Determining the Concentration of a Solution: Beer’s Law

Purpose
To determine the wavelength (color) of maximum absorbance for a copper (II) sulfate solution.
To examine the relationship between the absorbance and concentration of a copper (II) sulfate solution.
To determine the concentration of an unknown copper (II) sulfate solution.

Introduction
You will be using the Colorimeter shown in Figure 1. In this device, light from an LED light source will pass through the solution and strike a photocell. The CuSO₄ solution used in this experiment has a deep blue color. A higher concentration of a colored solution will absorb more light and transmit less light than a solution of lower concentration. The colorimeter will interpret the light received by the photocell and express it both as an absorbance and a percent transmittance value. 

Figure 1	Figure 2

You will prepare five copper (II) sulfate solutions of known concentration (standard solutions). Each solution will be transferred to a small, rectangular cuvette that is placed into the colorimeter. The absorbance of each solution will be measured and recorded. When a graph of absorbance vs. concentration is plotted for the standard solutions, a direct relationship should result, as shown in Figure 2. The direct relationship between absorbance and concentration for a solution is known as Beer’s Law. The concentration of an unknown CuSO₄ solution will then be determined by measuring its absorbance with the colorimeter and using the equation of the best-fit line to determine its concentration.

1. Obtain and wear goggles! CAUTION: Be careful not to ingest any CuSO₄ solution or spill any on your skin. Inform the professor immediately in the event of an accident.
        
2. Pour about 15 mL of 0.50 M CuSO₄ stock solution into a 50-mL beaker. Pour about 15 mL of deionized water into a different 50-mL beaker.
        
3. Into five clean, dry, and labeled test tubes, prepare 5.00 mL of the following solutions by dilution, using the 0.50 M CuSO₄ and the pipets available to you in the lab. (Note: If you are unsure of how to read the pipets, please ask. The most common mistake in this lab involves measurements with the pipets.) Prepare a table on Sheet 1 of an Excel spreadsheet that shows the amount of 0.50 M CuSO₄ and amount of water used in each test tube. Stopper and mix the contents of each test tube thoroughly.
    

   Table 1
   Test Tube	CuSO4 concentration (M)
   1	0.10
   2	0.20
   3	0.30
   4	0.40
   5	0.50

4. Save your spreadsheet using a filename convention of: Lastname1_Lastname2_Lab11.
        
5. Connect the colorimeter to the LabPro® interface, using an adapter, if necessary. Next, open the Logger Pro application from the desktop or the Start menu. From within Logger Pro, open the "Probes & Sensors" folder, then select the "Colorimeter" folder, and finally the "Absorbance-Conc" file.
        
6. You are now ready to use the colorimeter. Prepare a blank by filling a cuvette 3/4 full with deionized water. Place each of the of the solutions prepared in Step 3 above into separate cuvettes. The cuvettes should be filled approximately 3/4 full. Calibrate the colorimeter at l_max for the copper (II) sulfate solution.

   HANDLING CUVETTES: Cuvettes should be wiped clean and dry on the outside with a Kimwipe®. Do not use a paper towel! Handle the cuvettes near the top of the ribbed sides. Solutions should be free of bubbles. If a cuvette is not dry on the inside, a small amount of the solution to be used should be used to rinse the cuvette before filling. Always align the reference mark on the cuvette with the reference mark on the slot of the colorimeter.

    

   FINDING THE WAVELENGTH OF MAXIMUM ABSORBANCE (lmax): Before making any measurements with the colorimeter, the wavelength (color) of maximum absorbance must be determined. This may be done by following the calibration procedure below for each of the wavelengths available on the colorimeter. Once the colorimeter is calibrated at a particular wavelength, place a cuvette containing the most concentrated sample into the colorimeter. The wavelength that yields the greatest absorbance reading for this sample is called lmax and is the wavelength you should use for all of your measurements.

    

   CALIBRATION: Click the "LabPro" icon on the toolbar. Then click on the "CH1" icon and select "calibrate". Then click on the "Calibrate Now" button. Place the blank cuvette in the cuvette slot of the colorimeter and close the lid. Turn the wavelength knob of the colorimeter to the 0%T position. In this position, the light source is turned off, so no light is received by the photocell. Type "0" in the Reading 1 Value box. When the voltage stabilizes, click "Keep". Turn the knob of the colorimeter to the desired wavelength. In this position, the sample transmits 100% of the light. Type "100" in the Reading 2 Value box. When the voltage stabilizes, click "Keep", then click "Done".

7. Once, you have determined l_max and calibrated the colorimeter at l_max, measure the absorbance for each of the solutions in Table 1, as well as the absorbance of the deionized water blank, by placing them into the colorimeter, aligning the reference marks, closing the lid, and measuring their absorbances. Record the results on Sheet 2 of your spreadsheet that shows the molarity of CuSO₄ in each solution and each of their absorbances. Make sure to include the value of the wavelength at which you are making your measurements.
        
8. Using Excel, prepare a graph that plots absorbance vs. concentration. Obtain the equation of the best-fit line, setting the y-intercept to zero. (Why should the y-intercept be set to zero?)
        
9. Fill a cuvette about 3/4 full with your unknown sample of CuSO₄. Record your unknown number. Measure the absorbance of the unknown as before. If the absorbance is greater than any of the measurements used to prepare your trendline, dilute the solution and measure the absorbance again. Take note of the volumes used in your dilution.
       
10. Use the equation of your best-fit line to determine the concentration of the unknown solution (use a formula inside a spreadsheet cell). If you diluted your unknown, calculate the concentration of the undiluted solution and report the result. Clearly label the your unknown number and its concentration.
         
11. Discard the solutions as directed by your instructor. Clean your cuvettes. Do not use test tube brushes or paper towels, as they may scratch the surface of the cuvettes. Use Kimewipes® to dry the cuvettes.

Assignment

1. This will be a short report. In this report, you will need to include the title, experimental details, results (the table and graph you made above which can be embedded into a Word document), discussion, and references.
        
2. As a part of the discussion section, you will need to include the following:
   1. describe the preparation of 500.0 mL of 0.50 M CuSO₄, using solid CuSO₄ and water,
   2. using your trendline equation and Beer's Law, calculate the absorbance for 0.25 M CuSO₄ at l_max,
   3. any errors that occurred or other observations made.
           
3. Follow your instructor's directions for submitting this lab report. If you submit electronically, be sure to follow the filename convention mentioned earlier. If emailing, use "Chem 1061: Beer's Law Law" as the subject line. If you turn in a paper copy or do NOT embed your Excel file into your Word document, you will need to provide a sample calculation for determining the concentration of your unknown solution.

Written by Lance S. Lund, Anoka-Ramsey Community College. Portions of this lab courtesy of Kirk Boraas, Minneapolis Community and Technical College, and Vernier Software, "Chemistry With Computers".  Updated October 22, 2007.