UV/Vis Spectroscopy

The UV spectrum is usually taken on a very dilute solution (1 mg in 100 ml of solvent). A portion of this solution is transferred to a silica cell. A matched cell containing pure solvent is prepared, and each cell is placed in the appropriate place in the spectrometer. This is so arranged that two equal beams of light are passed, one through the solution of the sample, one through the pure solvent. The intensities of the transmitted light are then compared over the whole wavelength range of the instrument. The spectrum is plotted automatically as a log₁₀(I₀/I) ordinate and l abscissa. For publication and comparisons these are often converted to an e vs. l or log(e) vs. l plot. The l unit is almost always in nanometers (nm).

In general, organic compounds will have molar absorptivities (e) of around 10,000. Therefore, in order to obtain solutions that will have a maximum absorbance of 1, it is most likely that the concentration of the starting solution (the stock) to be 1 x 10^-4 M.

Preparing A Sample

The following steps can be followed to produce solutions that will give generally good results in the UV/Vis experiment. An example for 2-nitroaniline is worked out along the way.

1. Prepare a concentrated solution that is about 1 x 10^-3 M. This solution is made concentrated so that you can weigh out a reasonable amount of material (like 13 mg as opposed to 1.3 mg).
   
   For 2-nitroaniline (MW = 138.13 g/mol), weigh out about 0.0138 g of material. In this example, 0.0132 g were obtained, and diluting this amount of material to 100 mL in water produces a 9.556 x 10^-4 M solution.
    
2. Perform a 10:1 dilution to produce a solution roughly 1 x 10^-4 M. This will be the stock solution.
   
   For 2-nitroaniline, 10 mL of the concentrated solution was transferred to a new 100 mL volumetric flask, and diluted to 100 mL with water.
    
3. Prepare several more solutions that are dilutions of the stock.
   
   For 2-nitroaniline, solutions that are 80%, 60%, 40%, and 20% of the stock concentration are prepared as listed below:
   
    
   

   concentration	solution preparation
   9.556 x 10-5 M	stock
   7.654 x 10-5 M	20 mL of stock diluted to 25 mL
   5.734 x 10-5 M	15 mL of stock diluted to 25 mL
   3.822 x 10-5 M	10 mL of stock diluted to 25 mL
   1.911 x 10-5 M	5 mL of stock diluted to 25 mL

4. Take UV/Vis spectra for each solution and plot them.
   
   For 2-nitroaniline, the plot would look like that given below.
   
   
   
    

5. From the UV/Vis spectra, determine all l_max values, and produce a Beer's Law plot.
   
   For 2-nitroaniline, the Beer's Law plot would look like that given below.
   
   
    

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