CM123L ANALYTICAL CHEMISTRY LABORATORY

4th Term SY 2018-2019

Spectrophotometric Determination of Iron in Natural Water (Experiment 13)

Santos, Nanette D.1, Jalmasco, Caleb D.2

1Teacher, School of Chemical, Biological, and Materials Engineering and Sciences, Mapúa University; 2Student, CM123L/B9, School of Chemical, Biological, and Materials
Engineering and Sciences, Mapúa University, cdjalmasco@mymail.mapua.edu.ph

ABSTRACT

Water is one of the basics necessities of human life, making accessibility to a clean, pure, potable water a right for every
human. World Health Organization set its standard for acceptable amount of iron in a water sample, a ppm not greater than 3.0.
For this experiment, the experimenters determined the concentration and percent amount of iron in a natural water sample
through the uses of Beer-Lambert Law and spectrophotometric analysis. To be specific, an ultraviolet-visible (UV-VIS)
spectrophotometer was used for the experiment. The standard calibration method was used in the determination of the iron.
For the preparation of the calibration curve, five standards of the complex iron (II)-o-phenanthroline with ranging
concentrations from 0.5, 1.0, 1.5, 2.0, and 2.5 were used. The absorbance was read at 471 nm. From then, a regression line was
formed. The absorbance of the solution of whose Fe content was determined were tested also at 471 nm. The resulted
absorbance was 1.704 and its concentration is 2.1877 nm. The percent of Fe in the sample is 0.000218%. The obtained
concentration of iron lies within the range of the standard created by the World Health Organization. The experimenters were
introduced to instrumental analysis method and was able to determine the percent of iron in the natural water sample.

Keywords: natural water, iron, absorbance, Spectrophotometry, concentration

INTRODUCTION
The World Health Organization has established its
requirement before a water sample can be classified as
pure, the amount of iron must be less than 0.3 ppm (0.3
mg/L). (WHO, 2008) Levels of 0.3 – 3 ppm (mg/L) are still
acceptable according to them. Although iron is only
considered as a toxin if its highly concentrated, its amount
must be determined as it acts as an important surrogate
for heavy metals that are the real toxics.
passes through the sample. Depending on the range of the
wavelength of light source, spectrometers are classified as
UV-visible spectrophotometer or IR spectrophotometer.
UVVIS spectrophotometer uses light over the ultraviolet
range (185 – 400 nm) and visible range (400 -700 nm) of
electromagnetic radiation spectrum. While the other one
uses light over the infrared spectrum (700 – 15000 nm) of
electromagnetic radiation spectrum.

