CH312

ENVIRONMENTAL
CHEMISTRY
PRACTICAL 1
SPECTROPHOTOMETRIC
ANALYSIS OF IRON
IN WATER

AKARSANA KUMAR
S11132245
WEDNESDAY SESSION B
Abstract
This experiment was conducted to investigate the method of calorimetric analysis to determine
the iron concentration in the polluted water sample that was collected. Water sample containing
iron (II) was reacted with o-Phenanthroline iron (II) to form complex ion of tris o-Phenanthroline
iron (II). Observations were made on color change of the solution as the reagents were added
and heated. The intensity of absorption for the colored compound was measured using a
spectrophotometer. Results obtained were used to construct a calibration cure (absorbance versus
concentration) for iron (II), from which the concentration of the unknown iron was then
determined.

Introduction
Spectrophotometry is the science that deals with the quantitative study of the electromagnetic
spectrum, particularly the intensity of light. By using the ability of atoms to absorb light energy
of specific wavelength, it can be used in the calculation of the concentration of an unknown
solution. A spectrophotometer is the primary device used in spectroscopy. It is capable of
measuring the absorbance of a solution by quantifying the amount of light passing through a
solution placed in a specialized tube called cuvette (Diliman, 2007).
Iron is a stable ion. It forms an intense red colored compound with ortho-phenanthroline
solution, and is the basis for its determination. The red-orange complex that forms between iron
(II) and 1,10-phenanthroline (orthophenanthroline) is useful in determining iron in water
supplies. The reagent is a weak base that reacts to form phenanthrolinium ion, phenH+, in acidic
media. Iron (II) is quantitatively complexed in the pH range between 3 and 9. A pH of about 3.5
is ordinarily recommended to prevent precipitation of iron salts, such as phosphates. An excess
of a reducing reagent, such as hydroxylamine, was needed to maintain iron in the +2 oxidation
state. The complex, once formed, is very stable (J. Wiley, 2004)
This experiment specifically aims to determine the amount of iron in the polluted water sample
as a complex of ortho-phenanthroline by the spectrophotometric method of analysis.

Methodology
1. Calibration standard solution preparation
From 1000 ppm of Fe2+ stock solution, sub-stock solution of Fe2+ of 100 ppm was prepared
using the C1V1 = C2V2 formula. Hence, a working stock solution of Fe2+ was then prepared from
the sub stock solution of 100 ppm. Appropriate volume sof the working stock solution equivalent
to the following concentrations: 0 (blank), 0.2 0.4, 1.0 and 2 ppm (Fe2+) into 100 cm3 volumetric
flasks. 50 cm3 of distilled deionized water (DDW) was added to each volumetric flasks with the
following reagents in the fume hood.
  2 cm3 concentration HCI
 10 cm3 Ammonium acetate / Acetic acid buffer
 3 drops of 1M NaOH [the solution was then checked with the pH (litmus paper) to ensure
that the pH was around 3- 4.5]
 2 cm3 of o-Phenanthroline
The above flask were then made to the 100 cm3 mark with DDW. The absorbance of the
standards and the blank was then measured using UV spectrophotometer (UV 120) at 510 nm.
The absorbance of the blank was then subtracted from the absorbance of standards and
calibration curve was plotted.
2. Total iron determination
50 cm3of each water sample A & B was pipette out into 2 conical flasks. To each 5 cm3 of 1M
HCl was added. The conical flasks containing the water samples were then heated for 30 minutes
on the steam bath. The flasks was then removed from the steam bath, to which 2 cm3 of
Hydroxylamine hydrochloride (NH2OH.HCl) and 2 cm3 of conc. HCl was added in the fume
hood. The sample solutions were re-heated on the hot plate until they started to boil.
The flasks was allowed to cool, 25 cm3 aliquots of both the samples were pipette out into 50 cm3
volumetric flasks. 10 cm3 of the ammonium acetate and 2 cm3 of o-Phenanthroline solution
followed by 3 drops of 1 M NaOH was added to the flasks. The pH was tested using the litmus
paper. The volumetric flask was then made up to the 50 cm3 mark using DDW. The absorbance
of the samples were measured at 510 nm, which was then subtracted from the absorbance of the
blank determined in Part 1 and the concentration of the Total iron (Fe2+ and Fe+3) from the
calibration curve obtained in Part 1.
3. Ferrous iron (Fe2+) determination
25 cm3 aliquots of water sample A & B were pipette out into 50 cm3 of volumetric flasks. 5 cm3
of NTA solution was then added to the water samples, followed by 10 cm3 of ammonium acetate,
2 cm3 of o-Phenanthroline and 3 drops of 1 M NaOH. The solution was then made up to the 50
cm3 mark using DDW. The absorbance of the samples were then measured in the
spectrophotometer at 510 nm. The absorbance was measured from the absorbance of the blank
determined in Part 1. The concentration of Fe2+ was then interpreted from the calibration curve
obtained above.
4. Ferric Iron (Fe3+) determination
Iron (Fe3+) concentration was then determined in each of the two samples by difference.
Result & Discussion
In order to prepare the calibration standard solutions, appropriate volumes required from the
stock solution of 1000 ppm to prepare the sub stock solution of 100 ppm and hence the working
stock solution were calculated as shown below:

Sub stock solution:

C1V1 = C2V2
(1000 ppm) V1 = (100 ppm) (100 cm3)
V1 = 10 cm3
The above volume was then used to prepare the working standards that were to be used for
measuring absorbance as follows:

