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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


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-3g-1, the inner leaves the next highest with 4.83mg dm-3g-1, and the stem with the lowest iron content of 3.21mg dm-3g-1. In conclusion, the outer leaves of the cabbage had the maximum iron concentration. Word Count: 299

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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

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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

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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 myoglobin2. 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. 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. 2

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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].

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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.

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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.

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complex7, Fe2S2 ferredoxin8, and ferredoxin-NADP+ reductase9 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. 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. 8

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3. Methodology The following is the overall methodology for this investigation:

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

Quantification of iron(II) concentrations in outer leaves & stem 1. Prepare samples

2. Reduce iron(III) to iron(II) and measure absorbance

3. Calculate iron(II) concentration using standard regression line

Quantification of iron(II) concentrations in outer & inner leaves 1. Prepare samples

2. Reduce iron(III) to iron(II) and measure absorbance

3. Calculate iron(II) concentration using standard regression line

Scheme 1: Overall methodology of the investigation

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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. 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. 10

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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+ ď‚Ť 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 10-4M, 5.00 x 105

M, 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 cm3 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. Buffer reagent. 14 Excess reducing agent. 13

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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 10-5M, 2.50 x 10-5M, 1.25 x 10-5M, and 6.25 x 10-6M shown in figure 7 below.

From left to right: 1.00 x 10-4M, 5.00 x 10-5M, 2.50 x 10-5M, 1.25 x 10-5M, and 6.25 x 10-6M 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

0.393

0.211

0.108

0.054

0.028

Absorbance, A after 24 hours(a)

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.

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4.4 Data Processing The following is the graph of the standard calibration curve for iron(II) standard:

Absorbance against Concentration, c/ x 10-4 mol dm-3 0.45 0.4 y = 0.3894x + 0.0079 R² = 0.9986

Absorbance, A

0.35

0.3 0.25 0.2 0.15 (a) 0.1 0.05 0 0

0.2

0.4

0.6

0.8

1

1.2

Concentration of iron(II), c/ x 10-4 mol dm–3

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.

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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:

Preparation of samples 2. Burn until sample is white ash

1. Obtain dry mass

3. Add 4.0M HCl

Reduce iron(III) to iron(II) and measure absorbance 1. Spin solution in microcentrifuge

3. Dilute sample, measure absorbance using visible spectrophotometry

2. Add necessary solutions

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

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 170ď‚°C and the stem were heated for 30~40 minutes at 150ď‚°C in an oven to obtain dry mass15.

15

Mass of matter when it is completely dried and without any water content.

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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 (ď‚ą0.001g) 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

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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 (100–1000l). The tube is spun in a centrifuge machine shown in figure 11 to precipitate unwanted ash.

Figure 11: Microcentrifuge

2. 500l of solution is transferred into a 100.0cm3 beaker. 3. 250l of 5% trisodium citrate, 250l of 10% hydroxylammonium chloride solution, and 500l of 0.01M orthophenanthroline solution is added in that order. 500l 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. 100ď ­l of the solution is transferred into a cuvette using the micropipette. 1900ď ­l 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.

Triplicate samples of outer leaves

Triplicate samples of stem

Figure 12: iron(II) phenanthroline complex for triplicate samples of outer leaves and stem

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5.4 Data Collection Part of cabbage, 3.000g dry

Sample number

Absorbance, A of 10-fold dilution

1

0.311

2

0.316

3

0.307

1

0.075

2

0.071

3

0.078

Mean absorbance(a) ± SD

mass

Leaves

Stem

0.311 ± 0.005

0.074 ± 0.004

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.

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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.

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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

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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

Mean

Iron(II)

cabbage,

absorbance(a)

Concentration, Concentration(c), iron(II) per

3.000g dry

± SD

c/

c/ mg dm-3 for

gram of

x 10-4 (b) mol

3.000g dry mass

sample(d), c/

dm-3

of sample

mg dm-3 g-1

mass

Iron(II)

Amount of

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.

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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.

Triplicate samples of outer leaves

Triplicate samples of inner leaves

Figure 15: iron(II) phenanthroline complex for triplicate samples of outer and inner leaves

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6.2 Data Collection Part of cabbage, 3.000g dry mass

Sample number

Absorbance, A of 10-fold dilution

1

0.311

2

0.316

3

0.307

1

0.106

2

0.108

3

0.111

Outer Leaves

Inner Leaves

Mean absorbance(a) ± SD

0.311 ± 0.005

0.108 ± 0.003

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

Outer Leaves

0.311 ± 0.005

Iron(II) Concentration, c/ x 10-4 (b) mol dm-3 7.81

Inner Leaves

0.108 ± 0.003

2.60

Iron(II) Concentration(c), c/ mg dm-3 for 3.000g dry mass of sample 43.64 14.50

Amount of iron(II) per gram of sample(d), c/ mg dm-3 g-1 14.55 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.

