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UV-Visible Spectroscopy Iron in Dietary Supplements

Introduction
Ultraviolet-visible spectroscopy (UV/Vis) refers to the absorption spectroscopy in the ultraviolet-
visible spectral region. Many molecules absorb ultraviolet or visible radiation as they move between
energy levels. The wavelength of radiation that is absorbed directly affects the perceived colour of the
chemicals involved and is related to molecular structure. Different molecules absorb radiation of
different wavelengths; therefore an absorption spectrum can be used to qualitatively identify
compounds.

However, UV/Vis spectroscopy is mainly used in the quantitative analysis of compounds. When a
substance absorbs visible light, it appears coloured. The human eye does not see the colour that is
absorbed by the sample; however, what is seen is the complement of the absorbed colour. For
example, a solution of copper(II) sulphate appears blue because the absorption energy comes from the
orange region of the visible spectrum.

Iron is an essential human nutrient, as it has many roles within the body. A lack of iron can lead to the
development of iron deficiency anaemia. To combat this, many people use iron dietary supplements in
order to help maintain healthy levels of iron. To detect iron with the use of UV/Vis spectroscopy, the
reaction between Fe
3+
and thiocyanate irons (SCN
-
) must occur. This reaction gives an intensely red-
coloured product that can be used as a qualitative test to determine the presence of Fe
3+
. However,
most of the iron in dietary supplements is in its ferrous form and must be oxidised to the ferric form
with the use of hydrogen peroxide (H2O2). This is to ensure that the iron can be sufficiently bonded
with the thiocyanate irons, giving the red colour of the solution.

Aim
To determine the mass of Fe
3+
in a sachet of a dietary supplement using UV-Visible Spectroscopy.

Hypothesis
It is hypothesised that the mass of Fe
3+
found by using UV-visible spectroscopy is the same as the
mass of Fe
3+
stated on the sachet of the dietary supplement.

Materials
2.000 x 10
-4
M Fe
3+
standard solution
4 M HNO3 solution
10% KSCN solution
10% H2O2 solution
Iron dietary supplement (5mg/25mL)
Distilled water
5 x 25 mL volumetric flasks
250 mL volumetric flask
2 x small beakers
6 x Pasteur pipettes
6 x cuvettes
Autopipette
Spectrophotometer
Spectrometer
Safety goggles
Lab coat



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Method
Part 1: Preparation of the calibration curve
1. The beaker labelled iron standard solution was rinsed with a small amount of the standard
solution and then 40 mL of the solution was placed into the beaker.
2. Five 25 mL volumetric flasks were labelled with numbers 1 to 5.
3. An autopipette was used to transfer the amounts of iron standard solution listed in the table
to the flasks.
4. The 4M HNO3 and 10% KSCN solutions were added to each of the flasks.
5. The solutions were mixed by stoppering and shaking, then the colour variations in the flasks
were checked by eye.

Solution Fe
3+
solution 4 M HNO3
solution
10% KSCN
solution
Distilled
water
Fe
3+

concentration
1 0 mL 2 mL 2 mL To the line 0 M
2 1 mL 2 mL 2 mL To the line 0.000008 M
3 2 mL 2 mL 2 mL To the line 0.000016 M
4 3 mL 2 mL 2 mL To the line 0.000024 M
5 4 mL 2 mL 2 mL To the line 0.000032 M

6. 5 cuvettes were filled with each of the solutions using a clean Pasteur pipette each time and
were arranged in order of concentration.

Part 2: Frequency of light absorbed
1. A spectrophotometer was used to record the spectrum of the solution in flask 4 over a range of
approx. 400 700 nm. (refer to spectrum included)

Part 3: Determining the iron content of the supplement
(i) Preparation of the sample solution
1. The contents of the sachet were emptied into a small beaker.
2. An autopipette was used to add 1.0mL of this liquid into a 250 mL volumetric flask.
3. 1 mL of 10% H2O2 solution was added to the flask.
4. 10 mL of 4M HNO3 and 10 mL of 10% KNCS was added to the solution.
5. The flask was made up to the mark with distilled water using a clean plastic pipette.

