# Harry S Truman College Coversheet Iron Detection Method Lab Report

Lab 6 CoversheetPre-Lab: use only one page for pre-lab
Page 1 of 3
Lab 6 Coversheet
Page 2 of 3
Name:_____________________
Date:___________________
TA: _______________________
Partner(s):______________________
Unknown #: ___________________________
EDTA Concentration:_________________________
**Note: Everyone should do a “quick” trial and a “slow” trial. Include your “slow” trial and the
“slow” trials of 2 labmates who had the same unknown.
Trials
Total moles Ca2+ and Mg2+
1
2
3
a. Average:
standard deviation:
Rsd:
b. moles Ca2+ (by difference)
standard deviation:
Rsd:
Total Ca2+ and Mg2+
concentration, (moles/L) from
a.
Mg2+ concentration,
(moles/L) from b.
Sample Calculations:
Moles Ca2+
Lab 6 Coversheet
Post-Lab: use only one page for post-lab
Page 3 of 3
Lab 7: EDTA Titration
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12. EDTA Titration of Ca2+ and Mg2+ in Natural Waters
The most common multivalent metal ions in natural waters are Ca2+ and Mg2+. In this
experiment, we will find the total concentration of metal ions that can react with EDTA, and we
will assume that this equals the concentration of Ca2+ and Mg2+. In a second experiment, Ca2+ is
analyzed separately after precipitating Mg(OH)2 with strong base.
Research questions:
• What is the concentration of Ca2+ and Mg2+ in a natural water sample?
Learning Objectives:
Determine the elements of a titration that allow for quantitative analysis
Reagents
EDTA: Na2H2EDTA × 2H2O (FM 374.24), 0.6 g/student.
Buffer (pH 10): Add 142 mL of 28 wt% aqueous NH3 to 17.5 g of NH4Cl and dilute to 250 mL
with distilled water.
Eriochrome black T indicator: Dissolve 0.2 g of the solid indicator in 15 mL of triethanolamine
plus 5 mL of absolute ethanol. (Alternatively, Calmagite could be used by dissolving 0.05 g
in 100 mL of water. The color changes are the same for both indicators.)
Hydroxynaphthol blue indicator: This indicator may be sold as a neat solid or as a solid mixture
with ~99 wt% NaCl and 1 wt% indicator. If you have the neat solid, dissolve 0.4 g in 100 mL
of water. The 1% mixture with NaCl can be used as a solid. Hydroxynaphthol blue is
hazardous in case of skin or eye contact, ingestion, or inhalation. In case of skin contact,
flush with cool running water immediately. Cover the irritated skin with an emollient. If
irritation persists, seek medical attention. Wash contaminated clothing well.
Unknowns: Collect water from streams, lakes, or the ocean. To minimize bacterial growth,
plastic jugs should be filled to the top and tightly sealed. Refrigeration is recommended.
Lab 7: EDTA Titration
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50 wt% NaOH: Dissolve 100 g of NaOH in 100 g of H2O in a 250-mL plastic bottle. Store
tightly capped. When you remove solution with a pipet, try not to disturb the solid Na2CO3
precipitate. The solution is extremely corrosive to human tissue and clothing. In case of
contact, flush with cool running water immediately. Seek medical attention for serious burns.
Procedure
1. Dry Na2H2EDTA × 2H2O (FM 372.24) at 80°C for 1 h and cool in the desiccator. Accurately
weigh out ~0.6 g and dissolve it with heating in 400 mL of water in a 500-mL volumetric
flask. Cool to room temperature, dilute to the mark, and mix well.
2. a. Pipet a 1.000-mL sample of unknown into a 250 mL Erlenmyer flask and add 50 mL of
distilled water. To each sample, add 3 mL of pH 10 buffer and 6 drops of Eriochrome black
T indicator. b. Titrate with EDTA from a 50-mL buret and note when the color changes from
wine red to blue. Practice finding the end point several times by adding a little tap water and
titrating with more EDTA. Save a solution at the end point to use as a color comparison for
other titrations.
3. Repeat the titration with three samples to find an accurate value of the total Ca2+ + Mg2+
concentration. Perform a blank titration with 50 mL of distilled water and subtract the value
of the blank from each result.
4. For the determination of Ca2+, pipet two samples of unknown into clean flasks (adding 50
mL of distilled water if you use 1.000 mL of seawater). Add 30 drops of 50 wt% NaOH to
each solution and swirl for 2 min to precipitate Mg(OH)2 (which may not be visible). Add 6
drops of hydroxynaphthol blue indicator solution or ~0.1 g of solid 1 wt% hydroxynaphthol
blue to each flask. (This indicator is used because it remains blue at higher pH than does
Eriochrome black T.) d. Titrate one sample rapidly to find the end point and practice finding
it several times, if necessary. Remember that the titration of Ca2+ cannot require more EDTA
than you needed for Ca2+ + Mg2+. Save a solution at the end point to use as a color
comparison for other titrations.
