spectrophotometric determination of iron in cabbage
DESCRIPTION
Research done by my IB student Rachel Choi. Please cite and give proper reference to her on her work if you use this material.TRANSCRIPT
International Baccalaureate Diploma Program
Extended Essay
Chemistry
Determination of iron concentration in different parts and
layers of Brassica rapa ssp. Pekinensis using visible
spectrophotometry
Jung Youn Choi
Candidate Number: 002213-012
Word Count: 3225
Page 2 of 36
Abstract
Kimchi is a traditional Korean dish commonly eaten with every meal. It is
made with a main vegetable ingredient and seasoned with various vegetables. The
most popular form of Kimchi is made with napa cabbage (Brassica rapa ssp.
pekinensis) as the main ingredient.
This research examined the iron concentrations for the different parts of the
napa cabbage. The first part of the investigation compared whether the outer edge of
the leaves and the stalk of the leaves of the cabbage had a higher iron concentration.
The second part of the research compared whether the outer or inner leaves the head
of the cabbage contained more iron. The iron concentrations were quantified using
visible spectrophotometry. The absorbance readings were recorded at a max of
508.5nm, the wavelength the iron(II) orthophenanthroline complex exhibited a
maximum absorption peak.
First, a standard calibration curve for iron(II) of absorbance against
concentration was created. The standard curve had a good correlation (R2=0.9986)
between the concentration and absorbance. For each part of the investigation, 3.000
grams of dry mass was burned to white ash for triplicate samples of each cabbage part.
2.0cm3 of 4.0M hydrochloric acid was added to each sample to dissolve the ash and
form aqueous iron(III) solution. The iron(III) solutions were then reduced to iron(II)
and the absorbance of each sample was measured using a visible spectrophotometer.
Using Beer’s Law, the iron concentrations of the samples were then calculated by
plugging in the sample’s absorbance into the regression line of standard iron(II) curve.
Results showed that the outermost leaves of the cabbage had the highest iron
content of 14.55mg dm-3
g-1
, the inner leaves the next highest with 4.83mg dm-3
g-1
,
and the stem with the lowest iron content of 3.21mg dm-3
g-1
.
In conclusion, the outer leaves of the cabbage had the maximum iron
concentration.
Word Count: 299
Page 3 of 36
Table of Contents
1. Introduction ................................................................................................................... 5
1.1 Rationale for the study .......................................................................................................... 5
1.2 Aim........................................................................................................................................ 6
1.3 Kimchi ................................................................................................................................... 6
1.4 Iron ........................................................................................................................................ 8
2. Hypothesis ...................................................................................................................... 8
3. Methodology ................................................................................................................ 10
3.1 Quantification of iron samples using visible spectrophotometry ........................................ 11
4. Creating a standard calibration curve for Iron(II) ................................................. 13
4.1 Preparation of necessary solutions ...................................................................................... 13
4.2 Measuring the absorbances of iron(II) standard solutions .................................................. 13
4.3 Data Collection ................................................................................................................... 14
4.4 Data Processing ................................................................................................................... 15
5. Quantification of iron(II) concentrations in the outer leaves and stem ................. 16
5.1 Methodology for quantification .......................................................................................... 16
5.2 Preparation of outer leaves and stem samples ..................................................................... 16
5.3 Reducing iron(III) to iron(II) and measuring absorbance ................................................... 18
5.4 Data Collection ................................................................................................................... 20
5.5 Data Processing ................................................................................................................... 22
6. Quantification of iron(II) concentrations in the outer and inner leaves ................ 24
6.1 Methodology for quantification .......................................................................................... 24
6.2 Data Collection ................................................................................................................... 25
6.3 Data processing ................................................................................................................... 25
7. Data Presentation ........................................................................................................ 26
8. Data Analysis ............................................................................................................... 28
9. Evaluation .................................................................................................................... 30
9.1 Limitations and improvements ............................................................................................ 30
9.1.1 Obtaining dry mass and ashing .................................................................................... 30
9.1.2 Using colorimetry for determining iron concentration ................................................ 30
9.1.3 Number of samples ...................................................................................................... 31
9.2 Unresolved questions for further investigation ................................................................... 32
Page 4 of 36
9.2.1 Determining ascorbic acid concentration in Brassica rapa ssp. Pekinensis ................. 32
9.2.2 Determining iron concentration in Kimchi .................................................................. 32
10. Conclusion ................................................................................................................. 33
11. Appendix .................................................................................................................... 34
12. References .................................................................................................................. 35
Page 5 of 36
1. Introduction
1.1 Rationale for the study
It is important to study the iron concentration in food because iron is an essential
element in the metabolism of almost all living organisms[1]
. Iron is a critical
constituent of many important proteins and enzymes in the human body[2]
. For
example, iron can be found in the regulation of cell growth and differentiation,
hemoglobin1, and myoglobin
2. Iron helps strengthen the immune system, as it is a
major component of cells that fight infections, and provides the human body with
energy by being involved in chemical reactions that converts food into energy.
