ii PREPARATION AND CHARACTERIZATION OF CHITOSAN FOR HEAVY METAL IONS REMOVAL AĞIR METAL İYONLARININ UZAKLAŞTIRILMASI İÇİN KİTOSAN HAZIRLANMASI VE KARAKTERİZASYONU ANIES AWAD SATTI ELMAHDI Prof. Dr. SEMA BEKTAŞ Supervisor Submitted to Institution of Hacettepe University as a partial Fulfillment to the requirements for the award of the DOCTOR OF PHILOSLPHY In CHEMISTRY 2013 ii To my family iii ETİK Hacettepe Üniversitesi Fen Bilimleri Enstitüsü, tez yazım kurallarına uygun olarak hazırladığım bu tez çalışmasında; tez içindeki bütün bilgi ve belgeleri akademik kurallar çerçevesinde elde ettiğimi, görsel, işitsel ve yazılı tüm bilgi ve sonuçları bilimsel ahlak kurallarına uygun olarak sunduğumu, başkalarının eserlerinden yararlanılması durumunda ilgili eserlere bilimsel normlara uygun olarak atıfta bulunduğumu, atıfta bulunduğum eserlerin tümünü kaynak olarak gösterdiğimi, kullanılan verilerde herhangi bir değişiklik yapmadığımı, ve bu tezin herhangi bir bölümünü bu üniversite veya başka bir üniversitede başka bir tez çalışması olarak sunmadığımı beyan ederim. 03/07/2013 Anies Awad Satti EMAHDI iv ABSTRACT PREPERATION AND CHARACTERIZATION OF CHITOSAN FOR HEAVY METAL IONS REMOVAL ANIES AWAD SATTI ELMAHDI Doctor of Philosophy, Department of Chemistry Supervisor: Prof. Dr. SEMA BEKTAŞ July 2013, 122 pages Chitosan and cross-linked chitosan with epichlorohydrin beads was prepared with the aim of reversibly adsorb and release of heavy metal ions from aqueous solutions. The present work was orgnized in three main parts. During the first part of the study, chitosan beads was prepared by dissolving 3.0 g chitosan in 100 mL 2% acetic acid solution, and the solution was dropped into 2 M sodium hydroxide solution. The prepared chitosan beads puted into a flask with 100 mL sodium hydroxide (pH 10), and some amount of epichlorohydrin was added as cross-linking agent and stirred at 60 C for 6 h. the chitosan and cross-linked chitosan beads washed extensively with distilled water and stored for use. The characterization of the prepared chitosan and crosslinked chitosan was carried out by using Swelling Test, Elemental Analysis method, Scanning Electron Microscopy (SEM) image, specific surface area measurements, and Fourier Transform Infrared Spectroscopy (FTIR) techniques. The surface area of chitosan beads was 1.15 m2/ g while the surface area of cross-liked chitosan beads was 0.55 m2/g. In the secondpartof the study, adsorption of metal ions from aqueous solutions onto chitosan and cross-linked beads was investigated in batch processes. The effect of the v initial metal ions concentration, pH of the medium, temperature, weight of adsorbent materials, rate of stirring, and adsorption time on the adsorption capacity was examined. The last part of this study the selectivity ofchitosan and cross-linked chitosan by epichlorohydrin for removing of heavy metal ions from natural water samples was investigated. The maximum adsorption capacity of chitosan beads for aqueous solutions of selected metal ions (Cu(II), Hg(II), As(III) and As(V)) was obtained by experiments as 204 mg/g (3.21 mmol/g), 550mg/g (2.74 mmol/g), 100 mg/g (1.33 mmol/g), and 200mg/g (2.70 mmol/g) respectively.The maximum adsorption capacity of cross-linked chitosan beads was128mg/g (2.02 mmol/g), 84 (0.42mmol/g), 50 mg/g (0.66 mmol/g), and 181mg/g (2.42 mmol/g) for Cu(II), Hg(II), As(III) and As(V) respectively. Competitve adsorption studies were done with the solution that contains all four heavy metal ions. The adsorbtion capacity of Cu(II) ions are higher than the Hg(II), As(III) and As(V). The adsorption capacities for Cu(II), Hg(II), As(III) and As(V) ions were found to be 25 mg/g (0.39mmol/g), 20mg/g (0.10 mmol/g), 13mg/g (0.18 mmol/g), and 18 mg/g (0.23 mmol/g), respectively for chitosan. And 17 mg/g (0.27 mmol/g), 5.07mg/g (0.02 mmol/g), 9.28 mg/g (0.12mmol/g), and 12.37 mg/g (0.16mmol/g), respectively for cross-linked chitosan. Chelation behavior of heavy metal ions could be modelled using both the Langmuir and Freundlich isotherms. The results were fitted to the Langmuir isotherms. In order to examine the controlling mechanism of adsorption process, kinetic models were used to test experimental data. The result suggested that the pseudo-second order adsorption mechanism is predominant and that the overall rate of the adsorption process appeared to be controlled by chemical process. Key words: Heavy metal removal, Chitosan, Cross-linked chitosan, AAS. vi Özet AĞIR METAL İYONLARININ UZAKLAŞTIRILMASI İÇİN KİTOSAN HAZIRLANMASI VE KARAKTERİZASYONU ANIES AWAD SATTI ELMAHDI Doktora, Kimya Bölümü Tez Danışmanı: Prof. Dr. SEMA BEKTAŞ Temmuz 2013, 122 Sayfa Kitosan ve epiklorohidrin ile çapraz bağlanmış kitosanpartikülleri sulu ortamdan ağır metallerin tersinir biçimde adsorpsiyonu ve uzaklaştırılması için hazırlandı. Bu çalışma üç ana bölüm olarak organize edildi. Çalışmanın ilk kısmında, kitosan partikülleri 3.0 g kitosanın %2 asetik asit içeren 100 mL çözeltide çözünmesi ve ardından 2 M sodyum hidroksit çözeltisine aktarılmasıyla hazırlandı. Hazırlanan kitosanpartikülleri 100 mL sodyum hidroksit (pH 10) ile bir cam balona boşaltıldı ve bir miktar epiklorohidrin çapraz bağlayıcı madde olarak eklendi ve bu karışım 60 °C de 6 saat karıştırıldı. Kitosan ve çapraz bağlanmış kitosanpartikülleridistile su ile bolca yıkandı ve kullanım için hazır bekletildi. Hazırlanmış kitosan ve çapraz bağlı kitosankarakterizasyonu Şişme Testi, Elementel Analiz yöntemi, Taramalı Elektron Mikroskopu (SEM) görüntülemesi, spesifik yüzey alanı ölçümleri ve FourierTransformInfrared (FTIR) spektroskopisi tekniği ile gerçekleştirildi.Kitosanpartiküllerinin yüzey alanı 1.15 m2/g iken çapraz bağlı kitosan partiküllerinin yüzey alanı 0.55 m2/g olarak tespit edildi. Çalışmanın ikinci kısmında, sulu çözeltilerden metal iyonlarının kitosan ve çapraz bağlı kitosanpartikülleri üzerine adsorpsiyonubatch sistemde incelendi. Adsorpsiyonkapasitesiüzerine başlangıç metal iyonu derişimi, ortam pH’ ı, sıcaklık, adsorbent miktarı, karıştırma hızı ve adsorpsiyon süresinin etkileri incelendi. Çalışmanın son kısmında, kitosan ve epiklorohidrin ile çapraz bağlanmış kitosanın doğal su örneklerinden ağır metal uzaklaştırılması için seçiciliği test edildi. Kitosan partiküllerinin sulu çözeltilerde seçilmiş metal iyonları (Cu(II), Hg(II), As(III), As(V)) için maksimum adsorpsiyon kapasitesi sırasıyla 204 mg/g(3.21 mmol/g), 550mg/g(2.74 vii mmol/g), 100 mg/g (1.33 mmol/g) ve 200mg/g(2.70 mmol/g) olarak elde edildi. Çapraz bağlı kitosan partiküllerinin maksimum adsorpsiyon kapasitesi ise sırasıyla 128 mg/g(2.02 mmol/g), 84mg/g(0.42 mmol/g), 50 mg/g (0.66 mmol/g) ve 181mg/g(2.42 mmol/g) idi. Ağır metal iyonlarının şelatlaşma davranışı hem Langmuir hem de Freundlich izotermleri kullanılarak modellerdirildi. Sonuçlar Langmuir izotermlerine uygun bulundu. Adsorpsiyonprosesinin kontrol mekanizmasını incelemek amacıyla deneysel verilerin test edilmesi için kinetik modeller kullanıldı. Sonuçlar yalancı ikinci derece adsorpsion mekanizmasının daha baskın olduğunu ve bütününde adsorpsiyonprosesininkimyasal bağlanma kontrollü göründüğünü önermektedir. Yarışmalı adsorpsiyon çalışmaları bütün bu dört ağır metal iyonlarını içeren çözelti ile gerçekleştirilmiştir. Anahtar kelimeler: Ağır metal uzaklaştırılması, Kitosan, Çapraz bağlı kitosan, AAS. viii ACKNOWLEDGEMENT I wish to express my deepest appreciation to mysupervisor Prof. Dr. Sema BEKTAŞ, for her valuable guidance, suggestions and encouragement throughout the research and full support. Iam very greatly obliged and indebted to Prof. Dr. Adil DENİZLİfor his encouragement and helps during my studies. My great thanks to Assoc.Prof.Dr. Çiğdem Arpa ŞAHİNand Dr. İlknur DURUKANfor their professional advise, and helps during my studies. Special thanks to Assoc. Prof. Dr. Lokman UZUN,Dr. Nilay BERELI,Dr. Kubilay TEKIN, Dr. Murat AKGUL, Dr. Deniz TURKMEN, PhD student Mitra JALILZADEH and all members of Biochemistry research group. I would like to thanks all members of Atomic Absorption Research Group, Prof. Dr. Nuray Şatıroğlu,Res. Assis. Melek Guçoğlu, Res. Assis. Melis EFEÇINAR, for their helps and full support. My thanks also to my brothersEssam ELSHAHABY,PhD student in organic chemistry section. I would like to thank specialist chemist Beray TEMELLİ for their help in my studies. I am deeply grateful to all in chemistry department, Hacettepe University. I am deeply grateful to Sudan Government and Turkey Government and all in theEmbassy of Sudan in Ankara for their encouragement and support. I would like to express my special thanks and grateful to my mother for her undeniable support throughout years. Special thanks to my wife Hiba HUSSEIN and my sons for their encouragement during my studies. ix TABLE OF CONTENTS ETİK .................................................................................................................................. iii ABSTRACT ....................................................................................................................... iv ÖZET ................................................................................................................................. vi ACKNOWLEDGMENT ...................................................................................................... vii TABLE OF CONTENTS .................................................................................................... ix LIST OF FIGURES ............................................................................................................ xiii LIST OF TABLES ............................................................................................................. xvii 1. INTRODUCTION ........................................................................................................... 1 2. THEORATICAL ............................................................................................................. 4 2.1. Heavy Metals.............................................................................................................. 4 2.1.1. Toxicities of Heavy Metals ....................................................................................... 4 2.1.2. Effects of Heavy Metals on Human Health .............................................................. 5 2.1.2.1. Copper ................................................................................................................. 6 2.1.2.2. Mercury ................................................................................................................ 7 2.1.2.3. Arsenic ................................................................................................................. 12 2.2. Adsorption .................................................................................................................. 14 2.2.1. Adsorption Kinetics .................................................................................................. 15 2.2.2. Adsorption Equilibrium ............................................................................................ 16 2.2.3. Adsorption Isotherms .............................................................................................. 16 2.2.3.1. Freundlich Isotherm .............................................................................................. 17 2.2.3.2. Langmuir Isotherm ............................................................................................... 18 2.2.3.3. BET Isotherm ....................................................................................................... 18 2.3. Biosorption of Heavy Metals ....................................................................................... 19 2.4. Polymeric Adsorbents ................................................................................................ 