Dr. Beenish Zaki, Department of Biochemistry

 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

Approximate wavelength(nm)Color of light absorbedColour of light Reflection Violet (Vi)Yellowish Green Blue (B)Yellow Green (G)Red Yellow (Y)Blue Orange (O)Greenish Blue Red (R)Bluish Green

 Source of light: An electric lamp example tungsten lamp is used as a source of light.  Filters: The light from the electric lamp is passed through a filter which converts polychromatic light to monochromatic light of a specific wave.  Cuvettes: These are plain glass tubes or plastic containers. Can be round or rectangle in shape these are used to hold the solutions.  Photo cell or tube: Converts the light energy into electrical energy  A measuring device: The potential difference generated in photocell or tube is measured by a sensitive galvanometer this is generally calibrated to read directly either transmittance or absorbance or both.

 Beer’s Law  Lambert’s Law

 States that the absorbance of light is directly proportional to the concentration of the coloring substance (chromogen) Absorbance or Optical density α Concentration of the solution

 States that the absorbance of light is directly proportional to the path length. Absorbance or Optical density α to the path length

 For each test a blank should be prepared containing the reagents.  The cuvettes containing the blank should be put in the slot provided for it and the instrument should be adjusted to zero absorbance.  The blank should be replaced successively by the standard and the unknown solutions and their absorbance read.  The instrument should be switched off at the end of the experiment.

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

Name of Parts Present in Colorimeter Light sourceLED bulb FilterPrism CuvettesPlastic

Objective Structured Practical Examination (OSPE) Time Allotted: 5 Minute/Station Marks Allotted: 5  Identify the instrument and answer the following in the space provided in the response sheet:

S.No QuestionResponse Marks 1.Name the instrument kept in your station Colorimeter1 2.Name the law which states “Amount of light transmitted through a coloured solution decreases exponentially with increase in concentration of the coloured substance” Beers Law1 3.What is the function of filter used in colorimeter Converts polychromatic into monochromatic light 1 4.General formula to calculate the unknown concentration in the test sample O.D of test/ O.D of Std x Conc. Of std 1 5Calculate the serum total protein if O.D of test is 0.9 O.D of standard is 0.6 Concentration of standard 5gm/dl 0.9 X 5 = 7.5gm/dl 0.6 1