This document discusses colorimetry and spectrophotometry techniques. It explains that Beer's law and Lambert's law describe how the absorption of light by a colored solution depends on concentration and path length. A colorimeter uses a light source and filter to measure absorption, while a spectrophotometer uses a prism or grating to provide a narrow range of wavelengths, allowing distinction between closely related absorptions. Common applications include determination of substances in biological samples.
2. When a monochromatic light passes through a
colored solution, amount of light transmitted
decreases exponentially with increase in
concentration of colored substance.
I.e. the amount of light absorbed by a colored
solution is directly proportion to the conc. Of
substance in the colored solution.
Beer’s law
3. The amount of light transmitted decreases
exponentially with increase in path length
(diameter) of the cuvette or thickness of colored
solution through which light passes.
I.e. the amount of light absorbed by a colored
solution depends on path length of cuvette or
thickness or depth of the colored solution.
Lambert’s law
4. Expressed as amount of light transmitted
through a colored solution decreases
exponentially with increases in conc. Of colored
solution & increase in the pathlength of cuvette
or thickness of the colored solution.
Beer’s- Lambert’s law
5. 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 colorimetrically,
must be colored or it should be capable of
forming chromogens (colored complexes)
through the addition of reagents.
Colorimetry
6. Colored substance absorb light in relation to
their color intensity.
The color intensity will be proportional to the
conc. Of colored substance.
The instruments used in this method are
colorimeter or photometer or absorptiometers.
7. Colored solutions have the property of absorbing
certain wavelength of light when a monochromatic
light is passed through them.
The amount of light absorbed or transmitted by a
colored solution is in accordance with two laws:
Beer’s law
Lambert’s law
Principle
8. Is a sophisticated type of colorimeter where
monochromatic light is provided by prism.
The band with of the light passed by a filter is
quite board, so that it may be difficult to
distinguish between two components of closely
related absorption with a colorimeter.
A spectrophotometer is then needed.
The Spectrophotometer:
10. Colorimeter Spectrophotometer
Light source Tungsten Lamp Halogen Lamp
Monochromator Filter Prism /grating
Cuvaltte Glass Glass
photocell photocell Photomultiplier tube
Read out device Scale/ digital Scale/ digital
Component of colorimeter/ spectro.
11. 1. Different in component.
2. Different in band:
Spectro: narrow range of band.
Colorimet.: wide range of band.
Different between the 2 devices
12. 1- White light from a tungsten lamp passes through a
slit, then a condenser lens, to give a parallel beam
which falls on the solution under investigation
contained in cuvette. The cell is made of glass.
How colorimeter/spectro. works?
13. 2- Beyond the absorption cell is the filter, which is
selected to allow maximum transmission of the color
absorbed.
NOTE: The color of the filter is complementary to the
solution.
How colorimeter/spectro. works?
14. 3- The light then falls on to a photocell which generates
an electrical current in direct proportion to the
intensity of light falling on it.
How colorimeter/spectro. works?
15. 4- This small electrical signal is increased by the
amplifier which passes to a galvanometer of digital
readout to give absorbance reading directly.
How colorimeter/spectro. works?
16. 𝒂𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝒖𝒏𝒌𝒏𝒐𝒘𝒏 𝒄𝒐𝒄. = 𝒂𝒃𝒔𝒓𝒐𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝑺𝑻𝑫
Concentration of unknown conc. = concentration of STD
CUn. = A Un. × C St.
A St.
Calculation
17. Used in determination of amount of many
substances in blood, urine, saliva, CSF & other
specimens.
Example for common colorimetric assay are :
determination of blood glucose, blood urea etc.
Example for common spectrophotometric assay
is: determination of enzyme
Application of colorimetric/spectro.
assay