5. Titrate the other three samples carefully. After reaching the blue end point, allow each
sample to sit for 5 min with occasional swirling so that any Ca(OH)2 precipitate may
redissolve. Then titrate back to the blue end point. (Repeat this procedure if the blue color
turns to red upon standing.) Perform a blank titration with 50 mL of distilled water.
Lab 7: EDTA Titration
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6. Calculate the total concentration of Ca2+ and Mg2+, as well as the individual concentrations of
each ion. Calculate the relative standard deviation of replicate titrations.
Pre-Lab: This pre-lab is graded on completion. You need to answer every part of the
question for full credit. If you are not sure, it is OK to guess. You will not lose points so
long as you have answered every part of the question. Make careful observations in lab to
complete the post-lab!
1. EDTA titrations are sensitive to pH, and at high pH metal hydroxide species can be
removed by precipitation. Which species, Mg or Ca, do you expect to precipitate at high
pH? How can this be used in your titration? How could you know if you added
sufficient NaOH?
2. Steps 2 and 4 describe the titration. In which step will the total concentration of both
Ca2+ and Mg2+ be determined? Describe why are two titrations required.
3. A. For step 2, draw a picture and describe what is in the flask at step 2a. What will you
see with your eyes (be sure to consider the indicator)? What chemical species will be
present in the flask? B. Write out the chemical reaction(s) that occur during the titration.
C. Draw a picture and describe what will be in the flask at the end of the titration. What
is the titrant? What will you observe with your eyes at the end of the titration? What
chemical species will be in the flask? Be sure to indicate what will be the predominant
species, and what will only be present in small concentrations.
4. A. For step 4, draw a picture and describe what is in the flask at step 4a. What will you
see with your eyes (be sure to consider the indicator and any differences you expect from
step 2)? What chemical species will be present in the flask and in what physical state? B.
Write out the chemical reaction(s) that occur during the titration. C. Draw a picture and
describe what will be in the flask at the end of the titration. What is the titrant? What
will you observe at the end of the titration? What will be in the flask and in what
physical state? Be sure to indicate what will be the predominant species, and what will
only be present in small concentrations.
5. Be sure to prepare your notebook to perform the experiment. The data table in the
cover sheet is not sufficient for data collection. Go through the procedure and consider
what information is required for each step. Prepare a spot in your notebook to collect that
information so that you do not forget when you come to lab.
Lab 7: EDTA Titration
Page 4 of 5
Name:_____________________
Date:___________________
TA: _______________________
Partner(s):______________________
Unknown #: ___________________________
EDTA Concentration:_________________________
**Note: Everyone should do a “quick” trial and two “slow” trials.
Trials
Total moles Ca2+ and Mg2+
1
2
3
a. Average:
standard deviation:
Rsd:
b. moles Mg2+ (by difference)
standard deviation:
Rsd:
Total Ca2+ and Mg2+
concentration, (moles/L) from
a.
Mg2+ concentration,
(moles/L) from b.
Moles Ca2+
Lab 7: EDTA Titration
Page 5 of 5
Post-Lab: Revisit your pre-lab questions. If you made any errors in the pre-lab, you
should not alter your pre-lab, but correct them in the post-lab.
1. Write any corrections to pre-lab 1.
2. Include any corrections to pre-lab 2, but also answer the following questions A. For
at step 2a. What did you observe with your eyes? In what ways is this observation
consistent with what chemical species are present in solution? C. For at step 2b. What
did you observe with your eyes? In what ways is this observation consistent with what
chemical species are present in solution?
3. Include any corrections to pre-lab 3, but also answer the following questions A. For
at step 4a. What did you observe with your eyes? In what ways is this observation
consistent with what chemical species are present in solution? C. For at step 4b. What
did you observe with your eyes? In what ways is this observation consistent with what
chemical species are present in solution?
Lab component:
Pre-Lab (4 pts)
Sample Calculations
(8 pts)
Table 1 (10 pt)
Post-Lab (10 pt)
Notebook pages (8)
Description
Graded on completion. Must include drawings, chemical equations, and
descriptions.
Must include units, labels and be clear. For functions done by hand, provide
the equation and sample calculation.
Complete, calculations properly performed.
Graded on correctness. Must include drawings, chemical equations, and
descriptions.
