NOTE: Use Kim Wipes to clean the sides of the cuvette before placing it in the chamber of the spectrophotometer. This will remove any fingerprints or liquids from the side of the container. These can cause errors in your results. Always place the cuvette in the chamber in the same direction throughout the experiment to limit errors.

Rinse the cuvette with DI water and shake it out into the sink between Allura Red solutions.

Label all of the containers in this lab with the concentration of the solution.

Part A: Determining Maximum Wavelength for Absorption

1. Use a ruler to measure the inner diameter of the cuvette to the nearest 0.01cm. Record this information in your lab notebook.
2. Obtain approximately 40mL of Allura Red from the stock bottle on the table in the front of the classroom. Record the concentration of the solution in your lab notebook.
3. Fill a cuvette approximately 2/3 full of the stock solution.
4. Fill a cuvette approximately 2/3 with DI water. This will be your blank sample throughout this experiment. You want to “blank” the spectrophotometer every time you change the wavelength. Your instructor will show you how to do this at the start of class. These instructions will not tell you to “blank” the spectrophotometer between changes in wavelength. However, you need to remember to do this.
5. Set the spectrophotometer to the lowest wavelength for the color range that compliments red. Place the cuvette with Allura Red solution in the chamber. Record the absorbance and wavelength in your lab notebook. Repeat this procedure, changing the wavelength 10nm each time. Don’t forget to “blank” the spectrophotometer between each sample.

NOTE: The absorbance of your sample should not exceed one. If it does, talk to your instructor.

6. The absorbance of your sample should increase as you change the wavelength. However, you will notice the absorbance decreases when you change the wavelength at some point. This means you have identified a window for where the highest absorbance for the sample is. Once you have this 10nm window, test your sample for each wavelength in that window until you find the highest absorbance. Record the absorbance and wavelength in your lab notebook. Once you have identified the highest wavelength, check with your instructor to ensure you have identified the correct wavelength. Once you have found this wavelength with the highest absorbance, you will test all future samples using this wavelength.

Part B: Measuring Absorbance of Known Concentrations of Allura Red

1. You will need to complete a series of dilutions to create a total of 4 solutions of Allura Red. The blank sample (absorbance = 0) and the max absorbance you measured in Part A, Step 5 are your first two data points. Next, you will create four more solutions using the stock solution and obtain the absorbance of these four solutions.
2. Using the volumetric pipet, remove a 25mL aliquot (sample) of the stock solution from the beaker and place it in a 50mL volumetric flask. Add water to the line and carefully mix the solution by capping the flask with a stopper and inverting it, keeping your thumb on the stopper to hold it in place. Pour this solution in a clean, dry, labeled beaker. Rinse the volumetric pipet with DI Water and use the bulb to blow the remaining liquid out of the pipet.
3. Using the volumetric pipet, remove a 25mL aliquot of the new solution from the beaker and place it in a 50mL volumetric flask. Add water to the line and carefully mix the solution by capping the flask with a stopper and inverting it, keeping your thumb on the stopper to hold it in place. Pour this solution in a clean, dry, labeled beaker. Rinse the volumetric pipet with DI Water and use the bulb to blow the remaining liquid out of the pipet.
4. Repeat Step 3 two more times until you have made a total of four solutions of Allura Red.
5. Add some of the solution made in Step 2 to the cuvette and measure the absorbance of the sample. Record this information in your lab notebook.
6. Repeat Step 5 for each of the other Allura Red samples you created. There should be a total of 6 data points when you are done – the blank, the stock solution, and the four solutions you made from the serial dilution.

Part C: Creating a Beer’s Law Plot

1. Use your laptop or the computer in the front of the classroom to create a Beer’s Law Plot for your data. You must determine the new concentration for each of the four solutions you made using the dilution formula. You will want to look this formula up before class and include it in your lab notebook.
2. Plot the concentration of your six solutions and their absorbance (absorbance vs. concentration). Then, title your graph, label your axes, and include the equation for the line and R² value on your graph.
3. When you have completed your graph, let your instructor know so she can check your graph and print a copy for you and your partner. Finally, you will need to glue this graph in your lab notebook.

Part D: Determining the Concentration of Allura Red in Mouthwash

1. Obtain about 10mL of mouthwash from the table in the front of the classroom. Add this solution to the cuvette and measure its absorbance. Record this information in your lab notebook.

Waste Disposal

• Flush all solutions down the drain with lots of water.
• Place the volumetric flasks and pipets in the container on the instructor’s desk.
• Clean out the cuvettes and place them on the table in the front of the classroom.
• Wash any other equipment used with soap and DI water (the final rinse with DI water) before putting it away.