Caution Copper sulfate solution is may cause skin irritation and is extremely toxic to marine life. Dispose of all solutions in provided waste containers. Purpose To become familiar with using a spectrophotometer and gain an understanding of Beer s law and its relationship to solution concentration. Introduction Scientists use many methods to determine the identity and quantity of a substance in samples. Spectroscopy is a simple and powerful method for performing both qualitative and quantitative analyses. Each chemical species has a unique spectral fingerprint based on where electrons are located with respect to the nucleus. For example, a solution of sodium ions sprayed into a flame will change the flame s color to a bright yellow, while a solution of lithium ions will cause the flame to burn a deep red color. These flame tests reveal the solution s emission spectrum the wavelength (or color) of light revealed by the flame is due to excited electrons within atoms and ions in the solution relaxing to a lower energy state, emitting photons. A photon is a packet of light energy, the first indication that light may have particle-like properties. The flame provides the energy used to excite the electrons within the metal ions. The wavelength of radiation emitted can then be used to determine the energy lost by the electron as it relaxes. Since electrons can occupy only discrete energy states, the way radiation interacts with matter can indicate its chemical identity. Chemists commonly use absorbance spectroscopy, or how a substance absorbs photons of light, to obtain both qualitative (identity) and quantitative (amount) information. The quantitative measurement is achieved because each photon of light absorbed corresponds to the excitation of a single electron. Of course, in the laboratory, analyses are performed on large numbers of atoms or molecules, therefore a relationship must be established to obtain quantitative information. Initial spectrophotometric studies measured transmittance, which is defined as the fraction of light that passes through the sample: T I I 0 %T= T x 100 where I 0 is the intensity of the light passing through the solvent and I is the intensity of light that passes through the sample solution. Percent transmittance (%T) is simply the transmittance fraction multiplied by 100. A more useful quantity in performing analyses is the absorbance or the negative log of transmittance ( A = log T). A linear relationship exists between absorbance and concentration known as Beer s Law (A = ε b c), where b is the length of the path traveled by light through the sample, c is the concentration and ε is a molar absorptivity constant that depends on both wavelength and substance. This linear relationship between concentration and absorbance allows scientists to use spectroscopy for quantitative measurements of unknown samples. Using a calibration curve prepared from standard solutions (solutions of known concentration), the concentration of an unknown solution can be interpolated by linear regression. Revision Summer 2015 - CBB Page 1 of 7
Before Beer's law may be used as an analytical tool, it is necessary to select a suitable wavelength and determine whether Beer's law is valid (linear) at the wavelength selected. The most suitable wavelength is that at which a maximum absorbance is observed (called λmax). The λmax will always be at the same wavelength for a given species (even if the calibration on the instrument dial is in error) and can be found by any experimenter under any conditions. In this experiment a set of standard solutions of copper(ii) sulfate will be analyzed to obtain a Beer s Law plot (calibration curve). The concentration of copper in an unknown will then be determined using this plot. Procedure Preparation of Standard Solutions of Copper(II) sulfate 1. Obtain a 50 ml beaker and two burets. Deliver approximately 40 ml of 0.40 molar copper(ii) sulfate solution in a beaker. Place this in a clean buret. Fill the second buret with deionized water. 2. Label five (5) clean, dry test tubes 1-5. Using the burets, deliver the quantities of copper(ii) sulfate solution and deionized water to each test tube according to Table 1. Stir these solutions with a stirring rod, taking care to rinse and dry the rod thoroughly between test tubes. 