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• Electromagnetic radiation


  Has wave-like and particle-like properties
• Spectrum of electromagnetic radiation

  • Cosmic rays
  • Gamma rays
  • X-rays
  • Ultraviolet
  • Visible
  • Infrared
  • Microwaves
  • Radiowaves
• Wavelength


  The distance traveled by one complete wave cycle (distance between two successive crests) measured in nanometers (nm)
• The shorter the wavelength


  The greater the energy contained in the light, and the greater the number of photons
• Ultraviolet (UV) light


  Has very short wavelengths
• Infrared (IR) light


  Has very long wavelengths
• White light


  Results when all visible wavelengths of light (400-700 nm) are combined
• Visible color


  Wavelength of light transmitted (not absorbed) by an object
• Photons


  Particles of light
• What happens when an atom absorbs a photon

  1. An electron is moved to a higher energy level

  2. The mode of the covalent bond vibration is changed

  3. The rotation around its covalent bonds is changed
• Excited electron


  Is not stable and will return to ground state
• What happens when an electron returns to ground state

  It emits energy in the form of light (radiant energy) of a characteristic wavelength
• Absorption or emission of energy forms a line spectrum that is characteristic of a molecule and can help identify a molecule
• Spectrophotometer


  Measures light transmitted by a light-absorbing analyte in solution
• Components of a spectrophotometer

  • Power supply
  • Light source
  • Entrance slit
  • Monochromator
  • Exit slit
  • Cuvet/sample cell
  • Photodetector
  • Readout device
• Light source or exciter lamp


  Produces an intense, reproducible, constant beam of light
• Types of incandescent lamps

  • Tungsten
  • Deuterium
• When a lamp is changed in the spectrophotometer, the instrument must be recalibrated, because changing the light source changes the angle of the light striking the monochromator
• Monochromators


  • Glass filters
  • Interference filters
  • Diffraction gratings
  • Prisms
• Bandpass or spectral bandwidth


  The range of wavelengths in nanometers that is transmitted by the monochromator and exit slit between two points of a spectral scan where the light transmitted is one-half of the peak (maximum) transmittance
• Wavelength selection


  1. Entrance slit allows lamp light to enter

  2. Monochromator disperses the light into wavelengths

  3. Exit slit selects the bandpass of the monochromator that allows light of the selected wavelength to pass through the cuvet onto the detector
• Photodetectors


  Convert the electromagnetic radiation (light energy) transmitted by a solution into an electrical signal
• Readout devices


  Display the electrical energy from a detector on a digital display or readout system
• Atomic Absorption Spectrophotometry


  Ground-state atoms absorb light at defined wavelengths
• Line spectrum


  Refers to the wavelengths at which an atom absorbs light; each metal exhibits a specific line spectrum
• Atomic Absorption Spectrophotometry


  1. The sample is atomized in a flame where the atoms of the metal to be quantified are maintained at ground state

  2. A beam of light from a hollow-cathode lamp (HCL) is passed through a chopper to the flame

  3. The ground-state atoms in the flame absorb the same wavelengths of light from the HCL as the atoms emit when excited

  4. The light not absorbed by the atoms is measured as a decrease in light intensity by the detector
• The difference in the amount of light leaving the HCL and the amount of light measured by the detector is indirectly proportional to the concentration of the metal analyte in the sample
• Components of Atomic Absorption Spectrophotometry

  • Hollow-cathode lamp
  • Chopper
  • Burner head for flame
  • Monochromator
  • Detector
  • Readout device
• Hollow-cathode lamp


  Contains an anode, a cylindrical cathode made of metal being analyzed, and an inert gas such as helium or argon
• How a hollow-cathode lamp works

  1. Applied voltage causes ionization of the gas, and these excited ions are attracted to the cathode, where they collide with the metal coating on the cathode, knocking off atoms and causing atomic electrons to become excited

  2. When the electrons of the metal atoms from the cathode return to ground state, the characteristic light energy of that metal is emitted
• Vaporized metal atoms from the sample can be found in the flame. The flame serves as the sample cuvet in this instrument
• How the light from the hollow-cathode lamp is used

  1. The light produced in the HCL passes through a chopper and then to the flame, and the light is absorbed by the metal in the sample

  2. The light not absorbed will be read by the photomultiplier tube
• Flameless system


  • Employs a carbon rod (graphite furnace), tantalum, or platinum to hold the sample in a chamber
  • The temperature is raised to vaporize the sample being analyzed
  • The atomized sample then absorbs the light energy from the HCL
• Nephelometry


  The measurement of light scattered by a particulate solution
• The sensitivity of nephelometry depends on the absence of background scatter from scratched cuvets and particulate matter in reagents
• Turbidimetry


  Measures light blocked as a decrease in the light transmitted through the solution; dependent on particle size and concentration
• Turbidimetry uses a spectrophotometer for measurement, and it is limited by the photometric accuracy and sensitivity of the instrument
• Fluorescence


  A process where atoms absorb energy at a particular wavelength (excitation), electrons are raised to higher-energy orbitals, and the electrons release energy as they return to ground state by emitting light energy of a longer wavelength and lower energy than the exciting wavelength
• Fluorometry


  • Frequently UV light is used for excitation and is passed through a primary filter for proper wavelength selection for the analyte being measured
  • The excitation light is absorbed by the atoms of the analyte in solution, which causes the electrons to move to higher-energy orbitals
  • Upon return to ground state, light is emitted from the fluorescing analyte and that light passes through a secondary filter
  • The secondary filter and the detector are placed at a right angle to the light source to prevent incident light from being measured by the detector
• Advantages of fluorometry


  • It is about 1000 times more sensitive than absorption techniques and has increased specificity because optimal wavelengths are chosen both for absorption (excitation) and for monitoring emitted fluorescence