UNIVERSITY OF SANTO TOMAS

Faculty of Engineering

The Development of an Iron Detection Method in Liquid Samples

An Undergraduate Study
presented to the
Department of Chemical Engineering
Faculty of Engineering
University of Santo Tomas

In Partial Fulfillment
of the Requirements for the Degree of
Bachelor of Science in Chemical Engineering

by

Gallardo, Nadine Jenina A.

Monakil, Gemma Fae T.
Surla, Sheena I.

April 2019
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING i

APPROVAL SHEET

In partial fulfillment of the requirements for the Degree of Bachelor of

Science in Chemical Engineering, this thesis entitled “The Development of an Iron
Detection Method in Liquid Sample”, prepared and submitted by Gallardo, Nadine
Jenina A., Monakil, Gemma Fae T. and Surla, Sheena I., is hereby recommended
for approval.

__________________________________
Prof. Edna C. Quinto, Ph.D, ASEAN Eng
Thesis Adviser

Accepted as partial fulfillment of the requirements for the Degree of Bachelor of

Science in Chemical Engineering.

_______________________ _______________________
Engr. Cristina E. Tiangco, Ph.D Engr. Rose Ann Z. Tamolang
Department of Chemical Engineering Department of Chemical Engineering
Panelist Panelist

_______________________
Engr. Mildred A. Ebuengan
Entrepreneur, Owner EM6 Flight Enterprise
Panelist

_________________________
Engr. Mark Emile H. Punzalan
Chair, Department of Chemical Engineering
University of Santo Tomas
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING ii

ACKNOWLEDGEMENT

We would like to express our sincerest gratitude to the following people

who vitally assisted us in materializing our research study entitled, “Development
of an Iron Detection Method in Liquid Samples.”

To our thesis adviser, Prof. Edna C. Quinto, PhD., ASEAN Eng., the
accomplishment of this study would have not been possible without your
guidance, undying support, understanding, and precious time. We express our
sincerest gratitude for sharing us your expertise and for assisting our group in
every experiment that we had to do. We have been privileged with such an
adviser who is extremely patient especially during the times we had to stay until
late night in order to finish our experiments, and for never doubting us to do this
study.

To our panelists, Engr. Rose Ann Z. Tamolang, Engr. Cristina E. Tiangco,

Engr. Mildred A. Ebuengan, for generously exerting time and effort in assessing
our thesis, and for sharing your expertise, opinions, and suggestions in order for
us to improve our study.

To the Research Center for the Natural and Applied Sciences (RCNAS), for
aiding us during the experimentation phase of our research study and for giving us
permission in using the necessary equipment that we needed for our study.

To Mr. Florencio De los Reyes, M.Sc., for allowing us to use the UV-Vis
Spectrophotometer available in Lab 11 and work in the balance room especially in
urgent times. Also, for always reminding us the proper rules when working inside
the laboratory.

To our very hard-working laboratory technicians, for patiently waiting for

us to finish our experiments in the laboratory until the evening, and for providing
us all the necessary materials that we needed during our experiments.
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING iii

To our friends, for always motivating us to keep going and giving us

assistance during our hardships while conducting this study, and for reminding us
that we are not alone in this journey.

To our beloved families, we would like to express our sincerest gratitude

for your unending support, patience, understanding the times we cannot show in
family gatherings, providing everything especially our financial needs during the
study, and for trusting us.

Lastly, to the Almighty God, for surrounding us all these good people who
contributed to the completion of our study. We express our deepest gratitude to
You for always giving us the grace to continue this research despite all the
problems we have encountered. Thank you for guiding us all the way and thus, all
praises and the achievement of this study is given unto You.
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING iv

ABSTRACT

The study shows the response of standards of Iron (III) to excess ammonium

thiocyanate solution based on UV-Vis spectrophotometer. The method employs

3M thiocyanate solution as the recognition element for iron (III). A wavelength

scan in Lambda 25 Perkin Elmer spectrophotometer showed optimum reading at

the wavelength of 480nm. The method shows an RSD of 6.56% when a 20ppm

iron (III) standard was read continuously for 5 minutes. The signal is repeatable

with an RSD of 8.12% when the 20ppm standard is compared to a blank solution.

The detection method is sensitive with a calibration curve of y = 0.0044x-0.0423.

The calculated LOD is 3.38M/ΔAbs for the dynamic range of 10ppm to 35ppm. The

linearity, R2 is only 0.9274, this may be improved at different concentration ranges

close to the target samples for which the detection method may work. The figures

of merit of the method show promising application for iron (III) detection.

Preliminary steps were done to test the method on milk samples. However the

opacity of the milk or casein shows the reverse response. Probably other

components of milk are interfering with the reaction with the thiocyanate

reaction. Further steps are therefore recommended for the use of the method.
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING v

TABLE OF CONTENTS

List of Tables ………………………………………………………………………………………………...................vii

List of Figures …………………………………………………………………………………………….................….vii
Chapter 1: The Problem ………………………………………………………………………………………………..1

1.1 Introduction ……………………………………………………………………………………………….1

1.2 Objectives ………………………………………………………………………………………………….2
1.3 Significance of the Study ……………………………………………………………………………3
1.4 Theoretical Framework ……………………………………………………………………………..3
1.5 Hypothesis …………………………………………………………………………………………………4
1.6 Scope and Limitation ………………………………………………………………………………….4
1.7 Definitions of Terms ……………………………………………………………………………………5
Chapter 2: Review of Related Literature ………………………………………………………………………7
2.1 Oxidation of Ferrous Oxide …………………………………………………………………………7
2.2 Hydrogen Peroxide …………………………………………………………………………………….8
2.3 Effects of Iron in the Human Body ……………………………………………………………..8
2.4 Effects of Iron in Water on the Aquatic Life ………………………………………………..9
2.5 Sources of Iron and Its Effect on the Environment ……………………………………..10
2.6 Sensors ………………………………………………………………………………………………………12
2.6.1 Optical Sensor …………………………………………………………………………….12
2.6.2 Sensor Properties ……………………………………………………………………….12
2.6.2.1 Accuracy ……………………………………………………………………..13
2.6.2.2 Resolution …………………………………………………………………..13
2.6.2.3 Reliability ……………………………………………………………………14
2.7 Spectrophotometric Methods …………………………………………………………………….14
2.8 Performance Characteristics of a method of a Sensor ………………………………..14
2.8.1 Stability ……………………………………………………………………………………….14
2.8.2 Repeatability ……………………………………………………………………………….15
2.8.3 Sensitivity …………………………………………………………………………………….15
2.9 Other Methods of Iron Detection Published in Scientific Journals ……………….16
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING vi

