DESIGNING OF ION-IMPRINTED CRYOGELS AND THEIR USE FOR HEAVY METAL REMOVAL İYON BASKILANMIŞ KRİYOJELLERİN TASARIMI VE AĞIR METAL UZAKLAŞTIRILMASI AMACIYLA KULLANIMI MİTRA JALİLZADEH PROF. DR. SERAP ŞENEL SUPERVISOR Submitted to Institute of Science of Hacettepe University as a Partial Fulfillment to the Requirements for the Award of the Degree of Doctor of Philosophy in Chemistry 2014 ETHICS In this thesis study, prepared in accordance with the spelling rules of Institute of Graduate Studies in Science of Hacettepe University, I declare that • all the information and documents have been obtained in the base of ...............the academic rules • all audio-visual and written information and results have been presented ...............according to the rules of scientific ethics • in case of using others works, related studies have been cited in ...............accordance with the scientific standards • all cited studies have been fully referenced • I did not any distortion in the data set • and any part of this thesis has not been presented as another thesis ...............study at this or any other university. 30/01/2014 Mitra JALİLZADEH To my parents i ABSTRACT DESIGNING OF ION-IMPRINTED CRYOGELS AND THEIR USE FOR HEAVY METAL REMOVAL Mitra JALİLZADEH Doctor of Philosophy, Department of Chemistry Supervisor: Prof. Dr. Serap ŞENEL January 2014, 150 pages Heavy metals generally are toxic for animal and human body. Heavy metals due to stability and resistance to degradation are classified as environmental toxins. Therefore these metals should be removed from wastewaters. Cryogels are polymeric gel matrices. Cryogels generally have interconnected supermacroporos spongy structure, high pore density, and large pores. Due to their large surface area and low flow resistance, they can be used for fast and high capacity separation. Due to specific ion recognitions ion-imprinted cryogels were used as stationary phases in chromatography. Interaction of template molecule to functional monomer with non-covalent interaction is organized and provides solid materials which have functional groups, then by removing of template ions forms template ion specific regions in polymer. In present study, four different ion-imprinted supermacroporose 2-hydroxyethyl methacrylate (HEMA)-based cryogels: [PHEMA-N-methacryloyl-(L)-cysteine-Cd (II), PHEMA-N-methacryloyl-(L)-aspartic acid-Pb(II), magnetic PHEMA-N- methacryloyl-(L)-histidine-Cu (II) and non-magnetic PHEMA-N-methacryloyl-(L)- histidine-Cu (II)], were synthesized. These cryogels were characterized by surface ii area measurements (BET), swelling tests, scanning electron microscopy (SEM), EDX, and FTIR. The surface areas of ion-imprinted cryogels (106, 43.6, 92.0, and 78.5 m2/g ) were higher than those of non-imprinted cryogels (78.5, 18.6, 47.0, and 29.3 m2/g). These cryogels were used for adsorption of Cd(II), Pb(II) and Cu(II) ions from aqueous solutions, respectively. Adsorption of heavy metals by these cryogels and effects of pH, intial concentration, contact time, temperature on adsorption capacity were examined. The maximum adsorption capacities of the ion-imprinted cryogel membranes were 77.2 mg/g for Cu(II) by Cu(II) ion- imprinted non-magnetic cryogel, 182.7 mg/g by magnetic Cu(II) ion- imprinted cryogel, 86.7 mg/g for Cd(II) by Cd(II) ion-imprinted cryogel, and 122.7 mg/g for Pb(II) by Pb(II) ion-imprinted cryogel, respectively. Heavy metal ions adsorption behavior was examined by Langmuir and Freundlich isotherms. The adsorptions were best fitted by Langmuir isotherms. First and second-order kinetic models were applied for these adsorptions. Pseudo-second order kinetic model was suitable for these adsorptions and indicated that adsorption was chemically controlled. Thermodynamic values (∆Hº, ∆Gº, ∆Sº) were calculated and thermodynamic possibility of adsorption was evaluated. Selectivity studies for ion- imprinted and non-imprinted cryogels were studied. Competitive adsorption studies by eight metal ions (Cu(II), Cd(II), Pb(II), Zn(II), Ca(II), Co(II), Ni(II), Fe(III)) were examined. Adsorption and desorption studies were repeated for three times, and reusability of the cryogels was proved without a significant loss in adsorption capacity. Keyword: ion imprinted cryogel, PHEMA-N-methacryloyl-(L)-cysteine-Cd (II), PHEMA-N-methacryloyl-(L)-aspartic acid-Pb(II), magnetic PHEMA-N- methacryloyl-(L)-histidine-Cu, non-magnetic PHEMA-N-methacryloyl-(L)-histidine- Cu (II) iii ÖZET İYON BASKILANMIŞ KRİYOJELLERİN TASARIMI VE AĞIR METAL UZAKLAŞTIRILMASI AMACIYLA KULLANIMI Mitra JALİLZADEH Doktora, Kimya Bölümü Tez Danışmanı: Prof.Dr.Serap ŞENEL Ocak 2014, 150 sayfa Genelde ağır metaller insan ve hayvan vücudu için toksik madde olarak tanımlanmışlardır. Ağır metaller dayanıklılık, kararlılık ve kimyasal bozunmaya karşı direnç göstermek gibi özellikleri nedeniyle, çevresel toksinler olarak sınıflandırılmışlardır. Bu nedenle ağır metaller su kaynaklarından uzaklaştırılmalıdır. Kriyojeller, polimerik jel matrikslerdir. Kriyojeller genellikle birbiriyle bağlı süpermakrogözenekli süngerimsi bir yapıya, yüksek gözenek yoğunluğuna ve geniş gözeneklere sahiptirler. Geniş yüzey alanı ve düşük akış direnci sağladıklarından, yüksek kapasitede hızlı ayırma özelliğine sahiptirler. İyon baskılanmış kriyojeller spesifik iyon tanıma özeliği nedeniyle kromatografide sabit faz olarak kullanılırlar. Kalıp iyon etrafında fonksiyonel monomerlerin kovalent olmayan etkileşimlerle organize edilmesi ve kimyasal fonksiyona sahip katı malzemelerin oluşturulması sağlanır. İşlem sonrasında kalıp iyonun uzaklaştırılması ile yapıda kalıp iyona özgü bölgeler oluşmuş olur. Bu çalışmada, dört farklı iyon baskılanmış süpermakrogözenekli 2-hidroksietil metakrilat (HEMA) bazlı kriyojeller [PHEMA-N-metakriloil-(L)-sistein-Cd(II), iv PHEMA-N-metakriloil-(L)-aspartik asit-Pb(II), manyetik PHEMA-N-metakriloil-(L)- histidin-Cu(II) ve manyetik olmayan PHEMA-N-metakriloil-(L)-histidin-Cu(II)], sentezlenmiştir. İyon baskılanmış kriyojeller yüzey alanı ölçümleri (BET), şişme testleri, taramalı elektron mikroskobu (SEM), EDX ve FTIR çalışmaları ile karakterize edilmiştir. İyon-baskılanmış kriyojellerin yüzey alanları (106, 43.6, 92.0, ve 78.5 m2/g) iyon-baskılanmamış kriyojellerin yüzey alanlarından (78.5, 18.6, 47.0, ve 29.3 m2/g) daha yüksekdirler. Bu kriyojellere, sırasıyla sulu çözeltilerden Cd(II), Pb(II) ve Cu(II) iyonlarının adsorpsiyon çalışmaları gerçekleştirilmiştir. Ağır metal iyonlarının iyon baskılanmış kriyojel membranlar ile adsorpsiyonuna pH’ın, ağır metal derişiminin, adsorpsiyon süresinin ve sıcaklıklığın etkisi incelenmiştir. İyon-baskılanmış kriyojel membranların maksimum adsorpsiyon kapasiteleri Cu(II) için 77.2 mg/g Cu(II) iyon-baskılanmış kriyojelle, 182.7 mg/g Cu(II) için Cu(II) iyon- baskılanmış manyetik kriyojelle, 86.7 mg/g Cd(II) için Cd(II) iyon-baskılanmış kriyojelle, ve 122.7 mg/g Pb için Pb(II) iyon-baskılanmış kriyojelle elde edilmişdir. Ağır metal iyonlarının adsorpsiyon davranışları Langmuir ve Freundlich izotermleri kullanarak incelenmiştir. Sonuçların Langmuir izotermiyle uyumlu olduğu gözlemlenmiştir. Birinci ve ikinci derece kinetik modelleri bu adsorpsiyonlar için uygulanmıştır. Sözde-ikinci derecenin bu adsorpsiyonlar için daha uygun olduğu ve adsorpsiyonların kimyasal kontrollü olduğu gösterilmiştir. Termodinamik parametreler (∆Hº, ∆Gº, ∆Sº) bu adsorpsiyonlar için hesaplanmış ve adsorpsiyon proseslerinin olasılığı, termodinamik değerlere göre incelenmiştir. Seçicilik çalışmaları, iyon baskılanmış ve baskılanmamış kriyojellerle gerçekleştirilmiştir. Yarışmalı adsorpsiyon çalışmaları, sekiz metal iyonunun bulunduğu çözeltide (Cu(II), Cd(II), Pb(II), Zn(II), Ca(II), Co(II), Ni(II), Fe(III)) yapılmıştır. Adsorpsiyon ve desorpsiyon işlemi üç kez art arda tekrarlanmıştır ve kriyojel membranların adsorpsiyon kapasitesinde önemli bir kayıp olmadan tekrar kullanım için uygun olduğu gözlemlenmiştir. Anahtar kelimeler: iyon baskılanmış kriyojel, PHEMA-N-metakriloil-(L)-sistein- Cd(II), PHEMA-N-metakriloil-(L)-aspartik asit-Pb(II), manyetik PHEMA-N- metakriloil-(L)-histidin-Cu(II) ve manyetik olmayan PHEMA-N-metakriloil-(L)- histidin-Cu (II). v ACKNOWLEDGEMENT I would like to thank to my supervisor Prof. Dr. Serap ŞENEL for her help, encouragement, kindness, criticism, advice, and sensibility. I would like to express my deepness gratitude to Prof. Dr. Adil DENIZLİ for his kindness, advice, help, encouragement, and sensibility. In addition, I would like to express my deepness gratitude to Assoc. Prof. Dr. Lokman Uzun for his endless help, patience, advice, comments and suggestions about this research. He was always with me in during of my PhD education. I want to thank him for his helpful behaviors, enormous contribution, understandings, and effective advises. I would like to express my thanks to Assoc. Prof. Dr. Handan Yavuz Alagöz for her advice and kindness. I would like to thank my friends in Biochemistry Research Group and chemistry department, Recep Üzek, Mehmet Emin Çorman, Canan Armutcu, Turkan Mammadova, Esma Sari, Huma Ishaq, Dr. Fatma Yılmaz, Dr. Nilay Bereli, Dr. Müge Andaç, Dr. Ali Derazshamshir, Dr. Gözde Baydemir, Gülsu Şener, Bahar Ergün, Dr.Işık Perçin Demirçelik, Emel Tamahkar, Özlem Şahin, Masoomeh Mehrnia, Matin Yazdani, Mohammadreza Ghafarlu, Fatma Kartal, Dr. Veyis Karakoç, Mine Dursun, Çiğdem Çiçek, Dilara Saçlıgil, Yeşeren Saylan, Duygu Çimen, Ilgım Göktürk ,Tuğba Doğan, Gizem Uzunoğlu, Binaz Demirci, Erdoğan Özgür, Sevgi Aslıyüce, Kemal Çetin, Senem Çulha and Semra Akgönüllü for their help, trustful and providing me a friendly atmosphere to work in. I am especially grateful to Assoc. Prof. Dr. Selim Sanin, Dr. İlknur Durukan, Dr. Anies Satti, and Ömer Arslan for their help. My greatest thanks to my family, my brother, Ramez Jalilzadeh, my sister, Raana Jalilzadeh, my nephew, Hamed, and my nieces, Razieh, Ayshin, Elay for their kindness, patience and understanding... Mitra JALİLZADEH CONTENTS vi PAGES ABSTRACT………………………………………………………………………………..i ÖZET………………………………………………………………………………………iii ACKNOWLEDGEMENT…..……………………………………………………………..v CONTENTS ……………………………………………………………………………...vi FIGURE LEGENDS…………………………………………………………………......xi TABLE LEGENDS………………………………………………………….................xvii 1. INTRODUCTION………………………………………………………………….......1 2. GENERAL INFORMATION……………………………………………………….....4 2.1. Heavy Metals……………………………………………………………………......4 2.1.1. Heavy Metals Toxicity…………………………………………………………...4 2.1.2. Removal of Heavy Metals……………………………………………………......5 2.1.3. Conventional Refinement Techniques…………………………………...........5 2.1.3.1. Chemical Precipitation .....…………………………………….............….......6 2.1.3.2.2. Electrochemical process……………………………………………….........7 2.1.3.3. Liquid-Liquid Extraction…………………………………………….................7 2.1.3.4. Flotation..........................…………………………………………………......8 2.1.3.5. Coagulation and Flocculation …………………………………………….......8 2.1.3.6. Ion-exchange.................…………………………………………………….....8 2.1.3.7. Membrane Processes....…………………………………………………….....9 2.1.4. New Methods for Removing Heavy Metals…………………..…………..........9 2.1.4.1. Adsorption...............…………………………………………………….........10 2.1.4.1.1. Adsorption on Modified Natural Materials……………………………......13 2.1.4.1.2. Adsorption on Industrial by-Products…………….......………………......12 2.1.4.1.3. Adsorption on Polymers...........................……………………………......12 2.1.4.1.4. Modified Biopolymers...............................……………………………......13 2.1.4.1.5. Hydrogels.................................................……………………………......13 2.1.4.1.6. Cryogels…………………...................................................…………......14 vii 2.1.4.1.6.1. Cryogels in Treatment of Water and Wastewater………………........16 2.1.4.1.6.2. Composite Systems..............................