MICROANALYSIS OF VITAMIN C IN FRUIT JUICES

Lab MICRO 1
From Juniata College SIM

INTRODUCTION

Vitamin C is a water soluble vitamin that is required daily since it is not synthesized
by humans and cannot be stored by the body. Vitamin C is required for cellular
respiration, for enzyme function, as a component in collagen formation, and has been
reported to increase iron absorption within the body. Dr. Linus Pauling has found that
Vitamin C dosages in excess of the recommended daily allowance (RDA) appears to
reduce the severity and frequency of the common cold. The RDA of vitamin C in adults
is 60 mg per day. The vitamin is nontoxic even at high dosage levels. A dietary
deficiency in vitamin C may result in scurvy which is characterized by fatigue, shortness
of breath, muscle degeneration, gum bleeding, tooth loss, and even death. Just 10 mg of
vitamin C is enough to cure scurvy. Vitamin C, also known as ascorbic acid, is a weak
acid found mostly in citrus fruits and leafy green vegetables (broccoli, parsley, and
spinach). Apples, grapes, and peaches are low in vitamin C.
The method of measuring the amount of vitamin C in fruit juices in this lab utilizes
the ease at which vitamin C can be oxidized (forced to lose electrons). Iodine oxidizes
+ -
vitamin C. (C6H8O6 + I2 --> 2H + 2I + C6H6O6) After the reaction, excess I2 remaining
is detected by a starch indicator solution that turns blue - black in the presence of iodine.
Vitamin C has a long storage life; however, it rapidly decomposes when in solution
because it is oxidized by atmospheric oxygen. The decomposition is sped up by heat,
light, bases, oxidative enzymes, and traces of iron and copper. In the diet, vitamin C
concentrations are greatly reduced by cooking foods in water and then draining off the
water.

PURPOSE

The amount of vitamin C in various citrus juices is measured using an I2 / KI

solution and a starch indicator.

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 Vitamin C in Citrus Juices

EQUIPMENT/MATERIALS

1% starch solution 24 well spot plate

iodine solution 1.0 mg/mL Vitamin C solution
fruit juices medicine droppers for each juice & solution

SAFETY

• Always wear safety glasses in the lab.

• Iodine stains hands and clothes, so gloves and aprons are recommended.

• Do not drink the fruit juices after they have entered the lab. Dangerous
chemicals may dissolve into the juices after exposure.

PROCEDURE

1. Using a medicine dropper, place 5 drops of the standard vitamin C solution in 6 wells
(one row) of the spot plate. Each well used will act as a different trial.

2. Add two drops of the starch solution to each well containing the vitamin C solution.

3. Next, drop iodine solution into each well until the solution turns a purple to dark blue
color for at least 15 seconds. After each drop is added, gently shake the spot plate to
mix the iodine solution with the solution. Record the number of drops of iodine used
for each trial in the table on the data sheet.

4. Place 5 drops of a fruit juice in place of the vitamin C standard in each well of the
next row (6 wells) and follow steps 2 and 3.

5. Repeat step 4 for as many juices as your instructor requests.

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 Vitamin C in Citrus Juices

Name_______________________________
Name_______________________________
Period______________________________
Date________________________________

MICROANALYSIS OF VITAMIN C IN CITRIC JUICES

DATA TABLE
Number of drops of iodine solution used in each trial

1 2 3 4 5 6 Ave. Conc
Vit C

Standard

Juice 1

Juice 2

Juice 3

Juice 4

CALCULATIONS

1. Calculate the average number of drops of iodine solution required to react with the
Vitamin C standard. Repeat for each of the fruit juices. Fill in the appropriate spaces
on the table.

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 Vitamin C in Citrus Juices

2. Calculate the concentration of vitamin C in each of the fruit juices by comparing the
amount of iodine required for vitamin C and the amount needed for the fruit juice.
The concentration of the vitamin C standard is 1.0 mg / mL.

QUESTIONS

1. Which fruit juice contained the largest concentration of vitamin C?

2. List two nutritional sources of vitamin C other than citrus fruits.

3. Using the RDA for vitamin C listed in the introduction section of the experiment,
calculate the mL of each sample required to supply the RDA for vitamin C.

4. How many ounces of each sample provide the RDA for Vitamin C?

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MICROANALYSIS OF VITAMIN C IN CITRUS JUICES

TEACHER NOTES

Time: 30- 45 minutes

Preparations:
Time: 30 minutes

T: Make available a set of the equipment and materials for each lab group.

T: Prepare the following solutions:

Standard Vitamin C:
To make the 1 mg/mL standard, dissolve 100 mg of vitamin C in
100 mL of water (or use one 100 mg tablet of vitamin C). This solution
should be freshly prepared and refigerated.

