This activity quantitatively relates the concentration of a light-absorbing

CHEMISTRYType of Lab – SPECTROSCOPYBEER-LAMBERT LAW – EXPERIMENTAL PROCEDURELab format: this lab is a remote lab activityRelationship to theory: This activity quantitatively relates the concentration of a light-absorbingsubstance to the absorbance of light. Instructions for Instructors: This protocol is written under an open source CC BY license. Youmay use the procedure as is or modify as necessary for your class. Be sure to let your studentsknow if they should complete optional exercises in this lab procedure as lab technicians will notknow if you want your students to complete optional exercise. Remote Resources: Primary – UV/Vis Spectrometer, Secondary – Cuvette HolderInstructions for Students: Read the complete laboratory procedure before coming to lab. Under the experimental sections, complete all pre-lab materials before logging on to the remotelab, complete data collection sections during your on-line period, and answer questions inanalysis sections after your on-line period. Your instructor will let you know if you are requiredto complete any optional exercises in this lab. ContentsBeer-Lambert Law. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Learning Objectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Background Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Preparing to Use the Remote Web-based Science Lab (RWSL). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Introduction to the Remote Equipment and Control Panel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Prelab Questions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Experimental Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8LEARNING OBJECTIVES1. Measure and analyze the visible light absorbance spectrum of a standard solution to determinethe maximum wavelength of absorbance (λmax). 2. Create four standard solutions by mixing a stock solution and distilled water. a. Calculate the concentrations of these standard solutions using information provided bythe Laboratory Technicians. 3. Measure the absorbance of several standard solutions. 4. Create Tables to display observations. 5. Construct a standard curve for the standard solutions. Find the relationship between absorbanceand concentration. 6. Measure the absorbance of a solution of unknown concentration. Creative Commons Attribution 3. 0 United States LicensePa ge |1CHEMISTRYType of Lab – SPECTROSCOPY7. Calculate the concentration of the unknown solution using the standard curve that you derived. EQUIPMENTPaperPencil/penComputer with Internet accessPREPARING TO USE THE REMOTE WEB-BASED SCIENCE LAB (RWSL)Click on this link to access the InstallGuide for the RWSL: http: //denverlabinfo. nanslo. org/NANSLOLab-Info/content/get-ready-remote-lab-experienceFollow all the directions on this webpage to get your computer ready for connecting to the remotelab. INTRODUCTION TO THE REMOTE EQUIPMENT AND CONTROL PANELWatch this short tutorial video to see how to use the RWSL controlhttp: //denverlabinfo. nanslo. org/NANSLO-Lab-Info/video/absorbance_spectroscopy. htmlpanel: There are appendices at the end of this document that you can refer to during your lab if you needto remind yourself how to accomplish some of the tasks using the RWSL control panel. EXPERIMENTAL PROCEDURERead and understand these instructions BEFORE starting the actual lab procedure and collectingdata. Feel free to “play around” a little bit and explore the capabilities of the equipment before youstart the actual procedure. Once you have logged on to the Remote Lab, you will perform the following Laboratory procedures: 1. From the Cuvette Holder tab, turn on temperature controller. Ensure the temperature of thesystem is adjusted to 25. 0 degrees C. 2. Ensure that the stirring control is turned on. 3. From the Spectrometer tab, ensure the spectrometer’s light source is turned off. 4. Store a Dark Spectrum. 5. Ensure that cuvette 0 (the reference sample) is selected. 6. Turn on the light and you will see the spectrum of the light source7. Play around with the Boxcar Width and # Spectra to Average to get the least noisy (“jumpy”)spectrum that you can. 8. Store the Reference spectrum. 9. Ask the Lab Tech for information about the stock solution. You will use this information tocalculate the concentration of each standard solution. 10. Select cuvette 1 in the Qpod. This cuvette will be empty. 11. Inject some stock solution and some distilled water into the cuvette and record the volumes ofeach one that you dispensed. Creative Commons Attribution 3. 0 United States LicensePa ge |2CHEMISTRYType of Lab – SPECTROSCOPY12. After allowing the standard solution to mix for a minute or two, click on the Show AbsorbanceSpectrum button to view the Absorbance Spectrum. 