20 2.4.1. Polymer-Metal Complexes ...................................................................................... 20 2.4.2. Factors Affect Polymer-Metal Complex Formation .................................................. 21 2.5. Polysaccharides ......................................................................................................... 23 2.5.1. Starch and Cellulose ............................................................................................... 24 x 2.5.2. Alginic Acid .............................................................................................................. 25 2.5.3. Dextran .................................................................................................................... 25 2.5.4. Chitin ....................................................................................................................... 26 2.5.5. Chitosan .................................................................................................................. 27 2.5.5.1. Raw Chitosan ....................................................................................................... 29 2.5.5.2. Modification of Chitosan ....................................................................................... 29 2.5.5.3. Interaction Mechanisms ....................................................................................... 31 2.5.5.4. Chelation .............................................................................................................. 32 2.6. Characterization of Polymeric Adsorbents ................................................................. 33 2.6.1. Fourier-Transform Infrared Spectroscopy (FTIR) ................................................... 33 2.6.2. Scanning Electron Microscope (SEM) ..................................................................... 33 2.6.3. Elemental Analysis .................................................................................................. 33 2.6.4. Determination of Surface Area of Polymeric Adsorbents ........................................ 34 2.6.5. Optical Atomic Spectrometry ................................................................................... 34 2.6.5.1. Atomic Absorption Spectrometry .......................................................................... 34 2.6.5.1.1. Basic Instrument for Atomic Absorption Spectrometry ...................................... 36 2.6.5.1.2. Radiation Sources ............................................................................................. 38 2.6.5.1.3. Atomizers and Atomizer Units ........................................................................... 43 2.6.5.1.4. Interferences in Atomic Absorption Spectrometry ............................................. 48 2.6.5.1.5. Optics ................................................................................................................ 50 2.6.5.1.6. Detectors ........................................................................................................... 51 3. EXPERIMENTAL........................................................................................................... 52 3.1. Preparation of chitosan and cross-linked chitosan beads ......................................... 52 3.1.1. Materials ............................................................................................................... 52 3.1.2. Preparation methods ............................................................................................ 52 3.1.2.1. Preparation of chitosan beads .............................................................................. 52 3.1.2.2. Preparation of cross-linked chitosan beads by epichlorohydrin ............................ 52 3.2. Characterization of chitosan and cross-linked chitosan .............................................. 54 3.2.1. Swelling studies ....................................................................................................... 54 xi 3.2.2. Elemental analysis .................................................................................................. 54 3.2.3. Surface morphology ........................................................................................... 54 3.2.4. Measurement of surface area ................................................................................. 55 3.2.5. FTIR studies ............................................................................................................ 55 3.3. Adsorption studies of heavy metal ions by chitosan and cross- Linked chitosan ................................................................................................................. 55 3.3.1. Reagents and apparatus ......................................................................................... 55 3.3.2. Adsorption studies ............................................................................................... 56 3.3.3. Studies of adsorption isotherm ................................................................................ 57 3.4. Selectivity Experiments .............................................................................................. 57 4. RESULTS AND DISCUSSIONS.................................................................................... 58 4.1. Characterization of chitosan and cross-linked chitosan .............................................. 58 4.1.1. Swelling studies ....................................................................................................... 58 4.1.2. Elemental analysis .................................................................................................. 59 4.1.3. Surface morphology ................................................................................................ 59 4.1.4. Measurement of the surface area ............................................................................ 59 4.1.5. FTIR studies .................................................................................................... 60 4.2. Adsorption studies of heavy metal ions by chitosan and cross-linked chitosan .......... 64 4.2.1. Adsorption studies .............................................................................................. 64 4.2.1.1. Adsorption experiments of Cu(II) .......................................................................... 64 4.2.1.2. Adsorption experiments for Hg(II) ions ................................................................. 71 4.2.1.3. Adsorption experiments for As(III) ions ................................................................ 77 xii 4.2.1.4. Adsorption experiments for As(V) ions ................................................................. 83 4.3. Evaluation of sorption performance ............................................................................ 91 4.3.1. Adsorption isotherms ............................................................................................. 91 4.3.1.1. Langmuir adsorption model .................................................................................. 94 4.3.1.2. Freundlich isotherm model ................................................................................... 96 4.4. Adsorption kinetics1 ................................................................................................... 102 4.5. Competitive adsorption of metal ions ......................................................................... 109 5. CONCLUSION ............................................................................................................ 111 6. REFRENCES ................................................................................................................ 114 CURRICULUM VITAE ....................................................................................... 122 xiii LIST OF FIGURES Page Figure 2.1. Copper metal .................................................................................................. 6 Figure 2.2. Mercury metal ................................................................................................ 8 Figure 2.3. Mercury and environment ............................................................................... 10 Figure 2.4. Arsenic metal .................................................................................................. 12 Figure 2.5. Structures of starch and cellulose .................................................................. 24 Figure 2.6. Structure of alginic acid .................................................................................. 25 Figure 2.7. Strucure of dextran ......................................................................................... 25 Figure 2.8. Structure of kitin ............................................................................................. 26 Figure 2.9. Chitosan membranes ..................................................................................... 27 Figure 2.10. N-deacetylation of chitin ............................................................................... 27 Figure 2.11. Examples of chemical derivatization of chitosan .......................................... 30 Figure 2.12. Schematic representation for the crosslinking reaction of chitosan with Epichlorohydrin ................................................................................................................. 31 Figure 2.13. Atomic Absorption Process .......................................................................... 35 Figure 2.14. Diagram of Flame Atomic Absorption Spectrometer .................................... 36 Figure 2.15. Single-Beam Atomic Absorption Spectrometer ............................................ 37 Figure 2.16. Double-Beam Atomic Absorption Spectrometer ........................................... 38 Figure 2.17. Diagram of a Hollow Cathode Lamp ............................................................. 39 Figure 2.18. Hollow Cathode Lamp Emission Process ..................................................... 40 Figure 2.19. Electrodeless Discharge Lamp ..................................................................... 41 Figure 2.20. Deuterium Lamp .......................................................................................... 42 Figure 2.21. Premix burner optionally permitting the use of a flow spoiler or an impact Bead .................................................................................................................................. 45 Figure 2.22. Illustration of one kind of graphite furnace .................................................... 46 Figure 3.1. Schemmatic representation of chitosan cross-linked by epichlorohydrin ...... 53 Figure 4.1. FTIR Spectra of chitosan and crosslinked chitosan ........................................ 60 Figure 4.2. SEM image of chitosan................................................................................... 62 xiv Figure 4.3. SEM image of cross-linked chitosan .............................................................. 63 Figure 4.4. Effect of pH on the adsorption of Cu(II) by sorbents (amount of sorbent, 0.25g; Cu(II)concentration, 5mg/L; volume of Cu(II) solution, 50mL; rate of stirring,700rpm; contact time,120 min; at room temperature) ........................ 64 Figure 4.5. Effect of contact time on the adsorption of Cu(II) by sorbents(pH of the solution, 6; amount of the sorbent, 0.25 g; Cu(II) concentration, 5mg/L; volume of Cu(II) solution, 50mL; rate of stirring, 700rpm; at room temperature) ..................... 65 Figure 4.6. Effect of concentration on the adsorption of Cu(II) by sorbents (pH of the solution, 6; amount of the sorbent, 0.25 g; contact time 120 min; volume of Cu(II) solution, 50 mL; rate of stirring, 700rpm; at room temperature) .......... 