Working standard solutions:

C1V1 = C2V2
(100 ppm) V1 = (100 ppm) (100 cm3)
V1 = 10 cm3
Then appropriate volumes of the working standard solution of Fe2+ (10 ppm) required to prepare
the following concentration were calculated:
i) 0 ppm (blank) ii) 0.2 ppm
C1V1 = C2V2 C1V1 = C2V2
(10 ppm) V1 = (0 ppm) (100 cm3) (10 ppm) V1 = (0.2 ppm) (100 cm3)
V1 = 0 cm3 V1 = 2 cm3

ii) 0.4 ppm iv) 1 ppm

C1V1 = C2V2 C1V1 = C2V2
(10 ppm) V1 = (0.4ppm) (100 cm3) (10 ppm) V1 = (1 ppm) (100 cm3)
V1 = 4 cm3 V1 = 10 cm3

v) 2 ppm
C1V1 = C2V2
(10 ppm) V1 = (2 ppm) (100 cm3)
V1 = 20 cm3

Part 1: Preparation of the calibration standard solutions

Once the standard and unknown solutions were already prepared, their absorbance values were
then measured using a photometer.

Concentration Absorbance Blank – standard

0.0 ppm (blank) 0.112

0.2ppm 0.198 0.086

0.4 ppm 0.262 0.150

1.0 ppm 0.363 0.257

2.0 ppm 0.476 0.364

Table 1.0 absorbance of working standard (Fe2+) of various concentrations in the solution.
The intensity of absorbance obtained above was then used to construct the calibration curve as
shown below.

0.4
0.35 y = 0.1436x + 0.073
R² = 0.9588
0.3
Absorbance

0.25
0.2
0.15
0.1
0.05
0
0 0.5 1 1.5 2 2.5
Concentration (ppm)
Part 2: Total iron determination

Absorbance Sample absorbance –

Blank
Sample A 0.165 0.053
Sample B 0.143 0.031

y = 0.1696x + 0.1571
Sample A
0.053 = 0.1696x + 0.1571
x = 0.61 ppm

Sample B
0.031= 0.1696x + 0.1571
x = 0.74 ppm

Part 3: Ferrous Iron (Fe2+) determination

Absorbance Sample absorbance –
Blank
Sample A 0.171 0.059
Sample B 0.095 0.017

y = 0.1696x + 0.1571
Sample A
0.059 = 0.1696x + 0.1571
x = 0.57 ppm

Sample B
0.017= 0.1696x + 0.1571
 x = 0.82 ppm

Part 4: Ferric Iron (Fe3+) determination

Ferric iron = Total iron concentration – Ferrous iron concentration
Sample A:
= 0.61 – 0.57
= 0.03 ppm
Sample B:
= 0.74 – 0.82
= -0.08 ppm

One important principle involved in spectroscopy is the Beer’s Law. It directly relates the
concentration of a colored substance in a solution to the amount of light it absorbs.

A=abc (1)

Where A is the absorbance, a is the molar absorptivity constant (M-1 cm-1), b is the path length
(cm), and C is the analyte molar concentration (M). The molar absorptivity of both samples were:

Sample A
A=abc
0.059 = a (1 cm) (1.61 x 10-5)
a = 3.6 x 103 M-1 cm-1
Sample B
A=abc
0.017 = a (1 cm) (2.3 x 10-5)
a = 739 M-1 cm-1
Since the solutions are reddish orange, a blue-green wavelength of light (508 - 510) should be
selected for the spectrophotometer (file:///C:/Users/user/Downloads/29874799-
Spectrophotometric-Determination-of-Iron.pdf).
The reaction between ferrous ion and 1,10-phenanthroline to form a red complex serves as a
sensitive method for determining iron.
Fe2+ + 3 phen -> Fe (phen)+23 (2)
Structure of ortho-phenanthroline

Iron level in drinking water -10 milligrams per liter (mg/L).

While Iron level in remote areas- about 50–90 ng/m3; and
at urban sites - about 1.3 µg/m3
Iron colloids often tends to affect the composition of water as it controls the transportation and
availability of trace metals (anthropogenic and natural) by cycling. Due to their large surface area,
they tend to absorb large amounts of trace metals. Colloids, because of this property, remain in
colloidal suspension, but also changes state in response to certain conditions. Factors commonly
affecting the stability of colloidal suspensions include the solution chemistry, especially pH and
ionic strength, mixing conditions, number of particles and their size distribution (Clark, 2010).

Conclusion
This experiment has successfully facilitated an understanding of spectrophotometric analysis in
determining the concentration of iron present in polluted water sample in different states. For
better and accurate results, cuvettes must be accurately handled. Often two cuvettes are to be
used simultaneously, one for the "blank" solution and one for the samples to be measured. Any
variation in the cuvette (such as a change in the cuvette width or curvature of the glass, stains,
smudges, or scratches) will cause varying results.

Reference
- Diliman, 2007. Institute of Chemistry. Analytical Chemistry Laboratory Manual.
University of the Philippines. p. 81.
- Christian, G.D., Analytical Chemistry, 6th edition. New Jersey. John Wiley, 2004.
- file:///C:/Users/user/Downloads/29874799-Spectrophotometric-Determination-of-
Iron.pdf
- Clark, L. J. 2010. Iron(II) determination in the presence of iron(III) using 4,7-diphenyl-
l,l0-phenanthroline. Analytical Chemistry. 34(3):348-352