SPECTROPHOTOMETRY
A GUIDED-INQUIRY
SIMULATED EXPERIMENT
by
Gary L. Bertrand
Missouri University of Science and Technology

Proposed Studies                         Simulated Experiment

The instrument in this simulation is a Dual-Beam Recording Ultraviolet/Visible Light Spectrophotometer.  It compares the intensity of the light (I) that goes through a cell containing a sample (a colored dye in this case) to the intensity (Io) that goes through an identical cell containing only the solvent (water in this case), as the wavelength of the light is varied through the visible and/or ultraviolet regions. The output of the instrument may be selected as Transmittance [100 (I/Io)] or Absorbance [log10(Io/I)] . In this experiment, the instrument is set to scan wavelengths of light in the visible region, 400 to 680 nanometers (nm), and to record either Transmittance or Absorbance every 10 nm.  The experiment involves solutions of dyes of known concentration (ppm).  These solutions may be mixed with each other or diluted with distilled water, so that the concentrations are known in the final solutions.*  These solutions are placed in sample cells which may be placed in the Dual-Beam Spectophotometer and analyzed relative to distilled water in the Reference Cell. The results of this analysis may be viewed in a table of data and as a graph, either for Transmittance or Absorbance.  Some students may wish to transfer this data to a spreadsheet for further study**. *   It is important to record the volumes of dyes that are mixed for each solution to be studied, and the concentrations of the dyes.  The program does not save this data.  If you re-start the program, the concentrations may be different. **  You should record your data or print it out before dumping any solutions in order to continue the experiment.

PROPOSED STUDIES 1.  Prepare solutions of red dye and blue dye (and maybe even distilled water) and scan these solutions. Summarize your observations regarding the relationships between Transmittance, Absorption, and Wavelength for these solutions. 2. Prepare solutions of the red dye of three different concentrations and scan these solutions.  Concentrations should range from full strength to a substantial dilution (1:3 or 1:4).  Repeat this study with the blue dye.

Focusing on a few wavelengths at which the largest changes are observed, explore the relationships between Transmittance, Absorbance, and Concentration for each dye at specific wavelengths. 3.  Devise a strategy by which you could determine the concentration of either a red dye or a blue dye in an unknown sample. 4.  Devise a strategy by which you could determine the concentrations of red dye and blue dye in an unknown mixture of the two.