Determining the Concentration of a Solution: Beers Law

Objectives: Prepare solutions of known concentrations. Measure absorption for solutions of known concentration. Create a Beers Law Plot. Determine concentration of a solution of unknown concentration. Background: Many compounds absorb light. This experiment will focuses on the absorption of visible light. Beers Law, or more accurately named Beer-Lambert-Bouguer Law, provides a scientist a tool to determine a solutions concentration based on the level of light absorbed. In optics, the BeerLambert-Bouguer law is and empirical relationship in relating the absorption of light to the properties of the material the light is traveling through. Meaning, the more light a solution absorbs, the higher the solutions concentration. For this to work effectively the solution must be clear. Meaning there can not be any precipitate in the liquid. The diagram below shows a beam of light with power P o, (the o in Po stands for original) directed at a sample solution. Absorption takes place and the beam of radiation leaving the sample has radiant power P, a lesser amount of light leaving the solution.

Beer's law was independently discovered (in various forms) by Pierre Bouguer in 1729, Johann Heinrich Lambert in 1760 and August Beer in 1852. The amount of radiation absorbed may be measured in a number of ways: Transmittance: T = P/P0 % Transmittance: %T = 100 T Absorbance: A = log P0/P A = log 1/T A = log 100/%T A = 2 -log %T

The last equation, A = 2 -log %T , is worth remembering because it allows you to easily calculate absorbance from percentage transmittance data. Now let us look at the Beer-Lambert law and explore it's significance. This is important because people who use the law often don't understand it - even though the equation representing the law is so straightforward: A = bc A - absorbance - molar absorbtivity with units of Lmol-1cm-1 this is a measure of the amount of light absorbed per unit concentration or a measure of how well the substance absorbs light b - path length, width of the cuvette in which the sample is contained, with units of cm c - the concentration of the compound in solution, expressed in mol L-1 The A = 2 -log %T works best as it gives a linear plot, unlike %T = 100 T which gives a curved plot, both are depicted below.

This experiment uses the Vernier Colorimeter, figure 1, to determine the amount of light that is absorbed by the solution. In this device, red light from the LED light source will pass through the solution and strike a photocell. The NiSO4 solution used in this experiment has a deep green color. A higher concentration of the colored solution absorbs more light (and transmits less) than a solution of lower concentration. The Colorimeter monitors the light received by the photocell as either an absorbance or a percent transmittance value. The data gained by the Colorimeter, is used to create a Beers Law Plot, figure 2. After creating your Beers Law Plot you will be able to determining the concentration of the unknown be using the best fit equation, for the straight line of Absorbance vs. Concentration.

Procedure: 1. Obtain and wear goggles! CAUTION: Be careful not to ingest any NiSO 4 solution or spill any on your skin. If you do spill on yourself, wash your hands immediately. 2. Create a 0.40 M solution of NiSO4 using a volumetric flask large enough to generated a sufficient amount of solution to complete this lab. a. Nickel (II) Sulfate is a hydroscopic salt. Meaning it will always come with water attached to it. The formula will be NISO4 6 H2O b. Check your calculations with your instructor prior to making your 0.40 M solution. 3. Label six CLEAN, DRY, test tubes 1-5 and Unknown. Fill the tubes as indicated by the below table.
Test Tubes 1 2 3 4 5 Unknown Name: 0.40 M NiSO4 (mL) 2 4 6 8 ~10 Distilled H2O (mL) 8 6 4 2 0 Concentration (M) 0.08 0.16 0.24 0.32 0.40

4. For the unknown solutions your lab group will be creating another groups unknown solution. Record your volumes in the above table and calculate the concentration. Give this information to the instructor along with the test tube with solution of unknown concentration. 5. Connect the Colorimeter to the computer interface. Prepare the computer for data collection by opening the file 11 Beers Law from the Chemistry with Computers folder of Logger Pro. 6. Cuvette: a. You will use only ONE cuvette for the entire lab. b. You must not touch cuvette on is smooth sides, these sides must be very clean and scratch-free. c. A lid must always be on the cuvette PRIOR to putting the cuvette in the colorimeter. 7. You are now ready to calibrate the Colorimeter. Prepare a blank by filling a cuvette 3/4 full with distilled water. To correctly use a Colorimeter cuvette, remember: a. All cuvettes should be wiped clean and dry on the outside with a tissue.

b. Handle cuvettes only by the top edge of the ribbed sides. c. All solutions should be free of bubbles. d. Always position the cuvette with its reference mark facing toward the white reference mark at the top of the cuvette slot on the Colorimeter. 8. Calibrate the Colorimeter. a. Open the Colorimeter lid. b. Holding the cuvette by the upper edges, place it in the cuvette slot of the Colorimeter. Close the lid. c. Press the < or > button on the Colorimeter to select a wavelength of 635 nm (Red) for this experiment. d. Press the CAL button until the red LED begins to flash. Then release the CAL button. When the LED stops flashing, the calibration is complete. 9. You are now ready to collect absorbance data for the five standard solutions and water. a. Click and keep, entering 0 for the concentration of the water. b. Empty the water from the cuvette. c. Using the solution in Test Tube 1, rinse the cuvette twice with ~1 mL amounts and then fill it 3/4 full. d. Wipe the outside with a tissue and place it in the Colorimeter. e. After closing the lid, wait for the absorbance value displayed on the monitor to stabilize. f. Click the KEEP, type 0.08 in the edit box, and press the ENTER key. The data pair you just collected should now be plotted on the graph. 10. Follow the above steps for each of the solution standards. 11. Wait to do the unknown. Press Stop 12. In your Data and Calculations table, record the absorbance and concentration data pairs that are displayed in the table. 13. Examine the graph of absorbance vs. concentration. To see if the curve represents a direct relationship between these two variables, click the Linear Fit button, . A best-fit linear regression line will be shown for your five data points. This line should pass near or through the data points and the origin of the graph. 14. Obtain about 5 mL of the unknown NiSO4 in another clean, dry, test tube. Record the name of the unknown in the data table. Rinse the cuvette twice with the unknown solution and fill it about 3/4 full. Wipe the outside of the cuvette, place it into the Colorimeter, and close the lid. Read the absorbance value displayed in the meter. When the displayed absorbance value stabilizes, record its value in of the Data and Calculations table. 15. Print plot with Linear Fit equation while the unknown solution is in the colorimeter. Data and Calculations:
Test Tube 1 2 3 4 5 Unknown Name: Concentration (mol/L) 0.080 0.16 0.24 0.32 0.40 Absorbance

Conclusion:

1. Use the following methods to determine the unknown concentration. a. With the linear regression curve still displayed on your graph, choose Interpolate from the Analyze menu. A vertical cursor now appears on the graph. The cursors concentration and absorbance coordinates are displayed in the floating box. Move the cursor along the regression line until the absorbance value is approximately the same as the absorbance value you recorded. The corresponding concentration value is the concentration of the unknown solution, in mol/L. b. Use the Equation generated by the Linear Fit line and calculate the unknown concentration. Show all work. c. On your printed graph of absorbance vs. concentration, with a regression line and interpolated unknown concentration displayed manually with a ruler and mark the concentration of the graph. 2. What is the Y-intercept of your Beers Law Plot? The theoretical Y-intercept should be what value? 3. Why is it important to ensure you use the same cuvette for the entire experiment? Why is important to ensure you line up the cuvette with the same orientation for each trial? 4. Absorbance and % Transmittance have what type of relationship?