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Which Orange Juice Has the Most Vitamin C?

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Difficulty
Time Required Long (2-4 weeks)
Prerequisites Ideally you'd have your own citrus tree with ripe fruit for this project. The second-best option is to use citrus fruit from a store.
Material Availability Specialty items
Cost High ($100 - $150)
Safety Adult supervision required

Abstract

Which type or orange juice has the most vitamin C? In this project you'll learn how to measure the amount of vitamin C in a solution using an iodine titration method. You'll compare the amount of vitamin C in three different types of orange juice: home-made, premium not-from-concentrate, and juice made from frozen concentrate. Which do you think will have the most vitamin C?

Objective

The goal of this project is to determine which orange juice has the most vitamin C: home-made fresh-squeezed, premium not-from-concentrate, or juice made from frozen concentrate.

Credits

Andrew Olson, Ph.D., Science Buddies

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Last edit date: 2013-02-16

Introduction

In this project, you will determine the amount of vitamin C in various types of orange juice. You'll use a method called titration, which is a common technique in chemistry. Titration is a way to measure the unknown amount of a chemical in a solution (the titrant) by adding a measured amount of a chemical with a known concentration (the titrating solution). The titrating solution reacts with the titrant, and the endpoint of the reaction is monitored in some way. The concentration of the titrant can now be calculated from the amount of titrating solution added and the ratio of the two chemicals in the chemical equation for the reaction.

Let's go through the titration process with a specific example: the titration of vitamin C by iodine. The chemical name for vitamin C is ascorbic acid. When iodine and ascorbic acid are combined in solution, a chemical reaction takes place. In this chemical reaction, the ascorbic acid molecule loses electrons, which are transferred to the iodine molecule. Chemists call this type of reaction an oxidation/reduction reaction (or redox reaction for short). The ascorbic acid is oxidized to dehydroascorbic acid, and the iodine is reduced to iodide ions. Oxidation-reduction reactions always occur in pairs like this. The molecule that loses electrons is oxidized, and the molecule that accepts the electrons is reduced.

So how can you use the iodine-ascorbic acid reaction to determine the amount of ascorbic acid (vitamin C)? If you start with a known concentration of iodine, and carefully measure the amount of the iodine solution that you add, you can calculate how much ascorbic acid was present. How do you know when the iodine-ascorbic acid reaction is complete? You add an indicator to the solution. In this case, the indicator is soluble starch. When iodine reacts with starch, it turns the solution a blue-black color. If ascorbic acid is present in the solution, iodine will react with it, and not with the starch, so the solution will not change color. However, once all of the ascorbic acid has been oxidized, added iodine will be free to react with the starch, producing a distinct color change.

Which do you think will have more vitamin C (ascorbic acid): freshly picked oranges, or oranges that were picked one or two weeks ago? Does it matter if the oranges are stored at room temperature or in a refrigerator? You can find out for yourself with this project!

Terms and Concepts

To do this project, you should do research that enables you to understand the following terms and concepts:

  • Titration
  • Ascorbic acid
  • Iodine
  • Oxidation / reduction reaction
  • Indicator
  • Stoichiometry

Questions

  • What happens when iodine is added to a starch solution?
  • What happens when iodine is added to a starch solution that also contains vitamin C?

Bibliography

Materials and Equipment Product Kit Available

Supplies for this project are available in one convenient kit from the Science Buddies Store

To do this experiment you will need the following materials and equipment:

  • Samples of three different kinds of orange juice:
    1. Home-made fresh-squeezed (which means you'll need to buy some oranges)
    2. Premium not-from-concentrate juice (e.g. Tropicana® or Florida's Natural®)
    3. Made from frozen concentrate (following instructions on the concentrate can)
  • Juicer for extracting juice from oranges (manual or electric is fine)
  • Cheesecloth
  • Vitamin C tablets
  • Distilled water
  • Masking tape
  • Permanent marker
  • Small funnel (do not use for food after using it for chemistry)
  • Chemical safety goggles
  • Lab apron
  • Rubber (latex) gloves
  • 2% Lugol's iodine solution (30mL) available from Amazon.com
  • Soluble starch, 10 g
  • 50 mL graduated cylinder
  • 500 mL graduated cylinder
  • 50 mL Ehrlenmeyer flask
  • 50 mL buret
  • Ring stand
  • Buret clamp
  • Plastic transfer pipettes (or eyedropper) for adding drops of starch indicator solution
  • 1/4 teaspoon measuring spoon, or an electronic kitchen balance (accurate to 0.1 g)
  • Glass jars for iodine (300 mL) and starch solutions.
  • Optional: you can obtain more accurate results if you use a 250 mL volumetric flask for making your vitamin C standard solution.

