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Determination of iron in various food and environmental samples

(revised 3 March 2026)

Introduction

Importance of iron in the human body

Iron is one of many minerals required by the human body to function correctly.  Iron is necessary for the transport of oxygen in the blood by hemoglobin, for the storage of oxygen in the muscle cells by myoglobin, as well as for many other important metabolic processes which include the production of red blood cells, the supply of energy by the cytochromes in the electron transport system, and for deoxyribonucleic acid (DNA) synthesis.1 Hemoglobin and myoglobin are oxygen-carrying proteins found in the blood and muscle tissue, respectively, containing heme groups that bind the oxygen molecule.  Each heme group is composed of a photoporphyrin IX ring with an iron (Fe2+) atom at its center (see Figure 1 below).2

heme
Figure 1. Structure of heme consisting of the photoporphyrin IX ring with a bound iron atom in its ferrous (2+) state.2

In the lungs, the iron atom in the heme group of hemoglobin binds molecular oxygen (O2) and carries it to the cells of the body (e.g., muscle cells) so that they may carry out their normal functions. Thus, the role of iron in the heme group is vitally important, because, without it, oxygen would not bind to hemoglobin, and the cells of the body would not receive the oxygen they need to carry out their processes.  In short, the cells would die due to lack of oxygen.1 Iron deficiency leaves a person feeling tired, dizzy, and listless, and is the most common cause of chronic anemia.2 Iron, being essential for life, is found in many of the foods we eat. It is particularly abundant in red meat, and in smaller quantities in grains and vegetables.2 

Experimental objectives

To determine the amount of iron in a food or environmental sample (e.g. seaweed, broccoli, spinach, cereal), first the iron is extracted from the sample using the technique of dry ashing. Following this, you will determine the concentration of iron in your sample by spectrophotometry (also known as ultraviolet-visible spectroscopy, or UV-Vis spectroscopy) using the procedure you have determined for your prelab (see Prelab section below).

Extraction and analysis of iron

During the ashing procedure, iron in the ferrous (Fe2+) state in the sample is converted into the ferric (Fe3+) form by heating the sample over a Bunsen burner to a fine ash.3 The sample is ashed (burned or charred) to oxidize the iron contained within, ensuring that the extracted iron is in the ferric form. Hydrochloric acid is then added to the ashed sample to extract the iron from the ash in the form of a Fe3+(aq) ions, and the sample is filtered by gravity filtration. The filtrate is collected. This filtrate will be referred to as the original sample filtrate. As well, it is important to add hydrochloric acid to the sample to ensure that a solid precipitate of Fe(OH)2+ does not form through the process of hydrolysis. To determine the amount of Fe3+ in the sample, you will then follow the procedure you have presented in your prelab.

Ultraviolet-Visible (UV-Vis) spectrophotometry and Beer's law

You may find the video below about UV-Visible spectroscopy helpful.4



Theoretical and manufacturer's values for the amount of Fe3+ the seaweed samples

To find the scientific articles (minimum of two (2) that use the correct detection method)

    To find a procedure to determine the concentration of iron in your sample, the following list provides some important information:
  1. Search the Journal of Chemical Education and other online or print sources to find scholarly articles that present methods to analyze the amount of iron in ashing extracts by spectrophotometry (also known as ultraviolet-visible spectroscopy or UV-Vis spectroscopy). Standard spectrophotometric laboratory techniques exist to determine iron content in solutions. You must find two (2) credible sources that use your chosen method. Note that you will only have access to the PDF versions of the articles in the Journal of Chemical Education ONLY if logged in to the network at the Cégep (it will not work in the residence or at home).
  2. The method must detect from 10 to 80 µM Fe3+. The total volume of the solutions must be no greater than 15 mL.
  3. You have at your disposal the following solutions:
  4. Verify with your professor before continuing with the completion the different sections of the prelab (instructions are given below).

Safety

Pipet all solutions using a pipetting aid. Remember all solutions contain HCl. It can cause burns. Clean all spills and wash your hands with lots of H2O if you spill any HCl on them. Wear your safety glasses.

