Expt 054 -- Vitamin C in Fruit Juices

Ascorbic acid is titrated with iodine to the appearance of a starch endpoint color. A calibration curve is recommended.

Background

• Vitamin C (ascorbic acid) is required in the human diet.

  

• Just a small daily intake prevents disease (scurvy). Ascorbic acid is an excellent reducing agent; it is easily oxidized. When juices are titrated with iodine, it is assumed that the only reducing agent being oxidized is ascorbic acid. While this assumption is not correct, it is a very good approximation.

Safety

Iodine is toxic and stains skin and clothing. The room must be well ventilated. Avoid ingestion and inhalation of iodine. Wear goggles and apron. Wash spills immediately. Wash hands after the experiment.

Procedure

This procedure describes two alternate methods for using a standard. Three titrations of one standard solution may be used if the range of vitamin C in your samples is close to the concentration of the standard. Prepare a standard curve if the concentration of vitamin C varies considerably. Use the method suggested by your instructor.

One Standard-Three trials

1. Obtain a fresh solution of ascorbic acid from the instructor. the concentration should be known to 3 significant figures, and should be about 1 mg/mL.
2. Use a calibrated plastic pipet to add 1.00 mL (or 20 drops) of this solution to a well of a 24-well plate. Add 1 drop of starch solution.

   !!!Click here to See Movie.

3. Add iodine solution dropwise from a plastic transfer pipet with a restricted tip while counting the drops. Mix with a toothpick. Continue adding until a blue color appears that is not discharged by stirring.

   !!!Click here to See Movie.

4. Repeat this procedure two more times. Continue until consistent values -- titrations that agree within 1 drop -- are obtained.

Standard Curve

1. An alternative procedure is for 4 or 5 solutions of known ascorbic acid concentration to be available -- with concentrations in the range from 0 to 1 mg/mL.
2. Obtain a concentrated standard or 5 different standards from the instructor. If one concentrated standard is dispensed, make dilutions of the standard at 5 different concentrations. Titrate each of the 5 solutions to a blue endpoint.

   !!!Click here to See Movie.

3. Plot a graph of mg/ml of ascorbic acid versus drops of I₂ solution. Use a computer graphing program if one is available.

   !!!Click here to See Picture.

Juices

1. Use a calibrated plastic pipet to add 1.00 mL of juice to a well of a 24-well plate. Any juice in which a starch endpoint can be detected will be satisfactory. Add 1 drop of starch solution. Add iodine solution dropwise from a plastic transfer pipet with a restricted tip while counting the drops. Mix with a toothpick. Continue adding until a blue color appears that is not discharged by stirring. Repeat 2 more times.

   !!!Click here to See Movie.

   !!!Click here to See Picture.

2. Rinse the 24-well plate at the sink with running water.
3. Determine the number of mg ascorbic acid per drop of I₂.
4. Determine the number of mg ascorbic acid per mL of juice.

Questions

1. 0ne mL of a solution containing 1.13 mg ascorbic acid requires 53 drops of I₂ solution to titrate to a blue starch endpoint. A 1.00 mL sample of lemon juice required 24 drops of the same I₂ solution to titrate to a starch endpoint. Find the mg ascorbic acid/mL lemon juice.
2. A chemistry teacher prepares a fresh ascorbic acid solution of 1.08 mg/mL, and is about to make serial dilutions from this solution, when a fire drill occurs. The teacher decides to do something else for that day, and comes back in one week but uses the same solution. Predict the effect this will have on the concentrations of vitamin C reported by the students.

Handout

Name ___________________________ Class ________

Teacher__________________________

SmallScale 54 Vitamin C in Fruit Juices

Standard	Drops I2
Juice	Drops I2

mg ascobic acid/drop of I₂ = __________________

mg ascorbic acid per mL of juice = _____________

Handout Makeup

Name ___________________________ Class ________

Teacher__________________________

SmallScale 54 Vitamin C in Fruit Juices

Watch the movies, and use the data below to answer the questions.

Standard mg/mL	Drops I2
1.01	42
0.808	34
0.606	24
0.404	17
0.202	9

Plot a graph of mg/ml of ascorbic acid versus drops of I₂ solution. Use a computer graphing program if one is available.