66 Figure 4.7. Effect of sorbent weight on the adsorption of Cu(II), (pH of the solution, 6; contact time 120 min; volume of Cu(II) solution, 50 mL; rate of stirring, 700rpm; at room temperature) ..................................................................................... 67 Figure 4.8. Effect of stirring rate on the adsorption of Cu(II) by sorbents(pH of the solution, 6; amount of the sorbent, 0.25 g; contact time 120 min; volume of Cu(II) solution, 50 mL; rate of stirring, 700rpm; at room temperature) .................... 68 Figure 4.9. Proposed structures of Cu-Chitosan complex ............................................... 70 Figure 4.10. Effect of pH on the adsorption of Hg(II) by sorbents (amount of sorbent, 0.25g; Hg(II)concentration, 50mg/L; volume of Hg(II) solution, 50 mL; rate of stirring, 700rpm; contact time, 120 min; at room temperature ..................................... 71 Figure 4.11. Effect of contact time on the adsorption of Hg(II) by sorbents(pH of the solution, 5; amount of the sorbent, 0.25g; Hg(II) concentration, 50mg/L ; volume of Hg(II) solution, 50mL; rate of stirring, 700rpm; at room temperature) ....................................................................................................................... 72 Figure 4.12. Effect of concentration on the adsorption of Hg (II) by sorbents (pH of the solution, 5; amount of the sorbent, 0.25g; contact time 120 min. ; volume of Hg(II) solution, 50mL; rate of stirring, 700rpm; at room temperature) ........... 73 Figure 4.13. Effect of sorbent weight on the adsorption of Hg(II), (pH of the solution, 5; contact time 120 min; volume of Hg (II) solution, 50 mL; rate of stirring, 700 rpm; at room temperature) ............................................................................ 74 Figure 4.14. Effect of stirring rate on the adsorption of Hg(II) by sorbents (pH of the solution, 5; amount of the sorbent, 0.25g; contact time 120 min; volume of Hg(II) solution, 50 mL; rate of stirring, 700rpm; at room temperature) .......... 75 xv Figure 4.15. Effect of pH on the adsorption of As(III) by sorbents (amount of sorbent, 0.25 g; As(II)concentration, 50mg/L; volume of As(III) solution, 50mL; rate of stirring, 700rpm; contact time, 120 min; at room temperature ..................................... 77 Figure 4.16.Effect of contact time on the adsorption of As(III) by sorbents(pH of the solution, 6; amount of the sorbent, 0.25g; As(III) concentration, 50mg/L; volume of As(III) solution, 50 mL; rate of stirring, 700rpm; at room temperature) ...... 78 Figure 4.17. Effect of concentration on the adsorption of As(III) by sorbents (pH of the solution, 6; amount of the sorbent, 0.25 g; contact time 120 min; volume of As(III) solution, 50 mL; rate of stirring, 700rpm; at room temperature) .......... 79 Figure 4.18. Effect of sorbent weight on the adsorption of As(III), (pH of the solution, 6; contact time 120 min; volume of As(III) solution, 50 mL; rate of stirring, 700rpm; at room temperature) ..................................................................................... 80 Figure 4.19. Effect of stirring rate on the adsorption of As(III) by sorbents(pH of the solution, 6; amount of the sorbent, 0.25g; contact time 120 min.; volume of As(III) solution, 50mL; rate of stirring, 700rpm; at room temperature) ........... 81 Figure 4.20. Effect of pH on the adsorption of As(V) by sorbents (amount of sorbent, 0.25g; As(V)concentration, 50mg/L; volume of As(V) solution, 50 mL; rate of stirring, 700rpm; contact time,120 min; at room temperature) .................................... 83 Figure 4.21. Effect of contact time on the adsorption of As(V) by sorbents(pH of the solution, 6 for chitosan and 5 for cross-linked chitosan; amount of the sorbent, 0.25g; As(V) concentration, 50mg/L; volume of As(V) solution, 50 mL; rate of stirring, 700rpm; at room temperature) .......................................................... 84 Figure 4.22. Effect of concentration on the adsorption of As(V) by sorbents (pH of the solution 6 for chitosan and 5 for cross-linked chitosan; amount of the sorbent, 0.25 g; contact time 120 min; volume of As (V) solution, 50mL; rate of stirring, 700rpm; at room temperature) ...................................................................... 85 Figure4.23. Effect of sorbent weight on the adsorption of As(V), (pH of the solution, 6 for chitosan and 5 for cross-linked chitosan; contact time 120 min; volume of As(V) solution, 50 mL; rate of stirring, 700rpm; at room temperature) .................... 86 Figure 4.24. Effect of stirring rate on the adsorption of As(V) by sorbents(pH of the solution, 6; amount of the sorbent, 0.25g; contact time 120 min.; volume of As(V) solution, 50mL; rate of stirring, 700rpm; at room temperature) ..................... 87 Figure 4.25. Linear Langmuir isotherm of copper biosorption onto chitosan and cross-linked chitosan by epichlorohydrin ............................................................................ 89 xvi Figure 4.26. Linear Langmuir isotherm of mercury(II) biosorption onto chitosan and cross- linked chitosan by epichlorohydrin ................................................................. 92 Figure 4.27. Linear Langmuir isotherm of arsenic(III) biosorption onto chitosan and crosslinked chitosan by epichlorohydrin ......................................................... 93 Figure 4.28. Linear Langmuir isotherm of arsenic(V) biosorption onto chitosan and cross- linked chitosan by epichlorohydrin ................................................................. 94 Figure 4.29. Linear Freundlich isotherm of copper(II) biosorption onto chitosan and cross- linked chitosan by epichlorohydrin ................................................................ 95 Figure 4.30. Linear Freundlich isotherm of mercurry(II) biosorption onto chitosan and cross- linked chitosan by epichlorohydrin ................................................................. 97 Figure 4.31. Linear Freundlich isotherm of arsenic(III) biosorption onto chitosan and cross- linked chitosan by epichlorohydrin ................................................................. 98 Figure 4.32. Linear Freundlich isotherm of arsenic(V) biosorption onto chitosan and cross- linked chitosan by epichlorohydrin ................................................................. 99 Figure 4.33. For copper(II), pseudo-first-order kinetic of the exprimental data for the adsorbents ..................................................................................................... 100 Figure 4.34. For copper(II), pseudo-second-order kinetic of the exprimental data for the adsorbents ............................................................................................................. 104 Figure 4.35. Formercury(II), pseudo-first-order kinetic of the exprimental data for the adsorbents ..................................................................................................... 104 Figure 4.36. Formercury(II), pseudo-second-order kinetic of the exprimental data for the adsorbents ..................................................................................................... 105 Figure 4.37. For arsenic(III), pseudo-first-order kinetic of the exprimental data for the adsorbents ..................................................................................................... 105 Figure 4.38. For arsenic(III), pseudo-second-order kinetic of the exprimental data for Theadsorbents .................................................................................................................. 106 Figure 4.39. For arsenic(V), pseudo-first-order kinetic of the exprimental data for the adsorbents ..................................................................................................... 106 Figure 4.40. For arsenic(V), pseudo-second-order kinetic of the exprimental data for Theadsorbents .................................................................................................................. 107 xvii LIST OF TABLES Page Table 2.1. Effect of low dose mercury toxicity on various organ systems ........................ 11 Table 2.2. Spectroscopic flames for AAS with their properties ......................................... 44 Table 2.3. Advantages and disadvantages of both flame and electrothermal atomizers .. 47 Table 3.1. The working conditions for determination of Cu(II), Hg(II), As(III) andAs(V) in Flame Atomic Absorption Spectrophotometer ............................................................... 56 Table 4.1. Solubility effect of chitosan and cross-linked chitosan beads .......................... 58 Table 4.2. Swelling behaviour of chitosan and cross-linked chitosan beads ..................... 59 Table 4.3. Elemental analysis results of the chitosan and cross-linked chitosan .............. 59 Table 4.4. Measurement of the surface area of chitosan and cross-linked chitosan ......... 60 Table 4.5. Cu(II) adsorption parameters from studies with chitosan without cross- linking and cross-linked chitosan by epichlorohydrin ........................................................ 70 Table 4.6. Hg(II) adsorption parameters from studies with chitosan without cross-linking and cross-linked chitosan by epichlorohydrin ................................................ 76 Table 4.7. As(III) adsorption parameters from studies with chitosan without cross-linking and cross-linked chitosan by epichlorohydrin ................................................. 82 Table 4.8. As(V) adsorption parameters from studies with chitosan without cross-linking and cross-linked chitosan by epichlorohydrin ................................................. 88 Table 4.9. Langmuir and Freundlich adsorption isotherm constants for metal ions adsorbed by chitosanbeads .......................................................................................... 100 Table 4.10. Langmuir and Freundlich adsorption isotherm constants for metal ions adsorbed by chitosan cross-linked by epichlorohydrin beads ........................ 101 Table 4.11. The first and second order kinetic constants for the chitosan beads……….108 xviii Table 4.12. The first and second order kinetic constants for the cross-linked chitosan beads .......................................................................................................................................... 108 Table 4.13. Copmetitive adsorption of metal ions on chitosan .......................................... 109 Table 4.14. Copmetitive adsorption of metal ions on cross-linked chitosan ...................... 110 1 1. INTRODUCTION Heavy metal pollution in the aquatic environment has become a major concern because of the toxicity of heavy metals to aquatic life, human beings and ecological systems [1].Rapid industrialization and the increase in population have all contributed to the heavy metal pollution in the eco-system [2]. The importance of heavy metal pollution control has increased significantly in recent decades[3]. Metal ions at very low levels such as copper, iron, and zinc are considered essential for normal body functions. Metal ions are toxic at high concentrations. The difference between the ranges essentiality and toxicity is very narrow for heavy metal ions. The toxicity of heavy metals may be caused by mechanisms that include blocking essential functional groups of bimolecular and disrupting the integrity of biomembranes [4].These metal ions are metabolic poisons and enzyme inhibitors; they can cause mental retardation and semi- permanent brain damage [5].Toxic metals are released into the environment in a number of different ways: burning of fossil fuels,coal combustion, battery industry, mining and smelting of metallic ferrous ores, municipal wastes, electric device manufacturing among others [6], fertilizers manufacturing [7], sewage waste-waters, and automobile emissions…etc., the effective removal of heavy metal ions from water or industrial effluents is very important and has attracted considerable research and practical interest [8].Conventional methods that have been used to remove heavy metal ions from various industrial effluents usually include chemical precipitation, flocculation, membrane separation, ion exchange, evaporation, electrolysis [3], reverse osmosis, neutralization, cementation [9 -11], solvent extraction, and adsorption etc.. [5]; [8].The main limitation of these techniques is their low efficiency in the removal of trace levels of metal ions [2].Among these methods, adsorption is one of the most promising treatments for the removal of metal ions because it is simple, cost-effective, extensively used [12],easy handled[5]. A lot of adsorbents including agricultural wastes [13], natural zeolites [14], clays [15], and polymer adsorbents, have been widely reported to remove metal ions from aqueous solutions. However, the further applications of these materials are limited by their low adsorption capacities or efficiencies. 