Spectrophotometric quantification of iron in broccoli by wkkok19571

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									             International Baccalaureate Diploma Program
                        Sri KDU Smart School




                        Extended Essay
                          -Chemistry-


  Spectrophotemetric determination of iron content in
          broccoli plants Brassica oleracea




Which part of the broccoli plant contains the highest iron content and
   how different incubating temperature affects its iron content?




                           3990 Words Only




                                 By
                           Choo Ken Loon
                            002206-004
Abstract


        This extended essay is on spectophotometric determination of iron found in different parts of

the broccoli, Brassica oleracea and the effects of incubating temperature on the iron content.


        Spectophotometry quantification was adopted. Iron(II) was reacted with orthophenanthroline

solution to produce orange-red colour complex exhibiting a maximum absorption peak, λmax at 510nm.

Beer-Lambert’s law is applied where the absorbance reading is directly proportional to the

concentration of the sample. A standard calibration curve for iron(II) solution was plotted which

shows a significant (P<0.05) and good correlation (R2 = 0.9929) between concentration and

absorbance. Samples from different parts of the broccoli (buds, stems and base) was obtained, heated

to ash and dissolved in hydrochloric acid(4.0M) to form the required solution. Broccoli bud samples

were incubated in water of different temperatures (24.0°C, 60.0°C, 80.0°C and 100.0°C) for the

second part of the investigation.


        Statistical results show a significantly higher (P<0.05) iron content at the buds compared to the

stems and the base of the broccoli. The amount of iron in the buds and the stems are 2.90mg dm-3g-1 to

0.30mg dm-3g-1 respectively, indicating 90% higher iron content in the buds. Investigation on different

incubating temperature shows a negative correlation between incubating temperature and iron content.

Statistical results showed a significant decrease in iron content observed only when the incubating

temperature is at 100°C. Iron content for control sample(24.0°C), 8.30mg dm-3g-1 decreases to 2.65mg

dm-3g-1 at 100.0°C, implying a significant 61% drop. Incubating temperatures of 60°C to 80°C result to

a range of decrease of iron content between 1.8% and 25%. Statistical tests were carried out for all

results to support the significant difference between the samples (P<0.05).


        The conclusion was that the iron content is highest at the buds of the broccoli and

increasing incubating temperature decreases iron content.


(296 words only)



                                                                                                        2
                  Acknowledgement

                 I would like to thank:

Mr Lawrence Kok for his unending support and guidance

 My parents for supporting me and enduring my needs

       My friends for everything they have done

                          And

  All the other people who helped in this investigation




                                                          3
                                                                  Table of Contents
Abstract ............................................................................................................................................................................2
Acknowledgement ........................................................................................................................................................... 3
1.0          Introduction ..................................................................................................................................................... 5
    1.1          Rationale of study ......................................................................................................................................... 5
2.0          Hypotheses ....................................................................................................................................................... 7
    2.1          Investigation on the different parts of the broccoli plant .............................................................................. 7
    2.2          Investigation on the different incubation temperature................................................................................... 9
3.0          Methodology................................................................................................................................................... 10
4.0          Plotting a standard calibration curve for Iron(II) ...................................................................................... 12
    4.1          Requirements for the plotting of the standard calibration curve for Iron(II) ............................................... 12
    4.2          Procedure to prepare iron(II) phenanthroline complex and plotting the calibration curve .......................... 13
    4.3          Data Collection ........................................................................................................................................... 14
    4.4          Data Processing........................................................................................................................................... 15
5.0          Methodology for iron extraction from different parts of the broccoli plants ........................................... 16
    5.1          Preparing samples from different parts of the broccoli plant ...................................................................... 16
    5.2          Reducing iron(III) and measuring its absorbance ....................................................................................... 17
    5.3          Data Collection ........................................................................................................................................... 18
    5.4          Data Processing........................................................................................................................................... 20
6.0          Methodology adopted to investigate the effects of different incubation temperature on the amount of
             iron in broccoli buds ..................................................................................................................................... 23
    6.1          Preparing samples incubated in distilled water of different temperatures ................................................... 23
    6.2          Reducing iron(III) and measuring absorbance ............................................................................................ 24
    6.3          Data Collection ........................................................................................................................................... 24
    6.4          Data Processing........................................................................................................................................... 27
7.0          Data Presentation .......................................................................................................................................... 30
8.0          Data Processing: ANOVA and Tukey’s HSD Test ..................................................................................... 32
9.0          Data Analysis ................................................................................................................................................. 35
    9.1          Parts of the broccoli plant ........................................................................................................................... 35
    9.2          Incubating temperature ............................................................................................................................... 37
10.0         Evaluation ...................................................................................................................................................... 39
    10.1         Uncertainties and Limitations ..................................................................................................................... 39
    10.2         Ways of Improvement................................................................................................................................. 40
    10.3         Further Investigations ................................................................................................................................. 41
11.0         Conclusion ...................................................................................................................................................... 42
12.0         Reference ........................................................................................................................................................ 43
13.0         Appendix.........................................................................................................................................................45


                                                                                                                                                                                    4
1.0     Introduction

1.1     Rationale of Study

        Iron, one of the most abundant metals on Earth, is essential as it maintains good

health [1] including growth, reproduction and the human immune system. Iron is vital as a

component to form haemoglobin for oxygen transportation.


        Iron deficiency is the number one nutritional disorder [2], it causes fatigue and anaemia

where red blood cells with low concentration of haemoglobin fail to supply adequate amount

of oxygen to the body tissues. Thus, it was found worthy to study iron because it is directly

related to our daily diet and health.


        Questions were devised on how to maximize the iron intake when consuming

particular vegetable where certain variables were manipulated.


        The iron content in different parts of a specific vegetable was initially investigated.

Broccoli, Brassica oleracea was chosen due to high iron content (0.73mg/100g)[4] and a

source of many useful nutrition including high amount of calcium, beta-carotene, vitamin C

and fibre. This is to raise awareness regarding the proper part of the plant to be consumed

which gives us more iron. Further investigation was planned on the effects on iron content

due to the cooking procedure. It was proposed that the temperature of water used to incubate

vegetable would affect the nutrient content in the vegetable significantly. It is important to

create awareness on how simple preparation of vegetable would affect the nutrients found in

the particular vegetable as very often vegetables are overcooked and this affects the valuable

nutrients found in it.




                                                                                                  5
       The research is planned to be carried out on distinct parts of the broccoli plant

Brassica oleracea, namely the buds, stems and the base. The preparation method by soaking

broccoli in water was selected because it is a common and simple method of preparation.


       Hence, the precise research question is:


       “Which part of the broccoli plant contains the highest amount of iron and how

different incubating temperature affects its iron content?”




            Figure 1: The different parts of the broccoli plant the buds, the stems and the base.




                                                                                                    6
2.0      Hypotheses

2.1      Investigation on the different parts of the broccoli plant [5]


         Chlorophyll found in the chloroplast is responsible for light absorption during

photosynthesis. Ferredoxin1, cytochromes b6f2 and other electron-carrier proteins are involved

in the photosynthetic pathway while cytochrome c3, Fe –S clusters and other electron-carrier

proteins are present in mitochondrial electron-transport chain all which function as electron

carriers. All the listed protein complexes above have iron as part of its component.


