CHEM 1045 - Fall 24

Quantification of Iron in Whole Grain Cereal

Abdullah Shaikh
Submitted to: Sumanta Basak, November 15th

Honor Code Signature: Abdullah Shaikh

ABSTRACT
Broadly speaking this experiment’s objective was to demonstrate how spectrophotometry can be used
to determine the concentration of a specific substance within a mixture. In our specific experiment we aimed
to accurately determine the concentration of Iron within a whole grain cereal sample, using an Atomic
Absorbance Instrument. We initially extracted iron from the cereal by grinding it into a fine solution then
using a stir bar to collect residue and then reacted with Hydrochloric Acid. Following this, we used a
Spectrophotometer to determine the absorbance of Iron standards (0.5–5 ppm) at 522nm (λmax). These
values were then plotted on a graph to form a calibration curve which would subsequently be used to
determine the concentration in the cereal. Next, we measured the absorbance of the sample derived from
cereal which was 0.0609. Using the linear equation from the calibration curve we were able to solve for the
concentration of Iron which ended up equating to roughly 0.15ppm. Our calibration curve resulted in a high
r2 value indicating strong correlation and accurate results leading to us being confident in the iron
concentration we calculated. This method serves as a way to verify nutritional labels and help accurately
determine the concentration of specific substances within complex mixtures.

I. INTRODUCTION
In the food production industry, a vital aspect is transparency when it comes to nutrition inside items.
Maintaining an accurate description of nutritional values is not only required legally but ensures customer
safety and trust. One crucial nutrient when it comes to maintaining a health diet is Iron. Iron is an important
mineral that helps maintain healthy blood. A lack of iron is called iron-deficiency anemia, which affects
about 4-5 million Americans yearly. It is the most common nutritional deficiency worldwide, causing
extreme fatigue and lightheadedness1. Given the great importance of maintain a healthy amount of Iron in
one’s diet ensuring nutritional values for Iron are accurate is highly important. This experiment uses
spectrophotometry to determine the iron concentration within a commercial cereal aiming to verify the
nutritional values provided by the brand.
Spectrophotometry is a technique used in chemistry for determining the concentration of specific
substances in unknown mixtures. It accomplishes this by measuring their light absorbance across various
wavelengths. This method relies on the principle that different compounds absorb light at specific
wavelengths, and the amount of light absorbed is proportional to the concentration of the substance (as
shown by the Beer-Lambert Law). Through the analysis of the absorbance spectrum, we can identify and
quantify the components within a mixture2.
In our specific experiment where we aimed to quantify Iron in cereal. Fe2+ concentration is determined
spectrophotometrically by forming red Fe-dipyridyl complex, made by reacting ferrous ions with 2,2'-
dipyridyl. This complex exhibits maximum absorbance at 522nm (λmax) allowing for the creation of a
calibration curve to determine unknown iron concentrations based on absorbance measurements3. The
method's accuracy makes it suitable for analyzing trace amounts of iron in complex matrices, such as food
1
products4. The calibration curve derived from this experiment will have the absorbance of a set of known
concentrations against the concentrations (in ppm). Once this linear equation is derived, the concentration
of an unknown sample can be solved by rearranging. This method is widely used due to its simplicity and
accuracy in quantifying analytes in various samples5.

II. EXPERIMENTAL—METHODS AND MATERIALS

The experiment was centered around the spectrophotometric analysis of Iron to accurately quantify it’s

concentration within whole grain cereal.

The process began with preparing a cereal sample from which Iron could be extracted. Firstly, roughly

10g of whole grain cereal were obtained using a weighing boat and a balance. The cereal was then grinded

into a fine powder using an electric grinder to increase surface area and help release particles of iron

embedded into the cereal’s composition. The fine powder was then transferred to a 250 mL beaker where

approximately 150 mL of water was added. The mixture was then stirred magnetic stir plate for ten minutes

to achieve a smooth consistency. During the stirring iron particles which had been released from the cereal

following the grinding adhered to the magnetic stir bar.

Following the ten minutes the magnetic stir bar was extracted from the solution using a pair of tweezers

and placed into a clean test tube once being cleaned by deionized water of any cereal solution. In order to

dissolve the iron particles 6 mL of 6.0 M HCl was added to the test tube. The test tube was subsequently

placed into a water bath to boil for roughly ten minutes to ensure the complete dissolving of Iron. The

solution began to go slightly pale green indicating the formation of Iron Chloride. The solution was

transferred to a clean 100 mL volumetric flask and diluted to the mark using deionized water.