Spectrophotometry
In the determination of contaminants such as iron, a
method known as spectrophotometry is widely used. Due
to the investigations of chemical species, it was believed
that it involves the interaction between light and matter.
For absorbance spectrophotometry, it involves the transfer
of energy from a photon of light to an analyte to produce
excited state species. In an easier perspective,
spectrophotometry deals with the measurement of the
interaction of light with materials. (Germer et al., 2014). It
simply measures how much a chemical substance can
absorb the light through the measurement of light’s
intensity as a beam of light passes through the sample.
Figure 1 shows how spectrophotometer works. It is an
absolute idea that every chemical compound absorbs,
transmits, or reflects light within certain range of
wavelengths. (Shim, 2019)
In spectrophotometric approach, a spectrophotometer is
used to measure the amount of photon absorbed as it
Experiment 13│ Group No. 05│ July 20, 2019
Figure 1. Basic structure of spectrophotometers
Determination of iron through spectrophotometry
One of the easiest way to demonstrate the detection of iron
is on the use of the complexation of Fe2+ with 1,10-
phenanthroline or iron(II)-o-phenanthroline, that is
commonly in an intensely red-orange color. Like most metal
complexation reactions, the metal ion must compete with
H3O+ ions, and thus the metal complex will not form in
strongly acidic solutions. On the other hand, most metals
form insoluble metal hydroxides in basic solutions. For
these reasons the iron determination using o-
phenanthroline is carried out in a slightly acidic solution.
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In water samples, usually Fe3+ exists more making it
necessary to reduce it to Fe2+. This is done through the
addition of the reagent hydroxylamine hydrochloride
(NH2OH∙HCl). Another reagent, sodium acetate
(NaC2H3O2), is added to control the pH of the solution. The
pH is maintained because if the pH is too high, Fe2+ will be
oxidized to Fe3+, and if pH is too low, hydrogen ions will
compete with Fe2+ for the basic. (Pool & Copeland, 2016)
In the correlation of the absorbance with the concentration,
the Beer-Lambert Law will be used.
Beer-Lambert Law
The law relates light to the properties of the material of
which the light is passing thru. (Clark, 2019) The Beer-
Lambert law is shown in the equation
A = -log T = bc
Where A is the absorbance, T is the transmittance,  is the
molar absorptivity, b is the path length through the sample,
and c is the analyte concentration.
The transmittance is equivalent to
P In a 50-mL volumetric flask, transfer the unknown solution
T=
𝑃0
Where P is the amount of light that leaves the source and
passed through the sample and hits the detector, and P0 is
the amount of light that hits the detector when a non-
absorbing solution is in the beam path.
For the experiment, the objectives set were as follows. For
the experimenters to be familiar to the instrumental
methods of analysis. And at the end, be able to determine
the percent of iron in the natural water sample.
For this experiment, spectrophotometric method is the
technique that used for determining the amount of iron in
natural water.
MATERIALS AND METHODS
Reagents
Standard iron solution, hydroxylamine, double-distilled
water, sodium acetate, ortho-phenanthroline
Apparatus
100-m: volumetric flasks, 25-mL graduated cylinder,
volumetric pipettes (1 mL. 10 mL, 25 mL), cuvettes,
spectrophotometer
For the method of this experiment, there are two parts for
the procedure, namely the preparation of the calibration
curve for the standard Fe solution and the determination of
Experiment 13│ Group No. 05│ July 20, 2019
iron.
Preparation of the Calibration Curve (Standards)
Prepare five pieces of a 100-mL volumetric flask. For the
first flask, transfer 25 mL of the standard iron solution and
label. Then add 1 mL of hydroxylamine, 10 mL of sodium
acetate, and 10 mL of o-phenanthroline. Let it sit for five
minutes and dilute to mark. For the next four volumetric
flasks, add 20 mL, 15 mL, 10 mL and 5 mL of the standard
iron sample respectively and label. Add same amounts of
hydroxylamine sodium acetate, and o-phenanthroline. Let it
stand for 5 minutes and dilute to mark. Transfer to separate
test tubes and label, prepare the instrument. Start first by
determining the absorbance of a blank solution. Then fill the
cuvette with the highest concentration of the standard in
order to determine the peak and what wavelength will be
used. Be reminded to always clean the cuvette every after
use. After that, start with the lowest concentration, i.e. 5 mL,
increasing till 25-mL. Plot the calibration curve.
Determination of iron
and add 1 mL of hydroxylamine, 10 mL of sodium acetate,
and 10 mL of o-phenanthroline. Let it sit for five minutes
and dilute to mark. Label it and transfer to a test tube for the
examination. Following the five standards, after the 25-mL
standard, test the absorbance of the sample. It should be
within the range of the calibration curve. Report the
concentration of iron in the unknown in parts per million.
RESULTS AND DISCUSSION
Using all the procedures that are given to the student, the
solution was tested in the UV-VIS or the ultraviolet visible
spectrophotometer.
Table 1 shows the read absorbance of the
spectrophotometer in the sample. The absorbance was
read at 471 nm. This was determined by using the solution
with the highest concentration, and also the type of
wavelength that will be used.
Volume of mg/L or ppm Absorbance
Standard Fe After Dilution Reading at
Solution, mL 471 nm
5 0.5 0.373
10 1.0 0.766
15 1.5 1.179
20 2.0 1.566
25 2.5 1.936
Table 1. Preparation of the calibration curve
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Using the standard calibration method in finding the

concentration of iron, the experimenters were able to get
2.1877 ppm of phosphate.

