The Caliper

Determining the Concentration of an Unknown Protein Solution: An Application of Beer ’s Law

In this experiment, you can use a Colorimeter to determine the absorbance values of five protein solutions of known concentrations (standard solutions). When a graph of absorbance vs. concentration is plotted for these solutions, a direct relationship should result, as shown in the figure on the right. This relationship is known as "Beer’s law".

You can then determine the concentration of an unknown protein by measuring its absorbance, locating the absorbance of the unknown on the vertical axis of the graph, and finding the corresponding concentration on the horizontal axis (follow the arrows in the figure).

Materials
You will need the following materials:

• Vernier LabPro or CBL 2 interface

• two 10 mL pipets (or graduated cylinders)
• a computer, calculator, or Palm OS handheld
• two 100 mL beakers
• pipet or pipet bulb
• Vernier Colorimeter
• one cuvette
• distilled water
• five 20 x 150 mm test tubes
• test tube rack
• 30 mL of 1% albumin stock solution*
• stirring rod
• 5 mL of unknown albumin solution
• tissues (preferably lint-free)

Procedure

1. Add about 30 mL of 1% albumin stock solution to a 100 mL beaker. Add about 30 mL of distilled water to another 100 mL beaker.
2. Label four clean, dry, Test Tubes 1-5. (The fifth solution is the beaker of 1% albumin stock solution.) Pipet 2, 4, 6, and 8 mL of 1% albumin stock solution into Test Tubes 1-4, respectively. With a second pipet, deliver 8, 6, 4, and 2 mL of distilled water into Test Tubes 1-4, respectively. Gently, but thoroughly, mix each solution with a stirring rod. Clean and dry the stirring rod between stirrings. Add about 10 mL of the 1% albumin stock solution to Test Tube 5. Test Tubes 1-5 will have concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0% albumin, respectively.
3. Connect the Colorimeter to Channel 1 of the LabPro or CBL 2 interface. Start your data-collection program.
4. Set up the interface for the data collection. If you are using a computer, open an experiment file for absorbance vs. concentration in your data- collection program. If you are using a TI graphing calculator or Palm OS handheld, set up the data collection in the Event with Entry mode, using concentration as the x-axis entry.
5. Prepare a blank by filling an empty cuvette 3 /4 full with distilled water. Place the cuvette into the cuvette slot of the Colorimeter, and close the Colorimeter lid. If you are using a newer Colorimeter with auto-ID, while still on the main graph or main screen of the program, calibrate by simply pressing the CAL button on the Colorimeter. If you are using a non-auto-ID Colorimeter, use the calibration routine of your data-collection program to calibrate with a blank cuvette at 0 and 100 percent transmittance.
6. You are now ready to collect absorbance-concentration data for the five standard solutions.
   • Start the data collection. (Press the Collect button or choose Start on a calculator or handheld.)
   • Empty the water from the cuvette. Using the solution in Test Tube 1, rinse the cuvette twice with ~1 mL amounts and then fill it 3 /4 full (3.5 mL). Wipe the outside with a tissue, place it in the Colorimeter, and close the lid.
   • When the value displayed on the calculator screen has stabilized, collect this data point and enter “0.2” as the concentration in %. The absorbance and concentration values have now been saved for the first solution.
   • Repeat this procedure for the solutions in Test Tubes 2-5 (0.4, 0.6, 0.8, and 1.0%). Note: Wait until Step 7 to do the unknown. Click or choose Stop to end the data collection.
7. Determine the absorbance value of the unknown albumin solution. To do this, rinse the cuvette with some of the unknown albumin solution, then fill it 3 /4 full with the unknown. Important: For the unknown, you will simply monitor the absorbance value (on the meter window of the computer screen, or on the Main Screen of the calculator or handheld). Record this absorbance value.
8. To determine the concentration of the unknown albumin solution, display your graph of absorbance vs. concentration with a linear fit displayed, then use your program to interpolate along the regression line. When the interpolation cursor reaches the absorbance value you recorded for your unknown albumin solution, the corresponding concentration will be the concentration of your albumin unknown.