2 The necessity to reduce the amount of heavy metal ions to acceptable levels in wastewater streams and subsequent possible re-use of these metal ions, has led to an increasing interest in selective polymer adsorbents [5].The adsorption on polymer sorbents is reported to be the best method for removal of metal ions in a low concentration such as ppm level. Nonspecific sorbents, such as activated carbon, metal oxides, silica, and ion-exchange resins have been used[2].Activated carbons used in wastewater treatment are traditionally obtained from precursors such as wood, lignite and animal bones. However, the interest for the application of alternative and low-cost raw materials in the production of this adsorbent has increased during last years [16], specific sorbents have been considered as one of the most promising techniques. Specific sorbents consist of a ligand that interacts with the metal ions specifically and a carrier matrix that may be an inorganic material(e.g. aluminum oxide, silica or glass) orpolymer microbeads (e.g. polystyrene, chitosan, cellulose, polymaleic anhydride or polymethylmethacrylate)[2].The increasing number of publications on adsorption of toxic compounds by modified polysaccharides shows that there is a recent increasing interest in the synthesis of new low-cost adsorbents used in wastewater treatment [17]. Chitosan, a type of biopolymer, is a good adsorbent to remove various kinds of anionic and cationic dyes as well as heavy metal ions. Chemical modifications of chitosan that lead to the formation of chitosan derivatives, grafting chitosan and chitosan composites have gained much attention [18]. In this study two forms of chitosan biopolymer areprepared for the removal of heavy metal ions. Chitosan is a natural heteropolymer of glucosamine and N-acetyl glucosamine residues[19],Chitosan, is a non-toxic, environmentally friendly biopolymer[20],and has a wide variety of applications in the fields of biotechnology, biomedical, environmental, microbiology, and pharmaceutical studies [19], in addition, it is one of the most promising alternative adsorbents for removing heavy metal ions and dyes. Besides,chitosan is used in the preparation of some biotechnological materials such as cell-stimulating materials, antibacterial agents, blood anti-coagulants (heparinoids), photography, cosmetics,artificial skin, chitin and chitosan based dressings, food and nutrition, opthalmology, paper finishing, solid-state batteries, and drug-delivery systems [19],[20]. Chitosan, which is one of the most representative biopolymers, has recently received considerable interest for the removal of heavy metals due to its excellent metal-binding 3 capacities and low cost. Chitosan, poly (b-1-4)-2-amino-2-deoxy-D-glucopyranose, is produced by partially alkaline N-deacetylation of chitin, which can be widely found in the exoskeleton of shellfish and crustaceans as the second most abundant natural biopolymers after cellulose. It is known as an outstanding sorbent of extremely high affinity for transition and post transition metal ions selectively because the amino (-NH2) and/or hydroxyl(-OH) groups on chitosan chains serve as coordination sites [21]. Determination of trace ions in natural samples is of great interest because of its adverse effects on human health. Inductively Coupled Plasma Atomic Emission Spectrometry (ICP- AES), Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Electro- thermal Atomic Absorption Spectrometry (ETAAS), are employed in trace ions analysis[22], as well as Flame Atomic Absorption Spectrometry (FAAS)is widely used for these determinations due to the common availability of the instrumentation, the speed and simplicity of the procedures and the precision and accuracy of the techniques [23]. As a result (FAAS) is cheap and its usage is easier than other instruments [24]. In addition, most of the concentrations of the metal ions in environmental samples are readily determined using this technique. There are three main objectives of this research. The first one is to prepare chitosan and cross-linked chitosan by epichlorohydrin and study of characterization of these two polymers by Elemental Analysis, Fourier-Transform Infrared Spectroscopy (FTIR), determination of surface area of the chitosan beads and Scanning Electron Microscopy(SEM).The second one is to study the selectivity of chitosan and cross-linked chitosan for removing of heavy metal ions from aqueous solutions with different pH values, incubation time, temperature, concentrations and at different rate of stirring. The last objective is to study the selectivity of chitosan and cross-linked chitosan by epichlorohydrin for removing of heavy metal ions from natural water samples. 4 2. THEOREATICAL In the last years, environment contamination by heavy metals has gained much attention due to the significant impact on public health [25]. Industrialization and urbanization have resulted in increased releases of toxic heavy metals into the natural environment comprising soils, lakes, rivers, ground waters and oceans [26]. 2.1. Heavy Metals A heavy metal is a member of an ill-defined subset of chemical elements that exhibit metallic properties. Many different definitions of the term heavy metal have been proposed, based on density, atomic number, atomic weight, chemical properties or toxicity. Heavy metals are natural constituents of the Earth's crust. They are stable and cannot be degraded or destroyed, and therefore they tend to accumulate in soils and sediments [27]. Heavy metal solutions are widely used in industrial activities such as metal finishing, electroplating, painting, dying, photography, surface treatment, printed circuit board manufacture, etc... Most of the heavy metal ions are well-known toxic and carcinogenic agents, while metal residues in the environment pose a threat not only for human health, but also have serious detrimental effects for the aquatic eco-system. The presence of heavy metals in the aquatic environment has forced international environmental agencies to introduce strict regulations with regard to metal discharge, especially from industrial activities [9]. Heavy metals they are metabolic poisons that generally cause poisoning by binding to enzymes thereby decreasing their activity [28]. 2.1.1. Toxicities of Heavy Metals The symptoms of the toxic effects of heavy metals may vary widely at the physiological level, but the basic toxicity mechanisms at the molecular level may be limited. The toxicities of heavy metals may be caused by the following mechanisms: (1) Blocking the essential functional groups of biomolecules such as enzymes: Specific amino acid residues, such as serine-OH, cysteine-SH and histidine-N often constitute the active sites of enzymes. Hg(II), for example, binds strongly cysteine-SH s, blocking an enzymatic activity. 5 (2) Displacing essential metal ions from biomolecules: A metal ion may displace a native ion, if its affinity to the binding site is stronger than that of the native one. Often a biomolecule with a foreign metal ion loses its activity. (3) Modifying the active conformation of biomolecules, especially enzymes and perhaps polynucleotide’s: A coordination of a cation may change the conformation of a protein, rendering it non-functional. (4) Disrupting the integrity of biomembranes: A metal cation may bind the negatively- charged head(s) of phospholipids and the integral protein residues of the membrane. (5) Modifying some other biologically active agents: For example Cd(II) and Pb(II) appear to potentiate the endotoxins produced by bacteria. This might be due to their effect to block some enzymes which degrade to endotoxin. (6) Binding with bioanions, resulting in a decreased level of essential bioanions, especially PO4 3-or a displacement of an essential cation from biominerals: For example, Pb(II), having a size similar to that of Ca(II), could replace Ca(II) in a bone mineral. As a result the mechanical strength of the bone may be affected. The size and the electric charge would be an important factor in these effects. One of the basic toxic effects of Pb(II) is considered to be binding of PO4 3- , rendering its cytoplasmic level very low [29]. 2.1.2.Effects of Heavy Metals on Human Health Most of heavy metals (Fe, Zn, Cu, Mo, Co, Cr, V, Mn, and Ni) are essential nutrients for plants animals and microorganisms. Only in high concentrations they become toxic. All living organisms possess both mechanisms to store and transport metals for use in metallo- proteins or cofactors and to protect themselves against the toxic effects of metal excess. In a biological system the normal concentration range of each metal is narrow. Deficiency or excess of heavy metals causes pathological changes. According to a biological significance heavy metal ions may be arranged in the following order: iron > zinc > copper> molybdenum > cobalt > chromium > vanadium > manganese > nickel [30]. The heavy metals hazardous to humans include lead, mercury, cadmium, arsenic, copper, zinc, and chromium. Heavy metals toxicity and the danger of their bioaccumulation in the food chain represent one of the major environmental and health problems of our modern society [7]. The lack of Zn could result in a weakness immunity system, depression, 7 Copper is known as an important element in both industry and biological systems. It is an essential nutrient for all high plants and animals. It is critical for energy production in the cells, also involved in nerve conduction, connective tissue, the cardiovascular system and immune system. In addition, copper is closely related to estrogen metabolism and is required for women’s fertility and to main pregnancy. Dietary ingestion of copper is indispensable for good health. According to national surveys, the average dietary intake of copper in the US is approximately 1.0–1.1mg/ day for adult women and 1.2–1.6mg/day for adult men [33]. However, excess copper may be absorbed in the intestinal tissues that lead to intestinal disorders (nausea, vomiting, diarrhea and stomach cramps), impaired healing and reduced resistance to infections [32], serious lesions in the central nervous system and even permanent damage especially for children and liver or kidney damage or even death[34], Therefore, a precise, accurate and rapid measurement of copper is of great importance [33].The World Health Organization (WHO) recommended 1.5 mg/L as the maximum acceptable concentration of this metal in drinking water on account of its toxicity [35].Copper, as one of extensively used materials in the mechanical manufacturing industry, electroplating, light industry, architecture [36],electronics, automotive, and so forth, has caused many actual or potential sources of pollution. Effluent containing copper ions from many industries has posed a risk to both human beings and ecological environment. Therefore, copper ions removal from wastewater has become an important subject today [12]. 