2. PRESENTATION OUTLINE
INTODUCTION AND BRIEF HISTORY
PRINCIPLE
PARTS, COMPONENTS AND SCHEMATICS
CUVETTES AND TEST PROCEDURE
BEER-LAMBERT’S LAW
ADVANTAGES AND DISADVANTAGES
APPLICATION
CARE, USE AND MAINTENANCE
REFERENCES
4. COLORIMETER
It is the most common analytical technique
used in biochemical estimation in clinical
laboratory
It involves the quantitative estimation of color
A substance to be estimated colometrically,
must be colored or capable of forming
chromogens (colored complexes) through the
addition of reagents.
The color of light is the function of its
wavelength
5. COLORIMETER PRINCIPLE
When a monochromatic light passes
through a coloured solution, some specific
wavelengths of light are absorbed which is
related to colour intensity.
The amount of light absorbed or
transmitted by a colour solution is in
accordance with two law i.e. Beer’s &
Lambert’s Law.
10. SAMPLE HOLDER/ CUVETTE
•Cuvettes are rectangular cell , square cell or circular
one.
•Made up of optical glass for visible wavelength
(quartz or fused silica for UV).
•Optical path (length) of cuvette is always1cm.
•Capacity may be 3ml/2ml/1ml depending upon the
thickness of the wall of the cuvette.
•For accurate and precise reading cuvette must be
transparent, clean, devoid of any scratches and there
should be no bubble adhering to the inner surface of
the filled cuvette.
11. PREPARATION OF SOLUTION FOR
INVESTIGATIONS
In colorimetric estimation it is necessary to prepare 3
solutions
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BLANK(B)
STANDARD(S)
TEST(T)
12. BLANK
To eliminate the effect of light
absorption by the reagent used
Water BLANK
Reagent BLANK
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13. STANDARD
Solution of known concentration of the
substance
Known concentration
So concentration of
unknown can be
calculated
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14. TEST
Test solution is made by
treating a specific volume of
the test sample with reagents
As per
manufacturers
procedure
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15. Observed Color of
Compound
Color of Light
Absorbed
Approximate
Wavelength of Light
Absorbed
Green Red 700 nm
Blue-green Orange-red 600 nm
Violet Yellow 550 nm
Red-violet Yellow-green 530 nm
Red Green 500 nm
Orange Blue 450 nm
Yellow Violet 400 nm
17. LAMBERT’S LAW
•When a ray of monochromatic light passes
through an absorbing medium its intensity
decreases exponentially as the length of the
light path through light absorbing material
increases
BEER’S LAW
•The concentration of a substance is directly
proportional to the amount of light absorbed
or inversely proportional to the logarithm of
the transmitted light
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19. RELATIONSHIP BETWEEN
WAVELENGTH & COLOUR
Wavelength between 400nm to 700nm form the visible
spectrum of light
visible band of light in electromagnetic spectrum
21. COLORIMETER
Disadvantages
Cannot be used for colorless compounds.
It does not work in UV and IR regions.
We cannot set specific wavelength, as we have to set a range as a
parameter.
Similar colors from interfering substances can produce errors in
results .
22. APPLICATION
It is widely used in hospital & laboratory for estimation of
biochemical samples , like plasma, serum, cerebrospinal fluid (
csf ) , urine.
It is also used to quantitative estimation of serum components
as well as glucose, proteins and other various biochemical
compound.
They are used by the food industry and by manufacturers of
paints and textiles.
23. They are used to test for water quality, by screening for chemicals
such as chlorine, fluoride, cyanide, dissolved oxygen, iron,
molybdenum, zinc and hydrazine.
They are also used to determine the concentrations of plant nutrients
(such as phosphorus, nitrate and ammonia) in the soil or hemoglobin
in the blood and to identify substandard and counterfeit drugs.
24. USE, CARE AND MAINTENANCE OF
A COLORIMETER:
Read the user manual carefully.
Use the correct type of cuvette in the colorimeter as recommended by the manufacturer.
Make sure that the cuvette is clean and it’s optical surfaces are dry and free from finger marks
and scratches.
Clean the outside of the cuvette with tissue paper to remove any marks from the optical
surfaces.
To prolong the life of the lamp, switch off the colorimeter after use.
At the end of the day, disconnect It from the main switch and cover the colorimeter with its
protective cover.
25. 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.
WWW.Pharmatwiter.com
WIKIPEDIA
Light source: tungsten filament lamp
Slit :It is adjustable which allows only a beam of light to pass through. It prevents unwanted or stray light
Condensing lens: Light after passing through a slit falls on a condenser which gives parallel beam of light.
Filters : are usually made of colored glass. it is used for selecting narrow wavelength .they absorb light of unwanted wavelength and allow only monochromatic light to pass through
For e.g.: , a green filter absorbs all colours,except green light which is allowed to pass through.light transmitted through a green filter has a wavelength from 500-560nm.Filters used is always complimentary to the colour of the solution
Cuvette
-may be square,rectangular or round shape with fixed diameter and having uniform surface
-made up of plastic ,glass material
-solution in the cuvette absorbs a part of the light and the remaining is allowed to fall on the detector
Detector (photocell): The detectors are photosensitive elements which converts light energy into electrical signal .the electrical signal is directly proportional to the intensity of light falling on the detector
Output : the electrical signal generated in a photocell is measured by a galvanometer which displays transmission and optical density