……………………………......17 2.1.4.2. Membrane Filtration....................................……………………………......17 2.1.4.3. Electrodialysis.............................................……..……………………........18 2.1.4.4. Photocatalysis.............................................……………………………......18 2.1.4.5. Metal Chelating Method..............................……………………………......19 2.1.5. Cadmium........................................................……………………………......21 2.1.6. Copper........................................................…………………………….........21 2.1.7. Lead........................................................…………………………….............22 2.1.8. Polymer Based Adsorbents for Removal of Metals...........……………........23 2.1.9. Determination of Heavy Metals......................……………………………......25 2.1.9.1. Atomic Absorption Spectrometry................……………………………......26 2.1.9.2 Absorption Principles...................................……………………………......26 2.2. Molecular Imprinting Technology......................……………………………......27 2.2.1. Covalent Imprinting.......................................……………………….……......29 2.2.2. Non-Covalent Imprinting..............................………………………..……......29 2.2.3. Semi-Covalent Imprinting..............................……………………….……......30 2.2.4. Sythesis Methods of MIP..............................……………………….……......33 2.2.5. Surface Imprinting.......................................……………………….…..…......33 2.2.6. Novel Technologies for MIP..........................……………………….……......34 2.2.6.1. Controlled/Living Free Radical Polymerization(CLRP).....……….…….....34 2.2.6.2. Block Copolymer Self-assembly.........................……..………….……......35 2.2.6.3. Microwave-assisted Heating Method...............………………….…….......35 2.2.6.4. Ionic Liquid as porogen.....................................………………….……......35 2.2.7. Ion Imprinted Polymer(IIP)...................................………………….……......36 2.2.7.1. Different Approches for IIP................................………………….……......36 2.2.7.2. The Crosslinking of Linear Chain Polymers Carrying Metal-binding Groups...................................................................................................................37 2.2.7.3. Chemical Immobilization...................................………………….……......37 2.2.7.4. Surface Imprinting.............................................………………….……......38 viii 2.2.7.5. The History of Ion Imprinted Polymers..............………………….……......39 3. MATERIALS AND METHODS...........................................................................41 3.1. Materials.........................................................................................................40 3.2. Preparation of Ion Imprinted Cryogels............................................................40 3.2.1. The Cryogels for Cu(II) Ions........................................................................43 3.2.1.1. Synthesis of Functional Monomer............................................................43 3.2.1.2. Preparation of MAH-Cu(II) Pre-Complex..................................................43 3.2.1.3. Preparation of Cu(II) Ion Imprinted and Non-Imprinted Non-Magnetic Cryogels................................................................................................................43 3.2.1.4. Preparation of Cu(II) Ion Imprinted and Non-Imprinted Magnetic Cryogels................................................................................................................45 3.2.2. The Cryogels for Cd(II) Ions........................................................................46 3.2.2.1. Synthesis of Functional Monomer............................................................46 3.2.2.2. Preparation of MAC-Cd(II) Pre-Complex..................................................46 3.2.2.3. Preparation of Cd(II) Ion Imprinted and Non-Imprinted Cryogels.............47 3.2.2. The Cryogels for Pb(II) Ions........................................................................48 3.2.3.1. Synthesis of Functional Monomer............................................................48 3.2.3.2. Preparation of MAsp-Pb(II) Pre-Complex.................................................49 3.2.3.3. Preparation of Pb(II) Ion Imprinted and Non-Imprinted Cryogels.............49 3.3. Characterization Studies................................................................................51 3.3.1. Swelling Properties of Cryogels...................................................................51 3.3.2. FTIR Studies................................................................................................51 3.3.3. Surface Morphology....................................................................................51 3.3.4. X-Ray Analysis............................................................................................51 3.3.5. Surface Area Measurements.......................................................................52 3.3. Heavy Metal Ion Adsorption from Singular Aqueous Solutions......................52 3.3. Selectivity Studies for Metal ion Imprinted Cryogels......................................53 3.6. Desorption and Reuse....................................................................................54 3.7. Competitive Heavy Metal Adsorption/Enrichment..........................................54 4. RESULT AND DISCUSSION............................................................................55 ix 4.1.1. Swelling Characterization of Cryogels.........................................................55 4.1.2. FTIR Analyses.............................................................................................56 4.1.3. Surface Area Measurements.......................................................................58 4.1.4. Surface Morphology....................................................................................62 4.1.5. X-Ray Analysis (EDX).................................................................................63 4.2. Adsorption Studies with Ion Imprinted Cryogels.............................................63 4.2.1. Effective Parameters on Heavy Metal Adsorption Performances of Molecularly Imprinted Cryogels.............................................................................63 4.2.1.1. Effect of pH...............................................................................................63 4.2.1.2. Effect of Initial Concentration....................................................................67 4.2.1.3. Effect of Temperature...............................................................................71 4.2.1.4. Effect of Contact Time..............................................................................79 4.2.1.5. Adsorption Isotherms................................................................................82 4.2.1.5.1. Langmuir Isotherm.................................................................................83 4.2.1.5.2. Freundlich Isotherm...............................................................................84 4.2.1.6. Adsorption Thermodynamics....................................................................92 4.2.1.7. Adsorption Kinetics.................................................................................107 4.2.2. Magnetic vs Non-Magnetic Cu(II)-Imprinted Cryogels...............................116 4.2.3. Selectivity Studies.....................................................................................117 4.2.3.1. Ion Imprinted vs Non-Imprinted Cryogels...............................................117 4.2.3.1.1. Cu(II) Ion-Imprinted Magnetic vs Non-Imprinted Cryogels..................117 4.2.3.1.2. Cu(II) Ion-Imprinted vs Non-Imprinted Cryogels..................................118 4.2.3.1.3. Cd(II) Ion-Imprinted vs Non-Imprinted Cryogels..................................119 4.2.3.1.4. Pb(II) Ion-Imprinted with Non-Imprinted Cryogels...............................120 4.2.3.2. Competitive Adsorption by Ion-Imprinted Cryogels................................122 4.2.3.2.1. Adsorption of Cu(II), Cd(II), Pb(II), and Zn(II) by Cu(II) Ion-Imprinted Magnetic Cryogel.................................................................................................122 4.2.3.2.2. Adsorption of Cu(II), Cd(II), Pb(II), and Zn(II) by Cu(II) Ion-Imprinted Non-Magnetic Cryogel.........................................................................................123 4.2.3.2.3. Adsorption of Cd(II), Cu(II), Pb(II), and Zn(II) by Cd(II) Ion-Imprinted Cryogel................................................................................................................124 x 4.2.3.2.4. Adsorption of Pb(II), Cu(II), Cd(II), and Zn(II) by Pb(II) Ion-Imprinted Cryogel................................................................................................................126 4.2.4. Simultaneous Competitive Adsorption on Ion Imprinted Cryogels............126 4.4. Desorption and Reuse..................................................................................127 4.5. Comparison with Literature...........................................................................127 5. CONCLUSION.................................................................................................129 REFERENCES....................................................................................................133 CURRICULUM VITAE.........................................................................................150 xi FIGURE LEGENDS PAGES Figure 2.1. Schematic diagram of cryogels formation...........................................16 Figure 2.2. Methods for synthesis of functional polymers.....................................20 Figure 2.3. Schematic diagram of the molecular imprinting process....................28 Figure.2.4. Structure of commonly used functional monomers and cross-linkers 32 Figure 2.5. Schematic represention of IIP synthesis.............................................36 Figure 2.6. Schematic represention of surface imprinting.....................................39 Figure 2.7. Number of published articles about IIP in the last 10 years................39 Figure 3.1. Possible formula of MAH-Cu(II) complex............................................43 Figure 3.2. Possible formula of MAC-Cd(II) complex............................................47 Figure 3.3. Possible formula of MAAsp-Pb(II) complex.........................................49 Figure 3.4. Optic photos of ion imprinted cryogels in different shapes. (A) Pb(II), (B) Cu(II), (C) magnetic Cu(II), (D) Cd(II), imprinted cryogels...............................50 Figure 3.5. Atomic absorption spectroscopy instrument........................................53 Figure 4.1. Swelling ratio of cryogels (Asterisk superscript is involved for magnetic cryogel)..................................................................................................................55 Figure 4.2. FTIR spectra of Cu(II) ion imprinted poly(HEMA-MAH) cryogels........57 Figure 4.3. FTIR spectra of Cd(II) ion imprinted poly(HEMA-MAC) cryogels........58 Figure 4.4. FTIR spectra of Pb(II) ion imprinted poly(HEMA-MAAsp) cryogels.....58 Figure 4.5. SEM photographs of cryogels: A) Cu(II) ion imprinted; (B) Non- imprinted; (C) Cu(II) ion imprinted magnetic; (D) Non-imprinted magnetic;..........60 Figure 4.6. SEM photographs of cryogels: A) Cd(II) ion imprinted; (B) Non- imprinted; (C) Pb(II) ion imprinted; (D) Non-imprinted;..........................................61 xii Figure 4.7. EDX spectra of cryogels after desorption of metal ions: (A) Cu(II) imprinted; (B) Cu(II) non-imprinted; (C) Pb(II) imprinted; (D) Pb(II) non-imprinted; ...............................................................................................................................62 