LAB TESTS REPORT AND ANALYSIS
EXPERIMENT # 4
SPECTROPHOTOMETRIC DETERMINATIONB OF IRON IN NATURAL WATER
SAMPLE
AIM

To determine the concentration (molarity) of iron in water using a spectrophotometric
method.
INTRODUTION
Spectroscopy is the analysis of the interaction between matter and any portion of the
electromagnetic spectrum. One of the spectrophotometric methods is UV-Visible
spectrophotometry which uses light in the visible and adjacent near ultraviolet ranges. Solutions
of transition metal ions and highly conjugated compounds produce different colors depending on
the excitation of d electrons within the metal atoms. Absorbance of the solutions colour by
spectrophotometry results in determining the concentration of the individual components in the
solution.
Determination of iron in water depends on absorbance which is related to colour intensity and is
proportional to concentration of iron responsible for the colour. The reaction of iron with 2, 2
bipyridine (bipy) or 1, 10-phenanthroline is shown below:
3 bipy +
3 phen H + +
Fe (bipy)3 2+
Fe2+
Fe2+
Fe (phen)3 2+
or
+ 3H +
The intense colour produced by the above reaction results in determining the concentration of iron.
The absorbance used is obtained from results of a UV-visible spectrophotometry, which follows
the beer lambert law as follows:
Where A = Absorbance.
ε = Molar absorptivity
b = path length that light travels through analyte
c = concentration of the analyte in solution
P0 = Incident light
P = detected light
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MATERIALS
Apparatus:
Reagents:
6 Volumetric flask (100ml)
Ammonium iron (II) Sulphate- 6 - water
Pipette (10ml)
Hydroxylamine hydrochloride
Beaker (100ml)
Distilled water
Conical flask (250ml)
Sodium acetate
2 Volumetric flask (250ml)
1, 10 - phenanthroline
Spectrophotometer (UV-Vis)
3M sulphuric acid
Cuvette
Phenonthroline solution
Natural water sample
METHODS
About 0.002M stock solution of iron (II) was prepared in a 250ml volumetric flask. Thereafter a
standard solution was prepared by taking 25ml of the stock solution and poured in another 250ml
volumetric flask and 5ml of 3M H2SO4 was added into the same flask and diluted with distilled
water to the mark. A set of four 100ml volumetric flask was then prepared by pipetting 10, 20, 30
and 40ml of the prepared standard solution into each of the flasks. To each flask about 2ml of
hydroxylamine, 6ml of phenonthroline solution and 8ml of sodium acetate solution was added and
diluted with distilled water to the mark. A blank was then prepared by adding 20 drops of 3M
H2SO4 to another 100ml volumetric flask and adding 2ml of hydroxylamine hydrochloride, 6ml
of phenonthroline solution and 8ml of sodium acetate. Then the mixture was diluted with distilled
water to the mark. The mixture was let to settle for 5 minute so that the colour developed can be
stable. 10ml of water sample collected from the different sources was transferred to a 100ml
volumetric flask. The water sample was treated the same way as the standard solution by adding
2ml of hydroxylamine hydrochloride, 6ml of phenonthroline and 8ml of sodium acetate and the
dilute the mixture with distilled water to the mark. Now, using a UV-Visible spectroscopy, the
solutions prepared was then measured for absorbance. Starting with the blank and then the series
of standard solutions in ascending order then lastly the water sample (the cuvette was rinsed 4
times before measuring the water sample). The absorbance was recorded in a table as shown in
table of results.
RESULTS AND DISCUSSIONS
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The results after measurement were recorded in the table below:
Concentration (M)
Blank
0.00002
0.00004
0.00006
0.00008
0.00106
Standard
solutions
Water
sample
Figure 1: UV-Visible spectroscopy results.
