Spectrophotometric determination of iron in cabbage

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Research done by my IB student Rachel Choi. Please cite and give proper reference to her on her work if you use this material.

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Spectrophotometric determination of iron in cabbage

  1. 1. International Baccalaureate Diploma Program Extended Essay ChemistryDetermination 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
  2. 2. Abstract Kimchi is a traditional Korean dish commonly eaten with every meal. It ismade with a main vegetable ingredient and seasoned with various vegetables. Themost 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 thenapa cabbage. The first part of the investigation compared whether the outer edge ofthe 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 headof the cabbage contained more iron. The iron concentrations were quantified usingvisible spectrophotometry. The absorbance readings were recorded at a max of508.5nm, the wavelength the iron(II) orthophenanthroline complex exhibited amaximum absorption peak. First, a standard calibration curve for iron(II) of absorbance againstconcentration was created. The standard curve had a good correlation (R2=0.9986)between the concentration and absorbance. For each part of the investigation, 3.000grams 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 andform 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 byplugging 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 ironcontent 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 ironconcentration. Word Count: 299 Page 2 of 36
  3. 3. Table of Contents1. Introduction ................................................................................................................... 5 1.1 Rationale for the study .......................................................................................................... 5 1.2 Aim........................................................................................................................................ 6 1.3 Kimchi................................................................................................................................... 6 1.4 Iron ........................................................................................................................................ 82. Hypothesis...................................................................................................................... 83. Methodology ................................................................................................................ 10 3.1 Quantification of iron samples using visible spectrophotometry ........................................ 114. 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 ................................................................................................................... 155. 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 ................................................................................................................... 226. 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 ................................................................................................................... 257. Data Presentation ........................................................................................................ 268. Data Analysis ............................................................................................................... 289. 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 3 of 36
  4. 4. 9.2.1 Determining ascorbic acid concentration in Brassica rapa ssp. Pekinensis ................. 32 9.2.2 Determining iron concentration in Kimchi .................................................................. 3210. Conclusion ................................................................................................................. 3311. Appendix .................................................................................................................... 3412. References .................................................................................................................. 35 Page 4 of 36
  5. 5. 1. Introduction1.1 Rationale for the studyIt is important to study the iron concentration in food because iron is an essentialelement in the metabolism of almost all living organisms[1]. Iron is a criticalconstituent of many important proteins and enzymes in the human body[2]. Forexample, iron can be found in the regulation of cell growth and differentiation,hemoglobin1, and myoglobin2. Iron helps strengthen the immune system, as it is amajor component of cells that fight infections, and provides the human body withenergy by being involved in chemical reactions that converts food into energy.Inadequate iron consumption can result in iron-deficiency anemia, which can lead toextreme 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 irondeficiency was a problem in Korea, mostly among women and children[4]. Hence, Idecided to research about iron in a way that was connected to our diet. I began tothink about how Koreans could increase their daily dietary intake of iron in a way thatdid not deviate from their traditional diet.This made me think about Kimchi3. Koreans used to rely on Kimchi for theirvegetable intake during the winter when no fresh vegetables were available, andcontinue to eat Kimchi with every meal. Kimchi is low in calories while rich in ironas 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 redpepper, ginger, and garlic. Page 5 of 36
