1

Mau Jr. Brown

Conducted: January 26, 2016
Due: February 2, 2016
Lab 3
Colorimetric Determination of Iron
Introduction
Analyzing the title into simpler terms, colorimetry relates a substance and the amount of
color it displays in determining the concentration of iron. In this case, known solutions were
conducted and prepared to create a calibration curve to determine the concentration of identical
unknown solutions of iron. The concentrations of the known iron solutions can determine the
concentrations of unknown solutions by comparing the absorbance on a calibration curve.
The purpose of this experiment was to test the different amounts of iron solution with
constant amounts of ammonium acetate, hydroxylamine hydrochloride, phenanthroline solution,
and being diluted with distilled water. This lab also aided in comparing and contrasting the
known solutions in terms of color, amounts of volume of substances, absorbance, and
concentrations of each known solution. When iron is mixed with phenanthroline, an
orange/reddish solution is formed as a result of the reaction. The solutions color can be either
more or less concentrated depending on the amount of iron added. The increased amounts of iron
solution makes the solution more concentrated, thus making the solution appear darker.
Decreased amounts of iron solution makes a solution less concentrated, resulting in a light shade
of color, in this case a light orange-reddish color is displayed. The use of a spectrophotometer
established the amount of light each respective solution absorbed. In doing so, the sole purpose
of this experiment could be attained by using the colorimetric analysis of known solutions to
calculate the concentration of unknown solutions.
Procedure
In preparing the calibration curve, my partner and I obtained a 50 mL volumetric flask
and used three separate 1 mL pipets to pipet 1.00 mL of iron solution, 1 M ammonium acetate,
and 10% hydroxylamine hydrochloride in that specific order. Then, 10 mL of 0.30% ophenanthroline solution was added to the solution by using a 5 mL pipet. We then diluted the
solution with distilled water to fill 2/3 of the volumetric flask. The flask was then swirled gently
to ensure that the chemicals within the solution was uniformly dispersed within the flask. Then,
we resumed in diluting the solution to fill the flask up to the 50 mL mark on the volumetric flask.
The solution was then labeled and transferred in a 125 mL Erlenmeyer flask in order for the color
to develop. The time duration of 45 minutes was required before acquiring the absorbance of the
solution.
In determining the colorimetry of iron, the procedure was repeated 4 more times to obtain
4 more solutions in addition to the first, but required an extra 1 mL aliquot of iron solution per
trial. The amounts of the other chemicals namely ammonium acetate, hydroxylamine
hydrochloride, and phenanthroline solution remained the same. The amount of distilled water

used to dilute each solution varied, as long as it reached the 50 mL mark. When all solutions
were completely prepared, they were set aside for 45 minutes each to allow the development of
color to occur before using the spectrophotometer to determine the absorbance of each solution.
In determining or preparing three identical unknown solutions of iron, we weighed and
transferred 0.1 g of the unknown solution in a 50 mL volumetric flask. We then used an
eyedropper to add five drops of 6 M sulfuric acid. Then, the solution was diluted with distilled
water until the meniscus leveled at the 50 mL mark on the flask. Using a 1 mL pipet, we pipetted
1 mL of the solution into 3 separate volumetric flasks and repeated the procedure of adding the
given amounts of sulfuric acid and diluting with distilled water.
To find the absorbance of the known and unknown substances, the spectrophotometer
was used. After waiting 45 minutes per trial, we used separate eyedroppers to fill separate
cuvettes per known solution. In the same way, different eyedroppers were used to fill 2/3 of
separate cuvettes of unknown solutions. The cuvettes were filled 2/3 of the way and had a total
of 5 cuvettes of known solutions, 3 cuvettes of unknown solution, and 1 of distilled water. Before
using the spectrophotometer, it was calibrated by allowing the transmittance to equate to 100%
before putting the cuvettes in. Then, the wavelength was set to 510 nm as the cuvette of distilled
water was placed into the spectrophotometer. In the same manner, the absorbance had to be
tampered so the absorbance could equal 100 because distilled water should absorb all of the
light. Finally, each of the cuvettes containing known solutions were put in the spectrophotometer
to determine the absorbance. Then, the absorbance of each of the cuvettes containing unknown
solutions were determined.
Results
Table 3-1: Known Iron Solutions

Flask 1
Flask 2
Flask 3
Flask 4
Flask 5

Amount of 1
M
Ammonium
Acetate

Amount of
10%
hydroxylamine
hydrochloride

Amount of of
0.30% ophenanthroline

Amount of
iron
solution

Amount of
distilled
water

Total
Volume

1 mL
1 mL
1 mL
1 mL
1 mL

1 mL
1 mL
1 mL
1 mL
1 mL

10 mL
10 mL
10 mL
10 mL
10 mL

1 mL
2 mL
3 mL
4 mL
5 mL

37 mL
36 mL
35 mL
34 mL
33 mL

50 mL
50 mL
50 mL
50 mL
50 mL

Table 3-2: Determining Calibration Curve

Solution
Flask/Solution 1
Flask/Solution 2
Flask/Solution 3

Concentration (mg/mL)
0.05mg
1 mL= 50 mL = 110-3

Absorbance (No units)

0.231

2 mL=

0.1mg
= 210-3
50 mL

0.419

3 mL=

0.15mg
= 310-3
50 mL

0.618

Flask/Solution 4
Flask/Solution 5

4 mL=

0.2mg
= 410-3
50 mL

0.782

5 mL=

0.25mg
= 510-3
50 mL

0.972

Table 3-3: Stock/Unknown Solutions

Solution
Unknown Solution 1
Unknown Solution 2
Unknown Solution 3

Concentration (mg/mL)
0.0048
0.0042
0.0035

Absorbance (No units)

