Riboflavin Fluorescence Spectra
Experiment Three Report
Riboflavin Fluorescence Spectra
Name: Holly
Experimental Section: Include all details on preparation of standard solutions. Also include the vitamin tablet brand name; the listed mass of vitamin B2 per tablet; procedure used for extraction of the tablet and dilutions performed.
The experiment began by determining the best wavelength at which to excite the riboflavin molecule, by examining the graph given in the laboratory manual[1]. Because plastic cells that absorb UV light were being used to hold the solutions, the wavelength had to be >320nm. To ensure that we could clearly see the peak of the riboflavin fluorescence in the stock solution, the peak we chose to measure was the one approximated at ~450nm. To measure this, a few drops of the stock 50ppb solution was pipetted into the plastic cell and the fluorescence machine was calibrated using the settings in the laboratory manual[1]. Once the fluorescence spectrum was measured the peak of the spectrum was found, and using the wavelength value that the peak in the fluorescence spectrum occurred at and a scan range of 475nm-800nm, the excitation spectra could then be measured.
The next stage of the experiment involved determining how the fluorescence intensity varies with the concentration of riboflavin, using solutions diluted from 50 ppb to 40, 30, 20 and 10 ppb. The dilutions were made using 40mL, 30mL, 20mL and 10mL respectively and measured into 50mL volumetric flasks, using 20mL and 10mL pipettes, and all the solutions were remade, as the data measured was extremely different from the results expected and thus dismissed as incorrect. Once the solutions were remade, the data measured was much closer to the expected results. During this part of the experiment, when trying to measure how the fluorescence varies with concentration, the computer and fluorescence instrument had issues running the second file, chem2002b.mth, and so the first file, chem2002a.mth was
Riboflavin Fluorescence Spectra
Name: Holly
Experimental Section: Include all details on preparation of standard solutions. Also include the vitamin tablet brand name; the listed mass of vitamin B2 per tablet; procedure used for extraction of the tablet and dilutions performed.
The experiment began by determining the best wavelength at which to excite the riboflavin molecule, by examining the graph given in the laboratory manual[1]. Because plastic cells that absorb UV light were being used to hold the solutions, the wavelength had to be >320nm. To ensure that we could clearly see the peak of the riboflavin fluorescence in the stock solution, the peak we chose to measure was the one approximated at ~450nm. To measure this, a few drops of the stock 50ppb solution was pipetted into the plastic cell and the fluorescence machine was calibrated using the settings in the laboratory manual[1]. Once the fluorescence spectrum was measured the peak of the spectrum was found, and using the wavelength value that the peak in the fluorescence spectrum occurred at and a scan range of 475nm-800nm, the excitation spectra could then be measured.
The next stage of the experiment involved determining how the fluorescence intensity varies with the concentration of riboflavin, using solutions diluted from 50 ppb to 40, 30, 20 and 10 ppb. The dilutions were made using 40mL, 30mL, 20mL and 10mL respectively and measured into 50mL volumetric flasks, using 20mL and 10mL pipettes, and all the solutions were remade, as the data measured was extremely different from the results expected and thus dismissed as incorrect. Once the solutions were remade, the data measured was much closer to the expected results. During this part of the experiment, when trying to measure how the fluorescence varies with concentration, the computer and fluorescence instrument had issues running the second file, chem2002b.mth, and so the first file, chem2002a.mth was
References: The relevant literature used throughout the report must be appropriately referenced. In the text of the sections above, indicate a reference by a superscript number. [1]School of Chemistry & Molecular Biosciences 2013, CHEM2002 Physical Chemistry, Laboratory Manual, University of Queensland [2] J.A. Henderleiter et al., Chem. Ed., 1996 73 563-4. The analysis of riboflavin in urine using fluorescence. [3] M.G. Dauyvis et al., J Agric. Food Chem., 2002, 50, 1548-52. Role of Riboflavin in Beer Flavour Instability.