(ii) Analysis of the stock solution
1. The sample was transferred to a cuvette and the absorbance was measured at 473.0 nm (the
wavelength of maximum absorbance determined in part 2)

Part 4: Amount of light absorbed
1. The spectrometer was set to 473.0 nm.
2. The absorbance of each solution was measured. (refer to Table 1)
3. Using this information, a calibration curve was plotted. (refer to Graph 1)

Results
Table 1: The concentration of Fe
3+
in each solution with the corresponding absorbance
Solution Fe
3+
concentration (M) Absorbance
1 0 0
2 0.000008 0.026
3 0.000016 0.109
4 0.000024 0.144
5 0.000032 0.205
Sample unknown 0.098
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Graph 1: Calibration curve of the absorbance against concentration of Fe
3+


Spectrum 1: Absorbance spectrum (attached)

1. What is the iron concentration in the sachet solution (in the 250 mL flask)?
16 x 10
-6
M (according to the graph)

2. Assuming the sachet contains exactly 25.00 mL of iron supplement, what is the mass of iron
in the sachet?












3. Calculate the mass of Fe3(PO4)2 in the sachet.










0
0.05
0.1
0.15
0.2
0.25
0 0.000005 0.00001 0.000015 0.00002 0.000025 0.00003 0.000035
A
b
s
o
r
b
a
n
c
e

Concentration of Fe
3+
(M)
Graph 1: Calibration Curve
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Discussion
1. Briefly describe the method used in your analysis. A diagram illustrating the various steps
involved might be helpful.



















2. Briefly describe the principles of operation and the major components of the spectrometer
you used. A labelled diagram might be helpful.













3. Are there other parts of the instrument (aside from the sample) that could absorb light from
your light source? Has this affected your results? Why/why not?
Aside from the sample, no other parts of the instrument could absorb light from the light
source. If there was a part of the instrument that could absorb light, then the end results
would be different, making the amount of iron determined using UV/Vis would be different to
what was stated by the manufacturer.

4. Was the analytical procedure quantitative or qualitative, or both? Explain fully.
The analytical procedure was quantitative because it was used to determine the concentration
of Fe
3+
in the dietary supplement in terms of molarity.

5. What is the iron content as stated by the manufacturer?
The iron content as stated by the manufacturer was 5mg/25mL.



Standard solutions
were prepared
with varying
concentrations of
Fe
3+
.
Sample solution
was prepared.
Sample and
standard
solutions
were
transferred
into cuvettes.
A
spectrophotometer
was used to find the
absorption
spectrum.
A spectrometer was used
to find the absorption of
each of the samples.
From these results, a
calibration curve was
determined.
Using the
calibration curve,
the concentration of
Fe
3+
in the dietary
supplement was
found.
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6. What uncertainties (or errors) were involved in the procedure?
The nitric acid and potassium thiocyanate were not added to the flask with the 0 M Fe
3+
(flask
1). The contents of this flask were used to calibrate the spectrometer to 0 M concentration.
Fortunately, the results of the procedure were unaffected despite the nitric acid and
potassium thiocyanate not being present and the calibration curve produced was as expected.

7. Were there any unexpected results?
All the results were as expected and there were no unexpected results.

8. What are some other applications or uses of UV-visible spectroscopy that you investigated?
UV/Vis can be used in clinical analysis, measuring the concentrations of specific substances in
body fluids such as urine or blood. For example, the haemoglobin content and sugar levels in
blood can be found by using UV/Vis. In addition to this, like the experiment conducted,
UV/Vis can be utilised to identify the presence of metal ions; even if the metal ion itself is not
coloured, it is possible to be analysed if it is converted into a coloured compound. For
example, finding the amount of calcium in urine can be found using UV-Vis if an organic
complexing agent (eg. arsenazo III) is reacted with it to form a highly coloured liquid.

9. Why is iron particularly suited to this form of analysis? Do you think it would be detected by
the other instruments used in this workshop?
Iron is particularly suited to this form of analysis because UV/Vis is routinely used in
analytical chemistry for the quantitative determination of different substances like transition
metal ions. They can be detected because the solutions of these metal ions are often coloured.
The detection of iron could also be done by Atomic Absorption Spectroscopy (AAS) because it
is also a type of spectroscopy therefore meaning it is able to detect iron ions in a solution.

Conclusion
The amount of iron in a 25mL sachet of iron dietary supplement according the manufacturer is 5mg.
By using UV/Vis spectroscopy, the dietary supplement was tested and it was determined that there is
5.44mg of iron in the supplement. This result therefore supports the hypothesis that the mass of iron
found by using UV-Vis is the same as the mass of iron as stated on the sachet of the dietary
supplement. Five standard solutions were made with increasing concentrations of Fe
3+
with the
addition of nitric acid and potassium thiocyanate in order to make the solution a red colour. Then the
iron from the dietary supplement was prepared by oxidising the Fe
2+
ions to Fe
3+
using hydrogen
peroxide, then nitric acid and potassium thiocyanate were added to make this solution red as well. The
standard solutions and sample were then transferred into cuvettes, and an absorption spectrum was
found using a spectrophotometer and the solution in cuvette 4. The absorption spectrum clearly
showed the wavelength where the maximum amount was absorbed (473.0 nm), allowing us to use the
spectrometer to find the absorbance of each of the standard solutions and the sample. From these
results, a calibration curve was made and the concentration of the sample was able to be found.

