CHEMISTRY EXTENDED 2013-2014 ESSAY

Candidate Name: Ronit Banerjee Candidate Session Number: 002329-0398

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Word Count: 3992

CHEMISTRY EXTENDED

ESSAY

2013-2014

Ronit Banerjee

Candidate Session No.: 002329-0398

An investigation to optimize the heavy metal biosorbing

abilities of Pectin.


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Abstract Toxicity caused by waterborne heavy metals is a growing concern in third world countries as

these countries often lack the resources and technology to efficiently remove them. As such,

they are turning to biosorption as an inexpensive but effective means to do so. One such

biosorbent is pectin, a ubiquitous natural polymer. However, in the absence of chemical

modification, much of its biosorbent potential is unrealised. Thus, it is of interest to investigate

the following research question – Can the pH of the environment be optimised and the

molecular structure of pectin be modified through hydrolysis of its ester group so as to

produce a more competent and reusable biosorbent?

Pectin was extracted from orange peels using a water bath heating method, following which,

the pH of its environment was raised to study the effect of doing so on the efficacy of the active

sites (carboxylic acids). Separately, pectin was also subjected to acid catalysed hydrolysis of

ester groups in an attempt to increase the population of active sites. The biosorbent abilities

of pectin were quantified by measuring the decrease in concentration of copper(II) ions using

UV-visible spectrophotometry. Finally, the biosorptive capacity of pectin was recovered using

water, hydrochloric acid or EDTA solution.

This investigation revealed a direct correlation between increasing pH and metal biosorption.

Furthermore, hydrolysis also had a positive effect on biosorption. At experimentally

determined optimal conditions of pH 7 and 32% hydrolysis of ester groups, 10 g of pectin was

able to reduce the concentration of copper(II) ions from 0.05 moldm-3 to 0.035 moldm-3.The

most effective means of recovering pectin’s biosorbent abilities was EDTA solution (63% of

the original biosorption). Thus, the results prove that with chemical modification, pectin can be

a competent and reusable biosorbent for treating heavy metal contaminated water.

(294 Words)


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TABLE OF CONTENTS 1. Introduction ......................................................................................................................... 5

1.1. Metal Ion Removal through Biosorption ......................................................................... 6

1.2. Background Information of Pectin .................................................................................. 7

1.3. Motivation for Study and Research Question................................................................ 10

2. Methodology and Background ......................................................................................... 11

2.1. Choice of Method for Pectin Extraction ........................................................................ 12

2.1.1. Phase 1: Water Bath Heating Method to Extract Pectin ........................................ 13

2.2. Preparation of Pectin Stock Solution ............................................................................. 15

2.3. Ultraviolet-Visible (UV-Vis) Spectrophotometry ......................................................... 16

2.4. Experimental Protocol to Quantify Biosorption of Pectin ............................................. 17

2.4.1. Titrimetric Method to Verify Results from UV-Visible spectroscopy .................. 18

2.5. Phase 2: pH study .......................................................................................................... 21

2.5.1 Preparation of Buffers ............................................................................................. 21

2.6. Acid Catalysed Hydrolysis of Methyl-Ester in Pectin ................................................... 23

2.6.1 Method to Hydrolyse Pectin ................................................................................... 24

2.7. Drawback of Time as a Variable in the Hydrolysis Study ............................................ 25

2.8. Phase 3: Recovery ......................................................................................................... 27

3. Results and Discussion ...................................................................................................... 28

3.1. Verification of Copper(II) Sulfate λMAX ........................................................................ 28

3.1.1. Standard Curve for Copper Sulfate ........................................................................ 29

3.2. Results of pH Study ....................................................................................................... 31

3.3. Results of Hydrolysis Study .......................................................................................... 33

3.3.1. Further Results of Hydrolysis Study ...................................................................... 34

3.4. Verification of Results of Hydrolysis Study with Laboratory Grade Pectin ................. 36

3.4.1. Method of Obtaining Pectin ................................................................................... 37

3.4.2. Comparison Between Laboratory Grade and Self-Extracted Pectin ...................... 38

3.5. Regeneration of Biosorbent ........................................................................................... 39

3.6. Overview of Findings .................................................................................................... 40

4. Evaluation .......................................................................................................................... 41

4.1. Assumptions and Limitations ........................................................................................ 41

4.2. Method ........................................................................................................................... 41

5. Conclusion .......................................................................................................................... 42


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6. Bibliography ...................................................................................................................... 45

Appendix A- Chemicals and Apparatus.............................................................................. 49

Appendix B- Detailed Results............................................................................................... 51

B.1.Raw Data for Scanning Curve for Copper(II) Sulfate ................................................... 51

B.2.Raw Data for Standard Curve for Copper(II) Sulfate .................................................... 52

B.3.Raw Data for pH Study .................................................................................................. 53

Appendix C – Detailed Procedure ....................................................................................... 60

C.1.Preparation of Equipment .............................................................................................. 60

C.2.Extraction of Pectin from Orange Peels......................................................................... 60

C.3.Copper(II) Sulfate Calibration ....................................................................................... 63

C.4.Protocol to Determine Biosorbent Abilities of Pectin ................................................... 67

C.5.Titrimetric Verification (Section 2.3.1.) ........................................................................ 69

C.6.Protocol to Determine Total Number of Functional Groups in Pectin .......................... 72

C.7.Protocol to Determine Determine Amount of Carboxylic Acid Groups in Pectin ........ 74

Appendix D – MSDS ............................................................................................................. 76

D.1.MSDS for Sodium Hydroxide ....................................................................................... 76

D.2.MSDS for Potassium Iodide .......................................................................................... 77

D.3.MSDS for anhydrous Sodium Carbonate ...................................................................... 78

D.4.MSDS for Ethanol ......................................................................................................... 79

D.5.MSDS for Sodium Thiosulfate ...................................................................................... 80

D.6.MSDS for Copper(II) Sulfate Pentahydrate .................................................................. 81

D.7.MSDS for Anhydrous Citric Acid ................................................................................. 82

D.8.MSDS for 1M Hydrochloric Acid ................................................................................. 83

D.9.MSDS for Ethanoic Acid ............................................................................................... 84

D.10.MSDS for Pectin .......................................................................................................... 85

D.11.MSDS for Soluble Starch ............................................................................................ 86


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1. Introduction Metal ions are required in trace amounts by the human body for various metabolic1 processes

and for the creation of essential metalloproteins2 like haemoglobin and ferritin. However, when

in excess, they accumulate in the human body to form complexes with certain proteins. These

modified molecules lose their ability to function properly and result in the malfunction or death

of cells3, leading to organ damage4 and development of autoimmune and cardiovascular

diseases.

Heavy metal contamination is a widespread problem and due to it, more than 300 million

people did not have access to potable water in 2013.5 Efficient methods of removing heavy

metals from water such as reverse osmosis and distillation are expensive, whereas cheaper

methods such as sand filters are often unable to remove them.6, 7 Another method to remove

heavy metals is through chelating agents such as EDTA (ethylenediaminetetraacetic acid).

Although highly effective, when ingested, EDTA can leach iron from haemoglobin and calcium

from bones, thus, creating further physiological problems.

Biosorbents have also been used to remove heavy metals from water because of their low

cost and renewability. Biosorption8 is a passive non-metabolically mediated process of binding

ions by natural substances such as agricultural waste and its industrial by-products.

1 Regulski, Elizabeth E., Ryan H. Moy, Zasha Weinberg, Jeffrey E. Barrick, Zizhen Yao, Walter L. Ruzzo, and Ronald R. Breaker. Molecular microbiology [Print] 2008, 68(4), 918-932. 2 Springfield Technical College, Hemoglobin is a protein. http://faculty.stcc.edu/AandP/AP/AP2pages/Units18to20/blood/hemoglob.htm (accessed August 1, 2014). 3 Alissa, Eman M., and Gordon A. Ferns. Heavy Metal Poisoning and Cardiovascular Disease. Journal of Toxicology. [Online] 2011, 1-21. http://www.ncbi.nlm.nih.gov/pubmed/21912545 (accessed May 1, 2014). 4 Haga, A. European Journal of Cancer [Print] 1996 , 32(13), 2342-2347 5World Health Organisation Water, Health and Ecosystems. http://www.who.int/heli/risks/water/water/en/ (accessed January 12, 2014) 6 CreativeWater.Water Blogged RSS. http://www.cwsnaturally.com/blog/biofilm/ (accessed August 8, 2014). 7 Gilau, Asmerom M., and Mitchell J. Small. Designing cost-effective seawater reverse osmosis system under optimal energy options. Journal of Renewable Energy. [Online] 2008 33, no. 4, 617-630. http://www.cmu.edu/gdi/docs/designing-cost-effective.pdf (accessed January 12, 2014) 8 Parvathi, K., R. Nagendran, and R. Naresh Kumar. Lead biosorption onto waste beer yeast by-product, a means to decontaminate effluent generated from battery manufacturing industry. Electronic Journal of


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1.1. Metal Ion Removal through Biosorption

Biosorbents can remove heavy metals through a process termed chelation. Chelating agents

are substances that can form coordinate bonds9 with aqueous metal ions, thereby removing

them from the solution.

Extensive research has10 been carried out on common11 biosorbents such as chitosan and

cellulose to increase their biosorbent abilities by deliberately modifying their molecular

structure; by either increasing the number or the efficacy of the metal ion-binding sites. Once

the structure has been altered12, specific electron-rich groups (such as carboxylic acids) in the

molecule exhibit stronger Lewis base properties and thus donate their electrons to electron

deficient metal cations more readily.

This binding can take place as the stability constant13 of the biosorbent metal-ion complex is

greater than that of the aqua metal-ion complex.

[Cu(H2O)6]2+ + Biosorbent active sites ⇌ [Cu(Biosorbent actives site)]2+ + 6H2O

When the water ligands in the aqua metal cation complex are replaced with a single

polydentate ligand, the total number of species on the right of the equilibrium becomes greater

than that on the left. Thus, resulting in an increase in the disorder of the system and favouring

the forward reaction, in accordance with the second law of thermodynamics.

Biotechnology [Online] 2007, 10. http://www.ejbiotechnology.info/content/vol10/issue1/full/13/index.html (accessed May 1, 2014). 9 Idée, Jean‐Marc, Marc Port, Caroline Robic, Christelle Medina, Monique Sabatou, and Claire Corot. Role of thermodynamic and kinetic parameters in gadolinium chelate stability. Journal of Magnetic Resonance Imaging. [Online] 2009, 30(6), 1249-1258. http://onlinelibrary.wiley.com/doi/10.1002/jmri.21967/abstract;jsessionid=0942585E0695520D4E43287278C2BB58.f01t01?AccessCustomisedMessage=&userIsAuthenticated=true (accessed May 1, 2014). 10 Hosokawa, Jun, Masashi Nishiyama, Kazutoshi Yoshihara, and Takamasa Kubo & engineering chemistry research. 2009, 29(5), 800-805. 11 Wang, Jianlong, and Can Chen. "Biosorbents for heavy metals removal and their future. Biotechnology advances, [Online] 2009, 27(2), 195-226. http://www.sciencedirect.com/science/article/pii/S0734975008001109 (accessed May 2 2013 ) 12 Samuels, Robert Joel. Journal of Polymer Science: Polymer Physics Edition.[Print] 1981, 19(7), 1081-1105. 13 Bjerrum, Jannik. Stability constants of metal-ion complexes. Vol. 17. London: Chemical Society, 1964.