Iodine solution:
First make solution A by dissolving 0.6 g KI in 250 mL distilled H2O. Then,
make solution B by dissolving 0.6 g I2 in 25 mL ethanol. Next, add solution
A to solution B and dilute to 500 mL with deionized water.

1% starch indicator:
Add 1 g soluble starch in 100 mL distilled H2O, heat to dissolve and filter. 1
mL of laundry starch diluted to 100 mL with water can be substituted if
soluble starch is not available. Prepare fresh weekly.

V: The van can supply materials, including solutions, as requested.

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 Vitamin C in Citrus Juices

Answers to Questions:

1. Which fruit juice contained the largest concentration of vitamin C?

Answers will vary depending on data collected.

2. List two nutritional sources of vitamin C other than citrus fruits.

Answers will vary but may include fruit juices green leafy vegetables.

3. Using the RDA for vitamin C listed in the introduction section of the experiment,
calculate the mL of juice 1 required to supply the RDA for vitamin C.
Answers will vary depending on data collected.

Considerations:

The use of another indicator, Indophenol, is discussed in Chemtrek by Steven

Thompson, Allyn and Bacon, 1990. The starch endpoint color fades with time in the fruit
juices; the color is stable with the Vitamin C solution. The amount of the iodine solution
needed to produce an endpoint decreased when running 8 samples of a juice (or Vitamin
C) suggesting the Vitamin C was being oxidized by the air. Drops were easier to control
using medicine droppers, rather than Beral pipets. No difference in endpoints was
observed using either the 1% soluble starch solution or a 1% laundry starch solution.
The vitamin C solution should be freshly prepared and refrigerated. Also, fruit juices
should be bought on day of lab or 1 or 2 days before and refrigerated. The juices should
be refrigerated, if possible, throughout the lab, since the vitamin C concentration will
significantly change throughout the day if kept at room temperature.
Variations of the lab can include comparing temperature effects on vitamin C
concentration, storage time vs. vitamin C concentration, etc.

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 Vitamin C in Citrus Juices

Sample Data/Calculations:

DROPS IODINE SOLUTION

Sample 1 2 3 4 5 6 7 8 Ave.
Vit C 24 22 20 19 20 19 21 24 21
Orange 14 14 15 14 13 12 12 12 13
Juice
Grapefruit 10 9 10 9 8 8 9 9 9
Juice

Concentration of Vit C in OJ = # drops iodine used for OJ X Concentration of

Standard
# drops iodine used for Stand.

Concentration of Vit C in OJ = 13 drops X 1.0 mg/mL

21 drops

Concentration of Vit C in OJ = 0.62 mg/mL

Microscale Techniques

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 Vitamin C in Citrus Juices

Microanalysis of Vitamin C in Citrus Juices 12/4/02

For class of 24 students, 8 groups; 1 Demo

• 9-15mL glass dropper bottles labeled starch solution, filled

• 9-15mL glass dropper bottles of iodine solution, filled
• 9-Beral pipets labeled iodine
• 9-plastic bottles labeled vitamin C solution
• 9-Beral pipets labeled vitamin C solution
• funnel to fill bottles with vitamin C standard
• 1- 500mL bottle of deionized water to dilute ascorbic acid
• 1-wash bottle of DDI water to dilute the ascorbic acid
• 2- 250 mL vol flasks with lids into which 250 mg of ascorbic acid per flask has
been massed. Do not dilute to volume until ready to use. ***
• Styrofoam cups to put juice samples in (2/type of juice), plus several extra. Pack
20.
• 9-spot well plates
• 10-plastic droppers labeled for juices (done by Mobile Educator or Teacher
borrowing experiment
• 1- Sharpie pen for labeling pipets and Styrofoam cups
• 1- masking tape
** Note - The vitamin C and starch solutions must be prepped the same day as the lab.
This means that a volumetric flask containing the proper amount of vitamin C must be
taped shut and the mobile educator will add the water at the school. The size of the flask
sent will depend on the number of classes and class sizes.

Preparation of solutions:
Standard Vitamin C:
To make the 1 mg/mL standard, mass 250 mg (0.25 grams) of vitamin C in a
250 mL volumetric flask. Cover flask with lid. Pack enough DI water to
dilute to volume and include a wash bottle of DI water. This solution should
be freshly prepared the morning of the lab and refrigerated if possible.
Iodine solution:
First make solution A by dissolving 0.6 g KI in 250 mL distilled H2O. Then,
make solution B by dissolving 0.6 g I2 in 25 mL ethanol. Next, add solution
A to solution B and dilute to 500 mL with deionized water.
1% starch indicator:

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Add 2.5 g soluble starch in 250 mL distilled H2O, heat to dissolve and filter. 1
mL of laundry starch diluted to 100 mL with water can be substituted if soluble
starch is not available. Prepare fresh weekly

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