Riboflavin Fluorescence Spectra

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 used, however this issue, and having to redo the dilutions, cut into the experiment time. Using the data gathered in this part of the experiment, a graph of Fluorescence Intensity vs Concentration (in ppb) could be made.

After the Fluorescence Intensity vs Concentration was graphed, the experiment called for the extraction of 100mg of riboflavin from a “Nature’s Own” Vitamin B2 tablet, and interpolation from the graph to find the concentration, after the fluorescence was measured. This was done by measuring out 50mL of 25% acetic acid solution using a 100mL measuring cylinder to extract the riboflavin after it was crushed using a mortar and pestle and washed into a 50mL beaker. While washing the acetic acid and crushed pill solution from the mortar and pestle used, some spillage of the solution occurred, but the solution could not be remade due to a lack of time. This was then put onto a magnetic stirrer and stirred for approximately 10 minutes.

During the time while the solution was being stirred, the dilution calculations were performed to determine the amount of dilution required for the solution to fall between a 0-50ppb concentration, as the concentration could then be interpolated from the data gathered in the previous part of the experiment. Once the solution had been stirred for 10 minutes, it was removed from the fume cupboard and sat for a few minutes before the dilutions were

performed, making it possible that the grounds that had not been dissolved had time to settle to the bottom. The dilutions were performed using 50mL volumetric flasks and a 5mL automatic pipette and the fluorescence was measured, where it was discovered that the solution had an extremely high fluorescence intensity. It was discovered that the unit conversion done at the beginning of the dilution calculations was incorrect and as time was getting low, the solution was diluted twice more than originally thought necessary, before the fluorescence was measured with a reasonable result achieved. The calculations were then redone with the correct units. After this was done, the concentration of the solution with the crushed tablet could be interpolated from the graph done earlier and compared to our calculated value, and the mass of the tablet calculated.

Results Section:
Section 1. Examine the nature of absorption, excitation and fluorescence spectra. Include values for absorption, excitation and fluorescence spectra maxima. Upload your spectra in this section.

Figure 1. Fluorescence vs Wavelength graph of Riboflavin 50ppb Stock Solution The first part of the experiment involved graphing the fluorescence intensity of a 50ppb Riboflavin stock solution. The wavelength to begin measuring from was based on two factors, the fact that the plastic UV cells that were being used absorbed light up to 320nm, and that the molecule needed to absorb light very strongly, and needed to be seen clearly on the graph. Thus, reading the graph provided in the lab manual[1], it could be seen that there was a peak at around ~450nm which would easily be able to be seen without interference from the plastic cells. In the sample measured from the stock solution, the peak occurred at a  wavelength of 523.52nm and reached a peak of 115.52.

The excitation spectrum was then measured on the same graph. The wavelength at which the peak occurred on the fluorescence spectrum was entered into the program, and a scan range from 350nm to 800nm was used. The excitation spectrum had two peaks, the first occurring at a wavelength of 366.15nm and reaching a peak of 103.44 and the second at a wavelength of 442.18nm and a peak of 125.88 being reached.

Section 2. Examine how the fluorescence intensity varies with the concentration of riboflavin in solution. Include a plot of fluorescence intensity values as a function of riboflavin concentration. Include best fit and error calculations.

Figure 2. Fluorescence Intensity vs Concentration of Riboflavin The fluorescence intensity vs concentration graph of riboflavin above was created by diluting a stock solution of 50ppb, to 40, 30, 20 and 10ppb and measuring it in the fluorescence instrument. As can be seen in figure 2, as the concentration of riboflavin increases, so does the fluorescence intensity, and the graph is linear, in the form y=mx+c. The error in m=±0.133 and the error in c=±4.413 according to WinCurveFit. Section 3. Determine the concentration of riboflavin in your vitamin tablet. Include the calculation of the mass of riboflavin per tablet. Include error calculations. Determining the number of dilutions needed for our vitamin tablet to get it in the range of 050ppb. First the concentration of the vitamin tablet in ppb needed to be determined. First 100mg of riboflavin in the tablet (stated on the bottle) was diluted to 50mL with acetic acid. ppb=μg/L=100mg/50mL=0.1g/0.05L=100,000μg/0.05L= 2,000,000 ppb Working backwards, a concentration of between 0-50ppb is needed, so 20ppb should be a good value to get a reading, and easy to use in calculations. 5mL extractions were decided to enable ease of calculations and a lower error.

