Fluorometric Determination of Riboflavin (Vitamin B2)
Riboflavin is strongly fluorescent in 5% acetic acid solution. Excitation and fluorescence spectra will be obtained to determine the wavelengths of excitation and emission. This information will be then used to analyze standards, create a calibration graph, and determine an unknown concentration.
1-l volumetric flask
On the day of the experiment a 50.00 ppm solution will be prepared by diluting 50.00 mg of riboflavin into a 1-l flask and diluting with 5% (vol/vol) acetic acid. (CAUTION riboflavin is light sensitive. The flask should be wrapped with Al foil to prevent decomposition.) This solution will be subsequently diluted 1:5 in 5% acetic acid, to make a 10.00 ppm solution. Further dilute aliquots of this second solution with 5% acetic acid solution to prepare standards of 0.000 (blank), 0.200, 0.400, 0.600, 0.800, and 1.000 ppm riboflavin.
Using a chart recorder, obtain excitation and emission spectra of the 1.000 ppm solution to determine the best excitation and emission wavelengths. A suggested procedure is to set the emission wavelength to 520 nm and scan the excitation spectrum from 300 to 600 nm monitoring and noting at what wavelengths the peaks occur. Next, set the excitation wavelength to 390 nm and scan the emission spectrum form 300 to 600 nm noting the wavelengths at which the peaks occur. Using the wavelengths determined from the scans, fix the excitation and emission wavelengths. Obtain the maximum meter deflection by slowly rotating the excitation and emission wavelength selector. Once the maximum deflection has been reached, adjust the gain to give a reading 90% of full scale. Read the fluorescence intensity of all standards three times, using a new sample solution in the cell each time. Using this information, prepare a calibration curve. Obtain an unknown and check with your laboratory instructor whether the unknown may need to be diluted. If the sample needs dilution use the spectrophotometer to determine the absorbance. Set the wavelength of the spectrophotometer to the fluorescence excited wavelength. Make an appropriate dilution to reduce the absorbance to less than 0.05. Record your dilution. Measure the fluorescence intensity of the unknown three times, changing the solution in the cell every time. From the calibration curve calculate the mass (in micrograms and ppm) of riboflavin in your original (and diluted) unknown solution.
Report the concentration (in ppm) of your unknown and calculate the standard deviation of that value. Include absorption and fluorescence spectra in your report.
Question: Why is it necessary to maintain A<0.05? How would be the result affected if A>0.05?
Copied from a previous handout: 24 August 1998