Inadequate iron consumption can result in iron-deficiency anemia, which can lead to
extreme fatigue, weakening of the immune system, and tongue inflammation[3]
.
Knowing the importance of iron in our diet, I was surprised when I found out that iron
deficiency was a problem in Korea, mostly among women and children[4]
. Hence, I
decided to research about iron in a way that was connected to our diet. I began to
think about how Koreans could increase their daily dietary intake of iron in a way that
did not deviate from their traditional diet.
This made me think about Kimchi3. Koreans used to rely on Kimchi for their
vegetable intake during the winter when no fresh vegetables were available, and
continue to eat Kimchi with every meal. Kimchi is low in calories while rich in iron
as well as other nutrients such as vitamin A, vitamin C, calcium, and phosphorous[5]
.
1 The protein in red blood cells responsible for transporting oxygen. 2 The protein that carries and stores oxygen in muscle cells. 3 A traditional fermented Korean dish made of cabbages or radishes and seasoned with red pepper, ginger, and garlic.
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My goal is to find the iron concentration in different parts of the Kimchi cabbage and
thus determine which part of the cabbage should be consumed for the maximum
consumption of iron.
1.2 Aim
The aim of this Extended Essay is to investigate how iron content differs for different
parts of cabbage. In the first part of the investigation, I will determine whether the
same mass of cabbage leaves or the stem has a higher iron content. Using the results
from this investigation, I will then find out whether the inner or outermost leaves of
the cabbage has a higher iron content. This led me to my research question:
How will iron concentration differ for different parts and layers of Brassica rapa
ssp. Pekinensis?
1.3 Kimchi
Figure 1: Kimchi
Kimchi, shown in figure 1, is a traditional Korean side dish made through
fermentation. Leuconostoc mesenteroides, a type of lactic acid bacteria (LAB), and
yeasts initiate the fermentation. When the pH drops with the accumulation of organic
acids, other LAB such as Lactobacillus brevis, Lactobacillus plantaru, Streptococcus
faecalis, and Pediococcus cerevisiae carry on the fermentation[6]
.
Page 7 of 36
Figure 2: Napa cabbage (Brassica rapa ssp. pekinensis)
Although various vegetables such as radish, cabbage, and cucumbers are used to
make different types of Kimchi, cabbage Kimchi made with napa cabbage (Brassica
rapa ssp. pekinensis) shown in figure 2 is the most common. Napa cabbage is a type
of Chinese cabbage commonly found in East Asian cuisine. It is low in calories and is
an excellent source of zinc, iron, calcium, and vitamin C[7]
. Moreover, it prevents
infections and ulcers, and helps the body produce more antibodies[8]
.
The whole napa cabbage is used in the process of making Kimchi. Thus, I resolved to
investigate how iron content differed for different parts of cabbage, specifically the
stem, exterior leaves, and inner leaves, as shown in figure 3 below.
Page 8 of 36
1.4 Iron
There are two forms of dietary iron: heme iron and nonheme iron. Heme iron is found
in meat, while nonheme iron is in plants. Heme iron is part of the heme molecule, a
compound of the porphyrin4 class that forms the nonprotein part of hemoglobin,
myoglobin, and some other biological molecules. Most nonheme iron is iron(III) that
must be reduced to iron(II) before it can be absorbed[9]
. Hydrochloric acid and pepsin5
are used to release nonheme iron from food. A protein called transferrin then binds
and transports iron from the digestive system to the bloodstream[10]
.
2. Hypothesis
The light reactions of photosynthesis take place in the chloroplast of plants.