2.1.2.2. Mercury The heavy metal mercury, Hg, atomic number 80 and atomic weight 200.59 a silver white, heavy liquid at typical ambient temperatures and pressures. Mercurys melting point is - 38.89C under atmospheric pressure. Mercury is the only metal which is a liquid at room temperature[ 37];(Figure 2.2). 9 (2) Forms of mercury with relatively low toxicity can be transformed into forms with very high toxicity through biological and other processes; (3) Methyl mercury can be bioconcentrated in organisms and biomagnified through food chains, returning mercury directly to man and other upper trophic level consumers in concentrated form; (4) Mercury is a mutagen, teratogen, and carcinogen, and causes embryocidal, cytochemical, and histopathological effects; (5) High mercury content in some species of fish and wild life from remote locations emphasize the complexity of natural mercury cycles and human impacts on these cycles and (6)The anthropogenic use of mercury should be reduced, because the difference between tolerable natural background levels of mercury and harmful effects in the environment is exceptionally small. The toxicity of mercury depends strongly on its redox state. The most toxic form of mercury is the highly reactive Hg2+, which binds to the amino acid cysteine in proteins. In contrast, the danger of elemental mercury (Hg◦) and organo-mercury compounds lies in their transport routes. Mercury vapor is easily inhaled, enters the blood stream in the lungs and is thus distributed throughout the body. Within cells, it is oxidized to reactive Hg2+. The toxicity of monomethylmercury (MeHg+) or dimethylmercury (Me2Hg) is caused by its ability to penetrate membranes within seconds and also to cross the blood–brain barrier. Symptoms of mercury poisoning are mainly neuronal disorders but also damage to the cardiovascular system, kidney, bones, etc. The three major sources of Hg emissions are natural, anthropogenic and re-emitted sources. Urban discharges, agricultural materials, mining and combustion and industrial discharges are the principal anthropogenic sources of Hg pollution in the environment. Hg was used extensively in gold extraction; in the 1800s, it was used in the chloralkali industry, in the manufacture of electrical instruments, and as a medical antiseptic; and since 1900, it has been used in pharmaceuticals, in agricultural fungicides, in the pulp and paper industry and in the production of plastics. Mercury undergoes complex physical, chemical and biological transformations in the environment, being the principal ones: (a) the transport of Hg(II)through the atmosphere, its photochemical oxidation to reactive Hg(II) and subsequent deposition on soils, lakes, rivers 10 and the sea; (b) the methylation of Hg(II) by reducing bacteria in anoxic habitats, its uptake by aquatic organisms and accumulation in the food web, resulting in high mercury concentrations in fish and chronic low level exposure of humans. Hg contamination can be much more widespread than that observed for other metals, due to atmospheric transport, or to biomagnification through the food chain, reaching fish and humans [38]. Mercury is used in thermometers, barometers, manometers, and other scientific apparatus. Methylmercury (CH3Hg), It affects the immune system, alters genetic and enzyme systems, and damages the nervous system, including coordination and the senses of touch, taste, and sight. Methylmercury is particularly damaging to developing embryos, which are five to ten times more sensitive than adults. Exposure to methylmercury is usually by ingestion, and it is absorbed more readily and excreted more slowly than other forms of mercury. Elemental mercury, Hg0, the form released from broken thermometers, causes tremors, gingivitis, and excitability when vapors are inhaled over a long period of time.Short-term exposure to high levels of metallic mercury vapors may cause lung damage, nausea, vomiting, diarrhea, increases in blood pressure or heart rate, skin rashes, and eye irritation[41]. Although it is less toxic than methylmercury, elemental mercury may be found in higher concentrations in environments such as gold mine sites, where it has been used to extract gold. If elemental mercury is ingested, it is absorbed relatively slowly and may pass through the digestive system without causing damage. Ingestion of other common forms of mercury, such as the salt HgCl2, which damages the gastrointestinal tract and causes kidney failure, is unlikely from environmental sources[40]. Mercury, is ubiquitous in the environment. Figure 2.3. Atmosphere ( Mercury vapours ) vapour rain rain Hydrosphere (Biotransformation from inorganic to organic mercury sea, rivers, lakes and ground water) Geosphere (soil) ( Settles out as inorganic mercury) Figure 2.3 Mercury and environment 11 There have been numerous studies dedicated to the study of mercury toxicity. We have shortlisted a few below for the better understanding towards low dose mercury toxicity [42]. (Table2.1). Table 2.1.Effect of low dose mercury toxicity on various organ systems Nervous system Adults Children/infants Motor system Adult Children/infants Renal system Cardiovascular system Immune system Reproductive system Memory loss, including alzheimer like dementia, deficit in attention, hypoesthesia, ataxia, dysarthrea, subclinical finger tremor impairment of hearing and vision, sensory disturbances, increased fatique Deficit in language (late talking) and memory deficit in attention, autism Disruption of fine motor function, decreased muscular strength, increased tiredness Late walking Increases plasma creatinine level Alter normal cardiovascular homoeostasis Decrease overall immunity of the body, exacerbates lupus like autoimmunity, multiple sclerosis, autoimmune thyroiditis or atopic eczema Decreases rate of fertility in both males and females, birth of abnormal offsprings 13 hematopoieticand nervous systems. Two forms of inorganic arsenic are found in natural waters, depending on the redox potential: As(V) and As(III)[44], lower level exposure can cause nausea and vomiting, decreased production of red and white blood cells, abnormal heart rhythm, damage to blood vessels, and a sensation of ―pins and needles‖ in hands and feet, Ingestion of very high levels can possibly result in death and long-term low level exposure can cause a darkening of the skin and the appearance of small ―corns‖ or ―warts‖ on the palms, soles, and torso[41]. Arsenic is introduced in water through natural and anthropogenic sources: release from mineral ores, probably due to long term geochemical changes, and from various industrial effluents like metallurgical industries, ceramic industries, dye and pesticides manufacturing industries and wood preservatives. Two predominant species found in natural waters are inorganic forms of arsenic namely; arsenate As(V) and arsenite As(III) and their presence depend on the pH and redox conditions[45]. Arsenic is present in water as a result of both natural and anthropogenic activities. Inorganic arsenic can occur in the environment in several forms. In natural waters, and thus in drinking water, it is mostly found in trivalent (arsenic (III) (As(III))) or pentavalent (arsenic(V) (As(V))) states. Drinking water poses the greatest threat to public health from arsenic Arsenic dissolved in water is acutely toxic and can lead to a number of health problems. Usually arsenic is built up in the body through drinking water and food contaminated with arsenic and causes increased risks of cancer in the skin, lungs, liver, kidney, and bladder. Consumption of arsenic also leads to disturbance of the cardiovascular and nervous system functions and concentrations eventually lead to death. People drinking water contaminated with arsenic with equal to or greater than 50 ppb are prone to increased risks of lung and bladder cancer and of arsenic-associated skin lesions. The US Environmental Protection Agency (USEPA) in 2001 adopted a new standard for arsenic in drinking water at 10 ppb, replacing the old standard of 50 ppb. [46]. But the guideline of Asconcentration limit value recommended by WHO in drinking waters has been 10 g/L since1993. The European Union, USA, Canada, Japan, and Vietnam have accepted this value in their regulatory systems, but other countries (Bangladesh, Bolivia, India, etc.) still operate at present to the 50 g/L standard [45]. 14 2.2. Adsorption Adsorption is the accumulation of atoms, molecules, or ions at the surface of a solid or liquid as the result of physical (Van der Waals adsorption) or chemical forces(activated adsorption) depending on the type of forces between the adsorbate and the adsorbent. We can distinguish between two types of adsorption process depending on which of these two force types plays the bigger role in the process. In physical adsorption, the individuality of the adsorbate and the adsorbent are preserved. In chemisorption, there is a transfer or sharing of electron or breakage of the adsorbate into atoms or radicals who are bound separately [47]. It differs from absorption, in that an adsorbed substance remains at the surface while an absorbed substance spreads throughout the absorbing material. An adsorbed substance is termed an adsorbate while the material on which adsorption occurs is the substrate. The release of an adsorbate is termed desorption [48]. Adsorption is one of the most effective and simplest approaches to removing toxic and recalcitrant pollutants from aqueous systems, and activated carbon is one of the most widely used adsorbents for this purpose [49]. Activated carbon has undoubtedly been the most popular and widely used adsorbent in wastewater treatment applications throughout the world. Abundant information on the use of activated carbon for such purposes can be found in scientific literature Because of their great capacity to adsorb pollutants. This capacity is mainly due to their structural characteristics and their porous texture, which gives them a large surface area, and their chemical nature, which can be easily modified by chemical treatment in order to increase their properties. However, activated carbon presents several disadvantages. It is non-selective, quite expensive, and the higher the quality, the greater the cost. The regeneration of saturated carbon by thermal and chemical procedure is also expensive, and results in loss of the adsorbent. This had led many workers to search for more economic and efficient adsorbents. Due to the problems mentioned above, research interest into the production of alternative sorbents to replace the costly activated carbon has intensified in recent years. Attention has focused on various adsorbents, in particular natural solid supports, which are able to remove pollutants from contaminated water at low cost. Cost is actually an important parameter for comparing the 15 adsorbent materials. Starch and chitin may have potential as inexpensive, readily available materials and are classified as low-cost sorbents[17].However, their adsorption capacities, mechanical strength, and other properties need further improvement for wider application [50]. A large variety of nonconventional adsorbents have been examined for their ability to remove various types of pollutants from water and wastewater. The adsorption capacity of several low-cost-adsorbents, mainly biopolymers, which are obtained from renewable sources, has been investigated. Most of them, including chitosan, adsorb selectively several metallic ions [20]. 