Chapter 3: Methodology ……………………………………………………………………………………………… 17

3.01 Preparation of Reagents ……………………………………………………………………………18

3.02 Instrumentation System ……………………………………………………………………………19

3.0 Determination of Suitable Wavelength ……………………………………………………….20

3.1 Stability Test ………………………………………………………………………………………………..20

3.2 Repeatability Test ……………………………………………………………………………………….21

3.3 Sensitivity Test …………………………………………………………………………………………….22

3.4 Preparation and Preliminary Test for Real Sample ………………………………………23

Chapter 4: Data and Results ………………………………………………………………………………………….25

4.0 Determination of Suitable Wavelength ……………………………………………………….25

4.1 Stability Test ……………………………………………………………………………………………….26
4.2 Repeatability Test ……………………………………………………………………………………….28
4.3 Sensitivity Test ……………………………………………………………………………………………29
4.4 Preliminary Test Using Real Sample …………………………………………………………….33
Chapter 5: Conclusion and Recommendation ……………………………………………………………...36
5.1 Conclusion …………………………………………………………………………………………………..36
5.2 Recommendation ………………………………………………………………………………………..37
References …………………………………………………………………………………………………………………….39
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING vii

LIST OF TABLES

Table 3.1. Data of Milk Sample

Table 4.1 Summary of the features of the method

LIST OF FIGURES

Figure 3.1 Perkin Elmer UV/VIS Spectrometer Lambda 25 interfaced with a

desktop computer

Figure 4.0 Plot of absorbance vs wavelength (nm)

Figure 4.1.1 Stability Test

Figure 4.2.1 Plot of Repeatability Test (n=6)

Figure 4.3.1 Calibration Curve for ∆Abs Range: 0.0106 to 0.1235 (n=3)

Figure 4.3.2 Calibration Curve for ∆Abs Range: 0.0106 to 0.1235 with error bars

Figure 4.3.3 Visual observation of different concentrations of iron solution in ppm

Fig 4.4.1 Absorbance vs. Time of Nestogen milk sample

Fig 4.4.2 Absorbance vs. Time of Enfagrow milk sample

Fig 4.4.3 Absorbance vs. Time of Bonakid milk sample

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 1

CHAPTER 1
THE PROBLEM

1.1 Introduction

Iron is a vital nutrient that is present in almost all aspects of our lives. Its main

role is in the production of hemoglobin in red-blood cells and for oxygen transport

mechanism in the blood for all vertebrate and some invertebrate animals [1].

However, iron in excess can cause significant effects ranging from liver damage to

arthritis as a result of iron deposition in organs or joints and also, it will be an

environmental concern. Considering this fact, the presence of iron in different

environments needs to be carefully monitored. Although there are several existing

methods in detecting different mineral, these current techniques require the use

of expensive equipment and must be done by highly specialized personnel. In

addition, different samples may require different approaches in using existing

methods [2].

Due to the importance of heavy metals at trace level in the human health and

environment, the sensitive and accurate determination of the levels of heavy

metals in the environmental samples have been continuously carried out on the

analytical and environmental laboratories around the world. [3-5]. Among heavy

metals, iron in small amounts is an essential element for most life on Earth,

including humans and animals. It is well known that an iron deficiency is the most

common cause of anemia. On the other hand, too much iron can cause several
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 2

health problems. High levels of iron are associated with an increased risk for

cancer, heart disease and other illnesses such as endocrine problems, arthritis,

diabetes and liver disease. [6]

Several analytical techniques such as flame atomic absorption spectrometry

(FAAS), graphite furnace atomic absorption spectrometry (GFAAS), and inductively

coupled plasma mass spectrometry (ICP-MS) have been proposed for the

determination of trace and toxic metals in different environmental sample. The

determination of trace quantity of heavy metals in environmental samples, like

natural water and other real samples of environmental interest in which they are

found at very low concentrations, requires the use of pre-concentration methods

coupled to spectrometric methods, such as ICP-AES (inductively coupled plasma

atomic emission spectrometry) and FAAS. [7-9]

The present work describes the attempt for the determination of iron ions

from liquid samples using ammonium thiocyanate as the indicator. After

optimization of experimental variables and determination of analytical

characteristics, the method will be preliminarily tested for application in real

samples.

1.2 Objectives

1.2.1 To determine the working wavelength for this method

Chapter 1. The Problem

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 3

1.2.2 To measure the accuracy and reliability of the developed method

1.2.3 To determine the limit of detection

1.2.4 To prove the feasibility of the method

1.3 Significance of the Study

Iron is an element that is present only in minute amounts and is required

by both plants and animals. It is an essential in oxygen transport mechanism in the

blood for all vertebrate and few invertebrate animals [10]. There are several

existing methods in determining different minerals in liquid samples but we intend

to find an alternative that gives the result without the required protocol of

analytical methods. Thus, this study will be a significant endeavor in modifying the

existing methods used in detecting minerals in liquid sample, specifically iron.

1.4 Theoretical Framework

The ultraviolet-visible spectrophotometer is the device that will be used to

determine the changes in absorbance in the liquid samples. The principle behind is

known as the UV-Vis Spectrophotometry. As stated by Worsfold (2005)

“Ultraviolet–visible (UV–visible) spectrophotometry is primarily a quantitative

analytical technique concerned with the absorption of near-UV (180–390 nm) or

visible (390–780 nm) radiation by chemical species in solution” [11]. Some organic

and inorganic species inherently absorb radiation in the UV-vis range, and the

Chapter 1. The Problem

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 4

technique can be extended to non-absorbing analytes by exploiting a selective

reaction of the analyte with an appropriate reagent to form an absorbing chemical

species. [11]

Optical sensors are powerful analytic tools capable of providing analyte

information remotely. Compared to other types of sensors that use electronic,

electrochemical, or mechanical transduction, optical sensors are particularly

beneficial for remote and multimodal detection, which is preferred in chemical

and biological applications. In the development of optical sensors, it is important

to evaluate using common performance indicators such as sensitivity, selectivity,

and response time [12].

1.5 Hypothesis

Ammonium thiocyanate can be immobilized and be used as an active

reagent of an optical sensor for iron detection in liquid samples. Being the active

reagent of the optical method, ammonium thiocyanate will react and show visible

change in color of the solution when exposed to certain concentration of iron.

1.6 Scope and Limitation of the Study

This study will be conducted to develop an iron detection method in liquid

samples. Preliminary steps would be done to test the method on milk samples.