Chlorophyll a6, a pigment in the chloroplast, absorbs all visible light except green
during photosynthesis. The green light is reflected instead of being absorbed and thus
can be detected by the naked eye. This means the outer leaves contain the most
chlorophyll a because the outer leaves are green, while the inner leaves are yellow and
the stem is white.
During the light reactions of photosynthesis, electrons are excited to a higher energy
state when light strikes chlorophyll a. The energy from the excited electrons is
converted into ATP and NADPH by a process called photophosphorylation. In
photophosphorylation, electrons move through the photosynthetic electron transport
chain by several electron carriers that contain iron, including the cytochrome b6f
4 Pigments such as heme and chlorophyll whose molecules contain a flat ring of four linked heterocyclic groups. 5 Digestive enzyme in the stomach. 6 Type of chlorophyll present in photosynthetic organisms, including plants.
Page 9 of 36
complex7, Fe2S2 ferredoxin
8, and ferredoxin-NADP
+ reductase
9 as indicated in figure
4 below. Because the outer leaves have the greatest amount of chlorophyll a, the outer
leaves will have the most number of excited electrons. As a result, more cytochrome
b6f complex, ferredoxin, and ferredoxin-NADP reductase will be needed to transport
the electrons, increasing the amount of iron[11]
.
Figure 4: Iron-containing electron carriers in the chloroplast
Thus, the hypothesis for this investigation is the green outer leaves of the cabbage will
have the highest iron concentration of iron, followed by the yellow inner leaves and
finally the white stem.
7 A dimer comprised of cytochrome f, cytochrome b6, and Fe2S2 ferredoxin. 8 An iron-sulfur protein found in the chloroplasts of plants. 9 An enzyme comprised of reduced ferredoxin, NADP+, and H+. It reduces NADP+ to NADPH.
Page 10 of 36
3. Methodology
The following is the overall methodology for this investigation:
Scheme 1: Overall methodology of the investigation
Quantification of iron(II) concentrations in outer & inner leaves
1. Prepare samples2. Reduce iron(III) to iron(II)
and measure absorbance3. Calculate iron(II) concentration
using standard regression line
Quantification of iron(II) concentrations in outer leaves & stem
1. Prepare samples2. Reduce iron(III) to iron(II)
and measure absorbance3. Calculate iron(II) concentration
using standard regression line
Create iron(II) standard calibration curve
1. Prepare necessary solutions
2. Record absorbances of iron(II) standards
3. Graph absorbance against concentration, find regression line
Page 11 of 36
3.1 Quantification of iron samples using visible spectrophotometry
Since small concentrations of iron will be used for this investigation, visible
spectrophotometry was used to quantify iron because of its high degree of precision,
sensitivity and accuracy[12]
. A visible spectrophotometer, shown in figure 5, passes a
beam of light through the sample to determine the wavelength of light corresponding
to intensity of the sample’s color. This wavelength is then used to find how much
light the different iron(II) samples absorbed[13]
.
Figure 5: Visible spectrophotometer
In this investigation, the color of the samples was orange and a wavelength with an
absorbance peak at 508.5nm, the max, was used. It was at this wavelength the
maximum change in absorbance for iron solutions of any concentration occurs.
Beer’s Law, which shows a linear relationship between absorbance with cell path
length and sample concentration, was then used[14]
. The general equation is:
A = ε l c10
Cuvettes with the same size and shape were used for all samples, meaning all samples
were measured at the same wavelength with the same absorbance peak. Consequently,
l and ε are constants. This resulted in a direct variation between the sample’s
absorbance and its concentration.
10 A is the sample’s absorbance, ε is the molar absorptivity, l is the length of solution the light passes through, and c is the sample’s concentration.
