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Understanding Beer's Law Аас A = absorbance c = concentration of analyte There are specific conditions related to absorbance, such as, the wavelength, the wavelength- dependent molar absorptivity, and the path length of the cuvette. The wavelength-dependent molar absorptivity is a measure of how strongly a chemical species absorbs light at a particular wavelength. The path length is the width of the cuvette. a A = , bc A = absorbance E = wavelength-dependent molar absorptivity b = path length of cuvette c = concentration of analyte Each sample is irradiated with light (P.). If light is absorbed by the analyte in the sample, a lower intensity of light (P) is emitted from the cuvette. The intensity of light before and after passing through the cuvette is measured and the absorbance of the sample is obtained, as shown below. Î P P. A = -log ale A = absorbance P = the source light power after passing through the sample P. = the source light power 2
To generate a standard curve, a series of solutions with known concentrations will be prepared. The absorbance of each standard in the series will be measured (at a specific wavelength) and plotted (see Fig 2a). The best fit curve will be drawn through the data points (see Fig 2b). absorbance absorbance 0 concentration (mg/l) 0 concentration (mg/L.) A Best Fit Line is Drawn Fig 2a Absorbance is Measured Fig 26 The absorbance of the unknown sample is the measured at the same wavelength and compared to the known solutions (see Fig 3). absorbance concentration (mg/L) Fig 3 Absorbance of the Unknown is used to Determine the Concentration of the Unknown 3
Procedure Common dyes used as food coloring will be analyzed in this experiment. The widely used dyes. Red #40 or Blue #1, will be studied. Solutions of pure Red 1840 or pure Blue # I will be prepared and measured. Using these data, the quantity of Red 1840 or Blue #1 food coloring in commercial beverages will be determined. Preparation of the Standards for the Standard Curve The standard solutions of Blue #1 or Red #40 needed for the standard curve are shown in Table 1. Before the standard solutions can be prepared the dilution calculations must be completed. The equation CV - CV, will be used in the calculations. Table 1 Serial Dilution of Standards with Known Concentrations of Analyte Dye Sample Sample Sample Sample Sample Sample A B С D E o Blue #1 16 mg/L 8mg/L 4 mg/L 2 mg/L 1 mg/L Omg/L. (water blank) Red 40 48 mg/L 24 mg/L 12 mg/L 6 mg/L 3 mg/L O mg/L (water blank) Serial Dilutions of the FD&C Red #40 Standards To prepare 100 mL (V:) of Sample A (Cs) with a concentration of 48 mg/L. of Red #40 from the Stock solution (96-mg/L) (C), how much (V) of the Stock solution will be needed? Dilutions CV: C: V To prepare Sample A from the Stock Solution: |(96 ms) <; = ( 48 ms) (100 ml) V mg/ V, = 50 ml mg |(48 Ep) vi = (24 ms) (100 ml) Vi To prepare Sample B from mg Sample A: V = 50 ml Similar calculations will be needed to prepare Samples C, Dand E.
Preparing the FD&C Red #40 Standards by Serial Dilution Using a 100-ml beaker, measure out 60 mL of the Red #40 Stock Solution (96-mg/L) and bring the beaker back to your lab bench. Using a 25-ml plastic volumetric pipette, a pipette pump, and a 100-ml volumetric flask carefully prepare by serial dilution 100-ml solutions of all of the dye concentrations needed. Preparation of the Commercial Beverage Samples for Analysis using the Standard Curve Pour approximately 50 mL of the commercial beverage into a clean beaker and bring to your lab bench Sample U10 Using a labeled volumetric flask, prepare a 10/100 dilution of the commercial beverage Sample U25 Using a labeled volumetric flask, prepare a 25/100 dilution of the commercial beverage. Sample U00 Put a few milliliters of the commercial beverage (not diluted) into a labeled test tube. Table 2 Preparation of the "Unknown" Commercial Beverage Samples Analysis of Dye in Commercial Beverage Vi V, Sample U10 Commercial Beverage Diluted 10 to 100 10 ml 100 ml Sample U25 Commercial Beverage Diluted 25 to 100 25 mL 100 ml Sample UOO Commercial Beverage not diluted. Put a few milliliters of the unknown sample into a test tube.
