VITAMIN C (L-ASCORBIC ACID) DETERMINATION
OBJECTIVES
1. Learn a common technique used to determine the amount of vitamin C in foods.
2. Determine the vitamin C content in peach juice.
3. Compare this to the average amount of vitamin C in peach juice.
INTRODUCTION
Vitamin C (L-ascorbic acid) is a 6 carbon compound which is derived from glucose. Its structure and function are the same whether it occurs naturally in foods or it is prepared synthetically. Most plants and animals are able to synthesize their own ascorbic acid by the pathway shown below. Primates, guinea pigs, and a few other animals, however, are unable to carry out the last step in this pathway so ascorbic acid is a vitamin which must be included in the diet.
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Lack of sufficient vitamin C in the diet leads to the disease called scurvy. A major function of the vitamin is the formation hydroxyproline and hydroxylysine residues in collagen; the major structural protein of skin, bone, blood vessels, tendon, cartilage, and teeth. The symptoms of scurvy (muscle weakness, soft swollen gums, loose teeth, bruising and poor wound healing) are due to improper collagen synthesis.
A number of enzymes, in addition to the
prolyl hydroxylase
and
lysyl hydroxylase
needed for collagen synthesis, also require vitamin C. These include:
dopamine
b-hydroxylase
in the formation of norepinephrine from tyrosine
cholesterol 7
a-hydroxylase
in the formation of bile acids
p-hydroxyphenylpyruvate hydroxylase
in the degradation of tyrosine
homogentisate dioxygenase
in the degradation of tyrosine
tryptophan hydroxylase
in the formation of serotonin
The adrenal cortex also contains a significant amount of vitamin C which is depleted in response to ACTH. The role of the vitamin here is not known, but it may be involved in steroidogenesis.
The exact function of vitamin C in most of its metabolic reactions is not fully understood. Ascorbic acid undergoes facile and reversible oxidation to dehydroascorbic acid. This property suggests that it acts as a redox coenzyme. In some reactions vitamin C acts as an electron acceptor. In other reactions its role appears to be indirect. It helps maintain iron or copper ion in the hydroxylases in the reduced state.
The amount of ascorbic acid required by an average adult in order to avoid any deficiency symptoms is about 70 mg/day. A number of prominent scientists, Linus Pauling preeminent among them, recommend megadoses of 1 to 6 g/day to "maintain optimum health and maximum defense against infection." Evidence to support their recommendations is sparse. One rational is that vitamin C acts as an antioxidant and protects vital molecules such as nucleic acids from oxidation. This is undoubtedly true, however, there are also reports of toxic effects from megadoses of vitamin C such as dehydration, increased oxalate synthesis resulting in kidney stones, and increased uptake of heavy metal poisons.
Vitamin C is often determined not only for its own nutritive value, but as an indicator of other vitamins and minerals which may be lost during the preparation of foods. Nutrients can be lost from food by extraction into the cooking water, or they may be chemically destroyed by the cooking process. Vitamin C is easily oxidized to dehydroascorbic acid, especially in the presence of O2
and metal ions such as Fe+++
or Cu++. Our bodies are able to convert dehydroascorbic acid back to ascorbic acid so this oxidation does not destroy the vitamin. Ascorbic acid is also relatively stable to heat so cooking food does not destroy the ascorbic acid.
Dehydroascorbic acid, however, is converted to diketogluonic acid at elevated temperatures. Diketogluonic acid is a waste product and has no vitamin C activity.
When foods such as citrus fruits, tomatoes, and cabbage (all high in vitamin C content) are cooked, some of the vitamin C may be lost due to this conversion to diketogluonic acid. If some or all of the vitamin C is lost during the processing of your favorite junk food, it is probable that many other nutrients have also been lost.
Many nutrients require complicated procedures to assay, but, as you will see, vitamin C is relatively easy to measure without sophisticated instrumentation. An easy assay for ascorbic acid uses the reduction of iodine (I2) by ascorbic acid in the presence of starch where ascorbic acid is oxidized to form dehydroascorbic acid C6H6O6.
A potassium iodate (KIO3)
solution of know concentration is titrated into the analyte solution with an unknown amount of vitamin C with potassium (KI), starch and HCl. The KIO3
reacts with KI to form I2. As I2
is formed, it reacts more quickly with vitamin C than the starch. When all of the vitamin C is consumed, the remaining I2
then reacts with the starch turning the solution dark blue, indicating the end point. If the amount of KIO3
added to the vitamin C solution is known, the amount of vitamin C can be determined using a mole conversion.
KIO3
+ KI + 6 H+
----> 3 I2
+ 6 K+
+ 3 H2O
C6H8O6
+ I2
----> C6H6O6
+ 2 I- + 2 H+
ascorbic acid dehydro-
ascorbic acid
The assay can be done with a crude vitamin C extract without any elaborate purification steps. The vitamin C in a food is extracted into an oxalic acid solution for use in the titration. Oxalic acid is a good chelating agent that removes metal ions like Cu2+
and Fe3+
that would interfere with the assay by oxidizing the vitamin C. Because this is an virtual lab we will not do this step.
When doing a titration like this, generally the first thing that is done is a standard assay. A standard solution of the analyte (vitamin C in this case) with known concentration, is titrated to confirm or validate whether the procedure worked as expected. This is standard practice for any analytical assay and is called a standard assay or validation assay. Since this experiment is online we will not do a standard assay.