Page 12 of 36
All iron samples were reacted with orthophenanthroline11
in order to form an orange
colored iron(II) phenanthroline complex. The chemical equation of this reaction is:
3Phen + Fe2+
Fe(Phen)32+
Figure 6 below illustrates this chemical reaction using the structural formulas of
orthophenanthroline and iron(II) phenanthroline complex:
Figure 6: Reaction between orthophenanthroline with iron(II) to form iron(II)
phenanthroline complex
11 A heterocyclic organic compound that forms two or more coordination links with an iron ion to form a strong complex.
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4. Creating a standard calibration curve for Iron(II)
4.1 Preparation of necessary solutions
Iron(II) solutions with different concentrations were prepared. Reacting the solutions
with orthophenanthroline formed an iron complex and the absorbance of each
complex was measured. The necessary solutions12
were:
Iron(II) standard solutions with concentrations of 1.00 x 10-4
M, 5.00 x 10-
5M, 2.50 x 10
-5M, 1.25 x 10
-5M, and 6.25 x 10
-6M
5% trisodium citrate (Na3C6H5O7)13
solution
10% hydroxylammonium chloride14
(NH3OHCl) solution
0.01M orthophenanthroline solution
4.2 Measuring the absorbances of iron(II) standard solutions
1. 1.0 cm3 of 1.00 x 10
-4M iron(II) standard solution was transferred into a
cuvette using a micropipette.
2. 0.5 cm3 of 5% trisodium citrate solution was then added followed by 0.5
cm3 of 10% hydroxylammonium chloride solution and 1.0 cm
3 of 0.01M
orthophenanthroline solution.
3. The mixture was left for 24 hours for the iron complex to form.
4. The visible spectrophotometer for Logger Pro 3.7 was calibrated with a
cuvette containing a blank solution with the wavelength set at 508.5 nm.
5. The cuvette containing the mixture was inserted into the cuvette holder on
the visible spectrophotometer.
12 Refer to appendix for preparation procedures. 13 Buffer reagent. 14 Excess reducing agent.
Page 14 of 36
6. The absorbance was recorded at the max, 508.5nm
7. Steps 1 through 6 were repeated for iron(II) standards with concentrations
of 5.00 x 10-5
M, 2.50 x 10-5
M, 1.25 x 10-5
M, and 6.25 x 10-6
M shown in
figure 7 below.
Figure 7: iron(II) standards of different concentrations
4.3 Data Collection
Concentration,
c / mol dm–3
1.0 x 10 –4
5.00 x 10 –5
2.50 x 10 –5
1.25 x 10 –5
6.25 x 10 –6
Absorbance, A
after 24 hours(a)
0.393
0.211
0.108
0.054
0.028
Table 1: The concentration of Iron(II) standards with the corresponding absorbance readings.
(a) While conducting trial runs, it was observed that the absorbance readings fluctuated when the initial
absorbance was measured and stabilized after the mixture was left for 24 hours. Thus, the absorbance was
measured after 24 hours. The standard deviation for concentrations of standard iron(II) were not recorded as the
instruments used to prepare the standards have negligible uncertainty. Only one reading was recorded because it
was apparent that there was good correlation (R2=0.9986) between the absorbance and concentration after
graphing the data.
From left to right:
1.00 x 10-4
M,
5.00 x 10-5
M,
2.50 x 10-5
M,
1.25 x 10-5
M, and
6.25 x 10-6
M
Page 15 of 36
4.4 Data Processing
The following is the graph of the standard calibration curve for iron(II) standard:
Graph 1: Standard calibration curve for iron(II) standard
(a) Only one reading was recorded because of the good correlation (R2=0.9986) between the absorbance and concentration.
y = 0.3894x + 0.0079R² = 0.9986
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 0.2 0.4 0.6 0.8 1 1.2
Ab
sorb
an
ce, A
Concentration of iron(II), c/ x 10-4 mol dm–3
Absorbance against Concentration, c/ x 10-4 mol dm-3
(a)
Page 16 of 36
5. Quantification of iron(II) concentrations in the outer leaves
and stem
5.1 Methodology for quantification
The diagram below outlines the methodology used to quantify iron(II) in the outer leaves
and stem of the cabbage:
Scheme 2: Methodology to quantify iron(II) in the outer leaves and stem of cabbage
5.2 Preparation of outer leaves and stem samples
1. Random samples from the leaves and stem were taken from the outer green parts
of cabbage (Brassica rapa ssp. pekinensis), excluding the inner yellow leaves.
2. The leaves were heated for 15 minutes at 170C and the stem were heated for
30~40 minutes at 150C in an oven to obtain dry mass15
.
