A PROJECT PAPER ON

ANALYSIS OF IRON IN POND / TUBE WELL/

RIVER WATER.

For the partial fulfillment of degree of Bachelor of Science

Submitted by

BHAGYALAXMI DEHURY
Exam Roll-20CHE010
+3 3rd Year SCIENCE

CHEMISTRY HONOURS

Under the Supervision of

Dr. ANASUYA MISHRA
Asst. Prof. in CHEMISTRY
Govt. College (Auto), Angul
SESSION-2022-23
 DECLARATION

I hereby declare that the project entitled “Analysis of Iron in ground water”

submitted by me to the Govt. College (Auto), Angul is original and a bonafide

research work conducted by me under the guidance of Dr. Anasuya Mishra, Asst.

Prof. in Chemistry, Govt. College (Auto) Angul. All the persons who helped me in

completing this project have been duly acknowledged by me. I am entirely

responsible for any mistake in this project work. I further declare that this project

has not been submitted to any other University / College or Institution for any

degree or diploma.

Signature of the Candidate

2
 CERTIFICATE
This is to certify that the project paper entitled “Analysis of Iron in

pond/ tubewell/ riverwater.” is submitted by Bhagyalaxmi Dehury

bearing examinationRoll No. 20CHE010 and College Roll no. BSP20-068 in

partial fulfillment of the requirement for 6th semester of B.Sc.

examination (Chemistry) 2023. He has been successfully prepared this

piece of project work under my guidance and supervision.

Dr. Anasuya Mishra Dr. Nilachala Patel

Supervisor H.O.D.

3
 ACKNOWLEDGEMENT

In the preparation and completion of this piece of project work, I owe my

primary depth of gratitude and sincere thanks to Dr. Anasuya Mishra, Asst. Prof. in

Chemistry, whose keen supervision, immense help, stimulating suggestion and

valuable comments helped me to prepare this piece of project work.

I owe my deep sense of gratitude to Dr. Nilachal Patel (H.O.D), who gave

valuable advice to encourage me at each steps during the preparation of this piece of

project work.

4
Abstract
An easy, efficient and safe method was developed to determine iron in water
samples. Iron Cell Test kit (from Spectroquant) is used in which firstly all iron ions
are reduced to iron (II) ions by ascorbic acid. In a thioglycolate buffered medium,
iron (II) reacts with a triazine derivative to form a purple complex that is
determined photometrically. Calibration curve of iron standards was done with
concentrations of 0.50, 1.0, 2.0,3.0 and 4.0 ppm and it gave a R 2 value of 0.9989
and straightline equation y=0.4749x-0.046. Iron analysis was done on two sets of
water samples. Named as set I samples 1, 2, 3, 4, 5, and 6 and set II samples 1, 2,
3, 4, 5 and 6. They were acidified with 0.1% HNO3 and the absorbance was
measured in a UV-Visible Spectrometer at 565 nm. The concentrations were found
as 0.45, 0.13, 3.84, 5.64, 6.72, 5.78 ppm for set I samples and 0.11, 0.11, 0.14,
0.12, and 0.11 ppm, for set II samples respectively. The limit of detection (LOD) is
0.10 ppm, and, the limit of quantification (LOQ) is 1.0 ppm.

