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The Effect of Temperature and Air Exposure on the Ascorbic Acid Content of Orange Juice

Researched by Annie M.


The purpose of this experiment was to determine how the ascorbic acid content of pure orange juice is affected by air exposure and temperature.

I became interested in this idea last year while doing an experiment in class that involved measuring the vitamin C content of different fruit juices.

The information gained from this experiment would be beneficial to anyone who is health conscious, and could help consumers to decide how to store orange juice, in order to keep the highest level of ascorbic acid possible.



My first hypothesis was that the container exposed to less air would contain a higher level of ascorbic acid than the containers with more air exposed. My second hypothesis is that the juices that are heated would  contain less ascorbic acid than the juices that are chilled. 

I based my hypothesis on information found on Microsoft Encarta 2001, which states "The vitamin is easily destroyed by cooking or canning foods, and by exposure to air and light."



The constants in this study were:

  • The same volume of juice
  • The same surface area of the juice (when applies)
  • The same amount of time that the juice is left out (6 hours)
  • The same material of the container the juice is in (glass)
  • The same temperature that the juice is at when tested (70*, when applies)
  • The same day that the juice tested

The manipulated variables were the amount of air that the juice was exposed to, and the temperature of the juice.

The responding variable was the ascorbic acid content of the juice.

To measure the responding variable, put one drop at a time of the juice into 10mL of indophenol, and record the number of juice drops that it takes to bring the indophenol through titration.



45mL Indophenol
2 cartons Tropicana Homestyle 100% Fresh Orange Juice
1 500 mL flask
1 Celsius thermometer
Erlenmeyer Flasks
1 Small metal saucepan
1 Graduated Cylinder
1 Safety Goggles 
  Tap Water



A Fill a test tube with 10mL of indophenol.
B With an eyedropper, obtain some of the juice that is being tested.
C One drop at a time, add the juice into the indophenol.
D When the titration is finished, the juice will have gone from a bright blue to a clear light brown. Record the number of drops of juice needed to titrate the indophenol on data table.
E Repeat these steps for all trials.

Surface Area
1. Gather together the glass pie plate, the orange juice and the 500mL flask.
2. Open the juice and fill the flask to the 500mL mark.
3. Fill the graduated cylinder with 50mL of the same juice.
4. Pour the 50mL of juice from the graduated cylinder into the flask.
5. Using a washable marker, make a clear mark at the top of the juice on the glass marking 550mL. 
6. Pour the orange juice in to the flask into the pie plate. 
7. Fill the flask up to the 550mL line with juice. 
8. Now that both containers have 550mL of juice, set them on a flat surface  and check the ascorbic acid content.
9. Let juice sit out for 6 hours, and check ascorbic acid content at three and six hours. After six hours, dispose of juice.

4 degrees C
10. Adjust the thermometer in the refrigerator to 4*C (40* F).
11. Gather together 3 Earlenmeyer flasks and the 100% pure orange juice.
12.Take a small sample of juice and follow procedures A-E above three times, recording data on data table. 
13. Fill the flasks to the 100mL mark with juice.
14. Set the flasks of juice in the refrigerator.
15. Wait for 6 hours, every three hours measuring vitamin C content of each flask once, for three trials, and recording on data table. (Steps A-E, above)
16. After the final measurement at 6 hours, dispose of juice.
49 degrees C
17. Fill a small metal pot approximately halfway with tap water.
18. Place pot on the stove top, and turn stove to the low.
19. While water is heating, take a sample of the untested orange juice.
20. Follow steps A-E above 3 times with this orange juice.
21. Fill three Erlenmeyer flasks with 100mL of orange juice.
22. When the water on the stove is at 49* C place the Erlenmeyer flasks with juice in them in water.
23. Wait for juice to reach 49* C.
24. Leave like this for 6 hours, checking temperature every 15 minutes. If juice is less than 49*, add hot water untill temperature rises, or turn heat up a little. If juice is warmer than 49*,  take out and place in cool pan of water for a few minutes. Then move them back to pot.
After final measurement at 6 hours, dispose of juice.
93 degrees C
25. Fill the small metal pot approximately halfway with tap water.
26. Place pot on stove top and turn stove to high. 
27. While water is heating, take a sample of the untested orange juice.
28. Follow steps A-E above 3 times with this orange juice. 
29. Fill three Earlenmeyer flasks with 100mL of orange juice.
30. When the water on the stove is at 93* C place Earlenmeyer flasks with juice in them in water.
31. Wait for juice to reach 93* C.
32.  Leave like this for 6 hours, checking temperature every 15 minutes. If juice is less than 93*, add hot water untill temperature rises, or turn heat up a little. If juice is warmer than 93*,  take out and place in cool pan of water for a few minutes. Then move them back to pot.
33. After final measurement at 6 hours, dispose of juice.



The original purpose of this experiment was to determine how the ascorbic acid content of pure orange juice is affected by air exposure and temperature.