Following this 2.0 mL of the Iron Chloride solution was pipetted into a 10.0 mL volumetric flask where

the following reagents were added; 2 drops of 6.0 M HCl, 0.50 mL 10% hydroxylamine-HCl, 6 drops of

2.0 M sodium acetate and 1.0 mL of 0.1% 2,2-dipyridyl solution. Next these components were diluted to

the mark using deionized water.

Finally, now that an adequate solution, which can have spectrophotometric analysis conducted on, was

obtained a calibration curve needed to be constructed to solve for the unknown concentration. To plot the

curve we needed to acquire the necessary data which was the absorbance values of known concentrations

of Iron at 522nm (Because the Fe-dipyridyl complex exhibits maximum absorbance at 522nm). The

2
absorbance for the five Fe standard solutions; 0.5, 1, 2, 4, and 5 ppm was measured using the Atomic

Absorbance (AA) instrument. Lastly, the cereal sample’s absorbance was also measured under identical

conditions, allowing its iron concentration to be determined using the calibration curve.

III. RESULTS AND DISCUSSION

The experiment’s primary aim was to quantify the concentration of iron within cereal, and in order to

achieve this, the preciously mention experimental method/approach was used.

During the preparation of the cereal sample we stirred ground cereal and water for about ten minutes

with a magnetic stir bar to attract iron particles. Once we extracted the stir bar we noticed the small black

flakes indicating that there was iron present and it had been attracted by the stir bar, however there were

fewer flakes then we expected suggesting that this could be due to the cereal not being grinded enough

meaning some iron particles were still embedded within the cereal and hence unable to be attracted to the

bar. After washing off the excess cereal solution residue on the bar it was placed in a test tube with 6.0 M

HCl to dissolve the particles. The tube was subsequently heated in a boiling bath and slowly we noticed a

slightly pale green color appear in the solution. This indicated the formation of Iron Chloride confirming

that there was a presence of Iron ions a crucial fact given that they are needed to form the Fe-dipyridyl

complex which would be analyzed spectrophotometrically to help achieve our aim. However, the pale green

color wasn’t as vibrant as we expected and rather more muted, this was likely caused by the smaller amount

of Iron ions collected as previously mentioned meaning HCl was in excess.

We then subjected the solution to several dilutions, including preparation of the stock solution (diluted

to 100 mL) and the final analysis solution (diluted to 10 mL with reagents added). These dilutions were

essential to ensure measurable absorbance values at 522nm. The formation of the red Fe-dipyridyl complex,

facilitated by the addition of 2,2-dipyridyl and other reagents, was confirmed by the characteristic color

change, which maximized absorbance at 522 nm.

Once the Iron Chloride solution had been obtained and used to form the Fe-dipyridyl complex we could

begin the spectrophotometric analysis of Iron to determine its concentration in the cereal. To do this we

used the Atomic Absorbance instrument to determine the absorbance of a set of known Iron Concentrations.

3
We then did the same for the cereal sample. This data was then plotted to create a calibration curve of

absorbance vs iron concentration (in ppm/parts per million).

Table 1

Concentration (ppm) of Fe (Iron) Absorbance

0.5 0.0790

1 0.1234

2 0.1985

4 0.3470

5 0.3972

Cereal Extract Sample (Unknown Concentration) 0.0609

Figure 1

Calibration Curve (Absorbance vs Iron Concentration)

0.45
y = 0.0714x + 0.0505
R² = 0.996
0.4

0.35

0.3
Absorbance

0.25

0.2

0.15

0.1

0.05

0
0 1 2 3 4 5 6
Iron Concentration (ppm)

4
 Once we had obtained these values we were able to construct a calibration curve as shown in Figure 1.

Our results show a strong correlation (demonstrated by the high r2 value) between concentration and

absorbance in line with what we know about the Beer-Lambert Law which states that absorbance and

concentration are directly proportional6. This indicates that we can be fairly confident in the results we had

subsequently meaning that when we calculate the concentration in the cereal sample using the derived linear

equation we can be fairly confident in it. The linear equation obtained was y = 0.0714x + 0.0505 which we

rewrote as:

𝐴 = 0.0714𝐶 + 0.0505

where:

- 𝐴 is the absorbance

- 𝐶 is the concentration

The absorbance of the cereal sample at 522nm was observed to be 0.0609. To solve for the

concentration we simply substitute this value for absorbance and rearranged to find concentration which

ended up being 0.15 ppm.