5
 Table of Contents

1. Introduction 7

2. Method used for the experiment 8

2.1 Materials 8

2.2 Apparatus 8

3. Procedure 10

3.1 Preparation of calibration curve 10

3.2 Preparation of samples for measurement 10

3.3 Limit of detection (LOD) and limit of quantification (LOQ) 11

4. Results 12

5. Discussion 16

6. Conclusion 17

6
 1. Introduction
Pure water has no taste, but Water is a natural solvent. Most mineral from ground water,
Including iron will be absorbed by water. Larger amounts of iron in drinking water can give it an
unpleasant metallic taste. Iron is an essential element in human nutrition, and the health effects of iron in
drinking water may include warding off fatigue and anemia. “Iron is the second most abundant metal in
the earth's crust. Dissolved iron in water, causes the water to taste metallic”.
 Iron in drinking water
The water may also be discolored due to suspended solids containing minerals of iron that appear
brownish in color. Iron will leave red or orange rust stains in the sink, toilet and bathtub. It can build up
in your dishwasher and discolor ceramic dishes. It can also enter into the laundry equipment and cause
stains on clothing. “Even though the EPA says that the iron in the drinking water is safe to drink, the iron
sediments, other trace impurities may support bacteria that are harmful, and these bacteria are mostly
found in wells where the water has not been chlorinated”.
 Safety of drinking water
The safety of drinking water is utmost concern. World health organization has assigned well defined
standards for drinking water purity. U.S federal regulations, limit the amount of iron to less than 3ppm in
municipal drinking water. Iron is toxic at high concentration and acts as a surrogate for other heavy metals
whose presence in drinking water poses a danger to public health.
Human body requires iron but heavy doses are toxic. The Environmental Protection Agency considers
iron in well water as a secondary contaminant, which means it does not have a direct impact on health.
The Secondary Maximum Contaminant Level set out by the EPA is 0.3 milligram per litter, but this merely
a guideline and not a federal standard. Iron causes costly damage and other issues when present in high
concentration.
“In the drinking water supply, iron (II) salts are unstable and are precipitated as insoluble iron(III)
hydroxide which forms as a rust colored sediment”. When water is directly pumped from the well, the
water may contain iron (II) at concentrations of up to several milligrams per liter without any color or
turbidity. “When the iron levels are more than 0.05-0.1 mg/L turbidity and color develops in the pipe
system. Iron also promotes undesirable bacteria growth within a water works and distribution system
because of large deposition of iron minerals on piping.

“The iron concentration in rivers has been reported as 0.7 mg/L, and in groundwater, which is
anaerobic, iron is in the form of iron (II), with the concentration being usually 0.5-10 mg/L; and
sometimes, the concentration is found as high as 50 mg/L”. “The concentration of iron in water
should be less than 0.3 ppm (0.3 mg/L); however, it may be higher in countries where various iron
salts are used as coagulating agents in water- treatment plants and where cast iron, steel, and
galvanized iron pipes are used for water distribution”.

An experiment that mainly focuses on measuring iron content in river water, pond, and tube well
and determines whether the water meets the standards and may also suggest the presence of
other contaminants. Solutions containing iron are colorless at low concentration so the iron solutions
are tested by adding a completing agent that absorbs at a specific wavelength and is analyzed using a
spectrophotometer. Iron is used as a constructional material for drinking water pipes and for
structural support in automobiles, buildings and bridges. It is also use for treatment of iron deficiency
in humans. Various iron salts are used as coagulants in water treatment.
2. Method used for the experiment
For the determination of iron in the samples of various tap water, Iron Cell Test Kit from
Spectroquant is used. In the Test Kit all the iron ions present in the samples was reduced to Fe2+
ions by ascorbic acid. In the presence of the medium thioglycolate, a purple complex was formed
because of Fe2+ reacts with a trizine derivative. 11The complex was determined photo metrically
by using UV-Vis spectrophotometer.

A UV-Vis spectrometer is an instrument used to measure the amount of ultraviolet and visible
light absorbed by a solution. The light used in UV-Vis spectroscopy, is a very narrow portion of
electromagnetic spectrum. The instrument is designed so that the sample is placed between a
light source and detector. Depending on the sample, light may be absorbed causing electrons to
be promoted from one energy level to another. Since different metal ions, have different
absorption patterns, UV-VIS spectroscopy can be used to identify metal ions in solutions.

2.1 Materials
Iron metal, concentrated nitric acid, 6.0 M sodium hydroxide , 6.0 M hydrochloric acid , ascorbic
acid and triazine present in the Iron Cell Test kit.