3. Calculate the concentrations of all solutions before moving on to data collection. Test Tube # 0.40 M CuSO4 (ml) Deionized water (ml) 1 2 8 2 4 6 3 6 4 4 8 2 5 10 0 Preparation of a Beer s Law Plot 1. Connect a Colorimeter to Channel 1 of the Vernier computer interface. Connect the interface to the computer using the proper cable. 2. Start the Logger Pro program on the computer. Open the file 17 Colorimeter from the Advanced Chemistry with Vernier folder on the computer. 3. Calibrate the colorimeter. First place a cuvette containing deionized water in the cuvette slot of the Colorimeter and close the lid. Make sure one transparent side is facing the arrow at the top of the cuvette slot. Next, press the < or > buttons to select a wavelength of 635 nm. Finally, press the CAL button until the red LED begins to flash, then release. When the LED stops flashing, calibration is complete. Revision Summer 2015 - CBB Page 2 of 7
4. Measure absorbance values of the standard solutions. Click the COLLECT button to begin data collection. Using the solution in Test tube 1, rinse the cuvette with approximately 1 ml of solution, then drain and fill about ¾ full. Wipe the transparent sides with a tissue, and place it in the Colorimeter (properly aligned). Close the lid of the Colorimeter. When Absorbance readings stabilize, click the KEEP button, enter the concentration in the edit box, and then press ENTER. A point should show up on the data plot. 5. Discard the solution in the cuvette in a waste beaker, and rinse. Using the solution in Test tube 2, rinse the cuvette with approximately 1 ml of solution, then drain and fill about ¾ full. Wipe the transparent sides with a tissue, and place it in the Colorimeter (properly aligned). Close the lid of the Colorimeter. When Absorbance readings stabilize, click the KEEP button, enter the concentration in the edit box, and then press ENTER. A point should show up on the data plot. 6. Repeat Step 5 with the solutions in test tubes 3-5. Once all standard solutions have been measured, click STOP. 7. Look at the graph of absorbance vs concentration. Click the Linear Regression button and a best fit line will be shown for the data points. Write these absorbance values in your data table. 8. Obtain about 5 ml of the unknown CuSO4 solution in a clean dry test tube. Rinse the cuvette with small portions of the unknown solution, then fill ¾ full. Wipe the outside of the cuvette with a tissue and place it in the Colorimeter, closing the lid. 9. DO NOT press the collect button, as the absorbance readings should be visible on the screen. Once this value stabilizes, record it on the data table. 10. Dispose of all solutions in the appropriate waste container and clean all glassware and cuvettes. Graphs and Calculations Construct a Beer s Law Plot from the data in Table 2. See Figure 1 below for an example using KMnO4 solutions. Be sure to label your axes, provide a descriptive title, and include the equation and R 2 values directly on your graph. Calculate the concentration of your unknown samples by using your Beer s Law Plot. Use the equation of the best fit line and show a sample calculation. (Do not just estimate the concentration directly from the graph.) Figure 1: Sample Beer s Law Plot 0.8 0.7 0.6 Sara's Beer's Law Plot for KMnO 4 Absorbance 0.5 0.4 0.3 0.2 0.1 y = 2515.5x - 0.0002 R 2 = 0.9983 0 0 0.0001 0.0002 0.0003 Concentration (mol/l) Revision Summer 2015 - CBB Page 3 of 7
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Data Sheet Name: Lab Partner: Concentration Calculations: Table 2: Test Tube # 1 2 3 4 5 Unknown CuSO 4 Concentration (mol/l) N/A Absorbance Unknown concentration calculations: Revision Summer 2015 - CBB Page 5 of 7
Post-Lab Exercises Name: Lab Partner: 1. Why is absorbance used instead of transmittance in spectroscopic methods? 2. What physical process is being observed using the spectroscopic techniques used in this experiment? 3. What is the relationship between the color of a solution and the wavelengths the solution absorbs? Revision Summer 2015 - CBB Page 6 of 7
Name: Pre-Lab Questions A Beer s Law experiment yielded the following data: Concentration (mol/l) Absorbance 0.00 0.00 0.500 10-4 0.138 1.000 10-4 0.307 1.500 10-4 0.443 2.000 10-4 0.610 2. What is the equation for the best-fit straight line? 3. What is the R 2 value for this data? 4. What does the R 2 value tell us about this data? 5. What is the concentration of an unknown that has an absorbance of 0.524? Revision Summer 2015 - CBB Page 7 of 7