Although, other components of milk that might interfere and obstruct with the

Chapter 1. The Problem

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 5

reaction with the thiocyanate reaction, we would like to state that this aspect of

the study would be beyond the scope and limitation of the study. Furthermore, a

non-selective type of method would be develop, thus the iron present in a sample

will have to be isolated if possible. The actual data gathering of the study using a

Lambda 35 Perkin Elmer UV-Vis Spectrophotometer with UV-WinLab Software

would be conducted at the Research Center for the Natural Sciences of Thomas

Aquinas Research Complex of the University of Santo Tomas (UST). The

experimentation and data gathering would be conducted from October 2018 to

February 2019.

1.7 Definition of Terms

Iron. The most common metallic element in the universe. Pure iron is a soft,

lustrous, dark silvery-gray metal. Iron is a strongly reactive metal, being very

reactive with acids, and forms oxides, commonly known as rust, with air and

water.

Ammonium Thiocyanate. It is an acutely toxic, colorless crystalline solid that is

soluble in water. The odorless inorganic compound has many industrial and

consumer uses that include chemical analysis.

Chapter 1. The Problem

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 6

Sensor. It is a device that responds to a signal. These devices do not detect

substances. These are used in connection with other apparatuses to form a

detector.

Spectrophotometer. An instrument that determine the light absorbed and

transmitted by a sample solution. It is a device that is used to determine the

amount of a specific substance in a solution. It cannot be used to detect colorless

substances especially when the substance has turbidity.

Chapter 1. The Problem

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 7

CHAPTER 2
REVIEW OF RELATED LITERATURE

This chapter contains the main principles about optical sensing, its

properties, and spectrophotometric methods.

The oxidation of ferrous iron, the use of hydrogen peroxide, effects of iron

in the human body and in water on the aquatic life, as well as other sources of

iron and its effect to the environment are also discussed in this chapter. The

different methods presented in this chapter for determining the presence of iron

are those widely accepted and usually used by analytical chemists.

2. 1 Oxidation of Ferrous Iron

Iron in the ferrous oxidative state that is free to participate in Fenton

chemistry is a major source of oxidative stress in media. The reaction involves the

ferrous iron catalyzed conversion of hydrogen peroxide into a hydroxide ion and a

hydroxyl free radical with the concurrent oxidation of ferrous iron to ferric iron.

This reaction is profoundly important in cell culture, because when it occurs, it

creates the most damaging agent found in the cell culture system-the hydroxyl

free radical. This radical will react with virtually any molecule in the medium or

the cell. It is so reactive that it typically reacts very close to its site of formation. A

major action of hydroxyl free radicals in cell culture involves the initiation of lipid

peroxidation. Ferrous iron mediated lipid peroxidation can occur with iron in a
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 8

number of chelated complexes: the ferrous iron can be chelated to phosphate,

ADP, ATP, oxalate, and citrate. [13]

2.2 Hydrogen Peroxide

Hydrogen peroxide is an important reactant in natural aquatic systems,

environmental remediation technologies, and in biological systems. Reactive

oxygen species (ROS), which include hydrogen peroxide (H2O2) and hydroxyl

radicals (·OH) occur in rain and surface waters [14, 15]. The generation of ·OH, via

the decomposition of H2O2, degrades contaminants when methods involving

microbiological degradation are ineffective. H2O2 is relatively stable, in the

presence of ferrous iron, H2O2 forms ·OH via the Fenton reaction:

Fe (II) + H2O2 → ·OH + OH- + Fe (III) (1)

·OH is far more reactive than H2O2 and its reaction with aqueous species is

diffusion limited. Therefore, H2O2, in the presence of Fe (II), represents great

potential for reactivity [16].

2.3 Effects of Iron in the Human Body

Iron is vital for almost all living organisms by participating in a wide variety

of metabolic processes, including oxygen transport, DNA synthesis, and electron

Chapter 2. Review of Related Literature

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 9

transport. Disorders of iron metabolism are among the most common diseases of

humans and encompass a broad spectrum of diseases with diverse clinical

manifestations, ranging from anemia to iron overload and, possibly, to

neurodegenerative diseases. [17]

Heightened awareness in recent years of the adverse consequences of iron

deficiency has prompted renewed efforts to reduce the prevalence of this

common micronutrient insufficiency. One of the main reasons for the limited

success of programs to combat iron deficiency is the continuing uncertainty about

the optimal epidemiologic approach for identifying it and for measuring its

severity. In a 1985 World Health Organization (WHO) report, it was estimated that

15% to 20% of the world's population had iron deficiency anemia. Current

initiatives to reduce the high prevalence of nutritional iron deficiency have

highlighted the need for reliable epidemiologic methods to assess iron status.

[18].

2.4 Effects of Iron in Water on the Aquatic Life

Iron exists in two oxidation states in the aqueous environment: soluble or

reduced ferrous iron (Fe2+) and insoluble or oxidized ferric iron (Fe3+). In

oxygenated waters, ferrous iron (Fe2+) oxidizes to ferric iron (Fe3+) which is capable

of supporting aquatic life [19] [20]. It naturally occurs at a concentration of 1-3

parts per billion in ocean. It might likewise be discharged to water from natural

Chapter 2. Review of Related Literature

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 10

deposits, industrial wastes, and refining of ores. This discharge makes the level of

iron much higher and can cause harm in the marine ecosystem.

Iron is important to the marine ecosystem especially for mollusks and

green plants. The oxygen bound with iron travels in the blood transporting the

carbon dioxide out. Green plant uses iron for nitrogen binding while

phytoplankton depend vigorously on iron that the measure of iron present in

water restricts the amount of phytoplankton that can survive. However, when the

iron concentration is above 0.1 parts per million, this will potentially damage the

gills of the fish.

The toxicity of iron is dependent on the specie and the size of the fish.

Small amount of ferric iron particles can cause irritation of the gill which leads to

gill damage and secondary bacterial and fungal infections of fishes. Although the

extent of the damaged that can be caused by the physical presence of iron has

limited study as of the moment [20].

2.5 Sources of Iron and its Effect to the Environment

Iron is an important element and a fundamental part of the natural

environment and numerous important chemical reactions that includes being

transferred from other minerals, water or biological agents. Its environmental

Chapter 2. Review of Related Literature

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 11

impacts on physiology and ecology of aquatic organisms as well as with the

agroecosystems are an important issue [21].