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Despite their efficiency, biosorbents lose their efficacy14 once their active sites are saturated

with metal ions. Consequently, research15 has been done to develop protocols that enable the

recovery of the biosorbent abilities of a material after saturation.

1.2. Background Information of Pectin

Pectin (polygalacturonic acid) is a polysaccharide that occurs in very high quantities16 in the

peels and rinds of citrus fruits. It is insoluble17 in ethanol but partially soluble in water. It exists

predominantly in its methyl ester form (Figure 1), in which an ester or carboxylic acid group

takes the carbon-5 position. Each of the six-membered rings is linked to each other with an

ether linkage at the carbon-1 and carbon-4 positions, and an alcohol group is present at the

carbon-2 and carbon-3 positions.

Figure 1: Structure of Pectin in Citrus peels18

14 Gupta, Rani, Prerna Ahuja, Seema Khan, R. K. Saxena, and Harapriya Mohapatra. Current Science-Bangalore. [Print] 2009, 78(8), 967-973. 15 Zhenzhong, Liu, Deng Huiping, and Zhan Jian. Chinese Journal of Population Resources and Environment. [Print] 2007, 5(3), (2007), 23-30. 16 Obtaining Pectin with Separation Technology. http://www.westfalia-separator.com/applications/chemical-

pharmaceutical-technology/extraction/pectin.html (accessed 13 April 2013) 17 Garna, Haikel, Nicolas Mabon, Christelle Robert, Journal of food science [Print] 2007, 72(1): C001-C009. 18 Happi Emaga, Thomas, Nadia Rabetafika, Christophe S.Blecker, and Michel Paquot. Kinetics of the hydrolysis of polysaccharide galacturonic acid and neutral sugars chains from flaxseed mucilage. Biotechnol. Agron. Soc. Environ. [Online] 2012, 16(2), 139-147. http://popups.ulg.ac.be/1780-4507/index.php?id=8563 (accessed 14 June 2013)

4

3 2 1

5


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While ester, ether, and alcohol groups are electron rich, they are not as strong Lewis bases

as carboxylic acids.19 As such, much of pectin’s biosorbent abilities is unrealised due to the

dominance of esters over carboxylic acids. Consequently, if the population of carboxylic acids

were to be increased, it is likely that the biosorbent abilities of pectin will be ameliorated. This

can be achieved through acid or base catalysed hydrolysis of ester groups (Figure 2).

Figure 2: Acid and Base Catalysed Hydrolysis of Esters20

However, even if the number of active sites in pectin is increased, the biosorbent abilities of

pectin may not increase significantly because of the repulsion between the metal cation and

the partial positive charge on the hydrogen atom (As a result of being bonded to the highly

electronegative oxygen atom).21 This charge repels the incoming metal cation, (Figure 3) thus

limiting the efficacy of the carboxylic acid group in forming coordinate bonds with metal ions.

19 Snider, Barry B. Accounts of Chemical Research [Print] 1980, 13(11), 426-432.

20 Tutor Vista, Esterification. http://chemistry.tutorvista.com/organic-chemistry/ester.html (accessed August 1, 2014). 21 Hambright, Peter, and Everly B. Fleisher. Acid-base equilibriums, kinetics of copper ion incorporation, and acid-catalyzed zinc ion displacement from the water-soluble porphyrin. alpha.,. beta.,. gamma.,. delta.-tetrakis (1-methyl-4-pyridinio) porphine tetraiodide. Inorganic Chemistry [Online] 1970 ,9(7), 1757-1761. http://pubs.acs.org/doi/abs/10.1021/cr60203a005 (accessed 12 September 2014)

Cu2+ 𝛿+ δ-

Repulsion

Figure 3: Repulsion of Heavy metal ion due to partial

positive charge on hydrogen atom


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This repulsion can be overcome by deprotonating the carboxylic acid. Upon deprotonation, the

carboxylate ion will no longer repel metal ions. Furthermore, the electron density and Lewis

basicity of the active site would increase, and these active sites would be able to coordinate

with metal ions in a variety of modes (Figure 4). Therefore, it is likely that deprotonation will

result in an increase in the biosorbent abilities of pectin.

Figure 4: Multiple coordination for carboxylate ions22

Due the high electron density of the carboxylate ions and neighbouring functional groups like

alcohols and ethers, it is likely that they will come together to form a complex, modelled by the

egg in box diagram23 shown below24.

22 Arora, Himanshu, and Rabindranath Mukherjee. Coordination polymers using (2-pyridyl) alkylamine-appended carboxylates: magnetic properties. New Journal of Chemistry. [Print] 2010, 34(11), 2357-2365. 23 Leick, Sabine, Stefan Henning, Patrick Degen, Dieter Suter, and Heinz Rehage. Physical Chemistry Chemical Physics [Print] 2010,12(12) 2950-2958. 24 Self-drawn in Chem draw 12

Figure 5: Egg in box diagram showing possible coordination state of pectin


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1.3. Motivation for Study and Research Question The peels of citrus fruits such as orange are usually discarded; hence, in devising a method

to remove heavy metal ions from water, it would be ideal to repurpose waste products like fruit

peels to minimise costs and wastage. This would provide a cheap, renewable, easily

accessible and yet effective alternative to more costly or less effective means of purification of

water in third world countries. Being a consumer of oranges myself, I became interested in this

topic as it would allow us to utilise a seemingly useless part of the fruit to solve an important

environmental issue at a low cost.

As it is evident that the biosorbent abilities of pectin might be improved by increasing the

number or efficacy of the active sites, it is of interest to investigate the following research

question-

Can the pH of the environment be optimised and the molecular structure of pectin be

modified through hydrolysis of its ester group so as to produce a more competent and

reusable biosorbent?

My hypothesis is that both increasing pH and hydrolysis will increase the biosorbtive capacity

of pectin as they will increase the efficacy and the number of active sites respectively.


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2. Methodology and Background

In this investigation, the biosorbtive capacity of pectin will be enhanced by –

1. Increasing the population of carboxylic acid groups through hydrolysis.

2. Studying the correlation between the pH and biosorptive abilities of pectin.

After the biosorbent abilities of pectin have been maximised, a protocol to recover pectin’s

biosorbtive properties once saturated will be developed. This investigation will be divided into

three separate phases, outlined below.

Phase 1• Extraction of pectin from orange peels

Phase 2

• Increasing pectin's biosorbent abilities by-

• pH Study - Pectin will be buffered at various pHs and the pH at which maximum biosorption occurs will be determined

• Hydrolysis Study - The population of active biosorption sites will be increased and the subsequent effect on biosorption will be determined

Phase 3

• To enable the reuse of pectin the following will be carried out-

• Recovery of pectin's biosorbtive capacity - Once saturated, a protocol will be developed to recover the metal binding capacity of pectin

Figure 6: Flow chart showing overview and rationale of

investigation


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In this investigation, copper(II) ions will be used as a case study as they are one the most

common25 heavy metal ions in water. Furthermore, because of their dark blue colour in the

aqueous state, UV-visible spectrophotometry can be used to determine the concentration of a

large number of samples within a short period.

2.1. Choice of Method for Pectin Extraction

Before this investigation can proceed, it is important to find suitable a method to extract pectin.

Numerous methods26 are known, but the water bath heating method27, created by Erika

Kliemann and Karina Nunes de Simas was chosen as the only method to extract pectin as all

the required equipment and reagents were readily available in the laboratory.

Orange peels were chosen as the only source of pectin due to their high percentage by mass

of pectin28, which would provide a high yield during extraction.

25 Roychowdhury, Tarit, Hiroshi Tokunaga, and Masanori Ando. Survey of arsenic and other heavy metals in food composites and drinking water and estimation of dietary intake by the villagers from arsenic-affected areas. Science of the Total Environment. [Online] 2003, 308(1), 15-35. http://www.sciencedirect.com/science/article/pii/S0048969702006125 (accessed 13 November 2013) 26 Wang, Sijin, Fang Chen, Jihong Wu, Zhengfu Wang, Xiaojun Liao, and Xiaosong Hu. Optimization of pectin extraction assisted by microwave from apple pomace using response surface methodology. Journal of food engineering. [Online] 2007, 78(2), 693-700. http://www.sciencedirect.com/science/article/pii/S0260877405007582 (accessed 12 May 2013) 27 Kliemann, Erika, De Simas, Karina Nunes, Edna R. Amante, Elane Schwinden Prudêncio, Reinaldo F. Teófilo, Márcia Ferreira, and Renata DMC Amboni. Optimisation of pectin acid extraction from passion fruit peel (Passiflora edulis flavicarpa) using response surface methodology International journal of food science & technology [Online] 2009, 44(3), 476-483. http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2621.2008.01753.x/abstract;jsessionid=9D8C8D7C8AF560E937D06D4B18D53D48.f03t04?deniedAccessCustomisedMessage=&userIsAuthenticated=True (accessed 14 September 2013) 28 Guo, Xingfeng, Dongmei Han, Huping Xi, Lei Rao, Xiaojun Liao, Xiaosong Hu, and Jihong Wu. Extraction of pectin from navel orange peel assisted by ultra-high pressure, microwave or traditional heating: A comparison. Carbohydrate Polymers [Online] 2012, 88(2) 441-448. http://www.sciencedirect.com/science/article/pii/S0144861711011416 (accessed 12 November 2013)


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2.1.1. Phase 1: Water Bath Heating Method to Extract Pectin

The orange peels were dried in the oven at 800 C until they were completely dehydrated,

following which; they were ground into a fine powder. 10.000 g of this powder was then treated

with 100.0 cm3 of 1.5 moldm-3 citric acid for 30.0 minutes in a water bath at 80.00 C. The

resulting mixture was then filtered through a two-layer muslin cloth in the vacuum filter to obtain

the residue, to which ethanol was added to precipitate gelled pectin. Finally, the gel was heated

to dryness to obtain powdered pectin.29

29 For step-by step methodology refer to Appendix C.2.


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Figure 7: Overview of water-bath

heating method to extract pectin

from Orange peel powder

Dehydrated Orange Peels

Ground and

homogenised

10.000 g of peel powdered

peel added to 100.0 cm3 of

1.5 M citric acid

Beaker containing mixture

is immersed in water bath at

800 C for 30 minutes

Mixture filtered through 2 layer

muslin cloth then pectin is

precipitated with ethanol

Gel heated to dryness to

obtain pectin powder


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2.2. Preparation of Pectin Stock Solution

2.5 g of this powder was then made up to 250.0 cm3 in a volumetric flask.30

30 Image of bunsen burner adapted from - 604 Unit 2 Test. 604 Unit 2 Test. http://www.proprofs.com/quiz-school/story.php?title=604-unit-2-test (accessed August 1, 2014).

