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C O L O R I M E T R Y

C O L O R I M E T R Y. Prepared By Michigan Department of Environmental Quality Operator Training and Certification Unit. Note: A printed description of colorimetry is available in the OTCU Laboratory manual (Section 310) available on the OTCU website. C O L O R I M E T R Y.

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C O L O R I M E T R Y

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  1. COLORIMETRY Prepared By Michigan Department of Environmental Quality Operator Training and Certification Unit Note: A printed description of colorimetry is available in the OTCU Laboratory manual (Section 310) available on the OTCU website.

  2. COLORIMETRY Color Measurement

  3. COLOR Interaction between LIGHT and MATTER

  4. Matter “ORBIT” Energy Level of Electrons Nucleus Electrons

  5. Orbits = Energy Level Level 1 Level 2 Level 3 Each Electron Can Be In Only Certain Energy Levels

  6. (time) LIGHT Photon - “Energy Packet” wavelength -  (lambda) Wave frequency -  (gamma)

  7. TheEnergy (E)of the Photon is Related to thewavelength and thefrequency of the Wave () () LIGHT hc  E = h = Where: h = Planck’s Constant c = Velocity of Light

  8. hc  E = LIGHT Constants Every wavelength (l) has a specific Energy level.

  9. Summary Each Electron Can Be In Only Certain Energy Levels Every wavelength has a specific Energy Level.

  10. COLORIMETRY 600 nm 650 nm 700 nm

  11. COLOR RESULTS WHEN RADIATION IS ABSORBED BY AN ELEMENT OR BY A COMPOUND FORMED THROUGH A REACTION RED YELLOW RED ABSORBED YELLOW W H I T E GREEN BLUE BLUE

  12. TRANSMITTANCE (T) RATIO OF THE INTENSITY OF LIGHT LEAVING SOLUTION (I) TO THE INTENSITY OF LIGHT ENTERING SOLUTION (IO)

  13. TRANSMITTANCE IO I I T = IO %T = T x 100

  14. Comparing Light Transmittance to Cell Length 1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0 I0 Transmittance I1 I2 I3 I5 In I4 0 1 2 3 4 5 Units of Optical Path

  15. LAMBERT’S LAW Relates the absorption of light to the depth or thickness of the colored liquid Each layer of equal thickness will absorb the same fractionof light which passes through it An arithmetic increase in thickness gives a geometric decrease in light intensity transmitted

  16. Comparing Light Transmittance to Concentration 1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0 I0 Transmittance I1 I2 I3 I5 In I4 0 1 2 3 4 5 Units of Concentration

  17. BEER’S LAW Relates the absorption of light to the concentration of the absorbing substance in the solution The fraction of light absorbed is directly proportional to the concentration of the absorbing substance An arithmetic increase in concentration gives a geometric decrease in light intensity transmitted

  18. COLORIMETRY Perform a Chemical Reaction with the Element to be Analyzed that Results in a Compound of that Element that Absorbs Light. How Do We Use This Principle? Measure the Amount of Light Absorbed.

  19. COLORIMETRY The Amount of Light Absorbed Is Related To: • The Chemistry Involved. • 2. The Length of Light Travel. • 3. The Amount (Concentration) of Absorbing Material.

  20. I I o T = THE COMBINED LAMBERT’S LAW AND BEER’S LAW T = 10 -abc Where: a = constant for particular solution b = length of absorbing layer (light path length) c = concentration of absorbing substance {- Sign Indicates an Inverse Relation}

  21. I Io T = TRANSMITTANCE Absorbance = A = - log T T = 10 -abc log T = log (10 -abc) log T = -abc -log T = -(-abc) = abc -log T = abc A =

  22. ABSORBANCE (A) A = - log T A = abc Where: a = constant for particular solution b = length of absorbing layer (light path length) c = concentration of absorbing substance

