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Spectrophotometric Determination of Iron

by E. L. Pool and D. Copeland (with edits by R. Sandwick and M. J. Simpson)

Learning Goals

  1. Practice handling hazardous chemicals safely;
  2. Use a bunsen burner to heat a solution;
  3. Vacuum filter a solution;
  4. Practice using a transfer pipet and a mortar and pestle;
  5. Manipulate experimental conditions to illustrate the concept of limiting and excess reagents;
  6. Use Beer’s Law to create and apply a calibration curve for quantitative analysis;
  7. Consider the consequences of the limitations of the spectrophotometer and the analytical balance.

Introduction

You will use spectrophotometry to determine the concentration of iron in a multivitamin to see if the manufacturer’s claim is correct. Iron itself is not a huge absorber of light, but when it (in solution in the Fe2+ form) binds to 1,10 phenanthroline (C12H8N2), it forms a highly stable red/orange-colored species. By quantifying the color with spectrophotometry, we can deduce the concentration of iron in the solution and back-calculate the amount of iron in the original pill. This can be compared to the manufacturer’s claim.

phenanthroline1,10 Phenanthroline

Background

The formation of the red/orange-colored iron-phenanthroline complex requires the iron to be in the Fe2+ form, and the procedure thus includes the reagent hydroxylamine hydrochloride (NH2OH ● HCl) that will reduce all iron in the sample to the ferrous form. Sodium acetate (NaC2H3O2) is used to control the pH of the solution since this also affects the absorbance.

The red/orange iron-phenanthroline complex absorbs light at 508 nm, which is green light. This should make sense if you remember the color lab. To quantify the intensity of the color, you can use the spectrophotometer to measure the “absorbance.”

Once you measure the absorbance of the pill solution, how will you know how to correlate the absorbance with a concentration?  One way is to know the extinction coefficient and to plug the appropriate numbers into Beer’s law. Another way is to prepare iron standards and to generate a standard curve of absorbance versus iron concentration. This latter technique is preferred as it accounts for any small changes one might have in her/his particular laboratory situation. We will use this method in our iron determination.

An online tutorial of this lab is available here.

Preparatory Experiment

A key assumption in this experiment is that all of the iron will be converted to the colored complex, meaning there must be excess phenanthroline present. In preparation for the experiment, you will vary the amounts of the two reagents that are directly involved in the production of the colored complex: the iron and the phenanthroline. First, you will vary the amount of phenanthroline to ensure it is present in excess. Then, you will vary the amount of iron to determine the range of iron concentrations that can be measured under those conditions.

Part 1: Vary concentration of 1,10-phenanthroline while controlling the iron concentration
  1. Dilute the prepared 1,10-phenanthroline solution: use a transfer pipet to add 1 mL of 1,10-phenanthroline to a 100 mL volumetric flask, then fill to the line with DI water, cover with either a ground glass stopper or parafilm, and invert 17 times to mix.
  2. Pour the diluted phenanthroline solution into a small-ish beaker and label clearly.

    A small-ish beaker

  3. In four more small beakers, take about 10 mL of each: sodium acetate solution, hydroxylamine HCl solution, iron solution, and DI water. Label clearly. Refill as needed throughout the experiment.
  4. Put a plastic pipet into each of your small beakers.
  5. Get 8 clean cuvettes in a cuvette tray. Use a small piece of label tape on the top half of the cuvette to label each cuvette with the number of drops of phenanthroline you will add: 0, 2, 4, 6, 8, 10, 12, and 14.
  6. Into each cuvette, add 2 drops of hydroxylamine HCl, 2 drops of sodium acetate, and 3 drops of iron. Pro-tip: to ensure consistent drop size, hold the dropper vertically and keep the dropper somewhat full to avoid air bubbles.
  7. Add the phenanthroline drops according to the label.
  8. The total volume of each cuvette must be equal. Add drops of DI water to ensure the total number of drops in each cuvette is equal. Record the number of drops of water you use in Table 1. This step is super important! Remember what you learned in lab 2.
  9. Cover the cuvette with parafilm. Mix thoroughly. Wait about 10 minutes for the reaction to complete. Pro-Tip: Don’t waste this time. You can do other things instead of just watching the clock.
  10. Measure the absorbance at 508 nm for each cuvette. Blank the spectrophotometer with the cuvette containing 0 drops of phenanthroline. Record data in Table 1.
  11. Empty cuvettes into the waste container. Wash the cuvettes. Save your solutions.
Part 2: Vary concentration of iron while controlling the 1,10-phenanthroline concentration
  1. Get 6 clean cuvettes in a cuvette tray. Ideally, these are the ones you just washed from part 1. Label them with the number of drops of iron you will add: 0, 1, 2, 3, 4, and 5.
  2. Add to each cuvette: 2 drops of hydroxylamine HCl, 2 drops of sodium acetate, and 12 drops of phenanthroline.
  3. Add the iron drops according to the label.
  4. The total volume of each cuvette must be equal. Add drops of DI water to ensure the total number of drops in each cuvette is equal. Record the number of drops of water you add in Table 2. 
  5. Cover the cuvette with parafilm. Mix thoroughly. Wait about 10 minutes for the reaction to complete. Pro-Tip: Don’t waste this time. You can do other things instead of just watching the clock.
  6. Measure the absorbance at 508 nm for each cuvette. Blank the spectrophotometer with the cuvette containing 0 drops of iron. Record in Table 2.
  7. Empty cuvettes and all extra reagents into the waste container. Wash the cuvettes and save them for next week. Leave your beakers labeled and save the corresponding pipets because you will need them next week.

