Student Activity: Quantitative Determination of Total Iron in Natural Waters

by Kevin Currie

PURPOSE

Iron, an important micronutrient for humans and aquatic organisms but a household (staining capacity) and water treatment problem at high concentrations, is quantitatively determined. Two versions of the lab are presented: a macroscale version and a small scale version.

DESCRIPTION

Ferric iron is reduced to ferrous iron and complexed with 1,10-phenanthroline. This complex has a maximum absorbance at 510nm and can be quantified spectrophotometrically or colorimetrically.

TIME

One 45-minute period.

MATERIALS

Macroscale version:

Chemicals

• hydroxylamine solution: 10g of NH₂OH·HCl in 100mL distilled water
• ammonium acetate buffer solution: Dissolve 62.5 g NH₄C₂H₃O₂ in 40mL distilled water, add 175mL glacial acetic acid
• sodium acetate solution: Dissolve 25 g NaC₂H₃O₂·3H₂O in 100mL distilled water
• phenanthroline solution: Dissolve 1.00g C₁₂H₈N₂·H₂O, 1,10-phenanthroline monohydrate in 1L distilled water by stirring and heating to 80°C. Do not boil. Discard the solution if it turns dark.
• The concentrations of the solutions other than the iron standard are not critical because all these reagents are added to the sample in excess. No volumetric glassware is necessary for other than standard preparation. All these reagents are stable for several months.
• standard Iron solution (1.00mL = 1.00 microgram Fe): slowly add 20mL concentrated H₂SO₄ to 50mL distilled water in a 1L volumetric flask. Dissolve 1.404 g ferrous ammonium sulfate, Fe(NH₄)₂(SO₄)₂·6H₂O. Dilute to one liter with distilled water. 1.00mL of this stock solution equals 200 micrograms Fe. Pipet accurately 50.00mL stock solution into a 1L volumetric flask. Dilute with distilled water. 1.00mL of this standard solution equals 1.00 microgram Fe.
• distilled water

Equipment

• spectrophotometer (or photodiode photometer) for use at 510 nm providing a light path, 1 cm or longer
• hot plate
• wash bottles
• 100mL volumetric flasks
• glassware (for reagents above, teacher prep) 2 1-L volumetric flasks-3 1-L reagent bottles
• 1 50-ml pipet
• 1 1-200mL beaker
• 1 10-ml pipet
• 1L beaker

Small Scale Version:

Chemicals

• hydroxylamine solution
• ammonium acetate buffer solution
• sodium acetate solution
• phenanthroline solution
• standard iron solution (1.00mL = 1.00 microgram Fe)
• distilled water

Equipment

• Beral transfer pipets
• 24-well plates
• hot plate
• 10 (13 x 100 mm) Pyrex or other borosilicate glass test tubes

SAFETY INSTRUCTIONS

Normal laboratory safety practice including wearing safety eye protection and aprons should be followed. Care should be exercised when handling concentrated HCl. Adequate ventilation, preferably a hood enclosure, should be provided. Report, neutralize, and clean up any chemical spills.

PROCEDURE

1. Macroscale Version:

   A1. Sample preparation.

   The procedure has been intentionally organized into several modular parts. Because the iron sample must be boiled for a time, its treatment is placed before and after the working standard preparation to save time.

   1. Mix the sample thoroughly and measure 50.0mL into a 125 Erlenmeyer flask.

   2. Add 2-ml concentrated HCl

   3. Add 1-ml hydroxylamine solution

   4. Add a few glass beads to ease boiling and heat to boiling. To insure complete dissolution of all the iron, continue boiling until the sample volume is reduced to 15 to 20mL.

   A2. Working Standard Preparation.

   1. To establish a range of standards from 0 to 100microgram Fe/100mL final solution, pipet 0.0, 2.0, 4.0, 6.0, and 8.0, and 10.0mL of the standard iron solution (1.00mL = 1.00ug Fe) into 100ml volumetric flasks. The flask with no iron solution will serve as the reference blank. It is treated with all of the reagents below.

   2. Add 1mL of the hydroxylamine solution to each flask.

   3. Add 1mL of the sodium acetate solution to each flask.

   4. Dilute each flask to approximately 75mL with distilled water.

   5. Add 10mL of the phenanthroline solution to each flask.

   6. Dilute each flask to the mark with distilled water, mix thoroughly, and allow them to stand for about 10 minutes.

   7. Measure absorbance of each standard against the reference blank and construct a calibration curve of absorbance versus microgram iron.

   A3. Sample Preparation (continued).

   5. After allowing the sample to cool to room temperature, transfer it to a 100ml volumetric flask, rinsing the Erlenmeyer with several small portions of distilled water. Add each rinse to the volumetric flask.

   6. Add 10mL of the ammonium acetate buffer solution.

   7. Add 2mL of the phenanthroline solution.

   8. Dilute to the mark with distilled water, mix thoroughly and allow at least 10 minutes for the maximum color development.

   9. Measure the absorbance of the sample and compare this absorbance with the calibration curve to determine microgram Fe in the sample volume taken.

2. Microscale version.

   Prepare working standards by adding the following reagents to test tubes labelled A-J as described below.

