Purpose: To measure the vitamin C (ascorbic acid) concentration in various vitamin C supplements and drinks by determining the amount of indophenol indicator needed to react with reference solutions of known vitamin C concentration.

Similar to the idea presented in the Science News article “Tainted supplements flood the market,” students will analyze the amount of a chemical in a supplement. However, the vitamin C sources used in the experiment will not contain the harmful chemicals mentioned in the article. Chemicals used in a laboratory setting should always be handled with caution. Eating, directly touching and smelling chemicals should never be permitted.

Approximate class time: One 45-minute class period.

Supplies:

• Student handout
• Gloves
• Goggles
• Indophenol powder (for example, from Home Science Tools)
• Purified vitamin C (for example, from Home Science Tools)
• Colorimetric pH measuring strips
• Scissors
• Disposable plastic pipettes
• Balances or scales
• Weigh paper
• Test tubes
• Test tube racks
• Wax pencils or other markers for writing on the test tubes
• Vitamin C tablets that contain vitamin C
• Drinks or juices that have natural or artificial levels of vitamin C (lemon juice, orange juice, pineapple juice, apple juice, fruit punch, tomato juice, milk with added vitamin C, etc.), plus some that do not for comparison (water, regular milk, tea, etc.)
• Pliers or mortars/pestles to crush and grind supplement pills
• Graduated cylinders
• Beakers
• Glass stirring rods
• Sinks to rinse out test tubes

Directions for teachers:

Have the students work in pairs or small groups if supplies are limited.

Before class, dissolve 500 milligrams (0.50 grams) indophenol in 1,000 milliliters water and mix thoroughly. Divide this 0.5 mg/ml indophenol solution among the lab tables. Unused indophenol can be stored in a sealed bottle at room temperature for a year or so before it becomes inactive.

Also before class, dissolve 500 mg (0.50 g) vitamin C (ascorbic acid) in 500 ml water and mix thoroughly to make a 1.0 mg/ml vitamin C solution.

To save time and eliminate redundancy, you should make 1.0 mg/ml solutions from at least five different vitamin C supplement pills before class. We recommend that supplements in the pill form be handled by the teacher only. Please remind students that all chemicals in the science lab should be handled with caution. Test the supplement solution using the indophenol test. If the sample has too little vitamin C to quantify, make another supplement solution with more supplement and/or less water. If the sample has too much vitamin C to quantitate, make another supplement solution with less supplement and/or more water. Divide the solutions among lab groups.

If appropriate for your class level, have students graph the reference vitamin C concentrations versus the number of drops of vitamin C solution required to turn the indophenol solution clear. Once these values are graphed, a line of best fit should be determined. This curve can be used to determine the approximate vitamin C concentration of unknown supplement or juice solutions.

Please feel free to edit these instructions depending on the samples available for students to test.

General information and reminders for students:

One ingredient in some supplements is vitamin C, or ascorbic acid. You will test different solutions made from vitamin C sources to obtain a relative quantity of vitamin C contained in the sources.

The pH test strips change color to indicate how acidic (pH<7) or alkaline (pH>7) a sample is. Cut the test strips in half lengthwise, dip a strip into the vitamin C solution for two seconds and then let it dry. Compare the strip’s color to the color code on the packaging to determine the sample’s pH.

Indophenol dissolved in water makes a blue solution. Adding vitamin C reduces the color to the point that the solution becomes colorless. You can evaluate how much vitamin C is in a sample by counting how many drops of the sample must be added to an indophenol solution to make it colorless.

Always test pH before you do the indophenol solution test. If samples are too strong or too colored, dilute them with water and write down by how much you diluted them.

To avoid cross-contamination, use separate pipettes for indophenol, vitamin C, drink/supplement samples and water. Add drops without touching the liquid to which you are adding the drops.

Chemicals:

0.5 mg/ml indophenol solution

1.0 mg/ml vitamin C solution

Various drinks/juices to test

Various vitamin C supplement solutions to test

Data table:

Read through the following procedure, and take note of all the quantitative and qualitative data that needs to be recorded. For each part of the lab, create a data table that you can use while completing the activity.

Part 1: Make and test vitamin C reference standards for comparison

1. Make five tubes of different vitamin C concentrations. Label the tubes on the side so you can keep track of them:

Tube A: 1.0 mg/ml vitamin C = 16 drops of 1.0 mg/ml vitamin C

Tube B: 0.5 mg/ml vitamin C = 8 drops of 1.0 mg/ml vitamin C + 8 drops of water

Tube C: 0.25 mg/ml vitamin C = 4 drops of 1.0 mg/ml vitamin C + 12 drops of water

Tube D: 0.125 mg/ml vitamin C = 2 drops of 1.0 mg/ml vitamin C + 14 drops of water

Tube E: 0.0625 mg/ml vitamin C = 1 drop of 1.0 mg/ml vitamin C + 15 drops of water

 

2. The pH test strips change colors to indicate how acidic (pH<7) or alkaline (pH>7) a sample is. Cut the test strips in half lengthwise, dip a strip into the vitamin C solution for two seconds and then let it dry. Compare the strip’s color to the color code on the packaging to determine the sample’s pH. Record all results in your data table.