  23. ABSORBANCE (A) A = - log T A = abc If: a = held constant by carefully performing the analysis b = held constant by controlling the light path length Then: A is Directly Related to c (conc. of absorbing substance) If we can measure A, then we can determine c

  24. COLORIMETRY Measurement of the amount of LIGHT ABSORBED by the COLOR DEVELOPED in a sample

  25. CONCENTRATION CAN BE COLORIMETRICALLY DETERMINED IF: 1. Able to chemically develop a color with that substance and only that substance 2. The developed color obeys (follows) Beer’s Law over a reasonable range of concentrations 3. The developed color must be stable for reasonable length of time, reproducible, and sensitive to small changes in concentration 4. All loss of transmitted light must be from absorbance by substance measured (developed color) 5. All of substance present in sample must be available for reaction with color developing agent 6. Able to measure amount of light absorbed

  26. Sample Preparation Dilution Solids Removal --- Coagulation --- Centrifuge --- Filter pH Adjustment Digestion

  27. DIGESTION Destroy Organics Release Combined Constituent Change Form of Constituent

  28. Colorimetry Color Development Color Must Be: Stable Reproducible Sensitive

  29. Color Development Must Control : pH Time Temperature Ionic Strength

  30. COLORIMETRY Measurement of the amount of LIGHT ABSORBED by the COLOR DEVELOPED in a sample

  31. Color Measurement Compare Sample Color to Known Standards “Color Comparators” O.K. For Control – Not For Reporting

  32. Color Measurement Compare Sample Color to Known Standards Spectrophotometer “Calibration Curve” (verified)

  33. Colorimetric Instruments

  34. Spectrophotometer Sample Cell Detector Monochromator Light Source Meter

  35. Light Source WHITE LIGHT Constant Controllable Voltage Regulation Diaphragm Fatigue Voltage Adjustment

  36. Color (wavelength) Band

  37. Monochromator APERATURE OR SLIT PRISM OR DIFFRACTION GRATING Must be CAREFULLY Adjusted

  38. Sample Cell The Light Path is affected by the Cuvette Cuvette

  39. Sample Cell Must be CAREFULLY Aligned Cuvette

  40. PHOTOELECTRIC TUBE “DETECTOR” Differing Response for Various Wavelengths Bausch & Lomb 33-29-71 340-600 nm 33-29-72 (w / filter) 600-950 nm 33-29-92 (w / filter) 400-700 nm

  41. PHOTOELECTRIC TUBE “DETECTOR” Differing Response for Various Wavelengths Must Use the Correct Combination of Filter and Phototube For Wavelength Of Analysis

  42. INDICATING METER Gives the Readout in Transmittance or Absorbance

  43. INDICATING METER Some Meters Give Readout Directly in Concentration Use Only those Readings Between the Lowest and Highest Standard of Calibration

  44. INDICATING METER Some Meters Have “Built-in” Calibration These Calibrations Should Be Verified Periodically Using a Series of Standards and Only those Readings Between the Lowest and Highest Standard of Calibration Should be Used

  45. Optical System Lenses Mirrors Apertures Occluders

  46. Optical System The Instrument Must be Carefully Handled, Protected From Dust and Vapors, and Serviced Only By Qualified Technicians

  47. Spectrophotometer Sample Cell Monochromator Detector Light Source Meter

  48. COLORIMETRY Instrument Operation: Warm-up Set Monochromator Set ∞ Absorbance Set Zero Absorbance w/Blank Re-adjust as Needed

  49. COLORIMETRY Instrument Operation: General Rule – Absorbance Between 0.100 and 0.700 Some Analyses More Restrictive Best Readings – Between Lowest and Highest Standards Used In Calibration Watch for Irregularities

  50. COLORIMETER CALIBRATION Calibration or Standardized By Measuring Absorbance Readings of a Series of Known Standards Comparison of These Readings to the Reading for a Sample 1. Computer Spreadsheet 2. Instrument with Internal Microprocessor 3. “Plotting” a Graph

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