15 Mass of matter when it is completely dried and without any water content.
Determine iron(II) concentration
1. Calculate mean absorbance of triplicate samples
2. Interpolate mean absorbance into standard regression line
3. Calculate iron(II) concentration, iron(II) per gram of sample
Reduce iron(III) to iron(II) and measure absorbance
1. Spin solution in microcentrifuge
2. Add necessary solutions 3. Dilute sample, measure absorbance using visible
spectrophotometry
Preparation of samples
1. Obtain dry mass2. Burn until sample is
white ash3. Add 4.0M HCl
Page 17 of 36
Figure 8: Outer leaves and stem samples before and after heating in an oven
3. Approximately 3.000g of the dry leaves and stem samples shown in figure 8 were
each weighed on the electronic balance (0.001g) and placed in a beaker.
Triplicate samples were prepared for each part of the cabbage.
4. The beakers were placed on a wire mesh and heated directly over a Bunsen burner
until the samples turned into white ash, shown in figure 9.
Figure 9: Ashing sample
Page 18 of 36
5. 2.0cm3 of 4.0M hydrochloric acid was added to each beaker to dissolve the ash
and form aqueous iron(III) solution as shown in figure 10 below.
Figure 10: aqueous iron(III) solution
5.3 Reducing iron(III) to iron(II) and measuring absorbance
1. 1.0cm3 of solution with ash was transferred into a microcentrifuge tube using a
micropipette (100–1000l). The tube is spun in a centrifuge machine shown in
figure 11 to precipitate unwanted ash.
Figure 11: Microcentrifuge
2. 500l of solution is transferred into a 100.0cm3 beaker.
3. 250l of 5% trisodium citrate, 250l of 10% hydroxylammonium chloride
solution, and 500l of 0.01M orthophenanthroline solution is added in that order.
500l of distilled water is added and the solution is mixed using the micropipette.
4. The mixture is left for 24 hours.
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5. 100l of the solution is transferred into a cuvette using the micropipette. 1900l
of distilled water is then added and the solution is mixed using the micropipette.
6. The absorbance of the sample is measured.
7. Steps 1 through 6 are carried out for triplicate samples of the outer leaves and
stem of the cabbage shown in figure 12 below.
Figure 12: iron(II) phenanthroline complex for triplicate samples of outer leaves and stem
Triplicate
samples of
outer leaves
Triplicate
samples of
stem
Page 20 of 36
5.4 Data Collection
Part of cabbage, 3.000g dry
mass
Sample number Absorbance, A of 10-fold dilution Mean absorbance(a)
± SD
Leaves
1 0.311
0.311 ± 0.005 2 0.316
3 0.307
Stem
1 0.075
0.074 ± 0.004 2 0.071
3 0.078
Table 2: Table of the absorbance readings for different parts of the cabbage after 24 hours and the mean absorbance for the triplicate samples.
(a) Mean absorbance ± Standard Deviation obtained for the triplicate samples.
Page 21 of 36
Qualitative Observations
After heating the samples for about 30 minutes, the samples became completely charred.
The samples began to turn into grey ash after about an hour and began to turn into white
ash after 4 hours of burning. This process is shown below in figure 13.
Figure 13: Ashing process
A black substance was found sticking to the bottom of the beakers after burning the
samples to white ash, shown in figure 14. This substance was dissolved together with the
ash in hydrochloric acid.
Figure 14: black substance in beaker after ashing
The color of the orange-red complex was more intense for the leaves samples than the
stem samples.
Page 22 of 36
5.5 Data Processing
Example calculation of iron(II) concentration in different parts of cabbage
Using the standard calibration curve of iron(II) on page 9, the equation that illustrates the
relationship between absorbance and concentration is given as A = 0.389c + 0.007
Because the graph was plotted as absorbance, A, against concentration, c x 10-4
, the final
c value must be multiplied by 10-4
.
Using the mean absorbance for the triplicate samples of the outer leaves (0.311), the
iron(II) concentration of the outer leaves can be determined:
A = 0.389c + 0.007
c = 7.81 x 10-5
mol dm-3
The final c value must be multiplied by 10 again because the samples were diluted by a
factor of 10 in order to measure the absorbance:
c = (7.81 x 10-5
) x 10 mol dm-3
c = 7.81 x 10-4
mol dm-3
Iron(II) concentration with a value 7.81 x 10-4
mol dm-3
is then converted into a value
with units of mg dm-3
by multiplying the relative atomic mass of iron, 55.85g mol-1
:
c = (7.81 x 10-4
mol dm-3
) x 55.85g mol-1
c = 43.64mg dm-3
Page 23 of 36
Finally, the c value was divided by the dry mass of the sample (3.000g) in order to
determine the amount of iron per gram of sample:
c = 14.55mg dm-3
g-1
The same calculations were repeated for the cabbage stem.
