I)= Є.C
Limitation of Beers Lamberts Law:
1. when different forms of the absorbing molecules are in equilibrium
as in keto –enol tautomers.
2. when fluorescent compounds are present.
3. when solute and solvent forms complex though some sort of
association.
10. 1-Source-: A continuous source of radiant energy covering the
region of spectrum in which the instrument is designed to work.
UV- Hydrogen Deuterium lamp
Visible- Tungsten lamp
2.Filter and monochromators-: A light filter is a device
that allow light of the required wavelength to pass but
absorbs light of the other wavelength wholly and
partially.
Filter-:filter are of two types
1 Absorption filter
2 Interference filter
12. Absorption characteristics of common filters
Colour of filter Approximate range of
absorption band in mu
Yellow 450
Orange 500
Red 575
Purple 450 – 650
Blue 480
Green 400 – 475 , 575 - 700
13. Monochromators-:The monochromrator is to dipersing the
radiation according to the wavelength.
a. dispersing element (a prism or grating)
b. Slits-: there are two types of slit
Entrance slit
Exit slit
3. Cell / sample holder -:
The cell holding the sample (usually a
solution should be transparent to the wavelength region
being recorded.
18. Power supply-:
The power supply serves as triple function-
a. It decreases the line voltage to the instrument
operating level with a transformer.
b. It convert A.C to D.C with a rectifier if direct is
required by the instrument .
c. It smooths out any ripple which may occur in the line
voltages in order to deliver a constant voltage to the
source lamp and instrument.
21. Application
1. Detection of functional group
2. Extent of conjugation
3. Identification of unkown compound
4. Elucidation of the structure of vitamins-
5. Qualitative analysis
6. Quantitative analysis
7. Detection of impurities
22. Reference
Instrumentation By B. K. Sharma
Biophysical Chemistry by Upadhyay & Upadhyay
Nath.
instrumental methods of chemical analysis by
chatwal
