Experiment33‐1 Analysis for Vitamin C A. Introduction Vitamin C, known chemically as ascorbic acid, is an important component of a healthy diet. In the mid-eighteenth century the British navy found that the addition of citrus fruit to the sailors' diet prevented the malady called scurvy. Humans are one of the few mem bers of the animal kingdom unable to synthesize vitamin C, resulting in the need for regu lar ingestion in order to remain healthy. The National Academy of Sciences has established the threshold of 60 mg/day for adults as the Recommended Dietary Allowance (RDA). Linus Pauling, a chemist whose many contributions to chemical bonding theory should be well-known to you, recommended a level of 500 mg/day to help ward off the com mon cold. He had also suggested that large doses of vitamin C are helpful in preventing cancer. The vitamin C content of foods can easily be determined by oxidizing ascorbic acid, C 6 H 8 O 6 to dehydro-L-ascorbic acid, C 6 H 6 O 6 : O O +2H + +2e - O O O O OHOH C H C C H C H H OH OH OHH OHH Vitamin C Dehydro-L-ascorbic acid This reaction is very slow for ascorbic acid in the dry state, but occurs readily when in contact with moisture. A reagent that is particularly good for the oxidation is an aqueous solution of iodine, I 2 . Since I 2 is not very soluble in water, we dissolve it in a solution of potassium iodide, KI, in which the I2 exists mainly as I 3 - , a complex ion. The reaction with ascorbic acid involves I 2 , which is reduced to I - ion: )(2)(2 2 aqIaqIe −− ⎯→⎯+ In the overall reaction, one mole of ascorbic acid requires one mole of I 2 for complete oxidation. When the red-colored I 2 solution is added to the ascorbic solution, the characteristic iodine color disappears because of the above reaction. Experiment33‐2 Although we could use the first permanent appearance of the yellow color of dilute iodine to mark the end point of the titration, better results are obtained when starch is added as an indicator. Starch reacts with I 2 to form an intensely colored blue complex. In the titration I 2 reacts preferentially with ascorbic acid, and so its concentration remains very low until the ascorbic acid is all oxidized. At that point, the I 2 concentration begins to go up and the reaction with the indicator occurs: )()( 22 complexIstarchstarchaqI −⎯→⎯+ yellow blue Because an I 2 solution cannot be prepared accurately by direct weighing, it is necessary to standardize the I 2 against a reference substance of known purity. We will use pure ascorbic acid for this reference, or primary standard. After standardization you can use the iodine solution for the direct determination of vitamin C in any kind of sample. B. Procedure B-1. Standardization of the Iodine Solution Obtain from the storeroom a buret and-an unknown vitamin C sample. Weigh out accurately on the analytical balance three ascorbic acid samples of approximately 0.10 g into clean 250-InL Erlenmeyer flasks. Dissolve each sample in approximately 100 mL of water. Clean your buret thoroughly. Draw about 100 mL of the stock I 2 solution from the carboy in the laboratory into a 400-mL beaker and add approximately 150 mL of water. Stir thor oughly and cover with a piece of aluminum foil. Rinse the buret with a few milliliters of the I 2 solution three times. Drain and then fill the buret with the I 2 solution. After taking an initial reading of the buret (you may find looking toward a light source will make it easier to see the bottom of the meniscus), add 1 mL of starch indicator to the first ascorbic acid sample and titrate with the iodine solution. Note the change of the I 2 color as you swirl the flask gently and continuously during the titration. Continue the addition of the iodine, using progressively smaller volume increments, until the sample solution just turns a distinct blue. After reading the buret, titrate the other tvio sample solutions-being sure to add the starch indicator and to read your buret before and after each titration. B-2. Analysis of an Unknown Containing Vitamin C Given your experience with the standardization reaction, you should be able to devise an analogous procedure to determine the vitamin C content of your unknown sample. You will need to select a sample size, and you may need to carry out an initial treatment of the sample. In particular, if your instructor assigns you a fruit juice sample, it will be desirable to first f ilter the sample through cheese cloth, followed by rinsing of the filter with water. It may be helpful in choosing the sample sizes to calculate an iodine solution Experiment33‐3 parameter called the titer-the number of mg of ascorbic acid which reacts with 1 ml- of iodine solu tion. This number is easily found from the I 2 concentration and the mass relationship in the reaction. It is desirable to have the volume of I 2 for each titration be at least 15 mL. Using a small initial sample will give you an indication of how much to scale up for your final titrations. Report your results in per cent vitamin C, if a solid sample was used. For liquid samples, report mg of vitamin C per 100 mL. In each case, calculate the size sample required to give the RDA of vitamin C. Experiment33‐4 C. Data and Calculations C-1. Standardization of Iodine Solution Sample I II III Mass of Ascorbic Acid Sample (g) Moles of Ascorbic Acid Initial buret reading (mL) Final buret reading (mL) Volume of I 2 added (mL) Moles of I 2 consumed Molarity of I 2 (M) Titer of I 2 (MM Ascorbic Acid = 176 g/mole) Experiment33‐5 C-2. Unknown Sample Mass or volume of unknown Initial buret reading (mL) Final buret reading (mL) Moles of iodine added Moles of vitamin C in sample Mass of vitamin C in sample (mg) Percent vitamin C in solid sample Concentration of vitamin C in liquid sample Amount of sample which will furnish RDA Experiment33‐6 D. Analysis for Vitamin C Pre-Lab 1. Write a balanced equation for the reaction between I 2 and ascorbic acid. Identify the oxidizing agent and the reducing agent. 2. A solution of I2 was standardized with ascorbic acid. Using a 0.1000-g sample of pure ascorbic acid, 25.32 mL of I 2 were required to reach the starch end point. a. What is the molarity of the iodine solution? b. What is the titer of the iodine solution ( mg asc/mL) ? 3. A sample of fresh grapefruit juice was filtered and titrated with the above I 2 solution. A 1 00 mL sample of the juice took 9.85 mL of the iodine solution to reach the starch end point. a. What is the concentration of vitamin C in the juice in mg vitamin C/100 mL of juice ( mg/ 100 mL) ? b. What quantity of juice will provide the RDA amount of vitamin C (mL)? Experiment33‐7 Acknowledgements Masterton, W. L.; Slowinski, E. J.; Wayne, C. W. Chem. Principles in the Laboratory. 1997, 257-261. . large doses of vitamin C are helpful in preventing cancer. The vitamin C content of foods can easily be determined by oxidizing ascorbic acid, C 6 H 8 O 6 . see the bottom of the meniscus), add 1 mL of starch indicator to the first ascorbic acid sample and titrate with the iodine solution. Note the change of