2.2.1. Adsorption Kinetics The adsorption on a solid takes places in several stages; (1) External diffusion; the mass transfer by diffusion of the adsorbate molecules from the bulk fluid phase through a stagnant boundary layer surrounding each adsorbent particle to the external surface of the solid. (2) Internal diffusion; transfer of the adsorbate to the interior of the particle by migration of the adsorbate molecules from the relative small external surface of the adsorbent to the surface of the pores within each particle and by the diffusion on the adsorbate molecule through the pores of the particles. (3) The actual adsorption process; the molecule in the pores are adsorbed from the solution to solid phase. This stage is relatively fast, compared to the first two steps: hence, local equilibrium is usually assumed between these two phases[51]. The adsorption process is affected by the following factors: Surface area of adsorbent Nature of adsorbate pH Temperature Solute concentration Time of contact Nature of contacting 16 2.2.2. Adsorption Equilibrium Adsorption capacity of the adsorbent can be determined by making a contact between the adsorbate and adsorbent. If adsorption is the removal mechanism, then the residual concentration will be reached that will remain unchanged with time, which is also known equilibrium, and the process is adsorption equilibrium. Two types of adsorption processes are mentioned: (1) Physical adsorption, which is a reversible phenomenon. It results from the action of van der Waals forces. It is usually dominant at low temperatures and is multilayered. (2) Chemisorption is generally irreversible, because chemical interactions are involved between the adsorbate and adsorbent moiety. Factors affecting the adsorption process are pH, temperature, adsorbent quantity, and particle size including other chemical properties of the adsorbate and adsorbent [52]. 2.2.3. Adsorption Isotherms It is the relationship between the amount of adsorbate adsorbed on the surface of adsorbent and equilibrium concentration of the adsorbate at a certain temperature and other condition. The equilibrium data is formulated into an isotherm model. Brunauer [53], classified adsorption isotherm in six types. These types may be monolayer, multilayer or condensation in pores/capillaries. An isotherm model is suitable tool to assess the adsorption capacities in batch and column study. Batch study consists of contacting an adsorbate with a definite quantity of adsorbent in batch stirred system. The mixture is agitated to facilitate the adsorption process. In column study, adsorbent is packed in a column reactor and almost no flow or movement of adsorbent takes place inside the column. Different theoretical and empirical models have been proposed to describe the different types of isotherms in batch study. Most commonly used models are discussed here which are generally used for the interpretation of adsorption isotherms [52]. The equilibrium isotherm is fundamental in describing the interactive behaviour between the solutes and adsorbent. It is also essential in the design of an adsorption system. Three of the most commonly used isotherms, namely, the Langmuir, Freundlich and BET isotherms. Thermodynamic parameters such as Gibbs free energy change ( G0), enthalpy changes 17 ( H0) and entropy change ( S0) can be estimated using equilibrium constants changing with temperature. The standard Gibbs free energy change of the sorption reaction is given by: G0=−RTln K lnK = S0− H0..…………………………………..2.1 R RT Where K is the equilibrium constant, which can be obtained according to the method reported by Ramnani and Sabharwal, [54]; H0 and S0 can be obtained from the slope and intercept of the plot of ln K against 1/T. The negative values of G0 reflect the feasibility of the process, and the values become more negative with increase in temperature. The positive value of H0 showed the endothermic nature of the adsorption. The positive value of S0 suggested an increase in randomness at the solid–solution interface during the adsorption of the element on polymeric material [1]. 2.2.3.1. Freundlich Isotherm The equilibrium relationship in absorbers can often be described by a Freundlich relationship. Freundlich provided there is: No association or dissociation of the molecule after they are adsorbed on the surface. A complete absence of chemisorption. In other words for Freundlich isotherm to be valid, the adsorption must be purely a physical process with no change in the configuration of the molecules in the adsorbed state. Freundlich proposed the equation: …………………………………………..2.2 where k, n are empirical constants dependent on the adsorption rate and on the temperature, c is the equilibrium concentration of the solute in the solution in mg/L, q is the amount of solute adsorbed on the unit mass of the adsorbent for certain period of time (mg/L) according to the Freundlich equation, the amount adsorbed increases infinitely with increasing pressure. 18 2.2.3.2. Langmuir Isotherm The Langmuir model was originally developed to represent chemisorption on asset of distinct localized adsorption sites. The derivation of Langmuir adsorption isotherm involves five implicit assumptions. (1) The adsorbed gas behaves ideally in the vapor phase. (2) The adsorbed gas is confined to monomolecular layer. (3) The surface is homogeneous, that is, the affinity of each binding site for gas molecules in the same. (4) There is no lateral interaction between adsorbate molecules. (5) The adsorbate gas molecules are localized, that is, they do not move around on the surface. The commonly quoted form is: …………………………………..2.3 Where n is a temperature independent constant which is supposed to represent affixed number of surface sites, K is a temperature dependent equilibrium constant. 2.2.3.3. BET Isotherm In 1938, Brunauer, Emmett and Teller showed how to extend Langmuir approach to multilayer adsorption and their equation has come to be known as the BET equation. The basic assumption that each molecule in the first adsorbed layer is considered to provide one site for the second and subsequent layers. That the molecules in the second and subsequent layers, which are in contact with other sorbet molecule rather than with the surface of the adsorbent are considered to behave essentially as the saturated liquid, while the equilibrium constant for the first layer of molecule in contact with the surface of the absorbent is different. The resulting equation for the BET equilibrium isotherm is [55]. ………….2.4 19 Where qm is the amount of adsorbate in a monolayer per unit mass of adsorbent, qe is the ratio of mass of adsorbed solute to that of adsorbent, Ce is the equilibrium adsorbate concentration C0 and Cn are respectively the initial and removable adsorbate concentration and k is a constant parameter[51]. 2.3. Biosorption of Heavy Metals Biosorption has recently received a great deal of attention due to the low cost of the materials used in these applications and for the environmentally friendly impact of the treatment of exhausted sorbents. Several types of biomass have been tested for the recovery of precious metals, including fungal biomass,algal biomass but also polymers of biological origin.[56], [57], the term biosorption refers to many modes of non-active metal uptake by biomass. There are many different types of biosorbents like: Active biomass belonging to algae, bacteria or fungi. Non active kind of biosorbent which is essentially a waste product or a byproduct of a fermentation process. Abundant natural materials or polymers. The advantage of biosorption is that the biomass used, could be a raw material which is either abundant, a waste from another industrial operation or could be cheaply available. There are certain broad range biosorbents which can collect all heavy metals from the solution with a small degree of selectivity. Metal sequestration can occur by complexation, chelating, ion-exchange or coordination. Other mechanisms are physical such as adsorption or precipitation. Any of these mechanisms may be important in immobilizing the metal on the biosorbent. Since the biomaterials that are used for sorption are complex a number of these mechanisms could be occurring simultaneously. Naturally abundant biosorbents such as chitin and chitosan are recognized as excellent metal ligands, forming stable complexes with many metal ions, and serving as effective protein coagulating agents. An overview of some of the literature follows. Reported adsorption capacities are noted when possible to give some idea of sorbent effectiveness. Sorption depends heavily on experimental conditions such as pH, metal concentration, ligand concentration, competing ions, and particle size [58].There are several chemical groups that could potentially attract 20 and sequester metal ions: acetamido groups in chitin, amino and phosphate groups in nucleic acids, amino, amido, sulfhydryl and carboxyl groups in proteins and hydroxyls in polysaccharides [59]. 2.4. Polymeric Adsorbents In the past decades, polymeric adsorbents have been emerging as potential alternative to activated carbon in term of their vast surface area, perfect mechanical rigidity, adjustable surface chemistry and pore size distribution, and feasible regeneration under mild conditions. Generally, polymeric adsorbents can effectively trap many of the ubiquitous organic pollutants. To further improve adsorption performance of a given polymeric adsorbent toward other pollutants such as highly water-soluble compounds (e.g., sulfonated pollutants) and heavy metal ions, surface modification or functionalization has proved to be an effective approach because the functional groups bound to the polymeric matrixes are expected to provide specific interaction with the target pollutants [49]. Biosorbents gain wide attention as these are available in large quantities worldwide and are eco-friendly. The use of adsorbents containing natural polymers has received reorganization, in particular polysaccharides such as chitin and its derivate chitosan [50]. 2.4.1. Polymer-Metal Complexes Polymer science has emerged as an active discipline of material science. This field impinges an area of commodity, engineering and specialty polymer, there by stimulating interest all over the globe in exploiting never domains. One such branch that has emerged is polymer–metal complex comprising an organic polymer containing coordinating sites, complexes with metals. This is of relatively recent origin and an interdisciplinary approach taking into its fold areas viz. chemistry, metallurgy, environmental, and material sciences. Polymeric materials with the ability to create complexeswith metal ions are very common, originatingfrom both natural and synthetic source. Recentprogress made in design and synthesis of novel coordinationpolymers has brought a variety of polymeric materialsthat exhibit the structural diversities and attractiveproperties which can be further utilized in various fields, like catalysis, sewage treatment, optics, luminescence and sensor technology or polymer drug grafts[60]. 21 Polymer metal complex is composed of polymer and metal ions; where the metal ions are bound to the polymer ligand by a coordinate bond. A polymer ligand contains anchoring site like nitrogen, oxygen, or sulphur obtained either by polymerization of monomer possessing the coordinating site or by a chemical reaction between a polymer and low molecular weight compound having coordinating ability. The synthesis results in an organic polymer with inorganic function. The metal atoms attached to polymer backbone and to exhibit characteristic catalytic behavior which are distinctly different from their low molecular weight analogue. Indeed many synthetic polymers –metal complexes have been found to possess high catalytic efficiency in addition to semi conductivity, heat resistance and biomedical potentials. Complexation reaction and their resultant coordination structure are studied mostly by spectroscopic, such as UV-Visible, FTIR, NMR, ESR, etc. These spectra often show characteristics specific for polymeric ligand structure. The stability of the polymeric complex usually differs from that of a monomeric complex [61]. 