Materials Included In Kit

Additional Materials Required

Prelab Preparation

• Oxalic acid solution: Prepare 1% oxalic acid solution by dissolving 3.0 g of oxalic acid in 300 mL of distilled or deionized water.
• Ascorbic acid reference solutions A–D: Prepare these solutions fresh before use and store in amber bottles to protect from light. The solutions may be stored in the refrigerator for up to one week. Prepare reference solution D (ascorbic acid concentration = 100 mg/100 mL) by dissolving 200 mg ascorbic acid in 200 mL of the 1% oxalic acid solution. For best results, measure the mass of ascorbic acid using a milligram balance and accurately measure the oxalic acid solution using a graduated cylinder or volumetric flask. Solutions A–C (50 mL each) are prepared by appropriate serial dilution of reference solution D with 1% oxalic acid solution, as shown in the table below. Accurately measure all liquid volumes using a graduated cylinder or pipet.
  {14040_Preparation_Table_1}
• 2,6-Dichloroindophenol, standard solution: Prepare this solution fresh before use. Dissolve 0.4 g of 2,6-dichloroindophenol, sodium salt, in 500 mL of distilled or deionized water. The solution may be stored in the refrigerator for up to one week. See the Lab Hints for a discussion of the properties of DCIP.

Safety Precautions

The reference solutions contain 1% oxalic acid, a skin and eye irritant. The solutions are slightly toxic by ingestion. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. All food-grade items that are 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 from the lab after use. Remind students to wash hands thoroughly with soap and water before leaving the laboratory. Please review 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. Contents of the well plates and leftover solutions may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b.

Lab Hints

• The laboratory work for this experiment can reasonably be completed within a typical 2-hour lab period. The Prelab Assignment may be assigned as homework in preparation for lab and should be reviewed during 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 ownership and can give interesting results.
• For best results, prepare the Vitamin C solution fresh in 1% oxalic acid solution the day of the lab. The instability of Vitamin C solutions is probably due to microbial contamination. Oxalic acid has been shown to be an effective preservative for Vitamin C if the solutions are kept refrigerated. To use consumer-grade Vitamin C tablets in place of reagent ascorbic acid, crush the tablets with a mortar and pestle. Dissolve 200 mg of Vitamin C in 200 mL of oxalic acid solution and mix thoroughly to dissolve. (Do not heat.) There will be a small amount of undissolved binder material remaining—this will not interfere with the titration.
• To analyze fresh-squeezed fruit juices, squeeze about 20 mL of juice from a fresh fruit into a clean and dry 50-mL beaker. Place several folds of cheesecloth in a funnel and strain the juice through the cloth into a test tube or directly into a well on the reaction plate. Avoid getting pulp in the sample.
• In addition to fruits and fruit juices, other beverages that may be tested include milk, Gatorade^®, Kool-Aid^®, Tang^®, etc. Many vegetables, especially green pepper and cabbage, are also excellent sources of Vitamin C. Homogenize vegetables in a Waring (laboratory) blender with 1% oxalic acid solution. Extract 5 g of the 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 redox titration of Vitamin C with 2,6-dichloroindophenol may also be carried out in the reverse manner by counting the number of drops of Vitamin C solution required to decolorize a known volume of the dye solution. We tested both modifications of the procedure and found that the results were more consistent when the Vitamin C was titrated with dye until the first trace of color persisted in the solution. In a literature study of the two methods, it was reported that a nonlinear calibration was obtained for drops of Vitamin C required to titrate different concentrations of dye.
• Vitamin C may also be titrated using iodine solution as the oxidizing agent. Add 2–3 drops of starch indicator to the Vitamin C solution, then titrate dropwise with iodine/potassium iodide solution until a permanent blue color change is observed.
• Most packaged juices contain Vitamin C. Natural apple juice is an exception. Find unfortified (no added Vitamin C) apple juice in your local grocery store and check the Nutrition Facts label to verify.
• The amount of Vitamin C in packaged juices is often reported as percent of the daily value (DV) per serving size. The DV for Vitamin C is 60 mg for an adult.
• DCIP (sodium salt) is an ACS reagent chemical and is used in a standard method of Vitamin C analysis. (See Procedure 43.065 in the Official Methods of Analysis published by the Association of Official Analytical Chemists.) This is what the official citation says about the stability of the dye: “Decomposition products that make end point indistinct occur in some batches of dry indophenol and also develop with time in stock solution. If dry dye is at fault, obtain new supply.” It is important to test the dye beforehand and adjust the concentration of dye to obtain a convenient volume for titration. In our experience we found that the optimum dye concentration was between 0.300 and 0.400 g DCIP in 500 mL water.

