1. spectrophotoMETER Dr. Beenish Zaki, Instructor
Department of Biochemistry
(2nd February 2010)
spectrophotoMETER

2. Learning objectives
At the end of the practical session the student should be able to:
Identify the instrument
Explain the principle of spectrophotometer
Difference between a Colorimeter and a spectrophotometer

3. spectrophotometer
Combination of two devices, a spectrometer and a photometer.
Spectrometer is used for producing light of any selected wavelength or color
Photometer is used for measuring the intensity of light.
It can work in the ultra-violet, visible and infrared region of light for quantitative analysis.

4. spectrophotometer

5. The electromagnetic spectrum

6. Principle:
When the white light passes through a coloured substance, a portion of it is absorbed by the colouring substance (chromogen) and the rest is transmitted.
Example: If a chromogen forms red solution, it means that the absorption of the red component is minimum where as the complementary colour green is absorbed to the greatest extent

7. Basic components
Source of light: A Tungsten- Halogen is used as a source of light.
Filters: diffraction grating or prisms in combination with entrance and exit slit
Cuvettes: These are plain glass tubes of round or rectangle in shape these are used to hold the solutions.
Sample Compartment: 4 samples can be read one after the other by the instrument
Photo Diode and DC amplifier: Converts the light energy into electrical energy and finally into a digital display

8. Schematic diagram

9. Law’s of Spectrophotometer
The relationship between the absorption (transmittance) of light and the concentration of the chromogen was described by Beer’s, and that between the absorption of light and the thickness of the solution was described by Lamberts.

10. Beer’s Law
States that the absorbance of light is directly proportional to the concentration of the coloring substance (chromogen)
Absorbance or Optical density ∞ Concentration of the solution

11. Lambert’s Law:
States that the absorbance of light is directly proportional to the thickness of the solution.
Absorbance or Optical density ∞ thickness of the solution through which the light passes.

12. Handling
Turn on the power switch. The lamp will light when the unit is on. Warm up the instrument for approximately 20 minutes.
Push the mode key to T position. When the sample chamber door is closed it shows 100.0%. Now push the mode key to A the display shows 0000A. Repeat this action for the spectra to be stable.
Put the cells which contain blank, standard and sample solution into the sample holder.
Turn the desired wavelength.
Push/Pull the handle of the sample holder to make the light beam pass through the sample being tested. The absorption value will be shown on the digital display.

13. General formula O.D of test x Concentration Of standard
O.D of Standard
O. D= Optical Density

14. comparision Component Colorimeter Spectrophotometer Lamp Tungsten
Tungsten-Halogen
Filters
Filters are used having a limited number of bands across the spectrum
Diffraction gratings are used to split the spectrum into a large number of bands

15. comparision Component Colorimeter Spectrophotometer Cuvette plastic
Glass
Sensitive
Expensive
Specificity
More specific

16. Questions