2. What is colorimetry?
In analytical chemistry, colorimetry is a technique "used to determine
the concentration of colored compounds in a sample solution” at visible region
(400-800 nm)
3. Principle
The intensity of color is directly proportional to the concentration of the
compound being measured
Colorimetry is the technique frequently used in biochemical
investigations,involves the quantitative estimation of colors.
Color can be produced by any substance when it binds with color forming
chromogens.
The difference in color intensity results in the difference in the absorption of
light.
4. Complemetary color
Wavelength between 400nm to 760 nm form the visible spectrum of
light
light passed through a solution which selectivity absorbs radiation
at fixed wave lengths,then the color of the transmitted light is
complementary to that of the absored light.
Wavelength Color
absorbed
Color
transmitted
400-450 Violet Green-yellow
450-500 Blue yellow
500-570 Green Red
570-590 yellow Blue
590-620 orange Green-blue
620-760 Red Green
V
B G
Y
R O
5. Criteria for satisfactory colorimetric estimations
Stability of color
Color may be fade of air oxidation, photochemical decomposition,
temperature.
Intensity of color
The color of the solution should be intense in order to detect small
amount of constituents and for making accurate result in low concentration.
Clarity of the solution
Substance under investigation should be completely soluble in the
solvent, since turbid solution, suspention or colloidal solution absorb as well
as scatter light.
6. Reproducibility
The intensity of the colored solution must be reproducible. The effect
of order of adding reagent, pH and other variable should be clearly studied
Specificity
Color produced should be specific for the desired constituent. If
other constituents interfere with color reaction they be removed or prevented
from or prevented from functioning through appropriate treatment like use of
other coloring agent, altering the oxidation state.
Validity of Beer’s law
The intensity of color should be proportional to concentration. It can
be easily assessed by plotting absorbance Vs concentration, where a straight
line passing through origin should be obtained.
7. Types
Colorimetric analysis is two types
Visual colorimetry
Photo-electric colorimetry
Visual colorimetry is one of the oldest form of
color measuring technique which is not used now day, natural
or artificial light is used as light is used as light source and
determinations are made with a colorimetry or color
comparator where human eye is used as detector.
8. Photoelectric colorimetry
Progree in the development of colorimetric
method has resulted largly due to the application of photoeletric
cell, which eliminates the difficultes of complicated visual
comparition. In this method human eye is replaced by suitable
photoelectric cell, to afford a direct measure of the light intensity.
Instruments employing photoelectric cell measure the light
absorption and not color of substance
10. Light source
1. Tungsten lamp
filament mode of tungstone sealed in a glass envelope
Filed with inert gas.
higer temperature shorter the wave length.
It operates temperature 2890 K.
Use full wave length region between 350 nm and 250 nm.
Increasing temperature 6000 K, than the wave length maximum
will be shifted to 500 nm.
Expensive, filament operated at high temperature, quartz envelope
required.
Life time is limited due to gaseous tungsten formed by sublimation.
11.
12. Carbon arc lamp
If sufficient intensity of light is not obtained from tungsten lamp then
carbon arc lamp can be use as a source for color measurement.
13. 2. Filters
filters will absorb light of unwanted wavelength and allow only
monochromatic light to pass through
For ex: A green filter absorbs all color, except green light
which is allowed to pass through. light transmitted through a
green filter has a wavelength from 500-560 nm.
Filter
Absorption filter Interference filter
Ex: Glass filter,Gelatin filter
Band pass Cut off filter
14. Absoption filters
1. Absorption filters
These filters are made up of glass, coated with pigments (or)
they are made up of dyed gelatin.
Selection:
If the colour of the solution is Red, we have to use green filter
and
15. Prisms
In a spectrophotometer, light from the source is directed
through a convergent lens into a entrance slit at the focal point of
the lens, then through a prism and a second convergent lens.
The dispersion of radiation involves angularly separating the
different frequencies in a wavefront. The mechanism involved is
the process of refraction by prisma
16. 2. Interference filter (or) Fabry-perot filter
It has dielectric spacer film
made up of CaF2, MgF2 or SiO,
between parallel reflecting silver
films.
The thickness of dielectric spacer
film can be 1/2 λ. The
mechanism is that, the radiation
reflected by the second film and
incoming radiation undergoes
constructive interference to give
a monochromatic radiation.
17. Sample Holder
Cuvette are rectangular cell , square cell or circular one
Made up of optical glass for visible wavelength.
Common one is square,rectangular to avoid refraction
artefacts.
Dimension of cuvette is 1cm.
18. Detectors
Detectors are the transducers, which convert light energy to
electrical enagery .A detector should be possess follwing characteristics
1.Should be sensitive
2.Should have linear response
3.Its noise level Should be low
4.Should have short response time
5.Should stable
Barrier layer cell
Photomultiplyer tube
19. Barrier Layer cell (BLC)
It consists of an iron plate A, upon which a thin layer of
semiconductor selenium is deposited.
This in turn covered by a thin film of transparent silver layer,
which is lacquered except for portion of collecting ring for electrical
contact.
The iron plate act as anode and silver layer acts as cathode.
20.
21. Reference
Instrumental method of chemical analysis,chatwal.
Instrumental method of analysis, N.Grey, M.Calvin.
Pharmaceutical analysis, Ashutoshkar.
Instrumental method of analysis, B.K.Sharma
Principles and application of ultaviolet and visible
spectroscopy, A.Rajasekaran.
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