2.4.2. FactorsAffect Polymer-Metal Complex Formation The factors which affect chelate formation include: (1) The basic strength of chelating group. There is relationship between the basicity of chelating group, as measured by pKa, and the stability of the chelate it forms. (2) The electronegativity of the donor atoms of the basic group in the chelating agent. Atoms of lower electro negativity tend to form stronger bonds, e.g. nitrogen and sulphur are better than oxygen. Thus diphenylthiocarbazone (dithozone) forms more stable chelates than does its oxygen analogue, diphenylcarbazone. (3) Ring size: Five or six membered rings are most stable, since these have minimum strain. The functional groups of the base must be so situated in the molecule that they permit the formation of a stable ring. Chelate stability increases with the number of rings that are formed, possibly owing to increasing number of water molecules that are displaced from their metal coordination sphere by one molecule of the poly functional reagent. 22 (4) The metal ion characteristics such as charge/ionic radius ratio (ionic potential). As the ratio increase, the stability of metal complex usually rises, provided the metal ion has available binding orbitals for the electron pairs it accepts. The electronegativity of the metal also plays part. The electronegativity, measured as the energy necessary to remove electrons from the metal atom expresses the electron – attracting ability of the metal ions. The approximate order of the stability of complexes of limited number of divalent ions towards a number of chelating agents has been given as; Pb >Cu >Ni >Co >Zn >Cd >Fe >Mn >Mg. (5) Resonance and steric effect. The stability of chelate structure is enhanced by contribution of resonance structure of the chelate ring, thus copper acetyl acetone has greater stability than the copper of salicylaldoxime, if the chelated complexes are used in separation processes, all of these factors must be taken into care. The success of the polymeric ligand is determined by the capacity when removing metal ions from wastewater or from toxic mediums, the polymeric ligands can be used to remove some special and important metal ions from medium for regaining. After purification, these metal ions can be used in either process. The important parameters during removing metal ions from dilute aqueous solution for regaining are; (1)The solubility of the polymer ligand that forms complex, (2)The capacity of the chelate polymer, (3)The concentration of the metal salt, Other methods such as ion exchange systems that are used for metal regaining from aqueous solution has disadvantage because these systems can make reactions in heterogeneous phase. The purposes of the employing polymers that include chelating groups in separation techniques as a suitable material are[51]. (1) These materials are stable. (2) It can be used for many times, they are reusable. 23 (3) The obtained yield is high by using low energy. (4) They are permeable. 2.5. Polysaccharides Polysaccharides have been proposed as the first biopolymers to have formed on Earth. Polysaccharides are complex carbohydrates formed from monosaccharides. A number of monosaccharide molecules such as those of glucose, become linked by glycosidic bonds with the elimination of a molecule of water for each monosaccharide added. When a polysaccharide has multiple molecules of the same type, it is described as homopolysaccharide. For example, starch and celluloseis composed of only glucose. When a polysaccharide molecule is formed by more than one type of monosaccharide molecules, it is described as a heteropolysaccharide,for example, chitin[62]. Polysaccharides, is stereoregular polymers of monosaccharides (sugars), are unique raw materials in that they are: very abundant natural polymers (they are referred to as biopolymers); inexpensive (low-cost polymers); widely available in many countries; renewable resources; stable and hydrophilic biopolymers; and modifiable polymers. They also have biological and chemical properties such as non-toxicity, biocompatibility, biodegradability, polyfunctionality, high chemical reactivity, chirality, chelation and adsorption capacities. The excellent adsorption behavior of polysaccharides is mainly attributed to: (1) High hydrophilicity of the polymer due to hydroxyl groups of glucose units. (2) Presence of a large number of functional groups (acetamido, primary amino and/or hydroxyl groups). (3) High chemical reactivity of these groups. (4) Flexible structure of the polymer chain. In spite of these properties and advantages some problems can occur. For example, chitosan is soluble in acidic media and therefore cannot be used as an insoluble sorbent under these conditions, except after physical and chemical modifications. 24 One of the most important and useful feature of chitin and starch is their good chemical reactivity. They possess a large number of reactive groups (hydroxyl and/or acetamido groups) present at the 2-, 3-, and 6-positions in the glucose unit. These groups allow direct substitution reactions (esterification or etherification reactions) or chemical modifications (hydrolysis, oxidation or grafting reactions, and enzymatic degradation), usually referred to as chemical derivatization, yielding different polysaccharide derivatives for specific domains of applications. The starch and chitin derivatives can be classified in three main classes of polymers: (1) Modified polymers such as cationic starches, carboxymethylchitin. (2) Derivatized biopolymers, including chitosan, cyclodextrins and their derivatives. (3) Polysaccharide-based materials such as resins, gels, membranes,composite materials. The modification of the existing polysaccharides is one possible method of obtaining more polar sorbents. The possible chemical derivatization of starch and chitin is also an interesting property because it is well known that the grafting of ligands can improve their adsorption properties. By incorporating some functional (hydrophobic) groups either into the backbone of the network structure or as pendant groups it is possible to prepare materials with strong adsorption properties. The chemical modification of the starch and chitin also allows preparation of two derivatized polysaccharides, cyclodextrin and chitosan, respectively[17]. 2.5.1.Starch and Cellulose Starch is a carbohydrate consisting of a large number of glucose units joined by glycosidic bonds, butcellulose is a polysaccharide consisting of a linear chain of several hundred to over ten thousand β(1→4) linkedD-glucose units,(Figure 2.5). O OH H OHH OH C H OHH H O O H H OHH OH C H OHH H OO Cellulose ) ( n O H OHH OH C H OHH H O H H OHH OH C H OHH H O Starch )( n OO 1,4 1,4 Beta Alpha Figure 2.5. Structures of starch and cellulose http://en.wikipedia.org/wiki/Carbohydrate http://en.wikipedia.org/wiki/Glucose http://en.wikipedia.org/wiki/Glycosidic_bond http://en.wikipedia.org/wiki/Glycosidic_bond http://en.wikipedia.org/wiki/Polysaccharide http://en.wikipedia.org/wiki/Glycosidic_bond http://en.wikipedia.org/wiki/Glycosidic_bond 25 2.5.2.Alginic Acid Alganic acid, or alginate, is an anionicpolysaccharide, It is a linear copolymer with homopolymeric blocks of (1-4)-linked β-D-mannuronate (M) and its C-5 epimer α-L- guluronate (G) residues, respectively, covalenôly linked together in different sequences or blocks, (Figure 2.6). O OH O OHO OH O O HO O OH n m O OH Figure 2.6. Structure of alginic acid 2.5.3.Dextran Dextran is a complex, branched glucan (polysaccharide made of many glucose molecules), The straight chain consists of α-1, 6glycosidic linkages between glucose molecules, while branches begin from α-1, 3 linkages (Figure 2.7). O OH OH OH CH2O O OH OH O CH2O m n - 1,6 - 1,6 + - 1,3 Figure 2.7. Strucure of dextran http://en.wikipedia.org/wiki/Anionic http://en.wikipedia.org/wiki/Anionic http://en.wikipedia.org/wiki/Copolymer http://en.wikipedia.org/wiki/Homopolymer http://en.wikipedia.org/wiki/Epimer http://en.wikipedia.org/wiki/Covalently http://en.wikipedia.org/wiki/Glucan http://en.wikipedia.org/wiki/Polysaccharide http://en.wikipedia.org/wiki/Glucose http://en.wikipedia.org/wiki/Glycosidic 26 2.5.4.Chitin Chitin is a long-chain polymer of a N-acetylglucosamine, a derivative of glucose, In terms of structure, chitin may be compared to the polysaccharide cellulose and, in terms of function, to the protein keratin, (Figure 2.8). O NH OH H2C OH O O OH H2C OH O nCH3 O NH CH3 O Figure 2.8. Structure of kitin Chitin is a modified polysaccharide that contains nitrogen; it is synthesized from units of N- acetylglucosamine (to be precise, 2-(acetylamino)-2-deoxy-D-glucose). These units form covalent β-1, 4 linkages (similar to the linkages between glucose units forming cellulose). (Chitin is a natural linear biopolymer,of N-acetyl-D-glucosamine linked by B(1–4) glycosidic bond).Therefore, chitin may be described as cellulose with one hydroxyl group on each monomer substituted with an acetylamine group. This allows for increased hydrogen bonding between adjacent polymers, giving the chitin-polymer matrix increased strength. The name chitinis derived from the Greek word chiton, meaning a coat of mail, and was apparently first used by Bradconnot in 1811. It is recognized to be the second most abundant biopolymer on earth, next to cellulose. It occurs primarily as a structural component in the exoskeleton of crustaceans, insects, in the pens of squids; it is also found to lesser extents in other animals, plants, fungi and bacteria[63].Deacetylation of chitin with concentrated strong alkaline produces poly-D- glucosamine or chitosan, which has a high density of amino group, and is soluble in weakly acidic solvents such as acetic acid or formic acid. It appears that the physicochemical properties of chitin and chitosan are widely different, which are governed by three principal factors, i.e. source of raw material, molecular weight and degree of deacetylation[63]. http://en.wikipedia.org/wiki/Polymer http://en.wikipedia.org/wiki/N-Acetylglucosamine http://en.wikipedia.org/wiki/Glucose http://en.wikipedia.org/wiki/Cellulose http://en.wikipedia.org/wiki/Keratin http://en.wikipedia.org/wiki/Polysaccharide http://en.wikipedia.org/wiki/Biosynthesis http://en.wikipedia.org/wiki/N-acetylglucosamine http://en.wikipedia.org/wiki/N-acetylglucosamine http://en.wikipedia.org/wiki/Glucose http://en.wikipedia.org/wiki/Cellulose http://en.wikipedia.org/wiki/Cellulose http://en.wikipedia.org/wiki/Hydroxyl http://en.wikipedia.org/wiki/Monomer http://en.wikipedia.org/wiki/Acetyl http://en.wikipedia.org/wiki/Acetyl http://en.wikipedia.org/wiki/Hydrogen_bonding http://en.wikipedia.org/wiki/Hydrogen_bonding http://en.wikipedia.org/wiki/Polymers 28 Chitosan is more efficient than chitin in terms of adsorption capacity due to the presence of a large number of free amino groups on chitosan chain. The efficiency of the polymeric sorbents depends on various physico-chemical parameters such as particle size, surface area, pore diameter, pore volume, degree of cross linking and particle size distribution. The fraction of deacetylation for commercial chitosan samples is usually lower than 95%. Higher deacetylation degree may be achieved at the expense of supplementary deacetylation steps, which contribute to partial depolymerization, and high costs when appropriate and sophisticated processes are employed for deacetylation. Highly deacetylated products are generally reserved for biomedical applications. The presence of acetylglucosamine and glucosamine units contributes to the existence of heterogeneities in the polymer. Amino groups are strongly reactive with metal ions. Indeed, nitrogen atoms hold free electron doublets that can react with metal cations. Amine groups are thus responsible for the uptake of metal cations by a chelation mechanism. However, the amine groups are easily protonated in acidic solutions. Due to its perfect properties of chitosan among potential applications and uses the following must be mentioned: (1) Medical (bandages and sponges, artificial blood vessels production, preparation of surgeon implants, artificial skin, tumor inhibition, skin burns treatment, eye fluid and lenses production), pulp and paper industry (surface treatment, photographic paper production, carbonless paper production). (2) Cosmetics (make-up powder, nail polish, moisturizer production). (3) Biotechnology (enzyme immobilization, protein separation, chromatography applications). (4)Food (removal of dyes and acids preservative, color stabilization agent, preparation of functional food, animal feed additive). (5) Agriculture (seed coating, hydroponic fertilizers production, controlled agrochemicals release). (6) Water treatment (removal of metal ions, flocculant/coagulant of proteins, amino acids, dyes, heavy metal ions, filtration) [68], [69]. 