13. Determine the location of λmax. 14. Record the Absorbance of the sample at λmax. Each student in the group must write themeasurement down for later use. 15. Another student should take control of the interface. 16. Change to the next cuvette and repeat steps 11 through 15 for cuvettes 2, 3 and 4. 17. Measure the absorbance of the unknown concentration solution in cuvette 5. 18. After each student has collected some data (and everyone has recorded the full data set), youcan log out of the lab and work on the data analysis portion. If you have time left in yourscheduled lab period, you can continue working with your lab partners to analyze the data. Data Analysis (to be done offline if necessary): Plot a standard graph using the concentration and Absorbance values for the standard solutions. Plot Concentration on the X-axis and Absorbance values on the Y-axis. Draw a best-fit line goingthrough the origin. From the Absorbance of the unknown solution, you can calculate theconcentration of the unknown solution using the line equation of the standard curve. In Excel, the best-fit line and its equation can be determined by this method: a. Insert a scatter plot of the data, making sure that absorbance is on the y axis and concentrationis on the x axis. If they are switched, then delete the graph, change the positions of theabsorbance and concentration columns and insert the scatter plot graph again. b. Right-click one of the data points on the graph and select Add Trendline. c. Make sure “Linear” is selected and check the box to set the intercept to zero and also the one todisplay the equation on the chart. d. You will now have the best-fit line and the equation for that line. You can use this line equationto calculate the concentration of the unknown solution. Questions (show all necessary calculations): A. What was the concentration of the Stock solution?a. ________________ molarB. What were the volumes and concentrations of your standard solutions?a. Cuvette 1: __________ mL Stock, __________ mL water, __________ molarb. Cuvette 2: __________ mL Stock, __________ mL water, __________ molarc. Cuvette 3: __________ mL Stock, __________ mL water, __________ molard. Cuvette 4: __________ mL Stock, __________ mL water, __________ molarC. What is the concentration of the unknown sample?a. ________________ molarCreative Commons Attribution 3. 0 United States LicensePa ge |3CHEMISTRYType of Lab – SPECTROSCOPYD. Why do you have to first take an absorbance measurement of a cuvette filled with distilledwater? Why does this measurement have to be subtracted from the measurements of the othersamples?E. Why didn’t we just measure one or two samples with known concentrations?F. How many significant digits can you report in the concentration of the unknown sample? Whatlimits the number of significant digits in this result?G. What is the energy, in Joules, of one photon of light at λ max?H. Use Figure 1 to determine what color light is being absorbed at λmax. λmax: _________________________________750I. 620600580500Figure 1 – Visible portion of EM SpectrumColor at450380Figure 1 demonstrates the relationship between absorbed and reflected colors of light. Absorbed is opposite of reflected on the wheel. For example, if a substance absorbs orangelight, it will reflect blue light, and therefore appear blue. Compare the color of the stock solutionto the color of the light it absorbs. Does it agree with the color wheel? What can you deducefrom this?Figure 1 – By Sakurambo at English Wikipedia [GFDL (www. gnu. org/copyleft/fdl. html) or CC-BY-SA-3. 0(http: //creativecommons. org/licenses/by-sa/3. 0/)], via Wikimedia CommonsJ. If a chemical solution was primarily orange in color, approximately what wavelength would youexpect λmax of the absorbed light to be? Why?Creative Commons Attribution 3. 0 United States LicensePa ge |4CHEMISTRYType of Lab – SPECTROSCOPYCreative Commons Attribution 3. 0 United States LicensePa ge |5CHEMISTRYType of Lab – SPECTROSCOPYThis product was funded by a grant awarded by the U. S. Department of Labor’s Employment and TrainingAdministration. The product was created by the grantee anddoes not necessarily reflect the official position of the U. S. Department of Labor. The Department of Labor makes noguarantees, warranties, or assurances of any kind, express orimplied, with respect to such information, including anyinformation on linked sites and including, but not limited to,accuracy of the information or its completeness, timeliness,usefulness, adequacy, continued availability, or ownership. Creative Commons Attribution 3. 0 United States LicensePa ge |6