Materials Included In Kit

Additional Materials Required

Prelab Preparation

Ascorbic acid (reference) solution: Prepare 1% oxalic acid solution by dissolving 1.0 g oxalic acid in 100 mL of distilled or deionized water. Accurately measure 0.100 g of reagent grade ascorbic acid and quantitatively transfer the solid to a 100-mL volumetric flask. Add 1% oxalic acid solution to the mark to dissolve the solid. Mix well before dispensing. Prepare this solution fresh before use and store the solution in an amber bottle. The solution may be stored in a refrigerator for up to one week. Note: If a milligram balance is not available, prepare a more concentrated solution using a centigram balance to mass the reagent and then dilute the solution.

2,6-Dichloroindophenol, standard solution: Dissolve 0.3 g of 2,6-dichloroindophenol, sodium salt, in 500 mL of distilled or deionized water. Prepare this solution fresh before use. The solution may be stored in the refrigerator for up to one week.

Safety Precautions

The ascorbic acid reference solution contains 1% oxalic acid and is a skin and eye irritant. The solution is slightly toxic by ingestion. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. All food-grade items that have been brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any materials in the chemistry laboratory and do not remove any remaining food items after they have been used in the lab. Remind students to wash hands thoroughly with soap and water before leaving the laboratory. Please consult current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All of the solutions may be disposed of down the drain with plenty of excess water according to Flinn Suggested Disposal Method #26b.

Lab Hints

• The laboratory work for this experiment can reasonably be completed in one 50-minute lab period. The Prelab Questions may be assigned as homework in preparation for lab or they may be used as the basis of a cooperative class discussion before lab. If possible, ask students to bring in fresh fruits and fruit juices for analysis. Having different groups test different juices will increase student involvement and can lead to some interesting class discussion.
• For best results, prepare the Vitamin C solution fresh in 1% oxalic acid solution the day of the lab. The instability of Vitamin C solution is probably due to microbial contamination. Oxalic acid has been shown to be an effective preservative for Vitamin C solutions if the solutions are kept refrigerated.
• In addition to fruits and fruit juices, other beverages that may be tested include milk, Gatorade^®, Kool-Aid^®, etc. Many vegetables, including green pepper and cabbage, are also excellent sources of Vitamin C. Homogenize vegetables in a laboratory blender with 1% oxalic acid solution. Extract 5 g of vegetable with 50 mL of oxalic acid, filter the extract, and dilute as needed to obtain a convenient concentration for analysis. The effect of various cooking or processing methods on the Vitamin C content may also be studied—compare the effect of boiling, steaming, microwaving, etc., on the amount of Vitamin C in fresh vegetables.
• The accuracy of the microscale titration of the microscale analysis of Vitamin C may be improved by titrating different concentrations of Vitamin C reference solutions and constructing a calibration curve to find the concentration of Vitamin C in an unknown solution.

Teacher Tips

• Most juices contain Vitamin C; the exception is apple juice. Find unfortified (no added Vitamin C) apple juice in your local grocery store and check the Nutrition Facts label to confirm there is no Vitamin C. Including apple juice among the test juices may serve as a check on students’ integrity in reporting their results.

Answers to Prelab Questions

1. The Nutrition Facts label on a carton of orange juice reports that the juice contains 100 mg of vitamin C per one-cup (240 mL) serving. What is the concentration of Vitamin C in mg per 100 mL?
   {12392_PreLabAnswers_Equation_2}
2. Name two reasons why the Vitamin C content might not be the same in fresh, frozen or canned orange juice.

   The Vitamin C in fresh (unprocessed) fruits is susceptible to air oxidation and to microbial metabolism. Most frozen or canned (processed) juices are fortified with Vitamin C to improve their nutritional content. Note: This is a good place to stress to students the importance of reading the label, both in the lab and at home!

3. In the reaction of Vitamin C with DCIP (see Equation 1), which compound is being oxidized and which is being reduced?

   Ascorbic acid is being oxidized to its dehydro- form, while the 2,6-dehydroindophenol is being reduced. Note: the traditional definition of oxidation and reduction reactions (loss and gain of electrons, respectively) is difficult to recog¬nize in organic compounds. Organic chemists often define redox reactions in terms of the loss and gain of an oxygen atom (as in the oxidation of acetaldehyde to acetic acid) or the loss of two hydrogen atoms (as in the oxidation of isopropyl alcohol to acetone). Conversely, reduction of an organic compound may be defined as either the loss of an oxygen atom (as in the reduction of formic acid to formaldehyde) or the gain of two hydrogen atoms (as in the reduction of an alkyne to an alkene).

4. Why would it not be possible to analyze grape juice or cranberry juice using the method outlined in this experiment?

   The color of the juice would mask the pink color of the dye and make it impossible to detect the endpoint.

Sample Data

Answers to Questions

1. Calculate the average number of drops of DCIP needed to titrate the reference solution and each juice. Record the results in the table.

   See Sample Data.

2. The reference solution contains 100 mg of Vitamin C per 100 mL of solution. Use the following equation to calculate the concentration of Vitamin C (mg per 100 mL) in each juice based on the average number of drops of DCIP required to titrate the juice versus the number of drops required to titrate the reference solution.
   {12392_Answers_Equation_3}
   Orange Juice: (16/40) x 100 = 40 mg per 100 mL
   Apple Juice: Does not contain Vitamin C. First drop of DCIP was not decolorized.
   White Grape Juice: (24/40) x 100 = 60 mg per 100 mL
3. Rank the juices tested in terms of their Vitamin C content, from highest to lowest.

