SPECTROPHOTOMETER

• 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

  • Cosmic Rays
  • Gamma Rays
  • X-rays
  • Visible
  • Ultra-Violet
  • Infrared
  • Radio, TV, Microwave, etc
• Wavelength

  • Is the distance between peaks as light is envisioned to travel in wavelike manner
• Wavelength

  • Is the distance between peaks as light is envisioned to travel in wavelike manner
  • It is expressed in terms of Nanometer (nm); Angstrom (A); and Millimicrons (mµ) 1 nm = 10 A 1 nm = mµ
• Kinds of Wavelength

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

  • Is the number of vibrations of wave motions per second
  • Derived from the “Plank's Formula”
• Frequency

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

  • Distance between peak and trough
  • The higher the amplitude, the more intense the light= the more light energy.
• 350-430; VIOLET - YELLOW

  430-475; BLUE - ORANGE

  475-495; BLUE GREEN - RED-ORANGE

  495-505; GREEN-BLUE - ORANGE-RED

  505-555; GREEN - RED

  555-575; YELLOW-GREEN - VIOLET-RED

  575-600; YELLOW - VIOLET

  600-650; ORANGE - BLUE

  650-700; RED - GREEN
• Two primary consideration in every colorimetric analysis

  • Quality of the color
  • Intensity of the color
• Kinds of Colorimetry

  • Visual Colorimetry
  • Photoelectric Colorimetry
• Visual Colorimetry

  • uses the eyes in determining end point
• Photoelectric Colorimetry

  • Spectrophotometric measurement
  • Photometric measurement
• Spectrophotometric measurement

  • Measurement of light intensity in a narrower wavelength. It uses a device (prisms and/or gratings) to disperse the source of light into a continuous spectrum.
• Photometric measurement

  • Measurements of light intensity of multiple wavelength. It uses filter to isolate part of the spectrum.
• Different Types of Spectrophotometers

  • Single Beam
  • Double Beam
  • Visible Light (400-700 nm)
  • Ultraviolet Light
  • Infrared Light
• Single Beam

  • In this type, 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.
  • This type of spectrometer is usually less expensive and less complicated.
• Double Beam

  • In this type, before reaches the sample the light source is split into two separate beams. From these one passes through the sample and second one is used for reference.
  • This gives the advantageous because at the same time the reference reading and sample reading can take place.
• Visible Light (400-700 nm)

  • Visible spectrophotometers can use incandescent, halogen, LED, or a combination of these sources and these spectrophotometers vary in accuracy.
  • Plastic and glass  cuvettes  can  be used for visible light spectroscopy.
• Ultraviolet Light

  • UV spectroscopy is used for fluids, and even solids. Cuvettes, only made of quartz, are used for placing the samples.
• Infrared Light

  • IR spectroscopy, which helps to study different structures of molecules and its vibrations. Different chemical structures vibrate in different ways due to variation of energy associated with each wave length.
  • For example, mid-range and near infrared (higher energy) infrared tends to cause rotational vibrations and harmonic vibrations respectively.
• Parts of the Spectrophotometer

  • Light Source
  • Entrance Slit
  • Monochromator
  • Exit Slit
  • Analytical Cell or Cuvette
  • Photodetectors
  • 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 Lamp
• Tungsten Iodine Lamp

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