Must include brief procedure, single-line cross-outs, all data collected in
notebook, labels/dates/names on top of the pages. Must include clearly
labeled data collection tables as described in the pre-lab.
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Evaluation of an Analytical Method for
Determination of Iron Part 1
Evaluation of an Analytical Method for Determination of Iron
1
Learning Goals
1
Research Questions
2
Experimental context:
2
Iron in the Human Body
2
Introduction to Iron Deficiency
2
Women Most Likely to Suffer from Anemia
3
Medical Injustice and Sexism in Medical Research
4
Background Videos!
5
Iron and Detection Methods:
5
Method Properties:
6
Pre-lab Questions:
6
7
Procedure:
7
Post lab Questions:
8
References:
9
Learning Goals:
1. Propose and execute experiments to answer specific scientific questions.
2. Articulate the types of considerations that are involved in the development of an
analytical method.
3. Successfully determine and evaluate figures of merit.
4. Argue using experimental results for which method is preferred over others.
1
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Research Questions:
1. Which is the best derivatizing agent for the determination of iron?
Experimental context:
Iron in the Human Body
Making up only 0.006% of the human body, iron is one of the most essential elements in
all living organisms.1 70% of the body’s iron is located in a hemoglobin, which is stored in red
blood cells. Ferritin, another iron-containing protein, mainly functions to store iron within the
body and release it when needed to regulate the body’s iron levels.2 When iron intake is
decreased, the body’s iron reserves can become depleted causing iron deficiency. Alternatively,
when iron intake is increased, the body can suffer from iron overload.2,3 The average woman has
enough iron stored for about six months, whereas the average man has enough stored for three
years; therefore, women are significantly more prone to iron deficiency, while men are more
Figure 1. Venn Diagram of populations at high risk for iron-deficiency anemia13
Introduction to Iron Deficiency
Iron deficiency can be the result of a variety of conditions. Low iron intake throughout
the diet is the easiest to resolve, and most common in vegetarians and vegans. Heme-iron, which
is the easiest form of iron to absorb, comes only from meat.7 Non-heme iron, which exists in
plants or grains, requires acid to metabolize into a form that can be absorbed by the body.7 Many
2
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people are born with poor iron-balancing systems, causing them to have difficulty
absorbing iron, as in celiac disease. Even if those suffering with irregular systems get the
appropriate amount of iron in their diet, they will still suffer from iron deficiency.1 Additionally,
iron deficiency can result from severe blood loss.1 The most common cause of iron deficiency
from blood loss is menstruation.1
Figure 2. Graphic depicting anemic blood spears and anemia symptoms12
Women Most Likely to Suffer from Anemia
Moderate to severe iron deficiency will often result in the development of iron-deficiency
anemia, which causes symptoms like fatigue, chest pain, poor circulation, shortness of breath,
dizziness, irregular heartbeat, and general weakness.8 Approximately 7% of the population
suffers from iron-deficiency anemia.4 Populations at especially high risk for iron-deficiency
anemia are pregnant women, women of reproductive age (around 15-49 years of age)5, elderly
persons, Hispanics, and non-black hispanics.4 35.6% of black women aged 80-85 suffer from
iron-deficiency anemia, which is more than 5 times the national average. Each of these
respective high risk populations have been historically marginalized in society, specifically when
it comes to medicine and medical treatment.6
3
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Figure 3. Percentage of women in cardiovascular disease clinical trials vs. deaths 14
Medical Injustice and Sexism in Medical Research
Historically, pharmaceutical developers have chosen trial groups as homogeneously as
possible, to avoid complications based on race or gender. This meant that when conducting
clinical trials, companies would test only white men. Researches were not required by the federal
government to include women or people of color in their studies until 1994.9 It wasn’t until 2004
that the National Institute of Health required animal trials to be conducted on subjects of both
sexes.11 Even today, pharmaceutical companies which do not receive federal funding are not
required to test and study inclusively.9 As of 2009, women made up 39% of research subjects,
even though the population is 51% female.11 Researchers claimed, and some continue to claim,
that women’s frequent hormone fluctuation would complicate the trials too much or be too
expensive.6 Following successful clinical trials on white men, pharmaceutical companies made
the assumption that women are biologically the same as men, just smaller in weight, when
marketing the drugs.6
4
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Figure 4. Bar graph depicting the diversity participation of the clinical trials for four prescription
drugs15
There have been many concerning and harmful consequences of this type of gender
disparity in medicinal research. From 1997 to 2001, eight prescription drugs had been pulled off
the market because they were shown to have much greater health risks to women. 10 These health
risks were not discovered during the drug’s clinical trials, and the harm they caused to patients
could likely have been avoided with proper clinical trials.10 In 1982, a study was conducted from
Rockefeller University in New York City discussing the effects of obesity on estrogen levels in
relationship to breast and uterine cancer.11 Every test subject in the study was male, even though
the diseased they were investigating primarily affected women.11
Background Videos:
Iron deficiency anemia – causes, symptoms, diagnosis, treatment, pathology – Comprehensive and
fun video on all aspects of Iron-deficiency anemia.