Figure.4.8. The effect of pH on adsorption of Cu(II) by Cu(II) ion-imprinted magnetic cryogel membranes. Concentration: 60 ppm; incubation period: 120 min; T: 25ºC..................................................................................................................65 Figure 4.9. The effect of pH on adsorption of Cu(II) by Cu(II)-ion imprinted non- magnetic cryogel membranes; Concentration: 60 ppm; incubation period: 120 min; and T: 25°C...........................................................................................................66 Figure 4.10. The effect of pH on adsorption of Cd(II) by ion imprinted cryogel membranes; Concentration: 60 mg/L; incubation period: 120 min; and T: 25°C...66 Figure 4.11. The effect of pH on the adsorption of Pb(II) by ion imprinted cryogel membranes; Concentration: 60 mg/L; incubation period: 120 min; T: 25°C..........66 Figure 4.12. The effect of initial concentration on adsorption of Cu(II) by ion- imprinted magnetic cryogel membranes; pH: 5.5; incubation period: 120 min and T: 25°C..................................................................................................................69 Figure 4.13.The effect of initial concentration on adsorption of Cu(II) by ion imprinted non-magnetic cryogel membranes; pH: 5.5; incubation period: 120 min and T: 25°C...........................................................................................................70 Figure 4.14. The effect of initial concentration on the adsorption of Cd(II) by ion- imprinted cryogel membranes; pH: 5.5; incubation period: 120 min; and T: 25°C.......................................................................................................................70 Figure 4.15. The effect of initial concentration in adsorption of Pb(II) by ion- imprinted cryogel membranes, pH: 5.5; incubation period: 120 min; and T: 25°C.......................................................................................................................70 Figure 4.16. The effect of temperature on adsorption of Cu(II) by ion-imprinted magnetic cryogel membranes; Concentration: 30 ppm; pH: 5.5 and incubation period: 120 min......................................................................................................72 Figure 4.17. The effect of temperature on adsorption of Cu(II) by ion-imprinted magnetic cryogel membranes; Concentration: 130 ppm; pH: 5.5 and incubation period: 120 min......................................................................................................73 Figure 4.18. The effect of temperature on adsorption of Cu(II) by ion-imprinted magnetic cryogel membranes; Concentration: 400 ppm; pH: 5.5 and incubation period: 120 min......................................................................................................74 xiii Figure 4.19. Effect of temperature on the adsorption of Cu(II) by Cu(II) ion imprinted non-magnetic cryogel membranes; pH: 5.5; incubation period: 120 min; and concentration (A): 30 ppm, (B): 130 ppm and (C): 400 ppm..........................76 Figure 4.20. The effect of temperature on the adsorption of Cd(II) by ion imprinted cryogel membranes; pH: 5.5; incubation period: 120 min;and concentration (A): 30 ppm; (B): 130 ppm; (C): 400 ppm..........................................................................77 Figure 4.21. The effect of temperature on the adsorption of Pb(II) by ion imprinted cryogel membranes; pH: 5.5; incubation period: 120 min; and concentration (A): 30 ppm; (B): 130 ppm; (C): 400 ppm.....................................................................78 Figure 4.22. The effect of contact time on adsorption of Cu(II) by ion-imprinted magnetic cryogel membranes; Concentration: 60 ppm; pH: 5.5; and T: 25°C......80 Figure 4.23. The effect of contact time on adsorption of Cu(II) by ion imprinted non-magnetic cryogel membranes; Concentration: 60 ppm; pH: 5.5 and T:25°C...................................................................................................................81 Figure 4.24. The effect of contact time on the adsorption of Cd(II) by ion imprinted cryogel membranes; Concentration: 60 ppm; pH: 5.5 and T: 25°C.......................81 Figure 4.25. The effect of contact time for adsorption of Pb(II) by ion imprinted cryogels; Concentration: 60 ppm; pH: 5.5; incubation period: 120 min; and T: 25ºC.......................................................................................................................82 Figure 4.26. Langmuir isotherm model for adsorption of Cu(II) by ion imprinted magnetic cryogel membranes...............................................................................86 Figure 4.27. Freundlich isotherm model for adsorption of Cu(II) by ion imprinted magnetic cryogel membranes...............................................................................87 Figure 4.28. Langmuir isotherm models for adsorption of Cu(II) by ion-imprinted non-magnetic cryogel membranes........................................................................89 Figure 4.29. Freundlich isotherm model for adsorption of Cu(II) by ion-imprinted non-magnetic cryogel membranes........................................................................89 xiv Figure 4.30. Langmuir isotherm model for adsorption of Cd(II) by ion imprinted cryogel membranes...............................................................................................90 Figure 4.31. Freundlich isotherm model for adsorption of Cd(II) by ion-imprinted cryogel membranes...............................................................................................90 Figure 4.32. Langmuir isotherm model for adsorption of Pb(II) by ion imprinted cryogel membranes...............................................................................................91 Figure 4.33. Freundlich isotherm model for adsorption of Pb(II) by ion-imprinted cryogel membranes...............................................................................................91 Figure 4.34. Langmuir isotherms of ion imprinted magnetic cryogels (Cu*-1, Cu*-2, Cu*-3) at different temperatures (4, 25, 32, 40ºC).................................................96 Figure 4.35. Langmuir isotherms of ion imprinted magnetic cryogels (Cu*-4, Cu*-5, Cu*-6) at different temperatures (4, 25, 32, 40ºC).................................................97 Figure 4.36. Langmuir isotherms of ion imprinted magnetic cryogels (Cu*-7, Cu*-8, Cu*-9) at different temperatures (4, 25, 32, 40ºC).................................................98 Figure 4.37. Van’t Hoff plots for adsorption of Cu(II) on the ion imprinted magnetic cryogels.................................................................................................................99 Figure 4.38. Langmuir isotherms for adsorption of Cu(II) by ion imprinted non- magnetic cryogel membranes at different temperatures.....................................101 Figure 4.39. Van’t Hoff plot for the adsorption of Cu(II) by imprinted non-magnetic cryogel membranes.............................................................................................102 Figure 4.40. Langmuir isotherms for adsorption of Cd(II) by ion imprinted cryogels at different temperatures (4, 25, 32, 40ºC)..........................................................103 Figure 4.41. Van’t Hoff plot for the adsorption of Cd(II) by imprinted cryogel membranes..........................................................................................................104 Figure 4.42. Langmuir isotherms for adsorption of Pb(II) by ion imprinted cryogels at different temperatures.....................................................................................106 xv Figure 4.43. Van’t Hoff plots for the adsorption of Pb(II) by ion imprinted cryogels...............................................................................................................107 Figure 4.44. Pseudo first-order kinetic model for adsorption of Cu(II) by ion imprinted magnetic cryogel membranes..............................................................110 Figure 4.45. Pseudo second-order kinetic model for adsorption of Cu(II) by ion imprinted magnetic cryogel membranes..............................................................111 Figure 4.46. Pseudo first-order kinetic model plots for adsorption of Cu(II) by ion imprinted non-magnetic cryogels.........................................................................112 Figure 4.47. Pseudo second-order kinetic model plot for adsorption of Cu(II) by ion imprinted non-magnetic cryogels.........................................................................113 Figure 4.48. Pseudo first-order kinetic model for adsorption of Cd(II) by ion imprinted cryogel membranes.............................................................................114 Figure 4.49. Pseudo second-order kinetic model for adsorption of Cd(II) by ion imprinted cryogels...............................................................................................114 Figure 4.50. Pseudo first-order kinetic model for adsorption of Pb(II) by ion imprinted cryogel membranes.............................................................................115 Figure 4.51. Pseudo second-order kinetic model for adsorption of Pb(II) by ion imprinted cryogel membranes.............................................................................116 Figure 4.52. Comparison of adsorption capacities of ion imprinted magnetic and non-magnetic cryogels; pH: 5.5; incubation period: 120 min and T: 25ºC..........117 Figure 4.53. Comparison of adsorption capacities of magnetic Cu(II) ion-imprinted with non-imprinted cryogels; pH: 5.5; T: 25ºC; and incubation period: 120 min..118 Figure 4.54. Comparison of adsorption capacities of Cu(II) ion imprinted with non- imprinted cryogels; pH: 5.5; T: 25ºC; and incubation period: 120 min................119 Figure 4.55. Comparison of adsorption capacities of Cd(II) ion-imprinted and non- imprinted cryogels; pH: 5.5; incubation period: 120 min; and T; 25ºC................120 xvi Figure 4.56. Comparison of adsorption capacities of Pb(II) ion imprinted with non- imprinted cryogels; T: 25ºC; pH: 5.5; incubation period: 120 min.......................121 xvii TABLE LEGENDS PAGES Table 2.1. Physical methods for the removal of heavy methods……………………6 Table 3.1. Summarizing the strategy for cryogel synthesis……………….………..42 Table 3.2. Composition of Cu(II) ion imprinted non-magnetic cryogels…………..44 Table 3.3. Composition of Cu(II) ion imprinted magnetic cryogels……..………....46 Table 3.4. Composition of Cd(II) ion imprinted cryogels………..………...………..48 Table 3.5. Composition of Pb(II) ion imprinted cryogels…………….…..………..50 Table 4.1. Swelling ratio of PHEMA cryogels……………………………………….56 Table 4.2. Surface area measurements (BET) of ion imprinted and non-imprinted cryogels……………………………………………………………………………..……59 Table 4.3. Langmuir and Freundlich models parameters for adsorption of Cu(II) by Cu(II) ion-imprinted magnetic cryogels……………………………..……..…………88 Table 4.4. Langmuir and Freundlich models parameters for adsorption of Cu(II) by ion imprinted non-magnetic cryogels……………………………………..….….