Part of
cabbage,
3.000g dry
mass
Mean
absorbance(a)
± SD
Iron(II)
Concentration,
c/
x 10-4 (b)
mol
dm-3
Iron(II)
Concentration(c)
,
c/ mg dm-3
for
3.000g dry mass
of sample
Amount of
iron(II) per
gram of
sample(d)
, c/
mg dm-3
g-1
Leaves 0.311 ± 0.005 7.81
43.64 14.55
Stem 0.074 ± 0.004 1.72 9.62 3.21
Table 3: Part of cabbage, mean absorbance, iron(II) concentration, and amount of iron(II) per gram of
sample.
(a) Mean absorbance ± Standard Deviation obtained for the triplicate samples.
(b) The graph is plotted as c x 10-4 so the final value of c is multiplied by 10-4. However, c is multiplied by 10 again
because the solution was diluted by a factor of 10.
(c) Calculated by multiplying the relative atomic mass of iron, 55.85g mol-1.
(d) Determined by dividing iron(II) concentration by the mass of sample used.
Thus, it was determined that the leaves contained more iron(II) per gram of sample than
the stem.
Page 24 of 36
6. Quantification of iron(II) concentrations in the outer and
inner leaves
Using the fact that the leaves had higher iron content than the stem of the cabbage, I
extended my research to investigate whether the inner or outer leaves of the cabbage
contained more iron.
6.1 Methodology for quantification
The same methodology for preparing the samples of the inner leaves of the cabbage,
reducing iron(III), and measuring absorbance was employed. However, random samples
of leaves from the yellow inner parts of cabbage (Brassica rapa ssp. pekinensis) were
taken instead of stem samples. The same procedure for calculating the concentration and
the amount of iron(II) per gram of sample was used. Figure 15 shows the triplicate
samples of the outer and inner leaves.
Figure 15: iron(II) phenanthroline complex for triplicate samples of outer and inner leaves
Triplicate
samples of
outer leaves
Triplicate
samples of
inner leaves
Page 25 of 36
6.2 Data Collection
Part of
cabbage,
3.000g dry
mass
Sample
number
Absorbance, A of 10-fold
dilution
Mean absorbance(a)
± SD
Outer Leaves
1 0.311
0.311 ± 0.005 2 0.316
3 0.307
Inner Leaves
1 0.106
0.108 ± 0.003 2 0.108
3 0.111
Table 4: Table of the absorbance readings for the outer and inner leaves of cabbage after 24 hours and the
mean absorbance for triplicate samples.
(a) Mean absorbance ± Standard Deviation obtained for the triplicate samples.
6.3 Data processing
Part of
cabbage,
3.000g dry
mass
Mean
absorbance(a)
± SD
Iron(II)
Concentration,
c/
x 10-4 (b)
mol
dm-3
Iron(II)
Concentration(c)
,
c/ mg dm-3
for
3.000g dry mass
of sample
Amount of
iron(II) per
gram of
sample(d)
, c/
mg dm-3
g-1
Outer Leaves 0.311 ± 0.005 7.81
43.64 14.55
Inner Leaves 0.108 ± 0.003 2.60
14.50 4.83
Table 5: Part of cabbage, mean absorbance, iron(II) concentration, and amount of iron(II) per gram of
sample.
(a) Mean absorbance ± Standard Deviation obtained for the triplicate samples.
(b) The graph is plotted as c x 10-4 so the final value of c is multiplied by 10-4. However, c is multiplied by 10 again
because the solution was diluted by a factor of 10.
(c) Calculated by multiplying the relative atomic mass of iron, 55.85g mol-1.
(d) Determined by dividing iron(II) concentration by the mass of sample used.
Page 26 of 36
7. Data Presentation
Graph 2: Graphical representation of iron(II) concentration in the outer leaves and stem.
(a) Error bars show 95% confidence interval of the triplicate samples for each part of cabbage.