Gender Inequality in Health | Heather Bowerman | TEDxBerkeley – Ted Talk by Heather
Bowerman, founder and CEO of DotLab, addressing the gender disparities in health care
Iron and Detection Methods:
In this experiment we will evaluate three analytical methods for the visible quantitation
of ionic iron. In day 1, we will only evaluate one method, using o-phenanthroline as a
derivatizing agent. In day 2, we will evaluate the other two methods and learn more about these
reactions. The o-phenanthroline reacts with the +2 state of Fe. The color arises from a charge
transfer electronic transition.
5
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O-phenanthroline
Fe2+ + 3 o-phen
[Fe(o-phen)3]2+
Method Properties:
In order to carry out a direct spectrophotometric determination of a substance, it must
absorb strongly in the spectral region employed. This is often the case with organic substances
when examined in the UV or IR range. However, to examine inorganic species, such as Fe +3, the
substance must be converted into a colored derivative. This is often done by reaction with a
chromogenic (color forming) reagent to form a complex product. These reagents are designed to
form products with the inorganic species which absorb in the visible region. Since relatively few
organic compounds absorb strongly in this region, there is a minimal possibility of interference.
For most inorganic ions of interest it is possible to find many color forming procedures in the
literature.
A good chromogenic reagent will have the following properties:
● The system should show a linear relationship between absorbance and concentration of
the species to be analyzed over a wide range. In other words, it should follow Beer’s
Law (A = ebc). This greatly simplifies the calibration of the system.
● The absorbing compound should have a high e (molar absorptivity). That is, there
should be a large absorbance from a small sample. This will give high sensitivity.
● The absorbance should be stable with respect to time. In other words, the system
should not be subject to any rapid reactions or decompositions.
● The system should not be greatly affected by small variations in variables like
temperature, pH and total ionic strength since these variables are difficult to control or
reproduce.
● The system should exhibit selectivity. The chromogenic reagent should not form colored
species with other ions as this would lead to an ambiguity in the origin of the response.
Pre-lab Questions:
1) For this experiment, we will focus on three figures of merit: sensitivity (section 5-1, under
specifications), linear range, and detection limit (Section 5-2 method validation) of the three
derivatizing agents. Review the textbook to understand these terms and how to evaluate
these terms.
a) How will you evaluate the sensitivity of your derivatizing agents?
b) How will you determine the linear range of your derivatizing agents?
6
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c) How will you determine the detection limit of your derivatizing agents?
2) Propose an experiment to determine the criteria above for o-phenanthroline. Give an
overview and put a step by step procedure in your notebook. As a starting point, you will be
provided a 0.01 M standard, and the detection limit is no lower that 0.0006 M. Complete the
table below with a proposed dilution strategy.
Concentration of Iron (M)
Volume of 0.01 M Iron Standard Volume of Water
(mL)
(mL)
3) Please bring a smart phone to class and load/learn how to use a color detecting app. You can
watch the video below on using color assist. Alternately, you can take a photo of each
sample and analyze using imageJ. Be prepared to analyze the samples partway through lab.
You may need to run additional samples after taking a quick look at your data/
1. How to set up your camera to consistently photograph samples:

2. How to use color detecting apps. Be sure to download and play with the app before
class, or refresh on how to use imageJ
3. Doing math with microfluidics
Procedure:
The first day we will develop procedures with o-phenanthroline only. Your assay will be
conducted on 0.5 inch disks punched with a hole punch out of Whatman filter paper. The
following will need to be deposited on each disk, this has been prepared and you will need to
punch out the disks from the prepared paper.
Ferrozine and O-phenanthroline: 20 uL of a solution containing:
● 5% acetic acid buffer
7
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● 10% Hydroxylamine hydrochloride
● 0.2% o-phenanthroline
Detection will be done using either Color Assist or ImageJ. Images can be collected on either
the green or blue channels. Be sure to collect all the images at the same time and with the same
setup and keep track of which image corresponds to each sample.
1. Apply your sample solution/water to the center filter paper. Allow 5 min for complete
derivatization.
