Name: ____________________________ Per: _____ Lab Group: ____ Performance: BIOCHEMISTRY LAB 3: QUANTITATIVE ANALYSIS TESTING FOR VITAMIN C BACKGROUND Quantitative analysis in a laboratory setting is when a laboratory technician runs a test to determine a specific amount of a particular substance. Blood tests are often carried out in this way in order to find the amount of blood cells, ions, vitamins, hormones and other parameters. The goal of this laboratory is to introduce you to the concept of quantitative analysis. To do this, we will be measuring the amount of vitamin C present in a sample taken from a commercial product. History of Vitamin C - Ascorbic Acid – Ascorbate Vitamin C or L-ascorbic acid is an essential nutrient for humans, in which it functions as a vitamin. Ascorbate (an ion of ascorbic acid) is required for a range of essential metabolic reactions in all animals and plants. Most animals can synthesize their own vitamin C. However, humans and other higher primates, guinea pigs, most or all bats, and some species of birds and fish lack an enzyme necessary for such synthesis and must obtain vitamin C through their diet. Deficiency in this vitamin causes the disease scurvy in humans. It is also widely used as a food additive. In living organisms, ascorbate is a reducing agent also called an anti-oxidant, since it protects the body against oxidative stress, and is a cofactor in several vital enzymatic reactions. Scurvy has been known since ancient times. People in many parts of the world assumed it was caused by a lack of fresh plant foods. The British Navy started giving sailors lime juice to prevent scurvy in 1795. Ascorbic acid was finally isolated in 1933 and synthesized in 1934. Scurvy is an avitaminosis (a disease caused by chronic or long-term vitamin deficiency) resulting from lack of vitamin C, since without this vitamin, the synthesized collagen is too unstable to perform its function. The uses and recommended daily intake (RDI) of vitamin C are matters of on-going debate, with RDI ranging from 45 to 95 mg/day. Proponents of megadosage propose from 200 mg/day to upwards of 2000 mg/day. A recent meta-analysis of 68 reliable antioxidant supplementation experiments, involving a total of 232,606 individuals, concluded that consuming additional ascorbate from supplements may not be as beneficial as thought. Scurvy leads to the formation of liver spots on the skin, spongy gums, and bleeding from all mucous membranes. The spots are most abundant on the thighs and legs, and a person with the ailment looks pale, feels depressed, and is partially immobilized. In advanced scurvy there are open, suppurating wounds and loss of teeth and, eventually, death. The human body can store only a certain amount of vitamin C, and so the body soon depletes itself if fresh supplies are not consumed. It has been shown that smokers who have diets poor in vitamin C are at a higher risk of lung-borne diseases than those smokers who have higher concentrations of vitamin C in the blood. Nobel prize winner Linus Pauling and Dr. G. C. Willis have asserted that chronic long term low blood levels of vitamin C or Chronic Scurvy is a cause of atherosclerosis. Western societies generally consume sufficient Vitamin C to prevent scurvy. In 2004 a Canadian Community health survey reported that Canadians of 19 years and above have intakes of vitamin C from food of, 133 mg/d for males and 120 mg/d for females, which is higher than the RDI recommendations. Name: ____________________________ Per: _____ Lab Group: ____ Performance: PRE- LABORATORY QUESTIONS (1). What is the purpose of a quantitative analysis? (2). Why can humans develop scurvy, while it is unknown in most animals? (3). The Rx of Vitamin C with iodine is a Redox Rx. Comparing the structure of Vitamin C with the oxidized Vitamin C, what indicates that an oxidation took place? (4). Similarly, how could you determine that the idodine has been reduced? (5). Why is it necessary for you to first standardize the iodine solution? (6). Why is it important to add the titrant slowly? (7). Define the following terms: Indicator, Analyte, Titrant, End-point. (8). Copy the image of the laboratory set up (page 3) for titration. Identify and locate where you would find the indicator, titrant, and analyte. (9). What is the scientific name for Vitamin C? Name: ____________________________ Per: _____ Lab Group: ____ Performance: Quantitating Vitamin C using TITRATION To quantitate the amount of vitamin C you will use a technique called titration. In a titration a reagent known as the titrant is slowly added, using a burette, to a measured volume of the analyte (the compound being analyzed). Once sufficient titrant has been added to completely react with the analyte the procedure is stopped. This point is known as the end point. A compound known as an indicator is added to the analyte to help determine the end point. When sufficient titrant has been added to completely react with the analyte - the indicator (usually) changes color to indicate that the end point has been reached. Once the end point has been reached it is possible to determine the exact amount of titrant that was added by reading the markings on the burette. Finally, the volume of titrant can be used, together