Chem 1B Dr. White 11 Experiment 2: The Beer-Lambert Law for Thiocyanatoiron (III) Objectives To use spectroscopy to relate the absorbance of a colored solution to its concentration. To prepare a Beer s Law Plot to determine the concentration of an unknown. Introduction Technique: Absorbance Spectroscopy (see Appendix 3 in your text for more information) The intensity of the color in solutions can be quantified by measuring the absorbance of light by the solution. Absorbance Spectroscopy involves measuring, via a Spectrometer, the intensity of light after it passes through a colored solution. The deeper the color, the more light will be absorbed. The UV- Vis Spectrometer we will use consists of a white light source, a sample compartment and a detector. Incident light from the source is focused on the sample. Depending on the color of the sample, some wavelengths of light are absorbed and some are transmitted into the detector. The color of the light absorbed is directly related to the color of the light transmitted; i.e., the color we see. The color absorbed is the complement of the color we see. A color wheel, which shows the colors of the rainbow, is shown below. On a color wheel, the complementary color is the color found directly opposite on the wheel. For example, the complementary color to red would be green. absorption spectrum is taken. The substance is exposed to a source of incident electromagnetic radiation at various wavelengths. The substance absorbs the incident radiation at those wavelengths that coincide with transitions of its electrons from lower to higher energy levels. The substance transmits that is, it does not absorb the energy of the wavelengths that do not correspond to its characteristic electron transitions. The wavelengths of transmitted radiation are recorded by a detector. A simple picture of the concept is shown below. White incident light is focused on the colored sample. The sample absorbs some wavelengths of light and transmits the complementary wavelengths of light. Incident Light (I 0 ) b The transmittance of light (T) is defined as: Transmitted Light (I) T = I I 0 (1) The absorbance of light (A) is related to the transmittance by: The table below summarizes the relationship between the color observed and the color absorbed as well as the wavelength of the absorbed light. Table 1: Table of observed color and the related absorbed color along with wavelength. Color Observed Color Absorbed Wavelength of Absorbed Light (nm) Violet Yellow- Green 570 Dark Blue Yellow 580 Blue Orange 600 Green Red 650 Yellow Blue 450 Red Blue- Green 490 To determine the wavelength of absorbed light, an A = logt (2) Besides the concentration, c, of the absorbing species, two other factors determine the absorbance of a sample solution. The further light must travel through a solution, the greater the absorbance. This distance is referred to as the pathlength and is denoted with the symbol l. The ability of the molecule to absorb light at the given wavelength, known as the extinction coefficient ε, a quantum mechanical effect, will also determine the absorbance. The relationship between these influences and the absorbance is given by the Beer- Lambert Law: A = εlc (3) The Beer- Lambert Law states that there is a linear relationship between concentration and absorbance. Generally, dilute solutions follow the Beer- Lambert Law
12 Chem 1B Dr. White quite well. The absorbance of a compound is usually measured at the maximum point (λ max) in its absorption spectrum. By choosing λ max, we choose the point where the detection of the molecule in the solution is the most sensitive, since ε is the largest. We can prepare a series of solutions of the same molecule in known concentrations. If we plot the linear relationship between absorbance and concentration for these solutions according to the Beer- Lambert law, the result should be a straight line. This graph can also allow us to determine the concentration of an unknown solution by correlating the absorbance of that solution to its corresponding concentration. We can also use the graph to determine the value for ε for a molecule at a particular wavelength. Today, we will make solutions of known concentrations thiocyanatioiron(iii) ion solution (a deep red solution). and determine their absorbance. Then a Beer s Law Plot will be made to show the relationship between the concentration of thiocyanatoiron (III) and its absorbance. This plot will be used in Experiment 3 to determine an equilibrium constant. When solutions containing the colorless iron (III) ion and colorless thiocyanate ion are mixed, an equilibrium is established in which the thiocyanatoiron (III) complex ion is formed: Fe 3+ (aq) + SCN - (aq) FeSCN 2+ (aq, blood red) In part A, a stock solution will be prepared by mixing solutions containing the colorless iron (III) ion and colorless thiocyanate ion. In preparing the stock solution, we will make the iron (III) ion concentration much larger than the thiocyanate ion concentration. This causes the equilibrium above to shift far to the right, therefore converting essentially all of the SCN - into FeSCN 2+. We will then assume that the equilibrium concentration of FeSCN 2+ equals the original concentration of the SCN -. In part B, the stock solution will be diluted to create 5 standard solutions. The concentrations of these standard solutions can be determined by the following equation: M CV C = M DV D (7) In Part C, the absorbance of each standard solution will be measured at the wavelength of maximum absorbance, λ max, by using a spectrometer. Graphing the absorbances of each standard solution at the λ max against the known concentration of the FeSCN 2+ in each standard solution produces a linear relationship. This calibration line relates the concentration of the FeSCN 2+ to its absorbance. Procedure Note: Dispose of all chemicals in the provided waste containers. Use a large beaker and collect all your waste which you can then dump in the waste container. Part A: 1. Prepare a stock thiocyanatoiron(iii) ion product (FeSCN 2+ ) solution, by mixing the following in a small beaker using graduated cylinders to measure volumes: 3.0 ml of 0.00200 M KSCN 10.0 ml of 0.200 M Fe(NO 3) 3 solution 17.0 ml of 0.50 M HNO 3 Part B: 2. Using the stock solution prepared above, prepare a blank and 5 standard solutions of the FeSCN 2+ solutions using your best pipeting technique (be sure to rinse the clean pipet with the solution to be used first) to measure the volumes according to the table below so that each tube has a total volume of 5.00 ml: Tube # Volume of FeSCN 2+ stock solution (ml) Volume of HNO 3 (ml) Blank 0.00 5.00 1 5.00 0.00 2 4.00 1.00 3 3.00 2.00 4 2.00 3.00 5 1.00 4.00 3. Mix thoroughly by vortexing the tube. Part C: 4. If it is not already open, start the Logger Pro program on your computer. 