Answers to Prelab Questions

1. 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!

2. In the reaction of ascorbic acid with 2,6-dichloroindophenol (Equation 1), which compound is being oxidized and which compound is being reduced?

   Ascorbic acid is being oxidized to its dehydro- form, while 2,6-dichloroindophenol is being reduced. Note: The traditional definition of oxidation and reduction reactions (loss and gain of electrons, respectively) is difficult to recognize in organic compounds. Organic chemists often define redox reactions in terms of the loss and gain of oxygen and hydrogen atoms. Oxidation of an organic compound may be defined as either the 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).

3. The Nutrition Facts label on a carton of orange juice reports that the juice contains 100 mg Vitamin C per one-cup (240 mL) serving. What is the concentration of Vitamin C in mg per 100 mL?
   {14040_PreLabAnswers_Equation_2}
4. Read the entire Procedure. Why is it important to deliver the same size drops of DCIP in titrating different samples? Describe two technique tips for ensuring uniform drops.

   Uniform-size drops are needed to achieve accuracy and precision in the titration results. To make sure drops are the same size, squeeze out any air bubbles in the DCIP pipet and hold the pipet vertically above the reaction well.

5. 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 blue, purple or pink color of the dye and make it impossible to detect the endpoint.

Sample Data

Laboratory Report

*Student data tables contain entries for two juices to be tested. Sample data are reported here for four juices to illustrate the range of results that may be obtained. It is recommended that the experiment be carried out in a collaborative manner with different student groups testing different juices and comparing results as a class.

Answers to Questions

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

   See the data table.

2. Plot the average number of drops of DCIP required to titrate each reference solution A−D (y-axis) versus the known amount of Vitamin C (x-axis) in the sample (see the Materials section).
   {14040_Answers_Figure_4}
3. Draw a best-fit straight line through the points, including the origin. Use the resulting calibration curve to determine the amount of Vitamin C in each juice that you tested. Report the results in mg of Vitamin C per 100 mL of juice.

   Orange juice: 33 mg/100 mL
   Apple juice: 25 mg/100 mL
   White grape juice: 60 mg/100 mL
   Lemon juice: 18 mg/100 mL

4. Consult the Nutrition Facts label for one of the juices tested, if available, and report the known Vitamin C content. Convert the reported amount of Vitamin C to the units mg per 100 mL.

   For apple juice, the Nutrition Label reports 60 mg Vitamin C per 240-mL serving size. This converts to 25 mg per 100 mL.

   {14040_Answers_Equation_3}
5. Calculate the percent error in the analysis of Vitamin C in this juice.
   {14040_Answers_Equation_4}
6. Briefly describe sources of experimental error that might have affected the accuracy.

   The overall accuracy is excellent. Possible sources of student error include inaccurate or imprecise volume measurements and poor transfer techniques. It is also difficult to deliver uniform-size drops of DCIP. Accuracy can be improved by using volumetric glassware, such as a buret.

7. Compare class results for all the juices tested and rank the juices tested in terms of their Vitamin C content, from highest to lowest.

   Grape juice > orange juice > apple juice > lemon juice

8. (Optional) Compare the amount of Vitamin C in a packaged juice versus fresh fruit juice. What factors may account for the difference in results, if any?

   In our testing, the amount of Vitamin C was found to be similar in refrigerated (bottled) orange juice and in fresh-squeezed orange juice.

Introduction

The importance of eating fresh fruits and vegetables to prevent disease has been known for a long time. British sailors were nicknamed “limeys” because they were given limes and lemons to eat during long voyages to prevent scurvy. The concept of vitamins—trace nutrients required to protect against so-called deficiency diseases—was introduced in the early 20th century. The chemical structure of Vitamin C was determined in 1933, and it was called ascorbic acid in recognition of its anti-scurvy properties. How much Vitamin C is present in fresh fruit juices?

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. The Recommended Dietary Allowance (RDA) of Vitamin C for young adults is 60 mg per day, which is provided by one 8-oz. glass of fresh orange juice. Many medical and dietary professionals believe that higher doses of Vitamin C benefit the immune system, helping to stave off infections such as the common cold. The symptoms of Vitamin C deficiency include bleeding gums, loose teeth, skin bruises, joint pain and muscle aches. All of these may be attributed to the breakdown of connective tissue in the body.

