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Determination of Iron in Breakfast Cereal Using Ultra Violet-Visible

Spectrophotometry

Amanda Wang

Lab Partner: Shu Yee Lau

2/22/18

Lab Section 008

Spring 2018
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Introduction

Iron plays an important role in human’s diet. Iron can be found in animal products as well

as dark leafy green vegetables, and poaches such as beans and lentils. The iron found in animals

contain both heme iron as well as non-heme iron; while iron found in vegetables and poaches are

non-heme. Heme iron is absorbed to the body in a faster rate than non-heme iron. Iron is needed

to produce proteins that aid in cell growth and repair. In addition to that, iron is stored in red

blood cells in the form of hemoglobin which helps with oxygen transport throughout the whole

body. [1]

Iron deficiency can cause anemia, and a decrease in immune system. Conversely,

excessive iron intake is toxic to the body as it damages organs. This increase in iron absorption

in the body is called hemochromatosis, causing it to accumulate in the liver and will eventually

lead to liver damage. [2] According to World Health Organization, iron deficiency is one of the

highest reported nutritional deficiency. More than 30% of the world are anemic, and is most

likely due to iron deficiency. [3]

The Recommended Dietary Allowance (RDA) of iron for women is 18mg, and for men is

8mg. [4] Cereal is an integral part to many breakfasts around the world, including the United

States. In this experiment, a sample of cereal was used and crushed, and dissolved in

hydrochloric acid. Standard solutions in increasing concentrations of iron were prepared for the

calibration curve, and samples of the cereal were prepared. The absorbance for standards and the

samples were recorded, and data were analyzed. This experiment is significant because the

amount of iron in cereal will contribute to RDA and may combat iron deficiency, or excessive

iron consumption, knowing amount of iron ingested.

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Experiments like this had been conducted to determine iron concentration. One example

uses spectrophotometry of iron to determine iron content in different types of black tea. Samples

of black tea were obtained and crushed, then filtered and dissolved in nitric acid and

hydrochloric acid. Standard solutions of iron were also prepared to obtain a calibration curve for

Beer-Lambert’s Law, and absorbance for the standard and samples were recorded. [5] This

experiment is significant because tea is also widely consumed across the globe. Knowing the

amount of iron present in tea could raise awareness towards its iron content and amount needed

to meet RDA for each individual.

This experiment used Agilent Technologies Cary 8454 UV-Vis Spectrophotometer to

determine the amount of iron in General Mills’ Wheaties breakfast cereal using the calibration

curve of Beer-Lambert’s Law. Beer-Lambert’s Law will show a linear relationship between

absorbance and concentration, but fails at higher concentrations, as observed later in the report.

The result was compared to the true value of iron in the breakfast cereal, and compared to the

Recommended Daily Allowance of iron for each serving of cereal, for both men and women.

Based on this, men will need to consume up to two servings of Wheaties, while women will need

to consume up to four servings.

Experimental

2.6881  0.0001 g of Wheaties cereal sample were weighed out. The sample was then

crushed and placed in a 100mL beaker. Next, 10  0.02 mL of 4M hydrochloric acid was added

into the beaker. This mixture was covered using a watch glass and boiled for 5 minutes, making

sure that the mixture was not left to dry.

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The sample mixture was then left to cool, and quantitatively transferred into a 50mL

volumetric flask. The solution was then diluted to the mark, and thoroughly mixed. The solution

was filtered using a filter funnel into a dry 100mL beaker. The brown color in the solution shows

that the solution absorbs visible light, which will be corrected using a “sample” blank.

10mL volumetric flasks were prepared. Five flasks were prepared for standard solutions;

three were prepared for samples from the cereal; one was the water blank; and the last one as a

sample blank. In each of these volumetric flasks, 200  2 L of hydroxylamine solution and 1.00

 0.01 mL of 1.2M sodium acetate solution were added. 500  5 L of phenanthroline solution

was also added into all volumetric flasks except for the sample blank.