……88 Table 4.5. Langmuir and Freundlich model parameters for adsorption of Cd(II) by ion imprinted cryogel membranes…………………………………………………….90 Table 4.6. Langmuir and Freundlich models parameters for adsorption of Pb(II) by ion imprinted cryogel membranes……………………………………………………91 Table 4.7. Langmuir model parameters for adsorption of Cu(II) by Cu(II) ion imprinted magnetic cryogels at 4ºC and 25ºC……………………………………..94 Table 4.8. Langmuir model parameters for adsorption of Cu(II) by Cu(II) ion imprinted magnetic cryogels at 32ºC and 40ºC……………………………………95 Table 4.9. Thermodynamic parameters for adsorption of Cu(II) by ion imprinted magnetic cryogels……………………………………………………………………100 xviii Table 4.10. Langmuir model parameters for adsorption of Cu(II) by ion imprinted non-magnetic cryogels at different temperatures…………………………………100 Table 4.11. Thermodynamic parameters for adsorption of Cu(II) by ion imprinted non-magnetic cryogels……………………………………………………………….102 Table 4.12. Thermodynamic parameters for adsorption of Cu(II) by ion imprinted non-magnetic cryogels………………………………………………………………..104 Table 4.13. Thermodynamic parameters for adsorption of Cd(II) by ion imprinted cryogels…………………………………………………………………………………105 Table 4.14. Langmuir model parameters for adsorption of Pb(II) by ion imprinted cryogels at different temperatures…………………………………………………..105 Table 4.15. Thermodynamic parameters for adsorption of Cd(II) by ion imprinted cryogels…………………………………………………………………………………107 Table 4.16. Pseudo first-order and second-order kinetic models parameters for adsorption of Cu(II) by ion imprinted magnetic cryogel membranes…………….112 Table 4.17. Pseudo first-order and second-order kinetic models parameters for adsorption of Cu(II) by ion imprinted non-magnetic cryogel membranes………113 Table 4.18. Pseudo first-order and second-order kinetic models parameters for adsorption of Cd(II) by ion imprinted non-magnetic cryogel membranes…….115 Table 4.19. Pseudo first-order and second-order kinetic models parameters for adsorption of Pb(II) by ion imprinted non-magnetic cryogel membranes…….…116 Table 4.20. Comparison of selectivities of ion imprinted with non-imprinted cryogels………………………………………………………………………………..121 Table 4.21. Competitive adsorption of heavy metal ions by Cu(II) magnetic ion imprinted cryogel from multi-metal ions solution…………………………………122 Table 4.22. Competitive adsorption of heavy metal ions by Cu(II) magnetic ion imprinted cryogel from multi-metal ions solution……………………………….....123 xix Table 4.23. Competitive adsorption of heavy metal ions by Cd(II) ion imprinted cryogel from multi-metal ions solution……………………………………………..124 Table 4.24. Competitive adsorption of heavy metal ions by Pb(II) ion imprinted cryogel from multi-metal ions solution………………………...…………………….125 Table 4.25. Simultaneous competitive adsorption of heavy metal ions by Pb(II), Cu(II), and Cd(II) ion imprinted cryogel from multi metal ions solution………….126 Table 4.26. Adsorption/desorption/regeneration cycles for ion imprinted cryogels…………………………………………………………………………………127 Table 4.27. Comparison of this study with other studies………………………….128 1 1. INTRODUCTION Most of heavy metals are necessary in small amounts for body but in high concentrations are toxic [1]. There isn’t any agreed definition of authority units, such as IUPAC for heavy metals, but elements such as Zn, Fe, Cu, Cr, Co, Pb, Cd, Ni, Pd, Ag, Hg, Pt, Au, As, etc. are classified in this group of metals [2]. Accumulation of these metals in living organisms causes serious health problems. These metals after being absorbed by body make connection to cellular units with vital important ones such as proteins, enzymes and nucleic acids and inhibit their functions [3]. Toxicity of these metals most commonly affects the brain and kidneys, but some metals such as arsenic clearly cause cancer [4]. Removal of heavy metals from water is necessary, because these pollutant materials can’t be changed to harmless material as organic pollutants. Many methods have been used for removal of heavy metals. There are many traditional methods, such as chemical precipitation, adsorption, electrochemical process, extraction, floatation, coagulation, ion exchange etc., but these methods are generally expensive or risky [5]. Nowadays, new adsorbents are used for adsorption. Purposes of adsorption with these materials are development of cheaper and more effective process for removal of heavy metals from water [6]. Adsorbents such as lignin, tannin, chitin, chitosan, dead biomass, zeolite, clay, by- products of agricultural industry, activated carbon, carbon nanotubes and synthetic polymer-based ones have been used for adsorption of heavy metals from water [7]. Adsorbent materials have usually a porous structure, and the sorption process generally takes place in the pore walls or in specific locations within the particles [8]. Modified biopolymers that have functional groups have been used because of their widespread presence in nature but, synthetic polymers have been preferred as new generation of adsorbents [9]. These synhetic polymers can be designed for removing specific metals. Hydrogels and cryogels have been used for removing of heavy metals because of their ability of expanding their volumes due to their high swelling capacity in the solvent. These polymers have advantages such as preparation in different shapes, combination with various materials for getting desired properties and resistant to degradation [10]. 2 Cryogels are supermacroporous hydrogels which are formed at subzero temperature by radical polymerization of monomers [11]. Due to their polymeric network with interconnected macropores, they show very low flow resistance and the solution can diffuse into these matrices easily. Generally the pore size depends on the concentration of monomer, ratio of crosslinker, their physicochemical properties and the freezing conditions [12]. Cryogels have a high density of pores. Macroporous structures of cryogels allow transportation of undesirable particles from containing fluid through the gels. Composite cryogels can be formed in order to increase the capacity of the cryogels in water and wastewater treatment applications [13]. Molecular imprinting technology (MIT) is a technique for preparing materials with cavities that are able to recognize a certain molecule according to its shape, size and chemical functionality. Polymerization occurs around the interested molecules called as template. After removal of template molecules, a three dimensional cavity is formed. Specific sites for many molecules such as metal ions, organic molecules, proteins in a synthetic polymer can be created by via MIT [14]. Ion imprinted polymers (IIP) are prepared by copolymerization of monomer, ligand-metal complex and cross-linker. After removing of the template ion, these polymers have high selectivity towards the target ion due to the affinity of the ligand for imprinted metal ions [15]. The first ion- imprinted polymers have been introduced in 1976, but the real development of IIP is more in the last 10 years [16]. This study consists of two main parts; (i) preparation of various ion imprinted cryogels (Cu(II), Cd(II) and Pb(II)) and Cu(II) imprinted magnetic cryogels and use of these cryogels for removing of specific metal ions from aqueous solutions; (ii) thermodynamic and kinetic parameters for these processes. For this purpose, the functional monomers N-methacryloyl-L-histidine (MAH), N-methacryloyl-L-cysteine (MAC) and N-methacryloyl-L-aspartic acid (MAAsp) were prepared by reacting appropriate amino acids with methacryloyl chloride and then, pre-complexes of these monomers with metal ions were prepared. Ion imprinted magnetic and non- magnetic cryogels were synthesized by bulk polymerization of metal ion pre- complex, 2-hydroxyethyl methacrylate (HEMA) and methylene bisacrylamide. The 3 cryogel membranes were characterized by surface area measurements, swelling test, scanning electron microscopy, and FTIR. These cryogels were used for adsorption of heavy metal ions from aqueous solutions in different concentrations, pHs, incubation times and temperatures and their effect on adsorption dynamics was studied. The thermodynamic values such as ∆Hº, ∆Gº, ∆Sº for adsorption were calculated and then thermodynamic possibility of adsorption was indicated. The selectivity of ion imprinted cryogels for each of ion imprinted cryogel and specific ion in presence of other ions was studied. Reuse of this cryogels was examined with repeated adsorption-desorption cycles. Kinetics of the processes was studied as well. 4 2. GENERAL INFORMATION 2.1. Heavy Metals For identification of heavy metals there aren’t any agreed criteria as density, toxicity and atomic weight or definition of authority units, such as the IUPAC. All transition elements, transuranium elements, elements which have metallic property except those required by body and all of p-elements are called toxic metals [1]. However in this group, there are also non-metals such as arsenic that is known as a semi-metal. Heavy metals are adsorbed in soils and sediments. Degree of adsorption depends on electronic structures, diameters, degree of hydration, pH, concentrations, adsorbent structure, oxidation and concentrations of other metals. Heavy metals have very different properties; some of them are used in machine manufacturing, electronics, different parts of our daily life or high- technical jobs [17]. Heavy metal ions in domestic and industrial waste water are very important because of their damage for health of living organisms and ecological systems. The accumulation of these non-biodegradable components in living organisms causes serious health problems. Heavy metals are grouped as toxic (Hg, Cr, Pb, Zn, Cu, Cd, Ni, etc.), valuable (Pd, Au, Ag, Pt, etc.) and radionuclides (U, Th, Ra, etc.) [3]. The trace amounts of heavy metals can be determined in waste water samples using flame atomic absorption spectrometer (FAAS), graphite furnace atomic absorption spectrometer (GFAAS), inductively coupled plasma optical emission spectroscopy (ICP-OES), inductively coupled plasma mass spectrometry (ICP- MS), laser-induced breakdown spectroscopy (LIBS) and anodic stripping voltammetry. 2.1.1. Heavy Metals Toxicity Many heavy metals are necessary in small amounts for the normal development of the biological cycles but in high concentrations are toxic. These metals which are called trace elements for example Zn, Fe, Cu, Cr and Co are necessary for body but the amount more than physiological concentration is known to have toxic side effects [1, 17]. There is another group of heavy metals such as arsenic, lead, cadmium and mercury that are highly toxic for human beings and other living beings even at trace levels [14]. Heavy metals generally are toxic but oxides of 5 heavy metals aren’t toxic. Presence of heavy metal ions in rivers and lakes can cause of several health problems for animals, plants and human beings.The human body can not dispose the metals as a result they are deposited in various internal organs. Large deposits may cause adverse reactions and serious damage to the body [18]. The reason of extreme toxicity of heavy metals in organisms is that they are water-soluble in ions or compounds form and are easily absorbable by living organisms in nature. Generally toxic effect of heavy metals is due to their complexation with organic compounds. These metals after being absorbed by body get connected to cellular units with vital importance such as proteins, enzymes and nucleic acids and inhibit their functions [3]. These metals react with - SH groups of proteins, maybe this event chemically seems simple, but this is important in metabolism and can cause death [19]. In animal body because of their homeostatic mechanism, if concentration of metals doesn’t go over the limit of tolerance or heavy metals don't affect metabolism for long time, metabolism can protect itself [19]. Toxicity of metals most commonly effects on the brain and kidneys but other manifestations can be appearing. Some metals such as arsenic clearly cause cancer [4]. 2.1.2. Removal of Heavy Metals Presence of heavy metals in environmental waters is dangerous for human and other living beings. Organic pollutants can be changed to harmless material with biodegradation, but heavy metals don’t change into harmless material with biodegradation [20]. Therefore removing of heavy metals from water is necessary. Many methods have been used for removal of heavy metals. Methods that are used widely for metals removal is shown below (Table 2.1). 