CLIN CHEM LAB - INSTRUMENTATION

• Infrared light:

  • Nernst Glower
  • Globar
• Types of light source include Incandescent tungsten/Tungsten-Iodide lamp or Halogen Quartz Lamp, Mercury/Hydrogen Lamp/Xenon Arc, Hollow Cathode Lamp, Deuterium, and Infrared light such as Nerst Glower and Globar.
• Types of Monochromators include Prism, Diffraction Gratings, and Interference Filters.
• Types of Cuvettes include Borosilicate, Quartz or Plastic, Aluminosilicate, and Soft Glass.
• Types of Photodetector include Barrier-Layer Cell (Photocell, photovoltaic), Photoemissive/Phototube, and Photomultiplier.
• Electromagnetic radiation (EMR) is also known as radiant energy
• A particle of electromagnetic radiation is known as a photon
• Electromagnetic radiation is spread in the form of electromagnetic waves
• Electromagnetic waves can cause charged particles to move up and down
• They are characterized by wavelength and frequency
• Wavelength is the distance between identical points on consecutive waves
• Frequency is the number of waves that pass a point per unit time
• Visible light:

  • Transmitted light and absorbed light are complementary
  • Objects appear in different colors because they absorb some colors and reflect or transmit others
• Different colors are due to the absorption and reflection/transmission of specific wavelengths
• Spectrophotometry is the measurement of light transmitted by a solution to determine the concentration of light-absorbing substances
• Beer-Lambert's Law states that the concentration of a substance is directly proportional to the amount of light absorbed and inversely proportional to the logarithm of light transmitted
• Absorbance is directly proportional to concentration: A = ε × b × c
• Transmittance is the relative amount of light passing through the sample
• Absorbance (A) is related to transmittance (T) and is derived mathematically
• Internal components of a spectrophotometer:

  • Light source provides energy that the sample will modify or attenuate
  • Entrance slit reduces stray light
  • Monochromator produces light of specific wavelengths
  • Exit slit allows only a narrow beam of spectrum to pass through the cuvette
  • Cuvette is the analytical cell/sample holder
  • Photodetector detects and measures radiation in the visible region
• Types of photodetectors include barrier-layer cell, photoemissive/phototube, and photomultiplier
• Photomultiplier Tube (PMT) key information:

  • PMT consists of a series of anodes known as dynodes
  • Dynodes give off secondary electrons when hit by single electrons
  • PM tube can amplify light energy, allowing it to measure very low light levels
  • PM tube is 200 times more sensitive than the phototube
• Read-out device key information:

  • Displays the amount of light transmitted
  • Types of read-out devices include meter, digital, and recorder
• Types of Spectrophotometry:

  Single-Beam Spectrophotometers:

  • Simplest types of absorption spectrometers
  • Designed to make one measurement at a time at one specified wavelength
  • The absorption maximum of the analyte must be known in advance

  Double-Beam Spectrophotometry:

  • Light is directed forward in two directions by a rotating chopper
  • One light path is directed toward the sample cuvette, while the other is directed towards the reference cuvette
  • Intensity of the two light beams is measured by one or two photodetectors
  • Sample beam is compared to the reference beam
• Flame Emission Spectrophotometry:

  • Involves measuring electromagnetic radiation emitted by excited atoms in the flame
  • Used to determine the concentration of Na+, K+, or Li+ (Group 1 metals)
  • Light source is the flame, which also serves as the cuvette

  Atomic Absorption Spectrophotometry:

  • Measures concentration by detecting the absorption of electromagnetic radiation by atoms
  • Group two metals with 2(+) charges
  • Components include a hollow cathode lamp, mechanical rotating chopper, burner, monochromator, detector, and read-out device
• Fluorometry key information:

  • Measurement of fluorescence
  • Emitted light has a longer wavelength than the absorbed light
  • Components include a light source (Mercury Vapor lamp or xenon arc lamp), excitation/primary monochromator, cuvette, emission/secondary monochromator, and photodetector
  • Relationship between Flame Emission Spectrophotometry and Atomic Absorption Spectrophotometry
• Luminescence key information:

  • Emission of light by a substance when an electron returns to the electronic ground state from an excited state
  • Three types of luminescence: fluorescence, phosphorescence, and chemiluminescence
• Nephelometry & Turbidimetry key information:

  Nephelometry:

  • Detects light scattered at various angles
  • Measures the amount of light scattered in a particulate suspension at a 90º angle

  Turbidimetry:

  • Measures a reduction in light transmission due to particle formation
  • Measures the amount of light blocked in a particulate suspension
  • Factors affecting light scatter in turbidimetry and nephelometry