0.946
0.822
0.712

Graph 3-1: Calibration Curve of Known Iron Solutions

Known Iron Solutions
Concentratio
n

Absorbanc
e

0.001

0.231

0.002

0.419

0.003

0.618

0.004

0.782

0.005

0.972

Calibration Curve of Known Iron Solutions

1.2
0.97

1
f(x) = 191.77x + 0.020.78
R = 1
0.62

0.8

ABSORBANCE (NO UNITS) 0.6

0.42

0.4
0.2
0

0.23
0
0

0.01 0.01

CONCENTRATION (mg/mL)

Table 3-4: Calculating the Concentrations of Unknown Solutions

y = 191.77x + 0.0242
R = 0.9977
Unknown Concentration 1
y ( absorbance ) 0.0242
x=
191.77

Unknown Concentration 2
y ( absorbance ) 0.0242
x=
191.77

Unknown Concentration 2
y ( absorbance ) 0.0242
x=
191.77

x=

0.9460.0242
191.77

x=

0.8220.0242
191.77

x=

0.7120.0242
191.77

x=

0.9218
191.77

x=

0.7978
191.77

x=

0.6878
191.77

x=0.0048 mg/mL

x=0.0042 mg/mL

x=0.0035 mg/mL

Table 3-5: Calculating % Fe in Solution for each trial and Average Value
% Fe in Unknown Soln 1

% Fe in Unknown Soln 2

% Fe in Unknown Soln 3

mg of Fe=

mg of Fe=

mg of Fe=

0.0048mg
50 mL
50 mL
1 mL
1mL

0.0042mg
50 mL
50 mL
1 mL
1 mL

0.0035mg
50 mL
50 mL
1 mL
1mL

mg of Fe= 12 mg

mg of Fe= 10.5 mg

mg of Fe= 0.2 mg

% Fe=
mgof Iron
100
100 mg of iron

% Fe=
mgof Iron
100
100 mg of iron

% Fe=
mgof Iron
100
100 mg of iron

12 mg
100
100 mg of iron

10.5 mg
100
100 mg of iron

8. 75mg
100
100 mg of iron

= 12% Fe

=8.75% Fe

=10.5 % Fe
12+10.5+ 8.75
=10.42
Average Value:
3

Discussion:
As we prepared the calibration curve from the absorbance and concentrations of the
known iron solutions, the first solution with 1 mL of iron appeared as a very light orange color.
The second solution with 2 mL of iron appeared a bit darker than the first, but both almost
seemed identical. The third solution containing 3 mL of iron exhibited yet a darker shade of
orange than the previous solution. It was evident that the fourth solution containing 4 mL of iron
had a darker shade of orange than the third, and the very last solution of 5 mL of iron was darker
than the rest. Color varied depending on the amount of iron that was put into the solution. Each
solution had 1 mL of ammonium acetate, hydroxylamine hydrochloride, and o-phenanthroline
while being diluted to 50 mL. In preparing the unknown iron solutions, some errors may have
been put to play, but we were able to completely acquire the required solutions of the unknown.
All three solutions contained 1 mL of stock solution, five drops of 6 M sulfuric acid, and was
diluted completely with distilled water to the 50 mL mark on the volumetric flask. Taking into
consideration that all of these are identical, the color displayed by all 3 solutions were similar to
the color of Solution 5 of the known solution. Also, an identical absorbance and concentration is
assumed among the unknown solutions because they all contained the exact same amount of
substances.
We used eyedroppers to transfer each solution (known and unknown solution) into
cuvettes. Then, we calibrated the spectrophotometer (as mentioned in procedure) and put the
cuvettes in one by one. The spectrophotometer then displayed the absorbance of each of the
known iron solutions as well as the unknown solutions. The amount of absorbance is the amount
of light the solution is able to absorb. The excess light that is not absorbed by the solution just
passes through. In creating a calibration curve, my partners and I used a graph to plot the values
of absorbance on the y-axis and concentration on the x-axis. The known solutions were graphed
first followed by the unknown solutions (only absorbance was acquired) on the calibration curve
to get an approximate value of concentration.
Conclusion
The known iron solutions increased absorbance, concentration, and darkened in color as
more iron was added. Furthermore, if less or a minimal amount of iron is added to a solution, it
decreases in absorbance, concentration, and lightens in color. The color change was caused by
the addition of o-phenanthroline, which is a colorimetric reagent for iron detection. The values of
absorbance of the known iron solutions increased per trial, as well as the concentration. Both of
these values were dependent on the amount of iron solution that was added to each solution.
Notice how solutions 1-5 had the amount of iron of which they were labeled (e.g known solution
1 had 1 mL of iron, known solution 2 had 2 mL of iron, etc..). With increasing amounts of iron in

the known solutions, the same went with the absorbance. Solution 1 had the lowest absorbance,
and it increased per trial concluding that solution 5 had the greatest absorbance and
concentration.
Among the unknown iron solutions, 2 of 3 had the same concentration of 0.004 mg/mL
(without rounding) which corresponds to the concentration of solution 4 among the known iron
solutions. One of the three had a concentration of 0.0035 mg/mL which corresponds to the
concentration of solution 3 of the known iron solutions. In conclusion, the concentrations of the
unknown iron solutions were definitely assimilated by using a calibration curve of the known
iron solutions. In other words, the sole purpose of this experiment was attained by using the
colorimetric analysis of known solutions to calculate the concentration of unknown solutions.