Introduction

We can use the spectrophotometer to measure the absorption/transmission of a particular solution at a specific frequency. this "value" gives us information as to the concentration of the solution.

This exercise is designed to provide us with information about the relationship between the wave length of light, lambda, and the absorption of a pair of solutions of different colors. The first solution is a blue-black color while the second solution is a yellow-brown color. When iodine or Lugol's solution is added to water, the resulting solution is yellow brown. When iodine or Lugol's solution is added to a starch solution, it turns blue black.

Your objective is to construct graphs of absorption as a function of wavelengths for each of these solutions. Find the transmission peak wavelengths for an iodine solution, and for a starch solution to which you have added iodine as an indicator. (Is the amount of "stock" solution added to each tube important in determining the concentration with which you are working? Have you noted this in your notebook? By evaluating the graphs up-side down, you will be able to determine which frequencies are not affected by the color changes. (Why would you not want to use these frequencies in your experiment tests?)

Materials and Methods

Materials

• Spec 20
• Spec 20 tubes (cuvettes)
• Iodine solutions (Indicator Serial dilutions)
• Starch solutions ( Starch Serial dilutions)
• Starch-iodine solutions (For Enzyme Serial dilutions)

Methods

1. Set the Spec 20 frequency to 490 nanometers.
   
2. Using the knob on the left front of the colorimeter, with the sample holder empty, and the lid closed, zero the meter needle on the left hand side.
   
3. Using a "blank" of water, test tube of water, insert the blank into the sample holder and use the right hand knob on the front panel to bring the needle to the 100 % T on the right hand side of the meter.
   
4. Place the first of your serial dilutions tubes, the darkest, in the sample holder, close the lid. If the needle stays at zero, remove the sample and use the next tube of the sequence. continue until the needle moves above zero. Note this concentration.
   
5. Using this same sample, move the wavelength dial, top right hand panel, rock the dial back and forth over its range while watching the meter for maximum transmission. Progreseviely decrease the rocking until you have determined the wavelength at which you have a maximum transmission. Having set the wavelength of maximum transmission, use the knob on the left front panel to adjust the meter needle value to 100 % Transmission.
   
6. Next, starting at a low wavelength, e.g., 400, record the transmission value. Note both of these values in your notebook in a table. Go up 10 nm, i.e., to 410 nm, record the transmission value, continue until you have covered the whole of the spectral range.
   
7. Finally, use your most dilute solution, this should be yellow in color. Repeat as for the blue black colored solution, i.e., as in #5
   .

Results

Graph both of these curves on the same graph and the same piece of graph paper in your notebook. Note these page numbers in your table of contents.

Conclusion

• Are there wavelengths satisfactory for use in the enzyme experiment?
• How do you know this?

Discussion

• What values of wavelength can you use for this experiment?
• Why?
• Will you have a problem with the absorption peak of iodine blocking your need to follow the change of absorption of the starch-iodine color?