1 Vitamin C Content of Fruit Juice Introduction Vitamin C Vitamins are organic compounds that have important biological functions. For instance, in humans they enable a variety of enzymes in the body to function. The human body cannot synthesize vitamin molecules, so they must be obtained in the diet. If a particular vitamin is lacking in the diet, a deficiency disease will result. Vitamin C, also called ascorbic acid (shown below), is a water-soluble vitamin that occurs naturally in many fruits and vegetables, such as citrus fruits, green peppers, tomatoes, and parsley. It is not stable to heat, so cooking fruits and vegetables destroys much of their vitamin C content. It is also easily oxidized (converted to a non-useful form) by certain compounds, such as oxygen in the air. Therefore, the vitamin C content in an orange will be reduced if the orange has been cut in half and left exposed to the air for a period of time. Although fruit juice is a good source of vitamin C, it is not the healthiest way to obtain the vitamin, since fruit juices are very high in sugar/simple carbohydrates. Juices contain either added sugar or sugars that occur naturally in the fruit and become highly concentrated in the juice. Eating a serving of fresh fruit is far healthier because the effects of the natural fruit sugars are moderated by the fiber in the fruit. Structure of Vitamin C: OH HO CH 2 CH O C C O H C C HO OH Vitamin C is essential for the action of an enzyme that is involved in collagen formation. (Collagen is a protein found in bone and connective tissue such as tendons and cartilage, as well as the cornea of the eye.) Vitamin C deficiency leads to a serious disease called scurvy, in which the body is unable to properly synthesize and repair collagen. The US government recommended daily amount of vitamin C necessary to prevent scurvy is 75 to 90 mg per day for an average adult. Interestingly, humans and other primates (chimps, gorillas, etc.) are among the few mammals that cannot synthesize vitamin C in their bodies. Almost all other mammals (cats and dogs, for instance) make their own vitamin C and do not require it in their diets. These mammals produce as much as 2000 mg to 10,000 mg of vitamin C per 150 pounds of body weight each day. This has led to the suggestion that humans actually need more vitamin C than the 75 to 90 mg a day recommended by the government. Vitamin C is an antioxidant which may reduce the risk of cancer and other diseases of aging, as well as alleviate symptoms of the common cold. Many people take supplementary vitamin C tablets for these purposes. Tablets typically contain 500 to 1000 mg of vitamin C. Vitamin C is generally regarded as safe, but there is some debate about whether or not large doses of vitamin C in tablet form are truly beneficial, or whether very large doses of the vitamin have any serious side effects.
2 Titration In this experiment you will determine the vitamin C content of a fruit juice sample using a method called a titration. A titration is an experimental procedure which allows you to determine the exact amount of one substance that will react with a specific amount of another substance. In this experiment, you will determine the exact amount of iodine that will react with all of the vitamin C in a 20 ml sample of fruit juice. You won t know how much vitamin C is contained in the 20 ml juice sample, but you will be able to calculate its amount based on the amount of iodine needed to react with it. In this experiment the titration will consist simply of adding an iodine (I 2 ) solution to the 20 ml juice sample as iodine is added to the sample, a reaction will occur between the iodine being added and the vitamin C in the juice sample. The iodine will be added to the juice slowly by delivering it from an instrument called a buret. The buret has volume markings so that it will be easy to determine the exact amount of iodine needed to react with the vitamin C in the juice. In this reaction, one mole of iodine reacts with one mole of vitamin C (1-to-1 ratio). See below: 1 vitamin C + 1 I 2 1 oxidized-vitamin C + 2 HI You will add iodine to the juice sample just until the amount of iodine added (in moles) is exactly equal to the amount of vitamin C (in moles) in the juice sample at this point there will be exactly enough iodine to just react with all of the vitamin C. The volume markings on the buret will enable you to easily determine the total amount of iodine that was needed for all the vitamin C to react. Since moles of iodine are equal to moles of vitamin C at this point, this amount of iodine added from the buret will be an indication of the amount of vitamin C (in moles) that must be present in the 20 ml juice sample. The point when the reaction is complete, when moles of iodine added are equal to moles of vitamin C, is called the endpoint of the titration. When the endpoint is reached, you will stop adding iodine since the reaction is complete and all the vitamin C has reacted. If you add extra iodine past the endpoint, there will be no vitamin C for it to react with (since all the vitamin C has already reacted with iodine and has been used up ). The problem is that the volume reading on the buret will reflect the total amount of iodine added you won t know how much of this total volume was needed to react with the vitamin C, and how much was extra. Since the amount of iodine added is meant to be used as an indication of the amount of vitamin C in the juice sample, an inaccurate total iodine volume will give an incorrect indication of the amount of vitamin C. One more detail of the titration procedure needs to be explained: How will you know when you have added an amount of iodine that is exactly equal to the amount of vitamin C (in moles)? In other words, how will you know when the endpoint of the titration has been reached and you should stop adding iodine? You will add a chemical called an indicator to the juice sample, and the indicator will produce a dark brown/black or dark blue/black color when the endpoint has been reached. When this color appears in the juice and lasts for about 10 seconds, the endpoint has been reached and you should stop adding iodine. The titration procedure is then complete, and the amount of vitamin C in the juice sample can be determined.