Iron is a trace element as well as an example of a heavy metal. Trace

elements are elements present at low at low concentrations (mg kg−1 or less) in

agroecosystems. They are also called micronutrients and are crucial for plant

growth. Trace elements in an agroecosystem are either inherited from soil parent

materials or inputs through human activities. Heavy metals when repeatedly used

may cause contamination to the environment at a large scale [21].

There are many iron sources in freshwater lakes. These may include: the

products of weathered rocks and soil around watersheds, controlled by many

factors, such as geological process, soil composition, environmental temperature,

precipitation, and hydrology [22]. Due to its low solubility, iron concentration in

water is quite low despite being the second most abundant metal and fourth most

abundant element in the Earth's crust. Generally, iron concentrations in natural

freshwaters do not exceed 1 mg L-1. However, due to population explosion, rapid

urbanization and growth of industries disparity of iron concentration and different

environmental problems occur. Thus, the structure and function of the different

bodies of water ecosystems are altered as a result of the variations of its iron

content [23].

Chapter 2. Review of Related Literature

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 12

2.6 Sensors

One should initially realize where there is an unnaturally huge measure of

specific substances. A sensor is typically characterized as a device that receives

and responds to a signal or stimulus. To be closer to the purpose of this study we

may define it as a device that receives a stimulus and responds with a beam of

light. [24]

2.6.1 Optical Sensor

Optical sensors depend on electromagnetic radiation's

collaboration with matter that results in modifying or changing a few

properties of the radiation. A portion of these varieties are varieties

intensity, polarization, and speed of light in the medium. The sorts of

chemical sample to be analyzed affects which wavelengths of light are

modulated [24].

2.6.2 Sensor Properties

Various properties and capacities must be fixed and set in order to

have a functional and good sensor. Properties like accuracy, resolution and

reliability should all be enhanced to have an exceptionally proficient

Chapter 2. Review of Related Literature

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 13

sensor. A sensor is optimized for an extensive stretch of time for it to have

its properties at acceptable levels [24].

2.6.2.1 Accuracy

Accuracy alludes to the deviation of a value represented by

a sensor from the ideal or true value from its input. Accuracy simply

means the closeness of the value given by the sensor to the true

value. The higher its accuracy, the better the data acquired using

the sensor. It is like contrasting the closeness of an experimental

value to a literature value [24].

2.6.2.2 Resolution

Resolution describes the smallest amount of stimulus it can

sense. Ideal sensors should be able to sense small amounts of

stimulus though it is not necessary to always have that resolution.

Depending on the intended use, a sensor can have a higher

resolution in it is meant to detect a certain species of chemical at

higher concentrations [24].

Chapter 2. Review of Related Literature

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 14

2.6.2.3 Reliability

Reliability is the capacity of a sensor to perform a required

function under stated conditions for a state period. It is the length

of time in which the sensor will perform to the best of its

capabilities or with minimal or acceptable error under the

conditions it was set for [24].

2.7 Spectrophotometric Methods

These sorts of techniques utilize light sensors so as to gauge absorbance of

liquids dependent on dissipating light as it goes through a cuvette that contains

the liquid sample. It works by passing a beam of light through the sample inside

the cuvette and estimating the intensity of light reaching a detector. Correlation

between the absorbance and concentration of standard examples could be used

for the determination of the absorbing species in a natural sample.

2.8 Performance Characteristics of a Method of Sensor

2.8.1 Stability

Stability expresses variation of the measured mean as a function of

time. The term stability has been defined as the stability of the samples

Chapter 2. Review of Related Literature

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 15

being analyzed in a sample solution. It is a measure of the bias in assay

results generated during a preselected time interval.

2.8.2 Repeatability

Repeatability is the ability of the sensor to represent a value under

identical conditions. It is the ability of the sensor to give the same values in

the conditions is not changed. If the sensor’s output changes even if the

sample and other parts and properties of the setup was not changed, then

the said sensor has low repeatability. A sensor should ideally give the same

output given the same conditions any number of times.

2.8.3 Sensitivity

Sensitivity identified with the test's ability to recognize positive

outcomes. Sensitivity analysis is the study of how the uncertainty in the

output of a scientific model or framework (numerical otherwise) can be

distributed to various sources of uncertainty in its inputs [25].

Chapter 2. Review of Related Literature

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 16

2.9 Other Methods of Iron Detection Published in Scientific Journals

Knowing the composition of an element is vital for the assessing its

biological activity. Iron, being an important trace element, speciation methods is

needed, because bioavailability and metabolism are strongly dependent on the

species. Different procedures and techniques based on spectrophotometry,

voltammetry or aerometry have been offered for the determination of iron on the

oxidation state [Fe (II)/ Fe (III)].

In the study of Weber, a combined chromatographic detection system is

proposed. This method allows the determination of different iron complexes by

combining the use of an electrochemical detector with an on-line flame-AAS

detector. While the element-specific AAS detector determines the total iron

content, the electrochemical detector then selectively detects Fe (II) by oxidation

to Fe (III) [26].

Another method for the detection of iron, is with the use of 1, 10-

Phenanthroline. A solution containing both iron (II) and iron (III) will turned a

reddish-orange iron (II) complex and yellow iron (III) complex when 1, 10-

Phenanthroline is added. Both iron (II) and iron (III) complex had the identical

absorbance coefficients of 396 mµ. A study conducted by Harvey, Smart and Amis,

enables the determination of iron (II) and total iron in the same solution by

simultaneous measurements of absorbance at 396 mµ and at 512 mµ [27].

Chapter 2. Review of Related Literature

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 17

CHAPTER 3
METHODOLOGY

This chapter describes and defines each research method that was

followed through the course of the study. From the preparation of reagents,

instrumentation system that was used for data collection, the determination of

suitable wavelength, the stability, repeatability and sensitivity tests, as well as the

preparation and preliminary test for the real sample all were clearly discussed in

this chapter. Lastly, the researchers also discussed the methods that were used to

analyze the data.

The type of research that was conducted was both qualitative and

quantitative in nature. It is said to be qualitative due to the fact that the

researchers analyzed the claimed amount of iron. On the other hand, it is

quantitative since the researchers detected the amount of Iron by the optical

response (absorbance) in the liquid samples with the use of a UV-VIS

spectrophotometer. In this study, the researchers used the experimental method

of research. The method was to create solutions of varying iron concentrations

and the addition of thiocyanate for the solution to show varying coloration.

Concentrations of both the iron solutions and thiocyanate solution are known. The

optical density of the solutions was then identified using the said instrument by

allowing both the iron and thiocyanate solution to turn red. Thus, generating a
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 18

calibration curve that determined if the liquid samples contain any iron as

claimed.