  6. 6. My goal is to find the iron concentration in different parts of the Kimchi cabbage andthus determine which part of the cabbage should be consumed for the maximumconsumption of iron.1.2 AimThe aim of this Extended Essay is to investigate how iron content differs for differentparts of cabbage. In the first part of the investigation, I will determine whether thesame mass of cabbage leaves or the stem has a higher iron content. Using the resultsfrom this investigation, I will then find out whether the inner or outermost leaves ofthe 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 rapassp. Pekinensis?1.3 Kimchi Figure 1: KimchiKimchi, shown in figure 1, is a traditional Korean side dish made throughfermentation. Leuconostoc mesenteroides, a type of lactic acid bacteria (LAB), andyeasts initiate the fermentation. When the pH drops with the accumulation of organicacids, other LAB such as Lactobacillus brevis, Lactobacillus plantaru, Streptococcusfaecalis, and Pediococcus cerevisiae carry on the fermentation[6]. Page 6 of 36
  7. 7. Figure 2: Napa cabbage (Brassica rapa ssp. pekinensis)Although various vegetables such as radish, cabbage, and cucumbers are used tomake different types of Kimchi, cabbage Kimchi made with napa cabbage (Brassicarapa ssp. pekinensis) shown in figure 2 is the most common. Napa cabbage is a typeof Chinese cabbage commonly found in East Asian cuisine. It is low in calories and isan excellent source of zinc, iron, calcium, and vitamin C[7]. Moreover, it preventsinfections 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 toinvestigate how iron content differed for different parts of cabbage, specifically thestem, exterior leaves, and inner leaves, as shown in figure 3 below. Page 7 of 36
  8. 8. 1.4 IronThere are two forms of dietary iron: heme iron and nonheme iron. Heme iron is foundin meat, while nonheme iron is in plants. Heme iron is part of the heme molecule, acompound of the porphyrin4 class that forms the nonprotein part of hemoglobin,myoglobin, and some other biological molecules. Most nonheme iron is iron(III) thatmust be reduced to iron(II) before it can be absorbed[9]. Hydrochloric acid and pepsin5are used to release nonheme iron from food. A protein called transferrin then bindsand transports iron from the digestive system to the bloodstream[10].2. HypothesisThe light reactions of photosynthesis take place in the chloroplast of plants.Chlorophyll a6, a pigment in the chloroplast, absorbs all visible light except greenduring photosynthesis. The green light is reflected instead of being absorbed and thuscan be detected by the naked eye. This means the outer leaves contain the mostchlorophyll a because the outer leaves are green, while the inner leaves are yellow andthe stem is white.During the light reactions of photosynthesis, electrons are excited to a higher energystate when light strikes chlorophyll a. The energy from the excited electrons isconverted into ATP and NADPH by a process called photophosphorylation. Inphotophosphorylation, electrons move through the photosynthetic electron transportchain by several electron carriers that contain iron, including the cytochrome b6f4 Pigments such as heme and chlorophyll whose molecules contain a flat ring of four linkedheterocyclic groups.5 Digestive enzyme in the stomach.6 Type of chlorophyll present in photosynthetic organisms, including plants. Page 8 of 36
  9. 9. complex7, Fe2S2 ferredoxin8, and ferredoxin-NADP+ reductase9 as indicated in figure4 below. Because the outer leaves have the greatest amount of chlorophyll a, the outerleaves will have the most number of excited electrons. As a result, more cytochromeb6f complex, ferredoxin, and ferredoxin-NADP reductase will be needed to transportthe electrons, increasing the amount of iron[11]. Figure 4: Iron-containing electron carriers in the chloroplastThus, the hypothesis for this investigation is the green outer leaves of the cabbage willhave the highest iron concentration of iron, followed by the yellow inner leaves andfinally 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 9 of 36
  10. 10. 3. MethodologyThe following is the overall methodology for this investigation: Create iron(II) standard calibration curve 1. Prepare necessary 2. Record absorbances of 3. Graph absorbance against solutions iron(II) standards concentration, find regression line Quantification of iron(II) concentrations in outer leaves & stem 2. Reduce iron(III) to iron(II) 3. Calculate iron(II) concentration 1. Prepare samples and measure absorbance using standard regression line Quantification of iron(II) concentrations in outer & inner leaves 2. Reduce iron(III) to iron(II) 3. Calculate iron(II) concentration 1. Prepare samples and measure absorbance using standard regression line Scheme 1: Overall methodology of the investigation Page 10 of 36