Procedure

Part 1. Ashing

Obtain the mass of a dry crucible with its lid.  Add as much of your chosen sample (broccoli, spinach or seaweed) to the crucible as possible. The mass should not exceed 5 g). Reweigh. Heat the sample over a Bunsen burner as shown in Figure 2 below.  Make sure the sample is heated to a fine grey ash.  Cool the sample and weigh the ash. Transfer the ash to a clean beaker. Add approximately 10.00 mL of 2.0 M HCl with a pipet.  Stir gently, by swirling the beaker for about 5 minutes.  Filter the mixture by gravity filtration through a very small filter paper into a 10 mL graduated cylinder. Gently squeeze the filter paper to obtain the maximum amount of filtrate. Be careful to not break the filter paper. Note the final volume. Keep this filtrate. Henceforth, it will be referred to as the original sample filtrate. If necessary, repeat for the second sample.  

Ashing setup
Figure 2. Ashing setup with a crucible and bunsen burner.

Prelab for Part 1. Ashing

Part 2. Analysis of Fe3+ in the original sample filtrate - preparation of the calibration curve

Prelab 
  1. Provide a list of laboratory equipment and solutions.
  2. Provide the reaction used for detection.
  3. Provide a procedure. In other words, describe how you will prepare the calibration solutions, measure the absorbance of these solutions, and construct the calibration curve.
  4. Prepare tables for the experimental results. One such table must contain the solution number, the all of the different solutions that must be pipetted and the volume of each to pipet, as well as the total volume. Also include in the table, the final Fe3+ concentration of each solution. Provide your calculations. 
  5. Provide your sources at the end of the prelab in the References section. Do not forget to cite your sources using the NLM style.
You will then follow this procedure that you have developed when performing the experiment in the lab. Include all modifications suggested by the professor.

Part 3. Determination the amount of Fe3+ in the original sample filtrate (ashed sample)

Prelab

  1. Provide a stepwise procedure.
  2. Prepare tables for the experimental results. 
  3. Write a stepwise procedure to determine the amount of Fe3+ in the original sample filtrate (see ashing procedure below) by spectrophotometry.
  4. Describe how do you plan to use the results that you obtaine to determine the amount of Fe3+ in your original sample. Will you create a graph, table, etc. What calculations will you perform? The amount of Fe3+ in your sample must be expressed in µg Fe3+/g sample dry weight before ashing, as well as in percentage of Fe3+ extracted from the sample dry weight before ashing. 
  5. How will you compare your value with the manufacturer's or theoretical value? 
  6. Provide your sources in the References section. Do not forget to cite your sources using the NLM style.
You will then follow this procedure that you have developed when performing the experiment in the lab. Include all modifications suggested by the professor.

References

  1. Devlin TM. Textbook of Biochemistry with Clinical Correlations, 6th edition. Hoboken, NJ: Wiley-Liss; c2006. Chapter 21, Iron and Heme Metabolism; p. 823
  2. Katzung BG, Masters SB, Trevor AJ. Basic and Clinical Pharmacology, 11th edition. Toronto; The McGraw-Hill Companies, Inc. c2009. Chapter 33, Agents used in anemias; hematopoietic growth factors; p. 569
  3. Jackson PJ. The determination of ferrous iron in pulverized fuel ash and slags from pulverized fuel-fired boilers. J App. Chem. 1957.
  4. Professor Davis, ChemSurvival. How a simple UV-Visible spectrophotometer works [Internet]. YouTube; 2014 [cited 16 Feb 2023]. Available from: https://www.youtube.com/watch?v=wxrAELeXlek 
  5. Gaeckle J, Extent and magnitude of nutrient and contaminant concentrations in eelgrass (Zostera marina L.) in Puget Sound. Olympia, Washington: Washington State Department of Natural Resources; 30 March 2016
  6. Certified Organic Dulse Flakes. Franklin, Maine: Maine Coast Sea Vegetables INC.; 2012
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