0.0609 = 0.0714𝐶 + 0.0505

0.0104 = 0.0714𝐶

0.0104
𝐶=
0.0714

𝐶 ≈ 0.15

In this experiment, we had to use volumetric glassware and this was crucial as it ensured precision

throughout the experiment. Given the many dilutions and transfers of solutions, the volumetric flasks and

pipettes helped give us accurate measurements of solutions and reagents, minimizing errors in dilution and

concentration calculations. These tools were essential for preparing solutions with consistent and

reproducible concentrations, which impacted the reliability of our calibration curve and it derived equation

which was used to calculate the iron concentration in cereal.

5
IV. CONCLUSION
The most significant result of this experiment was the successful quantification of iron concentration
in a cereal sample using spectrophotometry. Through our experimental procedure, the concentration of iron
in the sample was calculated as approximately 0.15 ppm. This result was determined by first extracting iron
particles from cereal and creating a constructing a Fe-dipyridyl complex for spectrophotometric analysis.
Following this calibration curve was formed based on the absorbance measurements of standard iron
solutions with known concentrations and using the derived linear equation to solve for the unknown
concentration after recording its absorbance. Given the high r2 value of the graph (strong correlation)
demonstrating consistency with principles of spectrophotometry (specifically the relationship between
absorbance and concentration), we can be fairly confident in the calculated concentration as it was solved
for using the derived linear equation.
Although our experiment was largely successful there were a few issues which may have hindered the
accuracy of our final calculated concentration. Specifically the small amount of iron particles that were
collected on the stir bar (as mentioned previously, likely due to cereal not being fully grinded meaning
some iron particles were still embedded and unable to be attracted), indicated by the less vibrant green color
of Iron Chloride. This had the knock on effect of having a lower concentration of Iron in the Fe-dipyridyl
complex we prepared, which had its absorbance measured and was used to calculate the concentration in
cereal. This likely indicates that our Iron concentration of about 0.15 ppm is lightly lower than expected.To
ensure a more accurate result in the future we could ensure a more thorough grinding process of cereal to
fully release all iron particles and also conducting multiple trials to identify any anomalies.
The real-world implications of this experiment are quite significant. Our results demonstrate a practical
means of verifying nutritional values in consumer foods, which can have massive implications in the health
industry and even governmental regulations when it comes to ensuring safe and transparent values for
customers and sanctioning inaccurate labels.
In conclusion, this experiment was successful in demonstrating the benefits of using spectrophotometric
analysis in food science. The findings were largely accurate despite a few minor hiccups leading to us being
fairly confident in our final results.

V. REFERENCES
(1)
Iron. The Nutrition Source. https://nutritionsource.hsph.harvard.edu/iron/.
(2)
Caprette, D. R. Principles of Spectrophotometry. Rice.edu.
https://www.ruf.rice.edu/~bioslabs/methods/protein/spectrophotometer.html.
(3)
Team, C. 327/329. Spectrophotometric Determination of Iron. Unizin.org.
https://wisc.pb.unizin.org/analyticalchemlab/chapter/spec-fe/ (accessed 2024-11-20).
(4)
Niedzielski, P.; Zielinska-Dawidziak, M.; Kozak, L.; Kowalewski, P.; Szlachetka, B.; Zalicka, S.;
Wachowiak, W. Determination of Iron Species in Samples of Iron-Fortified Food. Food Analytical
Methods 2014, 7 (10), 2023–2032. https://doi.org/10.1007/s12161-014-9843-5.
(5)
Harvey, D. 5.4: Linear Regression and Calibration Curves. Chemistry LibreTexts.
https://chem.libretexts.org/Courses/Providence_College/CHM_331_Advanced_Analytical_Chemistry
_1/05%3A_Standardizing_Analytical_Methods/5.04%3A_Linear_Regression_and_Calibration_Curv
es.
6
(6)
Clark, J. The Beer-Lambert Law. Chemistry LibreTexts.
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Suppl
emental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Electronic_Spectroscopy/El
ectronic_Spectroscopy_Basics/The_Beer-Lambert_Law.