The results of the surface area testing were as follows: During the first experiment, the juice with the surface area of 314 cm2 began with only 14.7 drops needed to titrate the indophenol. At three hours, It went up to 15.7, but after six hours, dropped down to 14. The juice with the surface area of 15 cm2 began the same as the large surface area at 14.7 drops, and lost a little ascorbic acid as it went up to 15.7. At the end, it lost more ascorbic acid, as it went up to 15.7 drops. On the second experiment, which tested the effect of temperature, all of the juice started out with 14 drops. After three hours, the 4* C juice went to 16 drops, and after six hours, went up to 16.3 drops. The 49* C went up to 16 drops at three hours, and then to 18 drops at six hours. 

View My Data and Graphs



My first hypothesis was that the container exposed to less air would contain a higher level of ascorbic acid than the containers with more air exposed. My second hypothesis was that the juices that were heated would contain less ascorbic acid than the juices that were chilled. 

The results indicate that the first hypothesis should be rejected, and the second hypothesis should be accepted.

Because of the results of this experiment, I wonder if the orange juices had been left in the heat, or exposed to air longer, if the ascorbic acid content of the juice would continue decrease rapidly.

If I were to conduct this project again, I would first of all change the number of trials that I did from three, to ten. I would also start out with more juice in the Erlenmeyer flasks when I heated them to be sure that it wouldn’t evaporate completely before the last measurement. I would also have a control group in both of the experiments, a room temperature test for temperature, and some juice with a lid on it for the air exposure test. Another change that I would make if I could redo this experiment would be to measure the ascorbic acid content in a different and more accurate way, instead of with indophenol, so that I could determine exactly how much ascorbic acid is actually in the juice.




One of the very important needs of humans is good health. In order to maintain good health, a person should supply himself with sufficient vitamin C. Vitamin C, or ascorbic acid, is one of the least toxic substances known to man, and acts as an antioxidant against the tissue damaging substances in our environment known as free radicals. Without a healthy supply of vitamin C, a person is in danger of diseases like scurvy. One good source of Vitamin C is in orange juice. Orange juice has a very high level of vitamin C, but the nutrients can be easily destroyed by cooking and canning, and also exposure to air and light. 

Vitamins and Nutrients In Oranges

An orange has many nutrients, which is why this fruit is one of the healthier fruit choices. The largest nutrient in the orange is vitamin C, or ascorbic acid, which prevents and cures scurvy. It is also important for the growth of bones, teeth, gums, ligaments, and blood vessels. Oranges also contain many other nutrients including Potassium, Folic acid, and Thiamin. The orange is considered one of the most healthful fruits.

Pests and Diseases

With a wide variety of pests and diseases among orange groves, growers use with pesticides, and various other prevention techniques. Scale insects suck sap from the fruit, leaves and twigs. Scale insects can be prevented with wasps, so some orange growers foster the growth of wasps. Other pests like Weevils and nematodes can attack the roots of the orange tree. Fungal diseases like melanose and greasy spot also extensively damage the fruit. Melanose produces raised bumps on the leaves, stem, and fruit. Greasy Spot causes oily blotches on the leaves and stem. Another virus disease called Tristeza can be deadly to fruit.

Types of Oranges

There are several types of oranges. One type is the Valencia. These are firm and heavy. They remain on the tree during warm weather, and the orange skin re-absorbs the Chlorophyll from the leaves. Another type of orange is the Blood Orange. These are also known as pigmented oranges. The blood orange has a dark reddish color, and are firm and heavy. Flavors of Blood oranges tend to have raspberry or Strawberry overtones, and are much less acidic than other types of oranges. A third main type of orange is the Navel orange. Navel oranges tend to also be firm and heavy, and are seedless.


Scurvy is a disease that may be deadly. It is caused by a lack of ascorbic acid, or vitamin C. It may take 3-12 months in adults before symptoms occur, and when they do occur, they may include: weight loss, lethargy, aching, and irritability. In babies and infants, the symptoms may include fever, loss of appetite, irritability, and failure to gain weight. The infant may be unable to move his limbs, because of the bleeding under the tissue that covers the bones called Periosteum.  The child may also become anemic. In infants, scurvy is called Barlow's disease. When a person has scurvy, their wounds may not heal properly, their mouth and gums may get sore, and their teeth become loose. Also, their blood vessels may become weak, and the person may have bone defects. When the disease begins to develop, bleeding will develop under the skin, and especially their nails. Treatment for scurvy includes large doses of Vitamin C until symptoms are gone, and a very balanced diet, including vitamin C. The cure for scurvy was found in 1753 by Scottish doctor, James Lind, proved that eating large amounts of oranges and lemons would cure and help prevent scurvy. People in a larger risk of vitamin C are smokers, and people living  in cities with high levels of carbon monoxide from traffic.

