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Colorimeter ppt BIOCHEMISTRY

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Colorimeter ppt BIOCHEMISTRY

  1. 1. •COLORIMETER
  2. 2. •Visible spectrum
  3. 3. •Colorimeter Colorimeter is a instrument used for the measurement of coloured substance in solution. The instrument is operative in the visible range of the electromagnetic spectrum 8/26/2013 12:23 AM
  4. 4. •Photometry Photometry means measurement of light. The colour of light is a function of its wavelength. As the wavelength changes alteration in colour is detected. 8/26/2013 12:23 AM
  5. 5. • Colorimeter - Principle • When a white light passes through a coloured solution, some specific wavelengths of light are absorbed which is related to colour intensity. 8/26/2013 12:23 AM
  6. 6. 8/26/2013 12:23 AM
  7. 7. Components of the colorimeter 8/26/2013 12:23 AM
  8. 8. •COLORIMETER
  9. 9. •COLORIMETER
  10. 10. Relationship between absorbance and transmittance 8/26/2013 12:23 AM OD %T
  11. 11. • The measurement of colour intensity of a coloured solution by photometry is governed by two laws Beer’s law Lambert’s law 8/26/2013 12:23 AM
  12. 12. BEER’S LAW • The concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light 8/26/2013 12:23 AM
  13. 13. 8/26/2013 12:23 AM Beer’s law Beer’s law
  14. 14. LAMBERT’S LAW • When a ray of monochromatic light passes through an absorbing medium its intensity decreases exponentially as the length of the light path through light absorbing material increases 8/26/2013 12:23 AM
  15. 15. •LAMBERT’S LAW 8/26/2013 12:23 AM
  16. 16. Preparation of solution for investigation • In colorimetric estimation it is necessary to prepare 3 solutions BLANK(B) STANDARD(S) TEST(T) 8/26/2013 12:23 AM
  17. 17. 8/26/2013 12:23 AM BLANK To eliminate the effect of light absorption by the reagent used Water BLANK Reagent BLANK
  18. 18. STANDARD Solution of known concentration of the substance Both O.D and concentration are known So concn of unknown can be calculated 8/26/2013 12:23 AM
  19. 19. TEST Test solution is made by treating a specific volume of the test sample with reagents As per procedure 8/26/2013 12:23 AM
  20. 20. •Derivation of the Formula • Combining the two laws • AαCxL • OR A=KxCxL • Let AT=absorbance of the test solution • CT=concentration of the test solution • AS=absorbance of the standard solution • CS=concentration of the standard solution 8/26/2013 12:23 AM
  21. 21. 8/26/2013 12:23 AM AT AS KxCTxL KxCSxL = AT AS CT CS = CT = AT AS X CS AS=KxCSxLAT=KxCTxL
  22. 22. 8/26/2013 12:23 AM CT = AT AS X CS Concentration of TEST solution Absorbance of TEST Absorbance of STANDARD Concn of STANDARDX = Concentration of TEST /100ml Absorbance of TEST Absorbance of STANDARD Concn of Std X 100X = Xml
  23. 23. 8/26/2013 12:23 AM Concentration of TEST /100ml Absorbance of TEST Absorbance of STANDARD X = Xml Concn of Std X 100 Concentration of TEST /100ml O.D of ‘T’- O.D of ‘B’ O.D of ‘S’- O.D of ‘B’ X = Volume of ‘T’ Amount of ‘S’ X 100 Concentration of TEST /100ml T - B S - B X = Volume of ‘T’ Amount of ‘S’ X 100
  24. 24. •VERIFICATION OF BEER’S LAW 8/26/2013 12:23 AM 3% Cobalt acetate in 1% HCl 1% HCl REAGENTS Colorimeter Cuvettes INSTRUMENT
  25. 25. 8/26/2013 12:23 AM B S1 S2 S3 S4 S5 S6 S7 T Volume of 3% cobalt acetate [in ml] - 0.5 1 2 3 4 5 6 - Volume of 1% HCl [in ml] 6 5.5 5 4 3 2 1 - - Conc. Of cobalt acetate in mg/6ml - 15 30 60 90 120 150 180 O.D at 490nm 0.01 0.03 0.06 0.12 0.18 0.24 0.30 0.36 0.21
  26. 26. •By Graphical methood 15 30 60 90 120 O.D 0.02 0.08 0.14 0.2 0.26 0 0.05 0.1 0.15 0.2 0.25 0.3 O.D Concentration in mg/6ml 8/26/2013 12:23 AM 0.17
  27. 27. - - THANK YOU

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