2.1.3. Conventional Refinement Techniques Since ancient times there are traditional methods for removing heavy metals from the environment but some of these methods are expensive and risky due to the possibility of generation of hazardous by-products [18]. For removal of heavy metal ions from wastewater, physical, chemical or biological refinement technique or combined techniques can be selected. Several parameters such as the content and quantity of waste, chemicals and energy requirements, process performance, 6 process economics are considered. Amongst the available techniques, adsorption, ion exchange and membrane processes are frequently used. Table 2.1. Physical methods for the removal of heavy metals Base Commonly used methods Dimension Filters, membranes, particles, gels Density Precipitation Charge Ion exchange resins, electrokinetic systems Specific Affinity Interactions between ligands with particles or macromolecules 2.1.3.1. Chemical Precipitation This method is one of the old methods that were applied for heavy metal removal from water. It is used when economic recovery is not important and complex chemical compounds are not involved. This method is a good choice for high concentrations of heavy metals, but it is not effective for concentrations of less than 50 mg/L [21]. The method is based on conversion of metal ions to the insoluble precipitate such as hydroxide, sulfide, phosphate or carbonate by suitable agents, and removing the precipitate from medium. Organic polymers and alum as coagulants can improve the performance of process. Besides the advantages of cheapness and simplicity, this method has the disadvantages of formation of a large volume of precipitate having a low density, amphoteric character of some metal hydroxides, and inhibition effect of reagents having the ability to form complexes in refining environment. To form the precipitate in hydroxide salt, alkaline reagents such as NaOH, Ca(OH)2 or NH4OH are used. Most metal hydroxide precipitates are formed in the pH range of 8.0-11.0. If Ca(OH)2 is used as precipitating agent, secondary precipitates such as CaSO4.2H2O may be formed. So, precipitation agent having capability to form soluble by-products should be preferred [22], especially when metal recovery is important. Precipitation agents such as FeS and H2S are used 7 for sulfide precipitates. Typically, the metal sulfide solubility value is less than that of metal hydroxide; therefore operation efficiency for metal sulfides gives the better result. To prevent H2S(g) formation, neutralization of acidic waste is required before the precipitation process. Carbonate precipitates that are obtained with Na2CO3 and CaCO3 have larger solubility values than that of hydroxide forms. Phosphate precipitates are formed by adding phosphate or soluble salts of hydroxyl apatite. Blais et al. used dithiocarbamate (DTC), xanthatediethyl dithiocarbamate (DDTC) as reagents to perform the precipitation due to chelate formation [18]. Dean et al. used precipitation method for removing metals such as copper, zinc, iron, manganese, nickel, and cobalt as the hydroxide [21]. 2.1.3.2. Electrochemical Process This method is one of the most interesting traditional methods. In this method, an electrochemical cell is formed that is placed in mixture containing metal ions. Metal ions are deposited on the mercury cathode. High selectivity is the advantage of this method but the long duration of action (slow rate of accumulation) is its major disadvantage [21]. This method is used for recovery of metal ions in the form of pure metal or pure salt solution by electrolysis. Migration of toxic components toward the electrode depends on diffusion, convection and electrolytical migration. The ions having less negative potentials than water in solution fed to the cathode are reduced to metal. The presence of oxygen or other reducible elements decreases the current efficiency and increases the consumption of energy. Controlling the parameters for electrolysis, selectivity for metal ions may be obtained. If the reduction of metal ions is controlled by mass transfer, process performance increases. The electrode area, the mass transport regime and presence of turbulence promoters affect the process [22]. Electrocoagulation and electroflotation are the other options for electrochemical processes. But their investment and operating costs are very high. 2.1.3.3. Liquid-Liquid Extraction This is the widely used method for heavy metal treatment. In this method, chelating reagent is added to the aqueous phase for creating ion-specific complex of metal ion. Solubility of this complex in water is very low; hence complex is extracted by using an organic solvent such as hydroxyquinoline, diethyl 8 dithiocarbamate, and ammonium dithiocarbamate pyrilidine. The major disadvantages of this method are the use of little amount of organic solvent for increasing the efficiency of the aqueous phase separation factor and the difficulty of effective separation of these two phases after extraction. 2.1.3.4. Flotation In flotation, a surface-non-active material with suitable surface-active agent is converted to removable surface-active material by means of gas bubbles formed in solution. Advantages of this method are simplicity and efficiency of the method. Ions that are converted to hydrophobic precipitates by surface-active agents form foam by connecting to gas bubbles. 2.1.3.5. Coagulation and Flocculation Coagulation is destabilization and initial coalescing of colloidal particles and flocculation is the formation of larger particles (floc) from smaller particles. In coagulation process, charge neutralization and bridging mechanisms are important [23]. Following these processes, treatment process is completed by sedimentation and filtration. Coagulation for hydrophobic colloids and suspended particles gives better results. Chemical requirement and tangle volume are disadvantages of this method. 2.1.3.6. Ion-Exchange This physical method is based on exchange of ions between the solution phase and the solid matrix. The method has advantages of high decontamination capacity, treatment efficiency, fast kinetics and cost effectivity. Several parameters such as temperature, pH, contact time and ionic charge effect on the rate of exchange. Ion-exchange resins that are insoluble in water and organic solvents can be synthesized by covalent binding of charged functional groups to the cross-linked polymeric matrices. The matrix is usually polystyrene that contains 3-8% divinyl benzene. The particle size is usually in the range of 20-25 mesh. For the strong acid cation exchanger (-SO3H), cation affinity increases with the cation charge and with the increasing atomic number for different ions having the same charge. Resins with weak acid type cation exchangers that have carboxyl functional groups follow a reversed affinity order for alkali and alkaline 9 earth metals. For strong base anion exchangers having quaternary ammonium (- N+R3) groups, the ion exchange capacity increases with increasing ionic charge. The affinity order for weak base anion exchangers having secondary or tertiary amine groups is similar to the order reported for strong base anion exchangers. Amphoteric ion exchangers, such as AMF-IT and AMF-2M that exchange cation or anion depending on the solution pH can also be used [16]. If original sample contains ferrous ions, the adsorption of organic material will cause the reduction in performance due to chlorine and the possibility to form ferric hydroxide are the disadvantages of the method. 2.1.3.7. Membrane Processes Membrane process is based on the recovery of metal ions from diluted wastewater with semi-permeable membranes. Membrane processes that are used for the removal of heavy metal ions are ultrafiltration (UF), reverse osmosis (RO), nano filtration (NF), microfiltration (MF) and electro-dialysis (ED). Pore sizes of the membranes for MF and UF applications are (0.1 to 3 µm) and (0.01-0.1 µm), respectively [5]. Higher metal recovery performance in NF applications can be achieved due to narrow pore size (0.001- 0.01 µm). In the RO, metallic or polymeric-based membranes are used, and pressure or electric current being the driving force for the passage of solution through the membrane. Less space of apparatus, performance of process and little need for chemicals are the advantages of the method. Requirement of high energy, blockage of membrane pores and the necessity of membranes to renew in about five years are the disadvantages. Juang et al. reported removal of Cu(II) and Zn(II) ions from synthetic wastewater by chitosan-enhanced membrane filtration [24]. 2.1.4. New Methods for Removing Heavy Metals Generally purposes of new methods are development of cheaper and more effective technologies eventually, to decrease the amount of by-product and to improve the quality of the treated water. These new methods are adsorption with new adsorbents, membrane filtration, electrodialysis, metal chelating, and photocatalysis [6]. 10 2.1.4.1. Adsorption This method is one of the common methods for metal removal from water. Adsorption is adhesion of particles to a surface. Adsorbed substance is adsorbate and the substance that having capacity or tendency to adsorb particle is adsorbent [25]. According to interaction between the adsorbent and adsorbate, adsorption is classified into three kinds; Physical, Chemical and Ionic adsorption. Physical adsorption is a result of attraction power between the solid surface and adsorbed substance. Here weak van der Waals forces are active and adsorption occurs as multilayer and reversible manner [26]. Chemical adsorption occurs when there is chemical interaction between functional groups on the solid surface and adsorbed substance. This type of adsorption is single-layered and not reversible. Ionic adsorptions happen when ions with electrostatic forces adsorb in complementary charged zones. In this type of adsorption, ions with high charge and small diameter are adsorbed better than the other ions. When an adsorption phenomenon takes place, there isn’t only one kind of adsorption, maybe there are two kinds or three kinds simultaneously. Adsorption also depends on structure of the adsorbed substance, concentration, pH, and temperature. A material in the case of one component in solvent adsorb better than multi components in solvent on the surface of adsorbent. Adsorption is similar to equilibrium reaction and continues until the concentration of substance on adsorbent surface and solution is balanced. After balancing, concentration of adsorbed substance is constant in solution. Adsorption depends on concentration of adsorbed substance and temperature.Generally adsorption process is performed at a constant temperature to examine the concentration effect in process. The amount of adsorbed substance is plotted against the concentration of adsorbed substance in solution and this mathematical expression is called adsorption isotherm.The Freundlich and Langmuir isotherms are the mostly used adsorption isotherms [25, 26]. Generally in adsorption method first metal ion forms a complex then this complex, is adsorbed on the adsorbent. This method is the sorption of components from gaseous or liquid phases onto the surface of the solid substrate. Besides of design and flexibility, lack of pollutant formation, low investment costs and regeneration of adsorbent are the advantages of this method. In this process, lignin, tannin, chitin, chitosan, dead 11 biomass, zeolite, clay, by-products of agricultural industry, activated carbon, carbon nanotubes and