Spectrophotometry 1

1
Spectrophotometry 1

• Synopsis
• Electromagnetic Radiation
• Radiation and the molecule
• The Spectrophotometer
• Sample handling, spectral bandwidth,
• measurement at ? max, stray light
• Source unit, monochromator, detector measuring
  system

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• Electromagnetic radiation
• many compounds selectively absorb light
• for biochemistry, important regions of light are
• UV (180-350 nm)
• visible (350-800 nm)

3

• Spectrophotometry
• versatile analytical tool for
• identification (absorption spectrum)
• quantification
• highly sensitive and accurate
• may be non-destructive
• may require chemical reaction to produce
  absorbing compound

4

• Why do things appear coloured?
• many compounds absorb light
• light absorption is selective,ie. maximal at
  particular wavelengths
• eg. why do leaves appear green?
• photosynthetic pigments absorb the other colours

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• Radiation and the molecule
• light energy excites valence electrons
• when a photon of light is absorbed, the molecule
  moves to a higher energy level
• a photon is only absorbed if its energy matches
  the difference between two energy levels

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• Sample Handling
• measurements made on samples in solution
• standard cell or cuvette is sample container
• cuvettes made of glass or silica with carefully
  prepared optical windows

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• Spectral Bandwidth
• determining factor in performance of a
  spectrophotometer is the resolution ie the
  minimum separation between narrow absorption
  bands that can be observed

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• Measurement at ? max
• errors due to either wavelength setting or
  instrument calibration will be minimum when
  measurements are made at the wavelength of
  maximum absorption

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• Stray light
• Radiation emerging from the monochromator of all
  wavelengths other than the bandwidth at the
  selected wavelength
• Significant levels of stray light reduces the
  observed peak height

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• Source unit
• dual source supplied to cover the whole UV/Vis
  region (190 nm to 900 nm)
• UV derived from deuterium light source (190
  nm-380nm)
• Tungsten lamp longer wavelengths

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• Detector
• 3 types
• Phototubes
• Photomultipliers
• Silicon diode detectors

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• Measuring System
• Meter indicates output
• Digital read outs
• Microprocessor linked
• Chart recorders equipped with wavelength scanning
  provide an absorption spectrum