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

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

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.

spectrophotometer

The electromagnetic spectrum

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

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

Schematic diagram

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.

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

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.

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.

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

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

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

Questions