Overview

In this experiment you will determine trace amounts of ferrous iron in a unique unknown sample using spectrophotometric methods.

To cite this lab manual: “Spectrophotometric Determination of Iron”. A Manual of Experiments for Analytical Chemistry. Department of Chemistry at UW-Madison, Summer 2024.

Visual Abstract

Background

The Chemistry

In solution, ferrous iron combines with 2,2’-bipyridyl to form an intensely red-colored complex that has a broad absorption band with a maximum absorbance at 522 nm (Fig. 1 and Fig. 2).

Moss and Mellon¹ have shown that the color is developed rapidly and is very stable in the pH 3-5 range. In fact, colored solutions prepared under these conditions have been kept for up to three years without any noticeable change in intensity. For a set range of concentrations, Beer’s Law will work for this system, and therefore visible spectroscopy is an easy, cheap, and effective means to perform a quantitative analysis of Fe.

The experiment you will work through is well-defined and gives you a chance to practice performing the various manipulations important to forming the complex necessary for quantitative analysis. You should develop a clear understanding of the chemical function of each ingredient and rationale behind the chosen concentrations. Knowing these details for any standard method (such as this one) will allow you to modify the procedure as necessary to accommodate diverse sample types.

Standards are made with a ferrous containing compound since the sample and standards must exist in ferrous form for the analysis. The chemicals/ingredients need to be mixed/added in the sequence outlined in the procedure. Fe(II) oxidizes easily in water to Fe(III). Thus, the ferrous solution must be made fresh, and will not store well without additional chemicals to stabilize the ferrous ion. HCl is added to lower the pH, which increases the solubility of iron in solution and reduces the rate of the oxidation reaction, which starts the minute ferrous ion is released into an aqueous environment. Therefore, any Fe(III) should be chemically reduced back to Fe(II). Of several reducing agents suitable for this purpose, hydroxylamine hydrochloride has been found to be remarkably effective (Eq. 1).

The complex forms in a finite 3-5 pH range, thus acetate buffer is added to ensure the pH conditions are met. Finally, the complexing agent (2,2’-bibpyridyl) is added and IF ferrous ion is present, the characteristic red color should develop in solution. Sometimes it takes time for a complex to “age,” which refers to the kinetic process necessary for the complexing agent to fully coordinate with the cations in solution. Some procedures call for solutions to sit for as long as 30 minutes to allow the complex for fully form, although no wait period is necessary for this particular complex formation procedure.

The success of this method of analysis depends on proper buffering of the solution, presence of an effective reducing agent, and precise laboratory techniques.

The Application

In this experiment, you will receive a sample to transfer quantitatively into a volumetric flask and dilute to the mark. Your TA will assign you a specific volume of the sample, which, when diluted to 100 mL, will serve as your unique control sample. You should report the concentration of this 100 mL solution, which is the solution you prepared after the quantitative transfer of the volume provided by the TA. Your grade will partially depend on how closely your reported concentration matches the actual value determined by your TA.

Lab Concept Video

Write down observations or notes from the video in your lab notebook.

Pre-lab Work

Lab Skills

Review these lab skills videos prior to lab.

Extra Resources:

• Using the LabQuest to Collect Spectrophotometry Data
• Making Scientific Plots

Prelaboratory Exercises

The following questions should be completed in your laboratory notebook before you take the quiz, and especially before you start the lab.

1. Write out the chemical reactions important to the formation of the Fe(II)-2,2-bipyridyl complex. Note the importance of each chemical component and the order in which they should be added.
2. Based on what you know about the chemistry, propose what might happen should you forget to add the hydroxylamine when preparing the solution?
3. In the procedure below, you are directed to add 1 mL of 1 M HCl to the mixture made in a 100 mL flask. What is the pH of the solution AFTER HCl is added and BEFORE the acetate buffer is added? Assume the addition of HCl will fully control the pH.
4. Based on what you know about the chemistry, propose what might happen should you forget to add the acetate buffer when preparing the solution.

Before You Take The Quiz on Canvas

• Review the calibration curve technique and how it is used in this experiment.
• Review the chemistry involved with forming the colored complex.
• Be able to calculate the precise concentration of iron(II) for a solution prepared from ferrous ammonium sulfate hexahydrate.
• Be able to calculate the precise concentration of the iron(II) standards given the precise concentration of the 100 ppm iron(II) stock solution.
• Be able to calculate the concentration of iron(II) in the undiluted unknown (solution prepared in step 3) from a calibration curve and the absorbance of the sample prepared from the unknown solution (‘Sample’ solution prepared in step 4).
• Be able to convert between units of transmittance and absorbance.