  11. 11. 3.1 Quantification of iron samples using visible spectrophotometrySince small concentrations of iron will be used for this investigation, visiblespectrophotometry was used to quantify iron because of its high degree of precision,sensitivity and accuracy[12]. A visible spectrophotometer, shown in figure 5, passes abeam of light through the sample to determine the wavelength of light correspondingto intensity of the sample’s color. This wavelength is then used to find how muchlight the different iron(II) samples absorbed[13]. Figure 5: Visible spectrophotometerIn this investigation, the color of the samples was orange and a wavelength with anabsorbance peak at 508.5nm, the max, was used. It was at this wavelength themaximum change in absorbance for iron solutions of any concentration occurs.Beer’s Law, which shows a linear relationship between absorbance with cell pathlength and sample concentration, was then used[14]. The general equation is: A = ε l c10Cuvettes with the same size and shape were used for all samples, meaning all sampleswere 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’sabsorbance and its concentration.10A is the sample’s absorbance, ε is the molar absorptivity, l is the length of solution the lightpasses through, and c is the sample’s concentration. Page 11 of 36
  12. 12. All iron samples were reacted with orthophenanthroline11 in order to form an orangecolored 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 oforthophenanthroline and iron(II) phenanthroline complex:Figure 6: Reaction between orthophenanthroline with iron(II) to form iron(II)phenanthroline complex11 A heterocyclic organic compound that forms two or more coordination links with an iron ionto form a strong complex. Page 12 of 36
  13. 13. 4. Creating a standard calibration curve for Iron(II)4.1 Preparation of necessary solutionsIron(II) solutions with different concentrations were prepared. Reacting the solutionswith orthophenanthroline formed an iron complex and the absorbance of eachcomplex was measured. The necessary solutions12 were:  Iron(II) standard solutions with concentrations of 1.00 x 10-4M, 5.00 x 10- 5 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 solution4.2 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.13 Buffer reagent.14 Excess reducing agent. Page 13 of 36
  14. 14. 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 concentrations4.3 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, Aafter 24 hours(a) 0.393 0.211 0.108 0.054 0.028Table 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 initialabsorbance was measured and stabilized after the mixture was left for 24 hours. Thus, the absorbance wasmeasured after 24 hours. The standard deviation for concentrations of standard iron(II) were not recorded as theinstruments used to prepare the standards have negligible uncertainty. Only one reading was recorded because itwas apparent that there was good correlation (R2=0.9986) between the absorbance and concentration aftergraphing the data. Page 14 of 36
  15. 15. 4.4 Data ProcessingThe 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 0.35 y = 0.3894x + 0.0079 R² = 0.9986 0.3 Absorbance, A 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–3Graph 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. Page 15 of 36
  16. 16. 5. Quantification of iron(II) concentrations in the outer leavesand stem5.1 Methodology for quantificationThe diagram below outlines the methodology used to quantify iron(II) in the outer leavesand stem of the cabbage: Preparation of samples 2. Burn until sample is 1. Obtain dry mass 3. Add 4.0M HCl white ash Reduce iron(III) to iron(II) and measure absorbance 1. Spin solution in 3. Dilute sample, measure 2. Add necessary solutions absorbance using visible microcentrifuge spectrophotometry Determine iron(II) concentration 1. Calculate mean absorbance 2. Interpolate mean absorbance 3. Calculate iron(II) concentration, of triplicate samples into standard regression line iron(II) per gram of sampleScheme 2: Methodology to quantify iron(II) in the outer leaves and stem of cabbage5.2 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 170C and the stem were heated for 30~40 minutes at 150C in an oven to obtain dry mass15.15 Mass of matter when it is completely dried and without any water content. Page 16 of 36
  17. 17. Figure 8: Outer leaves and stem samples before and after heating in an oven3. 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 17 of 36
  18. 18. 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) solution5.3 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. Page 18 of 36
  19. 19. 5. 100l of the solution is transferred into a cuvette using the micropipette. 1900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 Triplicatesamples of samples ofouter leaves stem Figure 12: iron(II) phenanthroline complex for triplicate samples of outer leaves and stem Page 19 of 36
  20. 20. 5.4 Data Collection Part of cabbage, 3.000g dry Sample number Absorbance, A of 10-fold dilution Mean absorbance(a) ± SD mass 1 0.311 Leaves 2 0.316 0.311 ± 0.005 3 0.307 1 0.075 Stem 2 0.071 0.074 ± 0.004 3 0.078Table 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 20 of 36
  21. 21. Qualitative ObservationsAfter 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 whiteash after 4 hours of burning. This process is shown below in figure 13. Figure 13: Ashing processA black substance was found sticking to the bottom of the beakers after burning thesamples to white ash, shown in figure 14. This substance was dissolved together with theash in hydrochloric acid. Figure 14: black substance in beaker after ashingThe color of the orange-red complex was more intense for the leaves samples than thestem samples. Page 21 of 36