Absorbance
0.000
0.275
0.599
0.930
1.231
1.652
Calibration curve
1.4
y = 15995x - 0.041
R² = 0.9996
Absorbance
1.2
1
0.8
0.6
0.4
0.2
0
Concentration (M)
Figure 2: Calibration curve
From the table of results, 4 standard solutions with different concentration were used to produce
the calibration curve. This calibration curve helps in determining the concentration of iron in water
sample as the equation produced from the graph is related to the absorbance of the UV-visible
spectroscopy. During the experiment sodium acetate was added in almost every solution which
was produced to act as a buffering agent by maintaining the pH range of 3 to 9 of the complex
Fe (phen)3 2+ . Buffering was necessary to prevent the oxidation of Fe(II) to Fe(III). The use of
hydroxylamine hydrochloride was for the same reason. A blank is a solution that contains all the
same components as the sample solution but no known analyte material is present in the solution.
A blank solution was used in this experiment to help in calibrating the instrument as the results
which were obtained from the instrument were measured with respect to the blank solution. This
is so because a blank contains all of the components of the original sample except for the analyte.
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The concentrations of the standard solutions and iron in sample are illustrated in the calculations
below on how they have been obtained.
The R2 in the calibration curve shows accurateness of the results. More accurate results are
illustrated by R2 which is very close to 1 and the less accurate results shows values far from 1.
During the experiment some errors were likely to occur. These errors include poor measurements,
spills and failure to operate the instruments, these errors were categorized as human parallax. Not
only that, but also poor calibration of instruments and broken glassware also contributed to the
errors which may be led to the slightly not accurate absorbance of the different concentrations of
the experiment.
CALCULATIONS
1. Concentrations
i.
Concentration of a 250ml standard solution: Given M1 = 0.002M, V1 = 25ml
M2 = ??, V2 = 250ml
Using M1V1 = M2V2
M2 =
ii.
𝑀1 × 𝑉1
𝑉2
=
0.002 × 25
250
= 0.0002M
Concentration of 10ml standard solution from a 250ml solution
M1V1 = M2V2
M2 =
iii.
M1 V1
V2
=
0.0002 × 10
100
= 0.00002 M
Concentration of 20ml standard solution from a 250ml solution
M1V1 = M2V2
M2 =
iv.
M1 V1
V2
=
0.0002 × 20
100
= 0.00004 M
Concentration of 30ml standard solution from a 250ml solution
M1V1 = M2V2
M2 =
v.
M1 V1
V2
0.0002 × 30
100
= 0.00006 M
Concentration of 40ml standard solution from a 250ml solution
M1V1 = M2V2
3
=
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M2 =
M1 V1
V2
=
0.0002 × 40
100
= 0.00008 M
2. Concentration of iron (II) in water sample using the calibration curve
Using an equation: Y = 15995X – 0.041
Substituting for Y = 1.651
∴ 1.651 = 15995X – 0.041
X=
1.651+0.041
15995
= 0.000106 M
Therefore: using M1V1 = M2V2 to find concentration of iron in undiluted 10ml water
sample from 100ml
M2 V2 0.000106 × 100
M1 =
=
V1
10
= 0.00106 M
3. Concentration of iron if absorbance was 0.23
Using the same calibration curve
Substituting for Y = 0.23
∴ 0.23 = 15995X – 0.041
0.23 + 0.041
X=
15995
= 0.0000169 M
CONCLUSION
At the end of the experiment, it was shown that the concentration of iron in water sample was
0.00106M by using UV-visible spectrophotometry. The iron concentration resulted after using a
calibration curve produced from a series of standard solution diluted from a 0.002M iron stock
solution. After measuring the absorbance of the standard solution a graph with a straight line Y =
15995X – 0.041 was plotted using excel. This equation helped in calculation the concentration of
iron by substituting for Y as absorbance.
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References
Skoog D. A, West D. M and Holler F. J (1996). Fundamentals of Analytical Chemistry, 7th Ed,
Saunders College Publishing, Orlando, Florida, USA.
Ham, B. M. & MaHam, A. (2016). Analytical Chemistry: A Chemist and Laboratory Technician’s
Toolkit, John Wiley & Sons, Inc., Hoboken, New Jersey.
Chang, R. (2010). Chemistry, 10th Ed, McGraw Publishing Inc., New York, USA.
Mawenda, U. (2017). Lecturer 1: [Power point presentation]. Blantyre, Malawi, University of
Malawi
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