14.55
3.21
0
2
4
6
8
10
12
14
16
18
Outer Leaves Stemiro
n(I
I) p
er
gra
m o
f sa
mp
le, c
/ m
g d
m-3
g-1
Part of cabbage
The parts of cabbage and the corresponding iron(II) concentration per gram of sample, c/ mg dm-3 g-1
(a)
Page 27 of 36
Graph 3: Graphical representation of iron(II) concentration in the outer leaves and inner leaves.
(a) Error bars show 95% confidence interval of the triplicate samples for each part of cabbage.
14.55
4.83
0
2
4
6
8
10
12
14
16
18
Outer Leaves Inner Leavesiro
n(I
I) p
er
gra
m o
f sa
mp
le, c
/ m
g d
m-3
g-1
Part of cabbage
The parts of cabbage and the corresponding iron(II) concentration per gram of sample, c/ mg dm-3 g-1
(a)
Page 28 of 36
8. Data Analysis
Graphs 2 and 3 show the iron concentration to be the highest for the outer leaves with a
concentration of 14.55mg dm-3
g-1
. The inner leaves have the next highest iron content
with 4.83 mg dm-3
g-1
, while the stem has the least iron concentration with
3.21 mg dm-3
g-1
.
There are two possible explanations for this result.
The outer leaves are green in color while the inner leaves are yellow and the stem is a
whitish color. This means that the outer leaves contain the most chlorophyll a, followed
by the inner leaves and the stem. The presence of more chlorophyll a means more light
energy can be absorbed during photosynthesis, exciting more electrons. More electron
carriers must be present for the photosynthetic electron transport chain, including those
that contain iron such as cytochrome b6f complex, Fe2S2 ferredoxin, ferredoxin-NADP+
reductase. As a result, the cabbage part with more chlorophyll a (outer leaves) will have
higher iron content.
Furthermore, the stem of the cabbage is comprised mainly of xylem and phloem, vascular
tissues that play an important role in food, mineral, and water transport[15]
. Thus, the
leaves contain more mitochondria than the stem. The mitochondrial electron transport
chain, involved in chemiosmotic phosphorylation16
in the mitochondria, consists of
various electron carriers analogous to those found in the photosynthetic electron transport
16 Pathway that produces ATP from inorganic phosphate and ADP molecule.
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chain. These include iron-containing electron carriers such as Fe–S clusters, cytochrome
b56017
, and the cytochrome bc118
complex as shown in figure 16[16]
. This explains the
higher iron concentration in the outer and inner leaves compared to the stem of the
cabbage.
Figure 16: Iron-containing electron carriers in the mitochondria
17 Hemeprotein in the enzyme complex succinate dehydrogenase of the mitochondrial electron transport chain. 18 Enzyme containing two b-type hemes (bL, bH), one c-type heme (c1), and a two iron, two sulfur
iron-sulfur cluster (2Fe2S).
Page 30 of 36
9. Evaluation
9.1 Limitations and improvements
9.1.1 Obtaining dry mass and ashing
It was difficult to control the temperature of the oven so that the samples did not contain
any water but did not lose any of their non-water mass from charring. Therefore, it was
highly likely that not all the samples had dry mass or retained their full iron concentration
in the process of being charred. Error may also arise from the process of ashing the
samples. After ashing, it was observed that there was a black substance at the bottom of
the beakers that could be unoxidized iron. Since iron is a volatile mineral, it is possible
that some of the iron in the samples was lost during ashing.
Dry ashing using microwave instruments can be used to improve this methodology.
These instruments are programmed to first obtain a dry mass of the sample and convert
this sample into ash. Microwave devices are an accurate as well as an efficient alternative,
as they are able to finish dry ashing in less than an hour[17]
.
9.1.2 Using colorimetry for determining iron concentration
Using a colorimetric method to determine iron concentration may have prevented an
accurate measurement of iron concentration. Other chemical substances present in the
cabbage might have reacted with the orthophenanthroline to form their own colored
complex, interfering with the formation of the orange iron(II) orthophenanthroline
complex and affecting the absorbance readings. This in turn has an effect on calculating
the iron(II) concentration and leads to an inaccurate calculation of iron(II) concentration.
