Spectrophotometry and Photometry
   Spectrophotometry and Photometry
    • Photometric instruments measure light intensity without 
      consideration of wavelength.
       – Most instruments use filters (photometers), prisms, or 
          gratings (spectrometers) to select (isolate) a narrow 
          range of incident wavelength.
    • Beer Law: Concentration of a substance is directly 
      proportional to right amount of light absorbed or inversely 
      proportional to logarithm of transmitted light.
    • Spectrophotometric Instruments: measure light 
      transmitted by a solution to determine concentration of 
      light-absorbing substance in solution.
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   Spectrophotometry and Photometry 
   Spectrophotometry and Photometry 
   (cont.)
   (cont.)
     • Components of a Spectrophotometer
        – Light source
        – Monochromators
        – Sample cell
        – Photodetectors
     • Spectrophotometer Quality Assurance
        – Wavelength accuracy
        – Stray light
        – Linearity 
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  Spectrophotometry and Photometry 
  Spectrophotometry and Photometry 
  (cont.)
  (cont.)
    • Single-beam spectrophotometer—see Figure 5.5
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   Spectrophotometry and Photometry 
   Spectrophotometry and Photometry 
   (cont.)
   (cont.)
     • Atomic Absorption Spectrophotometer
         –  Measures concentration by detecting absorption of 
            electromagnetic radiation by atoms rather than by molecules
         –  Sensitive, precise
         –  Routinely used to measure concentration of trace metals that 
            are not easily excited
         –  See Figure 5.11
     • Flame Photometry
         –  Measures light emitted by excited atoms
         –  Was used to determine concentration of Na, K, or Li
         –  No longer routinely used; replaced by ion-selective electrodes
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   Spectrophotometry and Photometry 
   Spectrophotometry and Photometry 
   (cont.)
   (cont.)
    • Fluorometry
        – Basic instrumentation
            • Measure concentrations of solutions that contain 
              fluorescing molecules.
            • Emits short-wavelength high-energy excitation light.
            • Mechanical attenuator controls light intensity.
        – Advantages and disadvantage of fluorometry
            • Advantages: greater specificity and sensitivity
            • Disadvantage: very sensitive to environmental 
              changes
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