2. Take a picture of the chips. No flash is recommended. Please refer to the directions for
capturing images here.
1. If using ColorAssist or Color Grab for data analysis, record all of the color
values.
2. If using ImageJ (directions here) for data analysis, use the picture of the bottom
3. Repeat steps 1-6 for each trial to create a calibration curve.
1. Note: before each new device trial, make sure to rinse the holder with water and
dry with a paper towel to prevent contamination across runs.
Post lab Questions: Note: you will need to enter each question in a separate
area in gradescope, so be sure to keep your notebook well-organized. Be sure
to attempt the post-lab before lab next week!
1. Results Summary
a. Make a table summarizing the findings for o-phenanthroline. Think about how to
present your results clearly and succinctly and include a table caption. This table
should not include all of the data, only a summary of your figures of merit.
b. Make one graph showing all the data. Be sure the graph is clearly labeled.
c. Make one graph that highlights the figures of merit (linear range, sensitivity, and
detection limit) for your agents. Be sure to consider appropriate data fitting on
this graph and be sure the graph is properly labeled.
2. Results analysis
a. Explain and/or show calculation for how you determined the sensitivity of each
derivatizing agent.
b. Explain and/or show calculation for how you determined the linear range.
8
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c. Explain and/or show calculation for how you determined the detection
limit.
3. This lab challenged you to design your own experimental protocols. Please reflect on
what you have learned about designing experiments. Are there any overall strategies you
discovered? What aspects do you feel you learned or grew in your ability to do? What
aspects still seem hard or unclear?
References:
1. “Chronic Diseases Affected by Iron.” Iron Disorders Institute , 2009,
www.irondisorders.org/chronic-diseases-affected-by-iron.
2. Dusenbery, Maya. “Medical Research Has a Woman Problem.” New York Post, New
York Post Holdings, Inc, 21 Apr. 2018, nypost.com/2018/04/21/medical-research-has-awoman-problem/.
3. Hoff, Caitlin. “Taking on Gender Bias in Clinical Trials.” National Women’s Health
Network, 26 Feb. 2019, nwhn.org/taking-on-gender-bias-in-clinical-trials/.
4. Le, Chi Huu Hong. “The Prevalence of Anemia and Moderate-Severe Anemia in the US
Population (NHANES 2003-2012).” PloS one vol. 11,11 e0166635. 15 Nov. 2016,
doi:10.1371/journal.pone.0166635
5. Reproductive Health Indicators: Guidelines for Their Generation, Interpretation and
Analysis for Global Monitoring; World Health Organization, Ed.; World Health
Organization: Geneva, 2006.
6. “Iron Deficiency Anemia: Causes, Symptoms, and Diagnosis.” Ferapro, Ferapro, 3 July
2019, www.ferapro.com/2018/12/20/iron-deficiency-anemia/.
7. “Iron-Deficiency Anemia.” National Heart Lung and Blood Institute, U.S. Department of
Health and Human Services, www.nhlbi.nih.gov/health-topics/iron-deficiency-anemia.
8. Miller, Ashley. “Iron-Deficiency Anemia -.” HealthScope, 18 June 2020,
www.healthscopemag.com/health-scope/iron-deficiency-anemia/.
9. “NIH Policy and Guidelines on The Inclusion of Women and Minorities as Subjects in
Clinical Research.” National Institutes of Health, U.S. Department of Health and Human
Services, 9 Oct. 2001, grants.nih.gov/policy/inclusion/women-andminorities/guidelines.htm.
10. “The Nutrition Source: Iron.” Harvard T.H. Chan School of Public Health, The President
and Fellows of Harvard College, 28 Oct. 2019,
www.hsph.harvard.edu/nutritionsource/iron/.
11. Schiebinger, Londa. “Designing Health & Biomedical Research.” Designing Health &
Biomedical Research | Gendered Innovations, National Science Foundation, Stanford
University, European Commission,
genderedinnovations.stanford.edu/methods/health.html.
9
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12. Schirber, Michael. “The Chemistry of Life: The Human Body.” LiveScience,
Purch, 16 Apr. 2009, www.livescience.com/3505-chemistry-life-humanbody.html#:~:text=Iron%.
13. Srinivasan, Sujata. “Clinical Trials In Need Of Diversity.” Connecticut Health
Investigative Team, 22 June 2017, c-hit.org/2017/05/10/clinical-trials-in-need-ofdiversity/.
14. UCSF Health. “Hemoglobin and Functions of Iron.” Ucsfhealth.org, UCSF Health, 6
Nov. 2019, www.ucsfhealth.org/education/hemoglobin-and-functions-ofiron#:~:text=Patient%20Education,Hemoglobin%20and%20Functions%20of%20Iron,the%20lungs%20to%20the%20tissue
s.