3 Buret The iodine solution used during the titration will be contained in a buret (pictured below). The iodine will be delivered out of the bottom of the buret by opening and closing the stopcock near the bottom. Volume markings along the entire length of the buret will enable you to determine how much iodine was delivered. Buret stopcock Iodine will be delivered by draining it from bottom of buret
4 Reading a Buret A buret is an instrument designed to deliver very precise volumes of a liquid reagent. Reading a buret is very much like reading a graduated cylinder in that you should take a volume reading from the lowest point of the meniscus the meniscus is the curved upper surface of the liquid in the glass cylinder. You should always estimate ONE more digit (between the lines) than you can read from the scale. The difference between a buret and a graduated cylinder is that 0.00 ml is at the TOP of the buret (see picture below at right) just the opposite of how a graduated cylinder is marked. Because of this, you can fill the buret completely (to the 0.00 ml mark at the top), deliver a certain volume of liquid out of the buret by opening the stopcock at the bottom, and then read the scale to determine what volume was delivered (drained out). In other words, a buret reading shows the volume that was delivered, rather than the volume that is contained in the buret (a graduated cylinder shows the volume contained in the cylinder). Therefore, you should read the scale on a buret in a downward direction. This is exactly the opposite of reading a graduated cylinder, in which case you read the scale in an upward direction. Take a look at the examples below before attempting to do the 3 buret questions on the prelab. Note that each reading has two digits after the decimal. 0.00 ml mark More examples of buret readings: 12.50 ml mark 25.00 ml mark
5 Vitamin C Content of Fruit Juice Prelab Name 1. Why must we obtain vitamins, such as vitamin C, in our diets? What is the scientific name for vitamin C? What role does vitamin C play in the body? 2. What is the one-word term for the experimental method you will use to determine how much vitamin C is contained in a fruit juice sample? What chemical will react with the vitamin C in the juice sample during this procedure? What is the name of the piece of laboratory glassware that you will use to measure the volume of the chemical you just named in the previous question? 3. What two quantities will be equal at the endpoint of the titration carried out in this experiment? How will you know when the endpoint has been reach (what will you see)? Prelab continued on next page
6 Vitamin C Content of Fruit Juice Prelab Name 4. Assume that the buret at the right was previously filled to the 0.00 ml mark. What volume of reagent has been delivered? (Remember to estimate between the lines to get one more digit than can be read from the scale.) 5. Write down a reading for each buret pictured below. 6. Assume that the buret below was previously filled to the 5.00 ml mark. What volume of reagent has been delivered?
7 Vitamin C Content of Fruit Juice Procedure Preparation of the Buret. 1. To ensure that your buret is clean and does not contain any chemical residue that will interfere with the titration, rinse it thoroughly with tap water. It can be awkward to try to rinse a buret at the sink, so you may want to ask the instructor for suggestions. Be sure to open the stopcock at some point to allow the rinse water to clean the tip of the buret as well. After thorough rinsing with tap water, rinse once with distilled water and allow all liquid to drain out of the buret. 2. Using a funnel, pour approximately 3 ml of 0.010 M iodine solution into the buret. With the stopcock closed, hold the buret in a nearly horizonatal position and roll the solution around to wet the entire inner surface of the buret. Then drain the iodine out of the buret by opening the stopcock. (This iodine can be discarded down the drain.) The purpose of this step was to use a little iodine to rinse out any remaining water which would otherwise dilute the 0.010 M iodine that will next be added to the buret. The iodine s exact concentration must be known to calculate the vitamin C content of the juice. 3. Clamp the buret to a ring stand on your lab bench using a special buret clamp. Be sure the stopcock is closed! Then use a funnel to add approximately 10 ml of 0.010 M iodine to the buret. 4. It is necessary to fill the lower tip of the buret with iodine in order to end up with accurate volume readings. To fill the tip with iodine, place a clean beaker under the tip of the buret to catch any liquid, and then open the stopcock all the way for just a second or two. After closing the stopcock again, carefully examine the tip of the buret to see if it is entirely filled with liquid or if there are some air bubbles trapped inside. If there are air bubbles present, open the stopcock full blast for a few seconds until the rapid flow of liquid causes the air bubbles to be pushed out. If you are not able to eliminate a stubborn air bubble, ask the instructor for help. Air bubbles in the tip will lead to inaccurate volume readings. You should begin the titration with about 10 ml of iodine solution in the buret to ensure that you have enough for the titration. If necessary, add a little more iodine to the buret so that it contains approximately 10 ml of solution. You are now ready to take a volume reading from the buret. Reading from the lowest part of the meniscus, take an initial volume reading and record it on the report sheet (#3 on report sheet). Remember to read and record the volume to two digits after the decimal. (For example: 10.16 ml)