Juice	Drops I2
1 mL (20 drops)	18
1 mL	18
1 mL	19

mg ascobic acid/drop of I₂ = __________________

mg ascorbic acid per mL of juice = _____________

Curriculum-

Use this experiment when studying oxidation-reduction, solution stoichiometry, assays, or biochemistry. The experiment works well in regular and in applied chemistry classes. Teachers recommend using this experiment with a calibration curve.

Safety-

• Iodine is toxic and stains skin and clothing. The room must be well ventilated. Avoid ingestion and inhalation of iodine. Wear goggles and apron. Wash spills immediately. Wash hands after the experiment.
• Treat iodine stains on skin or clothing with 5% aqueous Na₂S₂O₃.

Time-

Teacher Preparation: 15 minutes

Class Time: 30 minutes

Materials-

• 10 mL of 0.01 M Iodine/KI -- Dissolve 0.5076 g of I₂, iodine , and 0.332 g of KI, potassium iodide, in 120 mL water. Dilute to 200 mL with water.
• 10 mL of Ascorbic acid -- Dissolve 100 mg ascorbic acid in 100 mL water
• 2 mL of 0.1% starch. Spray starch (Spray some in a beaker the same day. Some brands work better than others.)
• 4 mL of orange juice -- squeezed from an orange
• 24-well plate
• toothpick
• graduated plastic transfer pipet
• plastic transfer pipet with constricted tip

Disposal-

The materials used in this experiment may be disposed of safely at the sink.

Lab Hints-

• Make the ascorbic acid solutions fresh. Although vitamin C tablets are usable, samples of pure ascorbic acid probably are better. The tablets have fillers, and are less pure.
• Use a 1 L or 500 mL volumetric flask if one is available. Then use a 100-mL cylinder to prepare the dilutions.

Data Table-

• The standard ascorbic acid solutions contained 1.01 g/mL. These were serially diluted by the instructor to give 1.01, 0.808, 0.606, 0.404, and 0.202 mg/mL respectively.
• 1.00 mL portions of these solutions required 42, 34, 24, 17, and 9 drops of I₂ solution for titration.
• Three 1.00 mL samples of fresh squeezed orange juice required 18, 18, and 19 drops respectively to titrate.

Data Analysis-

If a computer and a graphing program are available, plot the calibration data and use a line fitting feature to determine an equation for the line. Plot drops of I₂ solution as the x-axis, and mg ascorbic acid as the y-axis. See below.

mg AA = (0.0243 x # drops I₂) - 0.0056; R² = 0.997

For 18.3 drops I₂, 0.439 mg ascorbic acid /mL orange juice.

If not, calculate mg ascorbic acid per drop I₂ solution for each point as follows:

Average = 0.024 mg ascorbic acid/ drop I₂.

Then use this value as a constant for the determination of unknown juices.

18.3 drops I₂ x 0.024 mg AA/drop = 0.44 mg AA/mL

Answers-

Q1. 0ne mL of a solution containing 1.13 mg ascorbic acid requires 53 drops of I₂ solution to titrate to a blue starch endpoint. A 1.00 mL sample of lemon juice required 24 drops of the same I₂ solution to titrate to a starch endpoint. Find the mg ascorbic acid/mL lemon juice.

A1. 1.13 mg aa x 24 drops = 0.51 mg ascorbic acid/mL

53 drops I₂

Q2. A chemistry teacher prepares a fresh ascorbic acid solution of 1.08 mg/mL, and is about to make serial dilutions from this solution, when a fire drill occurs. The teacher decides to do something else for that day, and comes back in one week but uses the same solution. Predict the effect this will have on the concentrations of vitamin C reported by the students.

A2. The reported values will be too high. By the time the students get to use the "standard" solution, it will have considerably less ascorbic acid due to oxidation in the air.

CoopLearn-

This experiment lends itself to cooperative learning activities. The class can work in groups to study many fruits and vegetables, and pool their data to come up with food tables for vitamin C. Use the standard curve if several different juices are tested.

Reference-

Suggestions about this experiment were made by Robert Silberman, Cortland, NY, and Robert Becker, Kirkwood, MO.

Key Words 1-

calibration curve, oxidation, reduction, redox, solution stoichiometry, vitamin C, iodine, starch, assay, linear equation, volumetric

Elements-

C I