UV-VISIBLE SPECTROPHOTOMETER
INTRODUCTION :
The instrument that is used to study the absorbtion of electromagnetic radiation as a function of wavelength or
time is called spectrophotometer. Spectrophotometry is mainly concerned with the following regions of spectrum. U V radiation lies
in the region 190 – 350nm.Visible radiation lies between 360 – 860 nm.
PRINCIPLE:
When a monochromatic light is passes through a homogeneous medium a part of which is absorbed by the
medium and the remainder is allowed to transmit asmuch. If Io denotes the intensity of incident light,and Ir incident light,Ir in
the intensity of reflected light,Ia the intensity of absorbed light,It is the intensity of transmited light then,
I𝜊 = Ia+It+Ir
Two separate laws governing absorption generally known as Beer- Lambert’s law.
LAMBERT’S LAW:
When a beam of monochromatic light passes through a homogeneous medium, the amount of light absorbed
by the the analyte at a specific wavelength is directly proportional to the path length of light that travelled through
the medium or thickness of the medium.
ABS α ℓ
ℓ
Path length or thickness of medium.
BEER’S LAW:
The intensity of beam of monochromatic light decreases exponentially with the increasing concentration of the
absorbing substances.
ABS α C
2.
C
Concentration of the analyte.
Sum 1 and 2
ABS α ℯ ℓ
Or
ABS
= 𝛴ℯℓ
Where ′ℓ′ is the centimetres then ′ℯ′ is termed as molar absorption coefficient.
COMPONENTS:
SINGLE – BEAM SPECTROPHOTOMETER:
DOUBLE - BEAM SPECTROPHOTOMETER:
Light source:1.UV-radiation:
Hydrogen lamp or deuterium lamp.
2.Visible radiation: tungsten filament lamp.
Now a days laser beam is also used.
3.Monochromator:
It is a prism or grating is to disperse the heterochromatic radiation in to its components wavelength.
4.Slits:
Entrance slit and exit slit.
5.Sample holder:Glass or quartz cuvetts are used to take the sample.
For
UV radiation: Quartz
Visible: Glass
6.Detector:Photomultiplier tube.
TYPES OF SPECTROPHOTOMETER:

Single beam spectrometer:Measures the intensity of transmitted light of sample and displays absorbance of the
analyte at a particular wave length.

Double beam spectrometer:-
It detects the difference in intensity of light absorbed by sample and reference
sample at a specific wave length.
APPLICATION:
In determination of



Nutrients like N, P, S,K,Ca,Mg,Zn etc.
Organic compounds in biological matter.
Toxic elements like Cd,pb etc.
Glucose,fructose, proteins etc.
DIFFERENCES BETWEEN SPECTROPHOTOMETER AND COLORIMETER:
Sl
no.
UV – VISIBLE
SPECTROPHOTOMETER
1)
Measuring absorbance in UV and Visible region.
Measuring absorbance in visible region only.
2)
Grating
Filter type
3)
Quantification quality kinetics . There are automatic.
Where as there are manual.
4)
Meaning absorbance at any wavelength between
190 – 1100 nm.
COLORIMETER
Meaning absorbance at specific fixed wavelength.
5)
Least count is ± .001
Least count is ± .01.
6)
Automatic in high version
Manual.