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• • Fluorescence polarization - The incident light is polarized in a single plane
• • Polarized - light sent through a filter so all the light that is sent through the filter is polarized in the same plane 
• Fluorescence polarization

  • the radiant energy is polarized in the same single plane as the incident light by large molecules
• FPIA is a common chemistry lab immunoassay
• • Flame emission photometry
  • Not widely used
  • Heat is used instead of light to excite electrons
  • Na+, K+, and Li+ were most commonly tested ions
  • Different ions would glow different flames
• flame emission photometry has been replaced by ion selective electrodes
• • Atomic absorption spectrophotometry
  • Measures atoms rather than molecules
  • Measures trace metals in a vaporized state
• mass spectrometry - uses energy to determine the molecular mass of substances we want to quantify
• mass spectrometry in the clinical lab is used mainly for trace metals and drugs of abuse
• mass spectrum - read out or print out of sending sample through mass spectrometer
• mass spectrometry is often paired with gas chromatography
• mass spectrometry

  • Bombards compound with electrons, compound breaks down into fragments
• • Every compound will break down in same fragments every time it is bombarded with electrons in mass spectrometry
• • Iontophoresis - migrate or separate small ions; almost never used; seen in electrophoresis labs
• • Macromolecules of interest in electrophoresis - major serum proteins, immunoglobulins, hemoglobin 
• hemoglobin electrophoresis in the lab is largely used for hemoglobin A1C and abnormal hemoglobin types
• support materials for electrophoresis include cellulose acetate, agarose gel, and polyacrylamide gel
• Electrophoresis

  A) wick into buffer

  B) sample application point

  C) support media

  
• • Densitometer - measures density of each individual band and graphs it 
  • Similar in design to spectrophotometer
• • Instead of fluorophore, when it is oxidized, it gives off light (called chemiluminescence)
• chromatography is electrophoresis without the electric
• ID of components in chromatography is done through different ways: e.g. color or how far they traveled
• high performance liquid chromatography uses pressure to separate the components
• the column in high performance liquid chromatography must be able to withstand pressure and must be able to slow down components
• gas chromatography

  • Separates mixtures that are volatile or can be made volatile (relatively low boiling point)
• Cannot use gas chromatography if the compound’s boiling point is above a certain temperature; looking at compounds that evaporate easily
• gas chromatography

  • Column is a very small, thin, hollow tube made of some type of metal
  • inside tube is gel
• Gas chromatography separates components of mixture (usually by size), then goes through mass spectrometer, bombards with electrons, and breaks up into individual fragments
• • Usually gas chromatography is paired with mass spec or some other method
  • Gas chromatography will not by itself ID the components that are separated, need to be paired with something else