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APPLICATION OF COLORIMETRY IN TRACE MINERAL ANALYSIS

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APPLICATION OF COLORIMETRY IN TRACE MINERAL ANALYSIS

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APPLICATION OF COLORIMETRY IN TRACE MINERAL ANALYSIS

  1. 1. AAPPPPLLIICCAATTIIOONN OOFF CCOOLLOORRIIMMEETTRRYY IINN TTRRAACCEE MMIINNEERRAALL AANNAALLYYSSIISS CCoouurrssee :: VVBBCC 881122 AAddvvaanncceess iinn TTeecchhnniiqquueess iinn BBiioocchheemmiissttrryy Prepared By: Irshad A. PhD Scholar Department of MSC and Tech VCRI-Namakkal
  2. 2. WWHHAATT IISS CCOOLLOOUURR?????? • Colour is one aspect of appearance • Electromagnetic radiation between 380-780 nm • Colour = light source + object properties + eye + brain
  3. 3. OOBBJJEECCTTSS……....WWhhyy iitt llooookk lliikkee?? Objects are characterized by the amount Incident of light they emit and reflect or transmit Light at each wavelength of interest When light is incident on an object a part of it is absorbed, a part is reflected and a part may be transmitted The object may also emit light All these characteristics contribute to the observed colour/appearance Reflected Light Absorbed Light Transmitted Light
  4. 4. A compound will absorb light when the radiation posesses the energy needed to move an electron from its lowest energy (ground) state to some excited state. The particular energies of radiation that a substance absorbs dictate the colours that it exhibits. Conversely the colour of a compound can help us to determine its electronic configuration. White light contains all wavelengths in this visible region. When a transparent sample (like most aqueous solutions) absorbs visible light, the color we perceive is the sum of the remaining colours that are transmitted by the object and strike our eyes.
  5. 5. CCOOLLOORRIIMMEETTRRYY It is the most common analytical technique used in biochemical estimation in clinical laboratory. It involves the quantitative estimation of colour. A substance to be estimated colorimetrically, must be coloured or it should be capable of forming chromogens (coloured complexes) through the addition of reagents. Coloured substance absorb light in relation to their colour intensity. The colour intensity will be proportional to the conc. of coloured substance.
  6. 6. CCOOLLOORRIIMMEETTEERR The instruments used colorimeters or Photometers or Absorptiometers
  7. 7. PPrriinncciippllee ooff CCoolloorriimmeettrryy Colored solutions have the property of absorbing certain wavelength of light when a monochromatic light is passed through them. Light Intensity Change : By Absorbance or Transmittance Quantity : Using Absorbance
  8. 8. PPrriinncciippllee ooff CCoolloorriimmeettrryy The amount of light absorbed or transmitted by a colored solution is in accordance with two laws: BBeeeerr’’ss llaaww LLaammbbeerrtt’’ss llaaww “Lambert-Beer’s Law”
  9. 9. BBeeeerr’’ss llaaww  When a monochromatic light passes through a coloured solution, amount of light transmitted decreases exponentially with increase in concentration of coloured substance.  i.e. the amount of light absorbed by a coloured solution is directly proportion to the conc. of substance in the coloured solution. A α c
  10. 10. BBeeeerr’’ss llaaww The amount of radiation absorbed may be measured in a number of ways: 1. Transmittance, T = P / P0 2. Absorbance, A = log10 P0 / P So, if all the light passes through a solution without any absorption, then absorbance is zero, and percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and absorption is infinite.
  11. 11. LLaammbbeerrtt’’ss llaaww The amount of light transmitted decreases exponentially with increase in pathlength (diameter) of the cuvette or thickness of coloured solution through which light passes. i.e. the amount of light absorbed by a coloured solution depends on pathlength of cuvette or thickness or dept of the colored solution. Independent of the wavelength of incident light
  12. 12. LLaammbbeerrtt--BBeeeerr’’ss LLaaww • Amount of light transmitted through a coloured solution decreases exponentially with increases in conc. of coloured solution & increase in the pathlength of cuvette or thickness of the colored solution.
