Teacher Guide Determining the Concentration of Iron in Vitamin Supplements Background Information Colorimetry is the science of measuring color. Colorimetry is a useful technique for determining the concentration of a colored chemical in a solution. Colorimeters are the instruments used to measure the color intensity by measuring how much light of a given wavelength is absorbed by a solution. This process is illustrated below because the colorimeter includes a light source and a detector. When a sample is placed in the colorimeter, light enters the cuvette and any light that is not absorbed is transmitted to the detector. Vernier Colorimeter light source sample cuvette detector Therefore, two important spectrochemical terms to address are transmittance and absorbance. The transmittance of a sample measures how much light is able to pass through it. Absorbance is how much light is absorbed by a sample. Since an intensely colored solution will have high absorbance and a low transmittance, these properties can be estimated using a visual method as well as colorimetric analysis. This visual method requires a diffuse light source, preferably a light box, to determine the pathlength for each sample. In this method, the standard is compared to the unknown. Two cylindrical paper sleeves are prepared and slipped over the large test tubes in order to exclude side lighting. With the paper sleeves in place, the standard test tube and the other test tube are compared, as you look lengthwise through the solution toward the light source. Small amounts of the standard can be added or removed from the comparison test tube until the color intensities appear the same. The depth of each solution is then measured from the bottom of each test tube. However, this lab is intended to address spectroscopic concepts using the Vernier probes for colorimetry to analyze vitamin supplements for iron content. Students will be engaged in this process by first completing an exercise related to dietary health and the utility of taking a vitamin supplement. After becoming familiar with the function of vitamin supplements in their everyday lives, students will be introduced to the Vernier probes for colorimetry by first collecting data for a calibration curve of absorbance versus concentration. The wavelength of maximum absorbance for the iron complex is 470 nm, so the Vernier colorimeter must be calibrated at this setting. In this lab, the teacher will prepare the master standard solution and provide students with instructions for diluting this standard. Then, Calibration Curve
students will use the Vernier probes to collect data points for creation of the calibration curve. Since Beer s Law tells us that there is a linear relationship between absorbance and concentration then a plot of absorbances versus the solution concentrations should result in a straight line. Experimental error may require a best-fit line since data points may not fall exactly on the line. Finally, students will use the calibration curve to identify the iron concentration in the unknown multivitamin supplement. A calibration curve can be used to find this unknown concentration graphically or algebraically. Algebraically, use the equation of the calibration curve (y = mx + b) and substitute the measured absorbance for y, and solve for x, which is the concentration. It has been found that the linear behavior of absorbance versus concentration will be reliable only for absorbance values less than or equal to 1. One of the challenges of analyzing samples from real life is that the appropriate dilutions must be performed in order to obtain suitable absorbances. When students analyze their samples, of the vitamin supplement as well as other iron-rich foods in the extension, they must first figure out what dilutions to make in order to obtain a sample with a useful absorbance. In researching this topic, it was found that liquid vitamin supplements must be diluted by about 1000 in order to create an acceptable sample. In this lab, it is necessary to create a colored iron complex. To do this phenanthroline is used as an indicator. In order to demonstrate the role of phenanthroline and the formation of the iron-phenanthroline complex it is useful to show students the effect of this indicator. First, show students separate solutions of Fe 2+ and phenanthroline, which are both colorless. Then, combine these solutions to show students the immediate formation of the vivid red complex. This enables students to visualize the importance of the phenanthroline indicator in this experiment. Motivation for this lab This lab is intended to incorporate everyday practices the ingesting of vitamin supplements into the classroom. In addition it allows for the integration of technology so that Vernier colorimeters can be utilized. This method can be used to test for iron in a range of samples, however liquid vitamins are the easiest to prepare for analysis. Students may bring in another sample, such as water samples, enriched cereals or other iron rich foods as an extension to this lab. Prerequisite Knowledge Electromagnetic spectrum Wavelengths of light How visible light interacts with solutions Concentration (M) Interpreting results of graphical analysis Laboratory safety and disposal of organic compounds Assessment This experimental task is presented to students as if they were scientific researchers for a vitamin company. They are trying to determine whether the amount of iron in the sample is adequate, but not excessive for the target audience. Students groups will use distinct samples intended for different populations and compare their experimental data to the recommendations for iron intake. At the end, students will turn in a Vee diagram to the vitamin company, with a cover letter indicating a summary of their findings and recommendations for their vitamin
distribution. Additionally, students will include a copy of their calibration curve and any other experimental data that was collected. Evaluation will be done based on thorough and accurate completion of the Vee diagram and a rubric that evaluates their cover letter to the vitamin company. Finally, a group oral presentation will be prepared as the groups report out to the class their recommendations to the vitamin distributors. Equipment Vernier colorimeters with cuvettes Computers or Vernier interfaces Test tubes four for each student group Materials Various liquid vitamin supplements can be purchased at a local drug store or online at http://www.nextag.com/liquid-multivitamins-with-iron/search-html (try the horse supplements to really make it interesting!) Phenanthroline can be purchased from a scientific distributor (i.e. Aldrich or Fischer Scientific). Ferrous ammonium sulfate may also be purchased from a scientific supplier Ethanol Standard solutions o Master standard can be prepared by dissolving 0.03 g of Fe(NH 4 ) 2 (SO 4 ) 2 in water in a 100-mL volumetric flask and filling the flask to the line with deionized water. In a 250-mL volumetric flask, 0.125 g of phenanthroline should be dissolved in a minimum amount of ethanol. By pipette, 25 ml of the iron solution can be added to the phenanthroline solution, and the solution should be mixed before filling the flask to the line with deionized water. Unknown solutions o The unknown solution can be distributed to the students and prepared so that it is closer to the concentration they will eventually need. This can be prepared by dissolving 0.25 g of phenanthroline in ethanol in a 250-mL volumetric and adding 2.5 ml of the liquid vitamin supplement. The solution can then be mixed well, and the flask filled to the line with deionized water. Students should expect to find by trial and error that they need to dilute this solution approximately ten-fold in order to obtain an absorbance in the desirable range. o Solutions tend to decompose overnight, so they should be made fresh daily. Time Requirement Two 80-minute blocks or four 40-minute class periods.
