SPECTRO LAB

• SPECTROPHOTOMETER


  A MACHINE THAT MEASURE THE AMOUNT OF LIGHT ABSORBED BY A SAMPLE
• It tooks weeks for results to come out and most of the time, there was only 25 percent accuracy of the result


  In older times
• Scientist Arnold J. Beckman and his colleagues at the National Technologies Laboratory (NTL) invented the Beckman DU spectrophotometer


  1940
• Results come through simple process within few minutes and with 99.99% accuracy
• 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)
• Light Source


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


  • Produces energy wavelength from 340 to 700 nm (visible region)
  • It is used for moderately diluted solution
  • Contains small amounts of halogen such as iodine to prevent the decomposition of the vaporized tungsten from the very hot filament
• Quartz Halide Lamp


  • Provides energy source with high output in the UV range (down to 165 nm)
• Deuterium Discharge Lamp


  • Deuterium lamps are more stable and generates continuous or discontinuous spectral
• Infrared Energy Source


  • Used above 800 nm
  • Examples: 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
• Types of Monochromators


  • Prism
  • Gratings
  • Filters
• Prism


  • Wedge-shaped pieces of glass, quarts, NaCl, or some other material that allows transmission of light
  • 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
• 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
  • Band pass in 35 to 50 nm or more
  • Colored Filters
  • Utilizes the wave character of light to enhance the intensity of the desired wavelength by constructive interference and reflection
  • Band pass is 10 to 20 nm
  • Interference Filter
• 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
• 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 – for solution that do not etch glass
  • Quartz or Plastic – does not absorb UV radiation at wavelength below 320 nm
  • Alumina Silica Glass – good for 340 nm and above (visible)
• Photodetectors


  Electron tube amplifying a current that can convert transmitted energy into an equivalent amount of electrical or photoelectric energy
• Kinds of Detectors


  • Barrier Layer Cell (Photocell/Photovoltaic Cell)
  • Phototube (photoemissive tube)
  • Photomultiplier (PM) Tube
  • Photodiode
• Barrier Layer Cell (Photocell/Photovoltaic Cell)


  • Simpliest detector, least expensive; temperature sensitive
  • Composed of selenium on a plate of iron covered with transparent layer of silver
  • It requires an external voltage source but utilized internal electron transfer for current production – low internal resistance
  • It is used in filter photometers with a wide bandpass
• Phototube (photoemissive tube)


  • It contains cathode and anode enclosed in a glass case
  • It has a photosensitive material that gives off electron when light energy strikes it
  • It requires external voltage for operation
• Photomultiplier (PM) Tube


  • Most common type – measures visible and UV regions
  • Excellent sensitivity and rapid response – detects very low levels of light
  • Detects and amplifies radiant energy
  • It should never be exposed to room light because it will burn out
• Photodiode


  • Not as sensitive as PM; excellent linearity
  • Measures light at a multitude of wavelengths
  • It detects less amount of light
• Meter


  • Simplest method of displaying output of the detection system
  • Also called READ-OUT DEVICE
• Applications of Spectrophotometers


  • Detection of concentration of substances
  • Detection of impurities
  • Structure elucidation of organic compounds
  • Monitoring dissolved oxygen content in freshwater and marine ecosystems
  • Characterization of proteins
  • Detection of functional groups
  • Respiratory gas analysis in hospitals
  • Molecular weight determination of compounds
• The visible and UV spectrophotometer may be used to identify classes of compounds in both the pure state and in biological preparations