synthetic polymer-based adsorbents can be used. Adsorbents may be grouped as carbon adsorbents (activated carbon, activated carbon fibers, carbon molecular sieves, fullerenes, hetero fullerenes, nano- materials), mineral adsorbents (silica gels, zeolites, clay minerals, inorganic nano- materials, metal oxides, metal hydroxides, activated alumina) and other adsorbents (synthetic polymers, composite adsorbents and mixed adsorbents) [27,28]. Adsorbent materials have usually a porous structure, and the sorption process generally takes place in the pore walls or in specific locations within the particles. The overall rate of process is controlled by diffusion rate of adsorbed substances into the capillary pores and varies with the square root of contact time with the adsorbent [29]. Selection of adsorbent depends on sorption rate and capacity of adsorbent, surface area for particulate adsorbents, mechanical strength, low cost, regeneration and re-usability. Porosity can be obtained by emulsion polymerization of monomers in solvents capable to solve monomer but a poor swelling agent for polymer. In creating nitrogen, oxygen, or sulfur containing binding sites for the polymeric sorbent, a suitable ligand is attached to the support by means of a suitable activation procedure, or a copolymer of a suitable functional monomer having the desired functional coordination sites is synthesized. If the concentration of metal ions in aqueous solution is in the range of 1-100 mg/L, chemical precipitation and electrochemical process are not effective. Ion exchange, activated carbon adsorption and membrane technology are expensive for wastewater having large volumes and containing low concentration of metal ions. In recent years efforts have been made to search low- cost adsorbents that have metal-binding capacity [30]. In adsorption of heavy metals on adsorbent there are three steps, first, the transport of heavy metals to surface of adsorbent; second, adsorption on the adsorbent surface; and third, the transport of adsorbent and adsorbate from solution. 12 2.1.4.1.1. Adsorption on Modified Natural Materials Natural zeolites due to their ion exchange capability gained a significant interest. Clinoptilolite has high selectivity for certain heavy metal ions such as Pb(II), Cd(II), Zn(II), and Cu(II) [26,29]. In recent years synthetic zeolites for heavy metals removal have been reported [28]. Clay–polymer composites, different phosphates calcined at 900ºC, activated phosphate with nitric acid, and zirconium phosphate have been employed as new adsorbents for removal of heavy metals from aqueous solution [28,29,30]. 2.1.4.1.2. Adsorption on Industrial by-Products Chemical modification can improve removal performance of industrial by-products for metal removal from water. Alinnor et al. used fly ash for removal of Cd(II) and Ni(II) from synthetic solution [30]. Uysal et al. used 1, 5-disodium hydrogen phosphate modified sawdust for adsorption of Cr(VI) from ground water [31]. 2.1.4.1.3. Adsorption on Polymers Some polymers besides carbon and hydrogen atoms have other atoms such as oxygen (O), silicon (Si), nitrogen (N) or phosphorus (P) atoms in their chains, these polymers are called "inorganic polymers". Polymer chains can be in linear or branched structure. Increase in branching of polymers causes decrease in their solubility. If these branches bind another main chain, cross-linked polymers are formed. Poly(HEMA) is a polymer that is synthesized by adding methylol (- CH2OH) group to methyl methacrylate and polymerization of this monomer, 2- hydroxyethylmethacrylate (HEMA). Poly(HEMA) and poly(HEMA) with a small amount of cross-linking agent, ethyleneglycol dimethacrylate (EGDMA) can be used for heavy metal adsorption. Cross-linking prevents polymer from dissolving in water and this polymer is called “swollen hydrogel" [32]. One of the new approaches in metal removal methods is use of specific adsorbents [33]. These adsorbents consist of polymeric carrier matrix and ligands which can interact selectively with a metal ion (ion exchange or chelate-forming). Some examples for these specific adsorbents can be given as follows [33]; Poly (β-diketone) adsorbents 13  Polyhydroxyantraquinone adsorbents  Polyphenylalanine adsorbent  Macrocyclic polythioether adsorbents  Polymeric adsorbent that have nitroresorcinol group  Polymeric adsorbent that have oxime group  Polymeric adsorbent that have tris-dithiocarbamates group  Poly(allylamine) and polyacrylamide adsorbents  Polyethyleneimine adsorbents  Phosphorus-containing carbon adsorbents  Sirorez-resin adsorbent 2.1.4.1.4. Modified Biopolymers Biopolymers are used because of their ability to decrease transition metal ion concentrations to sub-parts per billion concentrations, widespread presence and non-detrimental nature to the environment; also having functional groups, such as hydroxyls and amines that increase the efficiency of metal ions uptake and the maximum chemical loading possibility. Cross-linked chitosan, cross-linked starch gel and immobilizing chitosan on the surface of non-porous glass beads are described as modified biopolymer adsorbents for the removal of heavy metals from the water [32]. Amine sites of chitosan are responsible for metal ion binding through chelation mechanisms. Chitosan is also a cationic polymer and it’s also possible to sorb metal ions through anion exchange mechanisms. 2.1.4.1.5. Hydrogels Hydrogels are cross-linked and hydrophilic polymeric structures that have ability of expanding their volumes due to their high swelling capacity in the water. Due to this capacity, they are widely used in the purification of wastewater [36]. Hydrogels are synthesized with polymerization of one or different kinds of monomers in large number. Hydrogels are insoluble because there are hydrogen bonds or van der Waals interactions between main chains. Hydrogels due to their superior properties have been used for medical application in last 30 years. Cross-linked poly(HEMA) is the most widely used hydrogel. Hydrogels have advantages such as resistant to degradation, not absorbed by body, sterilization by heat and preparation in different shapes and forms, also hydrogels can be 14 combined with various materials for getting desired properties.The first application of hydrogels was as lenses due to their mechanical stability, high oxygen permeability and appropriate refractive index. The other applications of hydrogels are artificial tendon materials, bio-adhesive agent in wound-healing, artificial kidney membranes, artificial leather and in advanced applications such as development of artificial muscles. Smart hydrogels can change electrochemical alerts into mechanical work, thus can function as human muscle tissue. In biotechnological applications, particularly bioactive hydrogel can be used in separation of proteins [10]. Various hydrogels were synthesized and applied for adsorption of heavy metals. Kesenci et al. prepared poly(ethyleneglycol dimethacrylate-co-acrylamide) hydrogel beads and used for removing of Pb(II), Cd(II) and Hg(II) from water [32]. Essawy et al. prepared poly(vinylpyrrolidone-co- methylacrylate) hydrogel and used for removal of Cu(II), Ni(II), and Cd(II) [29]. Barakt et al. prepared poly(3-acrylamidopropyl) trimethyl ammonium chloride hydrogel and used it for As(V) removal [33]. 2.1.4.1.6. Cryogels One of the polymer gels is ‘cryogel’ derived from Greek word Krios (Kryos) which means frost or ice. “Cryogels” are supermacroporous hydrogels which are formed at subzero temperature by radical polymerization of monomers [11]. Monomers, crosslinker, initiator and activator are dissolved in solvent such as deionized water. Mixture is immediately incubated under frozen conditions. At subzero temperature, the solvent freezes, forms ice crystals, and then these ice crystals interconnect to each other. Some part of solvent or solute molecules remains unfrozen. Monomers polymerize in liquid microphase, get crosslinked and form gel. After melting of cryogel, an interconnected polymeric network forms [34]. Due to interconnected macroporous structure indicating very low flow resistance, the solution can diffuse into these matrices easily. Pores give unique spongy structure to cryogels. Generally the pore size is 5-100 μm and depends on the concentration of monomer, ratio of crosslinker, their physicochemical properties, and the freezing conditions [12]. Cryogels were first reported 50 years ago and because of properties, soon attracted attention due to large surface area and high density of pores. Cryogels provide high-capacity removal and provide convenience in working in viscous medium such as heavy metals industrial waste. 15 The biomedical and biotechnological potential of these materials has now been recognized by Lozinsky et al. [35]. Cryogels have advantages over other polymers as cryogels can be produced in different sizes and formats like discs, sheets, or monoliths with varying dimensions. Interconnected macroporous structure of cryogels makes them appropriate for the biomedical and biotechnological applications [36, 37]. Intermolecular bonds in the junctions of polymer network are different in gels and cryogels. Bonds of cryogels are chemical bonds as covalent, ionic or non-covalent. Methods which are used for heat-induced (thermotropic) gels cannot be used for the preparation of cryogels. Steps in preparation of cryogels:  The polymer solution containing gel-forming agents is frozen at temperatures, a few degrees below the solvent crystallization point. The frozen system looks as a single solid block, but essentially is heterogeneous and contains unfrozen liquid microphase with the crystals of the frozen solvent.  The crystals of frozen solvent perform as a pore-forming agent. When melted, they leave pores. The surface tension between solvent and gel phase rounds the shape of the pores, making pore surface smoother.  After melting, to form a system of interconnected pores arises inside the gel. The dimensions and shape of the pores depend on many factors; the most important is the concentration of initial materials.  Micropores are formed in the polymeric phase. Thus, cryogels have both heterophase and heteroporous structure [38,39]. 16 Figure 2.1. Schematic diagram of cryogel formation. Cryogels are used in bioseparation ( in bed chromatography as stationary phase by Arvidsson et al. and Chase et al. [40-41]), for immobilization of biopolymers as enzymes by Kokufuta et al., polysaccharides by Hayashi et al., nucleic acids by Cocquemcot [42- 43], and as carriers for cell immobilization [44,45]. 2.1.4.1.6.1. Cryogels in Treatment of Water and Wastewater For removal of heavy metals from water, the conventional methods are not always sufficient. Macroporous structure of cryogels allows transportation of undesirable particles from containing fluid through the gels. Composite cryogels can be formed in order to increase the capacity of the cryogels in water and wastewater treatment applications. Wang et al. used hydroxyapatite –PVA composite cryogels for removal of cadmium ions [13]. A cryogel having thiol functionalized groups on their surface for removing arsenic (V) from water was used. Composite cryogels containing MIP adsorbents have been used for water and wastewater treatment. The selectivity of MIP particles and the macroporous structure of cryogels make a unique combination for preparation of composite cryogels [46]. Hajizadeh et al. used composite cryogels containing activated carbon for treatment of water (phenol adsorption) [47]. Tekin et al. used composite cryogels containing imidazole group for removal of heavy metal ions such as Pb(II), Cd(II), Zn(II) and 17 Cu(II) from water [48]. Papancea et al. used PVA cryogel membranes for removal of metal ions [Cu(II), Ni(II) and Pb(II)] from aqueous solutions [49]. 