THE SPECTROPHOTOMETRIC DETERMINATION OF IRON IN AN UNKNOWN IRON SAMPLE Overview Backround Prep: A series of dilutions of a standard iron solution are prepared. A colored iron complex is formed in each of these dilute iron solutions with 1,10-phenanthroline. Hydroxylamine (as the hydrochloride salt to increase solubility) is added to reduce any Fe3+ to Fe2+ and to maintain it in that state. Experimental Prep: To make a calibration curve, the light transmittance of each of the dilute iron solutions is read on a spectrophotometer and converted to absorbance. A graph is drawn; a linear regression analysis of the data gives the equation for the linear correlation between light absorbance and concentration of the diluted solutions. Unknown Prep: A solution of an unknown iron sample is prepared and its transmittance is determined on the specrophotometer. The concentration of iron in the unknown solution is determined by using the absorbance of the unknown sample and applying the linear regression analysis equation. Spectrophotometer A spectrophotometer (spectrometer) is an instrument designed to accurately measure the absorbance of monochromatic light by a substance placed in the beam (cell): → → → Light source ⎯⎯ Monochronometer ⎯⎯ Cell ⎯⎯ Detector ⎯⎯ Meter → → Incident Beam ( I o ) ⎯⎯ Absorbed Beam (I) The percent transmission, T, of the beam through the sample in the cell is defined as: I %T = ⋅ 100 Io Percent transmission is a linear function on the output meter of the spectrometer, and is therefore the preferred scale. The absorbance of the incident beam is a logarithmic function: A = log ( I o I ) = log (100 %T ) As you will note from the picture above, absorbance can also be read from the output meter of the spectrometer, but the numbers are logarithmically spaced and become very difficult to read for large values of A (see left side of dial; compare the top scale %T with the bottom A)). In this experiment you will read percent transmittance and calculate absorbance. Lambert -Beer Law This is not a "law" in the classical sense, but an expression of the relationship observed between the absorbancy of a solution and its concentration. It states that the absorbance of a solution (A) is proportional to its concentration (c), the path length (b) through the solution, and a constant (a or ε ) called the extinction coefficient. A = log ( I o I ) = abc = ε bc = kc Notice that as the concentration gets larger and larger, less and less light will pass through the solution, until the concentration gets to a point where no light will pass through and the "law" fails. It is always necessary to check whether Beer's law is obeyed and the plot of absorbance vs. concentration yields a straight line over the range of concentrations being studied before an analysis of an unknown sample can be made. Since neither cell path length b nor the extinction coefficient ε are changing over the course of the experiment, we can replace them with a constant k, which will be determined from the slope of your linear regression data for the calibration curve. PROCEDURE Calibration Curve (work in groups of five for this part only) The preparation of these standard dilutions and ultimately your unknown require use of volumetric glassware; your instructor should discuss the use of these. 1. Pipette 1.00 mL of standard iron solution into a 50.00 mL volumetric flask (the standard iron solution has 0.0500 milligrams of iron per milliliter). 2. Add 1.00 mL of 1.00 M ammonium acetate solution. 3. Add 1.00 mL of 10% (wt./vol.) hydroxylamine hydrochloride solution. 4. Add 10.00 mL of 0.30 % (wt./vol.) o-phenanthroline solution. 5. Dilute (make up to volume) to exactly 50.00 mL using DI water. This completes the preparation of your first concentration of diluted solution. At this point, you may pour the solution into any clean, DRY, labeled container to free the volumetric flask for preparing the next concentration of diluted solution. 6. Repeat steps one through five above, using 2.00 mL of standard iron solution. 7. Repeat steps one through five above, using 3.00 mL of standard iron solution. 8. Repeat steps one through five above, using 4.00 mL of standard iron solution. 9. Repeat steps one through five above, using 5.00 mL of standard iron solution. 10. Prepare a blank solution containing all the chemicals EXCEPT THE STANDARD IRON SOLUTION. The Use of Spectronic 20 (Spec 20) 1. Turn on the instrument and allow it to warm up for about twenty minutes. 