2.5 g of pectin powder,

obtained from drying gel

is measured out.

Due to pectin’s low solubility, it is heated

over the bunsen flame and small amounts

of pectin powder are added while stirring.

Solution is transferred to volumetric flask

with the aid of funnel and made up to the

250.0 cm3 mark

1% by mass solution of Pectin

Figure 8: Overview of method to make pectin stock solution


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2.3. Ultraviolet-Visible (UV-Vis) Spectrophotometry

When a monochromatic beam of light passes through a solution, some of the incident light

intensity (Io) is absorbed and the remaining is transmitted through (I1). The UV-Visible

spectrophotometer measures the absorbance of the solution, given by the negative logarithm31

of the ratio of the intensity of the transmitted light to the incident light on the sample. By finding

the absorbance of the solution, the concentration of the sample can be found using the Beer-

Lambert law.

𝐴 = log𝐼1

𝐼0

The Beer-lambert law32 states that the absorbance of a sample is directly proportional to the

concentration of the sample when the path length of the beam held constant.

Figure 9: Illustration of Beer-Lambert Law

A =kc

Hence, by plotting a graph of absorbance against known concentrations of copper(II) sulfate,

the concentration of unknown samples of copper(II) ions can be determined.33

31 Miyawa, John H., and Stephen G. Schulman. "Ultraviolet–Visible Spectrophotometry." Handbook of Pharmaceutical Analysis 2001 187. 32 Sheffield Hallam University. Beer’s Law. http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm (accessed Mar 1 2013). 33 For step-by step methodology refer to Appendix C.3.

I0 I1

B

C


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2.4. Experimental Protocol to Quantify Biosorption of Pectin

During the course of this investigation, copper(II) ions were frequently treated with pectin. After

each treatment, the biosorption of pectin was quantified in terms of the concentration of the

residual copper(II) ions. However, the concentration of copper(II) ions in the pectin-copper(II)

sulfate mixture could not be quantified immediately using UV-Visible spectrophotometry

because the copper(II)-pectin complex is cloudy, and therefore scattered light. Hence, a

protocol was created so as to separate the copper(II) ions from pectin solution.

100.0 cm3 of 1% by mass of pectin solution was added to 50.0 cm3 of 0.2 moldm-3 copper(II)

sulfate. Once biosorption was complete, the mixture was placed in a test tube and centrifuged

at 2000 rpm for 5 minutes. The copper(II) sulfate supernatant was then removed, and its

absorbance was determined using the UV-Visible Spectrophotometer.34


Figure 10: Protocol to Measure Concentration of Residual

Copper(II) Ions

50.0 cm3 of 0.2

moldm-3 CuSO4

100 cm of 1% by mass

of pectin solution

50 cm3 of pH 2 Buffer

Pectin gel

Copper Sulphate Supernatant

Allowed to biosorb

Spun in Centrifuge at

2000 rpm for 5 minutes

Supernatant removed

and absorbance

quantified using UV-

visible

spectrophotometer


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2.4.1. Titrimetric Method to Verify Results from UV-Visible spectroscopy

UV-Visible spectrophotometry is useful in quantifying the concentration of a large number of

samples quickly. However, it is difficult to remove all the pectin from the mixture due to its

partial solubility. Furthermore, since pectin also absorbs at the λmax (The wavelength at which

a substance absorbance is the greatest) of Cu(II) sulfate35, there may be some interference.

In order to verify the accuracy of the UV-Visible spectrophotometry technique, a titration was

done.

35 λmax for Copper(II) Sulfate experimentally determined to be 810 nm in Section 3.1

Figure 11: Scanning Curve for Pectin to Show that

pectin absorbs at λmax of Copper(II) Sulfate (810 nm)


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The supernatant from the protocol to quantify pectin’s biosorption (Figure 9) was subjected to

an iodometric titration to verify the accuracy of the UV-visible spectrophotometer. 36 50.0 cm3

of 0.2 moldm-3 potassium iodide was added to it and then titrated with thiosulfate with starch

as an indicator. The amount of copper(II) ions obtained using this method was compared to

that obtained from the UV-visible spectrophotometric method.

36 For step-by-step methodology see Appendix C.5

Figure 12: Titrimetric Method to Verify UV-Visible

spectrophotometry

Copper Sulfate

Supernatant

50cm3 of 0.2

moldm-3 KI

Copper iodide

precipitate and

iodine

Titrated with 0.2

moldm-3 Sodium

Thiosulfate


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The reaction involved in the titration is as follows

2𝐶𝑢2+ + 4𝐼− → 2𝐶𝑢𝐼 + 𝐼2

𝐼2 + 2𝑆2𝑂32− → 2𝐼− + 𝑆4𝑂6

2−

For the same solution, the value for the concentration of the copper(II) sulfate as measured

using the UV-visible spectrophotometer is (0.498±0.004) moldm-3 the value for the

concentration using the titration was (0.482±0.002) moldm-3.

𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑑𝑖𝑓𝑓𝑒𝑟𝑒𝑛𝑐𝑒 =0.498 − 0.482

0.482× 100

= 3.3%

𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑒𝑟𝑟𝑜𝑟 = 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑑𝑖𝑓𝑓𝑒𝑟𝑒𝑛𝑐𝑒 − 𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑟𝑎𝑛𝑑𝑜𝑚 𝑒𝑟𝑟𝑜𝑟

= 3.3 − 0.8

= 2.5%

As the percentage difference between the two values is less than 5%, the spectrophotometric

method is verified.


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2.5. Phase 2: pH study

To determine the conditions required for maximum biosorption, a study to investigate the

correlation between biosorption and increasing pH was carried out. During this study, the pH

of the solution containing pectin was varied between 1 and 7 (pHs above 7 are not considered

as copper(II) precipitates above pH 7). Since the pKa (acid disassociation constant) of pectin

is significant, (3.5137) a buffer is needed to maintain the pH of the solution at a constant value.

A buffer solution is a combination of a weak acid and its conjugate base. The buffer maintains

approximately the same pH when small amounts of either acid or base is added to the system.

2.5.1 Preparation of Buffers

An attempt was made to use the same ions in each of the buffers, however; this was not

possible given the large range of pHs being tested. The salts and the ions chosen were those

that are known to be inert towards copper(II) ions and pectin.

37R. KOHN and P. KOVÁC . Dissociation constants of D-galacturonic and D-glucuronic acid and their О-methyl derivatives. Institute of Chemistry, Slovak Academy of Sciences. [Online] 1977, 478-485, http://www.chempap.org/file_access.php?file=324a478.pdf (accessed May 1,2014).


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To create the buffers, 1 moldm-3 solution of the respective acid was added to a beaker and

then titrated with conjugate salt until the desired pH is reached.

Ion pKa pH Volume of salt/cm3 Acid/cm3

Citrate

3.14

1 NA NA

2 6.8 93.2

3 42.0 58.0

Acetate

4.76

4 14.8 85.2

5 63.5 36.5

Phosphate

7.2

6 5.9 94.1

7 38.7 61.3

Each of these buffers were then added to separate solutions of copper(II) ions and pectin in

the first step of the biosorption protocol (Figure 9). UV-visible spectrophotometry was then

used to trace the change in biosorption.

Table 1: List of Buffers and Reagents Used To

Make Them


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2.6. Acid Catalysed Hydrolysis of Methyl-Ester in Pectin

To further increase the efficacy of pectin as a biosorbent, it was subjected to acid-catalysed

hydrolysis. Acid-catalysed hydrolysis, rather than base-catalysed hydrolysis was used in this

investigation as any base present in the pectin solution would react with carboxylic acid

groups, forming a sodium-carboxylate salt that does not biosorp heavy metals.38

As seen below, acid catalysed hydrolysis converts the ester groups to carboxylic acid groups.

As the resulting carboxylic acid group is the active biosorption site, it is likely that this would

increase the biosorbent abilities of pectin.

After pectin is completely hydrolysed, pectin’s structure will be as in Figure 13. The earlier

ester groups have been hydrolysed to carboxylic acids.

38 Tessier, Andre, Pg GC Campbell, and M. Bisson. Analytical chemistry,[Print] 1979 51(7), 844-851.

C R

O

OR’

+ H2O

Catalysed by Strong Acid

C R

O

OH

Figure 13: General Equation for Acid Catalysed Hydrolysis

+ R’OH

Figure 14 Pectin before hydrolysis Figure 15: Pectin after hydrolysis

⇌

Hydrolysis


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2.6.1 Method to Hydrolyse Pectin

Pectin was treated with 100.0 cm3 of 1.0 moldm-3 hydrochloric acid at intervals of 12 minutes,

then, excess ethanol was added to precipitate the pectin. The mixture was then filtered to

obtain the pectin precipitate, which was then heated in a water bath to evaporate the ethanol

used earlier to precipitate it and then made up to a solution of concentration 1% by mass of

pectin.

After every 12 minutes of hydrolysis, pectin solution obtained from this process was tested for

biosorption. Then using the data, a graph of biosorption against time hydrolysed was plotted.

Figure 16: Overview of method used to hydrolyse pectin

100.0 cm3 of 1%

solution by mass of

pectin

100.0 cm3 of 1.0 M

moldm-3 HCl

Left to hydrolyse for

intervals of 12 minutes

Excess ethanol added

to precipitate pectin

Pectin is then filtered

out

Excess ethanol is

evaporated by heating

in water bath for 90

min at 60 degrees

Made up to original

volume

Tested for copper(II)

biosorption


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2.7. Drawback of Time as a Variable in the Hydrolysis Study

During the hydrolysis study, it was assumed that the rate of hydrolysis is constant throughout

the reaction. However, as the reactants are used up, the rate slows down. Hence, due to the

non-constant rate, time alone is not a good indicator of the correlation between hydrolysis and

biosorption. Therefore, it is important to quantify the extent of hydrolysis in order to gauge how

hydrolysis effects biosorption.