2.1.4.1.6.2. Composite Systems Forming the multi-phase materials by two or more materials with maintaining their characteristics and limits can be defined as composite system. The composite materials have all properties of their components [50]. A new concept in chromatography is the preparation of sorbents that have high flow path and high surface area by embedding polymeric particles in various macroporous gels. Preparation of composite systems is preparation of cryogel with a higher surface area, the larger pore size and desired properties. Cryogels that are known as macroporous gels can be effective in removal of heavy metals from viscous industrial wastewater because of providing lower flow resistance and higher separation capacity. Because of their high flow rate they can be used in chromatographic column structure. Composite system in the structure of column was prepared by embedding molecular imprinted particles in the macroporous gels. This composite system without clogging was used in the removal of template molecule from wastewater due to its high flow rate [50]. 2.1.4.2. Membrane Filtration According to new membrane processes, treatment of wastewater containing copper and cadmium ions was accomplished by both of reverse osmosis (RO) and nanofiltration (NF) technologies. The results showed high removal efficiency of the heavy metals (98% and 99% for copper and cadmium, respectively) [51]. Multiple membrane process was used for selective separation. Commonly these processes are comprised of three steps; first, UF and microfiltration (MF) are used for separation of organic and suspended matters then, electrodialysis (ED) for desalination lastly, NF and RO are separately used to treat the concentrate from ED [52]. Recently polymer-supported ultrafiltration (PSU) technique has been shown that is favorable for effective removal of heavy metal ions from industrial influent [53]. Complexation–ultrafiltration is another technique that is used as an effective removal method for heavy metals by Petrov et al. [54], Barakat et al. [55] and Trivunac et al. [56]. Ferella et al. used surfactants-enhanced ultrafiltration process for removal of lead and arsenic by using cationic (dodecylamine) and 18 anionic (dodecyl benzene sulfonic acid) surfactants [57]. Recently modified UF blend membranes based on cellulose acetate (CA) with polyether ketone [58], sulfonated polyetherimide (SPEI) [59] and polycarbonate [60] were used for heavy metals removal from waste water. New integrated process combining adsorption, membrane separation, and flotation was developed for the selective removal of heavy metals from wastewater. This process contains three stages; first, heavy metal bonding (adsorption) in a bonding agent, then, wastewater filtration to separate the loaded bonding agent by cross flow microfiltration for low- contaminated wastewater or a hybrid process combining flotation and submerged microfiltration for highly contaminated wastewater lastly, bonding agent regeneration [61,62]. In another new hybrid process, flotation and membrane separation were developed by integrating specially designed submerged microfiltration modules directly into a flotation reactor [63]. Klaassen et al. and Madaeni et al. reported the other hybrid processes for heavy metals removal [64,65]. 2.1.4.3. Electrodialysis Electrodialysis (ED) in fact is a membrane separation in which ions in the solution are passed through an ion exchange membrane by applying an electric potential. Membranes have anionic or cationic characteristics and are in the form of thin sheets of plastic materials. In this method, solution with ions passes through the cell compartments, the ions cross anion exchange and cation-exchange membrane, anions migrate to cathode and cations migrate to anode [66]. Tzanetakis et al. removed Ni(II) and Co(II) ions from a synthetic solution by a high-performance membrane [67]. It was found that performance of an ED cell is almost independent of the type of ions and only depends on the operating conditions and the cell structure [68]. 2.1.4.4. Photocatalysis This method is based on two stages; first, to form electron–hole pairs (e-/h+) in the conduction and the valence band of the semiconductor; second, migration of these charge carriers to the semiconductor surface then, reducing or oxidizing species in solution having suitable redox potential as organic pollute or heavy metal. This method was used with different semiconductors for removing different 19 heavy metals. Cu(II) was removed with TiO2 (semiconductor) [69]. Heterogeneous photocatalytic oxidation of arsenide to arsenate in aqueous TiO2 suspensions was applied for the remediation of arsenide contaminated water [70]. 2.1.4.5. Metal Chelating Method Specific polymeric adsorbents which are used in chromatography methods in view of conventional separation methods are gaining more and more importance with each passing day [71]. Metal chelating chromatography method in the removal of heavy metal ions from environmental waters is the best compared to other chromatography methods because of higher selectivity and sensitivity. Adsorbents in this method consist of a carrier matrix and a ligand which can attach selectively with the metal ions (chelate-forming) [72,73]. Commonly used first matrix carriers were inorganic (silica, aluminum oxide, glass, etc.), but in recent years synthetic polymers (polystyrene, polymethylmethacrylate, etc.) have been developed and reported [72]. These synthetic polymer matrices can be produced easily and in various forms (microsphere, membrane or fiber), also can be modified to various functional groups for effective interaction between metal and matrices. Ligands (materials which can be chelated) are attached to carrier matrices for specific interaction with metal ions and their removal from aqueous media. Usually chelate forming functional groups contain oxygen, nitrogen and sulfur atoms. Nitrogen in the structure of functional group can be in the form of primary, secondary and tertiary amine, nitro, nitrozo, azo, diazo, nitrile, amide, and other groups. Oxygen can be in the form of phenol, carbonyl, carboxyl, hydroxyl, ether or phosphoryl groups and sulfur in the form of thiol, thioester, and disulfide groups. These groups can be attached on the polymer with chemical modification of the ligand or synthesis of sorbents by monomeric ligands. Functional polymers are synthesized by two different ways [72]. 20 Reactive monomer Reactive polymer Modified Monomer Modified polymer Figure 2.2. Methods for synthesis of functional polymers. The heavy metal ions after chelating can be collected selectively by the sorbent in the result of fitting specific functional groups on the polymeric matrix [74]. In the carrier matrix (sorbent), non-specific interactions (electrostatic, etc.) are minimized, on the other hand these adsorbents are synthesized by an inert material (usually polymeric base) in the form of spherical particles. Some of commercially available matrices are produced in the form of porous materials to increase the surface area of interaction and to contain porous surface so that metal ions can enter easily. Besides mechanical strength and ease of use inside the column hydrodynamically, matrices should be inert, should not reduce specificity of specific adsorption and have suitable functional groups for ligand binding. Principles of metal chelate chromatography are given as follows: Adsorption of metal ions is provided by feeding the solution that contains metal ions through column that is filled with the adsorbent. The second step, desorption of adsorbed metal ions by changing pH or ionic strength. Selection of carrier matrix in metal chelate chromatography is very important. Briefly matrix should have these properties:  Chemically inert  Dimensions uniform, spherical and rigid  Easily can be derivatized and clamped with a ligand 21  In the column applications, resistant to 5 atm pressure and having suitable surface and size for adequate liquid phase flow rate in column. 2.1.5. Cadmium Cadmium is in the IIB group with an atomic weight of 112.41. This metal is present in the nature in the form of sulfide, carbonate or oxide in zinc, lead and copper ores. Density, melting point and boiling point of this metal are 8.65 g/cm3, 320.9ºC and 765ºC, respectively. This metal does not have any basic biological function for humans. Its biological half-life in human’s body is 10-35 years. The U.S. Environmental Protection Agency (EPA) categorizes this metal in first category between 126 primary pollutants [75]. This metal is similar to zinc element in chemical properties. Cadmium can replace zinc that is an essential trace element for many plants, animals and micro-organisms and can disrupt metabolic processes. Cadmium is an active enzyme inhibitor. Cadmium is used in photography, TV phosphors, metal coating, anti-corrosion agent, pigments, and as a stabilizer in Ni-Cd cells. Cadmium can be delivered into water by waste batteries, paints, metal refinery facilities, galvanized pipe corrosion, soil and rock errosion, etc. The maximum allowable concentration of cadmium in water is 5 ppb [90] and minimum allowable discharge amount by WHO is 0.01 mg/dm3. When rate of hydrolysis is negligible, cadmium is in Cd(II) form. This form is the predominant form in the acidic, neutral and slightly alkaline medium. This metal forms inorganic complexes with anions such as carbonate, chloride and sulfate and organic complexes with humic acids. In the high pH values besides soluble ions [Cd(OH)+, Cd(OH)3 -] there are insoluble molecules such as Cd(OH)2. Amount of cadmium transferred from industrial wastes to the environment is estimated as 680 tons per year [76]. Cadmium can cause some health problems such as, kidney damage, cardiovascular disease, and high blood pressure [75]. 2.1.6. Copper Copper is in the IB group with an atomic weight of 63.546. This metal is present in the nature in the form of sulfides, carbonate or oxide in the chalcopyrite, chalcocite, azurite, malachite and cuprite minerals. Density, melting point and boiling point of this metal are 8.96 g/cm3, 1084.62ºC and 2562ºC, respectively. Copper is essential to all living organisms as a trace element. The suggested safe 22 level of copper in drinking water for humans varies depending on the source, but tends to be pegged at 2.0 mg/L. The U.S. Environmental Protection Agency's Maximum Contaminant Level (MCL) in drinking water for copper is 1.3 mg/L. Cu is an essential micronutrient, is a component of several enzymes mainly participating in electron flow and is a catalyst in redox reactions. Copperiedus is an excess of copper in the body [77]. Copperiedus can occur from exposure to excess copper in drinking water or other environmental sources. Copper in body damages proteins, lipids, and DNA. Copper poisoning causes vomiting, hematemesis (vomiting of blood), hypotension (low blood pressure), melena (black "tarry" feces), coma, jaundice (yellowish pigmentation of the skin), and gastrointestinal distress [78]. Long-term exposure to copper can damage the liver and kidneys. According to the latest research at Sanford-Burnham Medical Research Institute, copper causes testicular cancer [79,80]. The copper is usually used in electrical wires, roofing, plumbing and industrial machinery. 