analysislab

• Ultraviolet-visible spectrophotometry


  Technique to measure the concentration of solute/s in solution by measuring the amount of light that is absorbed by the solution
• Spectrophotometry


  • Takes advantage of the dual nature of light (particle and wave)
  • Measures the intensity of a light beam after it is directed through and emerges from a solution
• Example with copper sulfate (CuSO4)


  • Red part of the spectrum has been almost completely absorbed, blue light has been transmitted
  • CuSO4 absorbs little blue light and therefore appears blue
• Sensitivity in spectrophotometry


  Greater sensitivity can be gained by directing red light through the solution because CuSO4 absorbs strongest at the red end of the visible spectrum
• Spectrophotometer operation


  1. Light source gives off white light

  2. Light strikes a prism, separating the light into its component wavelengths

  3. Light waves can be separated by frequency

  
• Spectrophotometer measurement


  • Measures the amount of light (of certain frequency) transmitted or absorbed by the solution
  • Light that has not been absorbed by the solution in the cuvette will strike the phototube
  • Photons of light that strike the phototube will be converted into electrical energy
  • Current produced is proportional to the amount of light which originally struck the phototube and is an accurate measurement of the amount of light which has passed through (been transmitted by) the sample
• Different compounds have characteristic absorption phenomena and absorption spectra
• Concentration of every component may be found from the spectrophotometer measurements and calibration curve made using the samples of known concentration
• Materials


  • Copper sulfate, 0.4 M
  • Copper sulfate (unknown concentration)
  • Tissues (lint – free)
  • Distilled water
  • Science workshop TM interface
  • Beakers,100 ml (2)
  • Colorimeter
  • Cuvette
  • Pipets, 10 ml (2) with pipet bulbs
  • Stirring rod
  • Test tubes, large (6)
  • Test tube rack
• UV VIS Start-Up


  1. Plug-in the UV VIS Spectrophotometer

  2. Turn the switch on

  3. Press the START button, then the Mode Menu will be displayed on the screen

  4. Choose the Photometric function by pressing 1 button

  5. Set the wavelength by pressing the GOTO WL button

  6. Key in the Lambda max of the analyte: 635nm
• Preparation of the instrument (calibration)

  1. Prepare a blank by filling a clean and dry cuvette ¾ full with distilled water

  2. Place the cuvette 3/4-filled with the solvent inside the spectrophotometer properly

  3. Close the lid of the instrument and Press the AUTO-ZERO button
• Preparation of the standard solutions

  1. Add about 30 ml of 0.4M copper sulfate stock solution to a 100 ml beaker

  2. Add about 30ml of distilled water to another 100 ml beaker

  3. Label four (4) clean dry test tubes 1 through 4

  4. Pipet 2,4,6 and 8 ml of 0.4 M CuSO4 solution into test tubes 1 to 4 respectively

  5. With a second pipet, deliver 8,6,4 and 2 ml of distilled water into test tubes 1 to 4 respectively

  6. Thoroughly mix each solution with a stirring rod

  7. Keep the remaining 0.4 M CuSO4 in the 100 mL beaker to be used in the fifth trial
• Spectrophotometric analysis


  1. Empty the water from the cuvette used during calibration

  2. Using the solution of test tube 1, fill the cuvette ¾ full of solution

  3. Wipe the outside of the cuvette with tissue and place the cuvette in the colorimeter

  4. Place the different standard solution and unknown solution respectively in the empty cuvettes available

  5. Record the absorbance of the standard solution as well as the unknown solution

  6. Using Beer-Lambert Plot, measure the concentration of the unknown solution