2. Rotate the wavelength selector to 510 nm. Approach the wavelength from the low end of the scale and stop at 510 nm. Don't search back and forth for the exact setting. The one-way trip to the correct wavelength will eliminate backlash in the drive mechanism, in case it is necessary to recover the exact setting later. 3. With the door of the sample holder closed, adjust the "Zero Adjust" knob to bring the needle to zero on the percent transmission scale. 4. Fill a cuvette about half or two-thirds full with the blank solution, wipe it clean, and insert into the sample holder. Close the sample door and adjust the "Light Control" knob until the meter reads 100% transmittance. 5. Replace the sample blank with the first of your diluted standard iron solutions and record its transmittance. Repeat with the rest of your series of diluted standard iron solutions. Recheck the zero and 100% transmittance setting, using procedures 3 and 4 above, to be certain no "drift" has occurred. If it has, redo the calibration solutions. 6. Record the spectrometer number on your report sheet, as this same spectrometer must be used for your unknown samples. Determination of the Percent Iron in an Unknown Sample This part of the experiment is to be done individually. Your grade for this experiment will be based on how close you come to the actual percent iron in your unknown. Be sure to write down the names of the members of the group responsible for calibrating the spectrometer and determining the linear regression analysis of the calibration data. 1. Weigh out about 0.05 grams of your unknown sample to the nearest 0.0001 milligram on an analytical balance. Record the weight of unknown. Transfer the sample to a clean 50 or 100 mL beaker. 2. Add 5 drops of 6 molar H2SO4 and a few mL of DI water. 3. Quantitatively transfer your dissolved sample into a 50.00 volumetric flask, using several rinsings. Make up to exactly 50.00 mL using DI water. When well mixed, transfer this solution to a clean, dry, storage flask to free the volumetric flask for step 4. This is your unknown stock solution. 4. Pipette 1.00 mL of the stock solution into a 50.00 mL volumetric flask and continue steps 2 through 5 in the calibration procedure. Using the same spectrometer you used in the calibration, read the transmittance of your unknown solution and record the data. This is your test solution. 5. Repeat Steps 1-4 for a second portion of your unknown sample; record the transmittance of the second sample. Repeat for a third time if your instructor requires. Sample Calculation (NOTE: these are ONLY samples!) Mass of unknown Fe sample: 0.0433 g Concentration of unknown stock solution: 0.0433 g = 8.66 × 10 –4 g / mL = 0.866 mg / mL 50.00 mL Concentration of unknown test solution: 0.866 mg 1 × 1.00 mL × = 0.017328 mg / mL mL 50.00 mL Transmittance of sample: 32.3% Absorbance: log (100/32.3) = 0.4907974 Equation from linear regression analysis of calibration data: A = 197.8 c + 0.0092 Concentration of Fe in sample, using above equation, (where test sample A = 0.4907974): 0.4907974 = 1978 c + 0.0092 c = 2.4347698 x 10-3 = 2.43 x 10-3 mg/mL Percent Fe in sample: (2.43 x 10-3/0.0 1732) x 100 = 14.03 = 14.0 % Fe Note: Do not round off until the final percentage is determined for the sample. This avoids the introduction of large round off errors associated with logarithims. Spectrophotometric Iron Results Name:_____________________ Members of Calibration Group:______________________________________________________ __________________________________________________________________________________ Concentration of Fe (mg/mL) Transmittance Absorbance 1. 2. 3. 4. 5. Linear Regression Equation: ____________________________________________ (Remember to attach a graph of your calibration data) Unknown Fe Sample Number:__________ Trial 1 Trial 2 Trial 3 Sample mass (g) Concentration of Unknown Stock Solution (mg/mL) Concentration of Unknown in test Solution (mg/mL) Transmittance Absorbance Concentration of Fe in Unknown Sample (mg/mL) Percent Fe in Unknown Sample Average Percent Fe (%):____________________ Questions: 1. What is the purpose of the hydroxylamine hydrochloride in this experiment and why was this important? 2. The reaction Fe +2 + 2 H 2O ⎯⎯ Fe(OH ) 2 + H + occurs readily. Why would this be a problem in the → determination of the amount of iron? What procedure was used in this experiment to prevent this reaction from interfering with the determination of the percent iron in the unknown sample? 3. Why is the reagent blank necessary? 4. Why must the determinations of the unknown be done on the same day the calibration is run? After all, as long as you use the same Spec 20, why should it matter?