3.01 Preparation of Reagents

Ferric Ammonium Sulfate (NH4Fe (SO4)2), obtained from Merck, Darmstadt

and Ammonium Thiocyanate (NH4SCN), obtained from AJAX Australia, were

prepared to be used as the standard solutions in the study.

Iron solutions of standard concentrations were prepared using ultra-pure

water. The standard iron solutions used were in the form of ferric ammonium

sulfate (NH4Fe (SO4)2). Ferric ammonium sulfate was dissolved in an ultra-pure

water to make a stock solution with a concentration of 1273 ppm. Other

concentrations of iron, 35, 30, 25, 20, 15 and 10 were derived from the stock

solution by serial dilutions.

Variously, the thiocyanate solution was prepared by dissolving the

ammonium thiocyanate crystals in ultra-pure water to obtain a concentration 500

ppm solution. The indicator was prepared according to its solubility to water.

1.3651 g of NH4SCN was weighed then dissolved using ultra-pure water

(Millipore). Further preparation of 3M ammonium thiocyanate was done by using

the prepared stock solution as its solvent. Ammonium thiocyanate preparation

was carried out in room temperature by manual mixing.

Chapter 3. Methodology
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 19

3.02 Instrumentation System

The instrumentation system for the analysis of iron is composed of a

personal computer connected to a Lambda 25 Perkin Elmer UV-Vis

Spectrophotometer. The equipment used was shown in Figure 3.1. The sample

was placed inside a cuvette and was then introduced into UV-Vis

Spectrophotometer. The spectrophotometer was interfaced with a personal

computer for processing. Data acquisition and off-line analysis were performed

using Microsoft Excel (Microsoft Corporation, USA). Spectra readings were taken

from the chosen range of wavelengths which was from 385 nm to 750 nm. All

spectroscopic experiments were performed at room temperature. HTL and

Transferpette® Micropipettes were used for accurate measurement of the

amounts of ammonium thiocyanate and ferric ammonium sulfate to be tested.

Figure 3.1 Perkin Elmer UV/VIS Spectrometer Lambda 25 interfaced with a

desktop computer

Chapter 3. Methodology
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 20

3.1 Determination of Suitable Wavelength

In order to determine the working wavelength, spectrum analysis was

done in two different conditions. In the first condition, the sample that was tested

was the 20 ppm iron solution. For the first condition, a cuvette was filled with 2mL

iron solution. Spectra readings were taken from 385 nm to 750 nm which is the

range for the visible region. The other condition was the addition of 150 μL of

ammonium thiocyanate in the 2000 μL iron solution. The absorbance readings

obtained in the two conditions were plotted against the wavelength range

considered (385 to 750 nm). The two resulting spectra were compared to plot

their difference. The wavelength that gave the largest difference between the two

absorbance readings was the working wavelength used in the succeeding sensor

tests. The working wavelength was determined graphically and was represented

by the abscissa with the largest vertical difference occurred. All of the succeeding

absorbance measurements throughout the experiments were read at the working

wavelength.

3.1 Stability Test

Stability was determined by plotting the absorbance with time. The stability

of the signal at the chosen wavelength was tested by measuring the absorbance of

the ferric ammonium sulfate solution using the middle concentration of the

diluted iron solution which is 20 ppm. An amount of 150 μL ammonium

Chapter 3. Methodology
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 21

thiocyanate was added to 2000 μL of the iron solution. This was tested for 120

minutes to check for its stability.

The stability was checked with the use of the relative standard deviation of

the absorbance readings during the 120 minute run of the sample. The relative

standard deviation was calculated by dividing the standard deviation by the

average absorbance reading of the sample. A range of ≤ 10% is acceptable from

literatures.

3.2 Repeatability Test

The repeatability test is a measure of how consistently the method will show

the same thread The 20 ppm concentration of the diluted iron solution of was

used. An addition of 150 μL of ammonium thiocyanate was made and absorbance

was read for about 300 seconds. The absorbance of this sample was also read for

a response time of 300 seconds each run. This step was repeated 6 times and the

data collected from the UV-Win Lab Data Processor would be plotted using

Microsoft Excel.

The repeatability depends on the constant procedure applied to each trial

run, making sure that what was done in the previous sample will also be done in

the next samples to be tested. The calculated RSD must be ≤ 10% for the proposed

method to be considered repeatable.

Chapter 3. Methodology
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 22

3.3 Sensitivity Test

The Sensitivity of the method proposed was validated by generating a

calibration curve with delta absorbance (y-axis) vs. the standard iron solution

concentration (x-axis).

Effectiveness of the ammonium thiocyanate solution in determining the iron

present in the standard solution was tested by getting the absorbance of the iron

solution before and after adding ammonium thiocyanate. The test was conducted

by starting from the smallest concentration of 10 ppm to that of the highest

concentration which is 35 ppm.

2000 μL of the standard iron solution was placed in the cuvette and

absorbance was read. After 5 minutes, 150 µL of ammonium thiocyanate with a

concentration of 3M was added to the iron solution and was read for another 5

minutes.

The same procedure was done to the other remaining concentrations of the

standard iron solution. Cuvettes were washed with ultra-pure water and with the

next solution to be used to make sure residues were removed from the surfaces of

the cuvettes.

After the absorbance measurement, the data were collected from the UV-

Winlab Data Processor and Viewer Software; it was then plotted using Microsoft

Excel. The best calibration curve should have an R2 value of 1.

Chapter 3. Methodology
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 23

The limit of detection was calculated by using the following formula:

(3 ⋅ 𝑆𝑡𝑑𝑒𝑣 𝑜𝑓 𝑠𝑡𝑎𝑏𝑖𝑙𝑖𝑡𝑦)
𝐿𝑂𝐷 =
(𝑠𝑙𝑜𝑝𝑒 𝑜𝑓 𝑠𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦)

3.4 Preparation and Preliminary Test for Real Sample

The commercial milk samples that were used in this research are Nestlé

Nestogen Two, Mead Johnson Enfamil Two A+, and Wyeth Bonamil. Table 1 shows

the quantity of powder to be dissolved in an ultrapure water to be able to prepare

the samples needed.

Table 3.1. Data of Milk Sample

Brand Amount of powder/ scoop Weight of Ultra-pure Water

(grams) (milliliters)

Nestogen 4.9610 30

Enfamil 4.4658 30

Bonamil 10.3501 60

Chapter 3. Methodology
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 24

Each milk sample was prepared according to the suggested amount of milk

powder to be dissolved in ultra-pure water. The prepared solution was filtered 5

times and was placed in the centrifuge for 10 minutes to improve its opacity.