CiVi=CfVf
Where Ci –Initial concentration
Vi – initial volume
Cf – final concentration
Vf – final volume

So to find the initial concentration:
Ci=CfVf/Vi
Ci=20ppb*50mL/5mL= 200ppb
This continues until the right original concentration is reached: Ci=200ppb*50mL/5mL=2,000ppb
Ci=2,000ppb*50mL/5mL=20,000ppb
Ci=20,000ppb*50mL/5mL=200,000ppb
Ci=200,000ppb*50mL/5mL=2,000,000ppb

This means that 5 dilutions are needed when taking 5mL and diluting it to 50mL each time. Once this was been done, a sample of the last solution was placed into a plastic cell and measured with the fluorescence spectrophotometer and the fluorescence intensity was measured to be 83.04. Using the equation from Figure 2, and substituting the fluorescence intensity for the riboflavin tablet solution measured into the equation, the concentration of riboflavin was determined. The mass was then calculated by working backwards from the final dilution concentration to find the initial dilution concentration, and the mass from that. Calculations are shown below:

Equation from Figure 2: y=3.2834x +9.294
Substitute y=83.04 into eqn.
83.04=3.2834x+9.294
x=22.46
Thus, the concentration of the final dilution is 22.46ppb.
CiVi=CfVf
Ci=CfVf/Vi
Ci=22.46*50/5
Ci=224.60ppb

Thus, the concentration of the previous dilution was 224.60ppb. This continues until the correct number of dilutions that were performed has had the concentration calculated:

Ci=224.60ppb*50mL/5mL=2,246.03ppb
Ci=2,246.03ppb*50mL/5mL=22,460.25ppb
Ci=22,460.25ppb*50mL/5mL=224,602.55ppb
Ci=224,602.55ppb*50mL/5mL=2,246,025.46ppb

Thus, the original concentration of the riboflavin tablet solution (and thus in the riboflavin tablet) was 2,246,025.46ppb

To find the mass of riboflavin in the tablet:
mass=concentration*volume
Remembering that ppb=μg/L
mass=2,246,025.46*10^-6g/L*0.05L
mass=0.1123g=112.3mg
Error calculations:
Fluorescence intensity results=±1%
Our eqn is: y=3.2834x+9.294 which is in the form: y=mx+c
We want to find the error in our concentration, x.
Δx=√((Δy^2)+(Δc^2)+((Δm/m)^2))
Δx=√((0.001^2)+(4.413^2)+((0.1330/3.2834)^2))
Δx=4.413

The errors in the mass calculated by working backwards from the concentration will be larger than the error in the concentration from the formula. The main sources of the error are from the spillage of the original
crushed riboflavin solution that occurred, letting the solution sit for a few minutes after stirring, dilutions being slightly less than 50mL in some cases, and other mistakes due to time constraints.

Discussion Section: You must state the overall (main) outcome of the experiment (e.g. riboflavin concentration in the vitamin tablets) and describe in words how it was obtained. Compare your mass of riboflavin to that claimed on the bottle (did or did you not get similar result, if not, why might that be?). Include a discussion of the main sources of experimental error.

The aim of this experiment was to determine the concentration of riboflavin contained in a “Nature’s Own” Vitamin B2 tablet by comparing the fluorescence intensity vs concentration of known concentrations of riboflavin to the unknown sample in the tablet. This was done by taking measurements of various dilutions of a 50ppb stock solution, and measuring them using the fluorescence instrument, and then crushing the riboflavin tablet with a mortar and pestle and washing it out with acetic acid. Once it was crushed, it was placed onto a magnetic stirrer for 10 minutes before being diluted five times, based on calculations done earlier.

The mass of riboflavin determined from the sample from the tablet, which was 112.3mg was very similar to the 100mg amount listed on the bottle, and the main sources of error included: spilling some of the riboflavin solution while washing the mortar and pestle out with acetic acid, which could have changed the original concentration; letting the solution sit for a few minutes after stirring, allowing the particles to settle back onto the bottom of the beaker, making the concentration throughout the solution not uniform; diluting the solutions to just under 50mL, making them more concentrated; and other various human errors. When these errors are included, it is seen that the mass of riboflavin calculated was a similar result to the mass stated on the bottle, and thus fairly accurate.

Conclusion Section: In a short paragraph link your findings to the experimental objective described in the introduction (laboratory manual). Riboflavin is a very fluorescent molecule, and can be found in many areas of
study including using fluorescence spectroscopy to analyse for riboflavin in urine[2], as well as being one identified in malt as one of the factors contributing to a stale flavour in “sun-struck” beer[3]. In this experiment a fluorescence spectrometer was used to determine the concentration of an unknown amount of riboflavin from a “Nature’s Own” vitamin B2 tablet. This was done by measuring and plotting different fluorescence intensities of known concentrations of riboflavin and then measuring the unknown concentration of a dilution of a solution containing crushed vitamin B2 tablet and acetic acid, and interpolating between the known data to find the concentration. It was then possible to calculate by working backwards through the dilutions performed on the unknown solution to find the mass of riboflavin in the tablet, which could then be compared to the stated mass of riboflavin and any discrepancies commented on.

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.