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Experimental Procedure

Note: Iodine solution is poisonous. Avoid skin and eye contact. See the Materials Safety Data Sheet for complete information. Wear chemical safety goggles and rubber gloves when handling the concentrated solution. Iodine solution will stain clothing; a lab coat is also recommended. For more information on how to properly use a balance and different titration techniques see Chemistry Lab Techniques.

  1. Do your background research so that you are knowledgeable about the terms, concepts, and questions, above.
  2. Wear gloves, chemical safety goggles, and a lab coat or apron when using the iodine solutions in this experiment.
  3. Dilute the Lugol's solution 1:10 in distilled water to make your iodine titration solution.
    1. Pour the 30 mL Lugol's solution into the 500 mL graduated cylinder.
    2. Add enough distilled water to bring the total fluid volume to 300 mL and mix.
    3. Store the solution in a clean, tightly covered glass jar that is clearly labeled. Store it in a location that is protected from light.
    4. Rinse and dry the 500 mL graduated cylinder.
  4. Make a starch indicator solution.
    1. This can be anywhere from 0.5 to 1.0%. The exact amount of starch is not critical.
    2. For a 0.5% solution, add 1 g (which is equivalent to 1/4 teaspoon) of soluble starch to 200 mL of near-boiling distilled water.
    3. Stir to dissolve, and allow to cool.
    4. When cool, store the starch solution in a clean, tightly covered glass jar that is clearly labeled.
    5. Rinse and dry the 500 mL graduated cylinder.
  5. Make a fresh vitamin C standard solution (1 mg/mL). Do this on each day that you make vitamin C measurements from orange juice.
    1. You will use this solution to "standardize" your iodine titration solution. You will measure how much of your iodine solution it takes to oxidize a known amount of vitamin C. You can then use your iodine titration solution to determine the amount of vitamin C from test samples of juice from oranges.
    2. Crush a 250 mg vitamin C tablet, and dissolve it in 100 mL of distilled water.
    3. Pour into a graduated cylinder and add distilled water to bring the total volume to 250 mL.
  6. Titrate 25 mL of vitamin C standard solution.
    1. Use a clean 50 mL graduated cylinder to measure 20 mL of vitamin C standard solution.
    2. Pour this into a 50 mL Ehrlenmeyer flask (the shape of this flask allows you to swirl the solution to mix it without spilling).
    3. Add 10 drops of starch indicator solution.
    4. Set up the 50 mL buret on the the ringstand.
    5. Use a funnel to carefully fill the buret with your iodine titration solution. Tip: the fluid level should not be past the graduated markings on the buret.
    6. Write down the initial volume of the iodine titration solution in the buret.
    7. Place the Ehrlenmeyer flask (containing the vitamin C and starch solutions) under the buret.
    8. Carefully release the spring clamp of the buret to add iodine solution drop by drop.
    9. Swirl the flask to mix in the iodine solution after each addition.
    10. The titration is complete when the iodine creates a blue-back color in the solution that lasts for longer than 20 seconds.
    11. Record the final volume of the iodine solution in the buret.
    12. The difference between the initial volume and the final volume is the amount of iodine titration solution needed to oxidize the vitamin C.
    13. Repeat this step three times. You should get results that agree within about 0.1 mL.
  7. Here's how to prepare fresh-squeezed orange for testing.
    1. Use a juicer to squeeze orange juice from two (or more) oranges.
    2. You need 20 mL of juice per titration, and you should do at least three titrations per storage condition, for a total of 60 mL.
    3. Filter the orange juice through cheesecloth to remove any pulp and seeds.
  8. Titrating an orange juice sample is quite similar to titrating the vitamin C standard. Here are the steps:
    1. Tip: if any of the orange juice samples contain pulp, filter them through clean cheesecloth before doing the titration.
    2. Use a clean 50 mL graduated cylinder to measure 20 mL of the fresh-squeezed juice.
    3. Pour this into a 50 mL Ehrlenmeyer flask (the shape of this flask allows you to swirl the solution to mix it without spilling).
    4. Add 10 drops of starch indicator solution.
    5. Set up the 50 mL buret on the the ringstand.
    6. Fill the buret nearly full with your iodine titration solution.
    7. Write down the initial volume of the iodine titration solution in the buret.
    8. Place the Ehrlenmeyer flask (containing the vitamin C and starch solutions) under the buret.
    9. Carefully release the spring clamp of the buret to add iodine solution drop by drop.
    10. Swirl the flask to mix in the iodine solution after each addition.
    11. The titration is complete when the iodine creates a distinct color change in the juice/starch solution. This color change will be harder to see than with the vitamin C solution, since the juice starts out orange. The color will change from orange to grayish brown when the endpoint is reached. If you continue to add iodine, the color will darken further. You want to note the volume of iodine added when the color first changes.
    12. Record the final volume of the iodine solution in the buret.
    13. The difference between the initial volume and the final volume is the amount of iodine titration solution needed to oxidize the vitamin C.
    14. Repeat this step three times. You should get results that agree within about 0.1 mL.
  9. For each juice (fresh, premium, or from-concentrate), calculate the average amount of iodine needed to titrate a 20 mL sample.
  10. You can calculate the amount vitamin C in your samples by setting up a proportion. Here's an example (with made-up numbers) to show you how:
    1. Let's say that it took an average of 8.5 mL of iodine solution to titrate 20 mL of 1 mg/mL vitamin C standard solution, which means 20 mg vitamin C total.
    2. Let's also say it takes an average of 6.8 mL of iodine solution to titrate a 20 mL test sample of orange juice.
    3. We'll call the amount of vitamin C in the orange juice sample x. You can find what x is with the following equation:

