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Analytical technique used commonly in laboratories

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  1. 1. Introduction • Photometry is the most common analytical technique used in the biochemical laboratory. It is designed to measure the intensity of a beam of light. • Photometric principles are applied to the several kinds of analytical techniques: (a) where absorbed or transmitted light is measured: • Colorimetry • Spectrophotometry • Atomic absorption, and • Turbidometry (b) where emitted light is measured: • Flame emission photometry
  2. 2. Introduction (cont.) • The components of most photoelectric colorimeters are basically the same and the basic method of operation is also similar for all the instruments. • In analytical chemistry, Colorimetry is a technique “used to determine the concentration of colored compounds (analytes) in sample solution” at visible spectrum of light (400 – 800 nm).
  3. 3. Colorimetry Principle: 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
  4. 4. 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.
  5. 5. Beer’s law Beer’s law
  6. 6. Lambert’s law : • 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.
  7. 7. Relationship between absorbance and transmittance OD %T
  8. 8. Preparation of solution for investigation • In colorimetric estimation it is necessary to prepare 3 solutions: BLANK(B) STANDARD(S) TEST(T) 3/18/2016 8:34 PM
  9. 9. BLANK To eliminate the effect of light absorption by the reagent used Water BLANK Reagent BLANK
  10. 10. STANDARD Solution of known concentration of the substance Both O.D and concentration are known So concentration of unknown can be calculated
  11. 11. TEST Test solution is made by treating a specific volume of the test sample with reagents As per procedure
  12. 12. Combined Beer’s- Lambert’s law • 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. • Combining the two laws: A α C x L A = K x C x L 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
  13. 13. AT AS K x CT x L K x CS x L = AT AS CT CS = CT = AT AS x CS AS = K x CS x LAT = K x CT x L
  14. 14. CT = AT AS xCS Concentration of TEST sol. Absorbance of TEST Absorbance of STANDARD Con. of STANDARDx= Concentration of TEST/100ml Absorbance of TEST Absorbance of STANDARD Concn of Std X 100 x= X ml = ODT ODS x CS
  15. 15. 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’ Concentration of TEST /100ml x= Volume of ‘T’ Amount of ‘S’T - B S - B x 100 x 100
  16. 16. Standard curve (calibration curve) • The standard curve is prepared to check whether the method of assaying a particular substance follows Beer’s Law, i.e. whether the absorbance of the substance increases in a linear way with its concentration. • The standard curve is constructed by plotting a vertical axis (y – axis, ordinate) for optical densities (absorbance) and a horizontal axis (x – axis, abscissa) the concentration of standard solution. • The concentration of the test/unknown can be measured from the graph (standard curve).
  17. 17. Verification of Beer’s Law • Prepare 1% standard solution of glucose, i.e. 1gm/dl 1000mg/100ml. • Make different dilutions of standard solution using the general formula given as following for obtaining different concentrations of a solution by dilution with diluent (DW): Tube no. Conc. (mg%) Amount of mL needed, (V1) C1 x V1 = C2 x V2 DW (mL) Total vol. V2 (ml) 1 50 1000 x V1 = 50 x 2, V1= 0.1 1.9 2 2 100 1000 x V1 = 100 x 2, V1= 0.2 1.8 2 3 150 1000 x V1 = 150 x 2, V1= 0.3 1.7 2 4 200 1000 x V1 = 200 x 2, V1= 0.4 1.6 2 5 250 1000 x V1 = 250 x 2, V1= 0.5 1.5 2 6 300 1000 x V1 = 300 x 2, V1= 0.6 1.4 2 7 350 1000 x V1 = 350 x 2, V1= 0.7 1.3 2 8 400 1000 x V1 = 400 x 2, V1= 0.8 1.2 2
  18. 18. 0.06 0.12 0.18 0.24 0.3 0.36 0.42 0.48 O.D2 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400 450 CONC.OFGLUCOSE(mg/dL) OD of solution Standard Curve / Calibration curve
  19. 19. Light spectrum and their wavelengths
  20. 20. Complimentary color • Wavelength between 400nm to 760 nm form the visible spectrum of light • Light passed through a solution which selectivity absorbs radiation at fixed wave lengths,then the color of the transmitted light is complementary to that of the absored light.