15. United States, Congress, Heinrich, Janet. Drug Safety: Most Drugs Withdrawn in Recent
Years Had Greater Health Risks for Women, Public Domain, 2001, pp. 1–8.
10
Page 1 of 7
Evaluation of an Analytical Method for
Determination of Iron Day 2
Evaluation of an Analytical Method for Determination of Iron
1
Learning Goals:
1
Research Questions:
1
Iron and Detection Methods:
2
Method Properties:
2
Pre-lab Questions:
3
4
Procedure:
4
Post lab Questions: Note: you will need to enter each question in a separate area in gradescope, so
be sure to keep your notebook well-organized.
4
5
References:
** Please bring a computer to lab in order to be able to complete some
analysis before leaving lab.
Learning Goals:
1. Propose and execute experiments to answer specific scientific questions.
2. Articulate the types of considerations that are involved in the development of an
analytical method.
3. Successfully determine and evaluate figures of merit.
4. Argue using experimental results for which method is preferred over others.
Research Questions:
1. What are the most important parameters for measuring iron colorimetrically?
2. Which is the best derivatizing agent for the determination of iron?
3. Which derivative should we use to test iron concentrations in a vitamin sample?
1
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Iron and Detection Methods:
In this experiment we will evaluate three analytical methods for the visible quantitation
of ionic iron. In part 1, we examined o-phenanthroline as a derivatizing agent. In part 2, we will
explore two additional agents. Each of these employs a color forming agent: namely SCN-, ophenanthroline and FerroZine. The o-phenanthroline and FerroZine react with the +2 state of Fe
while SCN- reacts with the +3 state of Fe. For all three products, the color arises from a charge
+3
transfer electronic transition. For Fe /SCN- the transfer is ligand to metal, and for the ophenanthroline and FerroZine complexes the charge is initially transferred from metal to ligand.
Both o-phenanthroline and FerroZinecontain two electron pair donor nitrogens which are
distinguished in the figure above with bold-faced lettering. Chelation to the iron ion occurs
through these Nitrogens. Both of these complexing agents, or ligands, are called bidentate
because they contain two coordination positions.
Thiocyanate
Fe3+ + SCN-
FeSCN+2
O-phenanthroline
Fe2+ + 3 o-phen
[Fe(o-phen)3]2+
FerroZine
Fe2+ + 3 FerroZine2-
[Fe(FerroZine)3]4-
The reaction conditions specified are necessary to provide the proper oxidation state,
appropriate degree of protonation, and, in the case of SCN-, establish a large excess of SCN- to
force the reaction to completion. These are the reactions that occur under these experimental
conditions.
Method Properties:
In order to carry out a direct spectrophotometric determination of a substance, it must
absorb strongly in the spectral region employed. This is often the case with organic substances
when examined in the UV or IR range. However, to examine inorganic species, such as Fe +3,
the substance must be converted into a colored derivative. This is often done by reaction with a
chromogenic (color forming) reagent to form a complex product. These reagents are designed to
form products with the inorganic species which absorb in the visible region. Since relatively few
organic compounds absorb strongly in this region, there is a minimal possibility of interference.
For most inorganic ions of interest it is possible to find many color forming procedures in the
literature.
A good chromogenic reagent will have the following properties:
2
Page 3 of 7
● The system should show a linear relationship between absorbance and
concentration of the species to be analyzed over a wide range. In other words, it should
follow Beer’s Law (A = ebc). This greatly simplifies the calibration of the system.
● The absorbing compound should have a high e (molar absorptivity). That is, there
should be a large absorbance from a small sample. This will give high sensitivity.
● The absorbance should be stable with respect to time. In other words, the system
should not be subject to any rapid reactions or decompositions.
● The system should not be greatly affected by small variations in variables like
temperature, pH and total ionic strength since these variables are difficult to control or
reproduce.
● The system should exhibit selectivity. The chromogenic reagent should not form colored
species with other ions as this would lead to an ambiguity in the origin of the response.
Pre-lab Questions:
1) Complete a DRAFT of the post-lab questions from part 1. It is important to complete some data
analysis and think a bit more about the experiment before continuing! Your TA will check your draft,
but you do not need to submit to gradescope.
2) Propose an experiment to determine the criteria above for each derivatizing agent. Give an
overview and put a step by step procedure in your notebook. Use your results from last week
as a starting point, but be aware the detection limit and linear range may be a little different
for the different reagents. Complete the table below with a proposed dilution strategy.