This can be determined by finding the percentage of carboxylic acid groups in pectin using

the equation

Extent of Hydrolysis =Number of moles of (−COOH)

Total number of moles of functional groups× 100


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The total number of functional groups in pectin was determined using base catalysed

hydrolysis (Base catalysed hydrolysis is irreversible and hence reaches completion). 100.0

cm3 of 0.1 moldm-3 sodium hydroxide was added to 100.0 cm3 of 1% by mass pectin solution

and thereafter, the solution was allowed to stand for 100 hours (to ensure that all the ester

groups in pectin are hydrolysed). After hydrolysis, the sodium hydroxide catalyst will react with

the carboxylic acid groups and the amount of sodium hydroxide will reduce. The amount of

base used in this neutralisation reaction can then be determined by back titrating with

hydrochloric acid. 39

A second titration determined the number of carboxylic acid groups at any given time during

the acidic hydrolysis. 100.0 cm3 of 0.1 moldm-3 hydrochloric acid was added to catalyse the

hydrolysis, after intervals of 12 minutes, 200.0 cm3 the 0.1 M sodium hydroxide was added,

firstly to neutralise the acid catalyst and to react with the carboxylic acid groups that have been


100.0 cm3 of 1% by mass

solution of pectin

100.0 cm3 of 0.1 moldm-3

NaOH

Allowed to hydrolyse for

intervals of 12 minutes

The amount of NaOH minus the

amount of HCl used to neutralise the

total number of carboxylic acid plus

ester groups in the solution

Back titrated with 0.1

moldm-3 HCl

Figure 17: Overview of method used to hydrolyse pectin


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formed through the hydrolysis. Then, a back titration was performed to determine the amount

of sodium hydroxide neutralised by carboxylic acids.40

Using the results of these two titrations, the extent of hydrolysis at any point during the

procedure can be determined.

2.8. Phase 3: Recovery In order to thoroughly investigate the reusability of pectin, it was sequentially subjected to

solutions of copper(II) ions until the pectin was saturated with metal ions and was no longer

usable. Following which EDTA, water, or acid were tested as means to recover the biosorbent

abilities of pectin.


Figure 18: Overview of Method to Determine Number of

Carboxylic Acid Groups in Pectin

100.0 cm3 of 1% by

mass solution of pectin

100.0 cm3 of 0.1

moldm-3 HCl

Allowed to hydrolyze

for intervals of 12

minutes

The amount of HCl used

to neutralise the solution

is equal to the amount of

carboxylic acid groups in

the solution

200.0 cm3 of 0.1 moldm-3

NaOH is added to

neutralize acid and react

with carboxylic acid

groups.

Back titrated with 0.1

moldm-3 HCl


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3. Results and Discussion

3.1. Verification of Copper(II) Sulfate MAX As the equipment used in the study had lower accuracy as compared to those used in the

literature consulted, variations in the λMAX of copper(II) sulfate could occur. Therefore, It was

necessary to verify the wavelength at which the absorbance of the samples is maximum. Since

copper sulfate is blue, it will absorb light that is in the red region, which is around 750 nm to

850 nm. The survey scans obtained are as follows.

Thus, the value for MAX was found to be 810±1 nm.

0.5

0.52

0.54

0.56

0.58

0.6

0.62

740 760 780 800 820 840 860 880 900 920

(Ab

sorb

ance

±0.0

05

)/N

o U

nit

s

(Wavelength±1)/nm

Graph of Absorbance against wavelength for 0.05 moldm-3 Copper(II) Sulfate

Graph 1: Survey Scan (700nm-900nm) of 0.05 moldm-3

copper(II) Sulfate solution


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3.1.1. Standard Curve for Copper Sulfate Copper(II) sulfate solutions of the respective concentrations were created.

0.050 moldm-3 0.035 moldm-3 0.017 moldm-3 0.025 moldm-3 0.013 moldm-3

Figure 19: Samples Used for Copper(II) Sulfate

calibration Curve


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A standard curve was obtained by plotting a graph of absorbance of copper(II) sulfate at 810

nm against concentration.41

A = kc

Thus, the value of the constant k was determined to be 12.174±0.006 moldm-3cm-1

41 Graph shows mean intervals only, for detailed data see Appendix A

y = 12.174xR² = 0.998

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.01 0.02 0.03 0.04 0.05 0.06

Ab

sorb

ance

/No

Un

its

Concentration of Copper(II) Sulfate/moldm-3

Calibration curve-Absorbance Against Concentration(Error bars show summation of instrumental uncertainty and standard deviation)

Graph 2: Copper(II) Sulfate Calibration


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3.2. Results of pH Study

In order to determine the correlation between pH and biosorption, pectin was buffered at

various pHs, and its biosrption was monitored at each value until no further change was

observed.42

A direct correlation was observed between increasing pH and metal ion biosorption. This is

consistent with the hypothesis that when pH increases, the concentration of H+ ions in the

solution will decrease. Consequently, by Le Chatelier’s principle, the carboxylic acid groups

will give up their respective proton to minimise the change; however, since the solution is

42 Graph shows mean intervals only, for detailed data see Appendix A

0.0400

0.0420

0.0440

0.0460

0.0480

0.0500

0 1 2 3 4 5 6 7 8Res

idu

al C

on

cen

trat

ion

of

Co

pp

er(I

I) io

ns/

mo

ldm

-3

pH

Graph of concentration of Copper(II) ions/moldm-3 after 100 min against pH

(Error bars showing standard deviation and instrumental uncertainty)

Initial concentration of copper(II) sulfate

Graph 3: Final Concentration of copper(II) ions after 100

minutes of treatment with pectin at various pH


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buffered, most of the carboxylic acid groups to give up their proton, hence resulting in greater

biosorption. However, even at pH 7, pectin’s biosorption is not very significant (14.4%). Thus,

pectin must be further altered to increase its biosorbent abilities.

Additionally, it was also noted that the maximum biosorptive capacity was realised at each pH

after a period of 100 minutes. Graph 4 shows how the concentration of copper(II) steadily

decreases with time at pH 7.

R² = 0.9917

0.0420

0.0430

0.0440

0.0450

0.0460

0.0470

0.0480

0.0490

0.0500

0.0510

0 20 40 60 80 100 120

Res

idu

al C

on

cen

trat

ion

of

Cu

(II)

ion

s/M

Time/min

Graph showing correlation between concentration of Cu(II) ions/moldm-3

and time/min at pH 7(Error bars showing standard deviation and instrumental uncertainty)

Graph 4: Correlation between final concentration of copper(II)

ions and time at pH 7


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3.3. Results of Hydrolysis Study

During acid hydrolysis, the concentration of the copper(II) ions was measured every 12

minutes to monitor the effect of hydrolysis on biosorption.

The data from this segment of the investigation shows that acid catalysed hydrolysis was able

to increase the biosorptive capacity of pectin to twice its original value. However, once the

duration of hydrolysis exceeded 30 minutes, there was a decrease in the biosorptive capacity

of pectin. From this experiment alone, this phenomenon cannot be explained due to the non-

constant rate of hydrolysis; therefore, the biosorption of pectin must be correlated with the

extent of hydrolysis to elucidate the cause of this phenomenon.

0

0.01

0.02

0.03

0.04

0.05

0.06

0 10 20 30 40 50 60 70 80 90

Res

idu

al C

on

cen

trat

ion

of

Cu

(II)

ion

s/m

old

m-3

Time hydrolysed/min

Graph showing variation of final concentration of Cu(II) ions/moldm-3

against time hydolysed/min(Error bars showing standard deviation and instrumental uncertainty)

Graph 5: Variation of the copper(II) ions concentration with time

hydrolysed/minutes. Readings taken after 100 min treatment with pectin at pH 7


%Δ concentration

= 34.2% %Δ concentration

= 68.0%


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3.3.1. Further Results of Hydrolysis Study Titrimetric analysis of pectin’s structure43 revealed that the concentration of ester and

carboxylic groups to be 0.0029 mol per gram of pectin. Once this value was determined, further

titrimetric44 analysis was carried out to determine the amount of carboxylic acid groups in

pectin at various stages of hydrolysis.

43 Refer to Section 2.6.1 for detailed methodology or Appendix C.6. for step-by step methodology 44 Refer to Section 2.6.2 for detailed methodology or Appendix C.7. for step-by step methodology

Duration of hydrolysis/min

Number of moles of base/mol

(Excess to neutralise catalyst and

carboxylic acid groups)

Number of moles of acid/moles

(Obtained by titrating to

determine the amount of base

used while neutralising

carboxylic acid)

Number of moles of

carboxylic acid/molg-1

Extent of hydrolysis measured by percentage of (-COOH) with respect

to the total population of ester and carboxylic acid

- X Y X-Y (0.0029 − (𝑋 − 𝑌)

0.0029∗ 100

0 0.0100 0.0100 0.000 0.0

12 0.0100 0.0094 0.0006 20.7

24 0.0100 0.0091 0.0009 31.0

36 0.0100 0.0088 0.0012 41.4

48 0.0100 0.0087 0.0013 44.8

60 0.0100 0.0086 0.0013 44.8

72 0.0100 0.0086 0.0014 48.3

84 0.0100 0.0086 0.0014 48.3

Table 2: Results of study to determine the extent

of hydrolysis of pectin at various time intervals


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The decrease in the biosorptive abilities of pectin when its extent of hydrolysis passes the 35%

mark contradicts the hypothesis that- as the number of active sites increase, so should the

biosorptive abilities of pectin. It is possible that pectin behaves somewhat similar as maleic

acid, shown below.

Figure 20: Figure Showing Large Difference Between pKa1 and pKa2

of Maleic Acid as a Result of Intra-molecular Hydrogen Bonding

0

0.01

0.02

0.03

0.04

0.05

0.06

0 10 20 30 40 50 60

Res

idu

al C

on

cen

trat

ion

of

Cu

(II)

ion

s/M

Extent of hydrolysis

Graph showing variation of final concentration of Cu(II) ions/moldm-3

against extent of hydrolyis(Error bars showing standard deviation and instrumental uncertainty)


Graph 6: Variation of the Copper(II) ions concentration with extent

hydrolysed. Readings taken after 100 min treatment with pectin at pH 7


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Maleic acid45 has two carboxylic acid groups in proximity. After the first deprotonation, a very

strong intramolecular hydrogen bond is formed between the undissociated proton and the

carboxylate ion, resulting in the second deprotonation at a much higher pka.

For pectin, as the extent of hydrolysis increases over 35%, the carboxylate ions and carboxylic

acid groups are much closer in proximity, and there may be a similar intramolecular hydrogen

bonding phenomenon. Thus, the lone electron pairs on the carboxylate ions are no longer

available to form coordinate bonds, consequently, the biosorbent abilities of pectin decrease.

3.4. Verification of Results of Hydrolysis Study with Laboratory

Grade Pectin

The data obtained in the previous segment suggests the presence of an intramolecular

hydrogen-bonding phenomenon in pectin once there are sufficient carboxylic acid groups in

proximity. However, this trend could be the result of the presence of some unknown substance

that was introduced during the extraction phase; as such, it is necessary to examine a system

that consists of only pectin and Cu(II) ions.

45 Hussain, Syeed, Surapong Pinitglang, T. BAILEY, J. REID, M. NOBLE, Marina Resmini, E. THOMAS, R. GREAVES, C. VERMA, and Keith Brocklehurst. "Variation in the pH-dependent pre-steady-state and steady-state kinetic characteristics of cysteine-proteinase mechanism: evidence for electrostatic modulation of catalytic-site function by the neighbouring carboxylate anion."Biochem. J 372 (2003): 735-746.


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3.4.1. Method of Obtaining Pectin The ideal system in this experiment should consist of Cu(II) ions and pure pectin. Medical

grade pectin is very expensive hence laboratory grade pectin manufactured by Sigma-Aldrich

was used instead.