2.2 Apparatus
Iron Cell Test kit, Hanna pH meter pH range: pH 0.00 to 14.00 operated at room temperature (~
20 oC). A UV-Visible spectrometer was used to perform qualitative analysis. The UV visible
spectrometer was operated with viewer vision 1.00.00 with wavelengths ranging from 700 nm to
300 nm using polystyrene cuvettes. HPLC filters 0.45 µm with a polyethylene or Teflon filter
material were used to remove interfering materials and fineparticles where stated, micropipettes
with differing levels of precision (20-200 µL, 2-20 µL, 100-1000 µL),glass volumetric flasks of
10, 20, 50, 100...500 mL, and plastic transfer pipettes. Iron Cell Test Kit from spectroquant.

8
 Figure 1. Steps involved in the principle chemical reaction12

Mechanism:13 Ascorbic acid contains OH group, the hydrogen present in this OH group is
taken up by the Fe3+ resulting in the formation of Fe2+. The mechanism involved is reduction
reaction. The thioglycolate present herein the reaction is acting as a buffer, when the reduction
reaction is taking place i.e. conversion of from Fe 3+ to Fe2+ this is indicated by the color change
conforming that reduction reaction.

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3. Procedure
The following is a narrative of the procedure that was developed. It is written in a style may
be used for the future (see below) researchers or students who wish to perform this
procedure.

3.1 Preparation of calibration curve

1. From a 1000 ppm stock solution of Fe3+ in 1% HNO314, solution of concentrations of 0.5,
1.0, 2.0, 3.0, 4.0 ppm was prepared

2. The standards were treated according to Iron Cell Test kit instructions and the absorbance
was measured for each sample at 565 nm.

Absorbance vs. concentrations was plotted and the y-intercept was obtained slope and
correlationwere measured for each sample at 565 nm.

3.2 Preparation of samples for measurement

1. Water samples were collected from the ground water using (a clean 100 mL
polyethylene containers provided by the instructor). For tap water, the water was collected
after the tap was run for 2 minutes. For another source of water, such as a pond or river, the
water was collected using nitrile glove to protect the sample from contaminated with dirt and
bacteria fromour hands.

2. The container was labeled with name, date, and location of the sample.

3. From the water samples, 10 mL was dispensed into a small beaker and the pH was
measured.The pH (must be within the range 1-10).

If necessary the pH was adjusted with sodium hydroxide (6.0 M) solution or (6.0M) optimally,
the pH was adjusted to 7.

4. Further work was performed on a 25 mL aliquot of the sample.

5. If there were suspended solids, the 25mL aliquot was filtered using a 0.45 µm polyethylene
or Teflon filter.

6. The 25 mL aliquot was treated with 0.1 mL of HNO3 (0.1%v/v).7. Then, 5.00 mL was pipette
into a pre-prepared test tube containing the buffer ammonium thioglycolate and thioglycolic
acid.

8. The test tube was tightly capped and mixed well until the reagent and sample were
completelycombined.

9. The samples were left for 3 min. If the iron was present we will observe the formation of

10
apurple solution.

10. The sample was measured in the UV- Visible spectrophotometer with absorbance at 565 nm.

11. The dissolved iron concentration was calculated from the above calibration curve.

12. For reproducibility check, the procedure was repeated with a 25mL aliquot of the
samplesolution.

3.3 Limit of detection (LOD) and limit of quantification (LOQ).

The LOD is defined as the lowest amount of the analyte in a sample that can be detected but not
necessarily quantified. The detection limit is determined by the analysis of sample with known
concentration of analyte and by establishing the minimum level at which the analyte can be
reliably detected.

A signal to noise (S/N) ratio analysis is performed by comparing measured signals from samples
with known low concentrations of the analyte and with the blank samples. By establishing
the minimum concentration at which the analyte can be reliably detected, a S/N ratio of 3:1
was usedin this study.

Where S = height of the signal, and, N = height of the noise.2

The LOQ was determined as the minimum concentration of analyte in a sample that can be
quantified with acceptable precision and accuracy under the stated operational conditions of
the method. The quantitation limit was determined by the analysis of sample with known
concentration of analyte and by establishing the minimum level at which the analyte can be
reliably quantitated.

