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.

1. Colorimetry and
spectrophotometry
Mohammed Siddig Ali
MSc. Clinical Chemistry

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:

9. 1- Visible spectrophotometer.
2- Ultraviolet (UV) spectrophotometer.
Types of 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

18. Thanks for attention