         It is hypothesized that broccoli buds contains the highest amount of iron because of

higher density of iron-containing electron-carrier proteins:


Photosynthetic pathway


      1. Broccoli buds are greener due to higher density of Chlorophyll a4 compared to the

        stems and base since it is needed for photosynthesis.

      2. More electrons are excited due to higher amount of energy absorbed by chlorophyll.

      3. More ferrodexin, cytochrome b6f proteins and other electron-carrier proteins are

        required for electron transportation.


Mitochondrial electron-transport chain


      1. Mitochondria are more abundant in broccoli buds compared to the stems and base of

        the broccoli plant because the stems and base consist of mostly xylem and phloem

        which functions in food and water transport.




1
  Acidic, low molecular weight, soluble iron-sulphur proteins
2
  Monomeric unit of the complex that contains six bound prosthetic groups, three hemes (f, two hemes b, bp
and bn), one [2Fe-2S] cluster, and one molecule each of chlorophyll-a and carotene
3
  Able to transition between ferrous and ferric states within the cell, therefore functioning efficiently.
4
  Magnesium-containing substituted tetrapyrroles.
                                                                                                             7
2. More cytochrome c, Fe –S clusters and other electron-carrier proteins are required to

  function for the transportation of electrons for the complex processes occurring in the

  mitochondria which involves electron transfer for energy production.




       Figure 2: Electron-carrier proteins in the form of ferrodexin and cytochrome b6f located in the
                                             chloroplast.




    Figure 3: Mitochondrial electron-transport chain where cytochrome c and Fe –S clusters are located.




                                                                                                         8
2.2        Investigation on the different incubating temperature

           It is hypothesized that iron content in the broccoli plant decreases as the incubating

temperature increases because at high temperature:

       1. Cell walls break down easily.

       2. Increased rate of denaturation of the protein membrane present at the cell membrane.

       3. Increased kinetic energy5 increases probability of the electron-carrier proteins

           diffusing out of the cell membrane.

       4. Proteins in the plant might denature and react with each other forming unknown

           products of altered physical properties such as the increased solubility and ability to

           diffuse through the cell membrane, resulting to iron leaching out of the plant.

       5. The number of molecules having higher energy increases, increasing rate of reaction

           between molecules present in the plant according to the Maxwell–Boltzmann

           distribution, thus iron-containing proteins reacts at a higher rate, resulting to a greater

           loss of iron from the plant through leaching.
             Fraction of molecules
              with a given speed




                       Figure 4: Maxwell-Boltzmann distribution on the distribution of molecular speed at different
                                                        temperature.




5
    Average kinetic energy, EK is directly proportional to the absolute temperature. Emean = 3⁄2kBT.
                                                                                                                      9
3.0      Methodology


Since the iron content in broccoli is in small amount, a sensitive and less complicated method

was used – Spectrophotometry [6]. The theory involved relates concentration of the solution

containing iron to its absorbance of light at a specific wavelength. A visible

spectrophotometer which passes a light beam with wavelength complement to the colour of

the sample was use to measure the amount of light each iron(II) sample of different

concentrations would absorb.


         For this investigation, the product was orange-red in colour, thus a light beam of

specific wavelength which exhibits an absorbance peak at 510nm, λmax was used.


         Beer’s Law[7] was applied, it can be expressed as:


                                                     A = εlc6


The same type of cuvette was used in all the measurements and the molar absorptivity is

constant for all the samples, therefore both ε and l are constants. Hence, this indicates that the

absorbance of a sample is directly proportional to its concentration, A  c and the relationship

between variables A and c is linear.


         In this research, iron solution is reacted with orthophenanthroline (ο-phen) (figure 7)

to form an orange-red iron(II) orthophenanthroline complex (figure 8) at optimum pH of 3.5

where the complex would be stable for at least 20 hours at pH 2 – 9 [8]. The chemical

equation is given as:


                                           Fe2+ + 3Phen ↔ Fe(Phen)3 2+



6
 A is the absorbance of the sample. ‘ε’ is the molar absorptivity, which is a constant. ‘l’ is the thickness of the
cuvette holding the sample. ‘c’ is the concentration of the absorbing species.
                                                                                                                  10
The stoichiometric ratio is 1: 3.




                              Figure 5: The structure of o-phenanthroline.




               Figure 6: The structure of iron(II) phenanthroline complex and the solution.




                                    Figure 7: Visible Spectrophotometer


                                                                                              11
4.0      Plotting a standard calibration curve for Iron(II)


4.1      Requirements for the plotting of the standard calibration curve for Iron(II)


Iron(II) solution with known concentration were prepared. The solutions were reacted with o-

phennanthroline to form an iron complex and the absorbance of each complex measured. The

required solutions7 are:


      1. Iron(II) standard solutions8 of concentrations 25.0x10-5M, 10.0x10-5M, 7.50x10-5M,

         6.00x10-5M, 5.00x10-5M and 2.50x10-5M.


      2. 10% hydroxylammonium chloride9, NH3OHCl solution.


      3. 5% of trisodium citrate10, Na3C6H5O7 solution.


      4. 0.01M Orthophenanthroline solution of 0.01M.




7
  See appendix 1 for procedures to prepare the required solutions.
8
  See appendix 2 for dilution of iron(II) standards
9
  Excess reducing agent.
10
   Buffer reagent.
                                                                                              12
4.2        Procedure to prepare iron(II) phenanthroline complex and plotting the

           calibration curve


       1. 1.0cm3 of 25.0x10-5M iron(II) standard was transferred into a 100.0cm3 container

           using a micropipette.

       2. 0.5cm3 of 5% trisodium citrate solution was added followed by 0.5cm3 of 10%

           hydroxylammonium chloride solution and finally 1.0cm3 of 0.01M phenanthroline

           solution.

       3. The mixture was left for 60 minutes for the orange-red complex to form completely.

       4. 1.0cm3 of the solution was transferred into a cuvette.

       5. The instrument was calibrated using a blank solution11 with wavelength setting at

           510nm.

       6. The cuvette was inserted into the cuvette holder inside the visible spectrophotometer.

       7. The absorbance reading was recorded at 510nm, λmax12, again after 90 and 120

           minutes.

       8. Steps 1-7 were repeated using iron(II) standards of 10.0x10-5M, 7.50x10-5M,

           6.00x10-5M, 5.00x10-5M and 2.50x10-5M.




                       Figure 8: Iron(II) phenanthroline complex at different concentration.


11
     A solution containing all reagents except iron.
12
     A wavelength that exhibits maximum absorption peak.
                                                                                               13
4.3         Data Collection


                                             Absorbance reading, A after                    Mean Absorbance(a)
  Concentration, c /
                                                                                            ± (95%) Confidence
          mol dm-3                60 minutes           90 minutes             120 minutes
                                                                                                   Interval13

          25.0x10-5                  0.801                 0.761                 0.721           0.761 ± 0.100

          10.0x10-5                  0.337                 0.373                 0.302           0.337 ± 0.090

          7.50x10-5                  0.228                 0.233                 0.232           0.231 ± 0.007

          6.00x10-5                  0.189                 0.206                 0.196           0.197 ± 0.020

          5.00x10-5                  0.059                 0.202                 0.107           0.123 ± 0.600

          2.50x10-5                  0.118                 0.062                 0.089           0.090 ± 0.070

Table 1: The concentration of iron(II) standards, the corresponding absorbance reading after 60, 90 and 120
minutes respectively and the mean absorbance.
(a)
      Mean absorbance ± (95%) Confidence Interval obtained at 60, 90 and 120 minutes.