The reason as to why there was a 0.44mg difference between the final result and the mass stated on
the sachet is likely to be because the manufacturer found it unnecessary to have the mass of iron in the
sachet to have a decimal point when their users are likely not to mind the miniscule difference.
Alternatively, another reason as to who there was a difference is because of inaccurate results. The
accuracy of the results could be improved if there werent any time restrictions, therefore allowing
more time to prepare more standard solutions and, in turn, a more accurate calibration curve.

In conclusion, it was learned that UV/Vis has many uses, i.e. finding the concentration of metal ions in
a compound. Finally, a greater understanding of the principles and applications of UV/Vis was gained
from the outcome of this experiment.


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Risk Assessment
Preparation/Provision
of
Acute Hazards Control Measures First Aid

Nitric acid

It is not combustible,
but can enhance
combustion of other
substances.
Gives off
irritating/toxic fumes.
If inhaled, it can cause
sore throat, coughing,
burning sensation,
headache, shortness of
breath and labored
breathing.
Can cause serious skin
burns with yellow
discolouration.
If it comes into
contact with eyes, it
can cause redness,
pain and burns.
If ingested, it can
cause abdominal pain,
sore throat, burning
sensation, shock or
collapse and vomiting.

Ensure the acid is well
away from flammable
substances and any
combustibles or
organic chemicals.
Keep in a well-
ventilated room that is
cool and dry.
Wear safety gear (lab
coat, gloves, protective
goggles) and do not
eat, smoke or drink
during work.
In case of fire, do not
use foam. Keep cool
by spraying with
water.
If inhalation occurs,
treat with fresh air
and rest in a half-
upright position.
Artificial respiration
may be needed. Refer
for medical attention.
If there is exposure to
skin, remove
contaminated clothing
and rinse skin with
plenty of water. Refer
for medical attention.
If there is contact with
eyes, rise with plenty
of water for several
minutes and refer for
medical attention.
If ingested, do not
induce vomiting. Rest
and give one or two
glasses of water to
drink. Refer for
medical attention.


Potassium thiocyanate It is not combustible,
but gives off irritating
fumes/gases in a fire.
If inhaled, it can cause
coughing.
If ingested, it can
cause confusion,
convulsions, nausea,
vomiting and
weakness.
Keep separated from
strong oxidants in a
dry, well-closed area.
Wear safety gear (lab
coat, gloves, protective
goggles) and do not
eat, smoke or drink
during work.

In case of fire, use the
appropriate
extinguishing
medium.
If inhaled, treat with
fresh air and rest.
If there is exposure to
skin, remove
contaminated clothing
and rinse skin with
plenty of water.
If there is contact with
eyes, rise with plenty
of water for several
minutes and refer for
medical attention.
If ingested, rinse
mouth. Give a slurry
of activated charcoal
in water to drink.
Refer for medical
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attention.
Hydrogen peroxide It is not combustible,
but it can ignite
combustible materials.
There is a risk of fire
on contact with heat
or metal catalysts.
Inhalation can cause
sore throat, cough
dizziness, headache,
nausea and shortness
of breath.
If it comes into
contact with skin, it is
corrosive and causes
white spots, redness,
skin burns and pain,
If it comes into
contact with eyes, it is
corrosive and causes
redness, pain, blurred
vision and severe deep
burns.
If ingested, it can
cause abdominal pain,
abdominal distention,
nausea and vomiting.
Ensure the acid is well
away from
combustibles,
reducing agents and
hot surfaces.
Store in a cool, dark
area in vented
containers separated
from combustible and
reducing agents, food,
strong bases and
metals. Wear safety
gear (lab coat, gloves,
protective goggles)
and do not eat, smoke
or drink during work.

In case of fire, use
water in large
amounts (water
spray).
If inhalation occurs,
treat with fresh air
and rest in a half-
upright position. Refer
for medical attention.
If there is exposure to
skin, remove
contaminated clothing
and rinse skin with
plenty of water and
rinse again. Refer for
medical attention.
If there is contact with
eyes, rise with plenty
of water for several
minutes and refer for
medical attention.
If ingested, rinse
mouth, do not induce
vomiting and refer for
medical attention.