# Part 2 fluorescence of riboflavin goal divide work

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Part 2: Fluorescence of riboflavin Goal Divide work amongst your group to create a calibration curve for riboflavin. Use the calibration curve to determine the concentration of riboflavin in Rockstar energy drink. Procedure 1.Obtain approximately 50 mL of riboflavin (approximately 25.00 mg/L). Record the exact concentration of the stock solution. You will need to create dilutions of this stock solution in order to measure the constant kin ࠵? =࠵?࠵?8࠵?. Buret and Flask Group work: Student #3 - Riboflavin 1.Using the buret and flask method (Appendix A), prepare one standard solution at a time, transferring each solution to a cuvette for analysis. Use the table belowas a guide for solution preparations. Use %1 acetic acid to dilute your solutions to total volume of 5-mL. 2.Record the exact volumes you actually dispense and use these to calculate your final riboflavin concentration. When reusing the same glassware or cuvettes, it is good analytical practice to proceed from low to high concentration. Why? Table 4.Riboflavin standards preparation Standard Initial riboflavin concentration (M) Target volume of riboflavin (mL) Actual volume of riboflavin dispensed (mL) Total volume (mL) Final riboflavin concentration (M) 9 1.0 5.00 10 2.0 5.00 11 3.0 5.00 12 4.0 5.00 13 5.0 5.00 14 6.0 5.00 15 7.0 5.00 16 8.0 5.00 3.Using a fluorescence spectrophotometer, measure the intensity versus wavelength (Appendix C) for each standard solution.
Experiment 7 Spring 2020 Page 15 of 19 For fluorescence measurements no blank is required. Why is this? 4.Record the intensity at the wavelength of maximum fluorescence emission for each standard solution (#10 - #16). Table 5.Riboflavin calibration data Standard Fluorescence intensity at λmax(λmax= _____ nm) 9 10 11 12 13 14 15 16 Analysis of energy drink Group work: Full group (students #1, #2, #3) 1.Obtain a sample of Rockstar energy drink and place into a cuvette (remember, you must use a cuvette that is clear on all four sides). 2.Record the fluorescence of the energy drink using the samespectrophotometer you used previously. See Appendix Cfor fluorescence spectrophotometer instructions. 3.Graph your data and use the calibration curve to determine the concentration of riboflavin present in Rockstar. Compare this to the concentration of riboflavin reported on the back of the can.
Experiment 7 Spring 2020 Page 16 of 19 Lab Report 1.Calculate the actual concentrations of all the dyestandards and riboflavinstandards you made. 2.Construct calibration plots of absorbance vs. concentration or fluorescence vs. concentration, and determine the extinction coefficient or it fluorescence counterpart. a.Dye Standards: i.Use concentration and the absorbance data to construct Beer’s Law Plots for all your samples. ii.Using the techniques of linear regression, determine the slopes of these curves. iii.Calculate the extinction coefficients for each dye studied. Report the wavelength used for each value. b.Riboflavin Standards: i.Use concentration and the fluorescence data to construct a linear calibration curve for all your samples. ii.Using the techniques of linear regression, determine the slopes of these curves. For fluorescence, what does the slope represent? 3.