2000 µL of the iron solution was placed in a cuvette and was placed inside

the UV-Vis for 5 minutes to get the absorbance reading. This solution was oxidized

by adding 100µL of hydrogen peroxide to correct the Fe 2+ to Fe3+. After oxidizing

the sample, 150 µL of ammonium thiocyanate was added to the solution and its

absorbance was read for another 5 minutes. This procedure was repeated 3 times

for each milk samples.

After the absorbance measurement, the data were collected from the UV-

Winlab Data Processor and Viewer Software; it was then plotted using Microsoft

Excel.

Chapter 3. Methodology
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 25

CHAPTER 4
RESULTS AND DISCUSSION

This chapter describes the result of the tests done in chapter 3. It starts

with the determination of suitable wavelength to work on during the tests. This

was followed by deciding on what concentrations of iron will be used for

calibration as well as deciding on indicator reagent to be used. After which,

stability test is done based on the chosen concentration of iron and indicator.

When the chosen concentration of iron and indicator is proven to be stable, we

test on its repeatability and finally its sensitivity and limit of detection.

When iron reacts with ammonium thiocyanate, a red solution will be

formed. During the experiment, it was observed that the higher the concentration

of iron that will react with ammonium thiocyanate, the more intense the observed

color.

The features of the sensor such as stability, repeatability, and sensitivity

tests for the proposed method in determining iron was done with milk samples in

mind to test the effectiveness of the sensor.

4.0 Determination of Suitable Wavelength

The results from the stability test were plotted as absorbance vs.

wavelength. Based on the graph shown in the Figure 4.0, the optimum difference
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 26

was found at the wavelength of 480 nm. This wavelength shows that if readings

were done here, there is a wider scale to be able to read the absorbance with

better accuracy. Therefore, the working wavelength that was used in the

proceeding experiments was 480 nm.

Wavelength vs. Absorbance

1.8
1.6
1.4
1.2
Absorbance

1 Iron
0.8 Iron + SCN
0.6 SCN
0.4
Water
0.2
0
385 485 585 685
Wavelength

Figure 4.0 Plot of absorbance vs wavelength (nm)

4.1 Stability Test

Stability test was done for 20 ppm because it is the value close to the

amount of iron in milk, our target sample. This test was done to prove the ability

of the sensor to give a constant absorption reading. This was also done to identify

potential fluctuations due to different electrical interferences.

The concentrations above 20ppm were not used for they contain more

iron. Higher iron content may cause precipitation, thus no stable reading can be

Chapter 4. Results and Discussion

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 27

done. Lower concentrations may also be used for the stability test and a stable

reading would surely be generated considering the concentrations of both ferric

ammonium sulfate stock solution and ammonium thiocyanate solution, but for

higher accuracy we opted for the higher possible value.

Figure 4.1.1 shows the result for stability test which generated the lowest

RSD. The stability test (Figure 4.1.1) for the standard iron solution using 20 ppm

that is 2000 μL with an added constant amount of 150 μL of ammonium

thiocyanate generated the figure.

The absorbance reading of the solution that was tested using the

spectrophotometer showed a constant reading in the 120 minute run. Its relative

standard deviation was measured to be 6.56%. The range for acceptable values for

this test should be less than 10% which is the limit set for the method to be

considered stable. The value was within the range and therefore acceptable [28].

Absorbance vs time
0.3

0.2
Abs

0.1

0
0 1000 2000 3000 4000 5000 6000 7000 8000
Time (s)

Figure 4.1.1 Stability Test

Chapter 4. Results and Discussion

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 28

4.2 Repeatability Test

A repeatability test (Figure 4.2.1) was performed to validate the method

proposed. This was done with six runs giving an RSD of 8.12%. The value was

within the acceptable range which is 10%.

0.45

0.4

0.35
Abs

0.3

0.25

0.2

0.15
0 1000 2000 3000 4000
Time (s)

Figure 4.2.1 Plot of Repeatability Test (n=6)

After performing the different validation tests, the method of analysis

proposed was proven to be convenient to use and it was intended. Reliability of

the method was confirmed by the results obtained.

Chapter 4. Results and Discussion

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 29

4.3 Sensitivity Test

The sensitivity test was done by measuring the change in absorbance of

the different iron concentration (Figure 4.3.1). From this result, the changes in the

absorbance were plotted versus the concentration of iron. The curves produced

from the average of the trials have fairly linear characteristics. The figure shows

the responses of the spectrophotometer at the different ammonium thiocyanate

complex for different standard iron concentrations.

Final Calibration Curve

∆Abs vs. Iron Conc. (M)
∆Abs RANGE: 0.0106 - 0.1235
0.14
0.12 y = 0.0044x - 0.0423
0.1 R² = 0.9274
Absorbance

0.08
0.06
0.04
0.02
0
0 5 10 15 20 25 30 35 40
Concentration

Figure 4.3.1 Calibration Curve for ∆Abs Range: 0.0106 to 0.1235 (n=3)

The square of correlation coefficient and the equation of the curve for

each calibration were calculated using Microsoft Excel. Linearity of the calibration

curve generated is proven by the value of R2 which is 0.9274. It is the best possible

correlation coefficient for linear equations. This means that there is a linear

Chapter 4. Results and Discussion

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 30

relationship between the absorbance and the ferric ammonium thiocyanate

complex in the different ferric ion standard solutions. A summary of the features

of the method is shown in Table 4.1

Table 4.1 Summary of the features of the method

R\% RSD

Stability test 6.56

Repeatability test 8.12

Dynamic Range 0.0106 - 0.1235 Molar concentration

Sensitivity test y = 0.0044x - 0.0423 R2= 0.9274

The calibration curve generated from the three trials performed was

applied with the use of error bars. Error bars in general, indicates estimated errors

or uncertainties to be able to perceive how precise a measurement is.

Chapter 4. Results and Discussion

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 31

Final Calibration Curve

∆Abs vs. Iron Conc. (M)
∆Abs RANGE: 0.0106 - 0.1235
0.18
0.16
0.14
Absorbance

0.12 y = 0.0044x - 0.0423

0.1 R² = 0.9274
0.08
0.06
0.04
0.02
0
0 5 10 15 20 25 30 35 40
Concentration

Figure 4.3.2 Calibration Curve for ∆Abs Range: 0.0106 to 0.1235 with error bars

Generally, shorter error bars implies that the values are more reliable in

comparison to longer error bars. As can be observed in the figure 4.3.2, at

concentration of 15 ppm the error bar is shorter compared to other

concentrations. The overlapping error bars for the difference concentrations

which are increasing as the concentration increases show that there may not be

significant difference between the two concentrations with regards to their

change in absorbance. [29] Therefore further refinement in the method may be

recommended.