8 Titration of the Vitamin C in a Fruit Juice Sample. 1. Clean your 250 ml flask you will carry out the titration in this flask. Ask the instructor to demonstrate the use of a volumetric pipette. Then use a 20.0 ml volumetric pipette to obtain 20.0 ml of juice and drain the juice directly into the 250 ml flask. Record the type of juice and the sample volume on the report sheet. Add 40 ml of distilled water to the juice in the flask. Then add 5 drops of 3.0 M HCl (hydrochloric acid) as a catalyst, and 10 drops of 2% starch solution to serve as the indicator. 2. Begin the titration by draining about 1 ml of iodine out of the buret into the flask containing the juice sample. Stop and swirl the flask vigorously to mix the solution. As you added iodine you may have seen the appearance of a dark brown/black or blue/black color (but not necessarily). However, after swirling the flask, the dark color should have quickly disappeared. IMPORTANT: Read this paragraph carefully before proceeding to add more idodine. In Step 3 you will continue to slowly add iodine in this manner while swirling the flask. As you add more and more iodine, the dark color may continue to appear temporarily and will remain somewhat longer as the flask is swirled. Eventually you will have added enough iodine that the dark color will appear and remain permanent, even after swirling the flask this is the endpoint of the titration, the point at which the moles of iodine added are equal to the moles of vitamin C contained in the juice. When the dark black color appears and remains for at least 10 seconds without fading away, you should stop the titration (the endpoint has been reached). It is important not to overshoot the endpoint. Therefore, you should add the iodine slowly, a few drops at a time, swirling the flask continuously during the process. 3. Complete the titration by slowly adding more iodine while swirling the flask vigorously. When you near the endpoint, the iodine should be added very slowly only a drop at a time so as not to go past the endpoint. When the endpoint has been reached, take a final volume reading from the buret and record it on the report sheet. Use the initial and final volume readings to calculate the total volume of iodine used in the titration; record this total volume on the report sheet. Discard the contents of the 250 ml flask down the drain and rinse the flask clean. 4. Prepare for the titration of a second juice sample by adding more iodine to the buret so that there are about 10 ml of solution in the buret. Take an initial volume reading and record it on the report sheet. Repeat steps 1 3 of the titration procedure for the second juice sample. 5. When both titrations are complete, discard any unused iodine solution from the buret down the drain and rinse the buret thoroughly with tap water followed by distilled water. Complete the calculations on the report sheet to determine the vitamin C content of the fruit juice.
9 Vitamin C Content of Fruit Juice Report Sheet Name Data First Titration Second Titration 1. Type of fruit juice 2. Volume of fruit juice sample ml ml 3. Initial volume reading of iodine in buret ml ml 4. Final volume reading of iodine in buret ml ml Calculations First Titration Second Titration 5. Total volume of iodine used ml ml 6. Mass of vitamin C in juice sample mg mg The iodine contained in 1 ml of the 0.010 M iodine solution will react with 1.76 mg of vitamin. In other words, every ml of iodine solution needed for the titration indicates the presence of 1.76 mg of vitamin C in the juice sample. Show work below: 7. Concentration of vitamin C in juice sample mg/ml mg/ml Mass of vitamin C in juice sample (in mg) volume of juice sample (in ml) Show work below: 8. Average concentration of vitamin C in the juice mg/ml
10 Vitamin C Content of Fruit Juice Postlab Name 1. What is meant by the endpoint of the titration? What chemical was the indicator? How did you know when you had reached the endpoint? (What did you see?) Why is it important not to add too much iodine and go past the endpoint? 2. Based on the average vitamin C content you determined for the fruit juice (#8), how many mls of juice would you have to drink each day to get the recommended 75 mg of vitamin C? Show your work. 3. Why is it important that the buret in the experiment be rinsed with a little of the 0.010 M iodine solution before actually filling it with iodine solution to be used during the titration? 4. On the structure of vitamin C below, circle two different functional groups and name each group. OH HO CH 2 CH H HO C C O C 5. If you leave an open container of fruit juice in the refrigerator, what might happen to the vitamin C content? C O OH