Experimental

Equipment Needed:

• 5, 10, 20, 25, 50 mL pipets
• 10 – 100 mL volumetric flasks
• 1000 mL volumetric flask
• 500 mL volumetric flask
• 9 – plastic storage bottles

1. 100 ppm Iron Solution (MAIN STOCK). In a 400 mL beaker, add 5 mL of 6 M HCl to about 250 mL of water. Dissolve 0.7022 g of ferrous ammonium sulfate (Fe(NH₄)₂(SO₄)₂·6H₂O) in the acidified water. Transfer to a 1000 mL volumetric flask and dilute to the mark with deionized water. Mix well.
2. 10 ppm Iron Solution (WORKING STOCK). Pipet 50 mL of 100 ppm iron solution into a 500 mL volumetric flask. Dilute to mark with deionized water and mix well.
3. Unknown Solution. Turn in a clean 10 oz. plastic bottle labeled with your name to your laboratory instructor. When you receive your unknown solution, quantitatively transfer to a 100 mL volumetric flask, dilute to the mark with deionized water and mix well. DO THE QUANTITATIVE TRANSFER FIRST. The resulting 100 mL solution serves as the “prepared sample” in Table 1.
4. Preparation of Standards, Blank and Sample. Label nine 100 mL volumetric flasks according to the following scheme (0.5 ppm, 1.0 ppm blank, sample). Prepare the nine solutions following the block outline in Table 1.
   

   Table 1. Preparation of ferrous standards, blank, and sample.

   Standard Solution Conc (right) Reagents (mL) (down, in order)	0.5 ppm	1.0 ppm	2.0 ppm	3.0 ppm	4.0 ppm	5.0 ppm	7.0 ppm	Blank	Sample
   Unknown solution (This is an aliquot of the 100 mL solution prepared in step 3.)	–	–	–	–	–	–	–	–	25
   10 ppm Fe2+ Sol (step 2)	5	10	20	30	40	50	70	–	–
   1 M HCl	1	1	1	1	1	1	1	1	1
   1% Hydroxylamine	5	5	5	5	5	5	5	5	5
   2 M Sodium Acetate	2	2	2	2	2	2	2	2	2
   0.1% Bipyridyl	10	10	10	10	10	10	10	10	10
   Water	Dilute to the mark

   BE SURE TO MIX WELL. Visually examine the solutions and place the sample solution next to the standard that appears to have the same intensity of color as the sample.

5. Transmittance Measurements.
   • If you have not previously used a spectrophotometer, consult the operating instructions and your laboratory instructor about the operation of the instrument assigned to you. Using the LabQuest to Collect Spectrophotometry Data contains instructions for using the Vernier visible spectrometer to collect absorbance or transmittance data. Proper technique for making this type of measurement is also explained in that document.
   • Rinse a 1 cm square cuvet several times with small portions of the blank and then fill 3/4 full. Set the wavelength at 522 nm. Using the blank solution, calibrate the instrument to 100% T. Discard the blank solution and refill the SAME cuvet with the standard or sample solution. Be sure to insert the cuvet in the same orientation as you did the blank. Record your readings in %T, which will be displayed on the LabQuest readout.
   • Take your data as much as possible by measuring from low to high concentrations. Do this by starting with the 0.5 ppm solution. Rinse the cell several times with 0.5 ppm solution. Then fill 3/4 full and measure the %T of the solution. Discard the solution in the cell and then rinse several times with small portions of the next (e.g., 1 ppm) solution, then fill 3/4 full and measure the %T of the solution. Repeat this procedure for the other standard and SAMPLE solutions proceeding from the lower to higher concentrations. Be sure to include the SAMPLE solution. Re-blank your spectrometer several times to check the 100% T setting is retained.
   • Repeat the transmittance measurements for all eight colored solutions again, starting with the lowest (0.5 ppm) and proceeding to the next higher concentration.

Post-Lab Work Up

Results/Calculations

Fill out the answer sheet for this experiment completely. Answer the following post-lab questions.

1. Convert the individual %T values to absorbance (A) values. Calculate an average A value for each measurement and use these average A values as you continue with your calculations.
2. Prepare a working curve of average absorbance (A) against exact concentration of iron. Make sure to include the 0.0 A point from the blank you prepared. Include this plot with materials you hand in to your TA.
3. Using the working curve, determine the concentration (ppm Fe) of the UNKNOWN solution you prepared in step 3.

Challenge Questions

Challenge questions are designed to make you think deeper about the concepts you learned in this lab. There may be multiple answers to these questions! Any honest effort at answering the questions will be rewarded.

1. Figure 3 displays the light intensity emitted by two sources common to spectrophotometry:
   • The deuterium lamp, used for experiments utilizing absorbance or transmittance phenomenon in the UV region of the spectrum
   • The tungsten lamp, common for applications in the Vis region.

   Suppose a spectrometer in your lab ONLY has a deuterium lamp. What modifications would you need to make to the procedure to allow you to complete the experiment? How would the quality of your measurements be impacted? For example, do you predict the transmittance or absorbance measured for the current concentrations of the standards will increase or decrease? Do you predict Beer’s Law will still work for the system? Justify your answers using principles from lecture, the textbook, and your own experience in lab.

Lab Report Submission Details

Submit your lab report on Canvas as 1 combined PDF file.  This submission should include:

1. The completed answer sheet.
2. Your lab notebook pages associated with this lab, which should include answers to the post-lab questions and challenge questions.

The grading rubric can be found on Canvas.

References:

1. Moss and Mellon, Ind. Eng. Chem., Anal. Ed., 14, 862 (1942).
2. R. A. Day, Jr., and A. L. Underwood, pp. 383 392; 396 399.
3. R. W. Ramette, pp. 138 140; 145 151.
4. D. A. Skoog and D. M. West, Chap. 20; pp. 539 546.
5. Harris, D. C. & Lucy, C. A. Quantitative Chemical Analysis, 10^th ed.; W. H. Freeman: New York, NY, 2020