CONCEPTS

• Spectrophotometry

  • Method of determining the concentration of substance in solution by measuring the amount of light absorbed by that solution after appropriate treatment.
• Theory of light waves:

  • Electromagnetic radiation
  • Wavelength
  • Amplitude
  • Visible light
• Electromagnetic radiation

  • photons travelling in waves.
• Wavelength

  • distance between two peaks
• Amplitude

  • distance between peak and trough.
• Visible light: 400-700nm
• Beer’s Law or Beer-Lambert’s Law

  • States that the concentration of the unknown substance is directly proportional to the absorbed light (absorbance or optical density) and inversely proportional to the amount of transmitted light (% transmittance)
• EEP - Emission Flame Photometry
• FES - Filter Photometry Flame Emission Spectrophotometry
• Emission Flame Photometry (EEP) or Filter Photometry Flame Emission Spectrophotometry (FES)

  • The measurement of emitted light when electrons in an atom becomes excited by heat energy produced by the flame.
  • Excited atoms return to ground state by emitting light energy.
  • It is used primarily to determine the concentration of sodium, potassium or lithium since these alkali metals are easy to excite.
• Purposes of the Flame in the EEP

  • Breaks the chemical bond to produce atoms.
  • Source of energy absorbed by the atoms to enter an excited state
• Components of the Flame Photometer

  • Gases
  • Atomizer or Burner
  • Interference Filters as Monochromator
  • Detector
• Gases

  • Using a mixture of hydrogen and oxygen gas (acetylene, propane or natural gas)
• Atomizer or Burner

  • Breaks up the solution into finer droplets so that the atom will absorb heat energy from the flame and get excited.
• Two types of Burners

  • Total Consumption Burner
  • Premix Burner
• Total Consumption Burner

  • Aspirate sample directly into the flame, the gases are passed at high velocity over the end of the capillary suspended in the solution.
• Premix Burner

  • Involves the gravitational feeding of solution through a restricting capillary into an area of high velocity gas flow where small droplets are produced and passed into the flame.
• Interference Filters as Monochromator

  • Na
  • K
  • Lithium
• Na Filter transmit yellow light (589nm)
• K Filters transmit violet light (767nm)
• Lithium transmit red light (761nm)
• Lithium

  • Preferred internal standard and also act as a radiation buffer
• Reasons why lithium is preferred

  • Its emission characteristics are similar to those of Na+ and K+
  • Normally present as a trace element in human tissues and does not present interferences in the determination
• Purposes of Using Lithium

  • To achieve stability where there is fluctuations caused by changes in fuel of air pressure which affects flame temperature and rate of sample aspiration.
• Criteria in Choosing the Internal Standard

  • Its concentration must be precisely the same in all samples and standard.
  • Energy required of the internal standard must be close to that required to excite the elements being measured.
  • Must not be normally found in solution being analyzed.
• In cases where lithium is the analyte, cesium is used as internal standard.
• Fluorometry

  • Measurement of the concentration of solutions that contain fluorescing molecules
• Xenon lamp

  • most common light source
• Fluorometry applications

  • Used to measure small particles such as DRUGS.
• Chemiluminescence

  • Chemical energy generated in a cehmiluminiscent reaction produces excited intermediates that decay to a ground state with the emission of photons; no excitation is required unlike in fluorometry.
• AAS - Atomic Absorption Spectrophotometry
• Atomic Absorption Spectrophotometry (AAS)

  • Measures concentration of element by detecting absorption of electromagnetic radiation by atom, rather than by molecules.
  • The element is not excited but they are dissociated from their chemical bonds and placed in the unionized, unexcited ground state.
  • It is ideal for alkali metals that are not easily excited (e.g. calcium, magnesium).
• Components

  • Light Source
  • Mechanical Rotating Chopper
  • Burner
  • Monochromator
  • Detectors
  • Meter or Read-Out Device
• Light Source

  • Hallow cathode lamp, which produces a wavelength of light, specific for the kind of metal in the cathode.
• Mechanical Rotating Chopper

  • Modulates light beam coming from the hollow cathode lamp.
• Burner

  • Uses flame to dissociate the chemical bond and form free, unexcited atom.
• Total Consumption Burner

  • Flame is more concentrated and can be hotter, thus lessening chemical interferences.
  • However, it produces large droplets in the flame and produces a high acoustical noise.
• Premix Burner

  • Unlike the total consumption burner, gases are mixed and the sample is atomized before entering flame and the large droplets go to waste and not in the flame.
  • It has less noisy signals with longer pathlength and greater absorption and sensitivity.
  • However, flame is less hotter and therefore cannot dissociate metal complexes.
• Monochromator

  • Selects the desired wavelength from a spectrum of wavelength which could either be a prism or a diffraction gratings.
• Detectors

  • Uses photomultiplier tubes to measure the intensity of light signal.