   Grape Juice > Orange Juice > Pineapple Juice >> Apple Juice

References

This activity was adapted from Flinn ChemTopic™ Labs, Volume 23; Chemistry of Foods, Cesa, I., Editor; Flinn Scientific Inc.: Batavia, IL (2006).

Introduction

Vitamin C (ascorbic acid) is water soluble and is a strong reducing agent. In this laboratory activity, determine the Vitamin C content in various fruit juices and observe its reducing properties.

Concepts

• Vitamin C
• Oxidation–reduction
• Titration
• Endpoint

Background

Vitamin C occurs naturally in most fruits and vegetables including citrus fruits, strawberries, tomatoes, cabbage, green leafy vegetables and potatoes. Most animals also produce Vitamin C naturally. Humans are one of the few organisms that do not produce Vitamin C—it must be supplied in our diet. Vitamin C is important for collagen production and wound healing. The symptoms of Vitamin C deficiency—bleeding gums, loose teeth, skin bruises, joint pain and muscle aches—result from the breakdown of connective tissue in the body.

The structure of Vitamin C, whose chemical name is ascorbic acid, is shown in Figure 1. Ascorbic acid is a highly polar, water-soluble compound, and a strong reducing agent.

It is easily oxidized to give dehydroascorbic acid (see Figure 2) via the loss of two hydrogen atoms from the –OH groups in the ring. Both ascorbic acid and dehydroascorbic acid occur naturally in foods and are considered active forms of Vitamin C. Further oxidation, however, which occurs during cooking or food processing, converts Vitamin C to an inactive form. The ease of oxidation of ascorbic acid provides the basis for a laboratory method to measure the amount of Vitamin C in foods. Reaction of ascorbic acid with a blue dye, 2,6-dichloroindophenol (DCIP), converts the dye to a reduced, colorless form (Equation 1). The amount of Vitamin C in fruit juices can be analyzed by titration with DCIP. A blue solution of DCIP is added dropwise to a known volume of juice. Any Vitamin C in the juice immediately reacts with the DCIP and turns it colorless. As soon as all of the Vitamin C in the juice has reacted, however, the next drop of DCIP will not react and will stay colored. This represents the endpoint of the reaction.

Experiment Overview

The purpose of this experiment is to measure the amount of Vitamin C in fruits and fruit juices by microscale titration with 2,6-dichloroindophenol (DCIP). The amount of Vitamin C will be analyzed by comparing the number of drops of DCIP required to titrate a known volume of fruit juice versus the number of drops needed to titrate the same volume of a standard solution that contains a known amount of ascorbic acid.

Materials

Prelab Questions

1. The Nutrition Facts label on a carton of orange juice reports that the juice contains 100 mg of Vitamin C per one-cup (240 mL) serving. What is the concentration of Vitamin C in mg per 100 mL?
2. Name two reasons why the Vitamin C content might not be the same in fresh, frozen or canned orange juice.
3. In the reaction of Vitamin C with DCIP (see Equation 1 in the Background section), which compound is being oxidized and which is being reduced?
4. Why would it not be possible to analyze grape juice or cranberry juice using the method outlined in this experiment?

Safety Precautions

The reference solution also contains 1% oxalic acid, which is a skin and eye irritant. The solution is slightly toxic by ingestion. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical resistant apron. All food-grade items that have been brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any materials in the chemistry laboratory and do not remove any remaining food items after they have been used in the lab. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Read the entire Procedure before beginning the experiment.

1. Obtain a 24-well reaction plate and place it on top of a piece of white background paper.
2. Obtain 20 mL of DCIP solution in a clean dry beaker and place a labeled pipet in the solution.
3. Using a clean, graduated pipet, place about 3 mL of the ascorbic acid reference solution into well A1 of the reaction plate.
4. Using a clean, graduated pipet, carefully transfer 0.50 mL of the ascorbic acid reference solution from well A1 into each well A2, A3 and A4.
5. Slowly add the DCIP solution one drop at a time to the ascorbic acid sample in well A2. Stir the solution with a toothpick to mix the solution between drops. For best results, hold the pipet in a vertical position to obtain drops of a uniform size.
6. Count the number of drops of dye solution required to give the solution a slight pink color that does not fade with mixing and that persists for at least 30 seconds. This is the endpoint of the titration. Record the number of drops of the DCIP added in the data table.
7. Repeat steps 5 and 6 two more times with the ascorbic acid samples in wells A3 and A4. Record the results for each trial in the data table on the Vitamin C Analysis Worksheet.
8. Place 3 mL of one of the juices to be tested into well B1 of the reaction plate. Record the identity of the juice in the data table.
9. Using a clean, graduated pipet, transfer 0.50 mL of juice from well B1 into each well B2, B3 and B4.
10. Using the DCIP pipet, slowly add the dye solution one drop at a time to the juice sample in well B2. Stir the solution with a toothpick to mix the solution between drops. For best results, hold the pipet in a vertical position to obtain drops of a uniform size.
11. Count the number of drops of dye solution required to give the solution a slight pink color that does not fade with mixing and that persists for at least 30 seconds. This is the endpoint of the titration. Record the number of drops of DCIP added in the data table.
12. Repeat steps 10 and 11 two more times with the juice samples in wells B3 and B4. Record the results for each trial in the data table.
13. If desired, repeat the testing procedure (steps 8–12) with other juices in rows C and D, respectively. Remember to use a new graduated pipet before testing each new juice. Record the results in the data table.

Student Worksheet PDF

12392_Student1.pdf