Standard deviation for standard iron(II) concentrations were not recorded as uncertainty due

to the apparatus used for the preparation of the standards is assumed to be insignificant.




Due to the instability of the instrument, the absorbance readings fluctuate throughout the

absorbance measuring process. Mean absorbance was calculated using the absorbance

measured at intervals of 60, 90 and 120 minutes and the confidence interval was calculated.




13
      See appendix 3 for further calculations on (95%) confidence interval.
                                                                                                              14
4.4                    Data Processing



                                    Graph of Absorbance, A against Concentration, c / x10-4 mol dm-3
                        1.000
                                                                                                y = 0.3045x + 0.0055
                                                                                                     R² = 0.9929
                        0.800
                                                                                                       (P<0.05)
       Absorbance, A




                        0.600


                        0.400

                                                                           (a)
                        0.200


                        0.000
                                0               0.5                    1                       1.5                     2   2.5   3
                       -0.200
                                                                    Concentration of iron, c / x10-4 mol dm-3


Graph 1: The calibration curve for iron(II) plotted from the collected data.
(a)
      Error bars denote (95%) confidence interval.
(b)
      Significant correlation between concentration and absorbance (R2 = 0.9976, α = 0.05, P<0.05)14


14
      See appendix 4 for regression analysis.
                                                                                                                                 15
5.0        Methodology for iron extraction from different parts of the broccoli plants


The samples undergo dry ashing so that iron would be oxidized by air. Ash was dissolved in

(4.0M) hydrochloric acid15 to obtain an aqueous solution for absorbance measurement.


5.1        Preparing samples from different parts of the broccoli plant


       1. Random samples from the buds, stems and base were collected from a broccoli plant

           (Brassica oleracea). Triplicate samples were obtained from different broccoli plants.

       2. The samples were heated for 30 minutes in an oven on a heating tray to obtain dry

           mass16.

       3. 4.000g of samples from the buds, stems and base respectively was weighed using and

           electronic balance (±0.001g) and placed into a crucible. Triplicate samples were

           prepared.

       4. The crucibles without the lids on were heated directly in a crucible using a Bunsen

           burner for six hours.

       5. 1.0cm3 of concentrated hydrochloric acid (4.0M) was added to each of the crucible to

           dissolve the ash to form aqueous iron(III) solution.




         Figure 9: Samples from the broccoli buds, stems and base on a heating tray and during dry ashing.


15
     See appendix 5 for pictures of the ash dissolved in HCl acid.
16
     Mass of the dried matter which does not contains water.
                                                                                                             16
5.2      Reducing iron(III) and measuring its absorbance


      1. 1.0cm3 of solution together with ash was transferred into a microcentrifuge tube using

         a micropipette (100 – 1000)µl. The centrifuge machine is used to precipitate

         unwanted ash in the microcentrifuge tube.


      2. 1.0cm3 of solution is transferred from the microcentrifuge tube into a 100.0cm3

         container.


      3. 0.5cm3 of 5% trisodium citrate solution was added followed by 0.5cm3 of 10%

         hydroxylammonium chloride solution and finally 1.0cm3 of 0.01M phenanthroline

         solution.


      4. Absorbance of the samples was measured at time intervals 60, 90, 120 minutes.




                                                                                             17
5.3         Data Collection:


                                  Random                                                                                             Mean
                                                                     Absorbance reading, A after                                                        Mean absorbance(a) ±
 Part of the plant                 samples                                                                                     absorbance for             (95%) Confidence
                                (triplicates)                                                                                   each sample                     Interval
                                                           60 minutes          90 minutes              120 minutes

                                       1                     0.596                 0.669                    0.621                    0.629                    0.639±0.100
            Buds                       2                     0.602                 0.730                    0.616                    0.649
                                      3(b)                     -                      -                        -                        -
                                       1                     0.077                 0.152                    0.033                    0.087
           Stems                       2                     0.060                 0.089                    0.064                    0.071                    0.079±0.100

                                      3(b)                     -                      -                        -                        -
                                       1                     0.054                 0.118                    0.012                    0.061
            Base                       2                     0.089                 0.055                    0.075                    0.073                    0.070±0.020

                                       3                     0.014                 0.145                    0.070                    0.076
Table 2: Parts of the plant, random samples (n=3), the absorbance reading after 60, 90 and 120 minutes respectively and the mean absorbance with (95%) confidence
interval for triplicate samples.
(a)
      Mean absorbance ± (95%) Confidence Interval obtained for the random triplicate samples.
(b)
   / - Results were not included into the mean absorbance due to inconsistencies and irregularities. A 90% confidence interval Q test (rejection test) is performed to determine
the outliners in the data collected.17


17
      See appendix 7 for further calculations on Q test.
                                                                                                                                                                             18
Qualitative Observations


Some brown-black coloured substance18 was observed sticking on the side of the inner wall

of the crucible after heating. The black substance was scraped off using a sharp pointed

wooden splinter and was dissolved in hydrochloric acid (4.0M) together with the ash. The

intensity of the orange-red coloured complex decreases in the sequence of buds, stems and

base.




18
     See Appendix 5 for picture of the brown-black substance sticking on the wall.
                                                                                            19
5.4    Data Processing
With reference to the standard calibration curve, the concentration of iron(II) in the different

part of the broccoli plant was determined.


                                                                                                        Amount of
             Mean Absorbance(a)
Parts Of The                                         Concentration of         Concentration of         iron(II) per
             ± (95%) Confidence
  Broccoli                                          iron(II) ± SD19, c /      iron(II) ± SD, c /     gram of sample
                  Interval
   Plant                                              x10-4 mol dm-3              mg dm-3                ± SD, c /
                   (n = 3)
                                                                                                       mg dm-3 g-1
        Buds                0.639±0.100                  2.08±0.78                11.61±0.44           2.90±0.11
        Stems               0.079±0.100                 0.241±0.068                1.35±0.38           0.34±0.09
                                                        0.212±0.061
        Base                0.070±0.020                                            1.18±0.34           0.30±0.09
Table 3: Parts of the broccoli plant, mean absorbance with (95%) confidence interval and the amount of iron(II)
per gram of sample.
(a)
      Mean absorbance ± (95%) Confidence Interval obtained for the random triplicate samples.

SD = Standard Deviation




19
      See Appendix 8 for calculations for the standard deviation of the concentration of iron(II).
                                                                                                            20
Example calculation on the amount of iron(II) found in broccoli buds

From the standard calibration curve of iron(II), the regression equation relating absorbance to

concentration is given as A = 0.3045c + 0.0055 (refer to page 16). Since the graph is plotted

as absorbance, A against concentration, c x10-4, the final value of c is multiplied by 10-4. The

mean absorbance for samples from the buds is 0.639.

                    ����−0.0055
Therefore: c   =(                ) x 10-4
                      0.3045


                    0.639−0.0055
               =(                   ) x 10-4 = 2.08x10-4M
                        0.3045


The value in 2.08x10-4mol dm-3 is converted to units of mg dm-3 by multiplying with the

relative atomic mass of iron, 55.85g mol-1.


