EXP. 2 MICROSCALE DETERMINATION OF VITAMIN C
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1 University of Puget Sound Department of Chemistry Chem 230 EXP. 2 MICROSCALE DETERMINATION OF VITAMIN C LABORATORY OBJECTIVES AND ASSESSMENTS 1. Know what a titration is and how to use a titration to determine the amount of an analyte in a sample. a. Determine the amount of Vitamin C present in supplement tablets and a sample of fruit juice using a Rapid-Screening technique. b. Describe the titration process using the concepts of excess and limiting reagents, equivalence point and indicators. 2. Understand how to use statistics to describe the accuracy and precision of the titration method for determining Vitamin C. a. Use a spreadsheet to calculate relevant statistics for a data set. b. Calculate the standard deviation of each titration experiment. c. Use the Grubbs test to demonstrate and remove any outlying data points. d. Calculate the confidence interval of the Vitamin C tablet and compare to a true value using a Student s t-test. e. Identify sources of random error that contribute to the observed uncertainty 3. Understand how to standardize a solution. a. Describe and demonstrate how to prepare a Vitamin C standard solution using the proper analytical tools (analytical balance, weighing by difference, volumetric flask, etc.) b. Describe and demonstrate how to use the Vitamin C standard solution to standardize the titrant (I 3 - ) solution. 4. Understand how to use a standardized titrant solution for determination of an unknown. a. Describe and demonstrate how to use the standardized triiodide titrant (I 3 - ) solution to determine the amount of Vitamin C in known and unknown samples. INTRODUCTION Vitamin C, ascorbic acid, plays an essential role in the enzyme-catalyzed reactions that your body uses to produce strong connective tissue, including your skin. Humans and guinea pigs cannot make their own ascorbic acid; they have to get it in their diet. Ascorbic acid deficiency leads to the disease called scurvy. Among the symptoms of scurvy are weakness, skin lesions, and slow wound healing. All these effects are a result of weak connective tissue. To maintain good health, the current recommended daily allowance (U.S. RDA) of vitamin C is 75mg for adult women and 90 mg for adult men. Foods like fruits and vegetables with a high water content often contain large amounts of vitamin C. It is very soluble in water. If high doses of vitamin C are ingested, much of it is rapidly excreted in urine. Ascorbic acid (C 6 H 8 O 6 ) is a reducing agent that is readily oxidized to dehydroascorbic acid (C 6 H 8 O 7 ). See Harris (pg 367) for structures. C 6 H 8 O 6 + H 2 O + I 2 C 6 H 8 O 7 + 2H + + 2I - (1) Ascorbic acid reacts with a number of oxidants and several of these oxidation reduction reactions are the basis for methods to determine the ascorbic acid content of samples. The oxidizing agent you will use is iodine (I 2 ). In iodide containing solutions, c230 Exp. 2-Microscale Determination of Vitamin C 1
2 iodine (I 2 ) is in equilibrium with the triiodide ion (I 3 - ). (We will use the terms iodine and triiodide interchangeably.) I 2 + I - I 3 - (2) Since the oxidized form I 3 - is colored and the reduced form is colorless it is well suited for a titration method for determining ascorbic acid. The color can be amplified by adding starch to the solution because triiodide forms an intensely colored blue-black complex with starch that is easy to see. I Starch Starch I 3 - complex (3) (Blue-black color) The titrations in this lab will be rather different from future titrations. Instead of dispensing your titrant (I 3 - ) from a buret, you will be counting drops from a micropipet. How accurate is this method? To answer, you'll have to use statistical ideas like confidence intervals and the student s t-tests. The second difference in this titration lab has to do with how you standardize the I 3 - titrant. Instead of finding its molarity, you'll measure the number of drops you need to react with a known mass of ascorbic acid, frequently called a titer. The mass of ascorbic acid titrated by a drop of I 3 - is called the ascorbic acid titer, C(mg VitC /drop I 3 - ) about which we will have more to say in the Calculations section below. EXPERIMENTAL PROCEDURE RECORD ALL DATA IN YOUR LABORATORY NOTEBOOK. Please read Harris, pp (4-1 and 4-3) on some basic statistics before coming to lab. YOU WILL WORK WITH A PARTNER TO MAKE THE SOLUTIONS BUT WORK INDIVIDUALLY ON TITRATIONS 1. Preparation of the ascorbic acid Standard Solution (Work with a partner) a. Dissolve ~100 mg (weigh by difference using the analytical balance) in a clean 250-mL volumetric flask. Record mass in notebook. Fill the flask about half full with DI water. Shake and swirl the flask until the solid is dissolved. b. Fill to the line with DI water. After thorough mixing, transfer to a labeled flask and set aside while you go on to the next procedure. 