SPECTROPHOTOMETER (LAB)

• Spectrophotometer

  • A MACHINE THAT MEASURE THE AMOUNT OF LIGHT ABSORBED BY A SAMPLE.
• History:

  • In older times, it tooks weeks for results to come out and most of the time, there was only 25 percent accuracy of the result.
• Arnold J. Beckman

  • invented the Beckman DU spectrophotometer in 1940.
• National Technologies Laboratory (NTL)

  • Arnold J. Beckman and his colleagues at what laboratory they invented Spectrophotometer.
• History

  • Results come through simple process within few minutes and with 99.99% accuracy.
• Beer's Law or Beer's 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)
• Parts of Spectrophotometer

  • Light Source
  • Collimator (Lens 0
  • Monochromator (Prism or Grating)
  • Wavelength Selector (Slit)
  • Sample Solution (Cuvette)
  • Detector (Photocell)
  • Digital Display or Meter
• Light Source

  • Provides radiant energy in the form of visible or non-visible light that may pass through the monochromator.
  • The light of proper wavelength will be made incident on the analytical cell.
• Types of Light Source

  • Tungsten Iodine Lamp
  • Quartz Halide Lamp
  • Deuterium Discharge Lamp
  • Infrared Energy Source
  • Mercury Vapor Lamp
  • Hallow Cathode Lamo
• Tungsten Iodine Lamp

  • Produces energy wavelength from 340 to 700 nm (visible region).
  • It is used for moderately diluted solution
• Quartz Hallide Lamp

  • Contains small amounts of halogen such as iodine to prevent the decomposition of the vaporized tungsten from the very hot filament.
• Deuterium Discharge Lamp

  • Provides energy source with high output in the UV range (down to 165 nm)
  • Deuterium lamps are more stable and generates continuous or discontinuous spectral.
• Infrared Energy Source

  • Used above 800 nm
• Merst Glower

  • an electrically heated rod of rare earth element oxides
• Globar

  • uses silicon carbide
• Mercury Vapor Lamp

  • Exits narrow bands of energy at well defined places in the spectrum (UV and visible)
• Hallow Cathode Lamp

  • Consists of a gas-tight chamber containing anode, a cylindrical cathode, and inert gas such as helium and argon.
• Entrance Slit

  • Minimizes unwanted or stray light and prevents the entrance of scattered light into the monochromator system
• Stray Light

  • refers to any wavelength outside the band transmitted by the monochromator, it causes absorbance error
• • Stray light limits the maximum absorbance that a spectrophotometer can achieve.
• • Stray light is the most common cause of loss linearity at high-analyte concentration.
• Monochromator

  • Isolate specific wavelength of light.
• Monochromator light

  • light radiation of a single wavelength
• Types of Monochromators

  • Prism
  • Gratings
  • Filters
• Prism

  • Wedge-shaped pieces of glass, quarts, NaCl, or some other material that allows transmission of light
• Prism

  • Disperse white light into a continuous spectrum of colors based on variation of refractive index for different wavelength
  • Can be rotated, allowing only the desired wavelength to pass through an exit slit
• Gratings

  • Has small grooves cut at such an angle that each groove behave like a very small prism
  • Separates white light into various color component
  • Based on the principle that wavelengths are bent as they pass a sharp corner
• 2 Types of Filters

  • Colored Filters
  • Interference Filters
• Colored Filters

  • Made of glass that absorb some portion of the electromagnetic spectrum and transmit others
  • Light energy is absorb by dye compounds on the glass and is dissipated as heat
• Colored Filters

  • Band pass in 35 to 50 nm or more
• Interference Filters

  • Utilizes the wave character of light to enhance the intensity of the desired wavelength by constructive interference and reflection.
• Interference Filters

  • Band pass is 10 to 20 nm
• Advantages of Gratings over Prisms

  • Produces linear spectrum and therefore maintaining a constant band pass which is simple.
  • Can be used in the regions of spectrum where light energy is absorbed by glass prism
• Exit Slit

  • It controls the width of light beam (bandpass) – allows only a narrow fraction of the spectrum to reach the sample cuvette. Spectral purity of the spectrophotometer is reflected by the bandpass – the narrower the bandpass, the greater the resolution.
• Bandpass

  • the range of wavelengths between the points at which transmittance is one half peak transmittance
• Exit Slit

  • Accurate absorbance measurement requires a bandpass <1/5 the natural bandpass of the spectrophotometer.
  • The degree of wavelength isolation is a function of the type of device used and the width or entrance and exit slit
• Analytical Cell or Cuvette

  • Used to hold the solution in the instrument whose concentration is to be measured.
  • It is made of glass, quartz or plastic.
• Types of Cuvette

  • Borosillicate Glass Cuvette
  • Quartz or Plastic
  • Alumina Silica Glass
• Borosillicate Glass Cuvette

  • for solution that do not etch glass
• Quartz or Plastic

  • does not absorb UV radiation at wavelength below 320 nm