Procedure

Note: do this lab with a partner.

Sample (Unknown) Preparation

The sample preparation requires crushing a pill, boiling the powder in acid to release the iron, and filtering the solution to remove particulates that could interfere in the spectrophotometry measurements.

  1. Each pair should obtain one vitamin pill. Determine the mass of the pill. Using a mortar and pestle, crush the pill into a fine powder. (You can take your angst out on the pill, but be careful none of it escapes the mortar.) Transfer the pulverized pill powder quantitatively to a 400 mL beaker. Determine the mass of the powder. In your final calculations, you will need to scale up your final iron concentration in the pill to account for any powder that you did not use.

    Apparatus to heat a solution

  2. Add approximately 150 mL of 1.0 M hydrochloric acid (HCl) [CAUTION!]. Add a few boiling chips. Use an apparatus similar to the illustration to heat the solution to near boiling (simmer) and keep heating at that temperature for 10 minutes. Do not leave your solution unattended. Avoid loss by splattering. Record observations about the appearance of your solution.
  3. Set up a vacuum filter apparatus with a Buchner filter, a vacuum flask, and a piece of filter paper. Hold the flask firmly or clamp it to a ringstand so it does not tip over. Pour 50 mL of DI water through the filter first, then turn on the vacuum and pour the hot solution through the filter. Your final solution should be clear and yellow. Record observations about the clarity of your solution in Table 3.
  4. Pour the filtered solution into a volumetric flask. Dilute to exactly 1.000 L with distilled water and mix thoroughly.

Solution Preparation

  1. Label 7 clean test tubes with the concentration of iron that they will contain (in units of mg/L): 0, 5, 10, 15, 20, 25, and the unknown vitamin solution.
  2. Label 4 small beakers with the reagents you will acquire from the stock solutions: sodium acetate, hydroxylamine HCl, 1,10-phenanthroline, and DI water. Fill these beakers with about 10 mL of their respective reagent. Put a plastic pipet in each. Pro-tip: hopefully you still have these beakers and pipets from last week because you anticipated needing them again.
  3. To each test tube, add 1 mL of each: sodium acetate, hydroxylamine HCl, and 1,10-phenanthroline. Note: this week you are using a more concentrated phenanthroline solution that ensures it is in GREAT excess.
  4. To each test tube, add appropriate amounts of water and/or 25 mg/L iron stock solution to make a total of 5 mL of the concentration of iron specified on the label. Record the amounts of water and iron stock solution you used in Table 4. In the vitamin solution test tube, put 5 mL of the vitamin solution. 
  5. Mix test tubes with gentle agitation and let the reaction complete for 10 minutes. Although you should use this time wisely, I advise against cleaning up, as you may need to remake a solution or two before the lab ends.
  6. Record observations about the color change and any trends you notice in Table 4. Note in your observations what concentration iron solution appears most similar to your vitamin sample. Pro-tip: these qualitative measurements should agree with your quantitative spectrophotometric measurements that you make in the next step. Your eyes are good spectrophotometers, and something is probably wrong if the quantitative measurements disagree with your visual inspection.

Spectrophotometric Analysis

  1. Get 7 clean cuvettes in a cuvette tray. Label them with the concentration of iron that they will contain (in units of mg/L): 0, 5, 10, 15, 20, 25, and the unknown vitamin solution. Transfer approximately 1 mL of each solution from its test tube to its corresponding cuvette.
  2. Using a Spectronic 401 at 508 nm, read and record the Absorbance (A508) of your solutions. Record this data into Table 4.

Clean up

Check that your data in Table 4 increases linearly from low concentration of iron to high concentration of iron before cleaning up any part of your experiment. Ask your TA for help neutralizing the pill solution, then pour it down the drain. Pour the contents of the test tubes and cuvettes into the waste container. Throw away the cuvettes.

Data Analysis

The absorbance measurements of the standard solutions are used to determine the concentration of iron in the pill solution, which is used to determine the amount of iron present in the original pill. You can plot the concentration versus absorbance, then perform linear regression to find the formula that relates concentration and absorbance.

With this formula, you can simply plug in the pill solution absorbance and calculate the unknown concentration of iron in the pill solution. Using the concentration of iron in the pill solution, calculate the amount of iron present in the original pill. (Note: you will have to scale up your value to account for mass lost between the original pill and the pill powder.) You can then compare this value to the amount listed on the bottle in terms of percent error. The percent error is the absolute difference between the experimental and theoretical value divided by the theoretical value, then multiplied by 100 to get a percent.

Report

Fill out this worksheet. Turn in either a paper or digital copy. Optional survey.

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