Therefore: c   = 2.08x10-4M x 55.85g mol-1


               = 11.61mg dm-3


Finally, calculations were made to determine the amount of iron per gram sample.

                   11.61
Therefore: c   =           = 2.90mg dm-3g-1
                    4


Similar calculations were performed for different parts of the plants.




                                                                                              21
Example calculation on the standard deviation of amount of iron(II) found in broccoli buds

Calculations for standard deviation were performed using Windows Excel 2007. The

formula20 used is as shown below:


          sr   1     1          (�������� − ���� )2
SX =
          ����
             { ���� + ���� +���� 2        (���� ���� − ���� )2
                                                     }1/2


          0.02305    1      1           (0.639− 0.290)2
      =
          3044 .14
                   { 2 + 6 +(3044.14)2 (3.26x10 −8 )}1/2


= 7.83x10-6mol dm-3


The value in 7.83x10-6mol dm-3 is converted to units of mg dm-3 by multiplying with the

relative atomic mass of iron, 55.85g mol-1.


Therefore: SX = 7.83x10-6M x 55.85g mol-1


                    = 0.437mg dm-3


Finally, calculations were made to determine the standard deviation for concentration, SX per

gram sample.

                         0.437
Therefore: c        =            = 0.109mg dm-3g-1
                          4


Similar calculations for standard deviation were performed for different parts of the broccoli

plant.


The results show that the broccoli buds contained the highest amount of iron, therefore,

broccoli bud was selected for the second part of the research question which investigates the

effects of incubating temperature on the amount of iron in broccoli buds.



20
     See appendix 6 for the explanations of the symbols used in the formula.
                                                                                             22
6.0      Methodology adopted to investigate the effects of different incubating

         temperature on the amount of iron in broccoli buds


6.1      Preparing samples incubated in distilled water of different temperatures


      1. Random samples from the buds were collected from broccoli plant (Brassica

         oleracea). Triplicate samples were obtained from different broccoli plants.


      2. Water baths at room temperature 24.0°C, 60.0°C, 80.0°C and 100.0°C were prepared

         using electric water baths.


      3. 4.000g of samples from the buds was weighed using an electronic balance (±0.001)

         into separate water baths. Triplicate samples were prepared for each temperature.


      4. The samples were soaked for 15 minutes and then filtered.


      5. The filtered samples were carefully transferred to crucibles without the lids on and

         then heated directly using a Bunsen burner for six hours.


      6. 1.0cm3 of concentrated hydrochloric acid (4.0M) was added to each of the crucible to

         dissolve the ash to form aqueous iron(III) solution.




                           Figure 10: Water baths used to incubate the samples.




                                                                                                23
6.2        Reducing iron(III) and measuring absorbance

Same procedures from part 5.2 (page 18) was carried out to reduce the iron(III) in the

samples and to measure the absorbance.

6.3        Data Collection


Due to the intense colour formed by the complex, the samples were diluted by a factor of 5 to

reduce the orange-red colour intensity formed, enabling the visible spectrophotometer to

measure the absorbance21.


1.0cm3 of sample was pipette out and diluted with 4.0cm3 of distilled water to create a 5.0cm3

solution.




21
     A deviation would occur if the intensity of the sample is too high.
                                                                                           24
        Incubating           Random samples                               Absorbance reading, A after
       temperature                                                                                                        Mean absorbance         Mean absorbance(a) ±
                                                                                                                          for each sample          (95%) Confidence
         (±0.1)°C               (triplicates)              60 minutes               90 minutes            120 minutes                                  Interval

                                       1                      0.289                     0.457                0.371               0.372
          Room
       Temperature,                    2                      0.356                     0.408                0.365               0.376                 0.369±0.020
         24.0°C
                                       3                      0.342                     0.463                0.270               0.358

                                       1                      0.340                     0.429                0.373               0.381

          60.0°C                       2                      0.316                     0.400                0.310               0.342                 0.362±0.050

                                       3                      0.316                     0.438                0.334               0.363

                                      1(b)                       -                        -                    -                   -

          80.0°C                       2                      0.221                     0.415                0.226               0.287                 0.280±0.030

                                       3                      0.241                     0.319                0.258               0.273

                                       1                      0.067                     0.050                0.082               0.066

         100.0°C                                              0.068                     0.119                0.096               0.094                 0.121±0.200
                                       2

                                       3                      0.180                     0.270                0.181               0.204

Table 4: Incubating temperature, the sample number, the absorbance reading after 60, 90 and 120 minutes respectively and the mean absorbance with (95%) confidence
interval for triplicate samples, all of which the samples were diluted by a dilution factor of 5.
(a)
      Mean absorbance ± (95%) Confidence Interval obtained for the random triplicate samples.
(b)
      / - Results were not included into the mean absorbance due to inconsistencies and irregularities.
                                                                                                                                                                     25
Qualitative Observations


After incubating the broccoli buds in water of 100.0°C, it is observed that the water turns

greenish in colour. The intensity of the greenish colour decreases as the temperature of the

water decreases. However, the intensity of the orange-red coloured complex decreases as the

temperature of the water for the sample increase.




         Figure 11: The greenish colour filtrate for the samples at 100.0°C incubating temperature.




                                                                                                      26
6.4             Data Processing


The absorbance for the diluted concentration and its actual concentration expressed in mg dm-3 g-1 for samples incubated at different temperature:


                                                                             Diluted concentration(a)                                                 Actual concentration

          Incubating                                                                                                            Amount of
                                                                 Diluted concentration                                                                Amount of iron(II) in
         temperature               Mean absorbance        (b)                                        Concentration of         iron(II) in one
                                                                       of iron(II)                                                                    one gram of sample ±
           (±0.01)°C                                                                                 iron(II) ± SD, c /      gram of sample
                                   ± Confidence Limit                  ± SD22, c /                                                                           SD, c /
                                                                                                         mg dm-3                 ± SD, c /
                                                                     x10-4 mol dm-3                                                                        mg dm-3 g-1
                                                                                                                                mg dm-3 g-1
          Room                           0.369±0.020                     1.19±0.06                      6.64±0.31                1.66±0.08                   8.30±0.38
      Temperature, 24.0
                                         0.362±0.050                     1.17±0.05
               60.0                                                                                     6.53±0.30                1.63±0.08                   8.15±0.38
                                         0.280±0.030                    0.901±0.062
               80.0                                                                                     5.03±0.35                1.25±0.09                   6.25±0.43
                                         0.121±0.200                    0.379±0.058
              100.0                                                                                     2.12±0.33                0.53±0.08                   2.65±0.40
Table 5: Incubating temperature, mean absorbance with (95%) confidence interval for triplicate samples, its corresponding concentration of iron(II) when compared to the
calibration curve, concentration of iron(II) in mg dm-3 and the concentration of iron(II) in one gram of sample. The data are separated as diluted concentration and actual
concentration.
(a)
      diluted by a factor of 5
(b)
      Mean absorbance ± (95%) Confidence Interval obtained for the random triplicate samples.