with other parameters, to determine the amount of analyte that was present in the sample. It is important to add the titrant slowly so that an accurate measure of the volume needed to react with the analyte can be obtained. If too much titrant is added, exceeding the end point, then there will be an error in the amount of analyte calculated...and you will need to start the laboratory over from the beginning. In this experiment you will detect vitamin C by reacting it with elementary iodine (I2) using starch as the indicator. Vitamin C is a reducing agent and will react with elementary I 2 and reduce it to the iodide ion (I-). Once sufficient elementary I2 has been added (slowly via a burette) to react with all of the vitamin C in the sample, excess elementary I2 will interact with the starch and produce a deep blue-black color indicating the end point. The iodide ion does not interact with starch and does not produce a blue-black color. Using the volume of elementary I2 that was added to completely react with the vitamin C in the sample you will be able to calculate the amount of vitamin C in the sample if you knew how much vitamin C reacts with 1 mL of the iodine reagent. CAUTION!!! Adding the iodine too fast can lead to the problem of over-exceeding the end point which in return will result in the over estimation of the amount of vitamin C in the sample! 1. Standardization of your iodine solution: in the first part of the experiment you will perform a titration using a measured amount of a solution containing a known amount of vitamin C to calibrate the iodine reagent and to find out how much vitamin C reacts with 1 mL of the reagent. 2. In the second part you will perform another titration, but this time using a measured amount of a fruit juice containing a known amount of vitamin C and compare it to the label. 3. Finally you will re-measure the amount of vitamin C in the sample after subjecting it to heat, to investigate the sensitivity of vitamin C to heat. Name: ____________________________ Per: _____ Lab Group: ____ Performance: A. STANDARDIZATION OF THE IODINE SOLUTION Iodine solution change rapidly – I2 is a halogen gas that evaporates quickly – therefore iodine solutions need to be standardized all the time and that’s what you do here. You have an exact amount of Vit. C vs the ml of your current iodine solution needed to titrate it. Materials: iodine reagent, 1% starch, vitamin C sample, burette, 50 mL beaker, flask, 6M acetic acid A 1. Your instructor will weigh about 100 mg of vitamin C and dissolve it in 100 mL of DI water (distilled water). Copy the current concentration into your Report sheet. Transfer 1.0 mL of vitamin solution to a 50 mL beaker using an Eppendorf pipette. Add 3 drops of the starch indicator and 2 drops of 6 M acetic acid to the standard solution of vitamin C. Add 10 mL of DI water – use the cylinder -to increase the volume of the solution for titration. This will not affect your results. Calculate the number of milligrams (mg) of vitamin C inside the flask record mg of vitamin C in 1 mL of solution = mg of vitamin C (weighed out by instructor) 100 mL 1 mL A 2. Obtain a 2 mL graduated pipette set-up for the titration (burette). Using a clean, 20 mL beaker, obtain about 10 mL of iodine solution. Your instructor will demonstrate how to correctly use the burette. Caution: Keep iodine reagent away from clothes and skin. Rinse the pipette set-up with iodine solution prior to beginning the titration. Insert the pipette tip into the iodine solution and draw up about 2 ml of solution. Remove the beaker and continue to draw the solution up above the 0.0 mL mark, but not into the rubber tubing or syringe. Discard the rinse into the appropriate waste container, and repeat for a second rinse. Fill the pipette with iodine solution to just above the 0.0 mL volume line. Depress the plunger gently until the meniscus is at the 0.0 mL line or below. The pipette and its tip should be full of iodine solution, and free of bubbles. Record the initial pipette reading of iodine solution to the nearest 0.01 mL. Start the titration by adding the iodine solution into the vitamin C solution in the flask while stirring the contents (swirl the flask continuously while adding the iodine solution). The end point of the titration is a dark blue color that forms and persists for at least 30 seconds. At the end point, all of the vitamin C has been oxidized and the next drop of iodine solution is not reduced. Record the final reading of the iodine solution in the pipette. Calculate the volume of iodine solution used in the titration. Repeat the titration using a second 1.0 mL sample of vitamin C solution. Calculate the average volume of iodine solution used in the titration of vitamin C solutions in both trials. A 3. Calculate the mass (mg) of vitamin C that reacts with 1 mL of iodine solution. mg of vitamin C oxidized by 1 mL of iodine reagent = mg of vitamin C in flask Average volume (mL) of iodine reagent Name: ____________________________ Per: _____ Lab Group: ____ Performance: B. ANALYSIS OF VITAMIN C IN JUICES AND DRINKS Materials: iodine reagent, 1% starch, juice sample, burette, beral pipette, 20 mL beaker, 50 mL Erlenmeyer flask, 6M acetic acid. B 1. Measure 1 mL of juice into a 