The S/N ratio is performed by comparing measured signals from samples with known low
concentration of analyte with those of blank samples and by establishing the minimum
concentration at which the analyte can be quantitated; an S/N ratio of 10:1 was used in this
study. After measuring the signal noise ratio which was 0.0002 mAu and then the
concentrations were calculate by using standard calibration curve and the absorbance of the
signal noise ratio the LOD and LOQ values are 0.05 ppm and 0.50ppm

11
 Figure 2. Signals for LOD and LOQ15

4. Results:

Figure 3. Peak of the real sample concentration 0.75 ppm15

12
 Figure 4. Peak of the standard concentration 1.0 ppm15

The results of sample set I that we measured in this study are shown in Table 1.

Table 1. Absorbance and concentration of sample 1 to 7 (set I)

Sample no. Absorbance (mAU) Concentration (ppm)

1. Standard 0.436 1.00
2. S-1 0.092 0.45
3.S-2 0.0201 0.13
4.S-3 0.842 3.84
5.S-4 1.24 5.64
6.S-5 1.48 6.72
7.S-6 1.27 5.78

The results of the samples set II that we measured in this study are shown in Table 2

Table 3. Absorbance and concentration of samples 1 to 5 (set II).

Sample no.b Absorbance (mAU) Concentration (ppm)

1. S-1 0.0073 0.11
2. S-3 0.0058 0.11
3. S-4 0.0194 0.14c
4a.S-5 0.0056 0.12
5. S-6 0.0074 0.11

13
 Initially sample 4 showed a high reading of .097 ppm of iron, but on observing the sample
solution which showed lot of sediments in it so, the sample was allowed to settle and then the
concentration of settled (unmixed) sample had dropped to 0.14 ppm. Then after mixing the
sediment in the sample the concentration of iron again raised to 0.97 ppm so, the sample was
filtered in order to get rid of the sediment which upon gave a concentration of 0.12 ppm.
This results ismathematically consistent with the associative properties of the y= mx+b equation
especially since the y- intercept term is Non- zero.
Table 4. Error analysis for samples (set II)

Conc Abs 1 Abs 2 Abs 3 Error

±0.0007
0.11 0.0061 0.0061 0.0073
±0.0003
0.11 0.0053 0.0059 0.0058
±0.0039
0.14 0.0127 0.0127 0.0194
±0.0006
0.12 0.0048 0.0059 0.0056
±0.0000
0.11 0.0074 0.0074 0.0074

Table 4 describes the error analysis for given set of samples that were analyzed.

Error was calculated using the following formula

Highest observed absorbance – Lowest observed absorbance

Square root of the no of measurements

Figure 5. Peak for sample 4 (set II) concentration 0.97 ppm

14
Figure 6. Peak for sample 4 (set II) after filtration

Figure 7. Calibration curve of iron standards samples.

15
5. Discussion:

Figure 6. Photograph of set I blank, standards and samples

Figure 6 Photograph of the set I blank, standards and samples treated according to the procedure in
this study. Arranged left to right: a) blank which contains purified water and the chelating agent, b)
sample 1 (D3 So), c) sample 2 (city) taken directly from the city , d) the 1.0 ppm standard solution, e)
sample 2 (C3 So), f) sample 3 (F1210), g) sample 4 (F1206), h) the 3.0 ppm standard, i) sample 5
(F1208), j) the 4.0 ppm of standard.

From the result set I samples except for the sample 1 (D3 So) and sample 2 (city) contain high
concentration of iron. Figure 6 shows 10 solutions that were analyzed in set I. The samples are
arranged according to the color and the trends shows the sample that contain more iron it appears
progressively deeper blue in color.

