Vitamin C (Ascorbic Acid) is a water-soluble vitamin important for maintaining a healthy organism. It is found naturally in citrus fruits and leafy green vegetables. Vitamin C may be used in soft drinks as an antioxidant for flavor ingredients, in meat-containing products for curing and pickling, in flour to improve baking quality, and in a wide variety of foods for vitamin C enrichment. Vitamin C is also found in stain removers, hair dye preparations, plastics, and photography chemicals. It is an antioxidant, appears to boost immune response, and is necessary for the formation and organization of collagen, the development of the walls of blood vessels, and tissue healing. Antioxidants help rid the body of free radicals, reducing the incidence of muscle damage, cataracts, and potentially cancerous cells. Vitamin C deficiency symptoms include bleeding gums and easy bruising or slow wound healing. Severe vitamin C deficiencies are rare today. However, before the widespread availability of vitamin C containing foods, scurvy (a severe deficiency) was quite common. Overdoses of Vitamin C are rare, but may contribute to the formation of kidney stones. Humans are one of the few animal species that cannot make their own vitamin C, utilizing beneficial bacteria in the gut. Today, people are most interested in vitamin C’s long term health benefits of reducing the risk of disease and increasing quality of life. The RDA, or Recommended Dietary Allowance, is the level of intake of essential nutrients considered to be adequate to meet known nutritional requirements for the majority of people. The Recommended Dietary Allowance of Vitamin C is 60mg. However, various scientists have suggested amounts as great as 300mg are needed to maintain a healthy lifestyle. Because Vitamin C is a water-soluble vitamin, the amount of the vitamin absorbed in a single day can vary based on the daily diet. In this experiment, you will determine the amount of Vitamin C (Ascorbic acid) in different fruit juices, by titration of the juice with a solution of iodine.

1. Obtain 150mL of each of the two assigned juice samples. From the labels provided, record the amount or percentage of vitamin C per serving in the juice, and the serving size. Filter the juice into a 250mL beaker. Measure about 1g of oxalic acid to the nearest 0.01g, and add to the juice. The oxalic acid stabilizes the ascorbic acid. Set aside the juice samples.
2. Remove the buret from the buret stand. Using a small beaker, pour about 5 mL of the iodine solution into the buret. Carefully tilt the buret and roll it between your fingers in order to rinse the entire length of the buret tube. Discard the wash solution in a waste container by allowing it to drain through the stopcock. Repeat this wash twice more. Fill the buret with the iodine solution and replace it on the buret stand. Place an empty beaker under the buret and open the stopcock to allow all of the air bubbles to escape from the tip of the buret. Adjust the level of the solution so that the bottom of the meniscus is at or slightly below the zero mark on the buret. Record the initial volume of the filled buret to two decimal places.

   Never hold a beaker with solution above eye level since spilling could cause you to get solution into your eyes.

   Record the initial volume of the filled buret to two decimal places.

    
3. Prepare a standard L-ascorbic acid solution by weighing 0.120 - 0.130g of L-ascorbic acid and dissolving it in 250mL of distilled water in a 250mL volumetric flask. 

This solution is used to "standardize" or determine the exact concentration of the iodine solution. Once the exact concentration of the iodine solution is known, it can be used to find the unknown concentration of vitamin C in the fruit juice. Calculate the molarity (moles/liter) of your standard solution and record the value in your notebook. See sample exercise 4.9.

3. For the first titration, pipette 50mL of the standard L-ascorbic acid solution into a clean 150mL beaker and add 15 drops of the starch iodine indicator.
4. Record the starting volume of the solution in the buret. Titrate your sample by adding the iodine solution to the ascorbic acid standard dropwise until the standard turns a blue-black color and remains that color. To reach the end point very accurately, you can use a technique called splitting drops.

Splitting Drops. Open the stopcock very slowly until a drop is suspended from the tip of the buret . Touch the side of the flask to the tip and wash the drop down into the solution with a stream of distilled water from your squeeze bottle. When splitting drops gives you a permanent color change (one that does not fade before 30 seconds), you have reached the endpoint. If you are not sure whether or not you have reached the endpoint, add another split drop.

Record the volume needed to reach the endpoint. 

5. Repeat the procedure for a total of three titration values. 

Record the volume needed to reach the endpoint for each titration.

These titrations are used to determine the exact concentration of the iodine solution. At the equivalence point when the solution turns color, the molar amounts of ascorbic acid and iodine are equal. The amount of Ascorbic acid can be determined by a redox titration with a standardized solution of iodine. The iodine is reduced by the Ascorbic acid to form iodide.

When all of the ascorbic acid is reduced, the indicator changes color. Use M₁V₁=M₂V₂ , where M₁= unknown molarity of iodine solution, V₁= volume of iodine solution from the buret, M₂= molarity of the ascorbic acid solution and V₂= volume of ascorbic acid determine the Molar concentration of the iodine solution in the buret. See sample exercises 4.14 and 4.15.

6. Pipette 50mL of your first filtered juice sample into a clean 250mL Erlenmeyer flask. Add 15 drops of starch iodine indicator. Add a stirring bar to the flask and place the flask on a magnetic stirrer. Position the flask beneath the buret. Make sure the buret is filled to the 30mL mark at a minimum, and record the exact volume of iodine solution in the buret.
7. Add the iodine solution to the flask slowly a drop at a time. Use the splitting drops technique used above to find the definite, permanent color change (to blue-black) in the solution.

Record the volume of iodine needed to complete the titration.

8. Repeat the procedure twice for your juice sample for a total of three titration values. Repeat the procedure for the second juice sample.

Record the initial and final volumes of iodine solution for each titration.

Use M₁V₁=M₂V₂ , where M₁= known molarity of iodine solution, V₁= volume of iodine solution from the buret, M₂= unknown molarity of the ascorbic acid solution and V₂= volume of ascorbic acid determine the Molar concentration of the vitamin C (ascorbic acid) in the juice. See sample exercises 4.14 and 4.15.

9. Dispose of your chemicals in the appropriate containers, as directed by your instructor.

1. Using M₁V₁=M₂V₂, and your results, determine the Molarity of the Vitamin C in your samples.
2. Calculate the number of milligrams of Vitamin C in the juice samples. (See sample exercise 4.9 in BLB text)
3. Compare and contrast the values for your two juices. Based on your results, calculate the amount of Vitamin C in the 150mL of juice, and for a serving size. How do these values compare to the recommended daily allowance of vitamin C for an individual? Does your juice sample meet the RDA value?