2. Preparation of the Vitamin C Tablet Solution (Record label information.) (Work with a partner) a. Weigh a vitamin C tablet and record the weight to at least the nearest milligram in notebook. Record the amount of ascorbic acid on the manufacturer s label and the lot number of the Vit. C bottle in notebook. b. Place the tablet in a clean 500-mL volumetric flask. Fill the flask about half full with DI water, shake and swirl until the tablet is broken down. The tablet may leave a cloudy solution due to fillers and binders used in the manufacture of the tablet. c. Fill to the line with DI water and mix thoroughly. d. Transfer the solution to a clean, dry flask, label and set it aside while you go on to the next procedure. c230 Exp. 2-Microscale Determination of Vitamin C 2
3 3. Preparation of Juice Sample (Record label information.) (Work with a partner) Record the serving size and stated amount of Vit. C per serving from the label in notebook. If filtering is needed use filter paper and a funnel to obtain ~5-10 ml of filtrate. FOR THE REMAINING TITRATIONS-WORK INDIVIDUALLY BUT USE THE SOLUTIONS PREPARED WITH YOUR PARTNER. CALIBRATION OF MEASURING-PIPET 4. Calibration of Measuring-pipet (the graduated-stem plastic pipet) Note: It is essential that you always hold your pipets in a reproducible VERTICAL position at all times. a. Fill a 250-mL beaker with DI water to stabilize to room temperature. Before calibrating your 1-mL Measuring-pipet, practice drawing up 1.00 ml and without any loss, deliver the ml to 50-mL flask. b. Record the temperature of DI water that has stabilized to a constant temperature. c. Measure and record the mass (±0.001g) of the 50-mL flask with a stopper. d. At your workstation, draw up and transfer 1.00 ml of DI water into the flask. Stopper, measure and record the mass in the table in your laboratory notebook. e. Repeat the procedure to obtain five trials. f. Enter the data into your Calibration section of your spreadsheet. (See Figure 1.) TITRATION OF VITAMIN C STANDARD 5. Standardization of the Titrant Solution I 3 - (e.g., Determination of Ascorbic Acid Titer) a. Obtain a few milliliters of the triiodide titrant in a clean, dry 13 x 100-mm test tube. Don t store the triiodide titrant in plastic or keep it in a plastic pipet for longer than required for a titration. The iodine is adsorbed by plastic and this will change its concentration. WARNING: Solutions containing iodine will stain your skin and clothing. The stains are harmless, except to your pocketbook, if you ruin some article of clothing. Handle with care. If applied in time, a rinse with sodium thiosulfate solution can sometimes prevent staining. b. Use your calibrated Measuring-pipet to put 1.00 ml of the standard ascorbic acid solution in a clean well of your 12-well plate. c. From a plain plastic pipet, add one drop of starch solution. d. Using a Titrating-pipet, titrate the ascorbic acid by adding dropwise the triiodide titrant solution, stirring constantly with a toothpick. Record the number of drops required to turn the colorless solution blue. e. Repeat the standardization titration until you have ten (10) determinations. f. Try one additional titration without the starch indicator to the first permanent color. How do the titrations compare? c230 Exp. 2-Microscale Determination of Vitamin C 3
4 TITRATION OF VITAMIN C IN TABLET 6. Determination of Ascorbic Acid in Vitamin C Tablet a. Use your calibrated Measuring-pipet to put 1.00 ml of the solution made with the Vitamin C tablet into a well of your 12-well plate. b. Add one drop of starch solution. c. Using your Titrating-pipet, titrate the ascorbic acid by adding the triiodide titrant solution dropwise, stirring with a toothpick as before. Record the number of drops required to turn the solution blue. d. Repeat until you have ten titrations. TITRATION OF VITAMIN C IN SERVING OF JUICE 6. Determination of Ascorbic Acid in Juice a. For these sample determinations, use your calibrated Measuring-pipet to put 1.00 ml of your clarified juice in a clean well of your 12-well plate. b. Add one drop of starch solution. c. Titrate the juice sample by adding dropwise the triiodide titrant solution (stirring with a toothpick) and record the number of drops required to turn the solution blue. NOTE: The number of drops of I 3 - solution should be between 35 and 45. If it is not, you may adjust the volume of your juice sample to get the titration in this range. d. Repeat until you have ten titrations. CALCULATIONS 1. Transfer the data for the experimental results from the tables in your notebook to the Excel spreadsheet(s) you created. See Figure 1 for example of the layout and formatting of Sheet 1 of the spreadsheet, the Calibration of the Measuring-pipet and the Titration of the Vitamin C Standard. See Figure 2 for example of the layout and formatting of Sheet 2 of the spreadsheet for the Determination of Vitamin C in Tablet and Determination of Vitamin C in a Serving of Juice. 