The laboratory grade pectin was subjected to experimentally determined conditions of pH 7

and the extent of hydrolysis of pectin was determined using the titrimetric method.46

46 Refer to Section 2.6.1 or Section 2.6.2 for detailed methodology

Figure 21: Laboratory Grade Pectin as Purchased from Sigma Aldrich


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3.4.2. Comparison Between Laboratory Grade and Self-Extracted Pectin

The biosorption of the laboratory grade pectin was then compared to the self-extracted pectin

to obtain the following graph.

Graph 7: Variation of the Copper(II) ions concentration with percentage hydrolysed for two

different types of pectin readings taken after 100 min treatment with pectin at pH 7

As a similar trend is observed in the laboratory pectin, it is likely that both types of pectin

experience a similar mechanism after the extent of hydrolysis increases beyond 35%. This

trend provides further evidence that there may be an intra-molecular hydrogen bonding

phenomenon.

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0 10 20 30 40 50 60

Res

idu

al C

on

cen

trat

ion

of

Cu

(II)

ion

s/M

Percentage hydrolysis

Graph showing variation of final concentration of Cu(II) ions/moldm-3 against percentage hydrolysis for two types of Pectin

(Error bars showing standard deviation and instrumental incertainty)

Self-Extracted Pectin

Lab Pectin


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3.5. Regeneration of Biosorbent

Pectin was sequentially subjected to solutions of copper(II) ions, and it was found that its

biosorbent abilities decreased considerably after sequential treatments.

Treatment number

Final Concentration of

Cu2+ ions after 100

hours/moldm-3

Percentage change in

concentration/%

1 0.015 70.0

2 0.023 54.0

3 0.029 42.0

4 0.047 6.0

5 0.052 -4.0

In order to recover the biosorptive abilities of pectin, it was treated to 1.0 M EDTA, 1.0 M HCl

and water for 30 minutes separately. Then the regenerated pectin was subjected to copper

ions and the new uptake for each method was determined.

0

10

20

30

40

50

60

70

80

EDTA HCL Water

% C

han

ge in

co

nce

ntr

atio

n o

f C

u2

+

Comparison between initial biosorption and various methods of recovery

HCl

Table 3: Results of saturation study, showing pectin’s biosorption after sequential

treatments with Copper (II) Sulfate of concentration 0.05 moldm-3

Graph 8: Figure Showing Comparison Between Various

Chemicals for recovery On The Biosorptive Abilities Of

Pectin


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3.6. Overview of Findings

Phase 1• Extraction of pectin from orange peel powder using Klieman-

Nunes water bath heating.

Phase 2

• Increasing pectin's chelative capacity by carrying out a-

• pH Study -At the optimal pH of 7, pectin's biosorption doubled from its original value.

• Hydrolysis Study - The correlation between percentage hydrolysis and biosorption is positive until 35% or 30 min of acidic hydrolysis, after which the biosorption gradually decreases as a result of intra-moelcular hydrogen bonding phenomena.

Phase 3

• To enable the reuse of pectin the following will be carried out-

• Recovery of pectin's chelative capacity -The most effective method of recovering Pectin's chelative capacity was through the use of EDTA (60% of origianl biosorption)

Figure 22: Figure Showing Summary of findings

from different parts of the investigation


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4. Evaluation 4.1. Assumptions and Limitations Several assumptions were made during this study. Firstly, it was assumed that the solubility

of pectin remained constant during hydrolysis. However, as the pectin is hydrolysed, it will

become less hydrophobic due to an increase in the number of carboxylic acid groups.

Consequently, it is unknown if the increased biosorption is as a result of more efficient active

sites or due to better interaction with copper(II) ions due as a result of pectin’s greater solubility.

While hydrolysing pectin, to remove the ethanol added to precipitate it, it was heated in a water

bath. Due to the compatibility of the intermolecular hydrogen bonds in ethanol and water, an

azeotropic47 mixture is formed in which water and ethanol boil at the same temperature. As a

result, there still would be some ethanol left in the mixture even after heating it in the water

bath for a long time. Hence, some of the pectin may still be in the precipitate form and thus the

experimentally determined biosorption may be an underestimate. This can be avoided in future

studies by heating the pectin in the oven at a low temperature to dryness.

4.2. Method Although the titrimetric method useful to explain the correlation between extent of hydrolysis

and biosorption, it may not be very accurate. The titration reveals that the initial amount of

carboxylic acid groups to be 0% for the self-extracted and the laboratory pectin. Given that,

there was bound to be some hydrolysis during extraction and the pH study, it is unlikely that

the actual extent of hydrolysis is zero - therefore raising doubts about the accuracy of the

method.

47 Chem Guide U. "non-ideal mixtures of liquids." non-ideal mixtures of liquids. http://www.chemguide.co.uk/physical/phaseeqia/nonideal.html (accessed August 3, 2014).


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5. Conclusion The research question was -

Can the pH of the environment be optimised and the molecular structure of pectin be

modified through hydrolysis of its ester group so as to produce a more competent and

reusable biosorbent?

This study revealed that the biosorbent abilities of pectin can be increased by increasing the

pH of its environment or through acid catalysed hydrolysis of its ester groups. At optimal

conditions of pH 7 and 35% hydrolysis, pectin was able to reduce the concentration of

copper(II) ions from 0.05 moldm-3 to 0.035 moldm-3. It was also determined that the most

effective method of recovering pectin’s biosorbent abilities is EDTA.

These findings are significant, as pectin has been shown to be a competent biosorbent that

can be reused. Given its low cost, it can potentially be used to substitute more expensive or

less efficient modes of removing heavy metal ions from water such as reverse osmosis,

distillation and sand filters.

Figure 23: Copper(II) Sulfate Calibration Before (Left) after (Right)

treatment with pectin for 90 minutes at optimal conditions


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Furthermore, due to its effectiveness in biosorbing metal ions at pH 7 even without chemical

modification, pectin can also be used in remote areas where the reagents required for

modification may not be available. By reducing consumption of heavy metal ions, people can

avoid contracting certain autoimmune and heart diseases, thus helping to create a healthier

and more productive society.

As a thorough investigation on the biosorbent abilities of pectin is yet to be carried out by the

scientific community, there is still scope for further study. Furthermore, this study has also left

several unresolved questions as it only considers a one-ion system, when in actuality there

are a myriad of ions with varying radii and charge present in contaminated water bodies.

Perhaps the possible synergistic or antagonistic effects of having competing cations on the

biosorptive capacity of pectin can be studied.

This investigation has also developed a protocol to create a poor biosorbent into a rather useful

one, perhaps the effectiveness of this method can be tested on other polymers that have ester

groups, such as polymethyl methacrylate.48

48 polymethyl methacrylate . Wikispaces. http://www.winsornewton.com/assets/Resource%20Centre/Articles/Acrylics%20science/poly_methyl_methacrylate_jpeg.jpg (accessed January 8, 2014).

Figure 24: Polymethyl methacrylate


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The next step in this in this investigation could be directly to use orange peels as a biosorbent,

as doing so would eliminate most intermediate steps. This would facilitate the creation of a

superior biosorbent that could be obtained at almost no cost, which in turn would allow a larger

proportion of the world’s population to be rid of heavy metal toxicity and its associated

problems.

3992 Words


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R. KOHN and P. KOVÁC . Dissociation constants of D-galacturonic and D-glucuronic

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Appendix A- Chemicals and Apparatus

Apparatus Quantity Uncertainty

250cm3Conical flask 12 -

Test tube 30 -

Test tube holder 3 -

Water Bath 1 ±0.1oC

Weighing bottle 4 -

Electronic Mass Balance 1 ±0.001g

100ml Beakers -

100µl-1000µl Micropipette 1 Setting Uncertainty 100 ±3.6% 500 ±1.2% 1000 ±0.8%

UV-vis Spectrophotometer 1 ±0.005 if reading<1 ±0.5% if reading≥1

Centrifuge 1 -

Wash Bottles with de-ionized water 3 -

Plastic Spatula 3 -

Parafilm - -

Pipette Filler 3 -

50ml Bulb pipette 1 ±0.1ml

10ml Graduated Pipette 1 ±0.1ml


Cuvette 14 -

Dropper 6 -

100ml Volumetric Flask 1 ±0.06m

1 L Volumetric Flask 2 ±0.3ml

White Tile 2 -

Rubber Test tube stoppers 6 -

Funnel 3 -

Stopwatch 3 ±0.01s

100cm3 Beaker 10 -

Glass Rod 1 -

Oven with thermostat 1 ±0.1oC

Bee-hive shelves 18 -

Scissors 1 -

Tweezers 1 -

Blender 1 -

Table A.1: List of Apparatus used in Investigation


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CHEMICAL CHEMICAL FORMULA AMOUNT USED

Citric acid C6H7O8 Excess

HCl HCl 200 g (minimum)

Sodium Hydroxide NaOH

Ethanol C2H5OH Excess

Ethanoic acid CH3COOH 300g

Copper(II) Sulfate CuSO4 200g

Potassium Iodide KI 17g

Sodium Thiosulfate Na2S2O3 16g

Table A.2: List of Chemicals and Quantities used in

Investigation


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Appendix B- Detailed Results

B.1.Raw Data for Scanning Curve for Copper(II) Sulfate

Table B.1: Table showing the absorbance for various wavelength for 0.050 M copper(II) Sulfate

Wavelength/nm Absorbance/no units

750 0.515

755 0.528

760 0.542

765 0.552

770 0.561

775 0.574

780 0.582

785 0.586

790 0.591

795 0.598

800 0.599

805 0.603

810 0.604

815 0.600

820 0.598

825 0.596

830 0.594

835 0.590

840 0.588

845 0.581

850 0.577

855 0.569

860 0.565

865 0.556

870 0.550

875 0.542

880 0.536

885 0.528

890 0.521

895 0.512


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B.2.Raw Data for Standard Curve for Copper(II) Sulfate

Table B.2: Table showing the absorbance for various concentrations of copper(II) Sulfate. These

values were then used to plot a standard curve of absorbance against concentration

Concentration (M)