SD Standard Deviation



22
      See Appendix 6 for calculations for the standard deviation of the concentration of iron(II).
                                                                                                                                                                              27
Example calculation on the amount of iron(II) found after incubating at 100.0°C


From the standard calibration curve of iron(II), the regression equation relating absorbance to

concentration is given as A = 0.3045(c x 10-4) + 0.0055 (refer to page 16). Since the graph is

plotted as absorbance, A against concentration, c x10-4, the final value of c is multiplied by 10-4.

The mean absorbance for samples incubated at 100.0°C is 0.121.

                    ����−0.0055
Therefore: c   =(                  ) x 10-4
                        0.3045


                    0.121−0.0055
               =(                      ) x 10-4 = 0.379x10-4M
                          0.3045


The value in 0.379x10-4mol dm-3 is converted to units of mg dm-3 by multiplying with the

relative atomic mass of iron, 55.85g mol-1.


Therefore: c   = 0.379x10-4M x 55.85g mol-1


               = 2.12mg dm-3


Calculations were made to determine the amount of iron per gram sample.

                   2.12
Therefore: c   =          = 0.53mg dm-3g-1
                    4


Finally, the value obtained was multiplied by 5 because the samples were diluted by a factor

of 5.


Therefore: c   = 0.53 x 5 = 2.65mg dm-3g-1


Similar calculations were performed for different incubating temperature.




                                                                                               28
Example calculation on the standard deviation of amount of iron(II) found after incubating at

100.0°C


Calculations for standard deviation were performed using Windows Excel 2007. The

formula23 used is as shown below:


          sr   1     1            (�������� − ���� )2
SX =
          ����
             { ���� + ���� +���� 2          (���� ����   − ���� )2
                                                         }1/2


          0.02305    1        1                (0.121− 0.290)2
      =           { + 6 +(3044.14)2 (3.26x10 −8 )}1/2
          3044 .14 3



= 5.84x10-6mol dm-3


The value in 5.84x10-6mol dm-3 is converted to units of mg dm-3 by multiplying with the

relative atomic mass of iron, 55.85g mol-1.


Therefore: SX = 5.84x10-6M x 55.85g mol-1


                    = 0.33mg dm-3


Calculations were made to determine the standard deviation for concentration, SX per gram

sample.

                         0.33
Therefore: c        =             = 0.08mg dm-3g-1
                          4


Finally, the value obtained was multiplied by 5 because the samples were diluted by a factor

of 5.


Therefore: c        = 0.33 x 5 = 0.40mg dm-3g-1


Similar calculations were performed for different incubating temperature.

23
     See appendix 6 for the explanations of the symbols used in the formula.
                                                                                            29
7.0                     Data Presentation


                                                                 The parts of the broccoli and the corresponding concentration of iron(II) per gram of
                                                                                                sample, c / mg dm-3 g-1
        Concentration of iron(II) in one gram of sample,



                                                           3.5


                                                            3                          (a)



                                                           2.5
                          mg dm-3 g-1




                                                            2
                               c/




                                                           1.5


                                                            1


                                                           0.5


                                                            0
                                                                                Buds                             Stems                          Base

                                                                                                 Parts of the broccoli plant


Graph 2: The graphical representation of the amount of iron(II) and in various parts of the broccoli plant.
(a)
      = Error bars denote (95%) confidence interval of triplicate samples of each part of the broccoli plant.
                                                                                                                                                         30
                                                     The incubating temperature, T / °C and the corresponding amount of iron(II) per gram of
                                                                                     sample, c / mg dm-3 g-1
                                                    10
        Amount of iron(II) in one gram of sample,



                                                                       (a)
                                                     9

                                                     8

                                                     7
                      mg dm-3 g-1




                                                     6
                            c/




                                                     5

                                                     4

                                                     3

                                                     2

                                                     1

                                                     0
                                                                  24                     60                         80                   100

                                                                                         Incubating temperature, T / °C

Graph 3: The graphical representation of the amount of iron(II) and at different incubating temperature.
(a)
      = Error bars denote (95%) confidence interval of triplicate samples for each incubating temperature.


                                                                                                                                               31
8.0      Data Processing: ANOVA and Tukey’s HSD Test24


Data processing is carried out using Analysis of Variance (ANOVA) and Tukey’s HSD

(honestly significant difference) test. ANOVA shows the variance between or within each

group.


A null hypothesis is first assumed where there is no difference between the means of different

groups. Then the F ratio is calculated.

                                          mean square between groups
                              F ratio =
                                          mean square within groups


The result of ANOVA shows whether the F ratio is greater than the F critical value at the

significance level of 0.05 (α=0.05), if so, the null hypothesis is rejected and there is one

group that is significantly different from others.


Three assumptions were made to carry out ANOVA:


      1. The observations are independent (the value of one observations is not correlated with

         the value of another).

      2. The observations in each group are normally distributed.

      3. Equal variance for all groups.


Tukey’s HSD post hoc analysis is then conducted to test the hypothesis that all possible pairs

of means are equal. Pairs with differences exceeding the HSD are considered to be

significantly different.




24
 See appendix 9 for ANOVA and Tukey’s HSD Test.
                                                                                               32
ANOVA is performed using Microsoft Excel 2007 while Tukey’s HSD25 test is calculated
manually. The results are tabulated as shown below:

        Variable                 F-value                    F-critical                         Indication



           Parts                 2012.24                       6.94                 ANOVA tests on both sets of
                                                                                   data show F-value > F-critical.
                                                                                    This indicates that there is a
                                                                                    group which is significantly
      Temperature                  23.80                       4.35
                                                                                     different from the others in
                                                                                     their own respective set of
                                                                                                 data.
Table 6: Results of ANOVA on two sets of data to determine whether there is significant difference between
the mean absorbance reading of samples obtained from different parts of the broccoli and for the variable of the
temperature of water which the broccoli buds were soaked in.




25
     See appendix 9 for ANOVA and Tukey’s HSD Test.
                                                                                                              33
Tukey’s HSD multiple comparison test was carried out and the results are tabulated as shown
below:

   Group Combination
    Mean absorbance                   Mean difference of
   reading of different                                        HSD critical value                 Implication
                                      absorbance reading
   parts of the broccoli
           plant
    Buds          Stems                     0.560                      0.029                Significant difference
      Buds              Base                0.569                      0.029               Significant difference
     Stems              Base                0.009                      0.029              No Significant difference
Table 7: Results of Tukey’s HSD test on the mean absorbance reading of samples from each part of the broccoli
plant to determine which group is significantly different than the other.

Significance test at α = 0.05.




     Group Combination, °C                          Mean
   Mean absorbance reading of                     difference         HSD critical
                                                                                               Implication
 broccoli plant soaked in different              absorbance            value
      temperature of water                         reading
       100                       80                 0.159                0.111           Significant difference

       100                       60                 0.241                0.111           Significant difference


       100                       24                 0.247                0.111           Significant difference


        80                       60                 0.082                0.111          No significant difference


        80                       24                 0.089                0.111          No significant difference


        60                       24                 0.007                0.111          No significant difference

Table 8: Results of Tukey’s HSD test on the mean absorbance reading of samples that have been incubated in
water of different temperature to determine which group is significantly different than the other.

Significance test at α = 0.05.