1.00  0.01 mL of distilled water was pipetted into one of the nine other flasks, other than

the sample, as a water blank. To the five standards, 0.75, 1.50, 2.25, 3.00, and 4.50ppm Fe were

added to each flask. The remaining four sample volumetric flasks were prepared by pipetting

1.00  0.01 mL of cereal solution. The mixtures were then left to stand for 5 minutes, and was

diluted to the mark with distilled water, and was mixed thoroughly. The reaction that took place

was as shown below.

𝐹𝑒 2+ + 3𝑝ℎ𝑒𝑛𝐻 + → 𝐹𝑒(𝑝ℎ𝑒𝑛)2+
3 + 3𝐻
+

The samples and standards were brought to the spectrophotometer to measure their

absorbance. The water blank solution was used to rinse the cuvet, and the absorbance was

recorded at 508nm. Next, the lowest standard of iron solution was used to rinse the cuvet, and the

absorbance is recorded. Absorbance was then read in ascending order of standard solution,

rinsing cuvet with small portions of the solution before taking measurements. The same steps

were repeated when taking measurements for the cereal solution samples. The recorded
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absorbance of the samples was then subtracted from the sample blank to correct the absorbance

in the visible region.

Results

Concentration (ppm Fe) Volume of 15 ppm Fe Absorbance

added (mL)
0.75 0.50  0.005 0.14736
1.50 1.00  0.01 0.30588

2.25 1.50  0.01 0.45977

3.00 2.00  0.01 0.60458

4.50 3.00  0.02 0.71880

Table 1 Concentration vs Absorbance

The concentration of iron in the standard was obtained using 15 ppm Fe standard stock

solution. The volume added was calculated using M1V1=M2V2.

0.8

0.7
y = 0.166x + 0.0406
R² = 0.96429
0.6

0.5
Absorbance

0.4

0.3

0.2

0.1

0
-0.5 0.5 1.5 2.5 3.5 4.5
Concentration (ppm Fe)

Figure 1 Graph of Absorbance vs Concentration of Fe in ppm, from 0 ppm to 4.5 ppm.

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0.6
y = 0.2029x - 0.0008
R² = 0.99979
0.5

0.4
Absorbance

0.3

0.2

0.1

0
0 0.5 1 1.5 2 2.5 3
Concentration (ppm Fe)

Figure 2 Graph of Absorbance vs Concentration of Fe in ppm, from 0 ppm to 3 ppm

Sample Absorbance Absorbance of sample Iron Mass of Iron per Mass of Iron
of Sample – Absorbance of Concentration gram (mg/g) per 27 g
sample blank (mg/L) serving (mg)
1 0.25858 0.240813 1.1869 0.2557 6.904
2 0.25635 0.238583 1.1759 0.2533 6.839
3 0.25683 0.239063 1.1782 0.2538 6.853
Average 0.25725 0.239486 1.1803 0.2543  0.0005 6.866  0.014
Standard 0.00117 0.001153 0.01487 0.001266 0.0342
Deviation
Table 2 Absorbance, Concentration of Iron, Mass of Iron per gram, and Mass of Iron per serving

in Three Samples.

The average of the absorbance was calculated as shown in (1), and the standard deviation

is calculated as shown in (2).

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0.240813+238583+0.239063
𝐴̅ = = 0.239486 (1)
3

(0.240813−0.239486)2 +(0.238583−0.239486)2 +(0.239063−0.239486)2

𝑠=√ = 0.0011737 (2)
3−1

The concentration of iron in the samples were calculated using the calibration curve. The

slope of the calibration curve is εb. The slope of the calibration curve in Figure 2 is 0.2029, and

since the width of the cuvet is 1 cm, the molar absorptivity ε is 0.2029. With this information, the

concentration of iron in the sample could be calculated as shown in (3) and (4).