Absorbance of

Sample/No Units

Mean Absorbance/No

Units

Standard Deviation

(Absorbance of Blank±0.005)/No

Units

Average Absorbance of Sample-

Absorbance of

blank[A]/No Units

Trial 1

Trial 2

0.050 0.603 0.617 0.610 0.007 0.003 0.607

0.035 0.412 0.430 0.421 0.009 0.003 0.418

0.025 0.323 0.324 0.323 0.001 0.003 0.320

0.017 0.208 0.218 0.213 0.005 0.003 0.210

0.013 0.139 0.151 0.145 0.006 0.003 0.142


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B.3.Raw Data for pH Study

(Time after which absorbance reading

was taken±0.01)/min

(Absorbance ±0.005)/No Units

(Mean Absorbance±0.010)/No

Units

Standard Deviation

Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

0.00 0.613 0.614 0.614 0.613 0.613 0.613 0.001

5.00 0.613 0.612 0.613 0.612 0.612 0.612 0.001

10.00 0.612 0.611 0.610 0.611 0.612 0.611 0.001

15.00 0.609 0.609 0.610 0.609 0.611 0.610 0.001

20.00 0.605 0.606 0.609 0.607 0.608 0.607 0.002

25.00 0.603 0.602 0.607 0.603 0.602 0.603 0.002

30.00 0.599 0.600 0.604 0.603 0.600 0.601 0.002

35.00 0.597 0.596 0.599 0.600 0.599 0.598 0.002

40.00 0.596 0.594 0.597 0.598 0.598 0.597 0.002

45.00 0.595 0.593 0.593 0.597 0.597 0.595 0.002

50.00 0.594 0.591 0.590 0.592 0.595 0.592 0.002

55.00 0.594 0.590 0.591 0.591 0.594 0.592 0.002

60.00 0.593 0.591 0.590 0.590 0.592 0.591 0.001

65.00 0.590 0.589 0.589 0.591 0.593 0.590 0.002

70.00 0.590 0.590 0.589 0.591 0.591 0.590 0.001

75.00 0.589 0.589 0.590 0.590 0.592 0.590 0.001

80.00 0.590 0.589 0.590 0.588 0.591 0.590 0.001

85.00 0.589 0.590 0.589 0.587 0.590 0.589 0.001

90.00 0.588 0.590 0.589 0.587 0.591 0.589 0.002

95.00 0.588 0.590 0.588 0.589 0.589 0.589 0.001

100.00 0.589 0.590 0.589 0.588 0.589 0.589 0.001

Table B.3: Table showing the absorbance of residual copper(II) ions after various time intervals at pH 1


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(Time after which absorbance reading was

taken±0.01)/min



Units

Standard Deviation


0.00 0.611 0.613 0.613 0.612 0.611 0.612 0.001

5.00 0.611 0.612 0.612 0.612 0.611 0.612 0.001

10.00 0.608 0.609 0.608 0.609 0.608 0.608 0.001

15.00 0.606 0.605 0.607 0.608 0.604 0.606 0.002

20.00 0.603 0.604 0.605 0.605 0.602 0.604 0.001

25.00 0.600 0.601 0.599 0.598 0.600 0.600 0.001

30.00 0.595 0.597 0.596 0.596 0.594 0.596 0.001

35.00 0.590 0.593 0.595 0.593 0.590 0.592 0.002

40.00 0.584 0.589 0.590 0.588 0.587 0.588 0.002

45.00 0.580 0.585 0.586 0.582 0.584 0.583 0.002

50.00 0.578 0.579 0.581 0.578 0.580 0.579 0.001

55.00 0.577 0.578 0.580 0.575 0.576 0.577 0.002

60.00 0.577 0.576 0.579 0.575 0.577 0.577 0.001

65.00 0.574 0.574 0.579 0.574 0.572 0.575 0.003

70.00 0.573 0.573 0.577 0.574 0.571 0.574 0.002

75.00 0.575 0.574 0.575 0.574 0.571 0.574 0.002

80.00 0.571 0.574 0.575 0.573 0.570 0.573 0.002

85.00 0.572 0.574 0.576 0.572 0.570 0.573 0.002

90.00 0.572 0.574 0.575 0.523 0.572 0.563 0.023

95.00 0.572 0.575 0.575 0.572 0.569 0.573 0.003

100.00 0.572 0.574 0.576 0.573 0.569 0.573 0.003



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taken±0.01)/min



Units

Standard Deviation


0.00 0.614 0.613 0.614 0.612 0.614 0.613 0.001

5.00 0.613 0.613 0.609 0.608 0.612 0.611 0.002

10.00 0.600 0.598 0.603 0.607 0.611 0.604 0.005

15.00 0.599 0.596 0.592 0.605 0.605 0.599 0.006

20.00 0.595 0.588 0.590 0.600 0.599 0.594 0.005

25.00 0.582 0.585 0.588 0.596 0.593 0.589 0.006

30.00 0.580 0.578 0.580 0.590 0.591 0.584 0.006

35.00 0.576 0.575 0.576 0.582 0.585 0.579 0.004

40.00 0.572 0.572 0.574 0.576 0.579 0.575 0.003

45.00 0.569 0.568 0.570 0.570 0.570 0.569 0.001

50.00 0.565 0.565 0.575 0.568 0.560 0.567 0.006

55.00 0.552 0.560 0.560 0.561 0.558 0.558 0.004

60.00 0.552 0.558 0.558 0.560 0.550 0.556 0.004

65.00 0.552 0.557 0.556 0.553 0.552 0.554 0.002

70.00 0.552 0.556 0.553 0.551 0.550 0.552 0.002

75.00 0.552 0.556 0.552 0.549 0.550 0.552 0.003

80.00 0.551 0.552 0.550 0.548 0.549 0.550 0.002

85.00 0.549 0.548 0.549 0.547 0.548 0.548 0.001

90.00 0.546 0.547 0.548 0.547 0.549 0.547 0.001

95.00 0.546 0.547 0.549 0.546 0.548 0.547 0.001

100.00 0.546 0.547 0.549 0.546 0.548 0.547 0.001



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taken±0.01)/min



Units

Standard Deviation


0.00 0.614 0.614 0.614 0.614 0.613 0.614 0.000

5.00 0.613 0.613 0.612 0.611 0.612 0.612 0.001

10.00 0.608 0.609 0.613 0.610 0.611 0.610 0.002

15.00 0.605 0.606 0.608 0.608 0.608 0.607 0.001

20.00 0.602 0.602 0.604 0.606 0.605 0.604 0.002

25.00 0.594 0.596 0.597 0.599 0.596 0.596 0.002

30.00 0.586 0.589 0.597 0.590 0.586 0.590 0.005

35.00 0.583 0.582 0.589 0.585 0.580 0.584 0.003

40.00 0.578 0.579 0.572 0.580 0.576 0.577 0.003

45.00 0.572 0.574 0.569 0.571 0.568 0.571 0.002

50.00 0.564 0.565 0.562 0.566 0.563 0.564 0.002

55.00 0.559 0.562 0.558 0.561 0.560 0.560 0.002

60.00 0.549 0.559 0.555 0.555 0.556 0.555 0.004

65.00 0.545 0.549 0.550 0.549 0.550 0.549 0.002

70.00 0.543 0.545 0.550 0.549 0.549 0.547 0.003

75.00 0.542 0.540 0.549 0.547 0.546 0.545 0.004

80.00 0.542 0.543 0.549 0.546 0.543 0.545 0.003

85.00 0.541 0.538 0.547 0.542 0.542 0.542 0.003

90.00 0.540 0.538 0.545 0.541 0.541 0.541 0.003

95.00 0.539 0.537 0.543 0.541 0.540 0.540 0.002

100.00 0.539 0.537 0.543 0.541 0.540 0.540 0.002



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taken±0.01)/min



Units

Standard Deviation


0.00 0.613 0.613 0.614 0.613 0.614 0.613 0.001

5.00 0.612 0.612 0.612 0.612 0.612 0.612 0.000

10.00 0.607 0.607 0.608 0.607 0.608 0.607 0.001

15.00 0.599 0.600 0.602 0.605 0.601 0.601 0.002

20.00 0.598 0.598 0.600 0.600 0.597 0.599 0.001

25.00 0.594 0.595 0.594 0.596 0.593 0.594 0.001

30.00 0.593 0.590 0.593 0.591 0.590 0.591 0.002

35.00 0.591 0.586 0.588 0.590 0.587 0.588 0.002

40.00 0.587 0.584 0.584 0.585 0.580 0.584 0.003

45.00 0.582 0.581 0.578 0.572 0.576 0.578 0.004

50.00 0.573 0.575 0.568 0.568 0.570 0.571 0.003

55.00 0.562 0.568 0.560 0.562 0.564 0.563 0.003

60.00 0.556 0.562 0.554 0.556 0.558 0.557 0.003

65.00 0.545 0.548 0.541 0.543 0.546 0.545 0.003

70.00 0.537 0.539 0.535 0.536 0.537 0.537 0.001

75.00 0.538 0.535 0.535 0.535 0.536 0.536 0.001

80.00 0.537 0.533 0.535 0.537 0.535 0.535 0.002

85.00 0.536 0.532 0.536 0.535 0.534 0.535 0.002

90.00 0.533 0.530 0.535 0.535 0.533 0.533 0.002

95.00 0.532 0.529 0.535 0.534 0.533 0.533 0.002

100.00 0.532 0.529 0.535 0.534 0.533 0.533 0.002



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taken±0.01)/min



Units

Standard Deviation


0.00 0.616 0.612 0.612 0.613 0.614 0.614 0.002

5.00 0.614 0.611 0.610 0.609 0.611 0.611 0.002

10.00 0.600 0.605 0.600 0.601 0.600 0.601 0.002

15.00 0.593 0.593 0.595 0.594 0.591 0.593 0.001

20.00 0.581 0.586 0.581 0.581 0.582 0.582 0.002

25.00 0.578 0.581 0.572 0.575 0.576 0.594 0.003

30.00 0.570 0.575 0.565 0.570 0.568 0.570 0.004

35.00 0.565 0.572 0.562 0.566 0.562 0.565 0.004

40.00 0.561 0.565 0.560 0.564 0.560 0.562 0.002

45.00 0.568 0.562 0.559 0.563 0.559 0.562 0.004

50.00 0.561 0.561 0.556 0.559 0.559 0.559 0.002

55.00 0.555 0.560 0.555 0.557 0.558 0.557 0.002

60.00 0.550 0.558 0.550 0.555 0.556 0.554 0.004

65.00 0.542 0.550 0.548 0.550 0.550 0.548 0.003

70.00 0.536 0.542 0.542 0.543 0.546 0.542 0.004

75.00 0.531 0.534 0.537 0.530 0.537 0.534 0.003

80.00 0.527 0.531 0.535 0.525 0.529 0.529 0.004

85.00 0.525 0.521 0.529 0.524 0.526 0.525 0.003

90.00 0.526 0.521 0.525 0.524 0.525 0.524 0.002

95.00 0.525 0.521 0.525 0.524 0.525 0.524 0.002

100.00 0.525 0.521 0.525 0.524 0.525 0.524 0.002



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Appendix C – Detailed Procedure

C.1.Preparation of Equipment All equipment used must be washed twice with tap water and rinsed once with de-ionized

water before usage. After such cleaning, the equipment must be left in an oven for

approximately 30 minutes for dehydration.