                                                                                                          34
9.0    Data Analysis

9.1    Parts of the broccoli plant


       Graph 2 indicates that iron content is highest in the broccoli buds with 2.90mg dm-3g-1

followed by the stems and base which is 0.34mg dm-3g-1 and 0.30mg dm-3g-1 respectively

indicating that the iron content in the broccoli buds is 88% ~ 90% higher when compared to the

stems and base.


       Statistical tests performed at α=0.05 showed a significant difference in iron content

found in broccoli buds and stems and broccoli buds and base as the mean difference of each

group exceeds the critical value (Table 7). There is no significant difference between the iron

content in broccoli stems and base. Therefore, results show that iron content is highest at the

broccoli buds.


       There are two possible explanations on why iron content is highest at the broccoli buds:


       The broccoli buds are greener compared to the stems and the base due to higher

chlorophyll density. Therefore, more light energy is absorbed for electron excitation during

photosynthesis. Specific iron-containing electron-carrier proteins like ferredoxin and

cytochrome b6f are required for electron transportation. In conclusion, broccoli buds are

greener due to high chlorophyll density which absorbs more energy for more electron

excitation. Thus, more iron-containing electron-carrier proteins are required for electron

transportation, resulting to highest iron content in the broccoli buds.


       Another possible explanation is that electron-transport chains are present in the

mitochondria. Mitochondria is found abundantly in the broccoli buds because they are

involve in energy production while the stems and base contains xylem and phloem are

involved in food and water uptake. Iron-containing electron-carrier proteins such as

                                                                                               35
cytochrome c and Fe –S clusters transports electrons in the mitochondrial electron-trasport

chain. In conclusion, this process occurs more frequently in the broccoli buds due to higher

abundance of mitochondria, resulting in a greater number of iron-containing electron-carrier

proteins found in the broccoli buds. Thus, iron content is highest in the broccoli buds.




                                                                                               36
9.2     Incubating temperature


        Graph 3 indicates that the iron content in broccoli buds samples incubated at 100.0°C

is the lowest followed by samples incubated at 80.0°C, 60.0°C and room temperature 24.0°C.

Results show that iron content in the control sample incubated at room temperature 24.0°C

and sample incubated at 100.0°C is 8.30mg dm-3g-1 and 2.65mg dm-3g-1 respectively

indicating a 68% decrease in iron content after incubating at 100.0°C.


        Table 5 shows that some data have been omitted due to inconsistencies and

irregularities compared to the other data. However, for this part of the data collected, Q

rejection test was not performed. The omission was based solely on observations as the

outlier data would influence the results of the investigation.


        Statistical tests performed at α=0.05 showed a significant difference in iron content of

broccoli buds samples incubated at 100.0°C when compared to samples incubate at 80.0°C,

60.0°C and room temperature 24.0°C where the mean difference of each group exceeds the

critical value (Table 8).


        Preliminary data suggest that iron content is the lowest when broccoli buds samples

are incubated at 100.0°C. Given a few possible explanations:


        Cell walls break down easily and rate of denaturation of the protein membrane present

at the cell membrane increases when exposed to high temperature. This enables proteins

found in the buds to leach or diffuse out of the buds more easily which includes the diffusion

of iron-containing electron-carrier protein complexes such as ferredoxin, cytochromes b6f,

cytochrome c and Fe –S clusters. High temperature increases kinetic energy of the electron-

carrier proteins, resulting to the increased probability of the protein molecules to diffuse out

of the cell membrane. This is further suggested by the qualitative observations as the sample

                                                                                               37
filtrate of 100.0°C incubating temperature is greener compared to other sample filtrate of

lower incubating temperature. This indicates that maybe chlorophyll diffuses out of the

sample, causing the green colouration. In conclusion, increased incubation temperature

causes increased rate of diffusion.

       It is also possible that the proteins present in the buds are denatured and reacts with

each other forming unknown products of possible physical property changes such as

increased solubility or the ability to diffuse through the cell membrane, resulting to higher

quantity of iron diffusing out of the broccoli buds. Maxwell–Boltzmann distribution indicates

that rate of reaction increases as temperature increases, thus temperature directly influences

the rate of iron diffusing out of the broccoli bud. Therefore, iron content is lowest after

broccoli buds samples are incubated at 100.0°C when compared to other lower incubating

temperatures.




                                                                                                 38
10.0    Evaluation

10.1    Uncertainties and Limitations


        Errors might occur during dry ashing of the samples using crucibles due to incomplete

combustion. This is evident as some black substance suspected as carbon and unoxidized iron

is still visibly sticking to the crucible. Since no lids were used, iron which is considered a

volatile metallic compound may be lost during combustion.


        When reducing iron(III) and measuring the absorbance, solutions were added in the

order: trisodium citrate solution, hydroxylammonium chloride solution and phenanthroline

solution. It is unknown whether the sequence in which the solutions were added would affect

the intensity of the orange-red complex formed. Simplified and modified procedures were

carried out for this investigation due to time constraint as it is solely for iron content

comparison and it does not reflects the actual amount of iron in the broccoli.[8]


        There might be interfering ions present in broccoli plants which would react with

phenanthroline solution to produce a colour complex, interfering with the intensity of the

orange-red complex produced by iron-phenenahroline complex.


        Due to time constraint, only triplicate random samples were performed in this

investigation (n=3). Although random sampling was performed, the sample size (n=3) is too

small to be a good representation of the general population of Brassica oleracea.


        Contamination in the samples might occur due to impurities left on the broccoli plants

after washing. This is evident as some of the outlier data were omitted due to inconsistencies

when compared to other samples.




                                                                                                 39
        Systematic error also arises due to the visible spectrophotometer. Fluctuations occur

frequently when the absorbance of the samples were being measured, giving inconsistent

absorbance readings.


        The dry mass of the samples might vary as there is difficulty in obtaining the dry mass.

Samples heated in the over for too long might be charred but samples heated for a short

duration might still contain water. Hence, this uncertainty affects the accuracy on the iron

content in the samples.


10.2    Ways of Improvement


        Methods can be carried out to improve dry ashing method used. Microwave furnaces

can be used to replace the crucibles used. Samples are placed in small chamber to undergo

combustion quickly and preventing the loss of volatile metallic compounds. Although

expensive, it provides an alternative method of dry ashing using crucible which would reduce

the systematic error present. [9]


        Atomic-absorption spectroscopy (AAS) can be used for more accurate quantification

of iron in various samples. Samples are first vaporized and absorption of visible light excites

the electrons to a higher electronic energy level, enabling the concentration of iron to be

accurately quantified. Although AAS instrument would provide accurate data, it is an

expensive equipment. [10]


        Other colorimetric reagents can be used to increase the sensitivity of iron

quantification such as 4,7-Diphenyl-1,10-phenanthroline as it extracts the iron in the reagents

and water used in the test and thus of reducing the blank to zero. [11]




                                                                                                40
       Sample size should be increased in order to improve the accuracy and reliability of the

data. Increasing sample size will reduce the standard deviation. The larger the sample size,

the more likely it is able to represent the general population of Brassica oleracea.


10.3   Further Investigations and Unresolved Questions


       Prior investigation with o-phenanthroline on spinach, Amaranthus gangeticus on iron

content in different coloured part of the leaves: red and green failed due to interfering ions

and inconsistent data collected. Thus, method of quantification other than using o-

phenanthroline will be needed for this investigation.