Concentration of Iron (M)
Volume of 0.01 M Iron Standard Volume of Water
(mL)
(mL)
4.) Propose an experiment to test the concentration of iron in the vitamin samples tested in week 8.
Consider any needed dilution factor.
3
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1. How to use color detecting apps
2. Doing math with microfluidics
Procedure:
Your assay will be conducted on 0.5 inch disks punched with a hole punch out of
Whatman filter paper. It is important to work on a non-absorbing surface (i.e. not a paper towel).
There are washers available in lab for this purpose, or you may want to work on a watch glass or
other surface. The following will need to be deposited on each disk, depending on which of the
3 derivatizing agents you are working with:
Thiocyanate:
● 20 µL of 1M ammonium thiocyanate
Ferrozine and O-phenanthroline: 20 uL of a solution containing:
● 5% acetic acid buffer
● 10% Hydroxylamine hydrochloride
● EITHER 0.2% o-phenanthroline OR 0.1 M Ferrozine
Detection will be done using either Color Assist or ImageJ. Images can be collected on either
the green or blue channels.
Select one agent to use to test the concentration of your vitamin sample from week 8. Repeat 3
measures.
Post lab Questions: Note: you will need to enter each question in a separate
1. Results Summary:
a. Make a table summarizing the findings for the three derivatizing agents. Think
about how to present your results clearly and succinctly and include a table
caption. (Hint: we do not need to see all the data, only a summary of your figures
of merit.
b. Make one graph showing all the data (use different colors for each derivatizing
agent and be sure to label everything properly. Alternately, you may use separate
graphs for each derivatizing agent.)
4
Page 5 of 7
c. Make one graph (or one graph for each derivatizing agent) that highlights
the figures of merit (linear range, sensitivity, and detection limit) for your agents.
Be sure to consider appropriate data fitting on this graph and be sure the graph(s)
is/are properly labeled.
2. Results analysis:
a. Explain and/or show calculation for how you determined the sensitivity of each
derivatizing agent.
b. Explain and/or show calculation for how you determined the linear range of each
derivatizing agent.
c. Explain and/or show calculation for how you determined the detection limit of
each derivatizing agent.
3. Which derivatizing agent did you choose for to test the iron sample? How did you use
your figures of merit to choose that agent?
a. Prepare a table to compare your results from the iron concentration determined
this week and the iron concentration determined in week 8. Consider both
precision and accuracy of your results.
References:
1. “Chronic Diseases Affected by Iron.” Iron Disorders Institute , 2009,
www.irondisorders.org/chronic-diseases-affected-by-iron.
2. Dusenbery, Maya. “Medical Research Has a Woman Problem.” New York Post, New
York Post Holdings, Inc, 21 Apr. 2018, nypost.com/2018/04/21/medical-research-has-awoman-problem/.
3. Hoff, Caitlin. “Taking on Gender Bias in Clinical Trials.” National Women’s Health
Network, 26 Feb. 2019, nwhn.org/taking-on-gender-bias-in-clinical-trials/.
4. Le, Chi Huu Hong. “The Prevalence of Anemia and Moderate-Severe Anemia in the US
Population (NHANES 2003-2012).” PloS one vol. 11,11 e0166635. 15 Nov. 2016,
doi:10.1371/journal.pone.0166635
5. Reproductive Health Indicators: Guidelines for Their Generation, Interpretation and
Analysis for Global Monitoring; World Health Organization, Ed.; World Health
Organization: Geneva, 2006.
6. “Iron Deficiency Anemia: Causes, Symptoms, and Diagnosis.” Ferapro, Ferapro, 3 July
2019, www.ferapro.com/2018/12/20/iron-deficiency-anemia/.
7. “Iron-Deficiency Anemia.” National Heart Lung and Blood Institute, U.S. Department of
Health and Human Services, www.nhlbi.nih.gov/health-topics/iron-deficiency-anemia.
5
Page 6 of 7
8. Miller, Ashley. “Iron-Deficiency Anemia -.” HealthScope, 18 June 2020,
www.healthscopemag.com/health-scope/iron-deficiency-anemia/.
9. “NIH Policy and Guidelines on The Inclusion of Women and Minorities as Subjects in
Clinical Research.” National Institutes of Health, U.S. Department of Health and Human
Services, 9 Oct. 2001, grants.nih.gov/policy/inclusion/women-andminorities/guidelines.htm.
10. “The Nutrition Source: Iron.” Harvard T.H. Chan School of Public Health, The President
and Fellows of Harvard College, 28 Oct. 2019,
www.hsph.harvard.edu/nutritionsource/iron/.