C.2.Extraction of Pectin from Orange Peels

C.2.1. Materials and Equipment Required:



Test tube 30 -

Test tube rack 3 -


Weighing bottle(large) 1 -


100ml Beakers 6 -

Centrifuge 1 -


Plastic Spatula 1 -

Parafilm - -

Pipette Filler 2 -




1 L Volumetric Flask 2 ±0.3 cm3

Rubber Test tube stopper 20 -

Table C.1.Table showing the equipment required for the extraction of pectin from orange

peels

C.2.2.Chemicals Required:


Citric acid C6H8O7 288.120 g


Table C.2.Table showing the chemicals required for the Pectin extraction


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C.2.3.Preparation of 1.5 moldm-3solution of citric acid:

1. Place a weighing bottle (large) on the electronic mass balance and set tare to zero.

2. With the aid of a spatula measure 288.120 g of C6H8O7.

3. Transfer the C6H8O7 to a 1L volumetric flask, wash repeatedly with de-ionized water

in order to ensure maximum transfer

4. With the aid of a wash bottle, make the solution up to the 1 L mark.

5. Shake well to homogenize the solution.

C.2.4.Preparation of Orange Peels for Extraction

1. Set the oven/dryer to 800 Celsius and distribute the orange peels equally throughout

the oven.

2. Turn the oven on and monitor the mass of the orange peels using an electronic

balance every 30 minutes, until a constant mass is achieved.

3. After complete dehydration, grind the peels into a fine powder using a food

processor.

4. Place the orange peel powder in a zip-locked bag and place the bag in the

refrigerator.

5. Repeat steps 1 to 4 until 300 g of orange peel powder is obtained.


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C.2.5.Extraction of Pectin from Orange Peels:

1. Add10 g of the orange peel powder to 100 cm3 of 1.5 M citric acid in a 250 cm3 beaker.

2. Then place the beaker in a water bath at 80 degrees Celsius for 30 minutes.

3. Add excess ethanol to the mixture. A cloudy grey precipitate should be observed.

4. Filter the mixture through a two layered muslin cloth to obtain the precipitate as the

residue.

5. Heat the gel to dryness.

6. Repeat these steps until 140 g of pectin powder is obtained


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C.3.Copper(II) Sulfate Calibration C.3.1. Materials and Equipment Required


Test tube 30 -

Test tube holder 3 -

Electronic Water Bath 1 ±0.1oC

Weighing bottle(small) 1 -



100ml Beakers 1 -

100µl-1000µl Micropipette 1 Setting Uncertainty 100 ±3.6% 500 ±1.2% 1000 ±0.8%

100µl-1000µl Micropipette tips 2 -

10µl-100µl Micropipette 1 Setting Uncertainty 10 ±4% 50 ±1.3% 100 ±1%

10µl-100µl Micropipette Tips 2 -



Plastic Spatula 1 -

Pipette Filler 1 -

10ml Graduated Pipette 1 ±0.1 cm3

5ml Graduated Pipette 1 ±0.05 cm3

Cuvette 6 -

Dropper 2 -

100ml Volumetric Flask 1 ±0.06 cm3


Rubber Test tube stoppers 6 -

Funnel 1 -

Stopwatch 2 ±0.01s

100cm3 Beaker 1 -

Glass Rod 1 -

Bee-Hive Shelves 18 -


Table C.3.Table showing the equipment required for the Copper(II) Sulfate calibration


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C.3.2.Chemicals Required


Copper Sulfate CuSO4 1.995 g

Table C.4.Table showing the chemicals required for the Copper(II) Sulfate Calibration

C.3.3.Preparation of 0.05 M Copper Sulfate Solution

1. Place a weighing bottle (small) on the electronic mass balance and set tare to zero.

2. With the aid of a spatula measure 1.995 g of copper(II) sulfate.

3. Transfer the copper(II) sulfate crystals to a 250 cm3 beaker.

4. Stir with a glass rod until the copper(II) sulfate are completely dissolved.

5. Transfer the solution to a 250 cm3 volumetric flask with the aid of a funnel.

6. With the aid of a wash bottle, make the solution up to the 250 cm3 mark.



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C.3.4.Preparation of the Solutions Used for Calibration

1. Prepare six test tubes containing the volumes of the stock solution and de-ionized

water shown in table C.5. with the aid of the 10.0 ml measuring cylinder, and dropper.

Test tube

Concentration /moldm-3

(Volume of stock solution±0.1)/cm3

(Volume of De-ionized

water±0.1)/cm3

(Total Volume±0.02)/cm3

Uncertainty in concentration of solution/moldm-3

1 0.050 20.0 0.00 20.0 ±0.01

2 0.035 14.0 7.0 20.0 ±0.01

3 0.025 10.0 10.0 20.0 ±0.01

4 0.017 6.8 13.2 20.0 ±0.02

5 0.013 5.2 14.8 20.0 ±0.02

Blank 0 0.000 20.0 20.0 ±0.01

Table C.5. Concentrations of Copper Sulfate used for calibration as prepared by dilution of

0.05 M stock solution.

2. Stopper the solutions and shake well to homogenize them.


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C.3.5.Plotting the Calibration Curve:

1. Transfer the blank and the solution to be tested into separate cuvettes using droppers.

2. Start up the UV-vis spectrophotometer.

3. Place the blank and the solution to be tested in separate slots of the spectrophotometer.

4. Use the spectrophotometer to measure the absorbance of the blank at 810 nm.

5. Measure the absorbance of the solution at 810 nm using the UV-vis spectrophotometer.

6. Repeat steps 1-5 one more time to obtain a total of two values of absorbance for this

concentration.

7. Repeat steps 1-6for the solutions with concentrations of 0.05 moldm-3, 0.025 moldm-3,

0.017 moldm-3 and 0.013 moldm-3 respectively.

8. Plot a graph of the average absorbance against the concentration using the values obtained.

9. Calculate the value of the extinction coefficient from the graph.


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C.4.Protocol to Determine Biosorbent Abilities of Pectin C.4.1. Materials and Equipment Required



Test tube 2 -

Test tube rack 3 -




100ml Beakers 6 -


Centrifuge 1 -


Plastic Spatula 1 -

Parafilm - -

Pipette Filler 2 -




Cuvette 6 -

Dropper 4 -



Funnel 3 -

Stopwatch 3 ±0.01s

100cm3 Beaker 10 -

Table C.6.Table showing the equipment required for the determination of biosorption of

Copper(II) Sulfate calibration



Pectin - -


Copper(II) Sulfate CuSO4 -

Table C.7.Table showing the chemicals required for the Pectin extraction


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C.4.3.Protocol

1. 100 cm3 of 1% by mass of pectin solution is added to 50 cm3 of 0.2 M copper(II) sulfate.

2. The mixture is then placed in a test tube.

3. The mixture is then spun in a centrifuged at 2000 rpm for 5 minutes, to separate pectin

from the copper(II) ions.

4. Then the copper(II) sulfate supernatant will be removed using a dropper

5. Its absorbance will be determined using the UV-Visible Spectrophotometer


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C.5.Titrimetric Verification (Section 2.3.1.)

C.5.1. Materials And Equipment Required



Weighing bottle 4 -



Plastic Spatula 3 -

Dropper 4 -

100ml Volumetric Flask 1 ±0.06m

White Tile 2 -

Funnel 2 -

100cm3 Beaker 10 -


100 cm3 Measuring Cylinder 1 ±1 cm3

Retort Stand 1 -

Table C.8.Table showing the equipment required for the titration



Potassium Iodide KI 3.320 g

Sodium Thiosulfate Na2S2O3 3.162 g

Starch 1.000 g

Table C.9.Table showing the chemicals required for the titration


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C.5.3.Preparation of Solutions

Preparation of 0.2 M KI

1. Place a weighing on the electronic mass balance and set tare to zero.

2. With the aid of a spatula measure 3.321 g of KI crystals.

3. Transfer the crystals to a 100 cm3 volumetric flask and wash repeatedly to ensure

maximum transfer.



6. Store the solution in the refrigerator when not in use.

Preparation of 0.01 M Na2S2O3


2. With the aid of a spatula measure 3.162 g of Na2S2O3 crystals.

3. Transfer the crystals to a 100 cm3 volumetric flask and wash repeatedly to ensure

maximum transfer.



Preparation of 1% solution by mass of starch


2. With the aid of a spatula measure 1.000 g of starch powder.

3. In a 250 cm3 beaker, set 100 cm3 to boil using a bunsen flame.

4. Add the starch little by little and ensure that is completely dissolved.

5. Transfer the solution to a 100 cm3 volumetric flask.




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C.5.4.Procedure for Titrimetric Analysis

1. With the aid of the 10 cm3 graduated pipette, transfer all in the centrifuged mixture of

copper(II) ions and pectin to a conical flask

2. Add approximately 50 cm3 of KI to the copper(II) Sulfate.

3. Mix gently and wait for the Copper(I) iodide to precipitate.

4. Label the flask and keep it aside.

5. Fill the burette to the 0.00cm3 mark with the 0.02 M Na2S2O3.

6. Attach the burette to a retort stand.

7. Place a white tile at the base of the stand and place the labeled flask on the tile.

8. Titrate the mixture in the flask against the 0.02 M Na2S2O3.

9. When nearing the endpoint, add a few drops of the starch indicator.

10. The endpoint is reached when one drop of Na2S2O3 turns the solution colourless

11. Repeat steps 1-9 twice more to get a total of three sets of data for this titration.


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C.6.Protocol to Determine Total Number of Functional Groups in

Pectin

C.6.1. Materials and Equipment Required



Test tube 30 -

Test tube rack 3 -




100ml Beakers 6 -

Centrifuge 1 -


Plastic Spatula 1 -

Parafilm - -

Pipette Filler 2 -




Cuvette 6 -

Dropper 4 -



Funnel 3 -

Stopwatch 3 ±0.01s

100cm3 Beaker 10 - Table C.10.Table showing the equipment needed for protocol



Pectin - -



Sodium Hydroxide NaOH

Hydrochloric acid HCl Table C.11.Table showing the chemicals required for the titration


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1. 100.0 cm3 of 0.1 moldm-3 NaOH was added to 100.0 cm3 of 1% by mass pectin solution.

2. Cover the beaker with parafilm and allow it to stand for 100 hours.

3. Fill a burette with HCl up to the 0.00 cm3 mark and then titrate the solution with an

indicator. (Phenolphthalein).

4. The amount of HCl needed to neutralise the solution is equal to the original amount of

carboxylate ions.


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C.7.Protocol to Determine Determine Amount of Carboxylic Acid

Groups in Pectin

C.7.1. Materials and Equipment Required



Test tube 30 -

Test tube rack 3 -




100ml Beakers 6 -

Centrifuge 1 -


Plastic Spatula 1 -

Parafilm - -

Pipette Filler 2 -




Cuvette 6 -

Dropper 4 -



Funnel 3 -

Stopwatch 3 ±0.01s

100cm3 Beaker 10 - Table C.12.Table showing the equipment needed for protocol



Pectin - -



Sodium hydroxide Table C.13.Table showing the chemicals required for the titration


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1. 100.0 cm3 of 0.1 moldm-3 HCl is added to 100.0 cm3 of 1% by mass of solution of pectin

in a 500.0 cm3 beaker.

