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Colorimeter

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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.

Publicado en: Salud y medicina
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Colorimeter

  1. 1. Tapeshwar Yadav (Lecturer) BMLT, DNHE, M.Sc. Medical Biochemistry
  2. 2. •COLORIMETER
  3. 3. •Visible spectrum
  4. 4. •ColorimeterColorimeter
  5. 5. •Photometry
  6. 6. Colorimetry • 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.
  7. 7. • Colored substance absorb light in relation to their color intensity. • The color intensity will be proportional to the conc. of colored substance. • The instruments used in this method are colorimeter or photometer or absorptiometers.
  8. 8. • Colorimeter - PrincipleColorimeter - Principle • When a monochromatic light passes through a coloured solution, some specific wavelengths of light are absorbed which is related to colour intensity. • The amount of light absorbed or transmitted by a colour solution is in accordance with two law i.e. Beer’s & Lambert’s Law.
  9. 9. • The measurement of colour intensity of a coloured solution by photometry is governed by two laws
  10. 10. Beer’s law : • 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.
  11. 11. Beer’s law Beer’s law
  12. 12. 09/12/15 08:51
  13. 13. Lambert’s law : • The amount of light transmitted decreases exponentially with increase in pathlength (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 pathlength of cuvette or thickness or dept of the colored solution.
  14. 14. LAMBERT’S LAW 09/12/15 08:51
  15. 15. • Combined beer’s- lambert’s law is thus expressed as amount of light transmitted through a colored solution decreases exponentially with increases in conc. of colored solution & increase in conc. of colored solution & increase in the path length of cuvette or thickness of the colored solution
  16. 16. 09/12/15 08:51
  17. 17. Relationship between absorbance and transmittance 09/12/15 08:51 OD %T
  18. 18. • Light falling on a color solution is either absorbed, reflected or transmitted. Io=It + Ia Absorption & transmittance of light Io It Ia
  19. 19. Components of the colorimeter 09/12/15 08:51
  20. 20. Parts of the colorimeter Light source : tungsten filament lamp Slit : it is adjustable which allows only a beam of light to pass through. it prevents unwanted or stray light Condensing lenses: light after passing through slit falls on condenser lense which gives a parllel beam of light.
  21. 21. Filter : • made of colored glass. Filters are used for selecting light of narrow wavelength. • filters will absorb light of unwanted wavelength and allow only monochromatic light to pass through. For ex: a green filter absorbs all color, except green light which is allowed to pass through. Light transmitted through a green filter has a wavelength from 500-560 nm. • Filter used is always complimentary in color to the color of solution.
  22. 22. Cuvette(sample holder) : the monochromatic light from the filter passes through the colored solution placed in a cuvette. • it is made up of special glass/plastic/quartz material. • it may be square/rectangular/round shape with fixed diameter (usually 1 cm)& having uniform surface. the colored solution in the cuvette absorbs part of light & remaining is allowed to fall on detector. • For ex : a solution of red color transmits red light & absorbs the complimentary color green.
  23. 23. Detector (photocell): • Detector are photosensitive elements which converts light energy into electrical energy. • The electrical signal generated is directly proportional to intensity of light falling on the detector. Output : the electrical signal generated in photocell is measured by galvanometer, which displays percent transmission & optical density.
  24. 24. COLORIMETER
  25. 25. Colorimeter (1)(1) Wavelength selection,Wavelength selection, (2)(2) Printer buttonPrinter button (3)(3) Concentration factor adjustment,Concentration factor adjustment, (4)(4) UV mode selector (Deuterium lamp)UV mode selector (Deuterium lamp) (5)(5) ReadoutReadout (6)(6) Sample compartmentSample compartment (7)(7) Zero control (100% T),Zero control (100% T), (8)(8) Sensitivity switchSensitivity switch
  26. 26. Approx. wavelength Colour absorbed (Filter) Colour of solution <4oonm Ultra violet (UV-rays) Not visible 400-420nm Violet Green-Yellow 420-500nm Blue Yellow 500-570nm Green Red 570-600nm Yellow Blue 600-630nm Orange Green-Blue 630-700nm Red Green >700nm Infrared (IR-rays) Not visible Relationship between the wavelength and colour
  27. 27. Preparation of solution for investigation • In colorimetric estimation it is necessary to prepare 3 solutions
  28. 28. 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
  29. 29. AT AS KxCTxL KxCSxL = AT AS CT CS = CT = AT AS X CS AS=KxCSxLAS=KxCSxL AT=KxCTxLAT=KxCTxL
  30. 30. CT = AT AS X CS Concentration of TEST solution Concentration of TEST solution Absorbance of TEST Absorbance of STANDARD Concn of STANDARDX = Concentration of TEST / 100ml Concentration of TEST / 100ml Absorbance of TEST Absorbance of STANDARD Concn of Std X 100X = Xml
  31. 31. 09/12/15 08:51 Concentration of TEST / 100ml Concentration of TEST / 100ml Absorbance of TEST Absorbance of STANDARD X = Xml Concn of Std X 100 Concentration of TEST / 100ml 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 Concentration of TEST /100ml T - B S - B X = Volume of ‘T’ Amount of ‘S’ X 100
  32. 32. VERIFICATION OF BEER’S LAW 09/12/15 08:51
  33. 33. 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
  34. 34. •By Graphical method 0.17
  35. 35. • It is widely used in hospital & laboratory for estimation of biochemical samples , like plasma, serum, cerebrospinal fluid ( CSF ) , urine. • It is also used to quantitative estimation of serum components as well as glucose, proteins and other various biochemical compound. • They are used by the food industry and by manufacturers of paints and textiles. Application of colorimeter
  36. 36. • They are used to test for water quality, by screening for chemicals such as chlorine, fluoride, cyanide, dissolved oxygen, iron, molybdenum, zinc and hydrazine. • They are also used to determine the concentrations of plant nutrients (such as phosphorus, nitrate and ammonia) in the soil or hemoglobin in the blood and to identify substandard and counterfeit drugs.
  37. 37. Advantage It is inexpensive . Very well applicable for quantitative analysis of colored compounds. Easily transportable. •COLORIMETER
  38. 38. Disadvantage Cannot be used for colorless compounds.  It does not work in UV and IR regions. We cannot set specific wavelength, as we have to set a range as a parameter. Similar colors from interfering substances can produce errors in results . •COLORIMETER
  39. 39. Use, care and preventive maintenance of a Colorimeter: • Read the user manual carefully. • Use the correct type of cuvette in the colorimeter as recommended by the manufacturer. • Make sure that the cuvette is clean and it’s optical surfaces are dry and free from finger marks and scratches. • Bring filter in to place before switching on the colorimeter. • Before reading the absorbance of a solution, check that it is clear, there are no air bubbles in it. • Remove the cuvettes from the instrument when not in use.
  40. 40. CONTD… • Clean the outside of the cuvette with tissue paper to remove any marks from the optical surfaces. • To prolong the life of the lamp, switch off the colorimeter after use. • At the end of the day, disconnect It from the main switch and cover the colorimeter with its protective cover. • At regular intervals check the mains power adapter and cable for wear and tear and replace if damaged. • Keep in cool place away from corrosive chemicals or fumes.
  41. 41. 12-09-15 08:51 AM THANK YOUTHANK YOU

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