      X = (6.8 mg/ml)*(20 mg)/(8.5ml) = 16.0 mg

  11. Did one type of orange juice have more vitamin C than the others? Can you explain your results?

Troubleshooting

For troubleshooting tips, please read our FAQ: Which Orange Juice Has the Most Vitamin C?.

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Variations

  • Measure the amount of vitamin C in fresh fruits and vegetables. Which ones have the highest vitamin C content? Which ones have the lowest? Puree a 100 g sample of the fruit or vegetable, and strain it through cheesecloth with about 50 mL of distilled water. Add distilled water to bring the total volume up to 100 mL. From your measurement, you can figure out how much vitamin C there is per 100 g of the fruit or vegetable you sampled. You can use typical weights of the fruit or vegetable to calculate how much total vitamin C there is in each fruit or vegetable you test.
  • Measure the vitamin C content in fruit juices. Do the amounts correspond to what is on the label? Design an experiment to find out.
  • Does the amount of vitamin C in fruit juice decrease during refrigerated storage? Does the type of container (e.g., glass bottle, or paperboard carton) matter? Design an experiment to find out.
  • For a experiment on how storage affects the amount of vitamin C in oranges, see the Science Buddies project Do Oranges Lose or Gain Vitamin C After Being Picked?

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Frequently Asked Questions (FAQ)

