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Iron is an essential mineral needed for life. Iron is found in foods as the
Fe 3+ or Fe2+ state . The Fe2+ is the form that is absorbed and used by the human body.
We will be analyzing the amount of iron found in some cereals and other foods by a technique called spectrophotometry. In this technique, we will use the fact that all substances will absorb light of one particular color, and reflect others. The amount of light absorbed is proportional to the amount of substance present in a sample.
A spectrophotometer is a device which can provide specific wavelengths of visible light that are passed through a sample. The amount of light that is passed through the sample will be measured and compared to what was sent into the sample. Thus, the absorbance of the sample is measured.
To determine the concentration of the desired substance, in this case iron (III), a calibration curve will be used. The calibration curve will be prepared by making up solutions of known iron concentration and measuring the absorbance of each.
Then absorbance as function of concentration will be plotted. Using the curve, a sample’s absorbance can be measured, and the corresponding concentration read off of the graph.
PROCEDURE:
You will weigh 3.00 grams of your (crushed) cereal sample into a crucible or evaporating dish. You need to know the exact mass of the sample. The sample will be strongly heated to drive off any water in the sample. The heating is continued until you burn the food down to a mineral ash. The ash you obtain should be gray-white in color. You need to run your samples in duplicate.
After allowing the sample to cool, you will carefully transfer the ash to a large test tube. Add 5 mL of 2.0 M HCl, using a little bit of the HCl to rinse out the evaporating dish, and combining that in the test tube with the ash.
Stopper the tube and shake vigorously for about 1 minute. Add 5 mL of distilled water. Both the water and the acid should be dispensed from the burets.
Place a filter paper into a funnel and filter the contents of the test tube into a clean beaker. Take a 5 mL sample of this filtrate and place it in another labeled test tube.
Finish all your food samples and get them to this point before proceeding.
PREPARATION OF THE CALIBRATION STANDARDS
Each of the standard solutions has been prepared to contain a specific amount of iron. Take a 5.0 mL sample of each standard and transfer it to its own labeled test tube. You will treat the standards just like the samples now. [Note: Instructions for the preparation of the iron standards is in the ChemCom teachers manual. They are prepared by serial dilution of a 5 g sample of FeCl3-6H20 which is diluted to 100 mL. This makes a 1% Fe3+ solution.]
Using a burette, add exactly 5 mL of potassium thiocyante solution, (KSCN) to the solution in each of the test tubes. Gently invert the test tubes.
A red color will develop in response to the amount of iron in the sample.
MEASUREMENT USING THE COMPUTER COLORIMETER
In order to determine [FeSCN2+]eq, you will use the colorimeter shown in Figure 1. The FeSCN2+ ion produces solutions with a red color. Because the red solutions absorb blue light very well, the blue LED setting on the colorimeter is used. The computer-interfaced colorimeter measures the amount of blue light absorbed by the colored solutions (absorbance, A). By measuring the absorbance of each standard solution, you can prepare the calibration curve.
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Figure 1
Prepare the computer for data collection. Connect the interface to the computer, turn on the interface, and then turn on the computer. Plug the Colorimeter DIN plug into Analog Channel A on the interface. Start ScienceWorkshop. In the Experiment Setup window, click and drag the analog sensor plug icon to Analog Channel A. Select "Colorimeter" from the list of sensors. Click OK to return to the Experiment Setup window. In the Experiment Setup window, click and drag the Digits display to the Colorimeter icon. Select "Absorbance" and click "Display". Also, click and drag the Table display to the Colorimeter icon. Again, select "Absorbance" and click "Display".
You are now ready to calibrate the colorimeter. Prepare a blank by filling a cuvette 3/4 full with distilled water. To correctly use a colorimeter cuvette, remember:
?• All cuvettes should be wiped clean and dry on the outside with a tissue.
?• Handle cuvettes only by the top edge of the ribbed sides.
?• All solutions should be free of bubbles.
• Always position the cuvette with its reference mark facing toward the white reference mark to the right of the cuvette slot on the colorimeter.
?In the Experiment Setup window, double-click the Colorimeter icon to open the Sensor Setup window. Place the blank cuvette in the cuvette slot of the colorimeter and close the lid. Turn the wavelength knob of the colorimeter to the 0% T position. In this position, the light source is turned off, so no light is received by the photocell. Click "Read" in the row for "Low Value". Type "0" in the % max box.
?For Reading 2, turn the wavelength knob of the colorimeter to the Blue LED position (470 nm). In this position, the colorimeter is calibrated to show 100% of the blue light being transmitted through the blank cuvette. Click "Read" in the row for "High Value". Type "100" in the % max box. Click OK to return to the Experiment Setup window.
You are now ready to collect absorbance data for the four equilibrium systems and the standard solution. Click "Record"
Empty the water from the cuvette and rinse it twice with ~1-mL portions of the Test Tube 1 solution. Then fill the cuvette 3/4 full with the solution from Test Tube 1. Wipe the outside of the cuvette with a tissue and then place the cuvette in the colorimeter. After closing the lid, wait for the absorbance value displayed in the Digits display to stabilize. Record all your data in the table.
?Discard the cuvette contents as directed by your teacher. Rinse the cuvette twice with the Test Tube 2 solution and fill the cuvette 3/4 full. Follow the same procedure to find and record the absorbance of this solution.
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| Standard #1 | |
| Standard #2 | |
| Standard #3 | |
| Food sample #1 | |
| Food sample #2 | |
| Food sample #3 | |
| Food sample #4 | |
PREPARE THE CALIBRATION CURVE:
Using all the techniques that you have learned, carefully prepare your calibration curve. Draw a smooth curve connecting the points.
Use the calibration curve to determine the amount of iron in the food sample.
DATA ANALYSIS and CONCLUSIONS:
Questions? Comments??
Patti Mason