Student Guide Vitamin trouble, Stuart replied. She took vitamin D when she needed A. She took vitamin B when she was short of C, and her system became overloaded with riboflavin, thiamine hydrochloride, and pyridoxine, the need for which in human nutrition has not yet been established. From Stuart Little, by E.B. White (1945) Why? Vitamins are nutrients you must get from food because your body can t make them from scratch. Many of us take a multivitamin supplement even if we have a healthy diet to begin with. Intake of vitamins above the minimum daily requirement may prevent heart disease, cancer, osteoporosis, and other chronic diseases. Iron is one of the most abundant metals on Earth and is essential to normal human life. Iron is a part of many proteins and enzymes that help us to maintain good health it aids in oxygen transport and helps regulate cell growth and differentiation. Too little iron can cause anemia, which leads to fatigue, poor work performance and a weakened immune system. In contrast, excess amounts of iron can lead to toxicity and even death. As a high school student between the ages of 14 to 18 years old you should seek to intake 11 mg/day as a male and 15 mg/day as a female. Since iron can be toxic in excess and lead to illness when lacking, it is extremely important that multivitamin supplements contain appropriate concentrations of this vitamin. Learning Objectives (Students Will Be Able To) Utilize colorimetry and Vernier colorimeters Create and interpret the calibration curve in order to correctly describe data Determine the iron concentration in a vitamin supplement Report the information to the vitamin company in a concise memo, utilizing the Vee diagram
MODEL 1: Table 1: Adequate Intake for Iron for Infants (0 to 6 months) Age (months) Males and Females (mg/day) 0 to 6 0.27 Age Table 2: Recommended Dietary Allowances for Iron Males (mg/day) Females (mg/day) Pregnancy (mg/day) Lactation (mg/day) 7 to 12 months 11 11 N/A N/A 1 to 3 years 7 7 N/A N/A 4 to 8 years 10 10 N/A N/A 9 to 13 years 8 8 N/A N/A 14 to 18 years 11 15 27 10 19 to 50 years 8 18 27 9 51+ years 8 8 N/A N/A Iron is bound and transported in the body via transferrin and stored in ferritin molecules. Once iron is absorbed, there is no physiologic mechanism for excretion of excess iron from the body other than blood loss i.e., pregnancy, menstruation or other bleeding. Key Questions: 1. What type of person needs the most iron? 2. What type of person needs the least iron? 3. What happens to the daily iron allowances during pregnancy? 4. In what ways can iron be lost from the body? 5. How is iron transported throughout the body? 6. Based on the model of iron absorption and excretion, why is it possible to obtain an excess of iron in the body?
MODEL 2 VitaSmart Multivitamin Full Strength Nutrition Shake, Chocolate Key Questions 1. Which product contains more Vitamin A? Vitamin D? Vitamin E? 2. Which product contains more iron? How much more iron is present in this product? 3. Why do you think that some values for percent daily value are greater than 100%?