If you are having trouble with this project, please read the FAQ below. You may find the answer to your question.
Q: Instead of the Lugol’s solution or the iodine solution for starch test, can I substitute a pure potassium iodine solution?
A: Quite simply, no. The iodine needs to be elemental (i.e. not an ion and not part of another compound), or else the proper reactions won't take place, and your titration won't work.
Q: When I make the starch indicator solution, it does not really dissolve, but is clumpy and/or milky instead. What should I do?
A: he starch solution should be clear and may need to be heated more. You could try continuing to heat the starch solution in a pan on the stove until the solution becomes clear, just before it boils.
Q: Why do I have to make a fresh vitamin C standard solution and titrate it every day?
A: Since the calculations in this project are based on the data you get from the vitamin C standard solution titration, it is very important that these data are accurate. Because of this, there are two main reasons why making and titrating a fresh vitamin C standard solution increases the accuracy of your measurements:
  • When exposed to air, vitamin C will oxidize, or break down; after any vitamin C solution is exposed to air it will have a lower, unknown concentration the next day.
  • If you test orange juice samples over multiple days, you must make and titrate a fresh vitamin C standard solution each day or else you cannot compare data from one day to the next. By titrating a fresh vitamin C standard solution each day you take measurements and by observing the variability in the amount of iodine needed to titrate the vitamin C standard solution, you can better compare your orange juice titration results to the day-to-day variations in your vitamin C standard titrations.
Q: Should I stop the titration when I first see a hint of color, or when the entire solution has completely changed color?
A: When you first seen the color change, you should stop the buret from dripping, pick up the flask, swirl it, and put it back down. Wait for 30 seconds. If the color goes away, then continue with the titration, but if the color stays, then stop the titration. The color change indicates that all of the substance being titrated (vitamin C, or ascorbic acid) has reacted with the titrating substance (iodine titration solution). Specifically, when there is no ascorbic acid left, the iodine can react with the starch, causing the solution to change color. Therefore, the first time color lasts after adding a drop of iodine solution and swirling it is the first time when there is no more vitamin C in the solution, and this is when you should stop the titration. Be careful to ensure that the color is lasting and isn’t temporary by always swirling the solutions well.
Q: Why should I add the iodine titration solution drop by drop?
A: The titration reaction is very sensitive. One drop of iodine solution can be enough to drive the reaction to completion (if it were near completion before the drop was added). If more than one drop is added at a time, the data will not be as accurate as it could be.
Q: Why should I swirl the flask each time I add the iodine titration solution?
A: Mixing the flask is important to ensure that the solutions have completely reacted with each other. When adding the iodine solution, you may see a temporary color change that goes away when you swirl the flask. The color change that is important for this experiment is the one that is not temporary and does not go away after mixing the solutions.
Q: What if my solutions never change color?
A: There are a number of reasons your titration may not work. Three of the most common problems that can lead to a solution not changing colors in a titration are:
  • Using the wrong substances. Make sure that you use Lugol's iodine solution or another iodine solution sold as a starch indicator (not a pure potassium iodine solution) and a true, soluble starch. (If you purchased the Science Buddies kit, this problem would not apply. The materials in the kit are correct for this Project Idea.)
  • Using incorrect concentrations. If your vitamin C standard solution is too concentrated or your iodine titration solution is too dilute, or your starch indicator solution is not between 0.5 to 1.0%, it may require much more than 50 mL of iodine solution to titrate the standard solution and/or orange juice. The most common problem here is an overly dilute iodine solution; sometimes, the Lugol's iodine solution sold in stores is already dilute, and you don't need to dilute it more.
  • Using too much orange juice. If there is a large amount of vitamin C in the orange juice that is being tested, it may require more than 50 mL of iodine titration solution for it to change color. You could try using a smaller volume of orange juice.
Q: How do I read the volume of iodine titration solution in the buret?
A: To accurately read the volume of a liquid in the buret, read where the bottom of the meniscus is. The meniscus is where the surface of the liquid forms a curve. For example, in the picture below, based on reading from the bottom of the meniscus, the volume in the buret is about 14.58 mL.

Image
(Image: G. Carboni, 2004)
Q: Can I use corn starch instead of soluble starch? What is soluble starch anyway?
A: It is theoretically possible to do this experiment using corn starch - but it will be more difficult. Starches are notoriously difficult to dissolve. In fact, starch is insoluble in cold water. Heat is required to change the starch's semi-crystalline structure so that some of the amylose molecules are more accessible, this in turn makes the starch soluble. Which means that one of the technical challenges in this project is the creation of a starch solution (corn starch dissolved in water). It also explains why the solution is made with hot water in the Procedure.

Soluble starch is starch that has been chemically pre-treated so that its structure is altered and it is already more soluble; it is not sold in the grocery store. The project calls for using this type of starch because it is much easier to make the starch solution. If you chose to substitute corn starch for the soluble starch you will need to make sure that you use very hot, even boiling water. Add no more than 1/4 tsp of cornstarch to 200 ml of the hot/boiling water. Stir vigorously with a fork for several minutes to dissolve as much of the corn starch as possible. Test your solution by mixing 1 drop of the starch solution with 1 drop of the iodine solution on a piece of wax paper, if your starch solution is well made the resulting mixture should be a dark blue-black color.

If you do substitute corn starch for soluble starch you should make your starch solution up fresh every day of testing because the corn starch is more likely to fall out of solution over time.

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