The document discusses colorimetry, including the principles of how colors are perceived and the function of a colorimeter, which measures the intensity of color based on light absorption and transmittance. It explains key concepts such as Beer-Lambert Law and outlines the steps for using a colorimeter, preparing standard curves, and conducting quantitative experiments. Additionally, it compares colorimeters to spectrophotometers, highlighting differences in sensitivity and measurement range.

1. Colorimetry
Prof Dr Viyatprajna Acharya
MD, PhD

2. Colorimeter
• Visual colorimeter
• Photoelectric colorimeter

3. Three primary colours
• Visible range- 380-700nm
• UV range <380nm
• IR range >700nm

4. Most of the biochemical tests are based on
colours!! How do we see colours??
• As sunlight — which is a combination of all wavelengths —
hits an object, some materials will absorb specific
wavelengths. The wavelengths that aren’t absorbed get
reflected. This reflected light then reaches our eyes and
makes us perceive the reflecting object as being a
particular color.
• Colorimeter measures the colour intensity
• Compared with a known standard

5. Absorbance Vs Transmittance light
• Inverse relationship
• In colorimeter transmittance light is measured
and calculated for absorbance
• Absorbance is directly proportional to conc. Of
analyte and transmittance is inversely
proportional
• Unit of absorbance is OD (Optical density)

6. A= -log T

7. % Transmission (T)= Ie/I0 x100
%Absorbance (A)= Log10 1/T
A= Log10 100/%T
= Log10 100- Log T
= Log10 102- Log T
=2Log10 10- Log T
A= 2- Log %T

8. Parts of colorimeter
1. Light source, usually a tungsten filament lamp (400-700nm)
2. A monochromator—a coloured filter which allows a monochromatic
beam of light of a particular wavelength.
3. Shutter—which is a door to allow the light to pass through
4. Cuvette cell—A slot provided to place a Cuvette of uniform size
5. Photo cell—a photosensor which light energy to electrical energy
6. Galvanometer—measures the electrical current generated in the
photo cell and amplifies it.
7. Display (Digital LED)

10. Principles of colorimetry
• Beer’s law- The amount of light absorbed by a coloured
solution is directly proportional to the concentration of
coloring substance (solute) present in the solution.
A α C
A= absorbance C= Concentration

11. • Lambert’s law- The amount of light absorbed by a
coloured solution is proportional to the depth or path
length through which the light passes i.e. the
diameter of the cuvette.
A α L
Combined Beer-Lambert’s law
A= k.CL k= constant for the coloured solution
Amount of light absorbed by a coloured solution is directly
proportional to the concentration of the solute if the path
length is constant.

12. How to use a colorimeter
1. Switch on the colorimeter and allow to warm up for 15 minutes.
2. Select the desired wavelength/ filter.
3. With shutter closed or inserting a dummy opaque Cuvette in the Cuvette cell set to
zero.
4. Select the transmittance mode.
5. Place the Cuvette containing distilled water in the Cuvette cell and set to 100%
transmittance.
6. Select the absorbance mode. The 100% transmittance should read 0.00 in
absorbance mode.
7. Now the instrument is ready to take reading.

13. Standard curve preparation
• A series of known concentration of potassium
permanganate solution is taken and their OD is measured
• Graph is plotted
• Unknown solution OD is measured and its concentration
can be extrapolated from the curve

14. Linearity
• At higher concentrations the curve diverts
• solute molecules can cause different charge distribution on their neighboring
species in the solution. Since UV-visible absorption is an electronic
phenomenon, high concentrations would possibly result in a shift in the
absorption wavelength of the analyte. Some large ions or molecules show
deviations even at very low concentrations. For e.g. methylene blue
absorptivity at 436 nm fails to observe Beer Lambert law even at
concentrations as low as 10μM.
• alter the refractive index (η) of the solution
• Chemical alteration of solute molecules
• Depends on band length chosen too

15. Quantitative experiments
• 3 test tubes are taken and labeled as B, S & T for blank,
standard and test solution.
• B= Reagent with water
• S= Standard of the parameter to be measured (known
concentration)
• T= Test solution (unknown concentration)
• OD is measured for all three
• Calculation-
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16. Spectrophotometer
• Uses a prism or grating
• UV and IR range also can be
measured
• Hydrogen and deuterium lamps are
used- 200-900nm
• More sensitive and accurate

17. www.drvpacharya.com