  21. 21. Colors and complimentary colors of visible spectrum Color of the solution/ solution color transmitted Filter used/ color absorbed Wavelength (nm) Yellow blue Violet 380 – 430 Yellow Blue 430 – 475 Orange Green blue 475 – 495 Red Blue green 495 – 505 Purple Green 505 – 555 Violet Yellow green 555 – 575 Blue yellow 575 – 600 Green blue Orange 600 – 650 Blue green Red 650 - 750
  22. 22. Colorimeter
  23. 23. Components of Colorimetry
  24. 24. Components of Colorimetry 1. Light source: The light source is usually a tungten lamp, for wavelength in the visible range (320 – 700nm) and a deutarium or hydrogen lamps for ultraviolet light (below 350nm). a) Tungsten lamp  Visible range b) Deutarium/hydrogen lamp (preferred)  UV Rays c) Black body radiators (Nerst glower)  Infrared radiations
  25. 25. Light source 1. Tungsten lamp:  filament mode of tungsten sealed in a glass envelope  Filed with inert gas.  Life time is limited due to gaseous tungsten formed by sublimation.
  26. 26. Light source Carbon arc lamp • If sufficient intensity of light is not obtained from tungsten lamp then carbon arc lamp can be use as a source for color measurement.
  27. 27. Monochromators/Filters • This is a means of selecting a sufficiently narrow wave band • Filter will absorb light of unwanted wavelength and allow only monochromatic light to pass through. – E.g.: a green filter absorbs all color, except green light which is allowed to pass through. Filter Absorption filter Interference filter Ex: Glass filter,Gelatin filter
  28. 28. Monochromators • Early colorimeters used Absorption filters (i.e. glass filter, Gelatin filter) that transmitted a wide segment of spectrum (50nm or more). • Newer instrument use Interface filters that consist of thin layer of magnesium fluoride crystals with a semitransparent coating of silver on each side. • Monochromator consists of: – Entrance slit – Absorption/ interface filter and – Prisms or diffraction grating for wavelength selection – Exit slit
  29. 29. Sample Holder/ Cuvette • Cuvettes are rectangular cell , square cell or circular one. • Made up of optical glass for visible wavelength (quartz or fused silica for UV). • Common one is square, rectangular to avoid refraction artifacts. • Optical path (length) of cuvette is always1cm. • Capacity may be 3ml/2ml/1ml depending upon the thickness of the wall of the cuvette. • For accurate and precise reading cuvette must be transparent, clean, devoid of any scratches and there should be no bubble adhering to the inner surface of the filled cuvette.
  30. 30. Photosensitive detectors • Detectors are the transducers, which convert light energy to electrical enagery. A detector should be possess follwing characteristics: 1.Should be sensitive 2.Should have linear response 3.Its noise level Should be low 4.Should have short response time 5.Should stable.
  31. 31. Photosensitive detectors • Different detectors used are: 1. Barrier layer cells (photocells) – simplest 2. Photoemmisive cells 3. Photomultiplier tube (for low intensity lights) 4. Photoconductive cells (photodiodes) – newest.
  32. 32. Read out devices • The detector response can be measured by any of the following devices: a) Galvanometer b) Ammeter c) Recorder d) Digital readout. The signal may be transmitted to computer or print out devices.
  33. 33. Criteria for satisfactory colorimetric estimations • Stability of color Color may be fade of air oxidation, photochemical decomposition, temperature. • Intensity of color The color of the solution should be intense in order to detect small amount of constituents and for making accurate result in low concentration.
  34. 34. Criteria for satisfactory colorimetric estimations • Clarity of the solution Substance under investigation should be completely soluble in the solvent, since turbid solution, suspension or colloidal solution absorb as well as scatter light. • Reproducibility The intensity of the colored solution must be reproducible. The effect of order of adding reagent, pH and other variable should be clearly studied
  35. 35. Criteria for satisfactory colorimetric estimations • Specificity Color produced should be specific for the desired constituent. If other constituents interfere with color reaction they be removed or prevented from or prevented from functioning through appropriate treatment like use of other coloring agent, altering the oxidation state. • Validity of Beer’s law The intensity of color should be proportional to concentration. It can be easily assessed by plotting absorbance Vs concentration, where a straight line passing through origin should be obtained.
  36. 36. Glass/gel filter is placed in the filter slot 3/4th of cuvette is filled with distilled water and placed in the cuvette slot Instrument is switched ‘on’ and allowed to warm-up for 4-5 minutes
  37. 37. Button is adjusted using ‘coarse’ and ‘fine’ knobs to give zero optical activity in the galvanometer Blank solution is placed in an identical cuvette and the OD is read (‘B’) Blank solution is transferred to the original test tube
  38. 38. Test solution is taken in the same cuvette and O.D. is read (‘T’) Test solution is transferred back to the original test tube Standard solution is taken in same cuvette and O.D. is read (‘S’) Standard solution is transferred back to the test tube Cuvette is washed
  39. 39. Applications Of Colorimeter • Estimation of biochemical compounds in blood, plasma, serum, CSF, urine, etc.: – Glucose – Urea – Creatinine – Uric Acid – Bilirubin – Lipids – Total Proteins – Enzymes [e.g. ALT, AST, ALP] – Minerals [Calcium, Phosphorus etc.] etc….