29 2.5.5.1. RawChitosan The principal characteristics of chitosan that may affect its sorption properties are its deacetylation degree, crystallinity and, to a lesser extent, molecular weight. The deacetylation degree controls the fraction of free amine groups that will be available for interactions with metal ions. The amine groups on chitosan are much more reactive than the acetamide groups on chitin. The free electron doublet of nitrogen on amine groups is responsible for the sorption of metal cations. The protonation of amine groups in acidic solutions is responsible for the electrostatic attraction of metal anions[57]. 2.5.5.2. Modification of Chitosan Chitosan can easily be modified by chemical or physical processes to prepare chitosan derivatives (obtained by grafting new functional groups),Figure 2.11, or to condition the polymer (by preparation of membranes, gel beads, fibers, hollow fibers). These processes may be used for controlling the reactivity of the polymer (improving the affinity of the sorbent for the metal, changing the selectivity series for sorption, changing the pH range for optimum sorption) or enhancing sorption kinetics. Recently, there has been a growing interest in chemical modification of chitosan to improve its molecular weight and its solubility to widen its application. Among various methods, copolymerization is the common way to improve chitosan properties such graft as increasing chelating or complexation properties, enhancing adsorption properties etc. The high adsorption capacities of modified chitosan for metal ions can be of great use for the recovery of valuable metals or the treatment of contaminated effluents[70]. 30 O OH H2C OH O O NH2 Chitosan O OH H2C OH O O N O OH H2C OH O O NHR O OH H2C OH O O NH3 O OH H2C OH O O NH2CH2COOH O OH H2C OCH2CH2SO3 O O NH3 O OH H2C OH O O NH O OH H2C OH O O NH Shiff base CHR N-carboxymethylchitosan Crosslinked chitosan Sulfoethylchitosan Chitosan salts N- alkylchitosan Figure 2.11. Examples of chemical derivatization of chitosan Chemical modification is more common, which involves either grafting a specific group onto the chitosan backbone or cross-linking. This is done to increase metal sorption capacities or improve selectivity of the polymer for a certain species. Modification is also done to prevent dissolving of the polymer when soluble chitosan is not required and the sorption is performed at acidic solutions. Cross-linking can be done using cross-linking agents such as glutaraldehyde, cyclodextrin and epichlorohydrin. However cross-linking step might reduce the uptake efficiency of chitosan especially in the case of glutaraldehyde. Reaction of the chitosan amine groups with glutaraldehyde leads to the formation of imine functional groups thereby reducing the number of amine groups. Epichlorohydrin and ethylene glycol can also be used since they react with -OH groups of chitosan,Figure 2.12,therefore they should not 31 affect the residual amine functional groups for binding. The adsorption capacity depends on the extent of cross-linking and generally decreases with increase in the extent of cross- linking. O O NH2 OH H2C OO O NH2 OH H2C OH O O O NH2 OH H2C OH O O O NH2 OH H2C O OCH H2C O H2C Cl CH2 CHOH CH2 O (1) (2) Figure 2.12. Schematic representation for the cross-linking reaction of chitosan with epichlorohydrin Epichlorohydrin is a cross-linking mono-functional agent used to form covalent bonds with the carbon atoms of the hydroxyl groups of chitosan, resulting in the rupturing of the epoxide ring and the removal of a chlorine atom. Figure 2.12;explain the reaction of chitosan (1) with valuable metals or the treatment of contaminated effluents. Epichlorohydrin in an acidic condition might be cross linked at hydroxyl groups to form the epichlorohydrin cross-linked chitosan product (2), therefore, in the present case, the pHs and molar ratios of epichlorohydrin to chitosan conditions established were appropriated for the cross-linking reaction between chitosan and epichlorohydrin in the homogeneous reaction [66]. 2.5.5.3. Interaction Mechanisms Despite the large number of papers dedicated to the sorption of metal ions, most of them focus on the evaluation of sorption performances and only a few of them aim at gaining a 32 better understanding of sorption mechanisms. However, it is accepted that amine sites are the main reactive groups for metal ions, though hydroxyl groups (especially in the C-3 position) may contribute to sorption. These reactive groups may interact with metal ions through different mechanisms depending on the metal, the pH, and the matrix of the solution. The free electron doublet on nitrogen may bind metal cations at pH close to neutrality (or weak acidity). On the other hand, the protonation of amine groups in acidic solutions gives the polymer a cationic behavior and consequently the potential for attracting metal anions. It is important to observe that the sorption of a metal may involve different mechanisms (chelation versus electrostatic attraction) depending on the composition of the solution, the pH, since these parameters may affect the protonation of the polymer (repulsion of metal cations) and the speciation of metal ions. The chelation of metal cations by ligands in solution may result in the formation of metal anions, which therefore turns the chelation mechanism on chitosan to an electrostatic attraction mechanism on protonated amine groups of the polymer[57]. 2.5.5.4. Chelation The theory of hard and soft acids and bases (HSB), as defined by Pearson, describes the ability of ions to interact or enter into coordinate bonding with other ions or with ligands and shows that this depends on the availability of their outermost electrons and empty molecular orbitals. This must be considered on top of any electrostatic effects due to ion-ion, ion- dipole, and ion-higher multipole interactions. The last type of effect is governed primarily by the charge and size of the ion. The first type of effect can be described by means of the softness parameters and the Lewis acid/base parameters of the ions. The HSAB concept provides a description of the capacities of ions to prefer ligands of the same kind (soft–soft and hard–hard) to those of different kinds when forming coordinative bonds. Softness of ions generally goes hand in hand with their polarizability, and hardness with their electrostatic field strength[57]. Metal chelating agents for the removal of metallic impurities in wastewaters is an excellent application for large-scale use of chitosan. Therefore, much attention hasbeen drawn to this polymer and to the possibilities of modifying it in order to improve its selectivity. Chitosan has been described as a suitable natural polymer for the collection of metals ions, since the 33 amine groups and hydroxyl groups on the chitosan chain can act as chelation sites for metal ions.Cross-linking of chitosan reduces the metal adsorption capacity, it enhances the resistance of chitosan against acid, alkali and chemical. Cross-linking treatment decreases adsorption performances involving a decrease in the number of free amine groups, a decrease in the accessibility to the internal sites or block a number of adsorption sites [8]. 2.6. Characterization of Polymeric Adsorbents 2.6.1. Fourier-Transform Infrared Spectroscopy (FTIR) Infrared spectroscopy is one of the most important methods for the identification and characterization of chemical structures. Its greatest use lies in its unique application to the identification of chemical functional groups from vibrational spectra [69]. 2.6.2.Scanning Electron Microscope (SEM) SEM is a very widely used technique to study surface topography. A high energy (typically 10 KeV) electron beam is scanned across the surface. The incident electrons cause low energy secondary electrons to be generated, and some escape from surface. The secondary electrons emitted from the sample are detected by attracting them onto a phosphor screen. This screen will glow and the intensity of the light is measured with a photomultiplier. Some of the incident electrons may strike an atomic nucleus and bounce back into the vacuum. These electrons are known as backscattered primaries and can be detected with a backscattered electron detector. Backscattered electrons can also give information on the surface topography and on the average atomic number of the area under the electron beam. The surface sensitivity of the SEM can be done by raising the voltage on the sample to just below the incident primary beam energy [70]. 2.6.3. Elemental Analysis Elemental analysis on carbon, hydrogen and nitrogen is the most essential - and in many cases the only - investigation performed to characterize and/or prove the elemental composition of an organic sample. 34 2.6.4. Determination of Surface Area of Polymeric Adsorbents The determination of specific surface by means of the BET theory is based upon the phenomenon of physical adsorption of gases on the external and internal surfaces of a porous material. The BET (Brunauer, Emmett and Teller) Theory is commonly used to evaluate the gas adsorption data and generate a Specific Surface Area result expressed in units of area per mass of sample (m2/g). 2.6.5. Optical Atomic Spectrometry Optical atomic spectrometry involves the monitoring of electronic transitions of gas-phase atoms to perform quantitative determination of elements in samples. The three types of atomic spectrometry are atomic emission, atomic absorption, and atomic fluorescence [71]. 2.6.5.1. Atomic Absorption Spectrometry Atomic absorption spectrometry (AAS) is the measurement of the absorption of optical radiation by atoms in the gaseous state[72]. it is a common technique used in many analytical chemistry protocols, as well applications requiring a high degree of precision and accuracy, such as food & drug safety, clinical diagnostics, and environmental sampling. Atomic absorption spectrometers may be used to analyze the concentration of over 70 different elements in a given sample solution[73], [74], and it is a mature analytical method, which is present in almost any analytical laboratory as a working horse for elemental determinations of metals. Innovation, however, is still going on with respect to the introduction of the sample into the atomizer and the increase of the analyte sampling efficiencies and residence times in the atomizer [75]. Atomic absorption spectrometry relies on the Beer-Lambert law to determine the concentration of a particular analyte in a sample. The absorption spectrum and molar absorbance of the desired sample el