The role of Vitamin C in preventing scurvy is directly related to its biochemical function. Vitamin C is an essential co-catalyst required for the synthesis of collagen, the protein fibers that make up all connective tissue (e.g., cartilage, skin, tendons, ligaments). Vitamin C also functions as an antioxidant, protecting other vital chemicals in the body against oxidation. The antioxidant function of Vitamin C may explain its effect in reducing the severity of colds and other infections.

The structure of Vitamin C, ascorbic acid, is shown in Figure 1. Ascorbic acid is a highly polar, water-soluble compound, and a good 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, converts Vitamin C to an inactive form. Due to its ease of oxidation, Vitamin C is easily converted to the inactive form during food processing or cooking. 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 called 2,6-dichloroindophenol (DCIP) converts the dye to its 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 that is added will not react and will remain blue. This represents the equivalence point or endpoint of the titration.

Experiment Overview

The purpose of this experiment is to analyze the Vitamin C content in fruits or fruit juices by microscale titration with 2,6-dichloroindophenol (DCIP). The amount of Vitamin C will be determined by comparison against titration data obtained for a series of ascorbic acid reference solutions A−D containing known amounts of Vitamin C. A calibration curve will be prepared by plotting the average number of drops of DCIP required to titrate each reference sample versus the amount of Vitamin C in the sample.

Materials

Prelab Questions

1. Name two reasons why the Vitamin C content might not be the same in fresh, frozen or canned orange juice.
2. In the reaction of ascorbic acid with 2,6-dichloroindophenol (Equation 1), which compound is being oxidized and which compound is being reduced?
3. The Nutrition Facts label on a carton of orange juice reports that the juice contains 100 mg Vitamin C per one-cup (240 mL) serving. What is the concentration of Vitamin C in mg per 100 mL?
4. Read the entire Procedure. Why is it important to deliver the same size drops of DCIP in titrating different samples? Describe two technique tips for ensuring uniform drops.
5. Why would it not be possible to analyze grape juice or cranberry juice using the method outlined in this experiment?

Safety Precautions

The ascorbic acid reference solutions contain 1% oxalic acid, a skin and eye irritant. The solutions are slightly toxic by ingestion. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves, and a lab coat or chemical-resistant apron. All food-grade items that are 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 from the lab after use. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Read the entire procedure before beginning the experiment.

1. Obtain about 20 mL of DCIP dye in a clean and dry beaker and place a labeled pipet in the solution.
2. Select two juices from those available for testing. Record the identity of the juices in the data table.
3. Place a 24-well reaction plate on white background paper. Note that wells are identified using a combination of a letter (rows A−D) and number (columns 1−6).
4. Using a clean disposable pipet or eyedropper for each liquid, add about 3 mL of ascorbic acid reference solution A−D or juice to the appropriate well A1−A6 in the first row of the reaction plate, as shown in the following diagram.
   {14040_Procedure_Figure_3}
5. Using a 1-mL syringe or serological pipet, carefully transfer 0.50 mL of reference solution A from well A1 into each well B1 and C1 directly below it.
6. Using the labeled DCIP pipet, slowly add DCIP one drop at a time to reference solution A in well B1. Stir with a toothpick to mix the solution between drops. For best results, make sure there are no air bubbles in the pipet and hold the pipet in a vertical position to deliver uniform-size drops.
7. Count the number of drops of DCIP required to impart 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 (trial 1).
8. Repeat steps 6 and 7 to titrate the reference sample in well C1 (trial 2).
9. Rinse the 1-mL syringe or serological pipet with two portions of distilled water and then carefully transfer 0.50 mL of reference solution B from well A2 into each well B2 and C2 directly below it.
10. Repeat steps 6−8 to titrate the reference samples in wells B2 and C2.
11. Repeat the testing procedure (steps 5–9) four more times to analyze reference solutions C and D as well as the two juices. Remember to rinse the 1-mL syringe or serological pipet twice with distilled water before transferring each new sample to be tested to the wells directly below. Obtain and record data for two trials per sample.
12. Dispose of the contents of the reaction plate down the drain with excess water.

Student Worksheet PDF

14040_Student1.pdf