11. Schiebinger, Londa. “Designing Health & Biomedical Research.” Designing Health &
Biomedical Research | Gendered Innovations, National Science Foundation, Stanford
University, European Commission,
genderedinnovations.stanford.edu/methods/health.html.
12. Schirber, Michael. “The Chemistry of Life: The Human Body.” LiveScience, Purch, 16
Apr. 2009, www.livescience.com/3505-chemistry-life-human-body.html#:~:text=Iron%.
13. Srinivasan, Sujata. “Clinical Trials In Need Of Diversity.” Connecticut Health
Investigative Team, 22 June 2017, c-hit.org/2017/05/10/clinical-trials-in-need-ofdiversity/.
14. UCSF Health. “Hemoglobin and Functions of Iron.” Ucsfhealth.org, UCSF Health, 6
Nov. 2019, www.ucsfhealth.org/education/hemoglobin-and-functions-ofiron#:~:text=Patient%20Education,Hemoglobin%20and%20Functions%20of%20Iron,the%20lungs%20to%20the%20tissue
s.
15. United States, Congress, Heinrich, Janet. Drug Safety: Most Drugs Withdrawn in Recent
Years Had Greater Health Risks for Women, Public Domain, 2001, pp. 1–8.
Lab component:
Pre-Lab (7
points)
Data collection
(10 points)
Results
summary (12
points)
Results analysis
(8 pt)
Description
Must be complete. Notebook must be ready for data collection.
Use sufficient trials to determine the sensitivity, linear range, and
detection limit. Include all trials and indicate which were used in your
analysis. Be sure to keep a separate table for each reagent or add a column
to indicate the reagent
Includes a table summarizing the findings (i.e. figures of merit, not all the raw
data.) Graphical data shows both all of the data collected and key figures of
merit (I,e, linear range.)
Complete, calculations properly performed. Each factor is explained
properly.
6
Page 7 of 7
Table 3
unknown
analysis (7 pt)
Notebook pages
(3)
Complete, calculations properly performed.
Must include brief procedure, single-line cross-outs, all data collected in
notebook, labels/dates/names on top of the pages.
7
Lab 9: Coversheet Iron Detection Method
Page 1 of 4
Name:___________________________
TA/Section:_______________________
Read the ENTIRE question in the lab handout!!
Prelab: Show calculations and provide explanations!
a) How will you evaluate the sensitivity of your derivatizing agents?
b) How will you determine the linear range of your derivatizing agents?
c) How will you determine the detection limit of your derivatizing agents?
2) (Procedure in notebook, not on cover sheet.)
Concentration of Iron (M)
Volume of 0.01 M Iron Standard
(mL)
Volume of Water (mL)
3) Propose an experiment to test the concentration of iron in the vitamin samples tested in week
8. Consider any needed dilution factor.
Lab 9: Coversheet Iron Detection Method
Page 2 of 4
Name:___________________________
TA/Section:_______________________
Data Collection:
Complete the table Use as many trials as needed to determine the sensitivity, linear range, and
detection limit. Include all trials and indicate which were used in your analysis. Be sure to keep
a separate table for each reagent or add a column to indicate the reagent!
Name of derivatizing agent:
Concentration of iron stock:
Trial Volume iron
#
standard (mL)
Volume water Standard
(mL)
Conc. (M)
Intensity
(green)
Intensity
(blue)
Used trial?
(y/n)
Intensity
(green)
Intensity
(blue)
Used trial?
(y/n)
Intensity
(green)
Intensity
(blue)
Used trial?
(y/n)
Name of derivatizing agent:
Concentration of iron stock:
Trial Volume iron
#
standard (mL)
Volume water Standard
(mL)
Conc. (M)
Name of derivatizing agent:
Concentration of iron stock:
Trial Volume iron
#
standard (mL)
Volume water Standard
(mL)
Conc. (M)
Lab 9: Coversheet Iron Detection Method
Page 3 of 4
Name:___________________________
TA/Section:_______________________
Results Summary:
a. Make a table summarizing the findings.
b. Make one graph showing all the data for each derivative.
c. Make one graph that highlights the figures of merit for each derivative.
Lab 9: Coversheet Iron Detection Method
Name:___________________________
Page 4 of 4
TA/Section:_______________________
Results analysis & Post-Lab: Provide explanations and calculations!
2. Results analysis
a. Explain and/or show calculation for how you determined the sensitivity.
b. Explain and/or show calculation for how you determined the linear range.
c. Explain and/or show calculation for how you determined the detection limit.
3. Which derivatizing agent did you choose? How did you use your figures of merit?
a. Prepare a table to compare your results …

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