In this lab you will be determining the concentration of iron in a variety of multivitamin supplements. As you have seen from the previous exercises, iron is extremely important. Not only is a certain amount of iron important for our survival, but excess iron can also have detrimental effects. Therefore, it is extremely important that the concentration of iron in multivitamin supplements is accurate to its consumer. The vitamin company has created five different multivitamins. During the process a mix up occurred and the chief executive officer has commissioned your class to clear up the problem. Each multivitamin was created at the factory, but none of the samples were labeled to designate their appropriate end user. It is your task to determine the concentration of iron in the multivitamin and determine for whom this vitamin supplement would be appropriate for, according to the daily allowances. To do this you will be performing a laboratory analysis of vitamin samples. However, before an analysis of the sample itself can be done, a calibration curve must first be created. In your team you will be using the Vernier colorimeter to record absorbance for known concentrations of iron. Following this practice, a concentration versus absorbance graph will be created. From this graph you will be able to determine the concentration of iron in your unknown. The multivitamin consumers of America are counting on you, so go to it! Materials Four test tubes 10 ml pipette Cuvette Standard iron-complex solution (prepared by teacher) Vernier colorimeter Laptop computer Unknown multivitamin solution Vee diagram template Instructions 1. With a marker, label four test tubes 1-4. 2. You must now create the appropriate dilutions to make a calibration curve. The chart below shows the amounts of the master standard and water that are needed to make each solution. Using a graduated 10 ml pipette, transfer the required amount of deionized water to each test tube. 3. Between each dilution, rinse the pipette with some of the master standard and discard this rinse solution. 4. Use the pipette to add the required amount of master standard to each tube. 5. Swirl the test tubes to mix the solutions.
6. Calculate the concentrations of the four solutions you have prepared using the known concentration of the master standard and the volume of this solution that you added to each test tube. Fill these concentrations in on the chart. Standard Number Master Standard (ml) Water (ml) 1 2 8 2 5 5 3 7 3 4 10 0 Concentration (M) Colorimetric Analysis 1. Attach the Vernier colorimeter and LabPro to your computer. Plug in the LabPro. Open the LoggerPro software. 2. Fill a cuvette with deionized water and place it in the colorimeter. It is important to use the same cuvette for this calibration and for all measurements. Make sure that the cuvette is facing the same way each time you put it into the colorimeter. The smooth surfaces should be facing left and right and the rough surfaces facing toward and away from you. 3. Set the colorimeter to 470 nm, the wavelength of maximum absorbance, which is green light, and click the Calibrate button. 4. After you have finished the calibration, you will not need to change the position of the colorimeter knob for the rest of the experiment. 5. Empty the cuvette, and rinse it with a bit of your first standard solution. Begin with the least concentrated solution. Pour out this rinse solution and fill the cuvette with the solution. Return the cuvette to the colorimeter. 6. Click the Collect button on the menu bar at the top of the screen. Click Keep to record the first data point. A box will appear to prompt you to enter the concentration for the first standard. You should enter the concentration that you calculated for this solution. 7. Remove the cuvette from the colorimeter and rinse with deionized water and with your next solution. Fill the cuvette with the new solution, and return it to the colorimeter. 8. Repeat step 7 until you have collected data points for all four of your standards. 9. Click Stop to end the data collection. 10. On the menu bar at the top of the screen, click the button marked R. This will calculate the slope and intercept of the best-fit line through the data points you have just collected.
This is your calibration curve. Notice that it can be represented by the equation y = mx + b. Values for m and b are given in the box attached to the best-fit line. 11. Save your work, and print a copy of your data and calibration curve for your records. Colorimetric Analysis of the Unknown 1. Obtain the multivitamin supplement with an unknown concentration of iron. 2. Calibrate the colorimeter once more by placing deionized water into the cuvette, placing the cuvette into the colorimeter and clicking Calibrate on the colorimeter. 3. Now empty the cuvette. Fill the cuvette with the unknown solution. The unknown vitamin supplement has already been diluted by a factor of 100. Keep this in mind when performing calculations of the original concentration. 4. Place the cuvette inside the colorimeter and close it. 5. Click the Collect button on LoggerPro to see the data. Record the data here: 6. If the absorbance is greater than 1, the solution must be diluted appropriately, and the absorbance taken again. Just as you did dilutions for the standards, dilute the multivitamin solution. Repeat Steps 3 and 4, until an acceptable reading has been obtained. 7. Use your equation from your calibration curve to determine the concentration of the unknown. The equation is in the form y = mx + b, where the absorbance is y and the concentration is x. 8. Record your data on the chart in the front of the classroom. Then record the class data in the chart below. Unknown Number Concentration 9. Now that you have all of your data, begin to draw conclusions about the appropriateness of each vitamin supplement. Remember to review the data in the models about the daily intake of iron. These numbers can also be interpreted as mg/day or mg/ml.
Your Task Now that you have performed the analysis and collected class results you must report your information to the company using the Vee diagram. Be sure to fill it out with as much detail as you can so that the company is fully informed of your methods and your conclusions. Furthermore, your calibration curve should also be attached. After you complete your Vee diagram you must construct a cover letter to the company. In this cover letter you must address the following points: What method did you use to analyze their vitamin samples What data did you and the class collect How did you use this data and your calibration curve to determine the concentration of the original sample. What are your recommendations for the labeling of their unknown samples and how did you come to this conclusion The Vee diagram can be completed within your lab groups. However, each student must complete the letter independently. Address the letter to the Vitality Vitamin Agency. Your response will be graded on the completeness of your response and the accuracy of your solution.