  13. 13. Standard CCuurrvvee,, CCaalliibbrraattiioonn ccuurrvvee
  14. 14. Limitation ooff LLaammbbeerrtt--BBeeeerr’’ss LLaaww
  15. 15. AApppplliiccaattiioonnss ooff CCoolloorriimmeettrryy
  16. 16. SSttrruuccttuurree ooff CCoolloorriimmeetteerr
  17. 17. Structure ooff CCoolloorriimmeetteerr--LLiigghhtt SSoouurrccee 1. Tungsten filament lamp - Wavelength Range : Part of the UV and the whole of the Visible range (350 ~ 2,500nm) 2. Deuterium Arc Lamp - UV Region, Wavelength Range :190~420nm 3. Xenon Lamp - Wavelength Range : 190~800nm
  18. 18. SSttrruuccttuurree ooff CCoolloorriimmeetteerr 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 lens which gives a parallel beam of light.
  19. 19. Structure ooff CCoolloorriimmeetteerr-- FFiilltteerr • made of coloured 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 colour, 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 colour to the colour of solution.
  20. 20. MMoonnoocchhrroommaattoorr
  21. 21. Structure of Colorimeter- CCuuvveettttee((ssaammppllee hhoollddeerr)) the monochromatic light from the filter passes through the coloured solution placed in a cuvette. it is made up of special glass/plastic/quartz/ fused silica material. • it may be square/rectangular/round shape with fixed diameter (usually 1 cm) & having uniform surface.
  22. 22. SSttrruuccttuurree ooff CCoolloorriimmeetteerr-- DDeetteeccttoorr Detector • 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.
  23. 23. SSttrruuccttuurree ooff CCoolloorriimmeetteerr-- DDeetteeccttoorr
  24. 24. HHooww ttoo eessttiimmaattee aa ssaammppllee....?? Use of test (T), standard (S) and blank (B) Test (T): this solution is prepared by treating a specific volume of specimen (blood,urine, CSF…etc) with reagents. Standard (S): prepared by treating a solution of the pure substance of known conc. with reagents. For ex : pure glucose is taken as standard in estimation of blood glucose.
  25. 25. HHooww ttoo eessttiimmaattee aa ssaammppllee....?? Blank : prepared for rule out colour produced by reagents alone. Two types of blank : A) Distilled water as blank B) reagent blank (reagent used in the estimation is taken as blank) Calculation : • conc. Of substance in mg /100mg or gm/100ml of sample. OD of test- OD of blank × conc. of standard × 100 OD of standard – OD of blank vol. of test sample
  26. 26. Application ooff ccoolloorriimmeettrriicc aassssaayy Used in determination of amount of many substances in blood, urine, saliva, CSF & other specimens. Ex for common colorimetric assay are : Determination of blood glucose, Blood urea, Serum creatinine, Serum proteins, Serum cholesterol, Serum inorganic phosphate, Urine creatinine Glucose in CSF
  27. 27. OOtthheerr MMeetthhooddss a. Spectrophotometry b. Molecular fluorescence technique c. Atomic spectroscopy I. Flame emission photometry- most common in trace mineral analysis II. Atomic fluorescence III. Plasma emission spectroscopy **Entirely restricted to visible region of the of the spectrum d. Atomic absorption spectrophotometry e. Magnetic resonamce spectroscopy- Study of molecular stucture and biochemical reactions
  28. 28. Flame emission photometry
  29. 29. FFllaammee eemmiissssiioonn pphhoottoommeettrryy
  30. 30. FFllaammee eemmiissssiioonn pphhoottoommeettrryy
  31. 31. AAttoommiicc AAbbssoorrppttiioonn SSppeeccttrroopphhootteemmeettrryy
  32. 32. RReeffeerreennccee David Holme and Hazel Peck (1998) Analytical Biochemistry, 3rd edition, Prentice Hall, p-504. Ayoola P.B. and Olayiwola A. O. (2014) Trace elements and major minerals evaluation of earthworm casts from a selected site in southwestern Nigeria. ARPN Journal of Agricultural and Biological Science 9(6)216-218 Lab. Manual 9, Manual of methods of analysis of foods food safety and standards authority of India, Ministry of health and family welfare government of India new delhi, 2012. Andzouana Marcel and Mombouli Jean Bievenu, (2012). Proximate, Mineral and Phytochemical Analysis of the Leaves of H. myriantha and Urera trinervis. Pakistan Journal of Biological Sciences, 15: 536-541.
  33. 33. DDiissccuussssiioonn

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