CONCEPT

• Electromagnetic radiation


  Photons travelling in waves
• Wavelength


  Distance between two peaks
• Amplitude


  Distance between peak and trough
• 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)
• Spectrophotometry


  Determining the concentration of substance in solution by measuring the amount of light absorbed by that solution after appropriate treatment
• Components of Spectrophotometer


  • Emission Flame Photometry (EEP) or Filter Photometry
  • Flame Emission Spectrophotometry (FES)

  
• Flame Emission Spectrophotometry (FES)

  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
• Atomizer or Burner


  • Breaks up the solution into finer droplets so that the atom will absorb heat energy from the flame and get excited
• Types of Burner


  • Total Consumption Burner
  • Premix Burner
• Sodium (Na) Filter


  Transmit yellow light (589 nm)
• Potassium (K) Filter


  Transmit violet light (767 nm)
• Lithium


  Preferred internal standard, also acts as a radiation buffer
• Fluorometry


  Measurement of the concentration of solutions that contain fluorescing molecules
• Components of Fluorometry


  • Xenon lamp- most common light source
• Application of Fluorometry


  Used to measure small particles such as DRUGS
• Chemiluminescence


  Chemical energy generated in a chemiluminiscent reaction produces excited intermediates that decay to a ground state with the emission of photons; no excitation is required unlike in fluorometry
• Components of Atomic Absorption Spectrophotometry

  • Light Source
  • Mechanical Rotating Chopper
  • Burner
  • Monochromator
  • Detector
  • Meter or Read-out Device

  
• Hallow cathode lamp


  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
• Monochromator


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

  
• Detector


  Uses photomultiplier tubes to measure the intensity of light signal

  
• Chemical Ionization


  Matrix Interferences
• Reflectrometry


  Measures quantity of light reflected by a liquid sample that has been dispensed onto a grainy or fibrous solid support

  
• Applications of Reflectrometry


  • Urine Dipstick
  • Dry Slide Chemical Analysis
• Turbidimetry


  Turbidimetric measurements are made with a spectrophotometer to determine concentration of particulate matter in sample. The amount of light blocked by a suspension of particles depends not only on concentration but also in size

  
• Applications of Turbidimetry


  • Detection of bacterial growth and culture
  • Antibiotics sensitivity
  • Coagulation studies
  • Protein concentration in CSF and Urine
• Nephelometry


  Light scatted by small particles is measured at an angle to the beam incident to the cuvet. Nephelometrers measure particles which are too large for spectrophotometry, such as antibody-antigen complexes (proteins) formed in enzyme immunoassays

  
• Electrochemistry Techniques


  • Potentiometry
  • Amperometry
  • Coulometry
  • Voltametry
• Potentiometry


  Measurmenet of potential (voltage) between two electrodes in a solution to measure analyte concentration
• Applications of Potentiometry


  • pH, pCO2, Na+, Ca2+, K+, NH4+
• Amperometry


  Measurement of the current flow produced by an oxidation-reduction reaction
• Applications of Amperometry


  • pO2 (Clark electrode), glucose, peroxidase
• Coulometry


  Electrochemical titration in which the titrant is electrochemically generated
• Applications of Coulometry


  • Cl-
• Voltametry


  Potential is applied to an electrochemical cell and the resulting current is measured
• Applications of Voltametry


  • Anodic stripping voltametry (for lead and iron)
• Electrophoresis


  Separation of charged compounds based on their electrical charge
• Components of Electrophoresis


  • Driving force (electrical power)
  • Support medium
  • Buffer
  • Sample
  • Detecting system