  22. 22. 5.5 Data ProcessingExample calculation of iron(II) concentration in different parts of cabbageUsing the standard calibration curve of iron(II) on page 9, the equation that illustrates therelationship between absorbance and concentration is given as A = 0.389c + 0.007Because the graph was plotted as absorbance, A, against concentration, c x 10-4, the finalc value must be multiplied by 10-4.Using the mean absorbance for the triplicate samples of the outer leaves (0.311), theiron(II) concentration of the outer leaves can be determined: A = 0.389c + 0.007 c = 7.81 x 10-5 mol dm-3The final c value must be multiplied by 10 again because the samples were diluted by afactor 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-3Iron(II) concentration with a value 7.81 x 10-4 mol dm-3 is then converted into a valuewith 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 22 of 36
  23. 23. Finally, the c value was divided by the dry mass of the sample (3.000g) in order todetermine the amount of iron per gram of sample: c = 14.55mg dm-3 g-1The same calculations were repeated for the cabbage stem.Part of Mean Iron(II) Iron(II) Amount ofcabbage, absorbance(a) Concentration, Concentration(c), iron(II) per3.000g dry ± SD c/ c/ mg dm-3 for gram ofmass x 10-4 (b) mol 3.000g dry mass sample(d), c/ dm-3 of sample 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 3.21Table 3: Part of cabbage, mean absorbance, iron(II) concentration, and amount of iron(II) per gram ofsample.(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 againbecause 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 thanthe stem. Page 23 of 36
  24. 24. 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 Triplicatesamples of samples ofouter leaves inner leaves Figure 15: iron(II) phenanthroline complex for triplicate samples of outer and inner leaves Page 24 of 36
  25. 25. 6.2 Data Collection Part of cabbage, Sample Absorbance, A of 10-fold Mean absorbance(a) 3.000g dry number dilution ± SD mass 1 0.311 Outer Leaves 2 0.316 0.311 ± 0.005 3 0.307 1 0.106 Inner Leaves 2 0.108 0.108 ± 0.003 3 0.111Table 4: Table of the absorbance readings for the outer and inner leaves of cabbage after 24 hours and themean absorbance for triplicate samples. (a) Mean absorbance ± Standard Deviation obtained for the triplicate samples.6.3 Data processingPart of Mean Iron(II) Iron(II) Amount ofcabbage, absorbance(a) Concentration, Concentration(c), iron(II) per3.000g dry ± SD c/ c/ mg dm-3 for gram ofmass x 10-4 (b) mol 3.000g dry mass sample(d), c/ dm-3 of sample mg dm-3 g-1Outer Leaves 0.311 ± 0.005 7.81 43.64 14.55 Inner Leaves 0.108 ± 0.003 2.60 14.50 4.83Table 5: Part of cabbage, mean absorbance, iron(II) concentration, and amount of iron(II) per gram ofsample.(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 againbecause 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 25 of 36
  26. 26. 7. Data Presentation The parts of cabbage and the corresponding iron(II) concentration per gram of sample, c/ mg dm-3 g-1 iron(II) per gram of sample, c/ mg dm-3 g-1 18 16 14.55 (a) 14 12 10 8 6 4 3.21 2 0 Outer Leaves Stem Part of cabbageGraph 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. Page 26 of 36
  27. 27. The parts of cabbage and the corresponding iron(II) concentration per gram of sample, c/ mg dm-3 g-1 18 iron(II) per gram of sample, c/ mg dm-3 g-1 16 14.55 (a) 14 12 10 8 6 4.83 4 2 0 Outer Leaves Inner Leaves Part of cabbageGraph 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. Page 27 of 36
  28. 28. 8. Data AnalysisGraphs 2 and 3 show the iron concentration to be the highest for the outer leaves with aconcentration of 14.55mg dm-3g-1. The inner leaves have the next highest iron contentwith 4.83 mg dm-3g-1, while the stem has the least iron concentration with3.21 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 awhitish color. This means that the outer leaves contain the most chlorophyll a, followedby the inner leaves and the stem. The presence of more chlorophyll a means more lightenergy can be absorbed during photosynthesis, exciting more electrons. More electroncarriers must be present for the photosynthetic electron transport chain, including thosethat 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 havehigher iron content.Furthermore, the stem of the cabbage is comprised mainly of xylem and phloem, vasculartissues that play an important role in food, mineral, and water transport[15]. Thus, theleaves contain more mitochondria than the stem. The mitochondrial electron transportchain, involved in chemiosmotic phosphorylation16 in the mitochondria, consists ofvarious electron carriers analogous to those found in the photosynthetic electron transport16 Pathway that produces ATP from inorganic phosphate and ADP molecule. Page 28 of 36
  29. 29. chain. These include iron-containing electron carriers such as Fe–S clusters, cytochromeb56017, and the cytochrome bc118 complex as shown in figure 16[16]. This explains thehigher iron concentration in the outer and inner leaves compared to the stem of thecabbage. Figure 16: Iron-containing electron carriers in the mitochondria17 Hemeprotein in the enzyme complex succinate dehydrogenase of the mitochondrial electrontransport chain.18 Enzyme containing two b-type hemes (bL, bH), one c-type heme (c1), and a two iron, two sulfuriron-sulfur cluster (2Fe2S). Page 29 of 36
