EXPERIMENT 13
Vitamin C
INTRODUCTION
Vitamin C is an essential component of the diet for humans and some animals because
we lack the enzymes to make this substance from its natural precursor, glucose. A deficiency of
vitamin C leads to a disease known as scurvy, characterized by hemorrhages throughout the
body, which are especially noticeable on the gums and around the mouth and other areas of the
skin with abundant vascularization (blood vessels). It is essential in the synthesis of the protein
collagen, which connects cells in the body, especially in the blood vessels.
The disease scurvy was described as early as 1500 BC. In the winter of 1535, a group of
110 French explorers with Jacques Cartier were stranded along the St. Lawrence River and
became afflicted with scurvy. Several of the men died and many were totally incapacitated by
the disease before the Native Americans came to their rescue and recommended a concoction
from a local evergreen tree that cured the survivors. The common name for the tree is
arborvitae, or tree of life. A surgeon named Lind performed a scientific investigation of the
causes of scurvy among British sailors in 1747. He found that sailors given citrus fruits regularly
did not develop scurvy, while sailors receiving various other dietary supplements did. He
concluded there was some "antiscorbutic" factor in the citrus fruits that prevented the disease.
The chemical structure for the active antiscurvy factor was not determined until the early
1930's and was named ascorbic acid because it was an acidic compound with ascorbutic
(antiscurvy) properties. Ascorbic acid is a chiral compound, meaning it has "handedness". The
natural vitamin is L-ascorbic acid, meaning it has a "levo" or left handed structure, as opposed
to D-ascorbic acid, which has a "dextro" or right handed structure. The recommended daily (or
dietary) allowance (RDA) for vitamin C is 60 mg for adults, and higher for pregnant and
lactating women. This is an amount that is sufficient to prevent scurvy, although there is a lot of
controversy over how much vitamin C is needed for a healthy individual. Some people claim
that large doses of vitamin C will help to ward off colds, although there is not complete
agreement on this, primarily because individuals usually respond differently to various drugs.
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When vitamins are used in megadoses, they should be considered drugs and not everyone will
respond the same way.
In this laboratory you will measure the amount of vitamin C in various beverages. Some
beverages will be supplied, but each student is encouraged to bring in a sample of his or her
choosing to analyze for its vitamin C content. You will use a titration method for the
determination, similar to the titrations you did for antacid tablets. In this case you will use iodine
solution as the titrant (in the buret), which reacts with ascorbic acid to produce iodide ion and
dehydroascorbic acid. The ascorbic acid is readily oxidized by oxygen in the air under neutral or
alkaline conditions, so you will be adding acetic acid to keep the medium acidic. Starch will be
added to the sample, so when the ascorbic acid has completely reacted with iodine, any excess
iodine added will form a deep blue color with the starch that is present. This color formation
between starch and iodine
was demonstrated in the
CH2OH
previous laboratory with
carbohydrates.
HO
CHOH
O
MATERIALS NEEDED
Three 125 mL
HO
CH2 OH
O
+ I2
O
Ascorbic Acid
CHOH
O
O
+
2 I-
O
Dehydroascorbic Acid
Erlenmeyer flasks, 10 mL
pipet, 50 mL buret, ascorbic acid standard solution (1.00 mg/mL), 1% starch solution, 6 M acetic
acid solution, iodine solution, buret, fruit juices or other samples to test for vitamin C.
PROCEDURE
Add 25 mL deionized water and 2 mL of 6 M acetic acid to each of three 125 mL
Erlenmeyer flasks. Using a pipet, add exactly 10.0 mL of 1.0 mg/mL ascorbic acid standard
solution to each flask and 2 mL of 1% starch solution to each.
Set up a 50 mL buret and fill it with the iodine solution (see Titration Apparatus,
Appendix I). Turn the stopcock of the buret to fill the tip before you start the titration, making
sure you have no air bubbles in the tip. Record the initial level of the iodine solution in the buret.
