For instructional purposes only • 1st Semester SY 2020-2021 55

Exercise No. 8:
Vitamins
Introduction
Vitamins are organic compounds that are not synthesized by the cells of the
body and must be provided in our diet. They often act as cofactors for certain
enzymes. The deficiency of a vitamin in a diet can affect the activity of an
enzyme bringing about a deficiency disease. A lack of vitamin C can cause
scurvy, while a deficiency in Vitamin A is associated with night blindness. A
diet low in vitamin D can cause rickets in children, and a deficiency in B 12 can
lead to anemia. Vitamins B and C which are soluble in water are called “water-
soluble vitamins”, while vitamins A, D, E, and K are “fat soluble vitamins”.

In 1979, the National Academy of Sciences recommended the following daily

dietary allowances for females 23-50 of age, 120 lb.
Vitamin A 800 μg Riboflavin 1.2 mg
Vitamin D 5 μg Niacin 13.0 mg
Vitamin C 60 mg Vitamin B6 2.0 mg
Thiamin 1 mg Vitamin B12 3.0 mg
Folic acid 300 μg

TESTING FOR VITAMIN C

Vitamin C by the body to fight infection and repair damaged tissues. It is
present in a variety of foods including citrus fruits. Since vitamin C is a
reducing agent, we can measure its presence by its reaction with iodine. The
indicator for this reaction is starch. When iodine is present, the starch turns a
deep-blue color. However, if vitamin C is present and it has reduced the iodine
to iodide, the starch does not react and no deep-blue color forms.

Vitamin C
I2 2 I-
Blue with starch no color with starch

Learning Outcomes
1. To identify a vitamin as water or fat-soluble.

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 56 CHEM 132.2: Biochemistry Laboratory

2. To determine the vitamin C content in a variety of citrus juices and

other solutions.
3. To determine the effect of heat upon vitamin C.

Materials
 Vitamins A, B, C, D, E (or as many as Fruit Juices: orange, grapefruit,
possible) powdered, etc.
 Methylene chloride 1% starch indicator

 Vitamin C tablet Erlenmeyer flasks (250-mL)

 50-mL buret Iodine reagent (16 g KI + 1.5 g I2

 6M HAc (Acetic Acid) in 1L solution)

Procedure

A. VITAMIN SOLUBILITIES
Place a small amount of each available vitamin in separate test tubes.
Add 2 mL of water to each sample. Mix and observe each solution. If
two layers form, the vitamin is not water soluble. If the vitamin
dissolves to give a clear solution, it is water soluble. Record your
observations. Repeat the procedure adding 2 mL methylene chloride
(CH2Cl2) in place of water to each vitamin sample. Record your
observations.

B. STANDARDIZATION OF VITAMIN C
B-1 Weigh a 250mL Erlenmeyer flask and record.
B-2 Crush a tablet of vitamin C (100 mg tablet) and place in the flask.
Record the mass of the flask and the vitamin C.
B-3 Add 25 mL distilled water and mix. Add 2 mL 6M HAc and 3 mL
starch. Set up a buret for fixation. Fill the buret with prepared iodine
solution. CAUTION: KEEP IODINE REAGENT AWAY FRON CLOTHES
AND SKIN. Record the initial reading of the iodine solution in the buret.
B-4 Add the iodine to the flask containing the vitamin C tablet until a
deep-blue color persists for 30 sec. This is the endpoint. Record the
initial reading of the iodine solution in the buret.
B-5 Calculate the mg vitamin C contained in the Erlenmeyer flask.
B-6 Calculate the volume of iodine solution.

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For instructional purposes only • 1st Semester SY 2020-2021 57

B-7 Calculate the mass (mg) vitamin C that reacts with 1 mL iodine
solution.
mg vitamin C
= mg vitamin C/1 mL iodine solution
mL iodine solution

C. DETERMINATION OF VITAMIN C IN FRUIT JUICES

C-1 Place a 20 mL sample of a fruit juice in a clean Erlenmeyer flask.
You may also use 1.0 g of a powdered fruit drink such as Tang.
Record the type of fruit juice used. Add 50 mL of water and 2 mL 6M
HAc. Add 3 mL 1% starch indicator.
C-2 Add iodine solution from your buret to the flask containing the
fruit juice and starch indicator until a deep-blue color is obtained.
Deeply colored juices may obscure this color. Record the volume of
iodine solution added to reach the endpoint of the reaction.
C-3 Calculate the mg vitamin C in the sample. Use the value of mg
vitamin C/mL iodine solution obtained in step B-7.
mg vitamin C
mL iodine solution x = mg vitamin C in sample
1 mL iodine solution

C-4 Repeat the titration again and average your results, or try other
fruit juices available in the lab. Record the results.

D. HEAT DESTRUCTION OF VITAMIN C

D-1 Obtain 20-mL of the juice that gave you the greatest amount of
vitamin C content. Place the sample in a 250-mL Erlenmeyer flask.
Add 50 mL of water. Using a low flame, gently boil the solution for 10
min. remove the flask and cool it in an ice- water bath. Add 2 mL HAc
and 3 mL starch indicator. Titrate with iodine solution to the deep-blue
endpoint.
D-2 Calculate the mg vitamin C present in the heated sample.
D-3 Calculate the mg vitamin C lost (destroyed) by heating the sample.
D-4 If vitamin C is still present, heat new 20-mL samples of the juice,
one for 20 min and the other for 30 min. Coat the samples and titrate
with iodine and starch indicator.

E. VITAMIN C IN URINE (OPTIONAL)

Carry out the titration with iodine and starch with 25-mL samples of
urine. Compare the vitamin C content in the urine of a student who
took a vitamin C tablet that morning, one who drank a citrus juice, and
one who took no source of vitamin C. You can also monitor the
output of vitamin C over the day if laboratory time is available. You will
be responsible for setting up your laboratory record and showing
calculations.

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 58 CHEM 132.2: Biochemistry Laboratory

Guide questions and instructions on how to prepare

and submit the laboratory

Additional Resources

Use References style here

References

Robyt, J.F. and B.J. White. 1987. Biochemical Techniques: Theory and Practice. Brooks/Cole
Pub. Co. : Monterey, California; pp.407
Timberlake, K. 1988. Laboratory Manual for Chemistry. Harper Collins Publishers Inc. : New
York, pp 277-372.

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