If necessary, you can make further dilutions or intermediate dilutions to test.

3. Make five new tubes with 16 drops of 0.5 mg/ml indophenol per tube.

4. As you can see from your indophenol solutions, indophenol dissolved in water makes a blue solution. Adding vitamin C reduces the color to the point that the solution becomes colorless. You can evaluate how much vitamin C is in a sample by counting how many drops of the sample must be added to indophenol to make it colorless. Complete the following steps below, and record in your data table the number of drops of vitamin C solution needed to turn each indophenol solution clear. Make sure to gently shake the test tube in between the addition of every drop to ensure the solutions properly mix.

Tube 1:  Add drops from Tube A to Tube 1 until Tube 1 becomes colorless.
Record how many drops of 1.0 mg/ml vitamin C make the indophenol become colorless.

Tube 2:  Add drops from Tube B to Tube 2 until Tube 2 becomes colorless. 
Record how many drops of 0.5 mg/ml vitamin C make the indophenol become colorless.

Tube 3:  Add drops from Tube C to Tube 3 until Tube 3 becomes colorless.
Record how many drops of 0.25 mg/ml vitamin C make the indophenol become colorless.

Tube 4:  Add drops from Tube D to Tube 4 until Tube 4 becomes colorless.
Record how many drops of 0.125 mg/ml vitamin C make the indophenol become colorless.

Tube 5:  Add drops from Tube E to Tube 5 until Tube 5 becomes colorless.
Record how many drops of 0.0625 mg/ml vitamin C make the indophenol become colorless.

5. Graph the reference vitamin C concentrations versus the number of drops of vitamin C solution required to turn the indophenol solution colorless. Check with your teacher to see what program you should use to graph your data, or if you should graph your data by hand on graph paper. Once these values are graphed, determine a line of best fit.

6. Rinse and dry all test tubes, so you can reuse them for the next part.

Part 2: Test vitamin C in five drink or juice samples

1. Add 16 drops of a drink or juice sample to a test tube. Do this for five different drinks or juices. Label each tube with the name of the drink. Record each sample name in your data table.

2. Use colorimetric pH strips to measure the pH of each sample. Record the pH values in your data table.

3. Make five new tubes with 16 drops of 0.5 mg/ml indophenol per tube.

4. Using a different pipette for each drink or juice sample, add each drink or juice sample one drop at a time to the indophenol tubes until the solutions become colorless. Make sure to gently shake the test tube in between the addition of every drop to ensure the solutions properly mix. In your data table, record how many drops it took for each drink or juice sample to turn the indophenol solution clear.

5. Using the line of best fit from your graph of reference data, determine the concentration of vitamin C in each unknown solution. Record your results in your data table.

6. Rinse and dry all test tubes, so you can reuse them for the next part.

Part 3: Test vitamin C supplement pills solutions

1. Add 16 drops of a vitamin C supplement pill solution to a test tube. Do this for five different supplement pill solutions. Label each tube with the name or letter of the supplement pill. Record each sample name or letter in your data table.

2. Use colorimetric pH strips to measure the pH of each sample. Record the pH values in your data table.

3. Make five new tubes with 16 drops of 0.5 mg/ml indophenol per tube.

4. Using a different pipette for each supplement solution, add each supplement solution one drop at a time to the indophenol tubes until each becomes colorless. Make sure to gently shake the test tube in between the addition of every drop to ensure the solutions properly mix. In your data table, record the how many drops it took for each supplement solution to turn the indophenol solution clear.

5. Using the line of best fit from your graph of reference data, determine the concentration of vitamin C in each unknown supplement solution. Record your results in your data table.

6. Clean your equipment and your lab area.

Part 4: Analysis questions

1. Which drink or juice had the most vitamin C?

Student answers will vary.

2.  Which drink or juice had the least vitamin C?

Student answers will vary.

3.  Which supplement pill had the most vitamin C?

Student answers will vary.

4.  Which supplement pill had the least vitamin C?

Student answers will vary.

5.  How did pH correlate with vitamin C concentration? What does this tell you about vitamin C?

As the vitamin C concentration increases, the pH decreases. This relationship indicates that vitamin C is acidic.

6.  Which result surprised you the most?

Student answers will vary.