The limit of detection (LOD) is the lowest concentration of a sample that

can be reliably detected, but not necessarily quantified, under the stated

conditions of the test. This was calculated using the standard deviation of the

stability test and slope of the generated calibration curve.

Chapter 4. Results and Discussion

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 32

The limit of detection was calculated as follows:

𝑠𝑡𝑑𝑒𝑣(𝑠𝑡𝑎𝑏𝑖𝑙𝑖𝑡𝑦) × 3
𝐿𝑂𝐷 =
𝑚(𝑠𝑙𝑜𝑝𝑒 𝑜𝑓 𝑐𝑎𝑙𝑖𝑏𝑟𝑎𝑡𝑖𝑜𝑛 𝑐𝑢𝑟𝑣𝑒)

0.004959121 × 3
𝐿𝑂𝐷 = = 3.38 𝑀/∆𝑎𝑏𝑠
0.0044

A value of 3.38 was obtained through the calculations. This value means

that at 3.38 M concentration of a sample, the method could produce one

absorbance reading change..

3M concentration of ammonium thiocyanate was added to the different

standard iron solution. Visual observations (Figure 4.3.3) were done. The color

appearance was captured as soon as 2 µL of ammonium thiocyanate was dropped

to the iron solution.

Figure 4.3.3 Visual observation of different concentrations of iron solution in ppm

(10, 15, 20, 25, 30, and 35) with 3M of Ammonium Thiocyanate

Chapter 4. Results and Discussion

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 33

4.4 Preliminary Test Using Real Sample

The effectiveness of the sensor was tested by analyzing the iron content of

different milk samples. Figures 4.4.1, 4.4.2, and 4.4.3 shows the absorbance vs

time relationship of the brands Nestogen, Enfagrow, and Bonakid, respectively.

The graph shows the response of the milk samples when ammonium thiocyanate

solution is added to it meaning, the absorbance decreases which is the opposite of

the expected data. There are several factors that may cause the opposite change

in the trend of the graph for the milk samples as compared to the calibration data.

Absorbance vs. Time

2.8

2.75
Absorbance

2.7

2.65

2.6
0 300 600 900 1200 1500 1800 2100
Time/s

Fig 4.4.1 Absorbance vs. Time of Nestogen milk sample

Chapter 4. Results and Discussion

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 34

Absorbance vs. Time

2.75

2.7
Absorbance

2.65

2.6

2.55
0 300 600 900 1200 1500 1800 2100
Time/s

Fig 4.4.2 Absorbance vs. Time of Enfagrow milk sample

Absorbance vs. Time

2.8

2.75
Absorbance

2.7

2.65

2.6

2.55
0 500 1000 1500 2000
Time/s

Fig 4.4.3 Absorbance vs. Time of Bonakid milk sample

Chapter 4. Results and Discussion

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 35

First the opacity of the milk may be diminished on the formation of the

thiocyanate complex. During these preliminary tests, various ways of removing

the casein of milk was tried to lessen the turbidity of milk. The use of a finer

filtering material: membrane filter polytetrafluoroethylene, (PTFE) 0.2 microns

supplied by Advantec (Japan, Toyo Roshi Kaisha). However, the turbidity

remained. We recommend further studies on this process. Aside from casein there

may be other nutrients existing in the milk sample that reacts with the indicator.

In summary, the iron detection method as a possible sensor was done with

commendable figures of merit.

Chapter 4. Results and Discussion

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 36

CHAPTER 5
CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

In this paper, a spectrophotometric method of detecting the presence of

iron in liquid samples based on its reaction with ammonium thiocyanate was

studied and found to be fairly valid.

The following results support the conclusions stated above:

The iron standards were dissolved in water and were first oxidized to the

Fe (III) form. This was reacted with ammonium thiocyanate resulting in a red

blood coloration which gives significant absorbance readings even in the most

dilute concentrations. The suitable wavelength for reading the absorbance was

found to be 480 nm.

The stability test was done at the chosen wavelength using the 20 ppm

iron solution. The calculated RSD value for this test was 6.56%. Repeatability test

using the difference in absorbance between a blank and an iron solution standard

was also performed with an RSD value of 8.12% for n=6.

The test for sensitivity as proven by the generated calibration curve has an

equation of 𝑦 = 0.0044𝑥 − 0.423 for a dynamic concentration range of 10 ppm

to 35ppm which have a correlation of R² = 0.9274 for n=3.

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 37

The limit of detection, LOD for the calibration curve was calculated to be:

0.004959121 × 3
𝐿𝑂𝐷 = = 3.38 𝑝𝑝𝑚/∆𝐴𝑏𝑠
0.0044

With the advantages presented in this paper, the method proposed could

be suitable for practical uses for the determination of iron present in samples.

After all the necessary tests and experiments, the study has come to

conclude that the method for the iron concentration determination is effective.

However, the method developed is nonselective. Hence, the iron present in a

sample must be isolated prior to the quantitative determination. The proposed

method for determining iron in liquid samples provides advantages of simpler

process compared to the analytical protocol requiring trained specialists.

5.2 Recommendations

Initial steps were done to test the method on real samples such as

powdered milk. Due to time constraints, the actual optical sensor for the purpose

of quantifying iron was not reliable. It is recommended that an improved

methodology may include centrifugation to eliminate the opacity of the milk

samples or using micro filtration.

Chapter 5. Conclusion and Recommendations

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 38

In addition to milk samples this method may find application in

determining the presence of carbon monoxide and hydrogen cyanide as a result of

tobacco smoke. Risk assessment and harm reduction is a field where thiocyanate

is a biomarker of exposure to these substances.

Chapter 5. Conclusion and Recommendations

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 39

REFERENCES

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[7] Bispo, M. S.; Morte, E. S. B.; Korn, M. G. A.; Teixeira, L. S. G.; Korn, M.; Costa, A.