2. After intervals of 12 minutes, 200 cm3 the 0.1 M NaOH is added.

3. The solution is then titrated with HCl, the amount of carboxylic acid groups is equal to

the difference between the amount of NaOH and the amount of HCl used to titrate it.


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Appendix D – MSDS

D.1.MSDS for Sodium Hydroxide

Name of Chemical Sodium Hydroxide

Chemical Formula NaOH

Hazards Corrosive

Causes Respiratory Tract irritation

Damages Lungs, Mucous Membranes, and Respiratory tract, Skin, Central Nervous System, Lens and Cornea.

Harmful if Swallowed

First Aid Measures Eye Contact- Flush with running water for at least 15 minutes. Seek medical attention

Skin Contact- Wash with plenty of water for at least 15 minutes before removing all contaminated clothing. Seek medical attention.

Inhalation-Allow victim to rest in a well-ventilated area. If victim is not breathing, give artificial respiration. If breathing is difficult give oxygen. Seek medical attention.

Ingestion-Do not induce vomiting. Seek medical attention.

Accidental Release Measures Small Spill- Dilute with water and mop up. Place in appropriate disposal container

Large Spill- Stop only if there is no risk. Absorb with non-combustible material. Call for assistance on disposal

Handling and Storage Do not ingest

Do not breathe vapor or mist

Keep in a cool well-ventilated area

Personal Protective Equipment Face Shield

Full Suit

Vapor Respirator

Gloves

Boots

Source: Sodium Hydroxide; MSDS No. SLS3298 [Online]; Science Lab .com: Houston, Texas, 2005. http://www.sciencelab.com/msds.php?msdsId=9924998 (accessed Oct 3 2013).


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D.2.MSDS for Potassium Iodide Name of Chemical Potassium Iodide

Chemical Formula KI

Hazards Irritant

Permeator

Toxic to Mucous Membranes



Skin Contact- Wash with plenty of water. Cover irritated skin with emollient. Seek medical attention.

Inhalation-Allow victim to rest in a well-ventilated area. Seek medical attention.


Accidental Release Measures Small Spill-Transfer to appropriate disposal container using suitable equipment.

Large Spill- Use shovel to transfer to appropriate disposal container. Spread water on contaminated surface and allow it to evacuate through sanitary system.

Handling and Storage Keep away from heat

Do not ingest

Do not breathe dust


Store away from oxidizing agents

Wear suitable protective clothing

Personal Protective Equipment Safety Glasses

Lab coat

Dust Respirator

Gloves

Boots

Source: Potassium Iodide Anhydrous; MSDS No. SLP2050 [Online]; Science Lab: Houston,

Texas, 2005. http://www.sciencelab.com/msds.php?msdsId=9927571 (accessed Oct 3

2013).


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D.3.MSDS for anhydrous Sodium Carbonate Name of Chemical Sodium Carbonate Anhydrous

Chemical Formula Na2CO3

Hazards Irritant

Sensitizer



Skin Contact- Wash with plenty of water. Cover irritated skin with emollient. If contact is serious was with disinfectant soap and cover skin with antibacterial cream. Seek medical attention.

Inhalation- Allow victim to rest in a well-ventilated area. If victim is not breathing, give artificial respiration. If breathing is difficult give oxygen. Seek medical attention.



Large Spill- Use shovel to transfer to appropriate disposal container. Spread water on contaminated surface and allow it to evacuate through sanitary system.


Do not ingest

Do not breathe dust


Store away from acids


Lab coat

Dust Respirator

Gloves

Boots

Source: Sodium Carbonate anhydrous; MSDS No. SLS 2481[Online]; Science Lab:

Houston, Texas, 2005. http://www.sciencelab.com/msds.php?msdsId=9927263 (accessed

Oct 5 2010).


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D.4.MSDS for Ethanol Name of Chemical Ethyl Alcohol

Chemical Formula CH3CH2OH

Hazards Irritant

Causes Respiratory Tract irritation

Causes gastrointestinal irritation

Inhalation may cause dizziness and suffocation




Ingestion-Do not induce vomiting. If victim is awake give 2-4 cups of water/milk. Seek medical attention.

Accidental Release Measures Absorb spill with inert material then place in suitable container.

Use spark proof tools.


Avoid contact with eyes, skin and clothing


Use spark proof tools

Keep way from oxidizing agents


Gloves

Boots

Source: Ethanol; MSDS No. S75119 [Online]; Fisher Scientific: Fair Lawn, NJ, 2001.

http://www.nafaa.org/ethanol.pdf (accessed Oct 5 2013).


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D.5.MSDS for Sodium Thiosulfate Name of Chemical Sodium Thiosulfate

Chemical Formula Na2S2O3

Hazards Irritant

Permeator

Toxic to kidneys, skin, liver and central nervous system.






Large Spill-Stop leak if without risk. Do not touch substance. Use water spray to reduce vapors. Call of assistance on disposal.


Do not ingest

Do not breathe dust


Store away from oxidizing agents


Synthetic Apron

Vapor and Dust Respirator

Gloves

Boots

Source: Sodium Thiosulfate, MSDS No. 202870000 [Online];Sigma Aldrich, http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=SG&language=en&productNumber=S7026&brand=SIGMA&PageToGoToURL=http%3A%2F%2F3Den (accessed Sep 7 2013).


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D.6.MSDS for Copper(II) Sulfate Pentahydrate Name of Chemical Copper(II) Sulfate Pentahydrate

Chemical Formula CuSO4.5H2O

Hazards Irritant

Toxic to kidneys, liver and central nervous system.



Inhalation- Allow victim to rest in a well-ventilated area. If victim is not breathing, give artificial respiration. If breathing is difficult give oxygen. Seek medical attention.



Large Spill-Stop leak if without risk. Do not touch substance. Use water spray to reduce vapors. Call of assistance on disposal.


Do not breathe dust


Store away from oxidizing agents and alkalis


Lab Coat

Dust Respirator

Gloves

Boots

Source: Copper (ll) sulfate pentahydrate; MSDS No. SLC3778 [Online]; Science Lab .com: Houston, Texas, 2005. http://www.sciencelab.com/msds.php?msdsId=9923597 (accessed Oct 5 2013).


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D.7.MSDS for Anhydrous Citric Acid Name of Chemical Citric acid

Chemical Formula C6H8O7

Hazards Irritant

Permeator

Toxic to kidneys, liver and central nervous system.





Accidental Release Measures Small Spill-Dilute with water or absorb with inert material and place in an appropriate container.

Large Spill-Use spark proof tools and non-combustible materials to absorb the spill.


Avoid contact with eyes, skin and clothing


Use spark proof tools

Do not inhale gas/vapor/spray

Keep way from oxidizing agents, reducing agents, acids and alkalis


Vapor respirator

Full Suit

Gloves

Boots

Source: Citric Acid; MSDS No.SLC5449 [Online]; Science Lab: Houston, Texas, 2005.

http://www.sciencelab.com/msds.php?msdsId=9923494 (accessed Sep 7 2013).


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D.8.MSDS for 1M Hydrochloric Acid Name of Chemical 1M Hydrochloric Acid

Chemical Formula HCl

Hazards Irritant

Harmful if swallowed or inhaled




Ingestion-Give plenty of water and induce vomiting immediately. Seek medical attention.

Accidental Release Measures Ventilate area of spill

Eliminate all sources of ignition

Absorb material with suitable absorbent and place in container for disposal


Do not breathe vapor or dust


Personal Protective Equipment Safety Goggles

Lab Coat

Gloves

Boots

Source: 1M hydrochloric acid; MSDS No. SLH1132 [Online]; Online]; Science Lab: Houston, Texas http://www.sciencelab.com/msds.php?msdsId=9925949 (accessed 20 June 2013).


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D.9.MSDS for Ethanoic Acid Name of Chemical Ethanoic acid

Chemical Formula CH3COOH

Hazards Irritant

Harmful if swallowed or inhaled




Ingestion-Give plenty of water and induce vomiting immediately. Seek medical attention.

Accidental Release Measures Ventilate area of spill

Eliminate all sources of ignition

Absorb material with suitable absorbent and place in container for disposal





Lab Coat

Gloves

Boots

Source: Ethanoic Acid; MSDS No. http://www.sciencelab.com/msds.php?msdsId=9923494 [Online].Science Lab .com: Houston, Texas http://www.sciencelab.com/msds.php?msdsId=9923494 (accessed 20 June 2013).


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D.10.MSDS for Pectin Name of Chemical Pectin

Chemical Formula -

Hazards Irritant

Harmful if swallowed

First Aid Measures Eye Contact- Check for and remove contact lens. Flush with running water for at least 15 minutes. Seek medical attention if irritation occurs.

Skin Contact- Wash with soap and water. Cover the irritated skin with an emollient. Get medical attention if irritation develops. Cold water may be used.

Inhalation- If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical attention.

Ingestion- Do NOT induce vomiting unless directed to do so by medical personnel. Never give anything by mouth to an unconscious person. If large quantities of this material are swallowed, call a physician immediately. Loosen tight clothing such as a collar, tie, belt or waistband.

Accidental Release Measures Use appropriate tools to put the spilled solid in a convenient waste disposal container. Finish cleaning by spreading water on the contaminated surface and dispose of according to local and regional authority requirements

Use a shovel to put the material into a convenient waste disposal container. Finish cleaning by spreading water on the contaminated surface and allow to evacuate through the sanitary system





Lab Coat

Gloves

Boots

Source: Pectin; MSDS No. http://www.sciencelab.com/msds.php?msdsId=9926411 [Online].Science Lab .com: Houston, Texas (accessed 20 June 2013).


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D.11.MSDS for Soluble Starch Name of Chemical Soluble Starch

Chemical Formula -

Hazards Irritant

Harmful if swallowed

First Aid Measures Eye Contact- Check for and remove any contact lenses. In case of contact, immediately flush eyes with plenty of water for at least 15 minutes. WARM water MUST be used. Get medical attention if irritation occurs.

Skin Contact- Wash with soap and water. Cover the irritated skin with an emollient. Get medical attention if irritation develops.

Inhalation- If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical attention.

Ingestion- Do NOT induce vomiting unless directed to do so by medical personnel. Never give anything by mouth to an unconscious person. If large quantities of this material are swallowed, call a physician immediately. Loosen tight clothing such as a collar,tie, belt or waistband.

Accidental Release Measures Use appropriate tools to put the spilled solid in a convenient waste disposal container. Finish cleaning by spreading water on the contaminated surface and dispose of according to local and regional authority requirements

Use a shovel to put the material into a convenient waste disposal container. Finish cleaning by spreading water on the contaminated surface and allow to evacuate through the sanitary system





Lab Coat

Boots

Source: Soluble Starch ; MSDS No. http://www.sciencelab.com/msds.php?msdsId=9925086 [Online].Science Lab .com: Houston, Texas (accessed 20 June 2013).

CHEMISTRY EXTENDED 2013-2014 ESSAY

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