SPECTRO

• Spectrophotometer


  An analytical instrument used to measure the intensity of light passing through a sample, and to determine the concentration of chemical species in the sample

  
• Analytical Methods and Instrumentation

  • Electromagnetic Energy
  • Wavelength
  • Frequency
  • Amplitude

  
• Electromagnetic Energy


  Radiant energy from short wavelength gamma rays to long wavelength radio waves. They are photons of energy traveling in a wavelike manner. The shorter the wavelength, the higher the electromagnetic energy.

  
• Types of Electromagnetic Energies

  • Visible spectrum – 400 – 700nm
  • Ultraviolet region (UV) - < 400nm
  • Infrared region (IR) - > 700nm

  
• Wavelength


  The distance between peaks as light is envisioned to travel in a wavelike manner. It is expressed in terms of Nanometer (nm), Angstrom (A), and Millimicrons (mµ).

  
• Frequency


  The number of vibrations of wave motions per second, derived from the Planck's Formula. The lower the wave frequency, the longer the wavelength.

  
• Amplitude


  The distance between peak and trough. The higher the amplitude, the more intense the light = the more light energy.

  
• The wavelength is inversely related to frequency and energy; the shorter the wavelength, the higher the frequency and energy and vice versa.

  
• The Visible Light Wavelength and Corresponding Absorbed/Transmitted Colors

  • 350-430 nm: Violet/Yellow
  • 430-475 nm: Blue/Orange
  • 475-495 nm: Blue Green/Red-Orange
  • 495-505 nm: Green-Blue/Orange-Red
  • 505-555 nm: Green/Red
  • 555-575 nm: Yellow-Green/Violet-Red
  • 575-600 nm: Yellow/Violet
  • 600-650 nm: Orange/Blue
  • 650-700 nm: Red/Green

  
• Colorimetry


  Two primary considerations: Quality of the color and Intensity of the color
• Kinds of Colorimetry


  • Visual colorimetry
  • Photoelectric colorimetry (Spectrophotometric measurement, Photometric measurement)
• Types of Spectrophotometers


  • Single Beam
  • Double Beam

  
• Single Beam Spectrophotometer


  All the light passes through the sample. To measure the intensity of the incident light, the sample must be removed so that all the light can pass through.

  
• Double Beam Spectrophotometer


  The light source is split into two separate beams. One passes through the sample and the other is used for reference.

  
• Types of Spectrophotometers by Light Source

  • Visible Light (400-700 nm)
  • Ultraviolet Light
  • Infrared Light

  
• Ultraviolet light Spectrophotometer


  Used for fluids and solids. Quartz cuvettes are used for placing the samples.

  
• Visible light Spectrophotometer

  Can use incandescent, halogen, LED, or a combination of these sources and vary in accuracy.

  
• Infrared Spectrophotometer


  Helps to study different structures of molecules and their vibrations.

  
• Parts of the Spectrophotometer

  • Light Source
  • Entrance Slit
  • Monochromator
  • Exit Slit
  • Analytical Cell or Cuvette
  • Photodetectors
  • Meter/Read-out Device

  
• Light Source


  Provides radiant energy in the form of visible or non-visible light that may pass through the monochromator.

  
• Entrance Slit


  Minimizes unwanted or stray light and prevents the entrance of scattered light into the monochromator system.
• Monochromator


  Isolates specific wavelength of light.

  
• Types of Monochromators


  • Prism
  • Gratings
  • Filters
• Exit Slit


  Controls the width of light beam (bandpass) - allows only a narrow fraction of the spectrum to reach the sample cuvette. Spectral purity is reflected by the bandpass - the narrower the bandpass, the greater the resolution.
• 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 Cuvettes


  • Borosilicate Glass Cuvette – for solutions 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


  • Barrier Layer Cell (Photocell/Photovoltaic Cell)
  • Phototube (Photoemissive tube)
  • Photomultiplier (PM) Tube
  • Photodiode
• Meter/Read-out Device


  Simplest method of displaying output of the detection system.
• 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).
• Quality Assurance in Spectrophotometry

  • Wavelength/Photometric Accuracy (Didymium glass, Holmium glass)
  • Absorbance Check
  • Linearity
  • Stray Light