       Investigation on the effects of different cooking methods on the iron content of the

vegetable such as stir-frying, steaming and microwaving should be explored in order to raise

awareness. It is believed that iron content would not decrease as significantly if broccoli is

steamed or microwaved because water is not directly in contact with the broccoli, thus

reducing leaching of iron from the broccoli.


       An investigation can be carried out on how pH condition affects the iron content in

broccoli. Broccoli can be incubated in water of different pH value for this investigation to

determine the effects of pH on the iron content.




                                                                                                 41
11.0   Conclusion


       With enough supporting statistical evidence, it can be concluded that broccoli

(Brassica oleracea) buds has the highest iron content at 88% ~ 90% higher than the iron

content at the stems and buds.


       After being incubated at temperature of 100.0°C, the iron content in the broccoli buds

significantly decreases by 68% when compared to samples incubated at room temperature

24.0°C. It is suggested that iron content decreases as the incubating temperature increases.




                                                                                               42
12.0   Reference
[1]    National Institutes of Health, 2004. Dietary Supplement Fact Sheet: Iron. [Online]
       (Updated 24 August 2007)
       Available at: http://ods.od.nih.gov/factsheets/iron.asp
       [Accessed 14 December 2008]

[2]    Office of Dietary Supplements, 2005. Facts About Iron. [Online] (Updated 7
       September 2005)
       Available at: http://ibdcrohns.about.com/cs/nutrition/a/fdairon_2.htm
       [Accessed 14 December 2008]

[3][4] USDA National Nutrient Database, 19??. Nutrient Data Labotory. [Online]
       Available at: http://www.nal.usda.gov/fnic/foodcomp/search/
       [Accessed 15 December 2008]

[5]    Garrett, R. H., & Grisham, C. M., 2005. Biochemistry. 3rd ed. Belmont (CA): Thomas
       Learning, Inc.

       Koolman, J. & Roehm K. H., 2006. Color Atlas of Biochemistry. 2nd ed. New York:
       Thieme Stuttgart.

       Institute for Protein Research, Osaka University, 200?. Ferredoxins... Ubiquitous Iron-
       Sulfur Proteins. [Online]
       Available at: http://www.protein.osaka-u.ac.jp/enzymology/Fd_Model/Ferredoxin.html
       [Accessed 27 March 2009].

       Carnegie Mellon University, 200?. Research Projects. [Online]
       Avalibale at: http://www.chem.cmu.edu/groups/hendrich/research/index.html
       [Accessed 27 March 2009].

       Darragh, F., 200?. Kinetics: Collision Theory, Maxwell-Boltzmann Distribution. [Online]
       Available at: http://www.bustertests.co.uk/studies/kinetics-collision-theory-maxwell-
       boltzmann-distribution.php
       [Accessed 1 July 2009].

[6]    Daniel C. H., 2002. Quantitative Chemical Analysis, 6th ed. s.l. s.n.
[7]    Anon. Spectrophotometric Determination of Iron in Vitamin Supplement Tablets.
       [Class Handout]
[8]    Krishna Murti, G. S. R.,Volk, V. V. & Jackson M. L., 196?. Colorimetric
       Determination of Iron of Mixed Valency by Orthophenanthroline, [online]. Abstract
       only.
       Available at: http://soil.scijournals.org/cgi/content/abstract/30/5/663
       [Accessed 30 March 2009]

[9]    Skoog, D. A, West, D. M., Holler, F. J., Crouch, S. R., 2004. Fundamentals of
       Analytical Chemistry, 8th ed. US: Thomas Learning, Inc.
                                                                                            43
[10]   Tissue, B. M., 1996. Atomic-Absorption Spectroscopy (AA). [Online] (updated 21
       August 1996)
       Available at: http://elchem.kaist.ac.kr/vt/chem-ed/spec/atomic/aa.htm
       [Accessed 8 July 2009]

[11]   GFS Chemicals, 199?. Organics - Phenanthrolines and Bipyridines. [Online]
       Available at:
       http://www.gfschemicals.com/statics/documents/technical/technical300af5f5bcce4870
       85c1ff0229dcac49.html
       [Accesed 8 July 2009]

Kuzma, J. W. & Bohnenblust, S. E., 2001. Basic Statistics for the Health Sciences, 4th ed. s.l.,
Mayfield Publishing Company.

Christian, G. D., 2004. Analytical Chemistry, 6th ed. s.l., Matrix Publising Services.

Lim, J. A., 2008. Antimicrobial Activity, extended essay.




                                                                                             44
13.0    Appendix

Appendix 1: Procedure to prepare the required solutions


1.     1000.0cm3 of 25.0x10-5M iron(II) solution was prepared using (0.049 ± 0.001)g of

       hydrated iron(II) sulphate, Fe(SO4)2(NH4)2∙6H2O, the solution contained 1.0cm3 of

       concentrated hydrochloric acid (4.0M). A series of dilution was carried out to obtain the

       iron(II) solution of concentrations 10.0x10-5M, 7.50x10-5M, 6.00x10-5M, 5.00x10-5M

       and 2.50x10-5M.


2.     10% hydroxylammonium chloride, NH3OHClsolution was prepared by dissolving

       (10.000 ± 0.001)g of solid in 100.0cm3 of distilled water.


3.     5% of trisodium citrate, Na3C6H5O7 solution was prepared by dissolving (5.000 ±

       0.001)g of solid in 100.0cm3 of distilled water.


4.     0.01M Orthophenanthroline solution was prepared by dissolving (0.198±0.001)g of the

       solid in 10.0cm3 of ethanol26 and 90.0cm3 of distilled water was added to form a

       100.0cm3 solution.




26
  Ethanol was added to dissolve the phenanthroline solids as it consists of polar molecules while ethanol is a
polar solvent.
                                                                                                             45
Appendix 2: Dilution of iron(II) standards

Steps were carried out to dilute the 25.0x10-5M iron(II) stock solution to the solutions of
lower concentration 10.0x10-5M, 7.50x10-5M, 6.00x10-5M, 5.00x10-5M and 2.50x10-5M. The
dilution table is as shown below:

    Concentration of                 Volume of 2.5x10-4M iron(II) solution                    Volume of distilled
  iron(II) standards /                   transferred using pipette / cm3                     water added to form

                   -3                                              3
                                                                                             the 100cm3 solution /
         mol dm                                       (±0.03)cm
                                                                                                        cm3
                                                                                                   (±0.08)cm3
         10.0x10-5                                       40.00                                         60.00


         7.50x10-5                                       30.00                                         70.00


         6.00x10-5                                       24.00                                         76.00


         5.00x10-5                                       20.00                                         80.00


         2.50x10-5                                       10.00                                         90.00


Table 9: Concentration of iron(II) standards, volume of 25.0x10 -5M iron(II) solution transferred to a 100.0cm3
volumetric flask using a graduated pipette and the volume of water added to form a 100.0cm 3 solution.

According to the table above, the required volume of 25.0x10-4M iron(II) solution is
transferred to a 100.0cm3 volumetric flask using a graduated pipette. Distilled water is added
until the bottom of the meniscus reaches the calibration mark.