Now slowly add the iodine solution (several drops at a time) to the ascorbic acid standard
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solution in the Erlenmeyer flask. You will see a deep blue color appear that slowly disappears as
you swirl the solution. Continue adding the iodine solution a little at a time and swirl the flask
after each addition until the blue color persists. As you get closer to the endpoint, it will take
longer for the blue color to disappear. That means you should try to add smaller and smaller
amounts of the iodine solution between swirling. When you have added enough, so the blue
color remains for more than 30 seconds, record the final level of iodine solution in the buret.
Subtract the initial level from the final level to determine the volume of iodine solution added.
Repeat the procedure with each of the other two flasks containing the ascorbic acid
standard solutions, making sure you have enough iodine solution in the buret before starting each
titration. Be sure to record the initial and final level of iodine solution in the buret before each
titration. Use the average amount of iodine solution needed for the titrations in your calculations
later. How much error did you have in your measurements?
Add 25 mL deionized water and 2 mL of 6 M acetic acid to a clean 125 mL Erlenmeyer
flask. Using a pipet, add exactly 10.0 mL of your juice sample to the flask and about 2 or 3 mL
of 1% starch solution. Titrate the sample in the same way that you did the ascorbic acid
standard, making sure there is enough iodine solution in the buret for each titration. The final
volume for the previous titration will be the initial volume for this titration unless you opened the
stopcock of the buret between titrations. Be careful not to allow the level of the iodine solution
to drop below the graduation marks on the buret. When you reach the endpoint for the sample,
record the final volume in the buret and determine the volume of iodine solution added as you
did above for the ascorbic acid standard.
To calculate the amount of vitamin C in your sample you will compare the amount of
iodine used to titrate the sample with the amount used to titrate the standard. Since the standard
solution contains 1 mg ascorbic acid per mL, the calculation is as follows:
Vit C conc (mg/mL) =
Vol I2 soln used for sample (mL)
))))))))))))))))))))))))))))))))))
Vol I2 soln used for standard (mL)
To determine the total amount of vitamin C in your sample, multiply the concentration
(mg/mL) by the total volume of sample, e.g., 12 oz = 375 mL, so multiply this number by 375
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mL for a 12 oz sample.
Repeat this procedure for other samples. You should analyze at least 2 different juice or
beverage samples. You may also test vitamin C supplement tablets from home by dissolving the
tablets in 90 mL of water and 10 mL of 6 M acetic acid. Crush the tablet into a powder before
dissolving it. After it has dissolved, add enough of this solution to give about 20 mg of vitamin
C in the titration flask, along with 25 mL deionized water, 2 mL of 6 M acetic acid and 2 mL of
1% starch solution. Check with the instructor to make sure your calculations are correct for the
tablet you are using.
YOUR DATA SHEET SHOULD LOOK LIKE THIS
Ascorbic Acid Standard (1.0 mg/mL):
Flask 1 Flask 2 Flask 3
Final level of iodine solution in buret (mL):
_____
Initial level of iodine solution in buret (mL): -____
Total volume of iodine used (mL):
_____
-____
_____
_____
-____
_____
Average:
_____
_____ mL
Juice Samples:
Final level of iodine solution in buret (mL):
_____
Initial level of iodine solution in buret (mL): -____
Total volume of iodine used (mL):
-____
_____
Average:
_____
_____
-____
_____
_____
_____ mL
Questions to answer in your notebook:
1.
What percentage of the RDA for vitamin C does each test sample provide in a normal
serving? How large is a normal serving?
2.
In the previous experiment you used an iodine solution to test for the presence of starch.
What is the purpose of adding starch to the ascorbic acid solution prior to titrating it with
the iodine solution in this experiment?
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3.*
What is the purpose of titrating the ascorbic acid standard solution prior to titrating the
juice samples? Could you obtain the amount of vitamin C in the juice sample without
first titrating the ascorbic acid standard solution?
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