50 mL beaker. Add 3 drops of the starch indicator, 2 drops of 6 M acetic acid and 10 mL of DI water to the juice sample. B 2. Fill the pipette and record the initial reading of the level of iodine solution. Place the beaker with the juice and starch mixture under the pipette and begin adding iodine solution until the indicator just turns a dark blue. Record the final pipette reading for the level of iodine solution. B 3. Calculate the volume of iodine solution used to reach the endpoint of the titration. B 4. Calculate the mg of vitamin C in the 1 mL juice sample. Use the value of the mg of vitamin C per 1 mL of iodine solution obtained in step A.3. mg of vitamin C in 1 mL of juice = mg of vitamin C 1 mL of iodine reagent mL of iodine solution used B 5. Calculate the amount of vitamin C in one serving of the drink. On the nutrition label find how many ml equal one serving size: Use the following equation to calculate the amount of vitamin C in one serving of the drink. mg of vitamin C in 1 serving = mL in 1 serving mg vitamin C 1 mL of drink C. HEAT SENSITIVITY OF VITAMIN C Materials: iodine reagent, 1% starch, juice sample, burette, beral pipette, 20 mL beaker, 50 mL Erlenmeyer flask, hot plate, ice, 6M acetic acid. C 1. Pipette out 1 mL of the juice sample into your beaker. Add 12 mL of DI water. Boil the juice sample for 10 minutes with an hourglass covering the beaker to prevent evaporation. After 10 minutes, remove the beaker (careful! Hot!) and place it in an ice-water bath to cool. DO NOT FORGET!!! Add 2 drops of 6M Acetic acid and 3 drops of starch indicator to the cool solution. Do titration as above. Record the final level of iodine solution. C 2. Calculate the volume of iodine solution used in the titration. C 3. Calculate the mg of vitamin C present in 1 mL the heated juice sample. C 4. Using the value in B.4 of the mg of vitamin C in the juice sample, calculate the mgs of vitamin C that are destroyed in 1 mL after heating the juice. C 5. Calculate how much vitamin C has been destroyed during 10 min of boiling. mg destroyed (C.5.) x 100 = % destroyed mg original (B.4.) Name: ____________________________ Per: _____ Lab Group: ____ Performance: REPORT SHEET A. STANDARDIZATION OF THE IODINE SOLUTION A 1. Instructor’s concentration of vitamin C solution ___________ = ____________ mg/ml 100 ml How many mg of Vitamin C are in 1 mL solution? ______________ mg Trial 1 Trial 2 A 2. Initial pipette reading ______________ mL ______________ mL Final pipette reading ______________ mL ______________ mL Volume of iodine solution used ______________ mL ______________ mL Average volume of iodine solution used ______________ mL A 3. Calculating iodine standard: Look at formula for A 3. This measure is how many ml iodine react with how many mg Vitamin C in our specific situation - record that number here. You will use this for calculations in B and C! _______________ mg Vitamin C / 1 mL iodine solution B. ANALYSIS OF VITAMIN C IN JUICE DRINK B 1. Type of juice B 2. Initial pipette reading ______________ mL Final pipette reading ______________ mL Volume of iodine solution used ______________ mL B 3. _________________________________________ Think: How much iodine did you use compared to part A. ______________ mL What does that mean with regard to how much vitamin C in 1 ml of juice vs. 1 ml of vitamin C solution? Is there more or less vitamin C in the juice? ______________ Name: ____________________________ Per: _____ Lab Group: ____ Performance: B 4. mg Vitamin C in 1 mL juice sample (show your calculations) ______________ mg B 5. mg of vitamin C in one serving size (show your calculations) ______________ mg C. HEAT SENSITIVITY OF VITAMIN C C 1. Type of juice _____________________________________________ Initial pipette reading ______________ mL Final pipette reading ______________ mL C 2. Volume of iodine solution used ______________ mL C 3. mg Vitamin C in heated 1 mL juice sample (show your calculations) ______________ mg C 4. mg Vitamin C destroyed (show your calculations) ______________ mg C 5. Calculate how much % Vit C have been destroyed by 10 min of boiling (show math) ________% destroyed Name: ____________________________ Per: _____ Lab Group: ____ Performance: QUESTIONS (1). While performing one of the trials to determine the amount of vitamin C in the juice drink you realize that you forgot to add the starch to the flask containing the sample. Is there any point in continuing on with this trial? Is there any way to salvage this trial? Explain. (2). You calculated the amount of vitamin C in one serving of the juice drink (B.5.). How does this compare with the amount listed by the manufacturer? Is the amount of vitamin C you calculated more or less than the amount listed by the manufacturer? If the manufacturer claims it contains 100% Vitamin C it should contain 60 mg or more as this is the required daily dose by the WHO. (3). Based on your observations in part C what can you say about the sensitivity of vitamin C to heat? Calculate how much % is lost per minute exposure to 100 o Celsius. (4). Many food items including fresh vegetables are a good source of vitamin C. Based on your observations in part C, what precautions should you take in the preparation of food items containing vitamin C?