Page 31 of 36
For improvement, other compounds that are highly selective colorimetric reagents for the
quantification of iron can be used. These compounds include 1,10-phenanthroline; 4,7-
diphenyl-1, 10-phenanthroline; 2,2'-bipyridyl; 2,6-bis(2-pyridyl)pyridine; 2,4,6-tris (2-
pyridyl)-1,3,5-triazine; and phenyl 2-pyridyl ketoxime[18]
.
Also, atomic absorption spectroscopy(AAS) can be used instead of colorimetry as it is a
more accurate, precise, and fast method for determining the concentration of a specific
mineral. Like the colorimetric method, samples must be first ashed and dissolved in an
aqueous solution. The sample is vaporized and atomized19
in the atomic absorption
spectrometer. A beam of UV-visible radiation is passed through the sample and absorbed
by the free atoms in the sample. This absorption of the radiation is measured at a
wavelength specific to iron. The location and intensity of the peaks in the absorption
spectra can then be used to determine the iron concentration[16]
.
9.1.3 Number of samples
Since only triplicate samples were performed for the different parts of cabbage, the
sample size is too small in order for the results to be a general representation of the iron
concentration for the Brassica rapa ssp. Pekinensis. Thus, the sample size must be
increased in order for the data to be accurate and applicable to the general population of
Brassica rapa ssp. Pekinensis.
19 To be separated into free atoms.
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9.2 Unresolved questions for further investigation
9.2.1 Determining ascorbic acid concentration in Brassica rapa ssp. Pekinensis
While researching about iron, it was discovered that ascorbic acid helps the body’s iron
absorption[10]
. Further investigation can be done on the ascorbic acid concentration in the
different parts of Brassica rapa ssp. Pekinensis that were examined (stem, outer leaves,
and inner leaves). This will help determine which part of cabbage should be consumed in
order for the body to absorb iron easily, as well as establish which part of cabbage has the
highest ascorbic acid concentration.
9.2.2 Determining iron concentration in Kimchi
This investigation focused on determining the iron concentration for different parts of
Brassica rapa ssp. Pekinensis, a type of cabbage commonly used to make Kimchi. One
way to extend the investigation is to determine the iron concentration in the stem, outer
leaves, and inner leaves of commercial Kimchi. The data from this experiment can be
compared with the data obtained from Brassica rapa ssp. Pekinensis. Thus, it can be
determined if the chemical reactions that take place during the process of making Kimchi
(i.e. fermentation) significantly affects the iron concentrations in the different parts of
cabbage. Additionally, this extended investigation can show if the order of cabbage parts
with the highest to lowest iron content remains outer leaves, inner leaves, and stem even
after being made into Kimchi.
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10. Conclusion
The first part of the investigation, which was to determine whether the outer leaves or the
stem of the cabbage contained more iron, it was discovered that the outer leaves had more
iron(II) per gram of sample with 14.55 mg dm-3
g-1
than the stem with 3.21 mg dm-3
g-1
.
In the second part of the investigation, which examined whether the outer leaves or the
inner leaves of the cabbage had higher iron content, it was discovered the inner leaves
had less iron(II) per gram of sample with 4.83 mg dm-3
g-1
.
Page 34 of 36
11. Appendix A
Preparation of necessary solutions for iron(II) quantification
1. 1000.0cm3 of 0.01M iron(II) solution was prepared using hydrated iron(II) sulfate
Fe(SO4)2(NH4)2∙6H2O and 4.0M Hydrochloric acid. Serial dilutions were carried
out to obtain iron(II) standard solutions with concentrations of 1.00 x 10-4
M, 5.00
x 10-5
M, 2.50 x 10-5
M, 1.25 x 10-5
M, and 6.25 x 10-6
M.
2. 5% trisodium citrate (Na3C6H5O7) solution was prepared by dissolving (5.000 ±
0.001)g of trisodium citrate in 100.0cm3 of distilled water.
3. 10% hydroxylammonium chloride (NH3OHCl) solution was prepared by
dissolving (10.000 ± 0.001)g of hydroxylammonium chloride in 100.0cm3 of
distilled water.
4. 0.01M orthophenanthroline solution was prepared by dissolving (0.198±0.001)g
of orthophenanthroline in 10.0cm3 of ethanol and 90.0cm
3 of distilled water was
added to form a 100.0cm3 solution.
Page 35 of 36
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