Vitamin C Healthwise

To grow and live efficiently, the human body needs correct doses of vitamin C, or ascorbic acid.  Vitamin C acts as an antioxidant against tissue damaging substances in our environment known as free radicals. It is vital for the growth of bones, teeth, ligaments, gums, and blood vessels. Different aged people should have different amounts of vitamin C. Infants should have 30-35mg a day, children up to age ten should have 40-45mg a day, people ages 11 and up should have 50-60mg a day, and pregnant women should have  about 70mg a day. The body needs more vitamin C after serious injuries, major surgeries, being exposed to extreme temperatures, or being burned. Vitamin C is an organic compound  of Carbon, Hydrogen, and Oxygen. Pure vitamin C is a white compound, made up from sugar and dextrose. Conflicting evidence shows that Vitamin C prevents colds vs. reducing severity of their syndromes. 

Orange Juice Processing

Orange juice can be processed in many ways. One way, is the following. The process begins when growers pick the oranges, which are then delivered to the plant in a truck. They are put onto conveyor belts, which carry them to the washing area, where the leaves, stems, and dirt are washed away. Damaged fruit is then removed, and then the fruit continues on the conveyor belt., and then is stored in large storage bins. Next, the Department of agriculture inspects the fruit to make sure that it meets state standards. Next, selected oranges are released to have a final washing and inspection. The oranges are automatically sorted by size and put into three extractor lines, which may squeeze 2000 oranges a minute, and 500,000 gallons of juice a day.  The extractors separate the juice, pulp, seed, and peel. These will then be processed into byproducts. Next, the juice is made into natural strength, not from concentrate juice. This is pasteurized, quickly heated, to destroy bacteria, and then rapidly chilled at 32 degrees Fahrenheit, and pumped into storage tanks. The rest of the juice is made into Frozen Juice concentrate. To do this, 85 % of the water must be removed. This is done with huge evaporation units that have the capacity to evaporate 20,000 gallons of water in an hour. Along with water, the heat included during this process releases essence and aromas that are later used for flavors and fragrance. The juice concentrate is now 1/6 it's original volume. After the evaporation process, the juice is chilled at 34 degrees Fahrenheit.  Next, the juice is pumped in tanks and blended to customer specifications. Until needed by a customer, the NFC juice and Frozen Concentrate wait in the refrigerated storage area. 

Other Products from Oranges

Besides juice, oranges can be made into many other products. One of these is cold pressed oils from the peel, which are used to flavor juices, beverages, and other foods. Another orange product are the essences and aromas, which come from the evaporation process, and are used primarily to flavor citrus beverages. A third product that comes from oranges is press liquor. When cold pressed liquids are extracted from the processed orange peels through a screw press, the result is Press Liquor. One other orange product is Citrus Molasses, which comes from the evaporation process of press liquor.


Oxidation is a process where a chemical reaction occurs, and an object loses electrons. The electrons released by oxidation must be captured by another substance. Oxidation is always accompanied by a reaction called reduction, which is when an object gains electrons. Electrons that are released in oxidation are immediately destroyed by sulfur atoms, resulting in the formation of sulfide ions. Sulfide ions combine with iron ions, to form the iron sulfide. Iron rusting is an example of the original meaning of oxidation. Iron combines with oxygen and moisture to form rust. Oxidation occurs in the human body when food molecules mix with oxygen to form carbon dioxide, water, energy, and vitamin C (ascorbic acid).


As you can see, oranges and vitamin C are very important. Vitamin C is vital for us to function efficiently, and to stay healthy. Without it, our teeth, gums, ligaments, blood vessels, and bones would not be healthy. Orange juice is a good source of vitamin C, and can help to prevent scurvy, a disease caused by lack of vitamin C. It is important for people to realize the importance of human health and how vitamin C contributes to maintaining good health.


"Ascorbic Acid." Microsoft Encarta 2001 ed. CD-ROM, Microsoft Corporation, 2001

"Blood Oranges" 12-5-01 Online at:

 Hapemen,  Cathleen J.  "Oxidation" The World Book Encyclopedia. 1999

 Navel Oranges" 12-5-01 Online at:

  Rogers, Quinton R.  "Scurvy"  The World Book Encyclopedia. 1991

"Scurvy"11-13-01 Online at:

"Southern Gardens Citrus" 10-19-01 Online at:

"Valencia Oranges" 12-5-01 Online at:

  Wardowski, Wilfred E. "Orange" The World Book Encyclopedia. 1999 


This project could not have been completed without the help and assistance of several people. I would like to thank each one for their help.
  •  Debbie Briesmeister at Tree Top, for answering questions, and providing me with information on measuring the ascorbic acid content of orange juice.
  • My parents, for purchasing many of the materials that I needed to conduct this experiment, and for helping me to create the display for my project.
  • My teacher Mr. Newkirk for helping me with my graphs and other parts of my project, and for making the indophenol that I needed to conduct this experiment.



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