5. Calibrate the spectrometer by filling a clean, dry cuvette ¾ full with the clear solution in the tube labeled blank. Use a grease pencil to make a small mark on the cuvette so that you can place it in the same orientation throughout the experiment. Wipe the cuvette with a KimWipe and place it in the spectrometer. Select Calibrate Spectrometer from the Experiment menu. The calibration dialog box will display the message: Waiting... seconds for lamp to warm up. The
Chem 1B Dr. White 13 minimum warmup time is one minute. Click Finish Calibration and then click. 6. Empty the blank cuvette and rinse it with a small amount of the solution in test tube 1, careful to coat the inside of the cuvette. Discard the rinse. Fill the cuvette ¾ full with the test tube 1 solution and place it in the spectrometer. Click. A full spectrum graph of the solution will be displayed. Note that one area of the graph contains a peak absorbance. Click to complete the analysis. Click the Configure Spectrometer Data Collection icon,, on the toolbar. A dialog box will appear. Select Abs vs. Concentration under Set Collection Mode. The wavelength of peak absorbance (λ max) should be automatically selected (it should be around 450nm). If it is not around 450nm, choose the λ max by clicking on the graph or by checking the box next to the desired wavelength. Record this value in your notebook. Click to proceed and then click Yes to store the latest run 7. Click When the absorbance reading stabilizes, click. Enter the concentration of the FeSCN 2+ in tube 1 (you need to calculate this) and click. Record the Absorbance values in your notebook. Pour the solution back into test tube 1 in case you need to use it again. Using the solution in tube 2, rinse and fill the cuvette ¾ full. Wipe the cuvette with a KimWipe and place it in the spectrometer. When the absorbance reading stabilizes, click. Enter the concentration. Repeat this procedure for test tubes 3, 4, and 5. When you have finished testing the standard solutions, click 8. To determine the best- fit line equation for the standard solutions, click the linear fit button,, on the toolbar. Write down the equation for the standard solutions in your notebook. The dialogue box will also display a correlation coefficient, which indicates the strength of the linear relationship between the two plotted variables. A correlation coefficient of 1.000 means the data points lie on a perfectly straight line. The closer your correlation coefficient is to 1.000, the stronger the linear relationship between absorbance and concentration, and the better your calibration line. 9. Click on the Autoscale button (the one with the A on it) to autoscale the graph. Arrange the floating boxes so they are not covering up each other, or covering up any part of your data. To save your data, from the menu bar select File, then Save, click on the pop down menu arrow next to the box labeled Save In:, and select desktop. Save your data as Exp 2,Your Name. 10. In Logger Pro, from the File menu, select Print, click Print Footer, type your name. The selected printer should be ISCI321000A. Click OK. The graph will be sent to the printer in the lab and printed there. Retrieve your graph and attach it to your lab report. 11. From the menu bar on the laptop, select Data, then Clear All Data. Close each of the floating dialogue boxes remaining on the graph by clicking the X in the upper left hand corner of each of the floating dialogue boxes. All excess solutions should be disposed of in the Chem 1B Waste Container, found in the fume hood at the back corner of the room.
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Chem 1B Dr. White 15 Name: Lab Day/Time: Experiment 2 The Beer-Lambert Law for Thiocyanatoiron (III) Data and Results Part A: Stock solution Concentration of Fe(NO 3) 3 Used to Make the Stock Solution M Volume of Fe(NO 3) 3 Used to Make the Stock Solution ml Concentration of KSCN Used to Make the Stock Solution M Volume of KSCN Used to Make the Stock Solution ml Volume of the Stock Solution ml 1 Concentration of Fe(NO 3) 3 in the Stock Solution M 2 Concentration of KSCN in the Stock Solution M 3 Concentration of Fe 3+ in the Stock Solution M 4 Concentration of SCN - in the Stock Solution M 5 Concentration of FeSCN 2+ in the Stock Solution M Show your calculations for Concentrations in the following boxes: 1 2 3
16 Chem 1B Dr. White 4 5 Part B and C Concentration and Absorbance of Standard FeSCN 2+ solutions: Wavelength of Maximum Absorbance (λ max): [FeSCN 2+ ] (M) Measured Absorbance Standard Solution in tube 1 Standard Solution in tube 2 Standard Solution in tube 3 Standard Solution in tube 4 Standard Solution in tube 5 Show your calculation for the [FeSCN 2+ ] standard solution in tube 1 below: Equation of Straight Line: Attach a copy of the standard curve your printed from Logger Pro.
Chem 1B Dr. White 17 Post Lab Questions 1. A group of students made 5 standard solutions and measured their corresponding absorbance values to generate the graph below. Absorbance of Standard Solutions to Find Concentration of Unknown X Solution Absorbance 0.50 0.40 0.30 0.20 0.10 0.00 y = 75.9x + 0.0045 R 2 = 0.9946 0 0.001 0.002 0.003 0.004 0.005 0.006 Concentration of X (mole/l) a) Using only the graph above, determine the extinction coefficient (ε) for a solution of X. Be sure to include units. Assume the student used a 1.00 cm wide test tube during analysis. (see equation 3 what constants does the slope relate to?). b) A student has an unknown sample of solution X (same X as in part a above). This solution was then inserted into the spectrophotometer and had an absorbance reading of 0.187. What was the concentration of their solution X in their unknown sample?
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