2.1877 𝑝𝑝𝑚
= 𝟎. 𝟎𝟎𝟎𝟐𝟏𝟖 % 𝒊𝒓𝒐𝒏
10,000

CONCLUSIONS AND RECOMMENDATIONS

Iron is an essential mineral in the nutrition of humans. WHO

suggests that a range of 10 to 50 mg or iron per day must
be intake by a person to ensure a good supply of iron in the
body (WHO, 2008) However, if it went overboard such as
intaking as low as 40 mg/kg of body weight, death is
Figure 2. Calibration curve for standard iron sample imminent. It can cause hemorrhagic necrosis and sloughing
of areas of mucosa in the stomach with extension into the
Base on figure 2, a proportional relationship between the submucosa. (WHO, 2008)
absorbance and concentration was shown. This is a
manifestation of the Beer-Lambert law, that states that Contaminants such as iron should be regulated and with
absorbance is directly proportional to concentration and UV-vis spectrophotometry, the determination of these are
length of light. made possible in an easy, efficient way. It measures how
the chemical compound transmits or absorbs light. Through
The regression line and the constant R2 is this, the determination if iron in the natural water sample
y = 0.7852x – 0.0138 were made simpler.
R2 = 0.9996
Based on the data gathered by the experimenters, the
Table 2 then tackles the second part of the experiment, the concentration of iron in the natural water sample was found
determination of the iron in the sample water. Using the to be 2.1877 ppm or mg/L. Using the criterion set by the
plotted calibration curve from the standard iron complex, the World Health Organization (WHO), the sample water is not
iron was determined in a natural water. classified as a pure water sample but the iron concentration
is still acceptable as it lies within the range of 0.3 – 3.0
Absorbance reading at 471 nm 1.704 mg/L or ppm. It implies that the iron concentration in the
mg/L or ppm Fe based on 2.1877 water is still not risky and that the water is still safe for the
standard calibration curve human and for the environment.
Table 2. Determination of Iron
However, it was observed that the iron concentration is
near its limit and that measures that can prevent an
To get the concentration of iron using the standard calibration
increase in iron concentration is highly recommended. More
method, the slope of the equation (eq.1.2) is used:
recommendations include the use of a potable water as a
𝑘𝐴 𝐶𝐴 𝑉𝑜 𝑘𝐴 𝐶𝑠𝑡𝑑 sample to test out the iron concentration and determining
y = mx + b or 𝑆𝑠𝑝𝑖𝑘𝑒 (𝑦) = + × 𝑉𝑠𝑡𝑑 possible measures that can lower iron concentration to a
𝑉𝑓 𝑉𝑓
(eq. 1.2) reasonable amount.
Substituting the absorbance reading (y) to the regression
line gives out It can be then concluded, based on the set objectives on the
1.704 − 0.0138 experiment, that the experiment was successfully done. The
𝑥= experimenters were made familiar to the instrumental
0.7852
methods of analysis. And that the percent of iron in the
x = 2.1877 mg/L or ppm natural water sample was determined to be 0.000218%.

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REFERENCES

Carleton University (n.d). Spectrophotometric

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carleton.ca/chemistry/wp-content/uploads/speclab.pdf

Clark, J. (2019). The Beer-Lambert Law. Retrieved from

https://chem.libretexts.org/Bookshelves/Physical_and_T
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copy/Electronic_Spectroscopy/Electronic_Spectroscopy_
Basics/The_Beer-Lambert_Law

Germer, T.A. & Tsai, B.K., (2014). Experimental Methods

in the Physical Sciences, Vol. 46. 11-66. DOI:
org/10.1016/B978-0-12-386022-4.00002-9

N.A (n.d). Spectrophotometric Determination of Fe in

Drinking Water. Retrieved from
chem.fsu.edu/chemlab/chm3120l/spectro/spectrophot_d
etn_of_Fe.pdf

N.A (2012). Water Quality 1 - Spectrophotometric

Determination of Iron in Drinking Water. Retrieved from
http://web.pdx.edu/~atkinsdb/teach/427/Expt-
IronSpec.pdf

N.A (2019). Spectrophotometry. Retrieved from

https://chem.libretexts.org/Bookshelves/Physical_and_T
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Reaction_Rates/Experimental_Determination_of_Kinetcs
/Spectrophotometry

Pool, E.L., & Copeland, D. (2016). Spectrophotometric

Determination of Iron

Truman State University (2017). Spectrophotometric

Determination of Iron. Retrieved from
chemlab.truman.edu/files/2015/07/ironspec.pdf

World Health Organization (2008). Iron in Drinking-water

Background document for development of WHO
Guidelines for Drinking-water Quality. Vol. 2, Geneva

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