Here set II, five water samples were given , labeled as S-1, S-3, S-4, S-5 and S-6. All samples were
acidified to a concentration of 0.1 % (v/v) with HNO3 and then treated with a chelating agent to yield
a violet colored solution. Detection of all samples were performed on UV-Visible spectrometer at
565 nm. Once the water samples concentrations were determined, the results were compared with
EPA guidelines for recommended amount of dissolved iron (< 0.3 ppm). For samples1-3 and 5, the
water was considered below the EPA guideline. However, sample 4 had a concentration of

16
0.97 ppm. In this sample, visible sediments were found. We took additional action in determining
the cause of the high concentration. We decided to further test sample 4. We filtered the sample
and this was analyzed to 0.11 ppm. Additionally, we obtained a sample in which the water was run
from the tap for 2 min. This we measured to give a concentration of 0.14 ppm.

6. Conclusion
The limit for dissolved iron in drinking water is 0.30 ppm. As per our result, all the samples
concentrations in set I are much higher than the limit of iron in drinking water except sample 2 and
all samples concentrations in set II are in range of 0.10 to 0.15 ppm. There was one exception in which
sample 4 contained suspended solids that initially gave a high dissolved iron concentration (0.97
ppm). However, after filtration and also after having water run from the tap for 2 min, the iron
concentration decreased to acceptable levels. This means all samples pass the EPA recommendation
for dissolved iron drinking water. In addition, our analysis indicates that water containing suspended
solids may result in high dissolved iron concentrations. Overally, if the water is left running for two
minutes, the sediments are able to be flushed and an sample that is within the EPA concentration
guideline is obtained. Finally, the water samples that tested were found to have dissolved iron concentration
belowthe EPA limit of 0.3 ppm.

17
References
1
Gunnar Nordberg; Bruce Fowler; Monica Nordberg. Handbook on the toxicology of metals, 4th ed.;
Amsterdam, Elsevier, 2014,Chapter 41, Iron. pp 879-902.
2
Ibrahim A.Q.; Onyenekwe P.C.; Nwaedozic I.M. An Efficiency Assessment of Lower Usuma
Water Treatment Plant in AbujaMetropolis, Nigeria.
3
U.S. EPA. Secondary Drinking Water Standards: Guidance for Nuisance Chemicals,
4
Fawell, J.K; Land.U; Mintz, B. Iron in Drinking water. Back ground document for development of
WHO Guidelines for Drinking Water Quality. (online); Geneva, 2003.
5
International Organization for Standardization, Water quality—determination of iron
6
Iron and water: reaction mechanisms, environmental impact and health effects,
7
. Weaver LC, Comparative toxicology of iron compounds 1961.
8
World Health Organization. Iron in drinking-water. Background document for preparation of
WHO Guidelines for drinking-waterquality, 2008. Geneva, World Health Organization .
9
U.S. EPA. Ground Water and Drinking Water: National Primary Drinking Water
Regulations.
10
Joint FAO/WHO Expert Committee on Food Additives, Toxicological evaluation of certain food
additives and food contaminants,
11
Spectroquant Iron Cell Test 1.14549.0001 Merck KGaA
12
Klepo, L.; Copra-Janicijevic, A.; Kukoc-Modun, L., A New Indirect Spectrofluorimetric Method
forDetermination of Ascorbic Acid with 2,4,6-Tripyridyl-S-Triazine in Pharmaceutical
13
Drits, V. A.; Manceau, A., A model for the mechanism of Fe3+ to Fe2+ reduction in dioctahedral
smectites. ClaysClay Miner. 2000, 48, 185-195. Environmental Geochemistry

14Preparation of Fe3+ standard stock solution: Fe wire (1.000 g) is weighed and dissolved in conc.
HNO3 (10 mL). If necessary, gently heat the solution until the wire is completely dissolved, cool to
room temperature and quantitatively transfer the solution to a 1000 mL volumetric flask. Dilute to
1000 mL with purified water.

15. Shrivastava, A., Gupta, V. Methods for the determination of limit of detection and limit of

quantitation of the analytical methods. Chron. Young Sci.

18