2. Calibration of Measuring-pipet: From your spreadsheet calculations determine the mean value of the volume delivered and the standard deviation for the ml Measuring-pipet. Use this value in all of your calculations. 3. Standardization: For this calculation and the following ones, we employ the following symbols. M(mg) = mass of ascorbic acid in a well plate (mg) N = number of drops of I 3 - used in a titration C(mg/drop) = ascorbic acid titer, mass of ascorbic acid titrated by each drop of I 3 -. For example, for the standardization step (5), the mass of ascorbic acid in the 1.00-mL samples from the Measuring-pipet you used for standardization will be called M std (mg). Using the True volume from the calibration of the Measuringpipet we can calculate M std (mg) of ascorbic acid, in mg, M std (mg) = Mean true V x (mg Asc. Acid/mL of Std Asc. Acid solution) (4) c230 Exp. 2-Microscale Determination of Vitamin C 4
5 For each titration, divide this by the number of drops of I 3 -, triiodide titrant, N std giving the ascorbic acid titer, c aa (mg/drop) for each titration, C(mg/drop) = M std (mg)/n std (5) Enter formula in your spreadsheet (Figure 1) to calculate C(mg/drop) for Ascorbic acid samples. Figure 1. Example of partial spreadsheet for the Calibration of the 1.00-mL Measuring-pipet and the spreadsheet for the Standardization of your ascorbic acid solution for the Vitamin C experiment. These will be on Sheet 1 of your Excel document. 4. Grubbs-test: Look carefully at the data from each of the titrations to see whether any titration result looks like an outlier. Run a Grubbs test for all trials with respect to C(mg/drop) with the spreadsheet you created following the steps below. Show one Grubbs test sample calculation in your lab notebook. Compare your results with the values in Table 4-4 in Harris (pp 92) to decide if you should keep or reject the data. If a data point was rejected drag the contents of that cell in the G column over to the H column. This removes the outlier from the calculation but does not delete it all together in case you need to consider it later. Q.1. STANDARDIZATION-Determination of C(mg/drop): Calculate the mean C(mg/drop), standard deviation, and 95% confidence interval of the mean using Excel functions and cell formula calculations. Report the confidence interval in two ways: as mean ± number with matching decimal places, and as an interval (number to number). 5. DETERMINATION of mg Vitamin C in Tablet: The number of drops used in titrating the tablet solution, N tab, will allow you to calculate the mass of ascorbic acid in each well plate using the ascorbic acid titer, C(mg/drop). m tab (mg)= n tab x C(mg/drop) (6) c230 Exp. 2-Microscale Determination of Vitamin C 5
6 Again C(mg/drop) is the mean value of ascorbic acid titer you obtained previously. The number of drops for these titrations, N tab, multiplied by C(mg/drop) equals the mass of Asc. Acid in each ml titrated, m tab (mg). The values of m tab (mg) can be used to determine the mass of ascorbic acid in the entire tablet. To find this value, multiply m tab (mg) by the ratio of the volume of the volumetric flask to the well plate volume (eq. 7). Enter the appropriate formula in the M entire tab column to calculate this value. M entire tab (mg/tablet) = m tab (mg) x (500.0mL/Volume Measuring-pipet ) (7) Determine the mass of ascorbic acid in the Vitamin C tablet for each trial and calculate the mean value, etc. (Q.2.) Figure 2. Partial layout and formatting for Sheet 2, Determining Vitamin C in a Tablet and Determination of Vitamin C in a Serving of Juice. Q.2. Vitamin C Tablet: Calculate the mean ascorbic acid (mg) in your vitamin C tablet, standard deviation, and 95% confidence interval of the mean. Report the confidence interval two ways as before. Compare, statistically using the student t- test, with the amount listed on the label. Is there a statistically significant difference between your results and the amount listed on the bottle? Based on your results, what percent by mass of the tablet is actually vitamin C? 6. Juice: The number of drops used in titrating each juice solution, N juice, will allow you to calculate the mass of ascorbic acid in each well plate using m juice (mg)= N juice x C(mg/drop) (8) c230 Exp. 2-Microscale Determination of Vitamin C 6
