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Do Oranges Lose or Gain Vitamin C After Being Picked?

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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

Are oranges highest in vitamin C when they are fresh from the tree (or, in a pinch, the grocery shelf)? In this project you'll learn how to measure the amount of vitamin C in a solution using an iodine titration method.

Objective

The goal of this project is to determine whether the amount of vitamin C in oranges (or other citrus fruit) decreases after the fruit is picked.

Credits

Andrew Olson, Ph.D., Science Buddies

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Last edit date: 2013-02-16

Introduction

In this project, you'll learn an interesting method for determining the amount of vitamin C in a solution. The technique you will be using is called titration.

Titration is used to determine the unknown concentration of a chemical in a solution. In a titration, a carefully measured amount of a second chemical is gradually added to the solution. The added chemical reacts with the original chemical, whose concentration is unknown. The original chemical whose concentration is unknown is called the titrant, and the added chemical whose concentration is known is called the titration solution. The titration solution reacts with the titrant, and the progress of this reaction is carefully monitored. When 100% of the original compound has reacted with the added chemical, the titration is complete. Now the concentration of the original chemical can be determined from the amount of titration solution that was added.

So you can better understand how a titration works, let's look at the specific example of determining the concentration of vitamin C. Vitamin C, also known as ascorbic acid, is the titrant in this case (because its concentration is unknown). You start with a measured volume of the titrant. The titrating solution that will added to the titrant is iodine. You will start out by using your iodine solution to titrate a known amount of vitamin C, using a solution prepared from a vitamin C tablet. You will carefully measure the amount of iodine solution need to titrate the known amount of vitamin C. You will know when the titration is complete because you will add a third chemical—soluble starch—to the solution. The starch acts as an indicator: the starch changes the color of the solution when the iodine/vitamin C reaction is complete. As soon as the solution changes color, you will stop adding iodine solution. Once you have calibrated your iodine solution with a known amount of vitamin C, you can then repeat the procedure to determine how much vitamin C there is in samples of fresh-squeezed orange juice.

The chemical reaction of iodine with vitamin C is called an oxidation-reduction reaction. (Chemists often use the shorthand 'redox reaction' to refer to this type of reaction.) 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.

How will different storage times affect the amount of vitamin C in juice squeezed from oranges? Get ready to do some titrations to find out for yourself.

Terms and Concepts

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

  • Titration
  • Ascorbid acid
  • Iodine
  • Indicator
  • Oxidation-reduction reaction
  • 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:

  • About 10–12 juicing oranges (or other citrus fruit)
    1. Ideally, you would have access to a citrus tree with ripe fruit for the duration of the project.
    2. The next-best option is to use a big batch of store-bought citrus fruit. Use the procedure described below to measure the vitamin C content of citrus fruit stored for various lengths of time.
    3. You'll get better juice yield if you buy juicing oranges, not eating oranges.
  • Juicer for extracting juice from oranges (manual or electric is fine)
  • Cheesecloth
  • Vitamin C tablets, 250-mg
    • Optional: As an alternative, you could purchase pure ascorbic acid.
  • Distilled water
  • Transfer pipettes
  • 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 available from Amazon.com
  • Soluble starch
  • 50 mL graduated cylinder
  • 500 mL graduated cylinder
  • 50 mL Ehrlenmeyer flask
  • 50 mL buret
  • Ring stand
  • Buret clamp
  • 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. For more information on how to properly use a balance and different titration techniques see Chemistry Lab Techniques.

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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.

  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. (Note: if you purchased the iodine solution for starch test, you can skip this step.)
    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 oranges.
    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 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. Pick (or buy) 10–12 juice oranges. You will measure the vitamin C content of two oranges on the day of picking (day 1) and on days 2, 4, 8, and 14.
  8. Prepare fresh-squeezed orange juice samples.
    1. Use a juicer to squeeze orange juice from two 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.
  9. Titrating an orange juice sample is quite similar to titrating the vitamin C standard. Here are the steps:
    1. Use a clean 50 mL graduated cylinder to measure 20 mL of the fresh-squeezed juice.
    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 ringstand.
    5. Fill the buret nearly full with your iodine titration solution.
    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 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.
    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.
  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 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 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)/(8.5 mL) = 16.0 mg

  11. From your results, which oranges had the most vitamin C?

Troubleshooting

For troubleshooting tips, please read our FAQ: Do Oranges Lose or Gain Vitamin C After Being Picked?.

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Variations

  • You can extend the experiment to compare different storage methods for juice oranges. What effect does refrigerated storage have on vitamin C levels? Is refrigeration better at preserving vitamin C than room-temperature storage? Design an experiment to find out.
  • Measure the amount of vitamin C in other 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 an experiment on investigating the amount of vitamin C in orange juice from frozen concentrate compared to the amount of vitamin C in not-from-concentrate juice, see the Science Buddies project Which Orange Juice Has the Most Vitamin C?

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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: The 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 each day I make Vitamin C measurements from orange juice samples?
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 every day that you take measurements 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 do not make and titrate a fresh vitamin C standard solution each day you titrate orange juice samples, 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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