𝐴 = 𝜀𝑏𝑐 (3)

𝐴 0.239486
𝑐 = 𝜀𝑏 = 0.2029×1.00 = 1.1803 𝑝𝑝𝑚 𝐹𝑒 (4)

To obtain the mass of iron in each serving of Wheaties cereal, the amount of cereal in the

solution, mass of cereal was divided by volume of solution in the 50mL volumetric flask as

shown in (5). The mass of iron per gram of cereal was calculated by dividing the concentration

of iron by the mass of cereal by the mass per volume of the cereal (6). The mass of iron per gram

was multiplied by amount of cereal in a serving (7).

𝑚𝑎𝑠𝑠 𝑜𝑓 𝑐𝑒𝑟𝑒𝑎𝑙 2.3211𝑔

= = 46.422𝑔/𝐿 (5)
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 0.05 𝐿

𝑚𝑔
( ) 𝐿 𝑚𝑔
1.18030.01487 𝐿
× 46.422 𝑔 × 10𝑚𝐿 = 0.2543  0.0005 (6)
𝑚𝐿 𝑔

𝑚𝑔 27𝑔
0.2543  0.0005 × 𝑠𝑒𝑟𝑣𝑖𝑛𝑔 = 6.866  0.014 mg (7)
𝑔

The standard deviation of the iron concentration in ppm was calculated using the

equation shown below in (8) and (9). Where 𝑠𝑦 is the error from the calibration curve, k is the

number of replicate measurements, and m is the slope of the curve.

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∑(𝑑𝑖 )2
𝑠𝑦 = √ = 0.02003 (8)
𝑛−2

𝑠𝑦 1 1 (𝑦−𝑦̅)2
𝑢𝑥 = |𝑚| √𝑘 + 𝑛 + 𝑚2 ∑(𝑥 −𝑥̅ )2 = 0.014873 (9)
𝑖

Figure 3 Absorbance of all standards and samples used in the experiment.

Discussion

The calibration curve was made using the data from the iron standards. Figure 1 shows

that at higher concentrations, Beer’s Law fails. The R2 value of Figure 1 was 0.96429, which was

not within the acceptable range of 0.995 to 0.999. To improve linearity, the last value of 4.5 ppm

Fe was removed. This was recorded in Figure 2, with R2 value of 0.99979, which is in the

acceptable range. The value of (0,0) was included in the curve because according to Beer’s Law,

if the concentration of iron is zero, the absorbance should also be 0. The slope of the calibration

curve in Figure 2 was used to calculate the concentration of iron in the sample as this curve was

more precise, and showed reproducibility.

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In this experiment, the two blanks that were used are water blank and the sample blank.

When recording the absorbance of the standards and the sample mixture solutions, the water

blank, which included the same solutions as the standards, with distilled water except for the iron

solutions, was first recorded at the wavelength of 508nm. This measurement was taken into

account in the software to take the difference of the measured absorbance of the standard and

sample cereal solutions, and the water blank.

The cereal sample was boiled in strong acid solution to form iron-phenanthroline

complex as the weak base phenanthroline forms phenanthrolium ion. The iron-phenanthroline

complex was used to analyze absorbance in the spectrometer, because it gives a deep red color,

which shows that it absorbs visible light. The sample blank was used to correct the brown visible

light that was found in the cereal solution.

The Percent Daily Value of iron found in Wheaties is 45%, which gives the value of

8.1mg of iron in a serving. From the experiment, the average mass of iron per serving was found

to be 6.866  0.014 mg. The student’s t-test was conducted to compare experimental value to the

true value for 95% confidence interval. Where t = 2.920, s = 0.014, and n = 3.
𝑡𝑠
𝜇 = 𝑥̅ ± (10)
√𝑛

The 95% confidence interval lies in the range of 6.866 ± 0.024. Comparing this value to the true

value of 8.1mg, the experimental value does not lie within the 95% confidence range.

The RDA for iron is 10mg for men, and 18mg for women. From the experiment, the

amount of iron in a serving of Wheaties cereal meets 68.66% the RDA for men and only 38.14%

for women. This shows that men will be able to obtain their daily intake of iron in less than two

servings of Wheaties, while women will need to consume almost twice the amount required for
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men. This is important because overconsumption or under consumption of iron will affect one’s

health.