7 Find m juice for each titration of the juice. Then for each juice trial, use the volume of the sample, volume of a serving and m juice to calculate the amount(mg) of ascorbic acid in a serving of your juice. Calculate the mean value, etc. (Q.3.) M(mg/juice serving) = m tab (mg) x (serving size/volume juice titrated) (9) Similar to the manner used for the tablet determination set up your spreadsheet to record your data and calculate the results for a serving of your juice. Q.3. Juice: Calculate the mean concentration (mg/ml) of ascorbic acid in your juice sample, standard deviation and 95% confidence interval of the mean. Report the confidence interval two ways as before. Based on your results, how much of your juice do you need to consume to have the RDA of Vitamin C? Based on the label, how much of your juice do you need to consume to have the RDA of Vitamin C? Compare these amounts (first get them in matching units!). Did the juice have the stated amount of Vit. C per serving? ADDITIONAL QUESTIONS Q.4. Calculate the mean amount of I 2 per drop of triiodide reagent, in mmol and mg. Q.5. List the three major sources of random error in this experiment? Q.6. An error of one drop in fifty drops results in what percent relative uncertainty? What was your relative uncertainty ( Std.Dev. 100% ) from the Standardization of the Ascorbic Acid? Mean c230 Exp. 2-Microscale Determination of Vitamin C 7
8 WHAT TO DO To Be Turned in From Exp. 1-Statistical Analysis of Volumetric Tools of the Laboratory: Please hand in the following items in a packet STAPLED and in the following order: a) Printout of Plot for Calibration of Buret, b) Page(s) of Questions (Copy pages from notebook OK), c) Copy page(s) from laboratory notebook of the completed table of volume and mass data. d) Stapled at the end-any remaining copy pages. Be Sure To Put Your Name on Every Page. For Experiment 2 Before Lab: Read this experiment and Harris and prepare your notebook as follows You must also include the following information: Completed title bar. Reagent Table containing ascorbic acid and iodine. See Laboratory Guideline #IV. GUIDELINES FOR MAKING TABLES INCLUDING A TABLE OF REAGENTS. BE SURE TO NOTE THE DISPOSAL PROCEDURES IN YOUR TABLE. Write a summary of the procedure in your notebook. Include a balanced equation for the overall titration reaction. Prepare a table in your notebook to record data in the laboratory divided into four sections. One section with the raw data for the calibration of the 1.00-mL Measuring-pipet. (masses, volumes, etc. for each trial.) One section with the raw data for the ascorbic acid standardization (masses, volumes and number of drops for each trial.) One section with the raw data for the tablet. One section with the raw data for the juice. LEAVE EXTRA SPACE IN YOUR NOTEBOOK TABLES WHEN PREPARING! Following the example given in Figures 1 and 2 generate an Excel spreadsheet file. Submit a printed copy of this spreadsheet as prelab. You should bring a copy on a flash drive or yourself an electronic copy to use during lab. Bring your copy of Harris to lab and your computer. Do not bring your calculator. With your lab partner bring a labeled juice sample with the nutritional information label. The TA will check the copies of these pages from your notebook at the beginning of the lab period. During the Lab: Carry out the procedure as written. Record all data as you collect in your notebook. Modify your procedure if you find it necessary, but be sure to record the modifications. Record all other pertinent observations and measurements as you do the experiment. Show sample calculations in your laboratory notebook. If time permits complete the spreadsheet with your data in lab. c230 Exp. 2-Microscale Determination of Vitamin C 8
9 To Be Turned In: NOTE: Specific pages and order of assignment to be turned in will be state on the Moodle page for the next experiment. Complete your calculations in your spreadsheet. Your report will consist of: Answers to the Questions. Summary table in notebook showing results (including comparison with label info for 3 and 4) from 1) Calibration, 2) Asc. Acid Standardization, 3) Vit. C in tablet and 4) Vit. C in juice sample. Completed spreadsheet with your results sent to your TA as an attachment. Completed spreadsheet printout. Copies of the relevant tables and pages from your notebook. c230 Exp. 2-Microscale Determination of Vitamin C 9
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