                                                                                                               46
Appendix 3: Confidence Limit


Confidence limit allows us to estimate the range within which the true value might fall,
within a given probability, defined by the experimental mean and the standard deviation. The
confidence limit is given by:
                                                                   ��������
                                       Confidence limit = ���� ±
                                                                    ����

where t is a statistical factor that depends on the number of degrees of freedom, v and the
confidence level desired. The number of degrees of freedom is one less than the number of
measurements, s is the standard deviation of the mean and N is the number of samples.

                          v                                               Confidence Level
                          1                                                    12.701
                          2                                                     4.303
                          3                                                     3.182
                          4                                                     2.776
                          5                                                     2.571
                          6                                                     2.447
                          7                                                     2.365
                          8                                                     2.306
                          9                                                     2.262
Table 10: Values of t for v Degrees of Freedom for confidence level of 95%.




                                                                                              47
 Appendix 4: Regression Analysis
 Regression analysis was carried out to determine that there is a significant correlation
 between the concentration of iron and absorbance used to plot the standard calibration curve
 for iron(II) in section 4.0. The regression analysis is carried out with α = 0.05.



        Regression Statistics
     Multiple R               0.996
      R Square                0.993
  Adjusted R Square           0.991
   Standard Error            0.0232
    Observations                6

    ANOVA
                          df                SS                   MS               F           Significance F
  Regression              1               0.302                 0.302           559.25          1.896x10-5
   Residual               4              0.00216             5.403x10-4
    Total                 5               0.304

                                    Standard                                   Lower        Upper           Lower     Upper
              Coefficients            Error         t Stat      P-value         95%          95%            95.0%     95.0%
  Intercept    0.00554               0.0153         0.362        0.736        -0.0370       0.0481         -0.0370    0.0481
Concentration   0.305                0.0129         23.65      1.90x10-5       0.269         0.340          0.269      0.340
 Table 11: Results for the regression analysis of standard calibration curve for iron(II) with α = 0.05.



                       Notation                                                    Meanings
                            df                                                Degree of freedom
                           MS                                                    Mean square
                            SS                                                  Sum of squares


 The analysis shows that the P-value is 1.90x10-5 (P<0.05). Therefore, there is a significant
 correlation between the concentration of iron and absorbance used to plot the standard
 calibration curve for iron(II).




                                                                                                                 48
Appendix 5: The ash was dissolved in acid. Black coloured substance can be sticking on the
side of the inner wall




                                                                                         49
Appendix 5: Q Test

Q test is carried out to determine whether a particular measurement should be rejected. The Q
test is a statistical test which is used when only a small number of results are obtained.
                                           | outlier −nearest neighbour |
                                 Qcalc =
                                                       range

The value of Qcalc is compared to a set of Q test rejection coefficients for 90% confidence
level.

  N          3         4         5           6        7          8        9         10        11        12
  Q        0.94      0.76      0.64        0.56     0.51       0.47     0.44       0.41      0.39      0.37
Table 12: Number of samples and the corresponding Q test rejection coefficients for 90% confidence level.

The outlying data can be rejected when Qcalc > Q test rejection coefficient.




                                                                                                            50
Appendix 6: Standard Deviation for concentration of iron(II)

The standard deviation for concentration of iron(II) is calculated using a statistic formula. An
assumption was made such that there is negligible systematic errors during preparation. The
formula is given as shown:

                                     sr                             2
                              SX =
                                     ����
                                         1    1             −
                                        {���� + ���� +���� 2(��������(���� ���� )���� )2 }1/2
                                                                −
                                                               ����


where: SX         = Standard deviation of the concentration of the sample

         sr       = Standard deviation of the standard calibration curve

         m        = Slope of the standard calibration curve

         n        = Number of calibration standards

         ��������     = Mean absorbance of the samples

         ����       = Mean absorbance of all standards

         ��������     = Concentration of the standards

         ����       = Mean concentration of the standards




                                                                                              51
Appendix 7: Calculations of ANOVA and Tukey’s HSD Test

A null hypothesis is first assumed where there is no difference between the means of different
groups.

                                    H0: μ1 = μ2 = μ3 = ... = μk

The theoretical basis for performing ANOVA is the partitioning of the variance of all
observations into two sources of variation: variation between the group means and variation
within each group. The sampling distribution used for testing is called the F distribution.
The notations of ANOVA and meanings are tabulated as shown below:

                   Notation                                                      Meanings
                           2
                  MSw or ��������                            Within-group variance / mean square within
                            2
                   MSb or ��������                                   Between-group variance / mean square
                                                                              between
                         df                                              Degree of freedom
                         k                                                Number of groups
                         n                                       Number of observation in each group
                         N                                           Total number of observation
                         α                                                Significance level
                      SSb                                           Sum of squares between group
                      SSw                                            Sum of squares within group
Table 13: Notations of ANOVA and meanings.

If MSB > MSW, the variance between group is greater than the variance within group, then
there is treatment effect. There is no treatment effect when MSB ≈ MSW.
                                                         MS b
                                             F ratio =
                                                         MS w

MSb has k – 1 degree of freedom, dfb.

MSw has N – k degree of freedom, dfw.

 Source of       Sum of            df             Mean                 F ratio      Critical F     P value
 variation       Squares                         Squares,                           (α = 0.05)
                                                   (s2)

 Between           SSb           k-1                     ����
                                                MSb = ����−1
                                                          ��������          MS����          Fk-1,N-k   Computer
                                                                        MS����                     generated
  Within           SSw           N-k                      ��������
                                                         ����
                                                MSw = ����−����
   Total           SSt           N-1

Table 14: One-way ANOVA table.

                                                                                                        52
Post hoc Tukey’s HSD test is performed when the F ratio is greater than the critical F
which means that there is significant difference between at least one pair. Tukey’s HSD test
is carried out to perform multiple comparisons. The formula for calculating the HSD value is
as shown below:

                                                       MS ����
                                 HSD = q (α, k, N-k)
                                                         ����

There is significant difference when the difference between the mean of the groups is
greater than the HSD value.




                                                                                          53
The table below shows the ANOVA table generated using Microsoft Excel 2007.


   1. The part of the broccoli which contains the highest iron content




     Groups             Count       Sum         Average       Variance
      Buds                2        1.278         0.639         0.0002
     Stems                2        0.158         0.079         0.0001
      Base                3         0.21         0.070        0.00006



    ANOVA
    Source of
                         SS          df              MS           F      P-value     F crit
    Variation
 Between Groups          0.457       2            0.228        2012.2    9.86x10-7    6.94
  Within Groups         0.0005       4           0.0001

      Total             0.4572       6
Table 15: ANOVA table




                                                     0.0001
                                    HSD = 5.04
                                                       3


                                           = 0.029




                                                                                     54
   2. The effects of incubating temperature on the amount of iron.



     Groups             Count      Sum         Average        Variance
 Room Temperature
                          3        1.106        0.369         8.933x10-5
     (24°C)
       60°C               3        1.086        0.362          0.0004
       80°C               2        0.560        0.280         9.80x10-5
      100°C               3        0.364        0.121           0.005



      ANOVA

 Source of Variation     SS         df           MS               F        P-value        F crit

  Between Groups        0.1192       3           0.040          23.80      0.0005         4.35
   Within Groups        0.0117       7          0.0017

        Total           0.1308      10
Table 16: ANOVA table




                                                     0.0017
                                   HSD = 4.68          3


                                           = 0.111




                                                                                     55

								
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