One possible source of error that caused the experimental value of the concentration of

iron to be lower than the true value could be due to the reaction time. The sample cereal mixtures

were supposed to be left to stand for 5 minutes after the addition of phenanthroline,

hydroxylamine, and sodium acetate, before addition of water. This is so that the iron from the

cereal solution could react with the phenanthroline to produce the iron-phenanthrolium ion.

However, in preparing the solution, water could have been added too early, causing some of the

iron to be unreacted. Because of this, the recorded absorbance in this experiment for the sample

cereal is lower, causing the calculated mass per serving to be lower than the true value.

Another possible source of error could be due to external stray light that entered the

monochromator in the spectrophotometer. This external light could cause a difference in the

absorbance and cause out data to be less accurate to the true value.

Besides UV-Vis Spectrophotometry, Flame Atomic Absorption Spectroscopy (FAAS)

could also be used to detect heavy metals such as iron. A research was conducted by

Thermofisher using FAAS to monitor the amount of minerals present in meat. In this experiment,

the standard was prepared using stock standard solutions and strong acid HNO3, which was

similar to the standard solutions that were prepared in this experiment. The samples were

prepared by digestion of beef, chicken, and pork using microwave, instead of digesting the

solution in a strong acid as stated in the experimental part of this report. The FAAS experiment

also included a spike of 2 ppm in their experiment, using similar solution as the sample solution.

This experiment showed that samples of meat had 1.387mg iron /kg of meat. [6] This shows that

there is less amount of iron in meat than in Wheaties cereal, but does not show that Wheaties
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cereal is a better source of iron than meat. This is because iron found in cereal is non-heme iron,

while iron found in meat is heme-iron, which is easier to absorb by the human body. [1]

Conclusion

The result of this experiment showed that there were 6.866  0.014 mg of iron present in

a serving of Wheaties brand cereal, showed that the concentration of iron in the cereal sample

were lower than the true value as reported by General Mills. The calculated 95% confidence

interval also did not agree with the true value of 8.1mg of Iron. Comparing it to the RDA, men

will need to consume almost two servings of Wheaties cereal to reach their RDA, while women

will need to consume almost four servings to reach their RDA. The values obtained from this

experiment was lower than the true value, and this could be a result of a shorter reaction time, as

well as stray light entering the monochromator, and changing the absorbance.

This report focuses on the concentration of iron in Wheaties cereal. This work could be

expanded by looking into different foods and their concentration of iron. Future work could also

seek a deeper understanding of heme and non-heme iron and their differences using different

types of detection such as FAAS, and UV-Vis Spectrometry. With this, there will be a greater

understanding about iron content in food as people today are increasingly health conscious.
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Reference

1. Avery, L. The Importance of Iron in the Diet

http://www/healthguidance.org/entry/16754/1/The-Importance-of-Iron-in-the-Diet.html

(accessed Feb 19,2018)

2. Brody, J. E. A Host of Ills When Iron’s Out of Balance

https://well.blogs.nytimes.com/2012/08/13/a-host-of-ills-when-irons-out-of-balance/

(accessed Feb 19, 2018).

3. Micronutrient deficiencies http:/www.who.int/nutrition/topics/ida/en/ (accessed Feb 19,

2018).

4. Office of Dietary Supplements – Dietary Supplement Fact Sheet: Iron

https://ods.od.nih.gov/factsheets/Iron-HealthProfessional/ (accessed Feb 19, 2018).

5. S., M.; B.:I., K.;M., M. Bulletin of the Chemists and the Techologists of Bosnia and

Herzegovina. 2015, pp29-32.

6. Gadzhieva, A. Iron and Magnesium Determination in Meat using Flame Atomic

Absorption Spectroscopy http://tools.thermofisher.com/content/sfs/brochures/AN-43190-

Iron-Magnesium-Determination-Meat-using-Flame-Atomic-Spectroscopy.pdf (accessed

Feb 21, 2018).