COLORIMETER - Basics and application
·
Colorimeter
is a device used in colorimetry
·
It means
measurement of absorbance at particular wavelengths of light.
·
It means the
quantity/ concentration of substance present in a liquid sample.
·
Widely used
in biological sample analysis.
Principle
·
It works by
the Beer–Lambert law.
·
The
absorption of light transmitted through the solution is directly proportional
to the solution concentration.
·
This is done
by passing light of a specific wavelength of the visible spectrum through the
solution in a photoelectric colorimeter instrument and observing the
galvanometric reading of reflection sensitizing the quantity of light absorbed.
· It = Io X e-kc
or
· It = Io X e-kT
· Here, Io = Intensity of initial
(original) light
· It= Intensity of transmitted light
· k = constant
· C = concentration
·
T= Thickness
of the solution
·
Based on the
nature of color compounds, specific light filters are used.
o
Three
types of filters are available — blue, green, and red (400 – 700nm)
If a substance is following the beer’s law,
we get a straight line which is
· Passing from the origin and the slope is
“ab”. But sometimes we do not get a straight line.
· If we are getting much more absorbance value than desired, then deviation is said as +ve deviation.
· But if we are getting much less absorbance value than desired, then deviation is said as -ve deviation.
Instrumentation
The
essential parts of a colorimeter are:
· a light source (often an
ordinary low-voltage filament lamp);
· an adjustable aperture (lens);
· a set of colored filters;
· a cuvette to hold the working
solution;
· a detector (usually a photoresistor) to
measure the transmitted light;
· a meter to display the output from the
detector.
·
instrument is set to 100% transmission (0% absorption) for cuvette
with the solvent only.
· Filters are
made of glass or gelatine - Color of visible region with wavelength
o
1. Violet - 380-470 nm
o
2. Blue - 440-490 nm
o
3. Blue (slightly greenish) - 490-500 nm
o
4. Green - 500-560 nm
o
5. Yellow (slightly greenish) - 560-580 nm
o
6. Yellow - 580-600 nm
o 7. Orange - 600-650 nm
o 8. Red - 650-750 nm
o 9. Infra-red – 1000 nm
· Cuvette -
otherwise called sample vessel
· Cuvette should not absorb the light
·
For
different region different cuvettes are used
o
For UV
range: Quartz cuvette
o
For visible
range: glass cuvette
o
For IR
range: NaCl, KBr, nujol cuvette
Detector: The light which is initiated from the source and pass through cuvette is measured by the detector. Absorption or transmission of the light can be measured by detector.
Mainly three type of detectors can be used:
1.
Photovoltaic Cell (Barrier layer Cell)
2. Photo tubes (Photo
emissive tube)
3. Photo multiplier tubes
Recorder: The measurement of absorption or transmission is recorded in digital form in the recorder.
|
(1) Wavelength selection, (2)
Printer button, (3)
Concentration factor adjustment, (4) UV
mode selector (Deuterium lamp), (5) Readout, (6) Sample
compartment, (7) Zero
control (100% T), (8)
Sensitivity switch, (9) ON/OFF
switch |
Applications
- Colorimeter and Spectrophotometer is used in many areas of science including microbiology, biochemistry, forensics, physics, and medical health.
- Colorimeters and Spectrophotometer are widely used to monitor the growth of a bacterial or yeast culture.
- Colorimeters and Spectrophotometer are used to measure and monitor the color in various drugs, foods and beverages, including vegetable products and sugar.
- Colorimeters and Spectrophotometer have many practical applications such as testing water quality by screening chemicals such as chlorine, fluoride, cyanide, dissolved oxygen, iron, molybdenum, zinc and hydrazine.
- Colorimeters and Spectrophotometer are also used to determine the concentrations of plant nutrients such as ammonia, nitrate and phosphorus in soil or hemoglobin in blood.
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