EXPERIMENT 7

 

ANALYSIS OF VITAMIN B2 TABLETS BY FLUOROMETRY

 

Objective:  To determine the amount of riboflavin in vitamin B2 tablets using the

fluorescence properties of riboflavin and UV spectrometry

 

Introduction:  

Riboflavin is the active compound in vitamin B2 tablets and it has fluorescence

properties.  Since at low concentrations, fluorescence is found to be directly

proportional to concentration, quantitative measurements of unknown samples can be

made with the aid of standard solutions and/or calibration curves.

 

Experimental

Reagents/Equipment

Vitamin B2 stock solution – Concentration: 0.30 mg/ml

10 ml Volumetric flasks

Pipets (2ml and 10ml)

Vitamin B2 tablet

250 ml Volumetric flask

Turner Filter Fluorometer

Cuvetts

 

Procedure:

 

1) Using the vitamin B2 stock solution, prepare 6 standard solutions (10 mL each) ranging from 0.03 mg/ml to 0.30mg/ml (suggested final concentrations: 0.03, 0.09, 0.15,

0.18, 0.24, and 0.30 mg/ml). 

 

2) Using H2O as a blank, determine and record the fluorescence reading of each solution.

The excitation wavelength should be set at 444 nm and the emission wavelength at 530

nm.  Set ‘Fixed Scale’ at 1.000 units and the emission slit at 2.5 (round knobs on right

and left hand sides of the instrument).

 

3) Perform steps 1- 2 a total of three times.  Record all of these values.

 

4) Prepare a table and plot of fluorescence versus concentration, and verify the direct

relationship between concentration and fluorescence. This plot will be referred to as the

standard curve.  Be sure to include the error bars in the graph from the three separate

runs.  Also acquire the equation for the line.

 

5) Obtain a sample of vitamin B2 tablet.  Weigh and reduce 1 tablet to a fine powder (mortar and pestle).  Weigh 10 mg of the powder and transfer quantitatively to a 250 ml volumetric flask.  Dilute to about 150 ml with cold H2O.  Dissolve the powder (not all of the particle will dissolve) and adjust the volume with cold H2O to exactly 250 ml.  Mix thoroughly.

 

5) Transfer exactly 2.0 ml of the solution to a 10ml volumetric flask and make up the volume with H2O.  This should be a transparently clear pale yellow solution. Do this step three separate times to obtain three different sample readings.  Read and record fluorescence intensity of the solutions.

 

6) Determine the concentration of the unknown solution from the standard curve.  Make the correlation between the fraction of the tablet present in the solution and the concentration (mg/ml).  Determine the mg/tablet of riboflavin.

 

Please include your sample calculation for making up standard solutions.  Be sure to also include the calculation for mg of riboflavin/tablet.

 

Question: (please answer the following questions in your laboratory notebook)

The tablet label claims that there is 5 mg of riboflavin per tablet.  Based upon your lab results, does the tablet contain the same amount of riboflavin as it says on the label? 

Calculate the standard deviation of the lab result in comparison to the amount on the label.

 

DATA SHEET

ANALYSIS OF VITAMIN B2 TABLETS BY FLUOROMETRY

 

Concentration of Vitamin B stock solution                        

 

Exicitation wavelength:                nm                  

Emission wavelength:                nm

 

Concentration of

Standard Solutions                                         Fluorescence Intensities

                                                                                                                                               

                                                                                                                                               

                                                                                                                                               

                                                                                                                                               

                                                                                                                                               

                                                                                                                                               

 

 

Mass of Vitamin B2 tablet:             g

Mg tablet used for this analysis:                

 

Concentration of Vitamin B2 in the unknown from the standard curve:

                                                                                                                                               

 

Mg of vitamin B2 per tablet:                                                                                     

 

Average mg of vitamin B2 per tablet (+/- std):                                                                                                                

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Notebook:                                                                                          Final grade:                        

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA NOTE-  USE 5mg not 100 mg tablets