   1. DROPS of standard Fe in test tubes A-J

      	A	B	C	D	E	F	G	H	I	J
      Hydrazine	1	1	1	1	1	1	1	1	1	1
      sodium acetate	1	1	1	1	1	1	1	1	1	1
      distilled H2O	70	70	70	70	70	70	70	70	70	70
      Phenathroline	10	10	10	10	10	10	10	10	10	10
      distilled H2O	17	16	15	14	13	12	11	10	9	8
      Final Fe conc (micrograms)	10	20	30	40	50	60	70	80	90	100

   2. Mix the sample and deliver 50 drops to 13 x 100 mm test tube.

   3. Add 2 drops of concentrated HCl.

   4. Add 1 drop of hydroxyamine.

   5. Add a few glass beads and boil the sample to approximately a third the original volume by placing it directly on a hot plate.

   6. Allow the sample to cool to room temperature.

   7. Add 10 drops of ammonium acetate solution.

   8. Add 2 drops of phenanthroline. It may be necessary to add an additional few drops to get complete color development but make sure not to add more than the well cell can hold.

   9. Allow 10 minutes for the color to develop.

   10. Transfer the entire sample to the well cell taking note of the number of drops.

   11. Deliver the same volume of each standard to the cell well that was measured as the final volume of the sample after boiling.

   12. Match the sample color to one of the standards. It may be necessary to dilute one of the standards such that the color of the sample matches exactly.

MODIFICATIONS/SUBSTITUTIONS

This experiment is designed for an AP level course. The activity should be sequenced within the course following discussions of spectroscopy, complexation, and redox chemistry.

There is nothing magical about the hydrochloride or hydroxylamine. Other reducing agents will work too. Ascorbic acid will do the trick. When making substitutions remember that the concentration given for using

hydroxylamine (roughly 0.15M) involves 8 Fe⁺³ for each hydroxylamine molecule used.

In the macroscale experiment, a Bunsen burner may be substituted for the hot plate: DO NOT heat the pyrex test tubes with a Bunsen burner.

Carborundum or other inert material may be used instead of the glass beads.

In the macroscale experiment volumetric flasks aren't absolutely necessary. For example, when diluting the stock solution of Fe, 50mL could be pipeted into a beaker or reagent bottle and 950mL distilled water added to make 1 liter. Note also that graduated cylinders may be substituted for pipets. Some accuracy will be sacrificed. If the glassware used in this experiment has been used with iron compounds it is probably

advisable to rinse it prior to use with concentrated HCl (6M will do a decent job).

DISCUSSION

This method analyzes total iron. In most natural waters soluble iron will normally be present as Fe⁺³. Phenanthroline complexes the Fe⁺² form. The hydroxylamine is added to reduce Fe⁺³ to Fe⁺² as indicated:

oxidation NH₃OH⁺ + 3H₂O NO₃^- + 10H⁺ + 8e-

reduction 8Fe⁺³ 8Fe⁺²

Net NH₃OH^- + 3H₂O + 8Fe⁺³ NO₃^- + 10H⁺ + 8Fe⁺²

Each molecule of hydroxylamine reduces eight ferric ions to ferrous iron. Iron is also present, depending on the pH, as soluble FeOH⁺² complex, Fe(OH)₄^- complex, solid Fe(OH)₃, solid FeCO₃, and solid Fe(OH)₂. The sample is boiled in acid during the reduction step to ensure complete dissolution of solid or colloidal iron compounds and to protect against the formation of the basic species above, none of which is readily complexed by phenanthroline.

There are several potential interfering substances which will either complex phenanthroline (mostly metals) or oxidize ferrous iron back to ferric iron. The addition of an excess of hydroxylamine eliminates errors caused by excessive concentrations of strong oxidizing agents. The excess phenanthroline replaces that complexed by interfering metal ions.

The sodium or ammonium acetate solution is added for two reasons. Both form buffer solutions which help eliminate precipitates and help complex Fe⁺² such that phenanthroline will complex with the iron rather that some interfering species.

Organic material (algae or their excretions, in addition to humic material or other organic material) are possible interferents, but are largely taken into account by using the sample as a reference blank solution.

Sampling bottles should be glass. If a tap water sample is being taken the water should be allowed to run for several minutes before taking the sample. This will flush any iron that has accumulated in the pipes-copper will interfere in the analysis.

The sample size may be greater or less than 50mL (or 50 drops for the small scale method). The only requirement is that the final volume after the boiling step allow addition of reagents and be within the containment volume (100mL for spectrophotometric method or roughly 100 drops for the micro method). It will probably be easier to adjust sample size than remake your standards if the sample falls out of the calibration range.

CALCULATIONS

1. Macroscale Version:

   The concentration of the sample is related by its original volume and absorbance to the calibration standard concentration at that absorbance. Specifically, for an unknown giving an absorbance corresponding to 24 microgram: standard volume (mL).

   
   Sample concentration = standard concentration x (microgram/100mL) sample volume (mL)

   std con = std con x microgram per 100mL = / F(20 microgram per 100 x 100mL,50mL) = 40microgram/100mL, which is usually reported as 0.40mg/L Fe.

2. Microscale Version:

   The concentration (in micrograms/100mL) of the sample will be equal (in micrograms/100mL) to that of the standard which matches in color (assuming equal number of drops of were delivered to the cell well).

DISPOSAL

Flush all standards and samples down the sink with large quantities of water.

REFERENCES

Staff and participants, 1989 Dreyfus Institute on Environmental Chemistry, Princeton University, Princeton, NJ.

Woodrow Wilson Leadership Program in Chemistry lpt@www.woodrow.org
The Woodrow Wilson National Fellowship Foundation webmaster@woodrow.org
CN 5281, Princeton NJ 08543-5281 Tel:(609)452-7007 Fax:(609)452-0066