Activity 6: Guide
Protocol for Relative Quantification of Riboflavin Content
Materials
3 Chicken Eggs
Distilled Water
Commercially available food supplements containing riboflavin (at least 3 samples)
1.5-mL microcentrifuge tubes
Protocol
A. Preparation of Egg White Titrant
Break the eggs and separate the yolks from the whites.
Add 500 mL of distilled water.
Stir magnetically for several minutes until a flocculent suspension is produced.
B. Preparation of Vitamin Samples
Dissolve each food supplement capsule contents/ tablet in 100 mL distilled water.
Stir magnetically until a homogeneous solution is obtained.
Record (% w/v) of the aqueous vitamin solutions.
C. Fluorometric Quenching Titration
1. For each aqueous vitamin solution,
a. Prepare the following dilutions:
|
MCT |
Aq. Vitamin Solution (uL) |
Distilled Water (uL) |
Egg White Solution (uL) |
|
1 |
300 |
2700 |
0 |
|
2 |
300 |
2400 |
300 |
|
3 |
300 |
2100 |
600 |
|
4 |
300 |
1800 |
900 |
|
5 |
300 |
1500 |
1200 |
|
6 |
300 |
1200 |
1500 |
|
7 |
300 |
900 |
1800 |
|
8 |
300 |
600 |
2100 |
|
9 |
300 |
300 |
2400 |
|
10 |
300 |
0 |
2700 |
b. Measure the fluorescence of all the solutions using an excitation wavelength of 280 nm and emission wavelength of 525 nm.
c. Plot fluorescence versus volume of egg white solution added.
d. Observe that as more egg white titrant is added, the fluorescence decreases until a plateau is reached. Determine this final fluorescence after equivalence point is reached
e. The differences of final fluorescence from the initial fluorescence are directly related to the amount of riboflavin in the original capsules/tablets and are directly comparable. Make sure to account for dilution factors.