spectrophotometer
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  • 1. An instrument that measures the amount of light that passes through (is transmitted through) a sample.
  • 2. Uses a type of light to detect molecules in a solution Light is a type of energy, and the energy is reported as wavelengths, in nanometers (nm).
  • 3.  Ultraviolet (UV) Spectrophotometers. Uses ultraviolet light of wave lengths from 200 nm to 350 nm.  Visible (VIS) Light Spectrum Spectrophotometers. Uses visible light (white light) of wave lengths from 350 nm to 700 nm.
  • 4. R O Y G B I V
  • 5.  Shines a beam of light on a sample.  The molecules in the sample interact with the light waves in of 3 ways: Absorb the energy Reflect the energy Transmit the energy between and through the atoms and molecules of the sample.
  • 6. Blue molecules absorb the other colors of visible light. Blue molecules are blue because they reflect blue light.
  • 7.  Consider blue molecules, all the wavelengths of light are absorbed, except for the blue ones.  The blue wavelengths are transmitted or reflected off the molecules. If these blue wavelengths hit a detector (such as in the spectrophotometer or the nerve cells in your eye), they appear blue.
  • 8.  Molecules are whatever color of light that they do not absorb.  Green molecules appear green because they absorb most wavelengths of visible light, except the green wavelengths.
  • 9.  The spectrophotometer measures the amount of light transmitted through the sample (Transmittance).  By using an equation (Beers law), it converts the transmittance data to an absorbance value. What kind of data is this?
  • 10.  The concentration of an unknown sample can be determined by comparing the absorbance data to standards of known concentration.  The data generated with the set of known standards is called a standard curve.
  • 11.  Inner parts Lamp Prism or grating that direct light of a specific wavelength.
  • 12.  Visible spectrophotometer Contains a tungsten lamp that produces wwhhiittee lliightght.  Ultraviolet spectrophotometer Contains a deuterium lamp that produces light in the UV light part of the spectrum.
  • 13.  Outer parts: Sample Holder Display Knobs or buttons used to calibrate the spec to measure the designated molecule. Wavelength Selection
  • 14.  Visible Spectrophotometer White light hits the prism or grating, it is split into the colors of the rainbow (Visible Spectrum). The wavelength knob rotates the prism/grating, directing different color of light toward the sample.
  • 15.  The wavelength of light produced by the tungsten lamp range from about 350 nm (Violet light) to 700 nm (red light).  The molecules in the sample either absorb or Transmit the light energy of one wavelength or another.
  • 16.  The detector measures the amount of light being transmitted by the sample and reports that value directly (% transmittance) or converts it to the amount of light absorbed in absorbance units (au) using Beers Law.
  • 17. A = 2 – log10%T
  • 18.  After collecting data for your concentration an absorption spectrum graph is created.  These can be used when attempting to identify unknown substances (e.g. CSI)
  • 19. The absorbance spectrum is a graph of a sample’s absorbance at different wavelengths.
  • 20. The spectrophotometer can measure the amount of absorbance or lack of absorbance of different colored light for a given molecule.
  • 21.  The concentration of molecules in a solution affects the solution’s absorbance.  Remember [ ] is a ratio – when we change one number – it affects the ratio If there are more molecules in one solution than in another, than there are more molecules to absorb the light.
  • 22.  Determines the presence and concentrations of samples.  Determines the purity of a sample.  Look at the change of samples over time.
  • 23. A. Measure the absorbance of standards containing known concentrations of the analyte B. Plot a standard curve with absorbance on the X axis and analyte concentration on the Y axis C. Measure the absorbance of the unknown(s) D. Determine the concentration of material of interest in the unknowns based on the standard curve