  30. 30. 9. Evaluation9.1 Limitations and improvements9.1.1 Obtaining dry mass and ashingIt was difficult to control the temperature of the oven so that the samples did not containany water but did not lose any of their non-water mass from charring. Therefore, it washighly likely that not all the samples had dry mass or retained their full iron concentrationin the process of being charred. Error may also arise from the process of ashing thesamples. After ashing, it was observed that there was a black substance at the bottom ofthe beakers that could be unoxidized iron. Since iron is a volatile mineral, it is possiblethat 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 convertthis 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 concentrationUsing a colorimetric method to determine iron concentration may have prevented anaccurate measurement of iron concentration. Other chemical substances present in thecabbage might have reacted with the orthophenanthroline to form their own coloredcomplex, interfering with the formation of the orange iron(II) orthophenanthrolinecomplex and affecting the absorbance readings. This in turn has an effect on calculatingthe iron(II) concentration and leads to an inaccurate calculation of iron(II) concentration. Page 30 of 36
  31. 31. For improvement, other compounds that are highly selective colorimetric reagents for thequantification 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 amore accurate, precise, and fast method for determining the concentration of a specificmineral. Like the colorimetric method, samples must be first ashed and dissolved in anaqueous solution. The sample is vaporized and atomized19 in the atomic absorptionspectrometer. A beam of UV-visible radiation is passed through the sample and absorbedby the free atoms in the sample. This absorption of the radiation is measured at awavelength specific to iron. The location and intensity of the peaks in the absorptionspectra can then be used to determine the iron concentration[16].9.1.3 Number of samplesSince only triplicate samples were performed for the different parts of cabbage, thesample size is too small in order for the results to be a general representation of the ironconcentration for the Brassica rapa ssp. Pekinensis. Thus, the sample size must beincreased in order for the data to be accurate and applicable to the general population ofBrassica rapa ssp. Pekinensis.19 To be separated into free atoms. Page 31 of 36
  32. 32. 9.2 Unresolved questions for further investigation9.2.1 Determining ascorbic acid concentration in Brassica rapa ssp. PekinensisWhile researching about iron, it was discovered that ascorbic acid helps the body’s ironabsorption[10]. Further investigation can be done on the ascorbic acid concentration in thedifferent 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 inorder for the body to absorb iron easily, as well as establish which part of cabbage has thehighest ascorbic acid concentration.9.2.2 Determining iron concentration in KimchiThis investigation focused on determining the iron concentration for different parts ofBrassica rapa ssp. Pekinensis, a type of cabbage commonly used to make Kimchi. Oneway to extend the investigation is to determine the iron concentration in the stem, outerleaves, and inner leaves of commercial Kimchi. The data from this experiment can becompared with the data obtained from Brassica rapa ssp. Pekinensis. Thus, it can bedetermined 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 ofcabbage. Additionally, this extended investigation can show if the order of cabbage partswith the highest to lowest iron content remains outer leaves, inner leaves, and stem evenafter being made into Kimchi. Page 32 of 36
  33. 33. 10. ConclusionThe first part of the investigation, which was to determine whether the outer leaves or thestem of the cabbage contained more iron, it was discovered that the outer leaves had moreiron(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 theinner leaves of the cabbage had higher iron content, it was discovered the inner leaveshad less iron(II) per gram of sample with 4.83 mg dm-3 g-1. Page 33 of 36
  34. 34. 11. Appendix APreparation 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. Page 34 of 36
  35. 35. 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 Sept2010. <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 ofKimchi (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 SciencesLibrary. 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 ChicagoBIOS 100 Laboratory. Web. 4 Jan 2011.<http://www.uic.edu/classes/bios/bios100/labs/plantanatomy.htm> Page 35 of 36
  36. 36. [16] McNeil, Stephen. “Mitochondrial Electron Transport Chain.” University of BritishColumbia 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://www-unix.oit.umass.edu/~mcclemen/581Ash&Minerals.html>[18] “Colorimetric Determination of Iron.” Freepatentsonline. Web. 15 Jan 2011.<http://www.freepatentsonline.com/3836331.html> Page 36 of 36

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