Vitamin C
Name ________________________
PRE-LAB EXERCISES
Section ________
1. Where does vitamin C come from and what is the other name for it?
-Vitamin C (L-ascorbic acid) is a 6-carbon compound which is derived from glucose.
2. Name 3 tissues whose synthesis requires vitamin C. Explain how vitamin C is involved in the synthesis of collagen.
-skin, teeth, and bone (epithelial, blood vessels, and connective tissue)
-A major function of vitamin C is the formation of hydroxyproline and hydroxylysine
residues in collagen.
3. Name the oxidized form of vitamin C. Is this oxidation reversible?
-Vitamin C is easily oxidized to dehydroascorbic acid; yes.
4. Explain how vitamin C can be inactivated by heat, even though ascorbic acid is relatively stable to heat.
-Ascorbic acid is also relatively stable to heat so cooking food does not destroy the
ascorbic acid. Dehydroascorbic acid, however, is converted to diketogluonic acid at
elevated temperatures. Diketogluonic acid is a waste product and has no vitamin C
activity.
5. Briefly explain the standard assay for vitamin C.
-The standard assay determines the vitamin C concentration in a solution by a redox titration using iodine. As the I2
is added during the titration, the ascorbic acid is oxidized to dehydroascorbic acid, while the I2
is reduced to iodide (I-) ions. Due to this reaction, the I2
formed is immediately reduced to iodide. When all of the vitamin C is consumed, the remaining I2
then reacts with the starch turning the solution dark blue, indicating the end point.
6. Explain why oxalic acid is used for extracting vitamin C.
-Oxalic acid is a good chelating agent that removes metal ions like Cu2+
and Fe3+
that would interfere with the assay by oxidizing the vitamin C.
7. A 5.00 g sample of spinach was extracted with oxalic acid, diluted with water and an excess of KI, starch and HCl were added. The titration required 20.6 ml and 19.8 ml of 0.073 M KIO3
to turn dark blue in duplicate titrations. How much vitamin C was in 5.00 g of spinach?
PROCEDURE
Part I. Use the link to go to the virtual lab and follow the instructions there and below in this document.
http://chemcollective.org/activities/autograded/130
Part II. Watch the primer video provided by your instructor on Moodle.
This will give you an overview of how to use the Chem Collective virtual lab software and help get you started on the lab.
Part III. Making the Stock KIO3
Solution.
In the virtual lab page, use the link to the Wikipedia page to determine the approximate amount of ascorbic acid in 100 g of peach. This corresponds to 10 mL of peach solution. Then you will use that to determine how much KIO3
and water you will need to make your KIO3
stock solution.
1. Do a mole conversion to calculate an approximate amount of KIO3
you should have in 52 mL of titration solution (a full buret). See the Primer Video on Moodle if you need help.
2. Use the molar masses of ascorbic acid and KIO3
as well as the mole to mole conversion factors from the reaction equations in the introduction section to convert from the average grams of ascorbic acid in 100 g of peaches to moles of KIO3. Note: Don’t forget to use the ratio of KIO3
to I2
as well as the ratio of I2
to ascorbic acid from the stoichiometric reaction coefficients (go back to the primer video if you need help).
Remember: g ----> moles ----> moles ----> g
3. Once you’ve calculated how much KIO3
should be in 52 mL of titrant solution, multiply this number by approximately ~12 and add that amount to a 500 mL Erlenmeyer flask. Add 500 mL of water. This will give you plenty of solution so you won’t have to remake it. This is your KIO3
stock solution. Record the mass of KIO3
you added below.
4. Calculate the exact molarity of your KIO3
solution so you can use this to determine your unknown later.
Record your data here:
a. Mass of KIO3
added to KIO3
stock solution: ___________
b. Molarity of KIO3
stock solution: ___________
Part IV. Titration of an Unknown Peach Solution.
1.
Make an Erlenmeyer flask of the unknown peach solution containing the amounts of reagents listed in the virtual lab.
Unknown Peach Solution:
5 mL concentrated KI
4 mL 2M HCL
3 mL starch
10 mL peach juice
2.
Right click on the flask and duplicate it.
3.
Fill your buret to the 50.00 mL mark with your stock KIO3
solution. Titrate the unknown till you see a color change and repeat at least once.
4.
Record the volumes titrated below:
Trial 1 Trial 2 Trial 3 (optional)
Volume of KIO3
titrated: _________ _________ _________
Mass of ascorbic acid in 10 mL of unknown Peach Solution: __________
Part V.
Calculations.
1. Using the volume of KIO3
titrated, calculate how much ascorbic acid was in your unknown peach solution. Write this at the bottom of part IV above. On the virtual lab page, enter this amount in the blank box at the very bottom left of the screen and press the green “Check” button.
2. If you got the correct answer you will be able to download a pdf for proof that you successfully completed the lab, save this and turn it in with your report. If you didn’t get the correct answer check you calculations and try again. If you still get the wrong answer email your lab instructor your calculations to get help.
Post Lab Questions
1. How does your measured amount of vitamin C compare to the average amount of vitamin C reported for 100 g of peaches?
2. If this were a real lab, what sources of
experimental error
would you predict could have caused your value to be different from the value reported on the Wikipedia page?
3. Look up the RDA for vitamin C and calculate how many peaches you would have to eat to get your daily allowance.
4. Why did we use the average vitamin C amount to calculate the concentration of KIO3
stock solution?
5. Why did we have to divide by 3 when calculating how much KIO3
to add?