Spectrometer Setup 1. Connect the spectrometer to the LabQuest2 using the USB cable and connect the power supply to the LabQuest2. Tap the red button on the top left of the LabQuest2 and it should tum on within 5 seconds. 2. When the LabQuest2 is on and connected to the spectrometer, a red rectangle that shows UBS: Abs should be visible on the screen 3. Setting-up Abs at Specific Wavelength Measurements a. On the right of the screen where Mode is shown, it should show Events with Entry. If it shows Full Spectrum, tap on Full Spectrum to go to the next page and using the arrow at the pull-down menu, change the Mode by selecting Events with Entry b. tap OK 4. Setting a Specific Wavelength a. tap anywhere on the red rectangle b. select Change Wavelength c. type in the 500 nm (for red dye) and 630 nm (for blue dye) d. tap OK 5. Calibrate Spectrometer a. tap on Sensors from the menu bar b. tap on Calibrate then select USB: Spectrometer cpause about 5 seconds while Dark Sample is taken d. wait 90 seconds for lamp to warm up c. When the warmup is complete, place a cuvette containing dcionized water into the cuvette holder, be sure to match up the arrow on the cuvette with the arrow on the spectrometer f. Tap Finish Calibration & When the message Calibration completed and the Sample Time in ms is shown, tap OK. 7. Begin Data Collection a Tap on the green arrow at the bottom left to begin data collection b. Leave the water blank in the spectrometer, 8. Set Screen to a Meter Readout 3. To read the Abs of each sample at a specific wavelength with a Meter readout, go to the Menu Bar at the top of the screen and tap on the Meter Icon (an analog meter), typically at the upper left of screen. b. Abs should be visible in the red rectangle on the screen. c. With the water blank still in the spectrometer, record the first data point, which should read 0.000 for Absorbance. d. Do NOT turn off the spectrometer 9. Collecting Abs Data of Known Standards and Unknown Samples a. Remove the cuvette from the cuvette holder b. Pipet the solution from the cuvette and add the next solution. c. Wipe off the sides of the cuvette. Match up the arrow on the cuvette with the arrow on the Spectrometer d. Record the Abs value. Do the same for all of the known and unknown samples. Do NOT turn off the spectrometer between readings. 10. End Data Collection a. When ALL of the Abs values have been recorded, tap on the red square at the bottom left to end data collection. b. Data collection is complete when the green arrow (not the red square) at the bottom is visible again. 6
U00 Measuring the Absorbances Measure the absorbances for the FD&C Red #40 standard solutions and the unknowns at 500 nm or measure the absorbances for the FD&C Blue #1 standard solutions and the unknowns at 630 nm a. Record the absorbances of the standard solutions in the table below. b. Record the absorbances of the unknown samples in the table below Table 3 Standards with Known Concentrations of the Analyte and Unknown Samples Sample A B С D E O U10 U25 Concor 48 24 12 6 3 0 Dye mg/L mg L mg/L mg/L mg/L mel TBD TBD TBD Abs mm TBD - to be determined from standard curve (and dilution calculations, if applicable) Clean-up Steps Rinse and RETURN all plastic pipettes, cuvettes, and stoppers to the bins on the reagent area. Remove all labels from the volumetric flasks. Rinse and return to the reagent area. Include in Lab Report 1. Plot the Standard Curves. From the data obtained, make a graph of the data from the standards (Samples A-E, and o) on the curve (absorbance vs. concentration, see Fig 2a and Fig 2b). 2. Analysis the Commercial Beverage. Only use samples with an absorbance less than 1. Determine the concentration of dye in the commercial beverage (Samples U10, U25, and 100) from the standard curve (see Fig 3), Report the quantity as mg/L. 3. Determine Concentration of Dye in the Original Commercial Beverage. Since samples U10 and U25 were diluted, dilution calculations must be performed to determine the concentration of dye in the original solution. Report the quantity as mg/L. Questions 1. Explain the importance of a water-blank in a standard curve. 2. Explain if the independent variable is placed on the xor y axis and if the dependent variable is placed on the x or y axis when preparing a standard curve 3. Hydrogen peroxide (H:09) is a substance with a rather short shelf-life and quickly decomposes over time. If one wanted to make a graph for the decomposition of H:02 over 90 days and chose to measure the amount of H:0; (moles) in a bottle every day for 90 days, how would the graph be designed, i.e. would time (days) be on the x axis and H-0: (moles) on the y axis or would time (days) be on the y axis and H:0 (moles) on the x axis? Explain why. 7
4. Data and Observations include data and observations from the experiment) U25 UOO Measure the absorbances for samples (A-O) and record. (see Table 3) (at 500 nm for the Red #40 solutions or at 630 nm for the Blue #1 solutions) Standards with Known Concentrations of the Analyte and Unknown Samples Sample A B с D E o U10 Conc of 48 12 6 3 0 Dye mg/L | mg/L mg/L mg/L mg/L mg/L TBD TBD Abs nm TBD = to be determined from standard curve (and dilution calculations, if applicable). Be sure to fill in the wavelength at which the absorbance was measured. 24 TBD 5. Results and Calculations a. Make a graph of the absorbance vs concentration of the standards. (see Figs 2a and 26) b. From the graph, determine the concentration of samples U10, U25, and U00. (see Fig 3) c. Since samples U10 & U25 were diluted, the concentrations found on the standard curve are the concentrations of the diluted samples. Using the equation CV = C2V2. calculate C (the undiluted concentration) of samples U10 & U25.
7. Conclusion for Experiment Discuss ways in which the data from this lab could be improved. If you were to come into the lab to perform this experiment, suggest ways in which this laboratory could be improved. 8. Post-Lab Questions a. Explain the importance of a water-blank in a standard curve. b. Explain if the independent variable is placed on the x or y axis and if the dependent variable is placed on the x or y axis when preparing a standard curve. c. Hydrogen peroxide (H:02) is a substance with a rather short shelf-life and quickly decomposes over time. If one wanted to make a graph for the decomposition of H2O2 over 90 days and chose to measure the amount of H2O2 (moles) in a bottle every day for 90 days, how would the graph be designed, i.e. would time (days) be on the x axis and H2O2 (moles) on the y axis or would time (days) be on the y axis and H2O: (moles) on the x axis? Explain why 9. Additional References (If additional references were used to complete this report, site references here.)