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iron(II) per gram of sample, c/ mg dm-3 g-1

7. Data Presentation

The parts of cabbage and the corresponding iron(II) concentration per gram of sample, c/ mg dm-3 g-1 18 16

14.55 (a)

14 12

10 8 6 3.21

4 2 0 Outer Leaves

Stem

Part of cabbage 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.

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iron(II) per gram of sample, c/ mg dm-3 g-1

The parts of cabbage and the corresponding iron(II) concentration per gram of sample, c/ mg dm-3 g-1 18 16

14.55

(a)

14 12 10 8 6

4.83

4 2 0 Outer Leaves

Inner Leaves

Part of cabbage 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.

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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-3g-1. The inner leaves have the next highest iron content with 4.83 mg dm-3g-1, while the stem has the least iron concentration with 3.21 mg dm-3g-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 (2Fe2S).

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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.

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For improvement, other compounds that are highly selective colorimetric reagents for the quantification of iron can be used. These compounds include 1,10-phenanthroline; 4,7diphenyl-1, 10-phenanthroline; 2,2'-bipyridyl; 2,6-bis(2-pyridyl)pyridine; 2,4,6-tris (2pyridyl)-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.

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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 10-4M, 5.00 x 10-5M, 2.50 x 10-5M, 1.25 x 10-5M, and 6.25 x 10-6M. 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 90.0cm3 of distilled water was added to form a 100.0cm3 solution.

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12. References [1] “Iron.” Linus Pauling Institute at Oregon State University, 2009. Web. 20 Sept 2010. <http://lpi.oregonstate.edu/infocenter/minerals/iron/> [2] “Dietary Supplement Fact Sheet: Iron.” Office of Dietary Supplements. Web. 20 Sept 2010. <http://ods.od.nih.gov/factsheets/iron/> [3] “What Does Iron Do?” A to Z of Health, Beauty and Fitness. Web. 22 Sept 2010. <http://health.learninginfo.org/what-does-iron-do.htm> [4] “Korea.” SPOON Foundation Adoption Nutrition. Web. 22 Sept 2010. <http://adoptionnutrition.org/nutrition-by-country/korea/> [5] “Kimchi, from food to science.” Korea.net. Web. 22 Sept 2010. <http://www.korea.net/detail.do?guid=28037> [6] Kim, Yong-Suk, Zian-Bin Zheng, and Dong-Hwa Shin. “Growth Inhibitory Effects of Kimchi (Korean Traditional Fermented Vegetable Product) against Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus.” Journal of Food Protection. (2008): 325-32. Print. [7] “Napa Cabbage.” Healthaliciousness.com. Web. 15 Dec 2010. <http://www.healthaliciousness.com/vegetables/napa-cabbage.php> [8] Posh, Linda. “The Health Benefits of Cabbage.” Ezine articles. Web. 17 Dec 2010. <http://ezinearticles.com/?The-Health-Benefits-of-Cabbage&id=78014> [9] “Diseases of Iron Metabolism.” The University of Utah Eccles Health Sciences Library. Web. 17 Dec 2010. <http://library.med.utah.edu/WebPath/TUTORIAL/IRON/IRON.html> [10] “Iron.” Veganhealth.org. Web. 17 Dec 2010. <http://www.veganhealth.org/articles/iron#fn3> [11] Sivakumar, S. “Photosynthesis.” Bio-Siva. Web. 4 Jan 2011. <http://biosiva.50webs.org/photo.htm> [12] Outlaw, William. “Use of Spectrophotometer.” Experimental Biology Laboratory. Web. 5 Oct 2010. <http://www.southernmatters.com/BSC_3402L/> [13] Heidcamp, William H. “Spectrophotometry.” Cell Biology Laboratory Manual. Web. 5 Oct 2010. <http://homepages.gac.edu/~cellab/appds/appd-g.html> [14] Blauch, David. “Spectrophotometry.” Davidson College Chemistry Resources. Web. 5 Oct 2010. <http://www.chm.davidson.edu/vce/spectrophotometry/BeersLaw.html> [15] Muller, Michael. “Plant Structure and Function.” University of Illinois at Chicago BIOS 100 Laboratory. Web. 4 Jan 2011. <http://www.uic.edu/classes/bios/bios100/labs/plantanatomy.htm> Page 35 of 36


[16] McNeil, Stephen. “Mitochondrial Electron Transport Chain.” University of British Columbia Department of Chemistry Course Documents. Web. 15 Jan 2011. <https://people.ok.ubc.ca/wsmcneil/bio/electronchain.htm> [17] McClements, Julian. “Analysis of Ash and Minerals.” Analysis of Food Products: Food Science 581 Class Notes. Web. 15 Jan 2011. <http://wwwunix.oit.umass.edu/~mcclemen/581Ash&Minerals.html> [18] “Colorimetric Determination of Iron.” Freepatentsonline. Web. 15 Jan 2011. <http://www.freepatentsonline.com/3836331.html>

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IB EE on spectrophotometric determination of iron in cabbage  

IB EE on spectrophotometric determination of iron in cabbage

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