2.1.7. Lead Lead is in the IVA group with an atomic weight of 207.2. Density, melting point and boiling point of this metal are 11.34 g/cm3, 327.46ºC and 1749ºC, respectively. Metallic lead is rare in nature and found in ore with zinc, silver and (most abundantly) copper. The main lead mineral is galena (PbS), cerussite (PbCO3) and anglesite (PbSO4) [81]. Lead is used in building construction, lead- acid batteries, bullets, pewters, fusible alloys, and as a radiation shield. Lead is a poisonous substance to animals and humans. Lead is a highly poisonous metal (regardless if inhaled or swallowed), affecting almost every organ and system in the body. Lead can accumulate both in soft tissues and the bones. The main damage of lead is in the nervous system both in adults and children. Long-term exposure of adults can cause decrease in functions of the nervous system, also long-term exposure to lead or its salts (especially soluble salts or the strong oxidant PbO2) can cause nephropathy, and colic-like abdominal pains. It may also cause weakness in fingers, wrists, ankles, small increases in blood pressure, particularly in middle-aged and older people or anemial. Exposure to high levels of lead can damage the brain and kidneys in adults or children at least cause death. In pregnant women, exposure to high level of lead may cause miscarriage. Chronic, high-level exposure has shown to reduce fertility in males [82]. Lead 23 enters in drinking water with corrosion of house hold plumbing systems and erosion of natural deposits. Lead leaches into water through corrosion by a chemical reaction between water and plumbing system, also lead can leach into water from pipes, solder, fixtures and faucets (brass), and fittings. The amount of lead in drinking water also depends on the types and amounts of minerals in the water, staying time of water in pipes, the pipes wearing, acidity of water and its temperature. EPA estimates that 10 to 20 percent of human exposure to lead may come from lead in drinking water [82]. The U.S. Environmental Protection Agency’s Maximum Contaminant Level (MCL) in drinking water for lead is 0.015 mg/L. 2.1.8. Polymer Based Adsorbents for Removal of Metals Biological ligands which can be used for metal ion removal are peptides, macrocyclic chelating ligands and nucleobases (nucleic acids). Transition metals form coordinate covalent bond with protein ligands by electron pair in protein ligands. Commonly these ligands have S, N, and O atoms. These ligands have functional groups such as SH, -SS, -NH2, -NH, -OH, -OPO3H or carbonyl. The most common metal bonding units are Cys, His, Asp, Glu and more rarely Met, Asn, Gln, Ser, Thr and Tyr [83]. The coordination number of metals changes from one to eight depending on metal species. Protein groups capable of binding metal are called metallothionein, important units are cysteine (Cys) and sulfhydryl groups in cysteine, are responsible for metal binding.Thiols that have low molecular weight as cysteine‘s sulfhydryl group are reactive and range of their pKa is 8-1. The use of ion-imprinted inorganic polymers that are prepared by sol- gel process is very interesting, but because of different functional groups such as amine, organosilanes and thiols, different metal ion bonding locations occur. Passerini et al. used silanized glass matrix with immobilized glutaraldehyde and the cysteine containing system for pre-concentration of Cd(II), Co(II), Cu(II), Hg(II), Pb(II), and Zn(II) ions, and capacity values for these metals were 12.48, 5.50, 7.86, 6.06, 11.66 and 7.88 mmol /g, respectively [84]. Cd(II) imprinted (3– mercaptopropyl) trimethoxysilane-SiO2 system prepared by sol-gel method was used for concentrations of 10, 20 and 50 mg/L of Cd2+ and could remove 80% Cd(II) [85]. Li et al. modified the Cd(II) imprinted mercapto-silica polymer with tetraethyl ortho silicate for removing of Cd(II) and reported the capacity value as 24 83.89 mg/g [85]. Buhani et al. reported capacity value for Cd(II) removal as 53.3 mg/g by VA / HA composite cryogel [86]. Silica sol-gel matrices that are modified with polyethyleneimine, polyacrylic acid or EDTA can remove Cd(II) in the range of %90 [87]. Removal capacity value of bentonite–modified cysteine for Pb(II) and Cd(II) was 0.503 and 0.525 mmol/g, respectively [88]. Single imprinted 3- mercaptopropyl–trimethoxysilane was reacted with Cd(II) -chitosan complex that was cross-linked by epichlorohydrin then particle was cross-linked by tetraethoxysilane and double-imprinted adsorbent was synthesized. Capacity value for Cd(II) single- imprinted one was 342 mg/g whereas that for double- imprinted was 172 mg/g [86]. Özkütük et al. prepared the ion imprinted poly(HEMA-MAC) composite magnetic particles and reported capacity value as 28.45 μmol/g for removal of Cd(II) [89]. Ion imprinted polymeric particles were synthesized by copolymerization of binary ligand (zz)-N, N'-bis (2-aminoethyl) but- 2-enediamide, Cd(II) complex and pentaerythritol triacrylate crosslinker. Adsorption capacity value was 32.56 mg/g for Cd(II) [90]. 4-vinylpyridine (monomer) and ethyleneglycol dimethacrylate (cross-linker) in the presence of Cd(II) and quinaldic acid (complexing agent) were copolymerized and capacity value as 45.0 mg/g for Cd(II) was reported [91]. Capacity value for solid phase extraction by phenol-formaldehyde-Cd(II) -2-(p-sulfofenilazo)-1, 8- dihydroxy naphthalene-3, 6-disulfonate system ion imprinted polymeric system was reported as 270 µg/g. This capacity value was more than the capacity value reported for non ion imprinted system [92]. Singh et al. prepared a new chemically modified cellulose microfiber through oxidation with sodium periodate and functionalized with N, N-bis(2-aminoethyl)-1, 2-ethanediamine and applied for removal of Cd(II) and reported the maximum sorption capacity as 4.59 mg/g [93]. Segatelli synthesized poly(EGDMA-HEMA) microspheres that used dye, Congo red as metal chelating agent and capacity value was obtained as18.3 mg/g for Cd(II) removal [94]. Activated carbon sourced from BASS Borassus aethiopum (seed shells) and CONS Cocos nucifera (shells) were used for removal of Pb(II) and Cd(II) from wastewater. The adsorption capacity value for Pb(II) was found to be 12.19 mg/g and 24.39 mg/g for activated BASS and CONS respectively, also for Cd (II) was found to be 10.20 mg/g and 25.797 mg/g for activated BASS, and CONS, respectively [95] . Abollinoa et al. studied the adsorption of Cd, Cr, Cu, Mn, Ni, Pb and Zn on Na-montmorillonite (clay) in the presence of ligands. The 25 capacity of Na-montmorillonite for Cu(II), Pb(II), and Cd(II) was 3.04, 9.58, 5.20, and 3.61 mg/g [96], respectively. Coal fly ash as a low cost adsorbent was applied for removal of lead, cadmium and copper from aqueous solutions and capacity value was obtained as 10.0 mg/g, 5.0 mg/g and 2.8 mg/g, respectively [97]. NiŃã et al. used calcium alginate (a natural polymer obtained from marine algae) microparticles for removal of Pb(II) and Cd(II) from synthetic wastewater samples and capacity values were 167 and 182 mg/g, respectively [98]. Zhang et al. synthesized a magnetic ion-imprinted polymer by using 3-(2-aminoethylamino) propyltrimethoxysilane (AAPTS) as the functional monomer, tetraethylorthosilicate (TEOS) as the cross-linker, and Pb(II) as the template and used for removal of Pb(II) from real environmental samples. The maximum adsorption capacity was found as19.61 mg/g [99]. Zhu et al. synthesized lead ion-imprinted micro-beads with combination of two functional monomers (1,12-dodecanediol-O,O'-diphenyl- phosphonic acid (DDDPA) and 4-vinylpyridine) and adsorbed lead ions by these polymers.The maximum adsorption capacity of the lead ion-imprinted micro-beads was calculated as 93.55 mg/g [100]. 2.1.9. Determination of Heavy Metals For determination of heavy metals in trace amounts in the water, wastewater, sediments, air, soil, plants, foods, fertilizers and animal tissues, there are various instrumental methods. Flame atomic absorption spectrometry (FAAS), electrothermal atomic absorption spectrometry (ETAAS), inductively coupled plasma atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS), instrumental neutron activation analysis (INAA) and cold vapor atomic absorption spectrometry (CVAAS) have been used in the analysis of samples [101]. Usually flame atomic absorption spectrometry (FAAS) is used for determination of metals in trace amount. This technique is typically used for determinations in the mg/L range, and may be decreased to μg/L for some elements. AAS can be used to determine over 70 different elements in solution or directly in solid samples. If the metal concentration is in the range of μg/L, electrothermal or graphite furnace atomic absorption spectrometry is used. Also hydride generation atomic absorption spectroscopy (HGAAS) should be used for determination of some metals such as As, Sb, Bi, Ge, Pb, Se, Te, and Sn. In this method, elements react with sodium tetrahydroborate (NaBH4) and form their 26 reduced hydrides. These hydrides are determined by hydride generation atomic absorption spectroscopy method [102]. 2.1.9.1. Atomic Absorption Spectrometry This method is based on absorption of optical radiation (light) by free atoms in the gaseous state and quantitative determination of elements. The electrons of the atoms that absorbed light can be promoted to the higher energy state for a short period of time (radiation of a given wavelength). Amount of absorbed light depends on amount of atoms [102]. 2.1.9.2. Absorption Principles Due to quantum theory, a photon with hט energy is absorbed by an atom and the electrons of atom in the base state are promoted to the higher energy state. Planck in 1999 described the energy difference between two energy states as: ∆E= Ei - E0= hט = hc/λ (2.1) Ei= Energy of electron in transition state, J E0= Energy of electron in base state, J h= Planck’s constant, 6.63 × 10-34 m2 kg/s Frequency of the absorbed light , Hz =ט C= Speed of light, 3 × 108, m/s λ= Wavelength of absorbed light, m Lambert in 1760 found when a photon passes through the homogeneous medium, the intensity of light decreases but ratio of the transmitted light intensity to the incident light intensity is independent of the intensity of light [102]. I= I0.e -xd (2.2) X is absorption coefficient and depends on concentration. X= k.c (2.3) After Lambert, Beer improved the Lambert’s law: 27 A= log I0/I= k.c.d (2.4) A= Absorbance I0= the incident light intensity I= the transmitted light intensity K= absorption coefficient (depends on wavelength and medium), L/mol.cm c= Concentration of absorbed substance, mol/L d= the path length, cm 2.2. Molecular Imprinting Technology Every time environmental, pharmaceutic and biotechnology areas need fast and efficient novel methods for controlling the process and obtaining true product, therefore researchers search for better, more selective and sensitive analytical methods. Molecular imprinting technology (MIT) is preparing materials with cavities that are able to recognize a certain molecule according to shape, size and chemical functionality. Polymerization occurs around the interested molecules called as template. After removal of template molecules, a three dimensional cavity is formed in solid matrices. Specific sites for many molecules such as metal ions, organic molecules, proteins, and large species (cell, bacterie, etc) in a synthetic polymer can be created via MIT. This technique has been widely applied in many fields such as solid-phase extraction, chemical sensors and chromatographic separation due to their physical/chemical stability, thermal stability, low cost and easy preparation [103]. The first molecular imprinting material (silica gels) was synthesized in the early 1930. Polymerization reaction is a very complex process and is affected by many factors such as type and concentration of the monomer, cross-linker, initiator, temperature, and time of polymerization, whether the presence or absence of magnetic field, and volume of the polymerization mixture. For obtaining ideal imprinted polymer, a variety of factors should be optimized. Sometimes preparation of imprinting polymer is a very time-consuming process [104]. 28 Figur