Colorimeter and Spectrophotometer.pptx

Colorimeter & spectrophotometer

1. Colorimetry and spectrophotometry Mo hammed Siddig Ali MSc. Clinical Chemistr y

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. E xpressed 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- L ambert’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. C olorimetry

6. Colored substance absorb light in relation to their color intensity . The color intensity will be proportional to the conc. Of colored substance . T he 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 P rinciple

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. Different in component. 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. Concentration of unknown conc. = concentration of STD C Un . = 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