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Costa, A. C. S.; J. Braz. Chem. Soc. 1997, 8, 621

[10] Cooke, K. Why total iron is important. Kentucky Water Watch. Retrieved from

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[11] Worsfold, P. (2005). SPECTROPHOTOMETRY | Overview. Encyclopedia of

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[12] Pan, J., Cha, T., Chen, H., & Choi, J. (2013). Carbon nanotube-based optical

platforms for biomolecular detection. Carbon Nanotubes and Graphene for

Photonic Applications. doi:10.1533/9780857098627.3.270

[13] Perez, N. (n.d.). Francisella Tularensis Ferric-siderophore and Ferrous Iron

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[14] Willey JD, Kieber RJ, Lancaster RD, Atmos J: Chem. 1996, 25: 149.

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10.1021/es00043a024.

[16] Pryor WA: Annu Rev Physiol. 1986, 48: 657-

10.1146/annurev.ph.48.030186.003301.

[17] Lieu, P. T., Heiskala, M., Peterson, P. A., & Yang, Y. (2001). The roles of iron in

health and disease. Molecular Aspects of Medicine,22(1-2), 1-87.

doi:10.1016/s0098-2997(00)00006-6

[18] Cook, J. D. (2003). The quantitative assessment of body iron. Blood,101(9),

3359-3363. doi:10.1182/blood-2002-10-3071

[19] Cadmus, P., Brinkman, S., May, M. (2018).Chronic toxicity of ferric iron for

north american aquatic organisms: derivation of a chronic water quality criterion

using single species and mesocosm data. Archives of Environmental Contamination

and Toxicology, 74(4), 605-615. doi: 10.1007/s00244-018-0505-2

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 41

[20] Rostern NT. (2017). The effects of some metals in acidified water on aquatic

organisms. Oceanography and Fisheries, 4(4), 555-645. doi:

10.19080/OFOAJ.2017.04.555645.

[21] He, Z. L., Yang, X. E., & Stoffella, P. J. (2005). Trace elements in

agroecosystems and impacts on the environment. Journal of Trace Elements in

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[22] (PDF) Iron biogeochemistry and its environmental impacts ... (n.d.). Retrieved

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d_its_environmental_impacts_in_freshwater_lakes

[23] Harris, J.E. (1992) Weathering of rock, corrosion of stone and rusting of iron.

Meccanica, 27, 233-250.

[24] Fraden, J. (2015). Handbook of Modern Sensors: Physics, Designs, and

Applications. Basingstoke, England: Springer.

[25] Fraden, J. (2004). Handbook of Modern Sensors. NY: Springer.

[26] Weber, G. (1991). Speciation of iron using HPLC with electrochemical and

flame-AAS detection. Fresenius Journal of Analytical Chemistry,340(3), 161-165.

doi:10.1007/bf00324473
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[27] Harvey, A. E., Smart, J. A., & Amis, E. S. (1955). Simultaneous

Spectrophotometric Determination of Iron(II) and Total Iron with 1,10-

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[28] Guidelines for standard method performance requirements AOAC Official

methods of Analysis (2002) Appendix F, p. 4

[29] Harvey Motulsky GraphPad Software

http://fisbio.biof.ufrj.br/restrito/bioEstatistica/90_top_especiais/errorbars_stat_si

gnificance. Date retrieved: Mar 19, 2019

UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 43

APPENDICES

APPENDIX A

A.1 Stock Solution of Iron Standard:

11 g FeNH4 (SO4 )2
mol 1 mol Fe 55.85 g 1000mg 1
∙ 12H2 O x x x x x
482.25 g 1 mol FeNH4 (SO4 )2 ∙ 12H2 O mol 1g 1L
= 1273.9243 ppm

A.2 Stock Solution of Ammonium Thiocyanate

1.3651 𝑔 1000 𝑚𝑔
𝑥 = 2730.2 𝑝𝑝𝑚
500 𝑚𝐿 𝐻2 𝑂 1𝑔

2730.2(𝑉) = (500 𝑝𝑝𝑚)(500𝑚𝐿)

Volume of 2730.2 ppm NH4SCN solution to prepare 500 ppm solution = 91.57 mL

A.3 Serial Dilution

M 1V 1 = M 2V 2

For 35 ppm:
1273.9243(𝑉) = 35(500)

V = 13.7371 mL

For 30 ppm:
35(𝑉) = 30(500)
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 44

V = 428.5714 mL

For 25 ppm:
30(𝑉) = 25(500)

V = 416.6667 mL

For 20 ppm:
25(𝑉) = 20(500)

V = 400 mL

For 15 ppm:
20(𝑉) = 15(500)

V = 375 mL

For 10 ppm:
15(𝑉) = 10(500)

V = 333.3333 mL

A.6 Computation of Iron Concentration in Real Sample

2 Nestogen

8.6 𝑚𝑔 𝐹𝑒 𝑥
= → 𝑥 = 0.8901𝑚𝑔 𝐹𝑒
100 𝑔 𝑃𝑜𝑤𝑑𝑒𝑟 10.3501 𝑔 𝑃𝑜𝑤𝑑𝑒𝑟

0.8901 𝑚𝑔𝐹𝑒 1000 𝑚𝑙

𝑥 = 14.835 𝑝𝑝𝑚
60 𝑚𝐿 𝐻2 𝑂 1𝐿

3 Bonamil
6.8 𝑚𝑔 𝐹𝑒 𝑥
= → 𝑥 = 0.3037𝑚𝑔 𝐹𝑒
100 𝑔 𝑃𝑜𝑤𝑑𝑒𝑟 4.4658 𝑔 𝑃𝑜𝑤𝑑𝑒𝑟
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 45

0.3037 𝑚𝑔𝐹𝑒 1000 𝑚𝑙

𝑥 = 10.1225 𝑝𝑝𝑚
30 𝑚𝐿 𝐻2 𝑂 1𝐿

4 Enfamil
8.4 𝑚𝑔 𝐹𝑒 𝑥
= → 𝑥 = 0.4167𝑚𝑔 𝐹𝑒
100 𝑔 𝑃𝑜𝑤𝑑𝑒𝑟 4.9610 𝑔 𝑃𝑜𝑤𝑑𝑒𝑟

0.4167 𝑚𝑔𝐹𝑒 1000 𝑚𝑙

𝑥 = 14.8928 𝑝𝑝𝑚
30 𝑚𝐿 𝐻2 𝑂 1𝐿
UNIVERSITY OF SANTO TOMAS CHEMICAL ENGINEERING 46

APPENDIX B

B.1 Lambda 25 Perkin Elmer UV-Vis Spectrophotometer

B.2 Clay Adams Compact II Centrifuge

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B.3 Ohaus Analytical Balance

B.4 HTL Micropipette

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B.5 Transferpette® Micropipette

B.6Millipore Ultrapure water Purification System